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PROCEEDINGS 


OF THE 


ROYAL SOCIETY OF LONDON. 


Series B 


CONTAINING PAPERS OF A BIOLOGICAL CHARACTER 


VOL. XCIII. 


LONDON: 


PRINTED FOR THE ROYAL SOCIETY anp Soup sy 
HARRISON AND SONS, LTD., ST. MARTIN’S LANE, 
PRINTERS IN ORDINARY TO HIS MAJESTY. 


JULY, 1922. 


LONDON : 
HARRISON AND SONS, LTD,, PRINTERS IN ORDINARY TO HIS MAJEi 
ST. MARTIN’S LANE. Tos yea 


CONTENTS. 


SERIES B, VOL. XCIII. 


No. b 649.—January 2, 1922. 


Address of the President, Prof. C. 8. Sherrington, at the Anniversary Meeting, 
Novem per S OSM OOM sa cesntseer seuodte senses ataae bed sesbne de onde aisdeaniitaapeiiasbiesiedanarcezene 


On the Optical Rotatory Power of Crystalline Ovalbumin and Serum Albumin, 
By Elrid Gordon Young, Ramsay Memorial Fellow. Communicated by Prof. 
Hep Grsvil op kan sei Sr eee Manteca aoncAe see dats dasehiadeenweseadenceicadddescdeamaaasesenneas anes 


Kxperiments on Amphibian Metamorphosis and Pigment Responses in Relation 
to Internal Secretions. By Julian 8. Huxley (New College, Oxford), and 
Lancelot T. Hogben (Imperial College of Science, London). Communicated 
Tb) denote: 1Din ha duuleyvol Baie Ke 108) obs ae hs ancedalts qari asonGn none nS adopbaendcddacoonconohonhencade 


Studies in Bacterial Variability.—On the Occurrence and Development of Dys- 
agglutinable, Ku-ageglutinable and Hyper-agglutinable Forms of Certain 
Bacteria. (A Report to the Medical Research Council.) By E. W. Ainley 
Walker. Communicated by Prof. Georges Dreyer, FURS. ........ccccccenee een eeees 


No. B 650.—February 1, 1922. 


The Titration Curve of Gelatine.—Report to the Medical Research Council. By 
D. J. Lloyd, Biochemical Laboratory, Cambridge, and C. Mayes, Physical 
Laboratory, Eton College. Communicated by Prof. F. G. Hopkins, F.R.S. 


The Hemolytic Action of Sodium Glycocholate. By Eric Ponder. Communicated 
DYASIPIBe Shake yec hater shabu Sn GeeecamocediGaccdasecuaarseserccesntssdsncesadeauariessace 


The Mechanism of Ciliary Movement. By J. Gray, M.A., Fellow of King’s 
College, Cambridge, and Balfour Student, Cambridge University. Com- 
MUUNEAted bys tenors IeiSy Gracin ev wks Sulessusse sceeasee ise /steate de «scsissahts ssemesl 


The Mechanism of Ciliary Movement. I1—The Effect of Ions on the Cell 
Membrane. By J. Gray, M.A., Fellow of King’s College, Cambridge, and 
Balfour Student in the University of Cambridge. Communicated by Prof. 
J. S. Gardiner, F.R.S. oo... FA eT Oo ON, Rea Ge ee saassaae 

G2 


PAGE 


15 


36 


54 


69 


86 


104 


122 


On the Hypertrophy of the Interstitial Cells in the Testicle of the Guinea-Pig 
under Different Experimental Conditious. By Alexander Lipschiitz, M.D., 
Professor of Physiology (in collaboration with Benno Ottow, M.D., Charles 
Wagner, Sc.D., and Felix Bormann). Communicated by F. H. A. Marshall, 
F.R.S. (Plates 1 and 2) ......i sc. .ccesescaeensenseenseesens SeponEDRESBHODODInAAGAGADADo2I0N%¢ 


On the Irritability of the Fronds of Aspleniwm bulbiferum, with Special Reference 
to Graviperception. By T. L, Prankerd, B.Sc, F.L.S. Lecturer in Botany, 
University College, Reading. Communicated by Prof. W. M. Bayliss, F.R.S. 
(ZENG) Yaeme eRe CeEERE rere ceeec perbocdo benno macoec’Sacecn so oscisascaocss00c Seqsoanadhicus co: ‘ido 


No. B 651.—March 1, 1922. 


The Dia-Heliotropic Attitude of Leaves as determined by Transmitted Nervous 
Excitation. By Sir Jagadis Chunder Bose, F.R.S., Director, Bose Institute, 
Calcutta. Assisted by Satyendra Chandra Guha, M.Sc., Research Student, 
Bose: Institute, Calcutta “cicciscctecscoucesscensoncesesiantiee si aacteeesiiaetesssuiee ssh eee tenes 


The Ultra-Violet Absorption Spectra and the Optical Rotation of the Proteins of 
Blood Sera. By S. Judd Lewis, D.Sc. (Tiibingen), B.Sc. (London), F.1.C. 
Communicated! by Profs dN. Collie WARES) tepesrssirsctaisasiestenehen neh eeeeeteeht emer 


The Colouring Matter of Red Roses. By Geotlrey Currey. Communicated by 
Prof. Py Keeble, (BARISY 3 Skis... Sui deeceeeeas ate eaae eat ehne siete eterna ae an eee aaa eeee 


The Kata-thermometer as a Measure of Ventilation. By Leonard Hill, F.R.S., 
EL Me Vernon’ and): Hargood-Ashy.s..csssssectenacenesecsaccrenence ereiee ae eae eneene 


On the Heating and Cooling of the Body by Local Application of Heat and Cold. 
By Leonard Hill, M.B., F.R.S., D. Hargood-Ash, B.Se., and J. Argyll 
Campbell vie Nees eceecees so'ayncaficaus velemaene ca aot aueoeecuemuame meh aL aus eden sadgeneeceeeeem 


On the Oxidation Processes of the Hchinoderm Egg during Fertilisation. By 
@yShearer, MIR S: 3. cicevetacsvaucdee savasossseteeameenee eee sameaeneaee as soiduseiccdeulsmeseueneee 


The Depressor Nerve of the Rabbit. By B. B. Sarkar. Communicated by 
Singh. Shanpey schaters hy Ro.) 5 Colabe)4) mereeresherseseere sseaeierieseeieenieae talevies nee 


PAGE 


132 


148 


178 


194 


198 


207 


213 


230 


The Coagulation of Protein by Sunlight. By Elvid Gordon Young, Ramsay - 


Memorial Fellow. Communicated by Prof. F. G. Hopkins, F.R.S. .............5. 


No. B 652.—April 1, 1922. 


On the Development and Morphology ‘of the Leaves of Palms. By Agnes 
Arber, D.Sc., F.L.8., Keddey Fletcher-Warr Student of the University of 
Tiondon. Communicated by Prof. B. W. Oliver, BiR.S!) iicccccccssssccuecesesseccense 


Studies in the Fat Metabolism of the Timothy Grass Bacillus. By Marjory 
Stephenson, Beit Memorial Research Fellow, and Margaret Dampier Whetham. 
Communicated) by; Prof. PG: Hopkins) HRA SY crermsssss-arenceeseceeeebeeteneiast see 


235 


249 


262 * 


Vv 


Recoil Curves as Shown by the Hot-Wire Microphone. By Lieut.-Colonel 
C. B. Heald, C.B.E., M.D., and Major W. 8. Tucker, R.E., D.Sc. Com- 
municated by Prof. C. S. Sherrington, Pres. R.S. (Plates 5-8)...........scceceeeee 


The Velocity of the Pulse Wave in Man. By J. Crighton Bramwell, M.B., 
MID RAC Mee, Biavel A, Ne, LEUNG INES: einen eo anepodenboncHecocdcooROcou Ge Anges Booc Ae uaCSBBEEEABEE 


No. B 653.—May 1, 1922. 


On a Remarkable Bacteriolytic Element found in Tissues and Secretions. By 
Alexander Fleming, M.B., F.R.C.5. Communicated by Sir Almroth Wright, 
IBLIS SSH ~ (HENS IS). Gaetan den dangbb ORS aC RRDRUE RS HOCO SRO SEER CERT RC eae a Hse Nan erin 


The Pigmentary Effector System. 1.—Reaction of Frog’s Melanophores to 
Pituitary Extracts. By Lancelot T. Hogben, M.A., D.Sc, and Frank R. 
Winton, M.A. Communicated by Prof. E. W. MacBride, LL.D., F.R.S. ...... 


Relationships between Antiseptic Action and Chemical Constitution with special 


reference to Compounds of the Pyridine, Quinoline, Acridine and Phenazine 
Series. By C. H. Browning, J. B. Cohen, F.R.S., R. Gaunt, and R. Gulbransen 


The Action of “Peptone” on Blood and Immunity thereto. By J. W. Pickering, 
D.Sc. and J. A. Hewitt, Ph.D. B.Sc. Communicated by Prof. W. D. 
Eta Mo ubonse BRA Ss) eee seueictt sess aoe saucer ach atone tie se acleuls cto tsaeeseccemen techereeeees 08 


No. B 654.—June 1, 1922. 


Active Hyperemia. By D. T. Harris (Beit Memorial Research Fellow). Com- 
MMUNIeabedabyperom Wei banliss, HMRAS. seisescossastsecmerteserice sche erceescuesssiasce 


The Acidity of Muscle during Maintained Contraction. By H. E. Roaf. Com- 
municatedsbyacm Cysrsherring bon, PLeswhuss) cs.csaieessececocceescnoersensccescsccese 


On the Heat Production and Oxidation Processes of the Echinoderm Egg during 
Fertilisation and Early Development. By C. Shearer, F.R.S. ........ccceeeceeeees 


Further Observations on Cell-wall Structure as seen in Cotton Hairs. By 
W. Lawrence Balls, M.A., Se.D. (Cantab.), and H. A. Hancock. Com- 
municated by, Dr ee ubeyblackmans Hon. Sa) 0 (late O))cesssssccse-seccccnsenses cose sos. 


Observations on the Distribution of Fat-Soluble Vitamines in Marine Animals 
and) Plants.) by7d/0 han Ey orb, Disc.) WOT WeMmiR.S: --c.ccssecccesssceseueacesaeseacss- 


No. B 655.—July 1, 1922. 


On Blood-Platelets: their Behaviour in “Vitamin A” Deficiency and after 
“ Radiation,” and their Relation to Bacterial Infections. By W. Cramer, 
A. H. Drew, and J. C. Mottram. Communicated by Prof. W. Bulloch, F.R.S. 


Development of the Calcareous Parts of the Lantern of Aristotle in Hehinus 
miliaris. By D. W. Devanesen, M.A. Communicated by Prof. E. W. 
Mae bridemishasunn (latest lil15)\c tis saicecerekeus Serceatutes sanwactaccecdsaecceteenseenes 


PAGE 
281 


298 


306 


318 


329 


367 


410 


val 


PAGE 
Origin and Destiny of Cholesterol in the Animal Organism. Part XI1I.—On the 
Autolysis of Liver and Spleen. By John !Addyman Gardner and Francis 
William Fox (Beit Memorial Fellow). Communicated by Sir W. M. Fletcher, 
BORGS) . hanseweenteanesonigscehstunnsarciasesassess apee.inqceereasre eeGkel secs see eh cette eee 486 


OBITUARY NOTICES OF FELLOWS DECEASED. 


Harlot sDucie (wath) portrait) oc. cascs<on ence de eeeee ne eee heen Seana eee pitts 1 
Aldiiant Brow 2a. cass simeecsaeagescs som assicsb's cee dees dares eeeee sep eases Ueees cee eee eee iil 
Mouis'Compton: Maal... sc ccecckcccieses cet os onneeossbennenmet ee keene et eaC eee aeaeeee x 
George Stewardson Brady (with portrait) .............. .cescoeecseesensadeoscesees x 
Brancis:ArthurtBainbridge: vec. Jsccevsscosns se eccsencceteske teste eee ee eee eee eeaeee XXIV 
Augustus’ Désiré, Wuller.¢. 3... .acciscevebs seeds maceteneeectees ade teense cence eee eee ee XXVIi 
Vex. ciscchevsesaeciicosansaens bastedsoaisicg gaultine taste c eadeve cee tee na neeeeie ae eens oe ares XXxi 


Senay i, PROCEEDINGS 


gs nee THE ROYAL SOCIETY. 


- Series B. Vol. 93. : No. B 649, 


BIOLOGICAL SCIENCES. 


Be ~ CONTENTS. 


~ Page 
Rives of the President, Prof. C. S. SHERRINGTON, at the Anniversary 
Meeting, Never DOR IIZ Ie: Sh tetap ee eee : os I 
On the Optical Rees Power of Crystalline Ovalbumin and Serum 


Albumin. a E. G. YOUNG, Ramsay Memorial Fellow. ; Be AS 


Experiménts on Augean Metamorphosis and Pigment Responses in © 
Relation to Internal Secretions. By J. S. HUXLEY (New College, 
Oxford), and L. T: HOGBEN (Imperial College of Science; London) . 36 


Studies in Bacterial Variability—On the Occurrence and Development of 

Dys-agglutinable, Eu-agelutinable and MHyper-agglutinable Forms of 

_ Certain Bacteria. (A Report to the Medical Research Council.) By 
E. W.A. WALKER . : ; Seer ; Q : : 54 


? 


PRINTED AND PUBLISHED FOR THE ROYAL SOCIETY By 
HARRISON & SONS, Ltd, 44-47, ST. MARTIN'S LANE, LONDON, W.C.2. 


Price Four Shillings. 


No. B 649. 
ae January 2, 1922. 


> 


NOTICE TO AUTHORS AND COMMUNICATORS. 


The Council have had under consideration the rapid increase of the Society's 
expenditure on publications. In view of the necessity for economy, authors of papers 
are urgently requested to see that their communications are put in as concise a form as 
possible. Delay in decisions regarding publication, as well as subsequent trouble to 
authors, is often caused by diffuseness or prolixity. MSS. must be type-written or at 
least written in a legible hand, and properly prepared as copy for press. Type-written 
‘transcript should in all cases be carefully revised by the author before being presented. 
It is desirable that authors should retain copies of their MSS. for reference. 

Every paper must be accompanied by a summary not exceeding 300 words in length. 

Authors are requested to refer to a Memorandum on Mathematical Notation 
published’ in these “ Proceedings, 1909, Series A, vol. 82, p. 14, and to adhere to the 
suggestions therein contained, so far as possible. 

Authors are further requested to send in all drawings, diagrams or other illustrations 
in a state suitable for direct photographic reproduction. They should be drawn on 
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Where the illustrations are numerous, much time would be saved if the authors would 
indicate in advance those which, if a reduction of their number is found to be required, 
might be omitted with least inconvenience. 


“It shall be the duty of each Fellow or Foreign Member to satisfy himself that any letter, report or 
other paper which he may communicate, ts suitable to be read before the Society.” —Statute 64, Cap. xii. 


NOTICES TO FELLOWS OF THE ROYAL SOCIETY. 


The Council have directed that the Minutes of the Meetings of the Society shall 
be sent out as an inset in the ‘ Proceedings,’ separately paged. In future they will not 
be republished in the ‘ Year-Book.’ 

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Szorron B.—Brotoeicat SCrenceEs. 


Address of the President, Prof. C. S. Sherrington, at the 
Anniversary Meeting, November 30, 1921. 


Since the last Anniversary Meeting the roll of the Society has lost by 
death fifteen Fellows and one Foreign Member : 


Sir William Abney. Lord Moulton. 

Mr. Spencer Pickering. Prof. A. W. Reinold. 
Dr. A. Muirhead. Prof. E. J. Mills. 

Sir Lazarus Fletcher. Colonel J. Herschel. 
Prof. W. Odling. Mr. G. W. Walker. 
Prof. L. C. Miall. Dr. H. Woodward. 
Prof. R. B. Clifton. The Earl of Ducie. 


Dr. F. A. Bainbridge. 


On the Foreign List 
Prof. G. Lippmann. 


The Anniversary Meeting affords appropriate opportunity for some spoken 
reference to them. 

The earliest loss was that of WILLIAM DE WIVELESLIE ABNEY, a Fellow of 
the Society for upwards of forty years. Much of his scientific work may be 
summarised as being the establishment, by experiment, of photography as a 
science. With Sir William Abney photography was not merely a means but 
in itself a scientific end. The building of the image both in the wet and 
in the dry plate were successfully studied by him. He was a pioneer in 
the photography of the infra-red region of the spectrum. He suggested 
more than forty years ago the charging of carbons with calcium salts to 
enhance the are-light beam, the flame ares of to-day. Later he passed, 
so to say, from the photographic plate to the retina and investigated the 
relative visual intensity of different portions of the spectrum. As Advisor 

VOL, XCIII.—B. B 


2 Annwersary Address by Prof. C. S. Sherrington. 


to the Board of Trade he obtained, partly in collaboration with the late 
W. Watson, data most valuably discriminating between various types of 
colour vision; he contributed accurate measurements of visual differences 
between the foveal and para-foveal regions of the retina. His measurements 
of the visual luminosity curve of the spectrum stand as classical data of 
reference. He is remembered in the Society as a man whose personality 
endeared him to everyone who knew hin. 

The death of SPENCER PERCIVAL UMFREVILLE PICKERING removed a 
chemist, who at the time of his election to the Society, was one of the most 
arduous and prolific of researchers. The main theme of his work was 
solution and hydrates. A man of original view he often collided rather than 
moved with the scientific trend of the time, but he spared himself no pains in 
the pursuit of observations. His association with the Society will be happily 
perpetuated by the bequest from him, to become a research fund bearing his 
name. 

ALEXANDER MUIRHEAD, whose name is connected with the duplexing of. 
submarine cables by the artificial line with distributed capacity, also con- 
tributed perseveringly to the practical establishment of electrical standards 
of capacity. Much of his work was accomplished against difficulties of 
health which would have disheartened any but a man of remarkable courage 
and resolution. 

Lazarus FLETCHER was for ten years Director of the Natural History 
Museum. Mathematically trained, his chief scientific interest lay in 
problems connected with the physics of crystals, though much of his time 
was, given to the great National Collection of minerals of which he had 
charge for nearly thirty years. He devoted much patient and accurate 
research to the meteorites in that collection. His papers that are probably 
best known are those on the dilatation of crystals by heat, and on the 
Optical Indicatrix and transmission of light in crystals. In the latter of 
these he showed how the optical characters of crystals could be simply 
developed from the geometrical properties of an ellipsoid (which he called 
the Indicatrix) independently of any hypothesis as to the nature of the 
ether. His method has now been adopted by almost all teachers of the 
subject. Those who knew Sir Lazarus Fletcher are not likely to forget his 
‘simplicity of manner, his quiet humour and his unfailing consideration for 
others. 

WILLIAM ODLING, for many years Professor of Chemistry at Oxford, died 
there this spring at the age of ninety-two, severing a link with the chemistry 
of the mid-Victorian time. It was under his Chairmanship of the Institute 
‘of Chemistry that that body was granted its Charter in 1885. 


Anmversary Address by Prof. C. S. Sherrington. 3 


Louris Compton MIAtt was a biologist ; a naturalist in the old sense of the 
word. He did good and lasting zoological research. He was one of a group, 
few in number but strong in personality and influence, who laid the foundation 
of the existing University of Leeds. He was an enthusiastic educationalist, 
and appreciated highly the calling and the opportunities of the primary 
schoo]-teacher ; he helped that calling in many ways. He himself was a 
strikingly suecessful teacher. Those who knew him will recall how he 
studied teaching as an art, and loved it for its own sake. 

Dying at Oxford a little later in the year than Prof. Odling, RoBERT 
BELLAMY CLirron had been Professor of Experimental Philosophy there 
from 1865 until 1917. His first duty for his Chair had been the super- 
intendence of the erection of the laboratory, the Clarendon Laboratory, of 
which Sir Richard Glazebrook writes in his obituary notice of Clifton: “ tt 
was the first built in Europe for the special purpose of experimental 
instruction in Physics.” The fittings and teaching apparatus were largely to 
Prof. Clifton’s designs, and he gave much time and thought to their con- 
struction, perfecting and re-perfecting them in detail. So strict a custodian 
of them did he become that it was sometimes humorously said they had 
become too precious to be very accessible for their original purpose. How- 
ever that may be, under his hospitality the laboratory he had erected gave 
a home to a great piece of experimentation in Prof. Boys’ determination of the 
eravitation-constant. Clifton was a man of genial personality, of much con- 
versational cift, shrewd and humorous, and of a nature full of kindly qualities. 

WILLIAM REINOLD was Professor of Physics in the Royal Naval College. 
He had been Demonstrator under Clifton in the Clarendon Laboratory. 
It was during his long activity at the Royal Naval College, and as a teacher 
there, that his main scientific life-work was accomplished. 

In March last died suddenly Lord Moutton or Bank. Not an actual 
investigator in Science, he was yet a very real servant to the cause of 
scientific progress in this country. He possessed remarkable power of 
acquisition of knowledge, seizing rapidly and broadly the lines of advance 
taken by knowledge. A facile expositor of scientific themes to a lay or 
semi-lay audience, and gifted with an enthusiasm that never failed, he 
promoted the public appreciation of scientific work. Foreseeing from the 
outset of the War the magnitude of the strain that it would involve, he 
had the courage to demand a mobilisation of scientific resources adequate 
to that strain. The country owed much to his insistence and unsparing 
effort. His was a virile persuasion. After the coming of the Armistice, he 
‘turned his energies and influence toward urging a more thorough liaison 
between science and the industry of the country. 

B 2 


4 Anmversary Address by Prof. C. S. Sherrington. 


EpmuNnD JAMES MILLS held the Young Chair of Technical Chemistry in 
the Glasgow and West of Scotland Technical College. His papers were 
numerous both on applied and theoretical chemistry, and not a few of them 
were contributed to this Society, the first of them now more than fifty years 
ago. Returning to London in later life, he was for many years a frequent 
attendant at the Society’s meetings. 

Colonel JoHN HERSCHEL, a son of Sir John Herschel, and at one time 
Deputy-Superintendent of the Great Trigonometrical Survey of India, had 
been a Fellow of the Society for fifty years. He was a spectroscopic 
observer of a solar eclipse as far back as 1868. 

GABRIEL LIPPMANN, the eminent French physicist, died while at sea on 
his way from Canada home to Paris. He had been a Foreign Member of 
the Society for five. and twenty years. His interest in physics lay largely 
in the philosophic aspect, though his name is most familiar in connection 
with the capillary electrometer and with colour photography. Lippmann’s 
capillary electrometer became, so to say, a household tool in every physical 
laboratory, and likewise in many biological laboratories. In animal physi- 
ology it proved of unique service for the observations of the slight and 
fleeting electromotive reactions of isolated nerve and muscle. Until the 
advent of the string galvanometer it was the only instrument which could 
really cope with them. 

Of Lippmann’s process for the reproduction of colour by photography, 
our Foreign Secretary, Sir Arthur Schuster, who knew him from a time 
when they were fellow students together, kindly writes me as follows :— 
“Lippmann’s work on colour photography well illustrates his great experi- 
mental skill. Independently of the late Lord Rayleigh, who, in 1887, had 
on theoretical grounds foreseen the possibility of the reproduction of natural 
colours by an interference method, Lippmann conceived the same idea; but 
the experimental difficulties were formidable. The method depends on 
establishing a periodic structure in a photographic film by the mterference 
of the direct light and its reflexion from a metallic surface. It was neces- 
sary for the purpose that the films unlike those in ordinary use, should 
be transparent. The production of such films appeared for many years to 
be an insoluble problem, but ultimately the difficulty was overcome, and 
in 1901 Lippmann obtained his first suceess; but it was several years 
before he could secure the equality of sensitiveness throughout the visible 
spectrum which is essential if the natural colours are to appear with 
their correct values. The photographs obtained by Lippmann cannot be 
reproduced in print, but may be shown with brilliant effect by projection 
on a screen.” 


Anniversary Address by Prof. C. S. Sherrington. 5 

GEORGE WALKER WALKER had, following on a career of high promise at 
Cambridge, been successively Lecturer in Physics at Glasgow University, 
Superintendent of Eskdalemuir Observatory, Director of the new Magnetic 
Survey of the British Isles, and finally chief scientific worker at the Royal 

‘Naval Mining School, Portsmouth: It was during work in that latter 
capacity that his fatal illness began. He united in a remarkable degree 
mathematical attainment and inventive capacity. By his death physical 
science lost, sadly early, a finely accurate experimental exponent. 

Henry Woopwarb, late Keeper of the Department of Geology of the 
Natural History Museum, was a distinguished paleontologist. His scientific 
reputation was especially as an authority on éxtinct representatives of the 
Crustacea. He was.one of the founders, and for over fifty years editor, of 
the ‘Geological Magazine. His example and personal contact were a 
stimulus to many others, and the encouragement given by him to amateur 
workers was one of the features of his official career. . 

FRANCIS ARTHUR BAINBRIDGE died last month in early middle age. He 
had been elected a Fellow in 1919. Of delicate physique, constantly 
struggling against ill-health, he nevertheless accomplished, besides much 
routine teaching, a great deal of accurate research, some in pathology, 
more in physiology. He contributed to the differential recognition of the 
several types of paratyphoid bacilli, a matter at once of theoretical interest 
and great practical importance. His work in physiology opened with 
investigation of lymph formation, following on that of Bayliss and Starling. 
Then came work on urinary and salivary secretion, all of it characterised 
by great clearness of objective, and definiteness of plan. One of his best 
papers is one of his most recent. Its subject is the acceleration of the 
pulse, which muscular exercise constantly and so quickly induces. Bainbridge 
showed that the increased filling of the venous chamber of the heart, and 
the consequent increase of pressure in it, itself acts as a stimulus which 
excites through the nervous system the more frequent beating of the heart. 
He traced this control in part to depression of the vagus, partly to 
stimulation of the nerves which accelerate the heart. Bainbridge was an 

experimentalist of exceptional dexterity. Always cheerful, he seemed at his 
cheeriest when busiest in the laboratory. 

Lorp Ducts, whose decease fell latest in the year, had been a Fellow for 
nearly 67 years. Interested in Science, he was also greatly interested in 
secondary education. Latterly he had given his time and abilities chiefly 
to the countryside where he resided. By virtue of the date of his election 
to the Society, 1855, he had become its Senior Fellow.. 

We may note that the Seniority of Fellowship of the Society has now 


6 Annwersary Address by Prof. C.S. Shanningtort 


passed to one who has been a member of Council on many occasions, a 
Foreign Secretary, and Secretary, our sometime President, Sir Archibald 
Geikie, known among usalso as the genial historian of the Royal Society Club. 

It is little more than two years since the death of the late Lord 
Rayleigh, and this afternoon in Westminster Abbey there has been unveiled 
the tablet to his memory, given by subscribers from this Society and from 
the University of Cambridge, of which he was Chancellor. At the presen- 
tation ceremony the Society and the donors generally were represented by 
the Chairman of the Memorial Committee, Sir Joseph Thomson. The 
Society will feel it peculiarly appropriate that their representative on such an 
occasion should be one so closely associated with the late Lord Rayleigh in 
the Society, in the University which was their common alma mater, and in 
the domain of physical science itself. The recollection of the late Lord 
Rayleigh’s personality is present with us all: to meet him was to receive 
the impression of true greatness. The legend on the mural tablet runs :— 
“An unerring leader in the advancement of Natural Knowledge.’ To-day has 
seen the fulfilment of a fitting tribute, in a fitting resting-place, to a memory 
veneration for which the lapse of time will but intensify. 

The Bakerian Lecture of the year was by Dr. T. M. Lowry and Mr. P. C. 
Austin on “Optical Rotatory Dispersion.” The Croonian Lecture was by 
Dr. Henry Head. It had for its theme the disturbance of action in the 
nervous system due to the impairment of one part reacting on the function 
of another. Not unnoteworthy concerning the lecture is that, to push 
further the enquiries underlying it, the lecturer had subjected to surgical 
severance and restitching nerves of his own arm. 

To Dr. Head the Society owes a most acceptable gift. The Society 
possessed no portrait of Lord Lister. Dr. Head, on learning this year that 
such was the case, offered to the Society a portrait of Lister, by Legros, in 
black and white, a portrait that had been given to Dr. Head by the poet 
Henley, in whose possession it long was,—Henley, the poet whose word- 
portrayal of Lister, under whom he was a patient, is extant in the famous 
sonnet familiar to us all. The gift was gratefully accepted by Council. 

The Anniversary Meeting is naturally an occasion for retrospect ; it is also 
one which invites some thought to the present. The present time has in it 
an element of considerable anxiety for those who regard the prosperity of 
Science. Although the recent past has, it is true, been not unfavourable. 

I mentioned just now a university building, the earliest constructed for 
systematic experimental teaching in Physics, and that just 50 yearsago. It 
is a satisfaction to note the multiplication of such laboratories since then. 
This year at the inauguration in London of the Institute of Physics Sir Joseph 


Annwersary Address by Prof: C. S. Sherrington. 7 


‘Thomson remarked that now, in contrast against the early years of the 
Cavendish Laboratory, the study of Physics, as regards the numbers to 
whom it gives opening for a livelihood, constitutes in facet a profession of 
its own. The same can be said of the Science of Chemistry, and of the 
Biological sciences. Cultivation of science has been a feature of the country’s 
progress. This has in part been adjunct to the movement for the founda- 
tion of new Universities. The number of the English Universities has 
doubled in the last quarter of a century. The new Universities have shown 
admirable energy in their departments of science. Following in the tradition 
of the best of the older Universities they have, in instance after instance, 
made their laboratories places of research. Only last year the Council of 
the Society stated that to increase the resources and equipment of the 
Universities is one of -the best ways of aiding research in pure Science. 
The Report of the University Grants Committee in February of this year 
indicated that the Universities were unable to meet their existing yvesponsi- 
bilities, and that their resources are inadequate to meet legitimate demands 
upon them. It is, therefore, a matter of grave concern that the Government 
Grant to the Universities is now to be cut down heavily. The maintenance 
of the Universities at the level of efficiency which they have struggled so 
resolutely, and with much service but poorly paid, to sustain, will thus 
receive a very severe blow. 

Regression is the more disappointing because, during the war, there 
came an awakening of the conscience of the nation in regard to Science. 
The national need for wider and deeper interest in, and understanding of 
Science came home to the community as it had not done hitherto. The import- 
ance to the nation of, for instance, the national Physical Laboratory, whose 
parent this Society may justly claim to be, began to receive more general 
recognition than before. Its importance to the State became cogent to the 
State. Six years ago saw the founding of the Advisory Council on Research 
to the Privy Council, and a year later the establishing of the Department of 
Scientific and Industrial Research. These were not created as part of the 
machinery for the war, though during that common need they, like every other 
national organization, made their contribution. They were brought into 
existence to remedy deep-seated shortcomings which the war revealed in the 
country’s organization for scientific research. Their full effect was only 
to be expected to come now, after the attainment of peace. It is, therefore 
gravely disquieting that their State support estimates are being now reduced 
by some 30 per cent. and that further reduction still is asked for. 

Again, if we turn to the domain of Biology, and take within that the field 
of Medical Science, the Medical Research Committee, as it then was, had been 


8 Anniversary Address by Prof. C. S. Sherrington. 


organised and started not long before the outbreak of the war. It had from 
its beginning shown its utility and brought evidence of the great field of 
usefulness before it. Its services during the war and since the ending of the 
war have been conspicuous, indeed inestimable. Public appreciation of it has 
enhanced. The Government has recently raised the status of the Committee, 
so that it is now the Medical Research Council under the Privy Council. 

Annual Reports indicate the quality and the volume of the work it is 
accomplishing. It is creating a new era of research in scientific medicine 

in this country. But its financial State aid is to be cut down for the coming 

year, and the extent of that reduction is a real anxiety to all who have at 

heart the progress of Medicine in this country and of the Sciences on which 

Medical Science itself rests. 

I may say that, broadly taken, the apparatus for prosecution of research 
in this country is made up as follows: (1) Scientific and Professional 
Societies and some institutions entirely privately supported; (2) Univer- 
sities and Colleges, with their scientific departments; (3) Institutions, using 
that term in the widest sense, directly subventioned by the State, such 
for instance as the Medical Research Council, the Development Commission, 
and the Department of Scientific and Industrial Research. Of these three 
categories, the first named, the Scientific Societies group, work without 
financial aid from the State, apart from the small though extremely useful 
two Government Grants distributed, mainly to individual workers, through 
this, the Royal Society. At the present time many of the Societies sorely 
need financial help to carry on their labours, and some are absolutely at a 
loss to know how to publish the scientific results that are brought to them. 
(2) The second category, the Universities and Colleges, depend in part upon 
Government aid. In the aggregate of twenty-one institutions of University 
rank, following Vice-Chancellor Adami’s figures, students’ fees and endow- 
ment provide about 63°5 per cent. of the total income; for the rest they 
are dependent on Government Grant. (3) The third category as said, draw 
State-support direct. 

This triple system may seem a somewhat haphazard and inco-ordinate 
assembly. Yet in reality it is an organisation with much solidarity, and its 
co-ordination is becoming more assured. Its parts dovetail together. The 
first group, the scientific and professional Societies, is provided with a medium 
of intercommunication and co-action, the Conjoint Board of Scientific 
Societies. As to the separate categories composing the triple system itself, 
they also are in wide touch one with another. Between the Scientific 
and Professional Societies on the one hand and the Universities on the other, 
contact and inter-relation are secured by some degree of free and rightful 


Annwersury Address by Prof. C. S. Sherrington. 9 


overlap, both as regards general subject matter of research and of their 
personnel. Finally, there is excellent contact between both these categories 
and the third, the State-subventioned institutions. A special feature of the 
policy and administration of these State organisations secures this, a feature 
which makes the whole of this subject the more cognate to the purview of 
our own Society. To exemplify I may turn, for instance, to the Development 
Commission. Its programme of Fishery Research, avoiding the terms “ pure” 
research and “applied” research, in view of the possible implication that 
pure research does not lead to practical result, directs research not alone 
to the solving of particular economic problems. It supports more especially 
what it terms “free ” research, investigation in this case of the fundamental 
science of the sea and of marine life. This term “free” research is set in its 
full light by words of the Lord President of the Council, Mr. Balfour, where 
he points out that while the State may aid research, it will only destroy 
research if it resolves too rigidly to control it. 

Again, with the Advisory Council of Scientific and Industrial Research, 
its programme, gradually defined during the past six years, is laid down 
as having four main points: (1) the encouragement of the individual 
research worker, particularly in pure science; (2) the organisation of 
national industries into co-operative research associations; (3) the direction 
and co-ordination of research for national purposes; (4) the aiding of 
suitable researches undertaken by scientific and professional Societies and 
organisations. It recruits researchers by giving financial opportunity to 
promising students to be trained in research attaching them to experienced 
researchers. In short, it apprentices to research a number of selected 
younger workers in Universities, Colleges, and other institutions scattered 
throughout the country. 

* So, similarly, the Medical Research Council. Its Secretary, Sir Walter 
Fletcher, in an illuminating presidential address to Section I of the British 
Association Meeting this summer, said, speaking of the nexus between 
scientific research and the progress of Medicine, “It is the accumulating 
knowledge of the basal laws of life and of the living organism to which 
alone we can look for the sure establishment either of the study of disease or 
of the applied sciences of Medicine.” 

It is evident, therefore, that with a policy based on such principles as 
these, the third category in the triple system constituting the organisation 
for scientific research in this country, is one which has common aim and 
solid touch with both the others, the Universities and the Scientific and 
Professional Societies. One sees in short that the organisation which has 
come into existence and is maintaining scientific research in this country, 


10 Anniversary Address by Prof. C. S. Sherrington. 


is a real organisation. It did not spring fully equipped from the head of 
Jupiter. It has grown up rather than been planned. In that respect it is 
an organisation essentially British, and it seems qualified to do its work for 
the country well. We hear of adventures, political and other, the offspring 
of the day. But these were no adventures, these, to my mind, welcome, 
long-overdue, steps forward by the State toward the succour of Science and 
its welfare, steps that help to strengthen and consolidate the organisation 
for research by such adjuncts as the Medical Research Council and the 
Department of Scientific and Industrial Research. One of the strengths of 
this organisation that has arisen is, in my view, that it interlocks with the 
educational system of the country. It is an organisation which proceeds 
on the wise premiss that, in the case of Science, the best way to get the 
fruit is to cultivate the tree. It is an organisation which is proving successful 
and economical. Its output has proved a more than liberal return on the 
funds at its disposal. | 

But essential to its own continuance is continuance of adequate financial 
support from the Government. A tripod cannot stand upon two legs. The 
State-contribution in this country is relatively not large, but 1t is most 
important. Important as it has been in the past, it has now an importance 
most especially great. The cost of investigation is now higher, much higher 
than it has been. Endowment funds carry less far than they did carry. 
Private benefactions and voluntary generosity, although willing, are less 
able to be found and less capable at this time; already gauged as 
inadequate of themselves alone before the War, they obviously cannot 
alone cope with the necessary undertakings now. The present is a time 
when a large-scale withdrawal of the Government’s financial support must 
prove most formidably crippling. Such erippling will be greater than the 
actual measure of the sum withdrawn. would entail in ordinary times. 

None can fail to see the urgent need for national economy. It may be 
objected that the plea to which I am speaking is, in fact, one for the 
preferential treatment of Science. That is not so. Faced with need for 
stringent economy, there must, of course, be a rigorous cutting’ down of 
expenditure that is unnecessary. But a first enquiry is the discrimination 
between expenditure upon the inessential and the essential. Otherwise the 
economies seemingly effected may be no economies. The savings may be 
made in a fashion most costly in the end. Conceded that there must be 
some reduction in the moderate State expenditure on research, it would be 
no true economy if that reduction were pushed to the point of causing 
collapse of the fabric for the production of much-needed knowledge or of 
whole compartments of that fabric. 


Annwversary Address by Prof. C. S. Sherrington. il 


The necessary supply of trained research workers cannot be retained or 
replenished except by a steady policy pursued. If the financial provision 
for research is too severely cut down, that will mean the extinction of various 
investigations which cannot be satisfactorily continued at all under narrower | 
limits of expenditure than are imposed at present. One feature of modern 
research is that it has become more largely team-work, the combined effort of 
an assorted group of individuals with special training. Want of volume has 
tended to be a weak point in our national research. Reduction of the 
support by Government will react most rapidly on the number of competent 
investigators available, the number that makes a fair volume of team-work 
possible. The Report of the Advisory Council states that the effect of a set- 
back of this kind will be long-continued and adds that it may be lasting. . 

To pull down under emergency what has been built up through years of 
careful experience and is proving efficient, can hardly be ultimate economy. 
It is to unlearn a useful lesson learnt. Curtailment of the State aid— 
relatively small in this country—given to scientific research must harm 
the scientific production of the country. Some curtailment, however, at this 
time seems unavoidable. Though extension of buildings and equipment and 
personnel is wanted, it may be necessary to withhold that extension at this 
time, maintaining broadly the statws quo ready for expansion when that is 
once more feasible. But if research be an indispensable factor in the 
rebuilding of the national life, sacrifices should not be required from it dispro- 
portionately greater than from other services of a similarly essential kind. 
Reduction of the State’s support on a scale to entail ruin to the existent 
organisation would be a wastage rather than an economy. Calmly viewed, 
what more reminiscent of the wastage of the War itself than for machinery 
actually constructed, assembled, and producing what is needful for a nation’s 
strength as a pillar in the industrial and intellectual temple of the world, to 
be now under temporary change abandoned or broken up; and at a time when 
industry as a whole stands convinced of scientific research as a necessity for 
its recovery and well-being. 

My hope would be that scientific research on its present maintenance will 
be considered part of the intellectual bread of the community, part of the 
bed-rock on which rests the efficiency, not to speak of the industrial equip- 
ment of the nation; that it will be treated as such in the measure of State- 
support continued to it; that the State will remember that that support has 
to embcace at least both the Universities on the one hand, and, on the other, 
the research institutions administered by the State, for this reason, namely, 
. that the country’s organisation for research, complex in origin, yet economical 
and effective, stands as an integral system, to whose entire existence is 


12 Annwersary Address by Prof. C. S. Sherrington. 


essential an adequate State provision for both these constituent elements, 
indispensable, since they are, to the whole structure of the system. 
I now proceed to the distribution of the Medals. 


The Copley Medal is awarded to Sir Joseph Larmor. 


Sir Joseph Larmor has long held a leading position in the British School 
of Mathematical Physics. There is hardly a branch of this subject to which 
he has not made contributions of distinct originality and great value. His 
earlier researches on Dynamics, on Optics, both geometrical and physical, and 
on Elasticity, are marked by keen insight and by the novelty introduced in 
the treatment of familiar subjects. In more recent periods he has written on 
problems of Geodynamics, with the same illuminating force. His contribu- 
tions to the Theory of Electricity, in its many ramifications, are numerous 
and profound. His treatise on ‘ Aither and Matter’ forms a distinct land- 
mark in the history of the subject. In this we have the foundation of electro- 
magnetic theory on the single principle of least action, with the electron 
taken into account as an ethereal structure. He was the first to establish 
(to the second order of velocity) the correspondence between moving and fixed 
electrical systems, and shares with Lorentz, the distinction of discovering 
the generality of this correspondence to any order. It may fairly be said 
that his preliminary work was of the utmost value in paving the way to the 
modern developments of the Theory of Relativity. In addition to his own 
researches Larmor has, as Lucasian Professor, stimulated the work of others 
with notable success. His intimate and extensive knowledge of the history 
as well as of the results of physical science marked him out as the appropriate 
editor of the works of Stokes, Kelvin, James Thomson, and Henry Cavendish, 
to which he has contributed most serviceable annotations. 


A Royal Medal is awarded to Dr. Frederick Frost Blackman. 

Dr. Blackman is distinguished for his contributions to plant physiology, and 
especially to knowledge of the process of photo-synthetic assimilation of carbon 
dioxide. In this connection he devised apparatus of great delicacy and 
accuracy. Later he proceeded to an exhaustive investigation on the rate of 
assimilation within the green leaf. He determined, under varied and con- 
trolled conditions, the inter-relationship of the external factors and their 
several and joint effects on the rate of assimilation, and has laid the founda- 
tion on which a good deal of subsequent work by other investigators has been 
rendered possible. 

He was thus led to his theory of limiting factors, which has exerted much 
influence in both plant and animal physiology. With the help of his 


7 Gyoivooonian tne tea ; 
\ SEP 30 1922 
| : Orr; y 
Anmversary Address by Prof. C. S. Sherrington. Syxteco piven 7 


co-workers he has importantly extended our knowledge of permeability, and 
of the influence of anesthetics on plants. He occupies a leading position 
amongst plant physiologists, not only by reason of the importance of his 
discoveries, but also on account of the effective stimulus he has given to the 
school of investigators who have been trained in his laboratory. 


A Royal Medal is awarded to Sir Frank Watson Dyson. 


Sir Frank Dyson is distinguished not only for his enlightened and energetic 
administration of the Royal Observatory, but by his many important con- 
tributions to Astronomy. He has devoted special attention to investigations 
of the movements and distances of the stars, and of the bearing of these upon 
the structure of the stellar universe. He has concentrated his energies 
particularly on the stars surrounding the north celestial pole, and has collected 
or determined for this region of the sky all the different data which seem 
likely to aid in the solution of the stellar problem. In a long series of papers 
he has shown himself able not only to conceive and execute large schemes of 
observation, but also to deduce by graphical and mathematical analysis the 
theoretical conclusions which are implicit in the mass of data. Some of his 
investigations are remarkable for the extensive data which have been utilised ; 
one of them involves the proper motions of 12,000 stars, and another of 
26,000 stars. These researches have given Sir Frank Dyson a place in the 
front rank of workers on stellar distribution and movements. 

He has also given much attention to the accurate determination of stellar 
magnitudes, and has successfully established a regular programme of work on 
stellar parallaxes which has yielded results of high precision for a large 
number of stars. 

Previous to this he had been conspicuously successful in obtaining records 
of the spectrum of the corona and chromosphere during eclipses of the sun; 
his publications on those subjects are among the most valuable sources of 
solar spectroscopic data. . It was mainly to his foresight and organizing 
ability that we owe the successful observations of the deflection of light by 
the sun’s gravitational field during the eclipse of 1919. 


The Davy Medal is awarded to Prof. Philippe Auguste Guye in recog- 
nition of his work on optically active organic substances, on molecular 
association and on atomic weights. 

In his early work on Organic Chemistry, Prof. Guye was led to investigate 
the question whether a quantitative relationship exists between the molecular 
rotations of optically active substances and their chemical constitution. 
Although the answer proved to be in the negative, the attempt to establish 


14 Anniversary Address by Prof. C. S. Sherrington. 


such a relationship was yet productive of much valuable research on optical 
isomerides in his own laboratory, and stimulated the efforts of many investi- 
gators in that branch of physical chemistry, particularly in this country. 
Shortly after he had put forward his theory of the “product of asymmetry” 
he was attracted by the problems connected with Van der Waal’s equation 
and the critical state, and, from his interest in these, two important lines 
of investigation opened out. The one had relation to the degree of 
molecular complexity of matter in the liquid state, and occupied his 
attention mainly between the years 1893 and 1911. The other led him 
at the beginning of the present century to advocate, with much energy and 
persistence, the advantages of the physical method of determining atomic 
weights. In this field of work he became one of the foremost investigators ; 
his work on the calculation of precise gas densities was followed by chemical 
studies of the atomic weights of nitrogen, silver and chlorine, and by 
inquiries into sources of error, hitherto little recognised, in atomic weight 


determinations. 


The Hughes Medal is awarded to Prof. Niels Bohr. 


Prof. Bohr is well known to all physicists as the author of the conception 
to which the name “ Bohr-atom ” has been attached. A decade ago it became 
clear, from the researches of Sir E. Rutherford and others, that the atom of 
any element is formed out of an excessively minute positive nucleus of 
electricity, round which circulate a number of negative electrons equal to the 
atomic number of the element. Bohr discovered a mechanism for the 
motion of these electrons, which solved immediately the long-standing puzzle 
of the Balmer series of hydrogen, and which, after development and discus- 
sion, appears likely to provide a complete explanation of the spectra of the 
various elements. In this way he has opened up a line of investigation 
which has already attracted to itself many of the ablest mathematicians in 
Europe, and of which the success, in the simplest cases of the two light 
elements hydrogen and helium, is even now little short of perfect. 


On the Optical Rotatory Power of Crystalline Ovalbumin and 
Serum Albumin. 


By Etrip Gorpon Youne, Ramsay Memorial Fellow. 
(Communicated by Prof. F. G. Hopkins, F.R.S.—Received July 6, 1921.) 
(From the Biochemical Laboratory, Cambridge University.) 


The need for a physical method by means of which it would be possible to 
recognise a chemical individual of the protein group of compounds has 
undoubtedly been one of the factors contributing to the difficulty of research 
into the chemistry of the proteins. The present methods of chemical analysis 
do not nearly approach sufficient accuracy to distinguish between successive 
recrystallisations of a protein substance. The first serious effort to prove that 
an individual protein could be isolated is to be found in the publication of 
Hopkins (1900). It was here shown that a protein could be prepared with a 
constant specific rotation for successive recrystallisations and for material 
obtained from different sources. Unfortunately, this desirable physical 
constant, which is independent of the degree of colloidal dispersion, has 
been shown to vary with variations in physical and chemical conditions. 
Thus Alexander (1896) found the specific rotation of certain globulins 
varied according to the concentration of the protein and of salt present. An 
investigation of this phenomenon by Pauli, Samec and Strausz (1914) con- 
firmed the observations of Hopkins (1900) and of Osborne (1899) that the 
presence of neutral salts has no influence on the optical rotation of a protein. 
The addition of acids and of alkalies they found to increase the rotation of 
polarised lght, while the degree of change depended upon the nature of the 
anion in the case of an acid and of the kation in the case of a base. These 
observations were made, unfortunately, with the mixture of proteins contained 
in ox or horse serum which had simply been dialysed until salt-free. The 
nature of these changes may find explanation in a tautomeric equilibrium of 
the lactam-lactim type in the protein main chain when in aqueous solution, 
as suggested by Robertson (1912) and Sorensen (1912), and to which view 
Pauli inclines. 

R—CO—NH—R = R—C(OH) = N—R. 
Lactam formula. Lactim formula. 

The determination of the specific rotation of crystalline ovalbumin as a 
means of discovering its purity, as Hopkins had shown possible, was called 
into question by Willcock (1908), on the basis of the test used to show when 
the analysis sample had been washed free from sulphate. Using a ring test with 


16 Mr. E.G. Young. On the Optical Rotatory 


BaClo, Willcock claims that a very much greater sensitivity can be obtained ; 
and by employing this method, specific rotations of different albumin erystalli- 
sations were found to vary considerably. If that fact were true, the biological 
chemist would still be without a means of recognising a chemical individual 
of the protein group, even if he had isolated one. 

The experimental history as described in the following pages was developed 
with the object of discovering whether a constant specific rotation could be 
obtained for crystalline ovalbumin, and what was the relation between the 
hydrogen ion concentration and the optical rotation with reference to the iso- 
electric point. It was thought that such a study might yield an explanation 
of the variability of the rotatory power and possibly some direct evidence on 
the question of a tautomeric equilibrium in the protein molecule. These 
experiments are described first. The investigation was extended to a study 
of the conditions governing the preparation of a pure crystalline serum 
albumin and the properties of this substance examined. 


EXPERIMENTAL. 


The Specific Rotation of Crystalline Ovalbumin. 


The values obtained for the specific rotation of hen ovalbumin by different 
observers at different times and with material under somewhat different 
physical conditions have been recorded in Table I. The earlier determina- 
tions on amorphous material are interesting in that they show a higher value 
than that observed for crystalline material. Osborne and Campbell (1900) 
have shown that the non-crystallisable albumin, which they have called 
conalbumin, has a higher value for «]p than the crystalline, and contamina- 
tion by this substance probably explains the higher value. 


Table I. 
Observer. Material. Specific rotation. 

| | ° 
| TaLE EE (USS) soccoanodsndate cor crses Amorphous —38 -08 
| Panormoff (1898) ............... 5 —36°2 
eanormofi, (L898) iieeeeteeeeeers Crystalline —23°6 
| Wuvomens: (QUE) co oneocucoocndoce osee es —26 ‘1 
| Bondzynski and Zoja (1894)... * —26°0 to —42 54 
| Oslomaag (IE) ssonosneovsaconen — 28 °42 
| Osborne and Campbell (1900) % — 28 -60 to —30 80 

Taloyel:oters (IMCD) eocoececineseconee 5 —30°7 

Willcock (1908) ......ceccseees- ip —30°3 to —31°6 


Only the last three determinations are of importance, as they were made 
on similar material which had been recrystallised .several times, and of these 


Power of Crystalline Ovalbumin and Serum Albumin, — 17 


Hopkins alone obtained a constant value for successive recrystallisations. 
The earlier figures were obtained from material crystallised but once, and by 
the inferior technique of Hofmeister in most cases. 

The last worker on the subject claims that the true value is higher chem 
—30:7° and attributes her average result of —31:0° as due to a more sensitive 
test for the presence of sulphate ions, thus allowing of more thorough 
washing of the coagulated albumin before drying and weighing. The tech- 
nique which I have adopted in obtaining the following observations involved 
the use of a 400-mm. polarimeter tube, so that very large rotations were 
observed. A large Hilger polarimeter was employed and observations were 
made both with the sodium flame and with the green band of the quartz 
mereury-vapour lamp. The solutions used were made as concentrated as 
possible, being about 10 per cent. strength. The analysis of these solutions 
for their content of albumin was carried out in the first series by the Devoto 
method, using accurately calibrated pipettes for volume measurement of a 
quantity of solution sufficient to yield about 0°5 grm. of dried coagulum, and 
Kahlbaum’s purest (NH4)2SO, in saturated solution as coagulating agent. 
This solution is naturally acid, having a Py of 5:5 or less. Coagulation was 
brought about by placing the solution to which the albumin had been added 
for 1 hour ona boiling water bath. After cooling, the precipitate was filtered 
off on to hardened filter paper and washed with cold water until free from 
sulphate. Finally, the precipitate was removed quantitatively to a platinum 
basin by means of a few cubic centimetres of distilled water, the excess 
water was evaporated on a water bath, and the protein dried until of constant 
weight in a Lothar Meyer air bath at 110° C. 

The removal of the last traces of sulphate from the precipitate is a 
difficult operation. I have tested the sensitivity of the BaCl, test for 
sulphates, applied as a ring and in the direct way, on a series of dilutions of 
normal H2SO,. When carried out as a ring test, using a saturated solution 
of BaCly and allowing the tubes to stand 15 minutes before decision, the test 
is sensitive to one part in one million. The same limit was found for the 
test when applied by adding five drops to about 20 cc. of filtrate and 
allowing to stand 15 minutes. An approximate idea of the quantity of 
sulphate being removed can be obtained in the final stage of washing by 
comparison of the turbidity obtained with that given by known strengths of 
H2SO,. By continuous washing, I have never been able to remove the last 
traces of sulphate in less than 4 days. This point is discussed below. If 
any protein goes back into solution during this long period of washing, the 
amount must be negligible, for I have repeatedly tested the filtrates for 
protein with negative results, and evaporated down quantities of the filtrate 

VOL. XCIII.—B. Cc 


18 Mr. E. G. Young. On the Optical Rotatory 


to dryness without obtaining any visible residue. Table IL gives the results 
of a series of crystallisations :-— 


Table II. 
| 

Crystallisation. Second. | Third. | Fourth. | Fifth. Sixth. | Average. 
Concentration (per cent.) ......| 7°31 5 43 11°17 9 25 11-09 
Tero  VRAS Soar annsueeabepadeebadoaesace 5°3 4:9 4°9 5 ‘1 4°9 
Cin\dedecsneopacinue donpecduabesboodccded —8°87°; —6-70°} —13°80°| —11-29°| —13 °72° 
C55) Conoocanpideanusesduousoe sabn0gncciso0 —10-°80 —8:'16 | —16°73 | —13-°77 | —16°73 
[TEA ee BERS | —30-34 | —30:82 | —30-87 | —30-61 | —30-89 | —30-81° 
[alae evceckisertscsnaestecasesnectoneen | —36°93 | —37°36 | —37°74  —37°34 | —37°63 | —37 53 


The values obtained for the specific rotation of successive recrystallisations 
is thus in very good agreement with that observed by Hopkins previously 
with similar material. 

On account of the difficulty of washing free from sulphate encountered in 
the Devoto method, I have considered it of interest to compare this 
procedure with the simpler one of coagulating the sample for analysis in a 
buffer mixture at the isoelectric point. The most convenient buffer mixture 
for this purpose is a solution of acetic acid and sodium acetate in equal 
molecular proportions. Thus, 5 ec. of a N/1 CH3COOH solution were 
mixed with 5 cc. of a N/1 CH3;COONa solution, and the whole diluted 
to 50 cc. This solution has a Py of 474. To this mixture was added the 
volume of albumin solution, containing about half a gramme of protein 
(5 to 10 c.c.). The containing vessel was then heated for half an hour on a 
boiling water bath, to coagulate the albumin completely. It was then 
allowed to cool, filtered off on to hardened filter paper, and washed until 
free from sulphate. The washing period is much briefer than when 
(NH4,)2SOx is used as coagulating medium. But, even with this procedure, 
sulphate ions are very slowly removed after the first day’s washing at the 
rate of about 1 mgrm. per 100 c.c. of filtrate. 

Table III represents the specific rotatory power observed in a series of 
crystallisations by the use of both methods of coagulation simultaneously 
carried out, and it also shows a beautiful agreement between the determina- 
tions of the albumin concentration by the two methods used. The specific 
rotations obtained are particularly interesting, in that they are constant in 
magnitude after the second crystallisation, yet slightly lower than the series 
reported in Table II. This suggests two possibilities. There might be slight 
molecular differences in the albumin of two different lots of eggs hitherto 
undetected by our crude methods of analysis, or, what is much more probable, 
the crystals obtained in the first crystallisation might be a slightly different 


Power of Crystalline Ovalbumin and Serum Albumin. 19 


Table IIT. 
Crystallisation. Second. Third. Fourth. Fifth. Average. | 

Concentration (per cent.) — 

(1) (NH,),SO, method ...) 987 10 95 15°43 12-64 

(2) Buffer method ......... 9°89 10 ‘96 15 “42 12-66 

TH sandeocoa0ad sedecossdeadaan6cd000 5 4 54 5°3 5°3 

(55: pap eenotnesa sob ebbesacdosocdodeon —11:96° —13 °22° —18 52° —15 *22° 
G55, cog neokod sopbegennnGseddgbo conc —14 66 —16°12 — 22 ‘58 —18 57 
Gis)... cape pp coeRERES Here RCE EScERE — 30°26 —30°17 — 30 ‘02 —30°10 — 30 °14° 
zis: ea eee en core ae —37:09 | —36-79 | —36-61 | —36-70 | —36-80 


salt from that obtained previously. This possibility, more fully discussed in 
the notes on the method of preparation, suggested the desirability of 
studying the variation of the specific rotation in relation to the hydrogen 
ion concentration of the solution. 


The Influence of Px on Rotatory Power. 


The condition in which the protein molecule is isoelectric to the electric 
current has such a profound influence upon its physical properties that it 
was thought that a study of the relation of this condition to the optical 
rotation might yield an explanation of the divergent values recorded in the 
literature. For the purpose of experimentation, crystalline material, which 
had been crystallised four to six times, was used. The last precipitate of 
crystals was centrifuged at high speed from its mother liquor and redissolved 
in distilled water. The resulting solution thus contained a small amount of 
(NH4)2SO4. The changes in hydrogen ion concentration of the solution were 
followed by means of various indicators, using the standard buffer mixtures 
devised by Clark and Lubs (1917). A series of reagents was prepared by 
the use of material which had been purified to the standard required and 
dissolved in water which had been twice distilled in glass, firstly from 
KMn0O, and H.2SO,, and secondly from Ba(OH):. All test-tubes employed 
were of uniform diameter, of clear, colourless glass, and had been steeped in 
dichromate cleaning mixture for 24 hours, and then steamed in superheated 
steam for 1 hour. 

After some experimentation, the following series of indicators was selected 
as most suitable for use in solutions containing both protein and salt :— 


Pu range. Indicator. 
12— 2°8 Thymol blue. 
44— 6-0 Methyl] red. 
64— 7:8 Neutral red. 
78— 84 Phenol red. 
8:-4—10°0 Phenolphthalein. 


20 Mr. E. G. Young.. On the Optical Rotatory 


To eliminate the possible errors due to the presence of proteins and salts, 
the method of dilution was adopted. Thus, 1 c.c. of fluid was pipetted into a 
test-tube, 4 ¢.c. of water (CO2-free) added, and a suitable quantity of the 
indicator. A comparator was employed in the case of any turbidity or 
foreign coloration. This method of dilution was later verified by the use of 
the hydrogen electrode and potentiometer, and found to give correct values. 
The values obtained with the above indicators were also verified by the 
electrical method on test solutions. Methyl red, neutral red and phenol 
phthalein give very trustworthy figures. 

Lxperiment 1.—A concentrated solution was quickly prepared from freshly 
crystallised material, filtered and placed in a 400-mm. tube. Its rotation was ° 
observed at intervals in a polarimeter for several days and found to remain 
absolutely constant from the first observation of —15°35°. The Py of this 
solution was found to be 4:9. To this solution was added normal H2SO, until 
the acidity was increased to Py 3°75. Within a few minutes a very finely 
divided precipitate began to form which remained suspended and could be 
neither filtered nor centrifuged off. Further addition of acid so that the 
Py became 2°7 merely increased the fine insoluble precipitate. A slow denatu- 
ration was apparently taking place and the solution was extremely sensitive 
to mechanical shock, immediately forming films of denatured material. 

Experiment 2.—A fresh solution of Py 49 was made alkaline by the 
addition of several drops of concentrated NaOH solution and the variation 
in the degree of rotation observed in a 200-mm. tube is recorded in 
Table LV :— 


Table LV. 
| a 
| Date. Temperature. Pu. H aR, 
Gh 9 
Mecember WO each kasmose eee eee | 11:0 4:9 —1°91 
S BY g.cchaneateea cies ermceeerenees 11:0 | 4°9 —1°91 
ST ae RI CE cA 11-0 | 4:9 —1-91 
5 nae a Ee ES 6 11-0 | 6°65 —1-72 
Bod OMG (seb? ecetucneanastomet eres 11°5 6°65 —1°78 
a LURES aoe ncicbonaibecan pata 12:0 84 —1°78 
EEO ite a lle, ROMNEOCR 12-0 rae itl —1-79 
‘ 2 ate senate duloocsemace bere 12:0 8-4 | —1°80 


A distinct though small drop in rotatory power was observed and on 
further increasing the hydroxyl ion concentration the value for the rotation 
increased slowly. 

The etfect of adding concentrated NH,OH to a fresh solution of albumin 
was next tried, employing a higher concentration of protein. Precisely the 


Power of Crystalline Ovalbuman and Serum Albunun. 21 


same phenomenon of a primary drop in rotatory power followed by a slow rise 
was observed as recorded in Table V :— 


Table V. 

Time interval. | Temperature. Pu, | ar, 

nC! | ® 
— | 150 4°85 — 4°25 
AMNOUNS Nec sahies-sclecs chic eeusactiones | 16°0 4°85 —4°26 
1@ sanvjatCHeD Bae oa tooaeeocensednnonooees 15 °0 9°15 —3°94 
SMM OUNSE sialic avis salos pe aeidatjonts tae sale 15-0 9°15 —3 94 
ji Gloy saceataea Je ane Renee | 15:0 9°15 | —3 95 
2 GESTS. aa te ree ee | 15-0 9°15 | —3-96 
DEGAS ins teetccetian th ickisaee aaa seeeaee | 15°0 9-15 —3:99 

; | 
| | 


Lixperiment 3.—The effect of both acid and alkali was demonstrated by 
means of the addition of very small quantities of HCl and NH,OH and the 
rotations observed are recorded in Table VI :— 


Table VI. 
Time interval. Temperature. Pu. | ap. | [a]p. 
| | 

= | 11 G8 =A, Dy —31°89 

DEMUNMU LES Meer sn sine oee scence 11 7°35 —4:07 — 30°76 

SOemunntesh yh. jsue eee 11 7 | HAOE —30°83 

DahoUNsteen ete nae 11 TB | See —30 83 

BPIMIMUbESinneece et eee ease ae 12 7:8 —4°10 — 31-00 

iL GER ee Oo Se 12 We Tes i ae — 31°89 
Beminutesl ey. .neee eee ee eeee 12 4-8 —4-40 —33 25 | 

Misti es ceeis ac ete 12 | 4:8 SB |) aR BSCS 
ARC ayiewees eS ote tegeane dae suice 13 4:8 | —4°41 —33 ‘33 

| 


The effect of the addition of alkali is most interesting and the prompt fall 
in the rotation followed by a slow rise to the original value is distinctly 
suggestive of a tautomeric equilibrium. I have observed this same pheno- 
menon in the case of denatured serum albumin. The effect of increasing the 
hydrogen ion concentration of the solution is an instantaneous increase in the 
rotatory power, which remains constant at the new level. 

The experiment was repeated in order to discover at what Py the change 
in the rotatory power occurred, and to discover further if the change were 
reversible from either side of the isoelectric point. The observations are 
recorded in Table VII :— 


Mr. E.G. Young. On the Optical Rotatory 


22 
Table VIL 
Date. Pu, aE, {aJr. Date. Pu. ar. | (ale. 
° | c fo} 9 
April 20...) 49 —8°37 — 37°79 April 20 4:9 —8 37 —37 "79 
» 20 ...| 4°65 | —8 538 —38 48 » 20 5 4 —8 09 +36 °50 
6) Sima Acasa) Ipecac Som 5-4 | =8-10 | eaeres 
yee 4°65 | —8-63 —38 94 a 22 54 —8:'1l — 36 -59 
” 33 4-3 | —8'74 | —39-43 23 7-6 | —si4 | Sages 
” 96 43 | —8-72 | —39°34 ” 28 76 | —8:15 | Seema 
39 a0 4°3 —8°73 —39 39 » 20 76 —8'14 — 36°73 
x) 0) | 6°45 —8 06 — 36 37 20 7°6 —8:20 — 37 C0 
May 2 6°45 | —8-05 — 36 32 » 7°6 —8°25 — 37°23 
rn, oan) saa |) Sa aR 80 7-6 | —8-25 | —37-2R 
RO A ee fa —§ 67 —39°12 
| 


Note on the Crystallisation of Ovalbumin. 


The réle of acid in the process of crystallisation of albumin has received 
some attention at the hands of a number of investigators since the procedure 
was first carried out by Hopkins and Pinkus. These investigators found that 
a 10 per cent. solution of acetic acid served admirably for the adjustment of 
the solution so that crystals were deposited. Osborne recommended HCl 
very shortly afterwards, while Krieger found H2SQ, to give the best results. 
But as Hopkins pointed out in 1900 almost any moderately strong acid will 
bring about crystallisation. In his extensive study of the emulsoid colloid 
using crystalline ovalbumin as material, Sorensen (1917) employed N/5 H2SO4 
with additional water and (NH,4) SOx, solution. 

In the course of my experience I have had to prepare many small lots of 
crystals and have employed several different acids of various strengths but 
always adjusting the reaction finally to about the same optimum hydrogen 
ion concentration. On several occasions I have found the method of Hopkins 
to fail to yield crystals at the beginning, although the two possible causes of 
failure which he mentions were excluded, viz., staleness of the eggs and 
insufficient whipping. If, after a heavy amorphous precipitate has been 
thrown down while standing after the usual adjustment, more acid be added, 
crystals sometimes appear on further standing. These failures I attribute to 
an increased alkalinity in the egg whites and inability to attain the proper 
hydrogen ion concentration for crystallisation before the protein was 
precipitated. 

On two occasions when the optimum conditions had been established for 
crystallisation and a generous sowing of crystals added, a heavy precipitate 
was obtained, which consisted entirely of round globules of various sizes— 
the globuliths of the Hofmeister method of preparation. These globuliths 


Power of Crystalline Ovalbunun and Serum Albumin. — 23 


could never be induced to crystallise, in spite of sowing and repeated agita- 
tion. They remained for two months in this condition, slowly coalescing into 
a hard gel formation at the bottom of the beaker. They were then redissolved 
aud reprecipitated by (NH4)2SO,, but again the precipitate consisted of 
nothing but globuliths of an enormous size, which again settled toa gel. This 
observation is of importance in the light of two recently published papers. 
Bradford (1920) claims to have crystallised gelatin, and Oswald (1915) 
albumin from human ascitic fluid, yet in both cases only globuliths were 
obtained. In Table VIII I have collected the amount of acetic acid used in 
crystallisation of several lots of ege white and expressed it in terms of cubic 
centimetres per 100 c.c. of filtrate :— 

Table VIII. 


Lot. Fluid volume. | Pu. Acid volume. | Product. 
| 
c.c. CECs 
1 500 49 3°32 | Crystals. 
2 275 5-1 2-94 | Crystals. 
3 600 4°8 3 “70 | Globuliths. 
4 650 5:0 8 56 | Globuliths. 
5 825 4°9 1°88 | Crystals. 
6 325 4°7 2:95 | Crystals. 
| | 


It is thus shown that the globulith mother liquors required more acid to 
adjust them to the conditions of precipitation than the normal. Now it has 
been observed by both Hopkins and Sdérensen that this is necessary for eggs 
that are not strictly fresh. The phenomenon thus suggests to my mind 
further evidence toward an explanation of the rdle of acid and sulphate in 
evystallisation. We are dealing essentially with an equilibrium between 
albumin, water, salt and acid. The function of the salt is one of dehydra- 
tion, as has been shown by Chick and Martin (1913). ‘The function of the 
acid is probably twofold. By virtue of the fact that albumin is an ampholyte 
and on the alkaline side of its isoelectric point, it is bound to form a salt 
with free acid. At the same time, the acid ions have dehydrating powers. 
It is thus an adjustment of the available water molecules between the protein 
or protein salt, on the one hand, and the ammonium sulphate with the free 
acid ions, on the ether. Sorensen (1917) has brought forward evidence from 
careful quantitative experiments to show that crystalline ovalbumin is a 
definite hydrate. The above-mentioned observations would be then compre- 
hensible if, due to slight autolytic changes, the protein had lost some NH, 
groupings, and thus at least some of its power of salt formation with acids, 
yet retained its ability to form a definite hydrate. The globuliths, on this 
view, would be egg-hydrate, but not potentially crystalline ovalbumin. 


24 Mr. E. G. Young. On the Optical Rotatory 


In the light of the above discussion it is of interest to consider the 
conditions under which Hofmeister first obtained crystals. He slowly con- 
centrated a half-saturated solution of ammonium sulphate containing albumin. 
Now, such a solution is naturally acid, due to the fact that (NH,4)2SOx is a 
salt formed by the union of a very strong acid with only a moderately strong 
base. Furthermore, on slow evaporation in open vessels some of this free 
NH,OH will be lost, and the solution will become more acid until such a 
point is reached at which the hydrolysis will become negligible. It would 
thus be conceivable that at one stage of evaporation globuliths would appear, 
and, at a later stage, when the solution had become more acid, crystals. The 
globuliths might then take up more acid and erystallise, since the phases 
differ in degree of their constituents and not in kind, and the disperse phase 
is more or less permeable to electrolytes in the continuous one. | 

In any case, both erystals and globuliths are precipitated again from a 
solution of (NH4)2SOx, which is much less concentrated than that required for 
ordinary amorphous material. The concentration of (NH,)2SO, is usually 
Slightly more than quarter saturation, while amorphous albumin in equal 
concentration requires considerably more than half saturation. This fact in 
itself, to my mind, is a very good reason for believing that both crystals and 
globuliths are hydrates of ovalbumin. The réle of (NH4)2SO, would thus be to 
remove the solvate water associated with the albumin molecular aggregates 
which kept them in solution, but to leave the hydrate water still attached. 
If the hydrate were not formed, then the concentration would have to be con- 
siderably increased in order to control this excess of free-water molecules, 


and an amorphous precipitate would result. 


Discussion of Lesults. 


From the experiments described in the preceding sections we have seen 
that there is a variation of the specific rotation of ovalbumin, depending upon 
the hydrogen ion concentration of the solution. The addition of acid to a 
solution at its isoelectric point causes an increase of its rotatory power, 
which remains constant. The addition of alkali to a similar solution causes 
a prompt fall in rotatory power, which slowly rises to the original value. 
Further addition of alkali has no effect. These changes are reversible from 
either side of the isoelectric point. If, however, the albumin sample be kept 
at the isoelectric point or thereabouts, without adding either acid or alkali, it 
is possible to obtain a constant specific rotation for successive recrystallisations. 

The maximum experimental errors are small. The polarimeter readings 
are accurate to 0°01 of a degree, and since rotations of ten degrees or more 
have been observed, the error from this determination would not be greater 


Power of Crystalline Ovalbumin and Serum Albumin. — 25 


than 0:02 per cent. The error in the estimation of the dried coagulum from 
‘variations observed might amount to 2°5 mgrm. in 05 grm., or a maximum 
error of 0:5 per cent. A fluctuation of 0°25 of a degree in the specific rotation 
‘value would be the maximum experimental error permissible. It will be 
found that the results agree more closely than this asa rule. The variations 
an specific rotation produced by variations of Py, as shown in Table VII, 
for example, amount to several degrees. There seem to be two possible 
explanations for this phenomenon. As Fischer has shown, the salts of 
-amino acids have different optical rotations from the free acids themselves. 
Now the isoelectric point must be taken as the natural point of neutrality 
-of the protein molecule; that is, the point at which the dissociation con- 
stants of the protein as an acid and as a base balance. By the addition 
-of acid or alkali, salts would be formed on amino acid groupings, and the 
measure of salt formation would be the governing factor in change of 
-optical rotation. This is certainly not so on the alkaline side, for beyond 
the first drop in rotation, further addition of alkali has no obvious effect. 
Likewise the addition of acid can change the rotation once, and once only, 
apparently. 

The second explanation, as suggested by Robertson, that a tautomeric 
-equilibrium exists in certain amide groupings is most forcibly emphasised by 
‘the slow change in optical rotation in alkaline solution, and, further, by the 
reversibility of the phenomenon generally from either side of the isoelectric 
point. That such a tautomerism is probable from other reasons has been 
~very fully shown by Robertson, in supporting his theory of protein ionisation. 
Furthermore, such an equilibrium would be governed by the hydrogen ion 
‘concentration, and not by the quantity of acid molecules present. The 
-slight changes in the acidity of the medium might thus account for the 
‘fluctuations of the specific rotations observed and recorded in the literature. 


Crystalline Serum Albumin. 


Crystalline serum albumin was first prepared by Giirber in 1894 by the 
‘direct application of Hofmeister’s method to horse serum. Merely from the 
-appearance of the crystals, Gtirber concluded that three albumins occur in 
serum, all crystallisable. Michel, in 1896, introduced a slight modification in 
the technique, and determined the coagulation temperature and specific 
‘rotation of the crystalline product. Hopkins and Pinkus (1898) mention 
‘the tact that it is possible to obtain crystals from serum by the acid method, 
-but the time required is one to several days. Krieger (1899) shortly after- 
wards suggested the use of H2SQO, in place of acetic acid without any 
‘Quantitative data. In his paper on pure ovalbumin, Hopkins mentions that, 


26 Mr. E. G. Young. On the Optical Rotatory 


in some attempts to purify serum albumin in like fashion, more difficulty 
was experienced in arriving at a homogeneous product by reason of pigment 
contamination. The last study of serum albumin was carried out by Hardy 
and Gardiner, and only published in abstract form. By means of absolute 
alcohol and anhydrous ether as extraction solvents and dehydrating agents at 
—4° C., a snow-white protein was obtained, which could be readily dissolved 
and crystallised. This mode of preparation serves to remove all traces of 
fatty material present in the original precipitate from serum, and has been 
used in part of the experimental work. The claim is made by these authors,. 
unfortunately on very little published experimental evidence, that the 
albumin in serum exists as a complex of albumin, cholesterol esters and two. 
pigments. This point is discussed later in the light of my experimental 
findings. 

The various values recorded in the literature for the specific rotation of 
serum albumin are given in Table IX. 


Table IX. 
Observer. | Material. Specific rotation. | 
= a : | 
Hired énicgl (SSO) eer eensee renee Amorphous — 56°07 to —58 °41 
Starker (SSI) ereeeeeeeeeeeneeres a —60 05 
Sebelienl (S85) ieee -peseeees seers rD —60°1lto —62°6 | 
Machel (S96) ian neccerenese seal Crystalline —61:°0 
Maximovitsch (1901) ............ * — 47°47 
| | 


Preparation of Material. 


Two methods of preparing albumin have been followed with a view to- 
contrasting the products obtained, the alcohol-ether method of Hardy andi 
Gardiner, and a procedure evolved from the general principles of the 
Hopkins-Pinkus method. The blood was drawn from the right jugular’ 
vein of a normal horse and collected in sterile winchesters, where it was. 
allowed to clot spontaneously. The serum was siphoned off after 2 or 
3 days. With one preparation the blood was first defibrinated, and then. 
centrifuged free from corpuscles. 

Mcthod 1.—The serum obtained was transported to cold storage chambers, 
where it was poured into three times its volume of 95 per cent. alcohol at a 
temperature of —4° C. The proteins thus precipitated were allowed to: 
stand for 24 hours, when filtration was commenced through folded filters.. 
The bulky cream coloured precipitate was washed at —4° C. as follows: 
(1) three times with 95 per cent. alcohol; (2) twice with absolute alcohol ; 


Power of Crystalline Ovalbumin and Serum Albumin. — 27 


(3) twice with absolute ether. The precipitate was next transferred to 
soxhlet thimbles, placed in a desiccator, and transported to the soxhlet 
apparatus. In this it was now extracted with absolute ether for 15 hours, 
after which there was no further loss in weight. The thimbles were quickly 
removed to a desiccator containing H2SO, and thoroughly dried im vacuo. 
On cooling, cholesterol esters crystallise out from the ether used in 
‘extraction. The latter apparently extracts some pigment as well, for it 
becomes coloured a bright yellow. The product obtained by this procedure 
is a very fine snow-white powder, which can be kept indefinitely if 
thoroughly dried. It is necessary to keep the precipitate well protected 
from moist air when in contact with either alcohol or ether at a tem- 
perature above 0° C. The precipitate will otherwise turn into a brown, 
brittle mass on drying, which is insoluble in water. From 3250 cc. of 
serum, 240 erm. of protein were obtained, representing approximately 
74 per cent :— 
Analysis of Material. 

The white powder in dry form, or as a concentrated solution from which the 
protein had been removed by means of dialysed iron, was examined for the 
following substances by the tests mentioned below with negative results in 
every case. 


(1) Cholesterol by Salkowski and Libermann-Burchard. 

(2) Lipoid by organic phosphorus, choline and sulphuric acid. 
(3) Fat by acrolein. 

(4) Protein hydrolytic products by biuret. 

(5) Glucose by Benedict’s picric acid. 


The powder was analysed for total non-volatile solids and for its ash- 
content by heating to constant weight in a platinum crucible and igniting at 
a low temperature. The average for three determinations of loss in weight, 
probably representing volatilisation of traces of alcohol and ether, was 
12°36 per cent. and of ash 1:34 per cent. on the dry weight. The samples of 
powder remained soluble in water in spite of having been subjected to a 
temperature of 112° for 8 hours. This was no doubt due to the thorough 
dehydration. 

By reason of the statement made by Hardy and Gardiner that the product 
obtained by them from horse serum alter a somewhat similar procedure showed 
the normal alkalinity of serum, which, they remark, is not due therefore 
simply to alkaline carbonates, it was deemed important to determine the 
alkalinity of the powder in solution. A solution of 13°9 per cent. strength 
was placed in a hydrogen electrode vessel of the Barendrecht type, installed 


28 Mr. E.G. Young. On the Optical Rotatory 


in a constant temperature chamber at 18°, connected up with a calomel 
electrode containing saturated KCl solution and the usual electrical appliances 
for the electrometric determination of hydrogen ion concentration. The 
Walpole medical research potentiometer was used with a Weston standard 
cell as standard. The E.M.F. observed was 689°5 millivolts which represents 
a hydrogen ion concentration of 2°45x107-§ or Py 761. The solution was 
next diluted with an equal volume of water and the difference of potential ° 
again determined. The result was the same as before. 

As the ash-content of the protein powder is quite appreciable, and as is 
evident from the experiments recorded below this ash is made up, at least in 
part, of fixed alkali, the alkalinity of the solution I would attribute to the 
inorganic constituents rather than to the weakly basic protein. 

In the following experiments on the albumin obtained by the above method 
the determinations of Py were made by the potentiometric method as used for 
the alkalinity determination. 

Experiment 1.—A 20 per cent. solution was prepared in dinvulee water. It 
may be noted here that in spite of its content of globulin the protein powder 
is entirely soluble in distilled water. It would thus appear that by this 
method of preparation the eu-globulin is so changed that it is now quite 
soluble in distilled water. To the mixed protein solution was added a 
saturated solution of Kahlbaum’s purest (NH4)2SO, in equal volume. As is 
the case with ovalbumin preparations a distinct odour of ammonia is per- 
ceptible, indicating the presence of fixed alkali. The Py of the medium was 
5°84. The globulin precipitate was allowed to settle for 24 hours. It was 
then filtered off and a portion of the filtrate made acid with acetic acid 
(10 per cent.) to the point of equilibrium found desirable in the erystallisa- 
tion of ovalbumin, «ae. a slight permanent precipitate. No crystals were 
formed and the solution had a Py of 4:16. Another portion of the filtrate 
was similarly treated with acid but to a less degree. Crystals appeared in 
12 hours. The Py of the mother liquor was 430. The remaining filtrate 
was treated with acid to a point when the first faint turbidity was visible. 
Crystals in abundance were deposited inside of an hour. The Py of the 
mother liquor was 4°54. The angles of the crystals were sharp and the 
crystals themselves large and single, resembling small hippuric acid crystals 
very closely. They were filtered off and dissolved in distilled water. There 
was absolutely no trace of residue remaining in the fluid. The solution was 
coloured a yellowish-brown. An attempt to recrystallise this preparation by 
addition of saturated (NH4)2SO, solution, added to the point of the first 
permanent turbidity, produced not a single crystal but only a bulky 
amorphous precipitate in about 30 minutes. 


Power of Crystalline Ovalbumin and Serum Albumin. 29 


Experiment 2—A 15 per cent. solution was prepared and the globulin 
removed as previously. Acetic acid was added until the Py was adjusted 
to 48. Crystals began to form in 2 minutes and by 5 minutes there was a 
heavy precipitate, consisting entirely of crystalline material. The amount of 
albumin which was obtained in crystalline form by this procedure was 
estimated at 70°4 per cent. The crystals were redissolved in water and 
(NH:z)2SO. solution added with great care at a very slow rate to a point 
somewhat short of a permanent precipitate. On standing, the solution 
deposited crystals in about an hour, and the yield was increased by slow 
addition of (NH.)2SO4 The crystals were again brought into solution and 
the liquid still showed a slight yellow colour. The rotatory power was 
observed and the concentration of albumin determined by the Devoto 
method, as previously described. The albumin was again recrystallised and 
the determinations repeated. Table X summarises the results, and it is thus 
evident that the specific rotation is constant after the first crystallisation :— 


Table X. 
| | 
Crystallisation uate a | [a]p38 | ap | [a]z.}8 
: ° concentration. = | 2 rf | are 
| | 
per cent. 2 2 5 e 
= 0-98 —1°13 — 58 ‘06 —1°36 — 69°26 
| 2°18 —2°74 —62 94 —3 41 —78 -20 
| 3°36 —4°29 —62 84 —5 26 —78 °32 
1°42 = |} =O 70 —2°22 —78°25 
PAV ELADE.. . .2shoacores | — _ — 62°83 — —78 26 


Method 2.-—The object of this experiment was to discover whether it was 
possible to obtain an albumin giving the same specific rotation, but prepared 
by a different method. The second method of obtaining serum albumin in a 
pure state was evolved from attempts to obtain crystals from serum by the 
exact application of the method of Hopkins for ovalbumin. This method has 
been used by several investigators as a means of obtaining relatively pure 
serum albumin for various purposes, but the procedure has never been care- 
fully studied, and it was still an open question whether an individual albumin 
could be obtained from serum. Furthermore, the fundamentally important 
relations of serum albumin to the lipoids, cholesterol, and cholesterol esters 
of the blood stream seemed possible of investigation by a contrast of the 
serum albumin obtained directly and by the isolation method previously 
deseribed. 

The method was constructed on the basis of a series of preliminary 


30 Mr. E.G. Young. On the Optical Rotatory 


experiments with both CH3;COOH and H2SO, in an endeavour to determine the 
optimum P, conditions. It soon became evident that any amorphous precipi- 
tate which was formed before crystallisation never became crystalline. This 
is in sharp contrast with the behaviour of ovalbumin. The conditions for the 
deposition are spread over « much wider range of concentration both of 
hydrogen ions and of ammonium sulphate. Crystals, moreover, will continue 
to form as very long narrow needles for several weeks after their first 
appearance. 

When the first crop of crystals has been obtained, centrifuged off and 
placed in distilled water, there is always a considerable residue, although no 
amorphous material can be seen amongst the crystals. The nature of this 
residue has been investigated and is discussed in a later section. In order to 
discover whether the insoluble residue remaining after the first crystallisation 
could be eliminated by previous washing of the serum with ether, this experi- 
ment was tried on a sample of serum with complete success. The crystals 
formed with greater ease, probably due to the lowering of the surface tension 
at the interfaces of growing crystal and mother liquor. The angles of the 
crystals were very sharp and the size was uniform; furthermore, the crystals 
dissolved rapidly in distilled water, leaving only a minute trace of insoluble 
matter. 

The procedure which I have found successful for obtaining a large uniform 
yield of crystals and for recrystallisation is as follows. 

The serum is washed with an equal volume of ether in several small 
quantities by the use of a separating funnel. Serum globulin is removed by 
addition of an equal volume of saturated (NH4)2SO, solution. The precipi- 
tate is filtered off after 4 or 5 hours. The orange-yellow filtrate is slowly 
acidified by means of acetic acid (10 per cent ) or sulphuric acid, N/3, to the 
point of the first trace of turbidity, the solution being constantly stirred. 
This point corresponds to a Py value of 6:0, and a pigment change from red 
to yellow somewhat precedes it. The cloudiness will gradually become more 
dense as further masses of crystals separate. After two hours the acidity is 
further increased by the addition of about half as much acid as previously 
added. Crystals will be found to form with much greater ease once their 
deposition has been induced at a low hydrion concentration. After another 
two hours a third portion of acid is added, equal to the second. This should 
establish the Pa of 4:9 to 5:1, below which it is not advisable to go. The 
yield of crystalline material may be further increased by addition of more 
(NH,)2SO, solution, a few cubic centimetres at a time (1 cc. per 100 ae. of 
fluid) until precipitation ceases. The crystals so separated go into solution 
practically without residue, but the solution is strongly pigmented. Crystalli- 


Power of Crystalline Ovalbumin and Serum Albumin. — 31 


sation can be repeated without much loss of material if care is used in adding 
the (NH,4)2SO, with sufficient slowness. This operation is more difficult than 
the correspnnding one with ovalbumin in that to produce a permanent 
precipitate would be quite fatal to crystal formation. (NH4)2SO4 as a 
saturated solution must be added to a point when the precipitate, which first 
forms temporarily, redissolves slowly. From this on it is safest to add only a 
few cubic centimetres every 20 or 30 minutes (1 cc. per 100 ce. fluid). By 
this method I have obtained 82 per cent. of the total albumin in crystalline 
form. I have crystallised serum which had remained for six months at —4°C., 
so that the factor of staleness would not appear to affect the crystallisation 
of serum albumin to the extent that it does that of ovalbumin. 

Again, I would emphasise that, as in the case of ovalbumin, we are 
obviously treating an equilibrium reaction which is difficult to force to 
completion. The albumin remaining in solution need not of necessity be 
looked upon as non-crystallisable or as essentially different from the 
erystalline. It may be that, if it were possible to control physically all 
the constituents of the reaction, and to alter these as they were changed by 
the deposition of crystals, then the albumin obtained in crystalline form 
would amount to nearly 100 per cent. The physical difficulties, however, 
become greater as the concentration of albumin in solution becomes less. 
The fraction of total albumin crystallised, viz. 82 per cent., is somewhat 
higher by this method than that obtained by the first method, viz., 70 per 
cent. In the first method, however, the concentration of albumin was much 
lower, owing to the small amount of material available. It was thus more 
difficult to control the distribution of water between remaining albumin, 
(NH.4)2SO,, and free acid, while maintaining the latter at its optimum 
concentration. 

By the above method, it has been possible to carry through a series of 
erystallisations with determinations of rotatory power. Table XI summarises 
the results obtained :— 


Table XI. 
Crystallisation, Temperature. Concentration. [a]p. ar, [ale 
: ma 
; cae per cent. ° © ° 

HUIS bie oS oak 20 5 00 — | —3°78 —74°78 
Necond rt... 17 5-41 — —4:°09 —75 ‘71 
AMasbeeles ce ayonrcnwan 15 6-08 —62 98 —4°77 —75 60 
ommnthiieeresscsn. 20 2°34 | —62°:70 | —3:08 —78 -24. 

AVCLaSe coae..... — — | —62°84 | — | —78-42 

| | | 


32 Mr. E.G. Young. On the Optical Rotatory 


The third and fourth values are practically identical when the limits of 
accuracy of the determination of the albumin are considered. It is, 
moreover, of great interest to note that the value coincides with that. 
obtained by the alcohol-ether method, as recorded in Table X. 


Discussion of Results. 


There has been much discussion for years past as to the possible chemical! 
alliance of proteins with fats or lipoids in the blood. This is particularly 
true of eu-globulin, and Hardy and Gardiner have briefly indicated that it is 
possible that serum albumin is chemically bound to cholesterol esters and 
pigments in serum. Now, it is necessary to recognise that, in determining 
the value for specific rotation, we really have only allied rotatory power 
with coagulable material. That is to say, that identical weights of 
coagulated material in undenatured form from the two preparations, if 
made into solutions of identical concentration, would have an identical 
rotation. If cholesterol were present in chemical union with one albumin, 
then it must of necessity have an inappreciable effect upon the total 
observed rotation, and be completely volatilised during the drying operation. 
By .reason of the fact that cholesterol is optically leevo-rotatory, these 
circumstances are, to say the least, improbable. One other possibility must. 
be mentioned. The cholesterol, or other fatty material with which the 
albumin was chemically associated in serum, might be split off in the act of 
crystallisation. The “milieu” for such a chemically conceived separation is, 
indeed, nothing more vigorous than a concentrated (NH,4)2SO, solution and 
very dilute acetic acid at a temperature of 20°. At the same time, 
proteins are extremely labile molecular structures. There is, furthermore, 
one piece of concrete evidence. On first crystallisation direct from serum, 
there is an insoluble residue of a fatty nature. The deposition of this 
residue is completely inhibited by previous ether extraction. The crystals 
themselves are most probably a pure protein, and of the same nature as. 
found for ovalbumin, although differing in their chemical structure of amino 
acids, as indicated by the difference in specific rotation. 

The solutions used for the determinations of specific rotation recorded 
were never entirely free from pigment. The solutions from the first method 
were much clearer than those from the second. In the latter, the successive 
recrystallisations diminished the pigmentation of the solution but little- 
I have tried many methods of removing pigment by adsorbents without 
success, ¢.g., kieselguhr, charcoal and freshly precipitated barium sulphate. 
If the crystals are produced very slowly, the first crop formed is always 
much more coloured than subsequent ones. The amount of pigment, 


Power of Crystalline Ovalbumin and Serum Albumin. 33 


however, must be minute, since concentrations of over 5 per cent: of 
albumin can be examined in 200 mm. polarimeter tubes with ease. The 
work of Palmer and Eckles (1914) tends to show that the pigments are 
earotin and xanthophyll of plant tissues. But since carotin, which pre- 
dominates greatly in horse serum, has such an intense colouring power, the 
amount in the solution used optically must be very small—less than 1 mgrm. 
in 100 ce. 

The true relationship between the fatty material or pigment and the protein 
in natural serum is best understood, to my mind, by the concept that in such 
- complex compounds as the proteins there is no sharp distinction between the 
so-called physical adsorption and chemical union of associated substances. As 

Langmuir has pointed out from inorganic studies adsorption is fundamentally 
chemical. 
The Insoluble Residue. 
I have examined the residues which remained after the first crystallisation 
of several lots of albumin from serum. It is very obvious that they differ 
markedly in texture. Occasionally they can be collected with difficulty on 
account of being an oil. As such it is impossible to centrifuge and difficult to 
filter. When separated it is found to be entirely and readily soluble in ether, 
except for a little contaminating protein. This solution shows a very heavy 
precipitate on adding acetone but an oil is deposited on slow concentration. 
At other times the residue is a solid which can be readily centrifuged off and 
washed free from pigment. I have dissolved this in ether and hot alcohol. 
On cooling the solution in alcohol deposits, needles of the appearance of chole- 
_sterol esters and the solutions give a very strong cholesterol reaction. 
Since no amorphous material is ever visible under the highest power of the 
compound microscope when the first crystalline product is examined, yet a 
very marked residue remains on attempting resolution, I examined the 
erystals while they were going into solution under the microscope. It then 
became clear why the albumin dissolved so slowly. As the highly refractive 
protein interior of the crystal disappeared there still remained the outline of 
the erystal. Soon the field became covered with these shells of the former 
erystals without any remaining protein. There was still no amorphous 
material. On agitating the slide, however, the crystal outlines rounded into 
fat-hke globules. It would thus appear that each crystal had had a thin 
covering of fatty material deposited about it. This would naturally interfere 
with the process of crystallisation and make the crystal sizes vary. It is 
noticeable that large crystals are first formed which are four or five times the 
mass of smaller ones appearing later. Furthermore the presence of this fat- 
hike material alters the shape of the crystal, obliterating angles and inhibiting 
VOL. XCIII.—B. D 


34 Mr. E. G. Young. On the Optical Rotatory 


rosette formation. When the serum is ether-washed previously to erystallisa- 
tion, the crystals show sharp angles, are of uniform size and more quickly 
deposited. 

The above observation shows how the nature of the medium may affect the 
structure of the crystals and is of interest in the consideration of all the 
work which has been done in the endeavour to link up crystalline form of 
proteins with plant or animal species from material which has been erystal- 
. lised but once from its natural medium. It is also interesting in showing the 
possible physical associations between lipoid and protein material in protoplasm 
and the difficulty of removing the one constituent without affecting others. 


SUMMARY AND CONCLUSIONS. 


Crystalline Ovalbumin. 


The specific rotation of crystalline hen ovalbumin is found to be —30°81° 
for [a]p 1° and —37-53° for [«],°. The value for [2] is constant within the 
limits of experimental error after the second crystallisation if recrystallisation 
be carried out about the isoelectric point (Px 4°9-5'1). 

At a lower hydrion concentration (Py 5°3-5'4) a constant lower specific 
rotation is obtained, [«], * = —3014° and [a], !® = —36:80°. The results 
were identical by two different methods of analysis. 

The optical rotation of an albumin solution at its isoelectric point remains 
constant. If it be made slightly acid a prompt rise in rotatory power is 
observed to a new constant level. If the solution be made alkaline to Py 49 
a prompt fall in rotatory power ensues which very slowly rises to the previous 
value. This phenomenon can be brought about from either side of the 
isoelectric point and is reversible. The variations in optical rotation are 
explained on the basis of a tautomeric equilibrium of the lactam-lactim type. 

Experiments are recorded which tend to show that globulith formation is 
no indication of the power of a protein to crystallise, but probably indicates 
hydrate formation. 


Crystalline Serum Albumin. 


Two methods have been used for the preparation of pure serum albumin 
(horse). The first method involves the precipitation and complete dehydration 
of the mixed proteins of serum at a temperature of —4° C., by means of 
absolute alcohol and ether, removal of fatty substances by Soxhlet extraction, 
and crystallisation of albumin from aqueous ammonium sulphate solution ; 
70-4 per.cent. of the total albumin was obtained in crystalline form. After 
the first crystallisation the product possesses a constant rotatory power, 
[ice Sel) ODS eiliete Bi SiSz. 


Power of Crystalline Ovalbumin and Serum Albumun. — 35: 


The second method involves the extraction of the serum by ether and the 
crystallisation of the albumin by ammonium sulphate and acid at Piz 6°0 to 
5:0; 82 per cent. of the total albumin is thus obtained crystalline. After the 
third crystallisation the specific rotation is constant, [a]p 1% = —62'8°, 
ae —78°4°. 

The insoluble residue from the first crystallisation from natural serum is 
shown to bea deposit of fatty material around the crystal surface. Its nature 
varies with different preparations, sometimes phosphatides predominate : 
sometimes cholesterol esters. The relation of fatty material and protein in 
serum is discussed. 


In conelusion I wish to express my sincere thanks to Prof. F. Gowland 
Hopkins for his valuable advise and criticism during the course of this 
research. 


REFERENCES. 


Alexander, A. C., ‘J. Exper. Med.,’ vol. 1, p. 304 (1896). 

Bradford, 8. C., ‘Biochem. J.,’ vol. 14, p. 91 (1920). 

Chick, H., and Martin, C. J., ‘ Biochem. J.,’ vol. 7, p. 380 (1913). 

Clark, W. M., and Lubs, H. A., ‘J. Bacter.,’ vol. 2, pp. 1 and 109 (1917). 

Giirber, A., ‘Sitzungs. Wiirzburger Phys.-med. Ges.,’ p. 143 (1894). 

Hardy, W. B., and Gardiner, Mrs. S., ‘Proc. Physiol. Soc., J. Physiol.,’ vol. 40, p. 68 
(1910). 

Hopkins, F. G., ‘J. Physiol.,’ vol. 25, p. 306 (1900). 

Hopkins, F. G., and Pinkus, 8. N., ‘J. Physiol.,’ vol. 23, p. 130 (1898). 

Krieger, H. Th., ‘ Inaug.-Dissertation,’ Strassburg, from Maly (1899). 

Michel, A., ‘ Verhand. Wiirzburger Phys.-med. Ges.,’ p. 29, No. 3 (1896). 

Osborne, T. B., ‘J. Amer. Chem. Soc.,’ vol. 21, p. 477 (1899). 

Osborne, T. B., and Campbell, G. F., ‘J. Amer. Chem. Soc.,’ vol. 22, p. 422 (1900). 

Oswald, A., ‘Z. physiol. Chem..,’ vol. 95, p. 102 (1915). 

Palmer, L. S., and Hekles, C. H., ‘J. Biol. Chem.,’ vol. 17, p. 223 (1914). 

Pauli, W., Samec, M., and Strausz, E., ‘ Biochem. Z.,’ vol. 59, p. 470 (1914). 

Robertson, T. B., ‘ Die physikalische Chemie der Proteiue,’ Dresden (1912). 

Sorensen, S. P. L., ‘Erg. Physiol.,’ vol. 12, p. 393 (1912). 

Sorensen, 8. P. L., et al., ‘Compt. Rend. Lab. Carlsberg,’ vol. 12 (1917). 

Willcock, E. G., ‘J. Physiol., vol. 37, p. 27 (1908). 


36 


Experiments on Amphibian Metamorphosis and Pigment Responses 
in Relation to Internal Secretions. 


By Junian 8S. Huxury (New College, Oxford) and Lancetor T. HoGBEn 
(Imperial College of Science, London). 


(Communicated by Prof. E. W. MacBride, F.R.S. Received July 26, 1921.) 


CONTENTS. 
PAGE 

1. Metamorphosis of the Axolotl by Thyroid Administration ...............:.00. 36 
2. Acceleration of Metamorphosis by Iodine with Triton and Salamandra Larve 42 
3. Negative Effects of Administration of other Ductless Glands and Iodine on 

Metamorphosis in! “ASxolopllycesesnsaes.eeerascnecsesessnesnseeseisnees ast eters eee 44 
4. Effect; of Thyroid Weeding on! Necturus) 2.2... .se<ccscensemeceseeeer eee s-seaene eee eee nee 46 
5. Pigmental Reactions of Axolotls to Adrenal and Pituitary Hormones ...... 48 
6. The Pineal Pigment Cycle im Frog Wad poles) 2-20. .c.nc0ee sc. osece-ceseeeeseeeeeeee 51 
SUMMALY. (oecescacessenesescsseoseddecoecobssarnece deter saetacascccn eect eet seca tc Eeeee eee ae 52 


The problem of metamorphosis in Amphibia attracts attention from many 
points of view. These organisms have, one may say, acquired interest as 
being indicators for the action of certain internal secretions; the underlying 
mechanism of metamorphosis is still imperfectly understood ; and the general 
biological problems involved, especialiy with respect to the neotenous and 
perennibranchiate forms, are remarkably fascinating. The experiments here 
recorded were undertaken with a view to elucidating these issues more fully, 
and in the course of them data relevant to the pigmental responses of Amphibian 
larvie also emerged. Jor various reasons it was deemed desirable to publish 
an account of the observations so far completed. Acknowledgment is made to 
Mr. D. F. Leney of New College for assisting in the care of animals, to the 
Royal Society and the Trustees of the Dixon Fund for grants in aid of the 
expenses incurred by the authors respectively, and to Prof. E. W. MacBride, 
F-R.S., for his kindness in reading the MS. 


1. Metamorphosis of the Axolotl by Thyroid Administration. 


(a) When a note (Huxley, ‘ Nature, 1920) on the metamorphosis of medium- 
sized but immature Axolotls by means of thyroid feeding was published by 
one of the authors a year ago, and confirmed a few months later (Hogben, 
‘P.ZS8., 1920), our attention had not been called to Jensen’s work (1916). 
Since this appears to be little known and is not readily accessible to English- 
speaking workers, it is proposed to give a short 7éswmé of some of his most 
important findings. . 


On Amphibian Metamorphosis and Internal Secretions. 37 


Hirst, however, a record of one of our own experiments will be given. 
Two black Axolotls, one male, the other female, and both sexually mature 
(though not quite full-grown), were kept in a large tank, together with two 
fine full-grown white Axolotls as controls. The controls were fed on worms, 
while the others were given pieces of ox-thyroid. In every case the feeding 
was controlled, the worm or piece of thyroid being kept near the animal’s 
mouth until swallowed ; if persistently refused, it was removed. The thyroid 
feeding began on November 30, 1919. On December 29, the thyroid-fed 
animals were past the critical stage of metamorphosis (see Boulenger, 1913); 
on January 13 no trace of gills was left; on January 19 the animals left the 
water, and the metamorphosis could, therefore, be considered as complete. The 
temperature was at first that of the room, varying from 8° to 15° C.; from 
January 2, when the water was warmed by an electric-light bulb, it rose to 
between 14° and 16° C. The length of the specimens at completion of meta- 
morphosis was 17-4 cm. (4) and 146 em.( 2); the male had the typical sien 
of maturity—enlargement of the lips of the cloaca—well-marked. The 
controls during the same period showed no changes. It is an interesting fact 
that the metamorphosed animals, although tending to come more frequently 
to the surface about the time that the critical stage was reached, showed no 
inclination to leave the water until after the morphological alterations had 
run their course. It is only when a few days have elapsed after the com- 
pletion of the visible external changes that the animals emerge from the 
water. 


(b) Jensen’s Work. 


Curiously enough, Jensen also believed that he was the first to obtain the 
metamorphosis of Axolotls by thyroid, and it was only after his paper was 
completed that he found that he toohad been anticipated—by Laufberger (1913). 
Jensen’s experiments, however, are much fuller. He used ealf-thyroid as 
diet. Two-week old larvee on this diet all died within a few days, without 
change of form. Four immature specimens (10 to 16 cm. in length) meta- 
morphosed in 17 to 25 days without exception. They were fed only three to 
eight times, with 1 to 3 grm. thyroid in all. An intra-abdominal injection of 
jodothyrin caused metamorphosis in the same length of time (10 specimens). 
The amount of iodothyrin injected had no influence upon the rate of meta- 
morphosis within the limits used (1 to 10°5 megrm.). One specimen fed upon 
iodothyrin started to metamorphose, but after 21 days of very slow change 
began to reverse the process, and became larval again. Four mature 
specimens, 22 to 24 cm. long, and two of them known to be over 6 years old, 
were also successfully metamorphosed. The time required was here greater, 


38 Messrs. J. S. Huxley and L. T. Hogben. 


varying from 27 to 32 days. Twoof these animals were females who had just 
started to lay eggs; the very interesting fact was noticed that oviposition 
ceased almost immediately the thyroid diet was started. (It would thus appear 
that the katabolic action of thyroid will not permit of the anabolic activities 
necessary for the production of eggs. On the other hand, the experiment 
does not prove that egg laying cannot proceed during metamorphosis, for the 
amount of thyroid given was certainly very much in excess of that necessary 
to produce metamorphosis. It would be of great interest to attempt work 
along these lines with amounts of thyroid (or iodothyrin or thyroxin) close to 
the critica] amount.) One immature animal was fed on thyroid while in deep 
water; in spite of this, metamorphosis proceeded at the usual rate. There 
were no failures to metamorphose (except the above-mentioned one on 
iodothyrin diet) among all the survivors, and the two that died under the 
treatment were metamorphosing regularly up to the time of their death. 
Salamandra maculosa larve were also metamorphosed by a thyroid diet. 
Eight-day larvee came out on land in 13 to 16 days, and the same period of 
time was taken by 30-day larve. Axolotls in 1/10,000 KI solution did not 
metamorphose, but on the contrary developed abnormally large gills. 

Experiments were also carried out by Jensen upon Necturus and Proteus. 
One adult Necturus was at first fed on thyroid, later injected with iodothyrin. 
The only changes noted were the resorption of one gill on one side, and a 
slight change in pigmentation. The animal died after some time. Four 
specimens of Proteus were also treated, some fed on thyroid, others injected 
with iodothyrin. The animals were 12 to 24 cm. long. In all cases, there 
was a distinct but slight atrophy of the tail-fin and of the gills, but the total 
shrinkage of the fin in height was not more than 1 to 15 mm., and after 
6 months it regained its original size. Thus, with animals of this size, only 
slight effects could be obtained, indicating that in these permanently neotenic 
forms, the tissues are not sensitive to thyroid in the same way as in normally 
metamorphosing or facultatively neotenic species. Similar treatment failed 
to produce metamorphic effects in the Ammoceete larva of the Lamprey. 

Jensen further notes that in many, but not all, of his treated Axolotls, 
pathological symptoms occurred as a result of thyroid or iodothyrin. The 
animals refuse to eat, are usually sluggish when stimulated, rush about 
madly for several minutes, and exophthalmos of a very pronounced - type 
appears. This exophthalmos, it is interesting to note, disappears with the 
other pathological symptoms from a few days before metamorphosis to three 
weeks after it has occurred. 


On Amphibian Metamorphosis and Internal Secretions. 39 


(ec) External Agencies, Age, and Rate of Metamorphosis. 


As will be seen later, our young Axolotl larve fed on thyroid, all 
metamorphosed in from 24 to 32 days. We are thus in a position to 
compare the time necessary for metamorphosis induced by thyroid-feeding 
with that induced by air-breathing. Marie von Chauvin’s classical experi- 
ments were done with great precautions, and the time taken was very large. 
Those of Boulenger (1913) will serve as our example, for it is not probable 
that the time for metamorphosis by this means can be much reduced beyond 
what was found by him. His results show .that the time required for 
metamorphosis by air-breathing is a function of the temperature. Those 
kept at 75° to 80° F. metamorphosed in 89 to 109 days, while those kept at 
55° to 60° F. took 116 to 120 days. The times required by animals fed on 
thyroid were always much smaller, varying from 17 to 50 days. The times 
recorded by Jensen are uniformly less than those found by us. This very 
probably is connected with a difference of temperature, since our experi- 
ments were all carried out in the depth of winter, at temperatures, be it 
noted, considerably below the lowest used in Boulenger’s experiments. 
Mature animals always require a longer time than do immature. Jensen’s 
results show that the minimum time for metamophosis was 10 days longer in 
adults than in immature specimens, the maximum 7 days longer; the 
minimum time for adults was thus greater than the maximum for immature 
specimens. The difference in our own experiments was still greater. For 
immature specimens, it does not appear to make any difference whether 
they are quite young (under 3 inches in length) or half-grown; the time 
taken is approximately the same. This appears to indicate a real difference 
between the metabolism of adult and immature specimens, and that the 
time-difference observed is not due merely to differences of size. We may 
sum up by saying that thyroid-feeding causes metamorphosis much more 

‘rapidly than does enforced air-breathing; and that the change to sexual 
maturity in Axolotls increases the time necessary for metamorphosis by 
25 to 50 per cent. 

Some Amblystoma used in our experiments were bred in the laboratory in 
the spring of 1920. The eggs were laid in the second half of April. Young 
specimens from these eggs were used for experiment later in the same 
year. The first experiments were made in the first week of July, the 
larvee then being not quite 3 months old, and measuring almost precisely 
50 mm.in length. Four of these were fed upon thyroid, and showed all the 
usual metamorphie changes ; the gills were reduced to mere stumps without 
filaments, the fin along the back was resorbed, and the eyes became 


40 Messrs. J. 8S. Huxley and L. T. Hogben. 


prominent. All four, however, died between the fourth and fifth weeks of 
the thyroid diet, when practically metamorphosed. It was not possible to 
say whether death was due to internal causes resulting from a too-precocious 
transformation, or whether it was due to the animals not being able to 
climb out of the water: that this latter suggestion might well be valid is 
shown by the fact that the animals’ legs at this stage were proportionately 
smaller, and especially slenderer, than at later stages, and were quite 
possibly too weak to support the creatures on land. On the other hand, 
other Amblystoma, transformed by thyroid, usually remained several days in 
water after completion of all. visible morphological metamorphic changes; so 
that death is most probably tc be ascribed to internal changes, especially since 
it is known that in Anura there is a lower limit of size below which thyroid 
diet induces an attempt at metamorphosis, but one which is always followed 
by death. 

A single larva was next put on thyroid diet on August 16, 1920, z.e., when 
about 4 months old and 59 mm. in length. The animal showed meta- 
morphic changes after 15 days, came out of the water after 26 days, and lost 
the last traces of gills after 29 days. Metamorphosis can thus be induced in 
Amblystoma that have not yet attained a length of 24 inches; it is possible 
from then onwards at any size or age. Three more larve were started on the 
thyroid diet on August 9, 1920, when nearly 5 months old. The first 
traces vf change were observed on the 17th day, the gill-filaments were all 
resorbed by the 23rd day, and all emerged from the water between the 30th 
and 32nd days. A slight diminution in size had taken place in the process, 
the original lengths of 68, 60, and 60 mm. being reduced to 64, 59, and 
58 mm. respectively. 

The following experiment was carried out to see whether immersion in 
dilute alcohol would accelerate or retard the metamorphosis caused by 
thyroid. Five dishes were prepared, one with larva in each. All contained 
600 c.c. of liquid. (P) contained water, and the individual in it was fed on 
meat as a control. (T) also contained water, but the animal in it was fed on 
thyroid. (Q) contained 1/3 per cent. alcohol, (R) 1/6 per cent. alcohol, and 
(S) 1/12 per cent. alcohol. The animals in (Q), (R) and (S) were all fed on 
thyroid. The alcohol solutions were changed thrice weekly. The experiment 
was started on November 5, 1920. It was very noticeable that the animals 
in the alcohol solutions were, especially at first, more sluggish than either of 
those in plain water. They did not at first react to stimulation, but, after 
the stimulus had continued for some time, they responded by abnormally 
violent movements. After 2 to 3 weeks, however, they apparently became 
acclimatised to the alcohol, for their sluggishness was not nearly so marked. 


On Amphibian Metamorphosis and Internal Secretions, 41 


(S) was distinctly less sluggish than the other two, between which not much 
difference could be noticed. 

_ The experiment was not wholly conclusive. The differences between the 
rates of metamorphosis of the four thyroid-fed animals was very slight. 
There was, however, a slight retardation seen in the specimens kept in 
alcohol, and a retardation which increased with the concentration. 

The experiment was therefore repeated with the following variations: 
(1) Control: meat-fed in water. (2)-(5) Thyroid-fed: (2) in water ; 
(3) in 1/3 per cent. alcohol; (4) in 2/3 per cent. alcohol; (5) in 1 per 
cent. alcohol; two animals in each dish. The same slight retardation of 
metamorphosis with alcohol, increasing with concentration, was observed. 
Again, larger numbers are really necessary before the retardation can be 
regarded as proved; but, so far as it goes, these experiments appear to be 
significant. 

All the alcohol specimens this time showed redness and congestion of the 
gills, increasing with concentration. The congested gills were not reduced 
till relatively very late, and then decreased remarkably rapidly in size. 
The 2/3 per cent. and 1 per cent. specimens all died suddenly just before 
they were due to metamorphose. Metamorphosis in the control took 
29 days; in the 1/3 per cent. 29 and 31 days. 


(d) Size of Thyrord. 

From all recent work upon the metamorphosis of Amphibia, it would 
appear certain that metamorphosis is normally associated with thyroid 
activity, taking place when certain substances produced by the gland reach 
a definite concentration in the body. In those forms, therefore, in which a 
typical metamorphosis rarely or never occurs, we should @ priort expect to 
find an abnormally small thyroid. 

In the Axolotl, however, the thyroid is not abnormally small; further, 
it presents a perfectly normal histological picture. It would only be 
possible to give definite data after a careful examination of the relation 
between thyroid-weight, body-weight, and, if possible, iodine-content (or other 
criterion of activity) of thyroid in a number of Urodela, including normally 
metamorphosing forms, the Axolotl, and some Perennibranchiates. There is, 
however, one observation which it is of interest to mention here. That is 
the abnormally large size of the thyroid in Siren. This has been recorded by 
Wilder in his anatomical account of the animal (1891); and his account we 
have confirmed from the dissection of two specimens preserved in spirit in the 
Oxford University Museum. The thyroid is not only as large as in a normally- 
metamorphosing species, but much larger. Of this fact there can only be 


42 Messrs. J. S. Huxley and L. T. Hogben. 


two explanations. Either the thyroid of Siren is no longer producing the 
same type of substances as in other Amphibia; or, if it is continuing to do 
so, the bedy of the organism does not respond to the substances produced in 
the same way as in other species. That this latter is a possibility is shown 
by the example of Proteus and Necturus, which cannot be transformed 
by thyroid-feeding. In either event, the large size of the thyroid is very 
peculiar and demands investigation; possibly it has taken on some new 
function. From the particulars given by Platt (1896) it does not appear that 
the thyroid is under-developed in Necturus. In Typhlomolge, on the other 
hand, Emerson (1905) reports the complete absence of a thyroid gland. 
Leydig (1853) describes the thyroid of Proteus as being small, median, and 
composed of but 3 to.15 vesicles, which, however, often contained colloid. 


(e) Exophthalmos Associuted with Metamorphosis. 


It is an interesting fact, which, so far as we know, has not been previously 
emphasised, that Amphibia before metamorphosis have their eyes flush with 
the surface of their head, but that in the adult state the eyes protrude con- 
siderably. In the case of the Axolotl the eyes remain flush with the surface 
so long as the animal remains in the aquatic form, even if it becomes sexually 
mature. The phenomenon appears to occur equally in Anura and Urodela. 
In view of the connection of the thyroid with Amphibian metamorphosis, 
this protrusion of the eyeballs becomes interesting when it is remembered 
that exophthalmos is one of the most prominent symptoms of Graves’s 
disease. Whether the exophthalmos in Amphibia at metamorphosis has any 
relation to that of exophthalmic goitre cannot be definitely stated. 


2. Acceleration of Metamorphosis by Todine with Triton and Salamandra 
Larevee. 

Following Swingle’s results (1919), a solution of iodine was made by shaking 
up an excess of iodine crystals in tap-water in a 2-litre flask, allowing to 
stand for 2 days, and then diluting as required with tap-water. Two larvee 
of Salamandra maculosa were put into each of a series of dishes containing 
350 c.c. of fluid each. 

No. 1 was the control (tap-water only), the rest contained a saturated 
solution of iodine, diluted respectively 10, 50, 100, 500, and 1000 times. 
The experiment was performed at room temperature in January, 1920. In 
all except the 1/10 solution the animals fed well. In the control and in the 
1/100 solution the gill-filaments were unchanged after a week. In the 1 /500, 
the 1/100, and the 1/50 solutions the gill-filaments were somewhat reduced 
after 5 days. For some reason the greatest reduction occurred in the 


On Amphibian Metamorphosis and Internal Secretions. 48 


1/100 solution. In the 1/10 solution the animals refused to feed at all. 
One (the smaller) died after one day. The other was more sluggish than 
normal animals. It showed distinct reduction of the gill-filaments after 
3 days. The filaments were nearly absent after 5 days; on this day 
the solution was diluted to 1/12, as the animal seemed ill at ease. On 
the eleventh day mere stumps of gills were left, and the tail-fin had been 
nearly resorbed. The animal was poorly, not reacting to stimuli properly, 
and on the twelfth day it was dead. The saturated solution contained 
0076 mgrm. of iodine per litre. 

Another series of experiments was started on January 26, 1920. Unfor- 
tunately, it had to be discontinued after 16 days, owing to the ill-health of 
Miss F. Peterson, who kindly helped with the work, and to whom grateful 
acknowledgment is made. Twelve sets of four animals were taken, there 
being two larve of S. maculosa and two of Triton vulgaris in each lot. These 
were distributed as follows, according to temperature and to strength of 
iodine solution :— 


| Strength of solution (in dilutions of a solution of iodine | 
saturated at room-temperature). | 
Temperature. | : 
Te | 2. | 3. | 4, 
| | | 

| 

th OES) CE | 1/20 1/50 1/125 Control. 

Bae =182.0.2..,... etennete: | 1/20 mee 1/50 1/125 e 

Sa DS Oa ae aa aaa ie | 1/20 1/50 | 1/125 fs 
| 


In Series C no animals metamorphosed during the 16 days. In Series B 
five metamorphosed after an average time of 11:6 days. Im Series A five 
metamorphosed after an average time of 6 days. This shows a very decided 
retarding effect of low temperature upon metamorphosis. 

All animals were here kept in the same volume of water, so that only two 
variables affecting gill-size remained. High temperature was found to favour 
gill-development, while high iodine-concentration had the reverse effect. This 
was well brought out in the results. The gills of those kept at 26° C. could 
be arranged in a graded series according to size, the controls having the 
largest filaments, those in the 1/20 solution the smallest. Those kept at 
2°_5° C. also showed a series which was identical, except for the fact that it 
started at a much lower level; in fact, the gills of the controls at the low 
temperature were slightly smaller on the average than the gills of those kept 
in the 1/20 solution at the high temperature. The 17°-18° C. series was 
intermediate, but rather nearer, as would be expected, to the high-temperature 
series. 


44 Messrs. J. S. Huxley and L. T. Hogben. 


Great diversity in the rate of metamorphic change was found, so much so 
that the original purpose of the experiment—viz., to get some quantitative 
data on the time-relations of metamorphosis to iodine concentration— 
had to be given up. It appeared that the prominence of the eyes 
which accompanies metamorphosis in these forms, might begin at high 
temperatures while the gills were still hyper-normal in size. Thus the 
effect of high temperature may be to mask the effect of iodine as far as the 
gills are concerned. When the resorption of the gills did begin at high 
temperatures, it went very quickly, the effect of the iodine overcoming the 
antagonistic effect of temperature. This is probably, according to Swingle, 
due to the power of all tissues of the body, but especially the thyroid, to 
manufacture from iodine some substance which causes the initiation of meta- 
morphosis, when it reaches a certain critical concentration inside the body. 

It, however, appears probable that the iodine may also have a direct effect 
on the tissues of the gills. The violent action of the 1/10th solution upon 
the filaments in the first experiment is very likely to be explained in this way. 
It is at any rate certain that the filaments are very sensitive to external 
agencies, as is also the tail-fin. Both of these structures, for instance, are 
much reduced when Amblystoma larve are kept in a very small amount of 
water or in damp moss, the mechanical alterations seeming to initiate the 
reduction (although of course the final complete metamorphosis which takes 
place in these circumstances must depend upon other, more deep-seated 
changes). We may say, therefore, that low temperature, exposure to air 
instead of water, and probably iodine solutions, have a directly unfavourable 
effect upon the gill-filaments, causing a certain amount of dedifferentiation 
and resorption, while the other agencies, such as thyroid diet and iodine 
accumulated in the body, exert an indirect effect by altering the internal 
environment to the point where metamorphic changes are started. Once these 
begin, the character of the gill-epithelium is altered, and the gills are rapidly 
reduced to mere blobs (fig. 4). 


3. Negative Effects of Administration of other Ductless Glands and of Iodine on 
Metamorphosis in Axolotls. 


Attempts were made to induce metamorphosis by administration of iodine 
in the medium and with the food to three animals 12 cm. in length. In the 
latter case, as in Swingle’s earlier experiments (1919), a small quantity of 
finely powdered iodine was used, being in this case dusted on to thin slices ~ 
of meat rolled into pellets. Such treatment does not produce obvious dis- 
comfort, nor is it poisonous, and with several animals was continued bi-weekly 
for 8 to 10 weeks without any diminution of gills and tail-fin or any evident 


On Amphibian Metamorphosis and Internal Secretions. 45 


toxic consequences. All our endeavours confirm the conclusion of Jensen 
that iodine free of organic combination is not efficacious in producing meta- 
morphic changes in the Axolotl. It is thus evident that in the neoteny of the 
Mexican Salamander it is not primarily the exogenous factors (available 
iodine supply and temperature) contributing to normal Amphibian meta- 
morphosis that are significant. 

Hence it became desirable to test the effect of administering other ductless 
glands. In this connection the results of D. I. Macht (1919), who has claimed 
to accelerate transformation in frogs by prostate-feeding, as also Bennet 
Allen’s experiments (1920) on the part played by the pituitary gland (anterior 
lobe), demanded some attention. On the other hand the experience of 
Gudernatsch (1914) and others does not indicate the likelihood of influencing 
Amphibian metamorphosis by administration of pituitary gland per os. 

Both young (4 to 6 months) and old (18 months) larvee were fed with pituitary 
gland (anterior lobe). In the experiments glands of both old animals (ox) and 
of calves were employed and the treatment was continued for three months 
without producing metamorphic phenomena. As an illustration, particulars 
of the following experiment will serve. Fresh ox pituitaries were obtained 
from a slaughter-house, and the posterior and anterior lobes separated. On 
October 2, 1920, four vessels were prepared with four larve in each, which 
were nearly 6 months old. Those in vessel A were fed on thyroid; those 
in B on pituitary anterior lobe (henceforth called pituitary for brevity’s sake, 
since posterior lobe was never employed in any of these experiments); in C on 
thyroid and pituitary on alternate days; and in D on raw meat, as controls. 

Those fed on thyroid showed the first visible signs of transformation on the 
14th day, their gill-filaments were resorbed by the 21st day, and the meta- 
morphosis was morphologically complete on the 26th day. The controls 
showed no change throughout. Those fed on pituitary have showed no 
metamorphic changes at all; three were kept on the diet for 3 months, one for 
8 months. It is obvious that the diet has no effect on their transformation. 
Those fed alternatively upon pituitary and thyroid metamorphosed in a 
perfectly normal way; the process, however, took slightly longer, the first 
signs of change appearing two days later than in those fed only on thyroid, 
the total resorption of the gill-filaments, and the morphological completion of 
the process, each taking place 3 days later than with the thyroid-fed ones. 

The rates of growth upon the different diets are illuminating; they may 
best be presented in tabular form :— 


46 Messrs. J. S. Huxley and L. T. Hogben 


Original length. | Increase in length in 31 days. 
| 
) | Average. | Average. : Range. 
| | | 
— — l = 
mm. mim. mm. 

Avy thyroid! diet e22,).<cersseeeeeeeose-e se 63 ‘0 1°75 0 5-30 
[pb smbltiitaryediebi. 5. act. eeeeee neces 620 150 14 ‘0-16 ‘0 
| C. Alternate thyroid and pituitary 62 °5 4°5 30-6 5 
| diet 
apr iMieat dict ort ere | 61-5 13-0) | © Sao-o-r7e 


It will thus be seen that thyroid diet in this experiment did not cause a 
diminution of size, but permitted a very small increase. The controls fed on 
meat grew rapidly, but their growth was definitely, if slightly surpassed, by that 
of the pituitary-fed animals. Those fed alternately on pituitary and thyroid 
showed only a small amount of growth, but it was almost three times that 
recorded for those fed on thyroid alone, whereas the proportion of delay of 
metamorphosis in (C) was by no means equally great. From this, as well 
as from (B), it would appear that pituitary has definite growth-promoting 
properties for Amblystoma larve. 

Half-a-dozen animals used in the pituitary-feeding experiments were taken 
at intervals of time and transferred to thyroid diet. The time taken for 
metamorphosis in three cases was slightly longer (33 to 35 days to 
emergence) than in controls, so that there may have been a retardation. In 
any case, these larvee were clearly able to complete their metamorphosis.* 

One individual was also fed for 3 months on fresh prostate without 
showing any perceptible sigus of transformation, though subsequent thyroid 
treatment induced metamorphosis. 


4. Effect of Thyroid-feeding on Necturus. 


The suggestive, but inconclusive, experiments made by Jensen, with a 
view to elucidating more fully the significance of the Perennibranchiate 
condition in relation to the physiological processes underlying metamorphosis 


* One animal was continued on the pituitary diet for some time, a control in identical _ 
conditions being fed on ox muscle. After five months, the pituitary-fed specimen 
weighed 32°5 grm., the control 112 grm. Other animals of the same age fed on meat 
were all of about the same size as the control. It has since been suggested to me that 
if the control had been fed on more succulent diet, such as brain or liver, it would have 
rivalled the pituitary-fed specimen. Until the suggestion can be tested experimentally, 
I content myself with stating the facts. The growth of meat-fed specimens in the 
previous experiment, lasting 31 days, was about as great as that of the pituitary-fed 
animals. Later, the meat-fed animals’ growth became relatively much slower. Another 
noticeable feature was that in thyroid-metamorphosed specimens previously fed on 
pituitary the size of the limbs was greater than in those not so treated.—J. 8. H. 


On Amphibian Metamorphosis and Internal Secretions. 47 


in other Urodeles, indicated the advisability of extending such observations. 
It was not possible to obtain specimens of Proteus; but, through the 
courtesy of Prof. MacBride, three live medium-sized Necturus were secured, 
and submitted to thyroid treatment in his laboratory at the Imperial College 
of Science. 

The experiment began on November 10, 1920. One animal was kept as a 
control, and fed on small pieces of raw beef: the other two were given fresh 
thyroid (ox) gland tri-weekly, as in the case of the first experiments with 
Axolotls. Up till the time of writing, the treatment has been continued 
without interruption for 7 months. No pigmental changes have resulted ; 
and there has been in neither case any appreciable reduction of the tail-fin. 
As regards the condition of the external gills, observation is embarrassed by 
the fact that the filaments are in a very marked degree erectile, their 
length, when fully dilated with blood, being many times greater than 
when the animal is not actively respiring: they can, however, be induced to. 
extend by compelling the animal to perform muscular exercise, after which 
a rough estimate may be made of their maximum dimensions. Constant 
attention to this point showed very clearly that the filaments of the experi- 
mental individuals were relatively shorter even when fully extended ; but, 
for an obvious reason, it is not possible to interpret this as necessarily 
consequent upon a reduction in the actual amount of tissue; for it is well 
known that thyroid administration influences the blood pressure in Verte- 
brates, and the behaviour of the gills in Necturus is evidently a vaso-motor 
phenomenon. 

It does not seem likely, therefore, that an administration of thyroid in this 
Perennibranchiate form is effective in producing somatic modifications com- 
parable to those occurring at metamorphosis in other Urodeles. In view 
of the morphological data given by Platt, and of the experimental evidence 
available, it may therefore be stated that, if the Perennibranchiate forms 
like Necturus and Siren are not primitive in the invariable retention of 
the larval type of Urodele organisation throughout life, their failure to 
develop the predominantly adult characteristics is not due primarily to 
thyroid deficiency—this, of course, does not apply to Typhlomolge, and 
possibly to Proteus also—nor to a defective supply of iodine in the 
environment. There remain at least four possible interpretaticns, then, of 
the Perennibranchiate state :— 

(i) That these animals have never possessed genetic factors responsible for 
the structures of typical adult Urodela; «¢., that in this respect they are 
actually primitive. This appears to be negatived by the purely morpho- 
logical evidence available (see Gadow, 1901, pp. 65, 136). 


48 Messrs. J. S. Huxley and L. T. Hogben. 


(ii) That their thyroid mechanism is unable to make use of the available 
iodine supply so as to produce the requisite amount of active iodine compound 
required to stimulate metamorphosis. 

(iii) That endogenous factors involved in the maintenance of the thyroid 
in a condition of functional activity are not operating effectively. 

(iv) That the larval tissues concerned have collectively lost the power to 
respond to the thyroid activator. 

The second and last possibilities are emphasised by the fact that, whereas 
in normal Amphibia Swingle has shown that iodine free of organic combina- 
tion can suffice to induce metamorphosis not only in normal but in thyroid- 
ectomised larvee, in the case of the mature Axolotl, iodine alone is not an 
efficient substitute for the thyroid autacoid. The importance of the third is 
sufficiently demonstrated by the work of Uhlenhuth (1919) on the relation 
of growth to metamorphosis in Salamanders, and Allen’s recently published 
account of the inhibition of transformation in Anura by hypophysectomy. 
The state of affairs encountered in the Mexican Salamander is eminently 
suitable to a further analysis of what we have termed the endogenous factors 
in Amphibian metamorphosis; and it is hoped to obtain shortly in this 
connection data respecting the relation of the method of thyroid-feeding to 
enforced air-breathing as a means of bringing about the assumption of adult 
characters. 


5. Pigment Reactions of Axolotls to Pituitary and Adrenal Hormones. « 


While the feeding of Axolotls with pituitary gland did not prove productive 
of positive data in relation to metamorphosis, it yielded results which 
encouraged further enquiry into the reaction of Amphibian melanophores to 
the internal secretions. 

A few hours (6-12) after feeding Axolotls on pituitary (whole) gland, a 
marked darkening of the skin was observed; this was not noticed the first 
few days of pituitary diet, but became increasingly pronounced as the feeding 
continued. At first the darkening was very gradual, attaining its maximum 
intensity about 18 hours after feeding, which took place every 48 hours. 
Curiously enough, when this effect had quite passed off by the morning of 
the second day, the animals were of a ghostly pallor—considerably lighter 
than their normal shade, and remained thus until fed again. The rate of 
darkening progressively increased ; after 3-4 weeks of the pituitary diet the. 
maximum degree of expansion of the pigment cells was reached within an 
hour. The extreme subsequent paleness still occurred, appearing within 
24 hours of feeding. Finally, after about 3 months’ treatment the response 
diminished. 


On Amphabian Metamorphosis and Internal Secretions. 49 


These observations were based on a dozen medium-sized Axolotls (and as 
many controls) of the albino variety, in which the pigment cells are sparse 
and confined to the upper surface of the head and mid-dorsal region. The 
-yeaction was more pronounced when the whole gland or the posterior lobe 
alone was administered than when the anterior lobe only was used. While 
these experiments were in progress Bennet Allen (1920) issued a preliminary 
notification of experiments on pituitary removal and transplantation in 
tadpoles, mentioning inter alia that animals after pituitary removal display 
a silvery-white appearauce, in marked contrast to the coloration of the 
normal form. Thus the expansion of melanophores in reaction to the 
pituitary hormones would not appear to be confined to Axolotls.* 

The interest of this reaction is twofold: firstly in relation to Spaeth’s thesis 
(1916) that melanophores represent a modified form of smooth muscle fibre ; 
and secondly, in view of a suggestion by Fuchs (1906) that internal secretion 
may underlie the well-known phenomenon of “colour adaptation.” Spaeth 
instances, as intermediate in character between typical smooth muscle and 
pigment cells, the sphincter pupille, in which, in certain cases, the cytoplasm 
is densely charged with melanin granules; he points out that the melano- 
phores are mesodermal in origin; and draws attention to a remarkable 
parallelism between the reactions of the sphincter pupille fibres and melano- 
phores in response to electrical and light stimuli and chemical stimuli such 
as atropine. To this it may be added that both react in the same way to 
the pituitary hormone, for it has been shown by Cramer (quoted by Schafer, 
1913) that the latter induces dilatation of the pupil. 

The phenomenon of “colour adaptation” in reptiles, amphibia and fishes, 
has been provocative of much controversy and is little understood. It is 
widely known that these organisms can respond to the colour of their sur- 
roundings by pigmental changes. The most recent work of Laurens (1917) and 
others shows that, though the melanophores respond directly by expansion 
in bright hght, in the case of Axolotls which have been blinded there is no 
secondary modification of the response (partial contraction) after continued 
illumination, and no power to respond to their background, eg., to become 
darker when illuminated only from above in a blackened container. The 
pigmental responses of these animals thus appear to be under the control of 


* Allen’s (and also Smith’s) results indicate that it is the intermediate lobe of the 
pituitary which is concerned in pigmental control in Anura. Presumably the same 
holds good for the effect of a diet of Mammalian pituitary on the Axolotl. The 
intermediate lobe will remain attached to the posterior lobe unless specially dissected 
apart. Swingle’s recently published work (1921) confirms and extends the above- 
mentioned results. 


VOL, XCIII.—B. E 


50 Messrs. J. S. Huxley and L. T. Hogben. 


stimuli received through the organs of vision. How this control is exerted is 
at present an unsolved problem. 

Two possibilities invite consideration. Either the stimuli received by the 
eyes are transmitted entirely through the nervous system vid the fibres 
innervating the pigment cells—assuming that in all cases pigmental cells are 
innervated from the C.N.S.; or nervous stimulation of internal secretions 
efficient to produce the appropriate reaction may be involved. Obviously 
both mechanisms may operate concurrently. In order to interpret the 
pigmental responses in these animals in a manner consonant with the second 
hypothesis, it is first necessary to demonstrate that the melanophores react in 
one way to one type of internal secretion and in an opposite sense to another. 
It has been stated that a pituitary hormone causes the melanophores to 
expand; and the question arises whether other internal secretions can bring 
about the reverse effect. In this connection two observations provide a clue. 
McCord and Allen (1917) have recorded that after feeding tadpoles (Rana 
sylvatica) on pineal glands for 10 days, each subsequent meal was followed by 
transient, and complete, contraction of melanophores, noticeable in half-an- 
hour, reaching its maximum in about 45 minutes, and passing off after 2 to 
3 hours. Bigney (1919) again finds that by injecting adrenalin into the adult 
frog, the contraction of the pigment cells is produced, confirming earlier 
work of Lieben (1906). 

To test the reaction of the pigment cells in Axolotls to pineal treatment, 
eight medium-sized (9 month) larve were placed (November, 1920) in separate 
containers, of which the sides had been blackened, and illuminated from above. 
In this way maximum expansion of the pigment cells is brought about within 
a few hours. Four were fed on fresh pineal glands tri-weekly, and the 
remaining four (controls) were kept on a normal meat diet. The experiment 
was continued for 2 months and proved quite fruitless. No pigmental 
differences either of a permanent or temporary character could be observed 
in Axolotls, although, as will be seen later, McCord and Allen’s observations 
as to the effect of pineal feeding on tadpoles were afterwards confirmed. Two 
similar experiments with the same numbers were then repeated, with adrenal 
medulla instead of with pineal gland; whether administered as food or by 
adding fresh extract to the medium, a complete contraction of the pigment 
cells invariably ensued, with great vaso-dilatation of the gills.* 

It thus appears that the pigment cells of medium-sized Axolotls react in an 
opposite manner to pituitary and adrenal extracts, and the fact that in Allen’s 
experiments the removal of the pituitary was accomplished by melanophore 


* Probably the effect is in either case due to adrenalin acting on the skin wd the 


medium. 


On Amphibian Metamorphosis and Internal Secretions. 51 


contraction, is indicative that internal secretion underlies the mechanism 
by which the regulation of pigmental reactions is effected in normal life. 
In any case there would appear to exist a double compensating mechanism 
for the control of the behaviour of the melanophores: though we cannot 
yet legitimately infer that this constitutes the effective apparatus through 
which the organs of vision influence them. 


6. The Pineal Pigment Cycle in Tadpoles. 


In view of the lack of success with which efforts to induce a response 
on the part of the melanophores of the Axolotls were attended, it was 
decided to repeat McCord and F. Allen’s experiments upon pineal adminis- 
tration to Anuran tadpoles. About 500 tadpoles of Rana temporaria were 
employed for this purpose in glass containers during the spring of 1921. 
At first the controls (meat-fed), and the experimental animals which were 
fed on fresh ox pineal glands tri-weekly, were kept respectively in single 
containers. Later, for purposes of observation, the tadpoles were separated 
in glass bowls (placed on a white background) in colonies of twenty. 
Contemporaneously, tadpoles raised from eggs laid on the same day were being 
fed on suprarenal cortex, suprarenal medulla, corpus luteum, and anterior 
pituitary lobe for ulterior purposes, so that it was possible to compare the 
phenomena consequent upon pineal treatment with the results of adminis- 
tration of a more varied range of tissue extracts than were employed by 
the authors named above. 

In these experiments no change in the pigmental characteristics of the 
tadpoles was noticed during the first fortnight of pineal diet, which began 
about a fortnight after hatching. Before three weeks had elapsed, the 
phenomena of the pineal pigment cycle, as recorded by its discoverers, 
became evident. Within a quarter of an hour of feeding the tadpoles 
became visibly more pale, till, when the reaction reached its maximum, 
half an hour after the meal commenced, they assumed a quite unique 
appearance, by virtue of the contrast between the complete translucence 
of the head region and tail on the one hand, and the opacity of the 
visceral portion of the body on the other. This condition passes off after 
the lapse of five or six hours: till metamorphosis took place the same 
reaction followed each administration with the utmost regularity. No 
modification of the pigmental features occurred in the controls or in the 
additional cultures which were being fed synchronously with other glandular 
tissues. Histological preparations confirmed McCord and Allen’s conclusion 
that the behaviour of the melanophores is the significant element in the 
situation. After a pineal meal the melanophores are fully contracted, 

E 2 


52 Messrs. J. S. Huxley and L. T. Hogben. 


the pigment being congregated into compact masses in the centre of the 
Cellige , 

The time relations observed are somewhat different in these experiments 
from those obtained by McCord and Allen. ‘Thus the latent period was a 
little longer; the time required for the effect to manifest itself after feeding 
a little shorter, and the duration of the effect considerably longer. In the 
case of f. sylvatica, these investigators found that the reaction passes off 
in about two hours. Apart from these insignificant details, the experiment 
recorded entirely confirms their results. It may be added that extracts were 
also employed and produced rapidly (less) corresponding effects to those 
produced by administration per os. 

As regards the cortex-fed cultures, no difference was seen in the pigmenta- 
tion of tadpoles fed continuously from hatching till metamorphosis on supra- 
renal cortex as contrasted with controls. We take the opportunity of 
putting this observation on record, because Gudernatsch (1914) stated that 
cortex-fed tadpoles show progressively less pigment after five weeks’ treat- 
ment, an observation which, if confirmed, would seem significant in relation 
to the ztiology of Addison’s disease. 

That the pineal gland of Amphibia does actually function in relation to 
pigmental responses, although it is probable, cannot be legitimately contended 
on the basis of evidence so far available, as mammalian pineal glands were 
employed in the experiments. What can be definitely stated is that pineal 
tissue is specifically distinguished by the possession of a physiologically active 
substance, a conclusion which goes far to establish its claim to be classified 
as an endocrine organ. 

Summary. 

A. Metamorphosis. 

1. Salamandra and Triton larvee may be metamorphosed by immersion in a 
dilute solution of iodine. Metamorphosis is retarded by low temperature. 
High temperature at first causes increased growth of the gills. 

2. Sexually mature Axolotls can, as Laufberger and Jensen originally 
showed, be made to undergo metamorphosis by means of a thyroid diet. 

3. Metamorphosis is accompanied by exophthalmos, apparently in all 
Amphibia. 

4. In the case of the Axolotl, the time required for metamorphosis 
induced by enforced air-breathing is considerably longer than when induced 
by thyroid-feeding; in the latter case, it is longer for sexually mature 
than for young larve; and is in all cases accelerated by increase of 
temperature. 

5, Administration of iodine free of organic combination, or fresh glandular 


On Amphibian Metamorphosis and Internal Secretions. 53 


substance of the prostate and pituitary anterior lobe, is without any effect in 
relation to the metamorphosis of the Axolotl. 

6. Thyroid-feeding continued for 7 months was not accompanied by any 
noteworthy somatic changes in Necturus. 


B. Pigmental Responses. 


7. Pituitary feeding (posterior lobe or whole gland) produces a marked 
temporary dilatation, followed later by excessive contraction, of the dermal 
melanophores in albino Axolotls. 

8. Adrenal medulla extract produces temporarily complete contraction of 
the dermal melanophores in the Axolotl. 

9. Pineal administration (as extract or as food) rapidly brings about a 
striking transient contraction of the dermal melanophores in frog tadpoles: 
McCord and F. Allen’s observations in this connection are fully confirmed. 
It has, however, no effect upon the melanophores of the Axolotl. 


LITERATURE. 
Allen, B., ‘Science,’ 1920. 
Bigney, ‘J. Exptl. Zool.,’ vol. 27 (1919). 
Boulenger, ‘ Proc. Zool. Soc.,’ 1913. 
Von Chauvin, ‘ Zeit. f. Wiss. Zool.,’ vol. 27 (1877), and vol. 41 (1885). 
Detwiler, ‘J. Exptl. Zool.,’ vol. 31 (1920), especially pp. 149-151. 
Emerson, ‘ Proc. Boston Soc. Nat. Hist.,’ vol. 32 (1905). 
Fuchs, ‘ Biol. Centralbl., vol. 26 (1906). 
Gadow, H., ‘“‘ Amphibia and Reptiles,” ‘Cambridge Nat. Hist.,’ vol. 8 (1901). 
Gudernatsch, ‘ Am. Journ. Anat.,’ vol. 15 (1914). 
Jensen, ‘ Meddelelser f. d. Kgl. Veter. og. Landb.,’ vol. 44 (1916). 
Laufberger (in Czech), ‘ Biclogi¢ke Listy ’ (1913). 
Laurens, ‘J. Exptl. Zool.,’ vol. 23 (1917). 
Leydig, F., ‘Anatomisch-histologische Untersuchungen iiber Fische und Reptilien’ 
(Berlin, 1853). 
McCord and F. Allen, ‘J. Exptl. Zool.,’ vol. 23 (1917). 
Platt, ‘ Anat. Anzeiger’ (1896). 
Schafer, ‘ Lane Medical Lectures’ (1913), California. 
Smith, P. E., ‘Amer. Anat. Mem.,’ vol. 11 (1920). 
Spaeth, ‘J. Exptl. Zool.,’ vol. 20 (1916). 
Swingle, ‘J. Exptl. Zool., vol. 27 (1919). 
Swingle, ‘J. Exptl. Zool.,’ vol. 34 (1921). 
Uhlenhuth, ‘ Journ. Gen. Phys.,’ vol. 1 (1919). 
Wilder, ‘ Zool. Jahrb. (Anat. Abt.),’ vol. 4 (1891). 


54 


Studies in Bacterial Variability.—On the Occurrence and Develop- 
ment of Dys-agglutinable, Eu-agglutinable and Hyper- 
agglutinable Forms of Certain Bacteria. (A Report to the 
Medical Research Council.) 


By E. W. AINLEY WALKER. 
(Communicated by Prof. Georges Dreyer, F.R.S. Received’October 12, 1921.) 
(From the Department of Pathology, University of Oxford.) 


Introductory. 


That different cultures of an agglutinable bacterium may exhibit wide 
differences in relative agglutinability when tested with the same agglutinating 
serum is a familiar fact. But the conditions on which these differences 
depend remain to a great extent obscure. Yet a number of facts which bear 
upon the problem have come to light in the course of investigations carried 
out by various observers. 

A good many years ago the present writer showed (1901) (1) that if a 
series of strains* of B. typhosus be employed in preparing a corresponding 
series of agglutinating serums, each such serum is found to act more power- 
fully upon its homologous culture than upon any of the heterologous strains. 
It was, therefore, stated that the serums were not only specific for the species 
of bacterium in question, but also special in each case to the particular strain 
employed in its production. It also appeared that, so far as the evidence 
went, the heterologous strains always fell into the same order of relative 
agglutinability when tested with the different “special” serums. 

Somewhat similar results have recently been published by A. D. Gardner 
(1920) (2), in connection with his investigation of Paratyphoid A. serums, and 
he has shown that the peculiar sensitiveness of a given strain to agglutination 
by serum prepared with that particular strain, which I had indicated by the 
term “special” is, in part at least, a matter of velocity of reaction. The 
phenomenon (so far as it depends on reaction rate) was, therefore, spoken of by 
him as a “super-specific acceleration ” of reaction. 

Since my own experiments were made before the introduction of the present 
accurately standardised methods of determining agglutination titre, I had 

* Tn the use of the terms “strains” nothing is implied with regard to the cultures so 
spoken of save that they have been obtained from different sources, derived by different 
methods of cultivation, or selected in any way for a particular purpose. If they show 


differences, it is a matter for investigation in each case, whether such differences are 
permanent or fleeting. 


Studies in Bacterial Variability. 55 


re-examined the question in another relation (1918) (3) with four strains of 
B. typhosus. A rabbit was immunised successively with three of the strains, 
and on each occasion the titre of its serum rose toa higher level for the strain 
used as antigen on that occasion, than for either of the other three strains. 
Furthermore the four strains were seen to belong, two and two, to two sero- 
logical groups which were widely differentiated from each other by their 
relative sensitiveness to agglutination by different serums.* 

Somewhat similar serological differences are now well known in certain 
other organisations, ¢.g., Meningococcus, Pneumococeus; and they may at times 
be very marked indeed, so that particular strains are described as “ inagelutin- 
able ”—for example inagglutinable strains of B. typhosus. Yet it is found 
that on sub-culture these inagelutinable strains will sooner or later yield 
cultures which give good agglutination. Thus the inagelutinability appears 
to be only a phase, or temporary character. The inagglutinable form of: the 
organism cannot, therefore, on the existing evidence be regarded as mutant, in the 
proper sense of the term ; but it presents an example of bacterial fluctuation. 

Since the observations just referred to (1918) (3) raised a number of 
interesting questions the subject has been re-examined in detail during the 
progress of the present investigation by A. D. Gardner and Ainley Walker 
(1921) (4) who obtained inagglutinable strains of B. typhosus, and compared 
them serologically with ordinary “good agglutinators.” The existence of the 
two serologically different types of B. typhosus was fully confirmed. It was 
shown that they corresponded to a motile and a non-motile phase respectively 
of the bacillus. And it was further shown that from a single culture of the 
bacillus colonies could on occasion be isolated by plating which would on 
cultivation give rise to populations differing as widely, both serologically and 
as regards motility, as any strains obtained from different sources. 

The serological difference between the agglutinable (motile) form and the 
inagglutinable (non-motile) form of the bacillus was an antigenic difference. 
For if a serum were prepared with the inagglutinable form, it agglutinated its 
own bacillus and other inagglutinable strains quite well, and to high titre. 
But in contrast with the rapidly-formed, large and fluffy floceuli produced in 
an ordinarily well-agglutinating culture, the clumps produced in inagelutinable 
suspensions are always small, compact and slowly formed. 

On this account, and in view of what is to follow, I propose the term 
dys-agglutinable+ as a more appropriate designation than “ inagglutinable ” for 


* A more complete record of this experiment was published by Gardner and Ainley 
Walker (1921) (4). 

+ This is a hybrid word; but so are a good number of other accepted and useful 
terms. 


56 Mr. E. W. A. Walker. 


2 I 
this phase of the bacilius. The ordinarily well-agglutinable phase may then 


be spoken of as eu-agglutinable (where distinction is necessary) and the term 
hyper-agglutinable employed for phases where agglutinability is found to be 
considerably increased. The differences in agglutinability, and the antigenic 
differences in strains from different sources, and in sub-cultures from different 
colonies studied by Gardner and myself arose without experimental inter- 
ference; that is to say they were naturally occurring differences. So also 
appear to be the differences recently described in B. dysenteric (Shiga and 
Flexner—Y), and several other organisms including B. typhosus, m an 
important paper by Arkwright (1921) (5). Vines also has described and 
studied strains of meningococcus “ hypersensitive” to agglutination (1918) (6) 
as naturally occurring types. But the results to be recorded show that it is 
possible to produce experimentally similar differences of agglutinability in 
many varities of bacteria. 

In the course of experiment carried out in 1901-2, I found that the agglutin- 
ability of B. typhosus was lessened by growing it in a succession of sub-cultures 
in typhoid agglutinating serum diluted with ordinary culture bouillon 
(1902) (7). This diminution of agglutinability, and certain other phenomena 
observed, were interpreted in terms of Erlich’s theory, with the conceptions of 
which I was then imbued, and that interpretation needs reconsideration and 
may require revision in the light of present knowledge. For it is evident that 
a diminution in the agglutinability of an organism occurring under these 
conditions is open to more than one possible explanation, since the production 
of a relatively inagglutinable race of bacilli might be brought about in several 
different ways. 

Thus it might be due (1) to the gradual training and education of the whole 
mass of the population in successive generations to resist the action of 
agglutinins ; for example by the formation of anti-agglutinins, as I formerly 
concluded; (2) or it might be due to a selective encouragement of the 
propagation of the less agglutinable individuals (in a population composed of 
elements differing widely in agglutinability) by some cause facilitating their 
multiplication in successive generations, to the gradual exclusion and elimina- 
tion of the more agglutinable individuals ; (3) or both processes might play a 
part. 

The first process would offer an example of the heightening and develop- 
ment of a selected character, originally latent, but more or less common to 
the individuals composing the bacterial population; the second, one of the 
selective propagation, from a population whose members differed widely 
among themselves in this respect, of particular individuals in which that 
character was already highly developed. Put briefly, the one would be the 


Studies in Bacterval Variability. 57 


selection of a character for artificial increase, the other the selection of individuals 
for multiplication. And it is obvious that both processes might be at work 
side by side. 

In view of these considerations, it seemed likely that information of interest 
would be obtained by inquiring what degree of parallelism, if any, existed 
between the differences in agglutinability to be observed in different strains 
(however derived), and those obtainable by repeated cultivation in agglu- 
tinating serum. In particular it was hoped to throw some light on the nature 
and origin of so-called “serological strains” in different species of bacteria. 
Accordingly experiments have been carried out as described below. Mean- 
while the experiments of Gardner and myself already referred to (loc. cit.) 
have shown that extremely wide differences in agglutinability, such as might 
well form a basis for either process of selection, do actually exist between the 
individuals which constitute the population of a single culture. 

In the account which follows, the experimental work has been summarised 
so far as possible, and detailed data are recorded only for B. typhosus, in 
order to save space. A general survey of the phenomena under investigation 
is given, but only a selection of the experiments is recorded in illustration. 
In certain directions the results are preliminary, and a number of points 
remain to be worked out in detail. 


The Experimental Production of Dys-agglutinable and Hyper-agglutinable 
Strains (or Phases) of Bacteria. 

By repeated subculture in specific agglutinating serums diluted appro- 
priately with culture bouillon, dys-agglutinable forms have been obtained of 
the following bacteria, ten in all, which were all taken from strains then in 
use in the department: B. typhosus, B. paratyphosus A., B. paratyphosus B., 
B. erirycke, B. enteritidis (Gaertner), B. coli, B. dysenteric (Shiga), B. dysenterie 
(Flexner), V, W and Z. 

On transference from the serum bouillon to ordinary culture bouillon, 
these dys-agelutinable forms yield cultures which agglutinate with difficulty, 
if at all, with corresponding ordinary agelutinating serums; and those of 
them that were originally motile organisms are found to have passed into a 
very feebly motile or entirely non-motile phase. After formolisation and 
dilution to the opacity of standardised agglutinable cultures, they yielded 
good and uniform suspensions for agglutination tests, though in a number of 
cases this result was not obtained immediately or very easily. The dys- 
agglutinable phase of B. typhosus and of B. dysenteriw (Flexner), V, W and Z 
readily yielded excellent suspensions on a number of occasions. 

In other cases difticulty sometimes occurred for one of two reasons— 


58 Mr. E. W. A. Walker. 


firstly, that in the case of the motile bacilli, the non-motile phase, when 
first obtained, often shows a tendency to revert very quickly to the motile 
eu-agglutinable phase in bouillon culture; and, secondly, because, when 
rapid reversion does not occur, organisms in this group, and also in the 
dysentery group, which have been grown in a succession of cultures in 
dilutions of the homologous agglutinating serum, most often grow in early 
bouillon subcultures in the form of a deposit with clear supernatant fluid. 
Although these deposits may often shake up to form perfectly good sus- 
pensions for formolisation and dilution to standard opacity, they yield on 
other occasions suspensions in which many of the bacilli remain more or less 
agglomerated. The latter suspensions are always undesirable for use in any 
kind of observations on serum agglutination, and they were put aside as 
quite unsuitable for experimentation, if, after the usual period of heating in 
the water-bath, followed by 24 hours’ standing at room temperature, there 
was any deposit whatever at the foot of the control tube, or more than a 
faint granulation to be seen (with a lens) in the fluid. 


B. typhosus. 


An actively motile agglutinable bouillon culture of B. typhosus (T.e.)* 
was taken from a strain then in use, and a stock of formolised agglu- 
tinable culture of standardised opacity was prepared from it. A subculture 
was preserved in agar stab, and a subculture was made in the agglutinating 
serum of rabbits immunised with T.e., diluted 1 to 9 bouillon. The 
latter culture grew in the form of flocculi of agglutinated bacilli, which 
sank to the foot of the tube. It was carried on at intervals of 48 hours in 
small tubes in a succession of cultures in the diluted serum through nine 
passages. The serum dilutions were tested for sterility before use by 
incubation for 48 hours at 37° C. 

The successive subcultures were always made from the top of the fluid, 
care being taken not to disturb the deposit. In the first and second 
cultures this fluid looked clear; but, in the later cultures, an increasing 
turbidity of the fluid appeared, in addition to the flocculent deposit at the 
foot of the tube. This appearance suggested that an increasing number 
of the bacilli present were insusceptible to the agglutinating action of 
the serum bouillon medium. Nevertheless, the microscope often showed 
the presence of small clumps among the free bacilli. 

From the fifth and ninth serum bouillon culture ordinary bouillon cultures 
were made, and, from these, subcultures (T.e.5 and T.e.9) were prepared 


* The same strain as that denoted T (E) in the paper by Gardner and myself already 
referred to (4). 


Studies in Bacterial Variability. 59 


24 hours later in 250-c.c. flasks containing 100 ec. of bouillon. The latter 
were grown for 24 hours at 37° C., and then well shaken up and formolised. 
They were placed in cold storage and shaken up several times a day 
for 3 or 4 days, and were subsequently diluted with formolised normal 
saline solution to standard opacity. Microscopie examination before 
formolisation showed that T.e.5 was almost entirely in the non-motile 
phase, T.e.9 short and very highly motile. Serums were prepared by 
inoculating rabbits intravenously with 1 cc. of these formolised cultures, 
and taking blood on the eighth day. Rabbit 1 received 1 cc. of culture T-e. 
and serum was prepared (serum I, 1); a month later it received 1 cc. of 
culture, T.e.5, yielding serum I, 2, and, after the lapse of a month more, it 
received 1 c.c. of T.e.9 and yielded serum I, 3. 

On the same day as the last of these inoculations, rabbits 2 and 3 received 
intravenous inoculations of 1 c.c. of cultures T.e.5 and T.e.9 respectively, 
and serums II and III were subsequently obtained from them by bleeding on 
the usual day (eighth). 

These serums, together with a 100-unit standard serum (Dreyer), were then 
tested out against the cultures Te, T.e.5 and T.e.9, using the series of 
dilutions 1 in 25, 50, 125, 250, etc., of Dreyer’s series, and recording the 
results in Dreyer’s notation of total (t), standard (s), trace (tr), and inter- 
mediate values (eg., s—, tr+) as required. The tubes were read after 
2 hours in the water-bath at 54° to 56° C., followed by 15 minutes’ standing 
at room temperature, and again after 24 hours at room temperature. 


Table I—Showing Dilutions of Serums yielding “ Trace” Ageglutination. 


| 
| 2 hours’ readings. 


24 hours’ readings. 


Culture. | T. 36 Weve meron lves On MroGr eae ehoniedtcen Os | 

peo oe ee | 

100-unit Standard serum...| 750) 800 | 10 | 1,290 | 1,200 | 1,000} 900, 2,500 | 

Smelt Wheto. lands. | — | 1,600! 20] 2,500} — | 1,900} 800' 4,000 | 

Bering 8. oud oe | Sa | 5,000 450| 4500 — | 6,000 | 1,900 5,500 | 

Sperm oes ee 4,500 | 450 | 4,500 — | 6,000| 1,900 9,000 | 

ioe | 900 } 1,900 100 | 2,200 | 1,800 | 2,200 | 2,000 2,600 | 
BSerrmM Ts bec su,.. | 18,000 | 10,000 | 20 |18,000 | 20,000 | 20,000 175 45,000 | 


Serum I, 1 from Rabbit 1, inoculated with 1 c.c. T.e. 
pp be] i 1, x » 1 c.c. T.e.5, a month later. 
1, = » lee. T.e.9, a month later still. 
Zayed UE 5 2; 5 me Licres duero: 
3, a ge eee 19. 


60 Mr. E. W. A. Walker. 


The complete record of these tests is omitted from considerations of space. 
But the end-point readings, after 2 hours and after 24 hours, are presented 
numerically in the subjoined Table (Table I), where the figure given repre- 
sents the dilution of the serum observed for a reading of trace (tr), or 
estimated from an observed end-point reading of trace plus (tr+) or trace 
minus (tr—). 

From this Table, and in connection with it, the following points emerge :— 

1. In the culture T.e. 5 the bacillus has become distinctly dys-agglutinable. 
The 2 hours’ readings show a very low agelutination titre even in the three 
serums prepared with this cuiture itself, either alone (serum IJ) or following 
a previous inoculation of T.e. (serums I, 2 and I, 3). 

In the 24 hours’ readings the end-point titre has advanced to a fair 
height, except in the case of serum III. But it is to be stated in this 
connection that, in the tests made on this culture (T.e.5), no tube in any 
series showed a reading higher than trace plus (tr+), even when as many as 
six or seven tubes in series all showed some agglutination, save in the case of 
the two serums I, 2 and I, 3, where, after two and three inoculations respec- 
tively, the serum of rabbit 1 gave marked agglutination (total or standard) in 
the first two dilutions (1 in 25 and 1 in 50) after 24 hours. Even in these 
cases the agglutination was of the “ dysenteric” type, described by Gardner 
and myself as characteristic of so-called “inagglutinable” strains of 
B. typhosus. That is to say, the clumps were small, compact, and slowly 
formed, in contrast with the rapidly formed, large and fluffy floceuli of 
ordinary typhoid agglutination. 

2. The culture T.e.9, obtained by sampling the supernatant fluid of the 
ninth successive subculture in serum bouillon, is obviously not at all 
dys-agglutinable. On the contrary, it is much more agglutinable than the 
original T.e. It would seem to follow that, at this stage at any rate, the 
change produced by growth in diluted agglutinating serum cannot be one of 
progressive diminution of agglutinability in the whole population. 

It is rather a mechanical separation (by clumping and sedimentation) of 
the more agglutinable individuals from the less agglutinable, probably 
accompanied by facilitation of the growth and multiplication of the latter, 
particularly in so far as they remain unclumped. 

But since, as already mentioned, the supernatant fluid often contains 
(microscopically) many small undeposited clumps, it might easily happen, 
and presumably did happen in the case of culture T.e.9, that the sample 
taken for subcultivation in ordinary bouillon contained enough (clumped) 
motile agglutinable bacilli rapidly to outgrow the dys-agglutinable non- 
motile individuals in the course of two successive subcultures in bouillon, 


Studies in Bacterral Variability. 61 


the second of which constituted culture T.e.9. This point is further 
illustrated in a later experiment. 

So far there had been obtained from the culture T.e. cultures T.e. 5 and 
T.e. 9, the former definitely dys-agglutinable, the latter a good deal more 
agelutinable than T.e., in fact, two and a half times as agglutinable when 
referred to standard serum. 

3. Culture T.e.9 produced in rabbit 3 a serum of very high agglutinating 
power for T.e. and T.e.9. This, however, does not indicate that in T.e.9 one 
has isolated a specially powerful antigenic form of the bacillus. It only 
shows that rabbit 3 was a particularly good subject for the production of 
agglutinins. For on looking at the serums obtained from rabbit 1, it is seen 
that T.e. 9, which was used for the third inoculation, did not in this animal 
cause any increase of T.e. agglutination above its previous level. 

On the other hand, it is very evident that T.e.9 was remarkably poor in 
antigenic power for the dys-agglutinable bacillus T.e. 5. For whereas 
serum III (T.e. 9) was nine or ten times as powerful (24 hours readings) in 
its action on T.e. 9 as serum I, 1 (T.e.), it was only about one-fifth as strong 
against T.e.5. That is to say that in culture T.e.9 the antigenic power of 
the original T.e. has been reduced to about one-fiftieth as concerns T.e. 5. | 

4, Serum II (T.e. 5) is seen to be much more powerful in its action on 
T.e.5 culture than either of the other serums from single inoculations, and as 
powerful (24 hours readings) as the serums I, 2 and I, 3, though these serums 
act two or three times as strongly against cultures T.e. and T.e. 9 as does 
serum II. Nevertheless, T.e.5, which has become markedly dys-agglutinable, 
still retains at this stage good antigenic power in forming agglutinins for T.e. 

There is, however, some evidence that its antigenic power for T.e. has 
undergone reduction, since though serum II (T-e. 5) is definitely stronger than 
serum I, 1 (T.e.) for the original culture T.e. (say 16 per cent.), it was 
perceptibly weaker in its action on culture T.e. 9 (say 7 per cent.). 

In view of the foregoing considerations, it would seem that by growing 
the bacillus ‘I.e. in agglutinating serum one was in process of separating out, 
whether by the mechanical action of clamping and sedimentation alone, or 
by this combined with an influence favourable to the preferential multiplica- 
tion of the less agglutinable members of the population, a dys-agelutinable 
form of the bacillus; and that incidentally one also isolated a particularly 
highly agglutinable form, T.e. 9. 

In this latter culture dys-agglutinable elements were much less well 
represented than in the original T.e. from which both had been derived, as 
shown by its feeble antigenic action in relation to culture T.e. 5, when used 
for the production of agglutinating serum. But that they were by no means 


62 Mr. E. W. A. Walker. 


absent is rendered probable by an interesting observation which now requires 
mention, namely, that in the numerous agglutination tests made with T.e. 9 
culture a faint haze of unagglutinated bacilli always remained in the super- 
natant fluid of tubes that would otherwise have been recorded as exhibiting 
“total” agelutination owing to the complete deposition of the large fluffy 
flocculi of agglutinated bacilli. 

It is of interest to note that, even at this comparatively early stage of 
differentiation both serum II (T.e. 5) and serum ITI (T.e. 9) act more strongly 
on their own homologous culture than upon the standard culture T. 36, 
though this derived itself (at a much earlier date) from the same original 
source as culture T.e. 

Further investigation of the facts observed was attempted by making a 
similar experiment in duplicate (in 1 to 3 dilution of serum bouillon) and 
endeavouring to lead cultures of the bacillus along two diverging paths by 
making each subculture in one series of tubes from the top of the fluid in 
the preceding culture, and in the other series making each subculture from 
the deposit of agglutinated bacilli at the foot of the preceding tube. 

The experiment was carried on in both directions through nine successive 
subcultures (18 days) in serum bouillon. Cultures in ordinary bouillon 
were made from both series at each stage and formolised after 24 hours’ 
incubation, in the hope of obtaining in the one series an increasingly dys- 
agelutinable culture, and in the other one increasingly hyper-agelutinable. 

But the experiment failed in this respect, owing to the fact (which it revealed) 
that not only did the supernatant fluid of these serum-bouillon cultures often 
contain a good many microscopic clumps of agglutinated bacilli, but also the 
deposit held many unagglutinated (dys-agglutinable) bacilli in the interstices 
of the flocculent mass. Thus the results yielded cultures no more advanced 
in the desired directions respectively than T.e. 5 and T.e. 9. 

The question of how far the effect of growth in agglutinating serum might 
consist in the mere mechanical separation of pre-existing agglutinable and 
dys-agglutinable elements in a given culture was next considered. It was 
found that it is sometimes possible to obtain a dys-agglutinable non-motile 
culture of &. typhosus at the first attempt by simply putting up, with due 
precautions, a 1-in-10 agelutination test with living culture T.e. in a plugged 
sterile centrifuge tube, incubating at 37° C. until agglutination was well 
advanced, and then centrifngalising down the agglutinated bacteria, and 
making a culture or plating from the supernatant fluid. 

From this it follows that, at any rate in a considerable number of cases, 
there must be numerous dys-agglutinable individuals present in an ordinary 
bouillon culture of B. typhosus. 


Studies in Bacterial Variability. 63 


Moreover, when this method fails to give a dys-agglutinable bacillus, as 
may occur either because dys-agglutinable individuals are relatively few in 
number in the original culture (or possibly absent), or because they rapidly 
revert on cultivation in bouillon, success may often be obtained by combining 
the method of successive cultures in serum-bouillon with severe centrifugali- 
sation of each culture to deposit all clumps before proceeding to the next sub- 
cultivation. 

In another experiment, the bacillus T.e. was grown in media containing 
agelutinating serum for a period of four months. The successive subcultures 
were made at first every second day, then once a week, and later still at 
intervals of two or three weeks. At the end of this period a subculture was 
made in ordinary bouillon. On microscopical examination, after 24 hours’ 
incubation the bacilli were quite non-motile. Many fields were examined and 
no motile individual seen. From this bouillon culture (A) platings were 
made ona set of agar plates (vide wmfra), and a flask containing a litre of 
bouillon was inoculated and incubated for 24 hours. The resulting growth 
(T. dys.) consisted entirely of non-motile bacilli so far as could be seen, and 
after formolisation and dilution to standard opacity it was found to be very 
highly dys-agglutinable. It showed characters similar to those described by 
Gardner and myself (loc. cit. (4)) in our so-called “inagglutinable” cultures, 
and gave no agglutination whatever with 100-unit standard serum even in 
1 in 25 dilution after 2 hours in the water-bath. 

It also fasled to absorb any appreciable quantity of agglutinins from standard 
serum. Thus two portions of a particular standard typhoid serum were taken 
and diluted 1 in 10 the one (a) with standardised agelutinable culture T. 36, 
the other (6) with the formolised culture T. dys. The tubes were kept in the 
water-bath at 54°-56° C. for 4 hours, and subsequently at the room 
temperature for 24 hours. In tube (a), T. 36 was totally agglutinated when 
first examined at the end of 2 hours. In tube (0), T. dys. showed no agglu- 
tination after 2 hours, but a trace (tr) at the end of the 4 hours in the water- 
bath, and 24 hours later it showed a weak standard (s—) agglutination. 

Both tubes were then centrifugalised, and the supernatant fluid removed 
and denoted serum (a) and serum (0) respectively, and tested along with the 
original standard serum on culture T. 36 with the results shown in detail in 
Table IL. 

The sample of serum absorbed with T. 36 is seen to have been reduced to 
something between one-fourth and one-fifth of its original agglutinating 
power for T.36; whereas that absorbed with T. dys. shows no measurable 
loss at all of agglutinins for T. 36, but on the contrary the appearance of a 
slight wnerease. That is to say T. dys. was not only highly dys-agglutinable, 


64 Mr. E. W. A. Walker. 


but it was also incapable of absorbing appreciably ordinary typhoid 


agglutinins. 
Table IT. 


eee eee 25 | 50 | 125 | 250 | 500 | 1000 | 2500 | 5000 | Controls. 


| ' 
| | | 
Standard 2hours | t t t t— 5— tr OF (0) 
serum 2a) 5 | t t t t— s+ tr OB ee) 0 
-} 
| | j 
Serum(a)| 2 ,, t s+ tr tr— (0) (0) 0; O (0) 
Reese ei t tr+ | tr— 0 0 ON eae OT | 
Serum ()| 2, | ¢ t ad Wee g tr | .0. 1os0 0 | 
Alans t t tieel| Wee t— s— OF RO 0 
| | 


Standard serum 100-unit standardised serum (Dreyer). 
Serum (a)—Standard serum absorbed with T. 36. 
Serum ()— 5 i of T. dys. 


It is important to add that T. dys. was subsequently brought back to the 
eu-agglutinating phase. After a long residence in bouillon with numerous 
subcultures its lineal descendants were found to have reverted to the motile 
phase, and gave good agglutination with standard typhoid serum. A very 
similar result was subsequently obtained with a highly dys-agelutinable 
culture of B. paratyphosus B. 

It is clear that observations of this character must profoundly modify our views 
regarding the meaning and importance of serological differences, and the results 
obtained by absorption methods, particularly when such differences are only — 
quantitative and not qualitative. They suggest that “serological strains” of 
bacteria, even when apparently permanent, may represent no more than 
particular phases of activity of the bacterial type concerned. As to how such 
phasic differences may arise we have at present little evidence. How they 
are maintained for long periods through successive generations is a problem 
that urgently demands investigation. But it must certainly be admitted as 
conceivable that changes which can be induced in test-tube experiments, may 
on occasion also be produced in the body of an infected individual. Jf growth 
in the presence of agglutinating serum under laboratory conditions can lead to 
serological changes in one or other direction, it must be accepted as possible that in 
the living body the agglutinin-containing fluids and agglutinin-producing tissues 
of the animal may also wnder suitable conditions exert similar influences. In 
this connection the occasional isolation of “inagglutinable” typhoid bacilli 
from cases of typhoid fever, and the serological diversity of the strains of 
dysentery (Flexner) isolated during recent epidemics of dysentery afford 


suggestive evidence. 


Studies in Bacterial Variability. 65 


The bouillon culture (A) of which T. dys. was an immediate subculture had 
been plated out on agar as already mentioned. From these plates 20 colonies 
were picked off into bouillon on each of the succeeding 3 days. They were 
incubated for 24 hours, and then examined for motility and formolised for 
agglutination tests. All these 60 cultures were highly dys-agglutinable, and 
they were all non-motile, with the exception of one out of the third set of 20. 
This culture which, it will be noted, had grown for 5 days on serum-free 
media and had been three times subcultivated (once on agar and twice in 
bouillon), had begun to revert,and showed a certain proportion (possibly as high 
as 20 per cent.) of motile individuals among the mass of non-motile elements. 
It is interesting to observe that this proportion of motile elements was not 
sufficient to render the culture eu-agelutinable. 

So far, then, as the evidence from the bouillon subcultures of sixty 
colonies goes, the original T. dys. culture consisted entirely of non-motile 
bacilli. 

It is not necessary on the present occasion to go into much detail in 
deseribing results which showed increase of agglutinability and the oceurrence 
of hyper-agglutinable forms. 

The culture T.e.9 exhibited a degree of hyper-agglutinability. It was 
about two and a half times as agglutinable by standard serum as T.e. or T. 36. 
Furthermore, the culture T.36, with which it was compared, was itself a 
highly agglutinable culture. It had an index of 7, that is to say, it was 
nearly three times as agglutinable as the original standard agglutinable culture 
on which Dreyer’s unit was chosen, whose index was 2°5. If, as I suggest, 
this original standard be taken as the mean standard of eu-agglutinability, 
the culture T.e. 9 was seven times as agglutinable as that standard, and may 
be regarded as distinctly hyper-agglutinable. In further experiments, a 
still more highly hyper-agglutinable culture (about twice as agglutinable as 
T.e.9) was readily obtained by growing the bacillus in serum bouillon, 
centrifugalising out the flocculent clumps at an early stage, and making the 
succeeding serum bouillon culture from the deposit, and so on, instead of 
working from the supernatant fluid, as in the pursuit of dys-agglutinable 
_ forms. 

Similarly, starting from a dys-agglutinable culture, one can frequently 
restore it to eu-agglutinability by the same procedure. Needless to say, all 
these operations demand rigorous precautions against accidental contamina- 
tion of the cultures. The method has a practical application which deserves 
mention. 

Strains are sometimes met with in the case of B. typhosus (and the same is 
true of a number of other bacteria), which are “bad agglutinators.” Or it. 

VOL. XCII.—B. F 


66 Mr. E. W. A. Walker. 


may be that a strain, which for a period (possibly for years) has yielded 
excellent eu-agglutinable cultures, begins, for totally unknown reasons, to 
give bad agglutinators. Sometimes these “ bad” cultures are really more or 
less dys-agglutinable. They probably contain a fairly large proportion of 
dys-agglutinable individuals, so that, in a series of agglutination tubes, a 
haze of opacity remains in those which should show total agglutination, and 
the series tails off in a succession of “traces” instead of ending sharply. 
Such a culture cannot be used for standardisation. At other times they are 
self-agelomerated (self-agglutinated, auto-agglutinated) growths, which may 
refuse to shake up into uniform suspensions. These difficulties can usually 
be overcome by repeated daily subculture in bouillon for 14 days or more, as 
recommended by Dreyer. But that plan does not always succeed. In such 
cases a eu-agglutinable culture can often be obtained without much diffi- 
culty by the methods already described, working from the deposits if the 
culture was dys-agglutinable, or from the supernatant fluids if it was ina 
self-agglomerating phase. In confirmation, it may be stated that Dr. A. D. 
Gardner, to whom I communicated the method, informs me that on one 
occasion it enabled him to obtain a good agglutinator from an otherwise 
intractable culture. A similar separation can also sometimes be obtained by 
mere plating and selection of colonies. . 


B. paratyphosus A., B. paratyphosus B., B. ertrycke B., B. enteritidis (Gaertner), 
B. coli, B. dysentcrie (Shiga), B. dysenterie (Flexner), V, W, X, Y, Z, 
Vib. cholere. 


All these organisms were tried during the summer and autumn of 
1920, at the same time as the earlier experiments with 2B. typhosus, 
by the method of successive subcultures in diluted agglutinating serums. 
A dys-agglutinable culture of B. paratyphosus A. was readily obtained; one 
of B. paratyphosus b., with greater difficulty, owing chiefly to a strong 
tendency to revert when brought into ordinary bouillon culture. &. cola 
also yielded a dys-agglutinable non-motile phase. JS. wrtrycke and B. enteri-. 
tidis (Gaertner), were found very difficult to manage. | 

On one occasion only, in each case, was a non-motile dys-agglutinable 
bouillon culture obtained in a test-tube. Massive subculture in flask. 
seemed to lead to immediate reversion to the motile eu-agglutinable phase, 
and some of the cultures were hyper-agglutinable as compared with standard 
cultures when tested with standard serum. In another attempt, made with 
the same strain of B. enteritidis (Gaertner), during 6 weeks of this year 
(1921), though non-motile or largely non-motile colonies were several times: 


Studies in Bacterial Variability. 67 


obtained on agar plates from serum bouillon cultures, transference to bouillon 
invariably led to immediate reversion. 

Vib. cholere.—No dys-agglutinable phase has been obtained at present in 
this vibrio. 

All the foregoing organisms show a tendency to the formation of «a pellicle 
in serum-bouillon culture. &. typhosus, occasionally only and very delicate ; 
B. paratyphosus A and B, and B. colt, fairly often and well-marked; 
Vib. cholerw, frequently; B. crtrycke and B. enteritidis (Gaertner), very 
frequent and very heavy, often appearing to contain as much growth in the 
pellicle as in the deposit at the foot of the tube after 48 hours. These state- 
ments apply to cultures made in narrow agglutination tubes. 

B. dysenterwe (Shiga) yielded a dys-ageglutinable culture after eleven 
passages in serum-bouillon of 1 in 4 dilution. 

B. dysenterie (Flexner), V, W, X, Y, Z.—These five strains of Flexner 
dysentery gave interesting results after six passages through 1 in 4 bouillon 
dilutions of their appropriate serums. Bouillon cultures were made in flasks 
from the sixth passages, and subsequently formolised and diluted to standard 
opacity. V, W, and Z cultures were dys-agglutinable ; X was not perceptibly 
altered; and the Y culture possessed markedly increased agelutinability, 
being four times as agglutinable as the standard culture against which it was 
tested. Of the three dys-agglutinable cultures, Z gave no agglutination at 
all in 1 in 25 standard Z serum at the end of 4 hours in the water-bath, and 
no more than a trace minus at the end of the subsequent 24 hours at room 
temperature. W gave trace minus (tr —) at 1 in 25 after 4 hours, trace 
plus (tr +) at 1 in 25 and trace (tr) at 1 in 50, 24 hours later. V, on the 
other hand, though giving no reading as high as trace plus (tr +) in 
any dilution, showed traces of agglutination in every tube up to 1 in 1000, 
thus reaching the same actual end-point with its “traces” as did the corre- 
sponding standard V culture, which gave totals up to 1 in 250, standard (s) 
at 500, and trace minus (tr —) at 1000. 

The discussion of the relation of these dys-agglutinable and well-agelutin- 
able forms of bacteria to the R and S forms recently described by Arkwright 
(loc. cit.), and the description of their morphological and other characters, is 
reserved for a future occasion. 


Summary and Conclusion. 


1, Evidence has been brought forward that in the enteric and dysenteric 
groups of bacteria dys-agglutinable and hyper-agglutinable forms or phases 
occur, and can be produced experimentaily by the methods described. 


68 Studies in Bacterial Variability. 


2. Both forms may be obtained from one and the same eu-agglutinable 
strain of a bacillus, and both may revert, or may be converted mutually 
the one into the other. 

3. In agglutination tests carried out in the ordinary manner, a highly 
dys-agelutinable bacillus may fail to agglutinate at all (at 1 in 25) witha 
serum that agglutinates the culture from which it was derived up to 1 in 
1000 or more. It may also entirely fail to absorb from the serum any appre- 
ciable quantity of the agglutinins specific to that culture. 

4, These results appear to necessitate a considerable modification of current 
theories regarding the value of absorption tests as a means of determining 
bacterial affinities, but may help to throw some light on the difficult problem 
of “serological strains.” They show how necessary it is to reserve one’s 
judgment, where conclusions are drawn regarding true differences of bacterial 
type, in cases where the differentiation rests solely on agglutination and 
absorption tests; since differences of such remarkable degree are shown to 
exist between different individuals among the population of a single culture. 


I desire to express my indebtedness to Dr. A. D. Gardner, who kindly 
made independent readings of a number of my agglutination tests, and to 
Miss Edith F. Stubington for frequent and willing help in many of the more 
laborious experiments. 


REFERENCES. 


(1) Walker, E. W. Ainley, ‘Journ. of Pathology and Bacteriology,’ vol. 7, p. 250 (1901 
(2) Gardner, A. D., ‘The Lancet,’ vol. 2, p. 494 (1920). 

(3) Walker, E. W. Ainley, ‘Journ. of Hygiene,’ vol. 17, p. 380 (1918). 

(4) Gardner, A. D., and Walker, E. W. Ainley, ‘ Journ, of Hygiene,’ 1921 (in the press). 
(5) Arkwright, J. A., ‘Journ. of Pathology and Bacteriology,’ vol. 24, p. 36 (1921). 

(6) Vines, H. W. C., ‘Journ. of Pathology and Bacteriology,’ vol. 22, p. 197 (1918). 

(7) Walker, E. W. Ainley, ‘Journ. of Pathology and Bacteriology,’ vol. 8, p. 34 (1902). 


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69 


The Titration Curve of Gelatine.—Report to the Medical Research 
Council. 


By Dorotuy Jorpan Lioyp, Biochemical Laboratory, Cambridge, and 
CHarLes Mayes, Physical. Laboratory, Eton College. 


(Communicated by Prof. F. G. Hopkins, F.R.S. Received September 26, 1921.) 


I. INTRODUCTION. 


During the course of some work on the swelling of gelatine gels in acid 
and alkaline solutions, it became increasingly evident that no fully satis- 
factory theory of swelling could be postulated until further information had 
been obtained as to the quantitative relations, both general and particular, 
holding between the gelatine base and its combined acid in acid systems, 
and between the gelatine acid and its combined base in alkaline ones 
respectively. The work described in the following paper is an attempt to 
study the problem in its simplest form; namely, in a fluid system in which 
all the components are in solution. Hydrochloric acid and sodium hydroxide 
were chosen as the acid and base to use in the investigation, since both form 
highly ionised solutions in water, and since none of the ions resulting carry 
more than a single charge, thus simplifying any considerations deduced 
from the law of mass action. It is still an open question how far the law 
of mass action can be applied to colloidal solutions. It has been shown 
by Procter (20), Procter and Wilson (21), Wintgen and Kruger(27), that 
the quantitative relations found by them to exist in the combination of 
hydrochloric acid with gelatine, under the conditions .of their experiments, 
fell within the general statement of the law, such combination being 
regarded as a simple case of salt formation. 

Procter considered that his results were explicable on two hypotheses :— 

(1) That gelatine had a molecular weight of 839, and combined with 
one molecule of hydrochloric acid to form gelatine hydrochloride ; or 

(2) That if gelatine had a larger molecular weight, some multiple of 839, 
say $39x, then the gelatine molecule combined with # molecules of hydro- 
chloric acid and the ionisation constants of the z hydroxyl groups involved 
must be the same. The second hypothesis he rejected as improbable. 

We still consider thut the molecular weight of gelatine must be greater 
than Procter has allowed, and we do not regard it as improbable that a 
number of the hydroxyl ions of the gelatine may have approximately equal 
ionisation constants. Our experimental results suggest that, up to a given 

VOL. XCIII.—B. G 


70 Miss D. J. Lloyd and Mr. C. Mayes. 


concentration of hydrogen ions, a group of hydroxy] ions having approximately 
equal ionisation constants is involved; beyond this concentration, and up 
to a second fixed value, a second group approximating to a second constant 
is involved ; and beyond this again there is slight evidence of a third group. 
The factors required in order to bring the second and possible third groups 
into conformity with the generalised statement of the law of mass action 
are not yet fully known. 


II. MATERIAL AND MeEtTuHop. 


The gelatine used in this investigation was Coignet’s Gold Label gelatine, 
purchased in 1914. It was purified by prolonged dialysis in dilute hydro- 
chloric acid at a reaction of Cy = 10746, and subsequent precipitation in 
strong alcohol. Details of the purification are given elsewhere (Jordan 
Lloyd, 7). It was dried with absolute alcohol and ether, and kept in a. 
desiccator over pure sulphuric acid. (It is possible to cause the gelatine 
to lose more water by heating to 100° over phosphorus pentoxide in vacuo.): 
When dried with absolute alcohol it is a white brittle substance, fibrous 
in appearance, and containing 0°00 to 0:06 per cent. of ash. It forms clear 
solutions in water, which set to opaque white gels on cooling. The clear 
solutions set to colourless, glassy, transparent gels in the presence of free 
acid or base. The solution referred to below as 1 per cent. gelatine contains 
1 grm. of this purified dry gelatine in 100 c.c. of freshly-boiled distilled 
water at room temperature. 

The object of our experiments was to determine the amount of hydro- 
chloric acid or sodium hydroxide which would combine with a constant 
weight of gelatine, and the method employed throughout was the electrical 
measurement of the concentration of the free hydrogen ions in solutions 
containing 1 per cent. of gelatine and known concentrations of hydrechlorie 
acid or sodium hydroxide. The change of hydrogen ion concentration from 
that of an equally concentrated system containing no gelatine, is a measure 
of the acid (or base) which has combined with the gelatine. The routine 
method employed was to take 5 c.c. of a freshly made solution of 2 per cent. 
gelatine which had cooled but not yet set; the requisite amount of acid 
(or base) was added from a pipette calibrated to 0°01 c.c., and sufficient freshly- 
boiled distilled water to make the final volume 10 c.c. The reaction was taken 
at once by means of a gas chain. A Tinsley potentiometer was used. 

The electrodes used were of a very simple modified Barendrecht type, and 
were made for us by Mr. A. W. Hall, of the Biochemical Laboratory. Their 
great advantage is the ease with which they are cleaned, a matter of great 
importance, as we found it essential to clean and re-coat the electrodes after 


The Titration Curve of Gelatune. 71 


every reading. A diagram of the electrodes is given below (fig. 1), and is 
self-explanatory. 


trom De 
Aydroge ' 
ydrogen ! 
ground fatinum 

Pay wire , 

glass fat. g 

6 
platinum fol sp) 
coated with ; 
Platinutn black 
t 

Pie, Il L 


The platinum foil was coated very thinly with platinum black according 
to the directions of Michaelis (16). Only sufficient platinum was deposited 
to hide the glint of the foil. With such electrodes it was possible to take 
readings immediately contact had been made with the experimental fluid 
This is an essential condition for accuracy. With a slow electrode and a 
delayed reading, values tend to be very irregular. This is particularly 
marked in alkaline solutions. The experimental fluid was placed in silica 
cups into which the electrodes dipped. Contact was made by means of a 
sliding joint. The solutions had a strong tendency to froth near the iso- 
electric point. All experimental readings were taken at 20° C. 


Il]. EXPERIMENTAL RESULTS. 
(a) The Gelatine-Hydrochloric Acid Curve. 

If N represents the normality of a solution of hydrochloric acid and « its 
degree of ionisation, the concentration of the hydrogen ion present may be 
represented as Na on the normality scale, or —log Ne on the logarithmic scale. 
If 1 per cent. of gelatine be introduced into such a system, the reaction (Py) 
is no longer given by the expression —log Na, but by some lower value. 
This can be determined by the hydrogen electrode. 

The foliowing experimental values were obtained for the variation of 
reaction (Piz) with total acid-content (N), gelatine concentration being kept 
constant at 1 per cent. temperature at 20°C. The constants used in caleula- 
ting the reaction (Py) from the observed electromotive force (E) are taken 
from Michaelis’ ‘ Wasserstoffionen Konzentration’ (16). The values for the 
concentration of the hydrogen ion are given on both the logarithmic scale 
Py, and in terms of normality [H]. From the values of [H] it is possible to 
calculate the concentration of un-ionised free hydrochloric acid present in the 

G 2 


7% Miss D. J. Lloyd and Mr. C. Mayes. 


system. If this is represented as [HCl], and « is the degree of ionisation of 
the acid, then 
[Hcl] = [H]/4—[H] (1) 
The values of « are taken from Lewis’s ‘Text-book of Physical Chemistry’ 
(3rd edition). Using Blasel and Matula’s formula (1), if »’ represents the 
concentration of acid removed from independent solution by the gelatine, then 
n’ = N—[H]/«, or n' = N—({H]+[HCl)). (2) 
Equation (2) only holds if the Value of « in (1) is the same both in the 
presence and absence of dissolved gelatine. In Table [, Column I gives the 
concentration of acid used, Column II the corresponding values of «, and 
Column III the values of —log Ne. Column IV gives the electrode 
reading in millivolts, and V the value of Py caleulated from the formula 


Pu = (E—248'8)/581. 
Columns VI and VII give [H] and n’ respectively. 
The two curves —log Na:N and Py: N from the values given in Table I 
are shown in fig. 2, and are designated as A and B respectively. 


06 


1:0 


5-0} 


Fic 2. : 
Hbse/ssae = Normality of acta. 
Oradindes Ff, 


40 


0 02 Ou 02 25 
Buffer curve of gelatine in hydrochloric acid. 


The Titration Curve of Gelatine. 73 


x, S04 
[ 
| 
f 
| 


] 
| 
U 
! 
! 
| 
| 
i 
! 
f 
I 
! 


>A 
FIG. Se 
i . t 
Abscissa = NOIMality Of Bose f | 
Oroinetes = Fy ) [2:4 
© 
(Sf 
O2 Ov oO 


Buffer curve of gelatine in sodium hydroxide. 


Blasel and Matula, in calculating 7’, the hydrochlorie acid fixed by gelatine, 
as n’ = N—[H]/a, assume that the value for « is the same when [H] = [Cl] 
as when [H]= [Cl], provided that the values for [H] are equal. This, 
however, is not true. A closer approximation is obtained by taking the 
square root of the product of [H] and [Cl] as the basis of the calculation. 
The value for [H] is obtained experimentally from hydrogen-electrode 
readings. The value for [Cl] is obtained by assuming that the gelatine 
chloride is completely ionised, and that therefore, [Cl] =[H]+’. ‘This 
assumption is also made by Procter and Wilson (21), though Bugarsky and 
Liebermann’s experimental figures on the concentration of the chlorine ions 
do not support it (3). The amount of un-ionised acid [HCl] depends, 
therefore, not upon [H] but on [H].o,, where 

[H] cor, = V ((H] x [Cl]), 
and a new value for the acid fixed, say N’, follows as before. 
Column VIII and is shown plotted in fig. 4. [In the values for n’ calculated 


This is given in 


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sRo FO SI[OAT] [LUT | ‘ 
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TITA | TIA TA “A | “AI aut Ui Tl 


74 
| 


TL Sete 


The Titration Curve of Gelatine. i 


from Blasel and Matula’s formula it is assumed that the gelatine hydro- 
chloride contributes no chlorine ions to the system; in those for N’ it is 
assumed that the gelatine hydrochloride contributes all its chlorine as free 


0:03 xx 
FiG. 4. 5 ea 


0:02 


—> 
iS) 
x 


x = Lxperiinenal POMS . 


O= Pas Taheh TOM Sino0oihed 
CULVE 11) FIC 2. 


O O05. O-/O OS 
Curve of acid fixed. Abscisse =[H]. Ordinates = Acid fixed or N’. 


chlorine ions to the system. The two curves, n’:[H] and N’:[H], there- 
fore form the limits within which the actual curve must lie. In hydrochloric 
acid of concentrations less than 0:02 grm. of free hydrogen ion per litre the 
difference between the two limiting curves is negligible] It can be seen 
that the curve is not a simple smooth curve, but that it consists of two, and 
possibly three, distinct regions. The deductions from this will be considered 
after the gelatine-sodium hydroxide curves have been described. 


(b) The Gelatine—Sodivm Hydroxide System. 

Let N represent the normality of the caustic soda and « its degree of 
lonisation at 20° C., then —log N« equals the hydroxyl] ion concentration of 
the system and 1413—(—logNz) is the hydrogen ion concentration. 
Values for « are given by Kohlrausch (9), Noyes (18) and Jones (6). 
Unfortunately they differ considerably. The values given by Noyes were 
taken to plot the (broken) curve 14:13 +log N z in fig, 3 (marked C in the 
margin). Noyes’ values only go to a concentration 0°05 N. The curve was 
extended beyond this region by taking Jones’s figures for « and adding to 
them the difference between his figures and Noyes’, which may be taken as 
0°05 if « is expressed as fractions of unity. N, « and 1413+log N are 
shown in the first three columns of Table II. The fourth column gives 
the readings obtained for E, the electro-motive force at the surface of the 
hydrogen electrode in a 1:00 per cent. solution of gelatine. The fifth column, 


Miss D. J. Lloyd and Mr. C. Mayes. 


76 


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*T.89/8.876 -H = "a 


SIS 


The Titration Curve of Grelatine. OU 


which is sub-divided, gives values for Py calculated from E, and read from 
the smoothed curve N: Py (marked D in fig. 3). The sixth column gives 
[OH] the concentration of hydroxyl ions on the normality scale, and the 
final column gives n’, which is equated to N—[OH]/z, and is the first 
calculation for the amount of base “ fixed” by the gelatine. 

In taking the “acid-fixed” values, the calculations in Table I were made 
directly from the experimental readings of KE. These lie so very closely to a 
smooth curve that it is safe to assume that the experimental error is slight. 
In the case of the values of E in alkaline solution, the error is obviously 
very much greater. Chemical destruction of the gelatine by hydroxyl in the 
presence of spongy platinum is probably the cause, and hydrolysis has also 
been shown to have a slight effect on the reading. For this reason, an 
arbitrary smooth curve (Py:N or D in fig. 3) was drawn through the 


‘08 
iGeor 
ae en esessac = (OF 


Orowneates = Bese rived 


O O05 0-10 
Curve of base fixed, 7’. 


78 Miss D. J. Lloyd and Mr. C. Mayes. 


experimental points,and the calculations of 7’ have been made from Py readings 
taken from this smooth curve and not from the Py readings given in 
Table II. Taking into consideration the poor quality of our experimental 
determinations of E in alkaline solutions, and the lack of agreement as to the 
values of « for caustic soda, it has not been considered worth while to correct 
the values of m’ obtained by the Blasel and Matula formula. Fig. 5 shows n’ 
plotted against [OH], and will be referred to later in Section IV (0). 


IV. THE CALCULATION OF THE JONISATION CONSTANTS OF GELATINE. 
(a) The Value of Ky. 

The combination of gelatine with hydrochloric acid between 0:00 and 
0°25 N concentration of acid may be represented as a curve with three 
sections. 

Assuming for the moment that gelatine in hydrochloric acid solution 
behaves as a monacidic base from 0:00 to 0:04 N HCl, and combines with 
the acid to form an ionisable salt (see Introduction), the system may be 
represented as follows :— 

Let [G(H)OH] represent the un-ionised gelatine base, and [GH’] and 
[OH’] the two ions of the ionised base; and assume that the salt GHC] is 
completely ionised ; now by the law of mass action for a weak base, 

[GH'] x[OH’] _ 


Paien ny age | Le 
“(G()0H] Ki where Ky = K,’ the hydrolysis constant of the base. 


Let C = the equivalent concentration of the gelatine. 
Then [GH’]+[G(H) OH] = C, é 

GH’ H . 

oe ™ iepae Ky @) 

[GH’] Bert ck 

[GH]+[G(H)OH]  H+Ky 

Now, in equation (1), [GH’] is equal to N’ and is known, H is known, and 
therefore there are two unknown quantities, C and Ky. If Procter’s (20) 
value of 839 be taken as the reacting weight of gelatine, C = 0-012. 
Wilson’s (26) later value of 768 makes C equal to 0°013. Wintgen and 
Kruger (27), using the catalysis of methyl acetate as a measure of the 
concentration of the hydrogen ion, obtain a molecular weight of 839 for 


or 


gelatine, while, calculating from the experimental results of Pauli and 
Hirschfeldt, they obtain the value 881'4. They give 2°7 x 107! as a value for 
K, at 25° C. In the calculations given below, C is taken as 0:0120, 


The Titration Curve of Grelatine. 79 


Substituting for N’, H, G(H)OH in equation (1), we get the following values 
for Ky :— 


N’. (H.] [G (H) OH.) | TK 
; : | 
0 00679 000316 0 00521 |. 9 00242 
0 00812 0 -00380 | 0 -00888 | 0 00184. 
0 -00898 0 00490 0 :00302 | 0 00165 
0 00939 0 00644. 0 -00261 | 0 -00180 


K, may therefore be approximated to 0:0018, 


4°] 
ky ss SS = = 048s 10-4, 4 
whence >= & = 90018 0 : x 10 (4) 


A comparison of this value with Procter’s value of 5°2x10~! shows that 
they are both of the same order. It is a workable hypothesis to suppose 
that gelatine in the presence of hydrochloric acid, the concentration of 
which lies between 0:00 and 0:04 N, behaves according to the law of mass 
action, like a weak base with a reacting weight of 839 and ionisation constant 


of 48x10, each reacting mass combining with one equivalent of acid. 
' Since we do not consider, on the chemical evidence at present available, that 


the molecular weight of gelatine can be less than 10,000, then it follows that, 


in its first stage of combination with hydrochloric acid, the gelatine molecule 


gives the following values for histidin, arginin and lysin :— 


has available thirteen points of attachment for acid, all with a chemical 
potential very close to 0'48x107~". Procter has considered this possibility, 
and rejects it as improbable, but, if we consider that the acid is attached to 
the free —NH> groups of the lysin and arginin, and possibly some other 
di-amino-acid, then it does not seem so improbable that the ionisation 
constants of these basic groupings might be of the same order. Kanitz (8) 


First ionisation Second ionisation | 

constant, . constant. | 

aed | 

Elis bicintaeen ere eee nee Diiexal Ome 5:0 x 10-8 

ASHENDNID  sogonendacooaon000 | 10x 1077 2)-2)* 105 !2 | 
IOS Soatacuteanee coh koe 1:0x 107 Ley 4 1O- 


| 


These values are for the amino-acids in the free state. With arginin and 
lysin the first and second ionisation constants are of the same order of 
magnitude, hence it might be expected that even when bound by one amino- 
group into the protein molecule, the free amino-groups of both acids would 
have ionisation constants of the same order of magnitude. Evidence for the 
binding of the acid at these groups is given below. 


80: Miss D. J. Lloyd and Mr. C. Mayes. 


[GEO aie 

C [H]+0-0018 
which GH’ increases with [H] and only attains a maximum at infinite concen- 
tration of [H]. If we assume an error of 1 in 1000,[H] may be regarded as at 
infinite concentration when [H]= 0°01 N. The experimental curve, however, 
continues to rise very rapidly at still higher concentrations of acid. In his 
earlier papers Procter considers this difficulty and supposes that a second 
ionisation constant of a lower value also exists. But equations of the form 


The curve given by equation (3), #.¢., gives a curve in 


a6 B 
US ar 
2+a “e+b 


where a and bare constants, give when plotted for x and y, a 


curve which rises rapidly at first and later more slowly, and it proved 
impossible to fit such a curve to the observations on fig. 4. These seem to 
indicate a maximum about N’ = 0-01, then a gradual rise to a possible 
maximum about N’ = 0:02 followed by yet another increase in N’. The 
observations for the latter part of the curve, however, are too uncertain to 
justify any definite conclusions. ; 


(b) Calculation of Ka. 


If J is the iso-electric point ofan amphoteric electrolyte, K, and Kg, its. 
ionisation constants, 


then i wh (z A Kw) 


Kab, 20°C = 0:86 x 10m K, for gelatine at 20° C = +8x107-¥ 
J = 10-6 (17), 
whence K, should equal 3°5 x 1077. 
Now for a weak acid G (OH) H, we have 


[H"] x [G(OH)’] _ 
[G (OH) H] 


Ke 


If [G(OH)’] is put equal to 7’, and C is put equal to [G(OH)’]+[G(OH)H J, 
i.¢., to the initial concentration of the gelatine, then 
nv Garbo lk 


C= Tee Ree 


But if 839 is taken as the reacting weight of gelatine, then C = 0012, 
hence 7’ should be less than 0:012. But m’ is already greater than 0°012 in 
0:020 N sodium hydroxide and as N increases, 7’ increases, with an ever- 
increasing rapidity. Hence C cannot be taken as 0:012, but must be con- 
siderably greater. ‘That is, the reacting weight in alkaline solution must be 
less than in acid solution, and hence different linkages must be involved. 


The Titration Curve of Gelatine. ill’ 


a K 
= = fOr. 
ih Cail TRG, [OH, 


or [OH]/Ke, where Ky = K,,/K, = 0°86 x 107/35 x 1077 = 25 x 107%. 


Then C = (K,+[OH])n’/[OH], and when we substitute the above value for 
Ks we see that n’ should be nearly equal to C at extremely small concen- 
trations of hydroxyl ion and should then remain appreciably constant. This 
is not the case in fig, 5; here n appears to rise abruptly to about 0:00d and 
there seeks a maximum only to commence rising again to give a very steep 
curve. 

Hence it is obvious that in alkaline solution gelatine does not behave simply 
as a weak acid dissociating in accordance with the law of mass action. It is 
possible that this abrupt rise accompanies some structural change of the 
protein molecule such as Dakin had shown to occur in strong alkaline 
solution (4). It must always be borne in mind that the hydrolysis of the 
—(C(OH):N— groupings with the formation of free carboxylic and amino-groups 
occurs very rapidly in alkaline solution as measured by formaldehyde titration. 
Now the hydrolytic breakdown of the gelatine is not accompanied by a greatly 
increased basic binding power in the system, for a 1 per cent. solution of 
gelatine in sodium hydroxide having a reaction of Py = 12:97 was found after 
3 hours at 100° C. to have changed to a reaction of 12°91. Further standing 
for 48 hours at room temperature was accompanied by achange of reaction to 
12:90. This change in reaction corresponds to an increased combination of 
gelatine and base to the extent of only 0:011 equivalents of sodium hydroxide 
to 10 grms. of gelatine. 


VY. MECHANISM OF FIXATION OF THE HYDROCHLORIC ACID. 


The most obvious points of attachment for acids in the gelatine molecule 
are the free amino-groups, and if hydrochloric acid forms salts with gelatine 
by addition of these groups, the salts should be regarded as hydrochlorides. 
Gelatine contains 18-0 per cent. of nitrogen in its total dry weight. Accord- 
ing to Van Slyke and Birchard (25) 3:16 per cent. of this 18 per cent. (equal 
to half the lysin) can be removed as nitrogen gas by the action of nitrous 
acid, and can therefore be regarded as existing in the molecule in the form of 
free amino-groups. If these groups are the only ones in the molecule which 
ean bind hydrochloric acid, then the maximum combining power of 10 grains of 
gelatine should be 0:0039 equivalents. Kossel and-Cameron (11) and Kossel 
and Kellaway (12) have also brought forward evidence to show that proteins 
such as clupein and salmin, which contain no lysin, but which contain arginin, 
have a free amino-group in the euanidin nucleus of the arginin they contain. 


82 Miss D. J. Lloyd and Mr. C. Mayes. 


This group does not give off nitrogen gas under the action of nitrous acid 
(Van Slyke, 23). Kossel (10) states that the acid binding power of salmin is 
exactly that of its guanidin groups, and Kossel and Cameron (11) also 
consider that the free amino-groups of clupein must be those of its guanidin 
groups. Bracewell (2) considers that in all proteins the mechanism of acid 
fixation is by means of free amino-groups, and that in proteins such as 
gelatine, which contain both lysin and arginin, the acid-binding power should 
be given by the sum of half the lysin nitrogen plus one-quarter of the 
arginin nitrogen, each nitrogen atom binding one equivalent of acid. From 
this he calculates that since lysin contains 6°32 per cent. of the total nitrogen, 
and arginin 14°70, the maximum binding power of gelatine for acids should 
be 000085 equivalents per gramme, 7.c., 0°0085 equivalents for 10 grm. By a 
titrimetric method he finds that 1 grm. of undissolved gelatine powder can 
remove 0:00070 equivalents of acid from a supernatant solution. 

By the method which we have employed in this work the acid-binding power 
of gelatine, calculated on its lysin and arginin-content (which we find equal to 
0:0086 equivalents for 10 grm. of gelatine), lies not at the true maximum of 
the N’:[H] curve (fig. 4), but close to the first apparent maximum. It is 
possible, then, that in solutions of hydrochloric acid less than 0°02 N gelatine 
binds hydrochloric acid by means of its free amino-groups, and that it is the 
average ionisation constants of these basic groups that is given by the value 
48x10-". But with increasing concentration of acid, more acid is bound 
than can be accounted for on this hypothesis, and it is therefore necessary to 
consider what part the imino-nitrogen of the peptide linkage (—~COHN—) 
could play. Robertson (22) states that the acid-binding power of proteins is 
not much increased by hydrolysis, and we have found that the reaction of a 
1 per cent. solution of gelatine, which was found to be Py = 1:13, had only 
changed to Py; = 1:12 after 7 hours at 100° C. This change is of the same 
order as the experimental error of the method, nevertheless hydrolysis of the 
gelatine had occurred during the heating in the strong acid solution, as was 
shown by the fact that the gelling power had been destroyed. It seems, 
therefore, that the peptide linkage can function as an acid-binding group, 

Calculating again from Van Slyke’s figures, if every amino-nitrogen atom 
of the free amino-acids of gelatine (iz., the amino-nitrogen from lysin and 
arginin + the imino-nitrogen from the peptide linkages) can act as a point for 
the fixation of an equivalent of acid, then 10 grm. of gelatine should be able 
to combine with 0:092 equivalents of hydrochloric acid. If only the di-amino 
acids (arginin and lysin, together with histidin) can do so, then the maximum 
value for acid fixed in a 1 per cent. solution of gelatine would be 0:020 equi- 
valents. It can be seen from fig. 3 that the value 9-020 for N— corresponds to 


The Titration Curve of Gelatine. 83 


the maximum level of the second branch of the curve. The curve, however, 
continues to rise to a height of 0-030, though later it falls again. The experi- 
mental error in determining Pq becomes enormously magnified on the third 
limb of the [H]:N curve, and therefore too much stress cannot be laid on its 
smoothed form. However, it seems clear that some of the —COHN— groups 
can act as basic groups combining with acids. What réle, if any, other groups 
(such as the hydroxyl groups of the hydroxy-acids) in the molecule play in 
acid fixation is still unknown. It will be necessary to follow experimentally 
the fate of the chlorine ion before final decisions are possible. At present 
our calculations of N’ involve the assumption that the gelatine hydrochlorides 
are present as completely ionised salts, an assumption that is liable to lead 
to an increasing error with increasing values of [H]. 


The Mechanism for the Fixation of Sodium Hydroxide. 

The theory that proteins fix bases by means of their free carboxyl groups 
has given way on the accumulation of evidence that there are not enough 
of the latter to explain the quantitative relations 

Brailsford Robertson (p. 236) suggests that the enolic imino-linkages 
a Si 

ae , are responsible and “ that the neutralisation of bases by the 


proteins is accomplished, at any rate for the greater part, by the dicarboxylic 
radicles which they contain.” He gives the formula for potassium protein 
compounds as 


OK H OH 
i Nw 
ne aR 
R 
No= Ne 
la: reac 
OK H OH 


According to the most recent analysis of gelatine (Dakin, 1920), the only 
di-carboxy acids present are aspartic acid (3-4 per cent.) and glutamic acid 
(58 per cent.). Calculating from these figures. the maximum combining 
capacity of a 1 per cent. solution of gelatine should be 0-0168 equivalents. 
On examining the curve of base fixed in fig. 4, it can be seen that this 
value for n’ is by no means a maximum, but that instead the curve inflects 
and rises with increasing eradient. If every —COHN— group in the 
molecule is considered capable of acting as a point of attachment for bases, 
the maximum value for 1’ should be 0°09; n’ however rises considerably 
above this figure. There must therefore be other means by which the 
gelatine molecule can fix sodium hydroxide. The possibility of linkage 


84 Miss D. J. Lloyd and Mr. C. Mayes. 


at some of the hydroxy-groups of the substituted amino-acids serine and 
hydroxyproline, is not to be ignored. Hydrolysis of gelatine by caustic 
soda has been shown to increase slightly its basic binding power, a fact 
which suggests that not all the —COHN— linkages are as potent as base 
fixers as the free --COOH— groups. Loeb (14, 15) has shown that bases 
react with gelatine at the same hydroxyl ion concentrations in equivalent 
proportions. This fact shows that the reaction is ionic, and that the com- 
pounds formed are of the nature of ionisable salts. Loeb only worked with ~ 
solutions whose alkalinity was less than Py = 9. His experimental values 
correspond very closely to our values over the same range. 

The long slow rise of »’ with increasing alkalinity is very striking. A 
feature in which the alkali-gelatine system differs markedly from the acid- 
gelatine system is illustrated by a different property of the gelatine, namely, 
the turbidity of the gel. Sols of 1 per cent. gelatine in distilled water set 
in a few hours to white, turbid gels. In the presence of 0:001 N hydro- 
chloric acid the gel is not turbid, but quite clear and glassy after 24 hours’ 
standing at 15° C. In the presence of caustic soda, however, the turbidity 
of the gel 24 hours old persists up to a concentration of 0°002 N soda. 
Thus there is both quantitative and qualitative evidence to show that in 
the same protein the mechanism of fixing acids is different from that of 
fixing bases. 

VI. Summary. 

1. Hydrochloric acid combines with gelatine in solutions whose acid 
concentrations are less than 0:04 normal, according to the law of mass action. 
K, for gelatine is 48 x 10” at 20° CO. if 839 be taken as the reacting weight 
of gelatine. : 

2. The theory is put forward that over this range of the curve of com- 
bination of gelatine with hydrochloric acid, the combination occurs at the 
free —NHz2 groups. These groups are present in the lysin, arginin, and 
possibly some other of the amino-acids of the gelatine. The ionisation 
constants at these groups are taken as approximately equal. The salts 
formed are hydrochlorides. ; 

3. In concentrations of hydrochloric acid greater than 0:04 normal, the 
proportion of acid fixed is greater than would follow from the combination 
of hydrochloric acid with a weak base, with an ionisation constant of 
48x10-. This is not due to the hydrolytic decomposition of the gelatine 
and release of further free —NH» groups. It seems possible therefore that 
combination is also occurring at the nitrogen of the peptide linkages. 

4. In dilute sodium hydroxide of a concentration less than 0-01 normal, 
the gelatine combines with the base less rapidly than would follow- by a 


~The Titration Curve of Gelatine. 85 


calculation from the value for K, and the value for the iso-electric point, 
if 839 is taken as the reacting weight. 

5. It is suegested that the number of positions of attachment for bases is 
not the same as the number of positions for acids, we., that the reacting 
molecular weight 
basicity (or acidity ) 

6. It has not been found possible to calculate a value for K,. 
7. It would appear that in concentrations of sodium hydroxide about 
0:1 N the structure of the molecule undergoes some change. 


weight = is not the same in acid and alkaline solution. 


BIBLIOGRAPHY. 


. Blasel and Matula, ‘ Biochem. Zeit.,’ vol. 58, p. 417 (1914). 
. Bracewell, ‘J. Am. Chem. Soc.,’ vol. 41, p. 1511 (1919). 
. Bugarsky and Liebermann, ‘ Pfliig. Arch.,’ vol. 72, p. 51 (1898). 
. Dakin, ‘J. Biel. Chem.,’ vol. 13, p. 357 (1912-18). 
. Dakin, ‘J. Biol. Chem.,’ vol. 44, p. 499 (1920). 
. Jones, ‘ Publ. Carnegie Inst., Washington,’ No. 60. 
. Jordan Lloyd, ‘ Biochem. Journ.,’ vol. 14, p. 147 (1920). 
. Kanitz, ‘ Zeitsch. physiol. Chem.,’ vol. 47, p. 476 (1906). 
9, Kohlrausch-Holborn, ‘ Leitvermiégen der Electrolyte,’ Leipzig, 1898. 
10. Kossel, ‘ Zeitsch. physiol. Chem.,’ vol. 37, p. 112 (1902). 
11. Kossel and Cameron, ‘ Zeitsch. physiol. Chem.,’ vol. 76, p. 456 (1912). 
12. Kossel and Kellaway, ‘ Zeitsch. physiol. Chem.,’ vol. 72, p. 486 (1911). 
13. Lewis, ‘A System of Physical Chemistry,’ 3rd edit., London, 1920. 
14. Loeb, ‘J. Gen. Physiol.,’ vol. 1, p. 379 (1919). 
15. Loeb, ‘J. Gen. Physiol., vol. 1, p. 487 (1919). 
16. Michaelis, ‘ Die Wasserstofhonen Konzentration, Leipzig, 1914. 
17. Michaelis and Grinieff, ‘ Bioch. Zeit.,’ vol. 41, p. 373 (1912). 
18. Noyes, ‘Publ. Carnegie Inst., Washington,’ No. 63. 
19. Pauli and Hirschfeldt, ‘ Biochem. Zeit., vol. 62, p. 245 (1914). 
20, Procter, ‘ Kolloidchemische Beihefte,’ vol. 2, pp. 6-7 (1911); ‘Trans. Chem. Soc.,’ 
vol. 105 (1914). 
21. Procter and Wilson, ‘Trans. Chem. Soc.,’ vol. 109, p. 307 (1916). 
22. Robertson, T. B., ‘The Physical Chemistry of the Proteins.’ 
23. Van Slyke, ‘J. Biol. Chem.,’ vol. 9, p. 185 (1911). 
24, Van Slyke, ‘J. Biol. Chem.,’ vol. 10, p. 15 (1912). 
25. Van Slyke and Birchard, ‘J. Biol. Chem.,’ vol. 16, p. 539 (1913). 
26. Wilson, ‘J. Am. Leather Chem. Ass.,’ vol. 13, p. 177 (1918). 
27. Wintgen and Kruger, ‘ Koll. Zeit.,’ vol. 28, p. 81 (1921). 


— 


oA oO £ w bp 


VOL. XCIII.—s. mi 


86 


The Hamolytic Action of Sodium Glycocholate. 


By Eric PONDER. 
(Communicated by Sir E. Sharpey Schafer, F.R.S. Received September 14, 1921.) 


(From the Department of Physiology, University of Edinburgh.) 


Introduction. 

This paper contains a detailed investigation into the action of sodium 
elycocholate, and into certain phenomena in which this salt plays an impor- 
tant part. The presentation of this research is a matter of some difficulty, 
since the observations recorded constitute merely the outlines of a very 
complex subject. It has been thought best, even at the expense of some 
lack of logical sequence, to present the problems more or less in the order in 
which they presented themselves for solution, the reader being thus taken 
over the several questions in the order in which they were investigated. ‘To 
avoid undue length, no detailed description of methods used is given, if such 
description is to be found in a previous paper, on the findings of which this 
work is based (1). 


The Physical Condition of Solutions of Bile Salts. 

Although there is no definite statement on the subject, the general opinion 
appears to be that sodium taurocholate and sotlium glycocholate form true 
solutions in water. If this be so, it is remarkable that they possess properties 
peculiar to colloids. Ii sodium taurocholate be dissolved in water, a clear 
yellow solution results. This clearness soon disappears, the solution becomes 
opalescent and, after about 12 hours, quite opaque. The opalescence is 
caused, presumably, by the taurocholate passing into a physical state other 
than that in which it was when first dissolved. The opalescent solution has 
all the properties of a taurocholate solution ; its hemolytic activity is as 
oreat as is that of a clear solution of the same strength. A difference appears 
on filtering the opalescent solution ; the filtrate has a less hemolytic power 
than the original solution. ‘This fact, together with the opalescence, suggests 
that the taurocholate has assumed a less dispersed form. 

The more dilute the taurocholate solution is made, the more rapidly does 
the opalescence appear ; in solution in 1 per cent. saline a similar occurrence 
takes place, but less rapidly than in aqueous solution, 

Sodium glycocholate behaves in a similar way, but the appearance of the 


The Hemolytic Action of Sodium G'lycocholate. 87 


opalescence is not so rapid. If in a 1 per cent. solution in saline, the 
opalescence takes days to appear. 

Both salts possess a property peculiar to colloids—they protect a gold sol 
against precipitation by electrolytes. This protective power is shown in the 
following way. Using a gold sol prepared by the formaldehyde method, 1 c.c. 
is precipitated by 0-1 cc. of 10 per cent. NaCl within 10 seconds. If small 
quantities of bile salts be added, we find that precipitation is prevented. This 
protective power is apparent before the solution becomes opalescent, and 
becomes less as opalescence proceeds, attaining a minimum after about 
36 hours. The smallest quantity of taurocholate and glycocholate respec- 
tively which will protect 1 ¢.c. of gold sol against 0:1 ¢.c. 10 per cent. NaCl is 
shown in the following Tables, the solutions being kept at 18° C.:— 


Table I. 
| : ame 1) 
Smallest protecting quantity. 
Hours after = SEL Jal aR Tose aaaini cA 
[DECI PEREHO Taurocholate Glycocholate | 
(1 in 1000). (1 in 1000). 
Oc c.c. 
1 hour 0:2 0°35 
12 hours 0°3 O-4 
24, | 0°35 | 0°55 
36, 0:4 0-6 
gas 0-4 06 


It will be observed that (1) the taurocholate is about twice as protective as 
the glycocholate, and (2) that, as opalescence becomes established, there is a 
loss of protective power, the minimum being about half the protective power 
of the fresh solution. 

A more complete study of the physical chemistry relating to this subject is 
being made. 

The Hemolytic Action of Sodiwm Glycocholate. 


Ina paper previously published (1), it has been pointed out that sodium 
elycocholate behaves in a manner similar to that found in the cases of sodium 
taurocholate, saponin, and other hemolytic agents. In dilutions higher than 
1 in 1000, the rapidity with which this salt produces hemolysis depends on 
the dilution, there being a relatively simple relation between the two. When 
one examines the action of the salt in concentrations of 1 in 100, 1 in 500, 
etc., a different behaviour is observed : in three respects especially. 

(1) There is great difficulty in obtaining consistent results; this difticulty 
does not ordinarily exist, for the time required for hzemolysis can, as a rule, 
H 2 


88 Mr. E. Ponder. The Hemolytic 


be observed correct to a few seconds, and readily reproduced with consistence. 
In the case of sodium glycocholate acting in these high concentrations, a 
variability in the time taken for hemolysis appears, even when factors such 
as temperature are controlled. 

(2) The salt heemolyses more rapidly in dilutions of 1 in 1000 and there- 
abouts, than in dilutions of 1 in 100. 

(3) A freshly prepared suspension of red cells seems to be less rapidly 
hemolysed by the salt in all dilutions up to 1 in 1000, and especially in 
dilutions of 1 in 100, and 1 in 50, than is a suspension which is 12 or 
18 hours old. This is not unexpected; the envelopes of these old cells 
being probably weak. The importance of using a freshly prepared suspension 
for quantitative hemolytic tests was insisted on in a previous communication. 

The following Table gives the times taken for complete hemolysis of 
0-2 c.c. of standard blood suspension, freshly prepared, by various dilutions 
of this salt, at 18° C.:— 


Table II. 
| 3 4h 3. | “y 
ie : : ——} 2 Sie 
| 50 25 minutes. | 400 | 40 minutes. 
100 90 5 | 600 30 a 
200 65 re | 1000 | 10 3) 
| 


It must be understood that this Table is merely representative of the 
general behaviour of the salt, and that the times given are not the same for 
all suspensions, but vary with the condition of the cells, etc. 

These results may be looked upon as unexpected—hzmolysis occurring 
more rapidly with a dilution of 1 in 1000 than with a dilution of 1 in 100. 
It is obvious that the hemolysis depends on some factor other than the 
simple solution of the lecithin and cholesterin envelope of the erythrocyte in 
the solution of bile salt (2). It will further be observed that sodiwn 
glycocholate is a feebly hemolytic agent compared with sodium taurocholate. 

It is with occurrences connected with the action of the glycocholate in 
these relatively high concentrations that this paper is concerned. 

It is convenient here to insert a note regarding terminology. The 
letter T is used to denote the time required for the complete hemolysis 
of the amount of blood suspension used. The letter 7 represents the tem- 
perature at which the experiment is conducted. The symbol 6 denotes the 
number of cubic centimetres which contain 1 grm. of a hemolytic agent, in a 
solution which is being used to produce hemolysis. For detail regarding 
this nomenclature, the reader is referred to a previous paper (1): im that 


Action of Sodium Glycocholate. 89 


paper the special technique employed in these hemolytic experiments is 
given in full. In the case of sodium glycocholate, when working at tem- 
peratures in the neighbourhood of 20° C., the exact temperature is very 
important, as in this region a slight rise of temperature greatly accelerates 
the hemolysis. Neglect of taking this into account is a fruitful source of 
error. : 

The Effect of Blood Serum on the Action 9f Bile Salts. 

Blood serum exerts a powerful inhibitory influence on the hemolytic 
action of sodium glycocholate and sodium taurocholate. This is illustrated 
by the following results, the experiment being carried out in the way 
indicated in a previous paper (1), and in this case at 18° C. :— 


Table IIT. 
AW 
Sodium taurocholate, 1in 1000 ............ 3 minutes. 
fe > O‘le.c. serum ...... 16 _ 
Sodium glycocholate,1in 1000 ............ 10 i 
+ 5 O‘le.c. serum ...... 37 33 


With a view to discovering which constituent of serum produced the 
inhibition, the inhibitory power, if any, of each constituent of serum was 
studied. This problem will be dealt with later: the subject for consideration 
at present being certain phenomena occurring when serum albumin is 
brought into contact with sodium glycocholate and a blood suspension. The 
fact that the bile salts cannot exert a hemolytic action in the presence of 
blood serum is of great interest, since it throws new light on the con- 
troversies regarding hemolysis and hemoglobinuria in jaundice (3). 


The Effect of Serum Albumin on the Hemolytic Action of Sodium Glycocholate. 


The following solutions are used :— 
(1) Solutions of sodium glycocholate in saline (0°95 per cent. NaCl). The 
strength of these solutions is shown in the following Table :— 


Table IV. 


Glycocholate. Final concentration. Value of 6. 


per cent. | 
2-5 i 1 in 100 100 
1-25 | 1 200 200 
0-833 | 1 300 300 
0-625 | 1 400 400 
075 | 1 500 500 
0°417 | 1 600 | 600 
0-25 | 1 1000 1000 


90 Mr. E. Ponder. The Hemolytic 


The second column gives the concentration of glycocholate in the tube if, of 
any of these dilutions, 2 cc. be taken, and have 2 cc. of saline, or saline 
solution of albumin, and also 1 c.c. of standard blood suspension, added. The 
third column gives the value of 6 for such a mixture. 

In all the experiments recorded below, for convenience, quantities one-fifth 
of these are used, the final concenfrations being the same, eg., instead of 
2 ¢.c. of glycocholate plus 2 ¢.c. of saline plus 1 ¢.c¢. of suspension, 0°4 c.c. of 
elycocholate, 0:4 c.c. of saline, and 0:2 c.c. of suspension are used. 

(2) A solution of serum albumin in saline. The serum albumin was 
prepared from blood, dried, and kept for some months before use. The 
strength of the solution is 1 per cent. 

(3) A standard blood suspension, as described in the previous paper (1). 
This is essentially a 5 per cent. suspension of thrice washed human 
erythrocytes in normal saline. 

If to 0-4 c.c. of 2°5 per cent. glycocholate be added 0:2 c.c. of suspension, 
and, after an interval of 5 seconds, 0°4 ¢.c. of serum albumin solution be 
added, hemolysis oceurs very rapidly, in about 30 seconds. It has been 
noted above that a 1 per cent. solution of glycocholate takes over 4n hour to 
produce hemolysis of this quantity of suspension. Since, after adding the 
albumin, the concentration of glycocholate is 1 per cent., and since hemolysis 
oceurs in about 30 seconds, it is obvious that the serum albumin solution 
has a powerful accelerating effect on the action of the bile salt. It may be 
observed that the serum albumin solution is of itself non-hemolytic: and 
that the rapid hemolysis is in no way explained by the fact that the blood 
cells remain in contact with a 2°5 per cent. solution of glycocholate for 
5 seconds, since the salt in this concentration will not produce any hemolysis 
in this short time. Control experiments, using saline instead of the serum 
albumin solution, render the accelerating action of the latter quite clear. 

The oceurrence of this rapid hemolysis depends on several factors. The 
rapid hemolysis occurs with a mixture of serum albumin, sodium glyco- 
cholate, and blood cells. These three substances may, however, be mixed in 
three different ways :— 

Method 1.—Put 0:4 c.c. of glycocholate solution, 25 per cent., in a tube, 
add 0:2 e¢.c. of blood suspension, and then, after an interval add 0°4 c.c. of 
serum albumin solution. 

Mcthod 2.—Put 0:4 ¢.c. of serum albumin solution in the tube, add 02 c.c. 
of blood suspension, and after an interval, add 0:4 ec. of 2°5 per cent. 
glycocholate solution. 

Method 3.—To 0-4 ec. glyeocholate solution add 04 cc. of the serum 
albumin solution, and then 0:2 c.c. of the blood suspension. 


Action of Sodium Glycocholate. 91 


The interval is, for convenience, 5 seconds. In all three cases the com- 
position of the final contents of the tubes is the same. Yet very different 
results appear. 

By method 1, hemolysis occurs in 30 seconds. 

By method 2, hemolysis occurs in 25 minutes. 

By method 5, haemolysis may occur in a short time as in method 1, or after 
a long time, as in method 2; the time is usually intermediate between the two. 

It is thus obvious that two different phenomena are being observed, 
according as to whether the glycocholate or the albumin is first brought into 
contact with the cells. It is further obvious that method 3 is of no use for 
the giving of consistent results,as the time taken to produce hemolysis varies 
under apparently the same set of circumstances. The results given by 
method (1) will first be considered, as being the more important. 

The effects produced by varying the quantity of sodium glycocholate in the 
above experiment, may now be investigated. 

In a series of tubes is placed 0°4 cc. of varying dilutions of sodium glyco- 
cholate, as mentioned above; to each tube is added 0°2 c.c. of blood suspension, 
and after an interval of 5 seconds, 0°4 cc. of 1 per cent. serum albumin 
solution is added. The observation of the 5 seconds interval is very 
important. The results are expressed in tabular form :— 


Table V. 

When 7 = 18. 
: | iy | . 1%, | 
| | | | 
A Sede, a —|| | 
‘ 100 | 1 minute. | 400 55 seconds. 
200 | 24 minutes. 600 5 5 | 
300 13 iy | 1000 6 minutes. | 

i 


It will be found that very rapid hemolysis occurs with all the dilutions of 
the glycocholate used. 

If the same suspension be tested in a similar way after it has stood for a 
few hours, a different state of affairs will be found; the blood cells have 
undergone a change on standing. ‘This surprising result occurs with great 
regularity and, with patience, the stages of the change may be made out. As 
an example, below is given in tabular form, the behaviour of a freshly 
prepared standard blood suspension, as time elapsed. The time for hemolysis 
of 0-2 c.c. of this suspension by 0°4 c.c. glycocholate of various concentrations 
plus 0:4 c.c. of 1 per cent. serum albumin, was estimated by method 1, as 
above, at intervals of 1 hour, 12 hours and 24 hours after preparation, as 
well as immediately after preparation. The results were as follows: 


92 Mr. KE. Ponder. The Hemolytic 


Table VI. 
When 7 = 18. 
Time after preparation. 
5 — wa ee = ——— = | = 
5 minutes. | 1 hour. 12 hours. | 24 hours. 
| 
100 13 minutes | 2% minutes 18 minutes 40 minutes. 
200 2 ” | 30 ” | 20 » 15 ” 
300 1 minute | A es B55 3 
400 50 seconds 50 seconds | 1 minute 1 3 
600 Zorg Lure Sot nee Thee 1 eas 
1000 2 minutes | 3 minutes 7 minutes 8 ri 


All blood suspensions exhibit this change, some in greater degree, some in 
less. The change is not one merely to be detected with care, but a very 
obvious one, which makes investigation into this subject very difficult, much 
experience being necessary to correctly interpret results. It will be seen 
from this Table—which gives a typical result—that the freshly prepared 
suspension is rapidly heemolysed by all the concentrations of glycocholate, on 
the addition of the serum albumin, it may be therefore termed “sensitive.” 
An old suspension, however, is not rapidly heemolysed except by dilutions of 
glycocholate in the neighbourhood of 1 in 500; it may, therefore, be called, 
compared to the fresh suspension, an “insensitive” suspension. This meaning 
will be attached to these terms in the following pages. 

At this point it will be convenient to deal with one essential difference 
between a sensitive and an insensitive suspension. When a standard blood 
suspension is prepared, blood is drawn into citrated saline, to prevent 
coagulation. The suspension is centrifuged, the cells washed thrice with 
saline, and the cells then added to normal saline (0°95 per cent. NaCl), to 
form a 5 per cent. suspension. This suspension is normally sensitive. If the 
blood be drawn into normal saline instead of into citrated saline, and the act 
of coagulation thus permitted, an insensitive suspension results after washing 
the cells, and preparing the suspension in the same way as before. This is a 
constant occurrence; the act of coagulation seems to determine that the 
suspension shall be insensitive. If the blood be drawn slowly from the 
finger and clotting thus be allowed to begin, the resulting suspension will be 
insensitive. This very interesting fact is of use; for, with reasonable care, a 
sensitive suspension can always be prepared, and also if an insensitive suspen- 
sion be required, it can with certainty be made. 

The changes through which a sensitive suspension goes on standing are 
very curious; further investigations, to be noted below, throw some light on 
these changes, It may be observed that there is no difference between a, 


Action of Sodium Glycocholate. 93 


sensitive and an insensitive suspension as regards the activity of either sodium 
glycocholate or sodium taurocholate when acting upon it; the difference 
exists only towards the mixture of glycocholate and albumin. The above 
mentioned phenomena occur not only with human erythrocytes, but with the 
red cells of dogs, cats, rabbits and guinea-pigs. The length of time for which 
a sensitive suspension remains unchanged varies. Some suspensions become 
insensitive within half an hour of preparation; others remain unchanged for 
as long as 12 to 20 hours. 

Having considered the effect of varying the amount of glycocholate used 
(Table V) when 0:4 cc. of 1 per cent. albumin solution is used to accelerate 
the hemolysis, it must now be considered what the effect is of varying the 
amount of serum albumin. The following Table shows this. The substances 
are mixed in the same way as that adopted for the drawing up of Table V, 
i.e., by method 1; 0-4 ¢.c. of albumin is added in each case. 


Table VII. 
When 7 = 18. 

Glycocholate. | 
ey San nnnnE TO , | 
| | | 

1 in 100. 1 in 400. 1 in 600. | 1in 1000. | 
: eG : | 
2 per cent. | 80 seconds | 20 seconds 35 seconds 5 minutes. 
1 Fi 28 minutes | 30 ,, SOtpney 15 “4 
0°5 eS 43 - 4 minutes | 20s 35 Pe 
0:2 . 56 93 12 3 13 minutes 17 Ss 
} 


From a consideration of this Table it is obvious that the phenomena are 
very complex. The subject will be left in the meantime, and referred to 
again later (Table XIV). It will be sufficient to note here that the quantity 
of serum albumin used in combination with various dilutions of glycocholate 
is of the greatest importance. In the above experiment the suspension used 
was an insensitive one. 

Certain of the occurrences met with when the sodium glycocholate, blood 
cells, and albumin are mixed by method 2 may now be considered. 

In this method, 0:4 ¢.c. of 1 per cent. solution of serum albumin is placed 
ina tube, 0:2 ¢.c. of standard suspension is added, and after five seconds, 
0:4 c.c. of any desired concentration of sodium glycocholate is added. The 
results of various dilutions of glycocholate may he given; the suspension 
used js sensitive, 


94 Mr. E: Ponder. The Hemolytic 


Table VIII. 


When 7 = 18 

| Method 1. | Method 2. | 
| 

| | i iy 

| | | 

| 

100 ie 5 seconds | 25 minutes. 
200 10 si | 30 seconds. 
| 300 "eee | 7 Oa 

| 400 20h | BH 

| 600 25 5s | 40 

| 1000 2} minutes 4 minutes. 


The hemolysis does not occur so rapidly with method 2 as with method 1. 
The difference is most marked when high concentrations of glycocholate are 
used. In investigating the sensitiveness of a suspension, therefore, it is very 
important that this difference be kept in mind. 


The Protective Action of Serum Albumin. 


It has been seen that while serum albumin, if added to a cell suspension in 
contact with sodium glycocholate, accelerates the hemolysis produced by the 
latter, if it be added to a cell suspension it will protect it against the action 
of the sodium glycocholate and serum albumin mixture. 

To investigate this further, one may put up four tubes, as follows :— 

Tube 1.—1 e.c. suspension 0-2 ¢.c. of 1 per cent. serum albumin. 

Tube 2.—1 cc. suspension 0:1 c.c. of 1 per cent. serum albumin. 

Tube*3.—1 e.c. suspension 0:05 cc. of 1 per cent. serum albumin. 

Tube 4.—1 ¢.c. suspension 0:02 c.c. of 1 per cent. serum albumin. 

The suspension used is a sensitive one. Allow all tubes to stand for 
5 minutes. Examine the suspensions to see if they are sensitive or insensitive. 
The following result is typical :— 


Table IX. 
When + = 18: 6 = 100; +0°4 ec. 1 per cent. albumin. 
Tube. an, | Tube. | 1. | 
if ae 
Control 15 seconds. 1 3 5 minutes. | 
1 40 minutes. 4 | 1 minute. | 
22 i | | | 
| 
| 


bo 


The effect, then, of adding a small quantity of serum albumin solution to a 
sensitive suspension is to render it insensitive, The degree of insensitiveness 


Action of Sodium Glycocholate. 95 


produced depends on two factors: (1) the amount of serum albumin added ; 
and (2) the time for which it remains in contact with the cells. A suspen- 
sion thus made insensitive is not resensitised by washing once in the centri- 
fuge: repeated washings may render it sensitive to some degree. 


The Protective Power of Blood Serwnv. 


As serum blood albumin has this effect, it might be supposed that blood 
serum itself had a similar effect. The following typical experiment shows 
this clearly :— 

Tube 1.—1 ¢.c. suspension 0:02 c¢.c. serum. 

Tube 2.—1 e.c. suspension 0015 e.c. serum. 

Tube 3.—1 c.c. suspension 0:01 ¢.c. serum. 

Tube 4.—1 ¢.c. suspension 0:005 c.c. serum. 

Allow the tubes to stand 5 minutes. The suspension used is a sensitive 
one. ‘Test the suspensions to see if they are sensitive or not. 

The typical result is as follows :— 


” 


Table X. 
When 7 = 18; 6= 100: 40:4 c.c. 1 per cent. albumin. 
we ia OY Paras SEI ES tia cami ila a ] z Para Sea PR TPG io eee | 
| | 
| Tube. h, Tube. mn | 
Control | 15 seconds. | 3 | 6 minutes. 
1 20 minutes. | 4 | 25 50 
| 2 15 | 


The serum thus renders a sensitive suspension insensitive. Such an insensi- 
tive suspension is however restored on washing the cells in the centrifuge ; an 
occurrence which is not found in the protection conferred by serum albumin, 
and suggesting that the protection is conferred in different ways in the two 
cases. The protection is conferred not by fresh serum only, but by preserved 
serum, kept for over 18 months by the method of Leers (4). 


Animal Haperiinents. 

It is contirmatory of these experiments that the protective action of serum 
albumin oecurs in vivo as well as in vitro. 

If a rabbit be taken and from a vein a small quantity of blood (0:08 c.c.) be 
drawn into citrated saline, the suspension resulting after washing these cells 
'—preferably once—is sensitive to the mixture of glycocholate and serum 
albumin. If now 5 cc. of a 2 per cent. serum albumin solution in saline be 
injected into a vein, and blood withdrawn from a distant vein about 
5 minutes later, the suspension prepared from this blood will be insensitive, 


96 Mr. E. Ponder. The Hemolytic 


Results for four rabbits may be recorded :— 


Table XI. 

When sis: 
| =o 
Rabbit. T, 1st sample. 1, 2nd sample. Albumin injected. 
| it 15 seconds 24 minutes O-l gramme, | 
| 2 AN on, Tepes | 0°04 ,, | 
3 Bg OE anh 0-08, 
4 30 Tigo We 0-04, 


The injection seems to cause no untoward effect on the rabbit, which is 
under chloroform ancesthesia. 

These phenomena will be found to be not peculiar to serum albumin : 
other similar substances act as powerful accelerators of glycocholate heemo- 
lysis and as protective agents, when used differently, just like serum albumin. 
Peptone is such a substance: its actions are exactly parallel with those of 
the albumin. On trying the effects of various animal extracts, similar 
properties were found to be possessed by both adrenalin and pituitrin (Parke, 
Davis preparations). Since it has been shown that pituitrin contains 
histamine, this at once suggests the possibility of the phenomena being due 
to histamine or histidine, appearing in both the albumin, the peptone, and the 
pituitrin (5). 

The Effect of Histamine on Glycocholate Hemolysis. 

A series of solutions of histamine (Burroughs Wellcome) is prepared, the 
following dilutions being convenient :—1 in 500, 1 in 1000, 1 in 2000, 
1 in 5000, 1 in 8000, and 1 in 10,000. These solutions are made in normal 
saline (0:95 per cent. sodium chloride). 

Histamine and histidine are, per se, non-hemolytic. If 0:4 ¢.c. of 2°5 per 
cent. glycocholate solution have 0:2 cc. of standard blood suspension added, 
and if, after 5 seconds, 0:4 ¢.c. of 1 in 1000 histamine be added, hemolysis 
occurs instantaneously. 

The histamine thus has the accelerating action found with the serum 
albumin, whose action was probably due to the histamine contained in it as 
an unavoidable impurity. This is a most interesting fact, for it enables 
many more exact observations to be made on the phenomena mentioned 
above in connection with serum albumin. Certain preliminary notes must 
first be made. 

The Effect of Histamine on Colloid Gold. 


In view of the fact that sodium glycocholate protects a gold sol, it is 
important to know the action of histamine on such a colloid. Histamine 


Action of Sodium Glycocholate. 97 


precipitates colloid gold, acting powerfully in this respect. Histidine acts in 
a similar way, but less powerfully. The precipitating action of a 1 in 1000 
solution of histamine is very marked, 0:1 c.c. precipitating | c.c. of the gold 
sol used in about 10 seconds. 

It is probable, therefore, that histamine acts as a disturber of all colloids, 
and therefore of sodium glycocholate. We have seen that the hemolytic 
action of this substance when in “combination ” with serum albumin or 
histamine is not to be accounted for by simple solubility of the envelope of 
the erythrocyte in the bile salt: it is possible that surface tension produces 
the effect, in which case the interaction of a ‘colloid like the glycocholate, 
and a precipitator of colloids such as histamine, would be of great interest. 
The consideration that the hemolysis may be due to changes in the physical 
state of the solution, connected with occurrences known to colloid chemistry, 
suggests that the acidity or alkalinity of the hemolysing solution will be of 
ereat importance: the phenomena perhaps being analogous to those of 
adsorbtion (6). 

It will therefore be necessary to investigate (1) the action of serum 
albumin, and of histamine, on a blood suspension subjected to the hemolytic 
action of various amounts of sodium glycocholate; and (2) the effect of 
acidity or alkalinity on this action. 

The following Tables contain such an investigation. The suspension used 
is an inactive one. The various concentrations of glycocholate are similar 
to those previously used (Table IV). The quantity of serum albumin added 
to each tube, in the columns relating to its action, is 0:4 cc. of a 1 per cent. 
solution in saline. The quantity of histamine added to each tube, in the 
columns relating to its action, is 0°4 c.c. of a 1 in 5000 solution in saline. 

“Acid histamine,” or “acid serum albumin,” is made by adding to 10 cc. 
of the histamine solution, 1 in 5000, or, to 10 c.c. of the 1 per cent. albumin 
solution, 0°1 ¢.c. of decinormal. HCl. “Alkaline histamine,” or alkaline serum 
albumin, is prepared by adding to 10 c.c. of either substance in the con- 
centrations mentioned above, 0:1 c.c. of 1 per cent. NazCQOs. 

The substances were mixed in the order referred to as method 1, i.2., the 
glycocholate first, then the blood suspension, of which 0-2 c.c. is used, and 
then, after 5 seconds, the serum albumin or histamine. 


98° Mr. E. Ponder. The Hamolytic 
Table XII. 
When 7 = 18. 


| | | 
| | Acid Alkaline Acid | Alkaline 


| e ao: FEDS UIA | albumin. albumin. histamine. | histamine. 

| | | 
100 65 mins. | 95 mins. 43 mins. | 105 mins. 55 mins. 110 mins. 

| 200 40 ,, i B40) 355 Ios Gian 40 secs. .| 73 ,, 

| 300 one | 90 secs. 30 secs. 58, 30. a oe 

| 400 nein, | go. 200i. fe 6 See Sane 
500 | 50 sees. | 310) 5. OD es IRS SOR 4 ,, 
600 | 1min. 35. RO, 4, Hom HO. <p 45 secs. 

1000 | 5 mins. | 3 mins. | 38% mins. | 23) ae 24 mins. 15 mins. 
| | 


This Table, which is representative of the general results obtained with an 
inactive suspension, expresses several important points :— 

(1) Histamine behaves similarly to the serum albumin, as an accelerator of 
the glycocholate heemolysis. 

(2) The rendering of the hemolysing solution acid causes hemolysis to be 
more rapid: if the hemolysing solution be alkaline, the hemolysis is 
retarded. The amount of deviation from neutrality is very small in the 
above case. 

(3) The relation between the speed of hemolysis under the various 
conditions and the amount of glycocholate used is expressed. Columns 1 
and 2 are confirmatory of the Tables illustrating the behaviour of an 
inactive suspension. 

The question of the reaction of the hemolysing fluid is obviously one of 
great Importance. A series of observations in which the Py is determined 
would be ideal: the difficulties attendant upon the use of buffer solutions in 
connection with these hemolytic experiments are at present, however, 
insuperable. The question is being investigated. 

It now remains to consider the effect of varying the quantities of 
histamine employed for accelerating the glycocholate in its hemolytic 
action. This is done in the following two Tables, the first Table illustrating 
the results when a sensitive suspension is used, the second illustrating the 
results in the case of an insensitive suspension. In each case, the tem- 
perature at which the experiments were conducted was 18° C.: the substances 
were mixed by method 1. 


Action of Sodium Glycocholate. 99 


Table XIII.—A. Sensitive Suspension. 


| Hugtaniine. | Glycocholate. 
| | | oy Paes 
lin | lin 100. | 1 in 200. | 1 in 800. | 1 in 400. | 1 in 500. | 1 in 600. | 1 in 1000. | 
| | | = 
| 500 5 secs. 5secs. | 8secs. | 8secs. | 10 secs. 25 secs. | 40 secs. 
eelhOCOF = Wt Mass | Ei i Gy Fe | LO? ce 35° ,, | 14 mins.: 
| 2,000 Sg AN MO gy We MD gy a BD ey a BS sp 30 ,, 23 
5,000 45 mins. _ 15 mins. | 30 ,, WeOl as | 28 ,, 20a tees ar 
| 8,000 DO | AUSae 68: AG: 5: AG ee Mh BO agp Bo 2 ining 
10,000 HOI; 24 ,, 24 mins. | 2 mins. 1 min. ata) 55 50 secs. 


Table XIV. —B. Insensitive Suspension. 


Histamine. | Glycocholate. 
E | s Be Agta borin reingh 
| lin | 1in 100. | 1 in 200 | lin 300. 1in 400. | 1 in 500 | 1 in 600 | 1 in 1000 
| 
| s wan Sal ariel a see le ef 
500 5 sees 5 secs. | 3 secs 8 secs 10 secs. 15 sees 40 sees 
1,000 Hitt pte | LONDS LS ba iy| MESiEs YS 5. 13 mins. | 
20005. 17-85, LOM WG pe 20s eee 30} 2s 
5,000 | 85 mins 40 mins 1 min 30. (, 25) oes Zan, Mee ad 
pie78,000 | 90: ,, 42 ,, 5 mins. | 45 ,, PP BO Bp RS os lmin. | 
| 10,000 (105 ,, 45 ,, (hats 2 mins. | 1 min. 30, 50 secs. | 
| 


From these somewhat complicated Tables very little new is to be learnt, 
except that the occurrences which take place when the action of glycocholate 
of sodium is accelerated by histamine are exceedingly complex. Several 
points may, however, be noted :-— 

(1) If these times be plotted on graph paper, a series of curves of definite 
character will be obtained. The character of the curves, however, does not 
suggest any generalisation. 

(2) The difference between the sensitive and the insensitive suspension may 
be seen in columns 1, 2and 3 of the respective Tables. At the other dilutions 
these differences diminish. This is confirmatory of the observations made 
with serum albumin. 

(3) From an inspection of columns 1 and 2, it appears that there is a very 
great disproportion between the effect produced by a 1 in 2000 histamine 
solution and a 1 in 5000 solution. This suggests that the occurrences met 
with when 1 in 500 to 1 in 2000 histamine is used are different in kind from 
those met with when more dilute histamine solutions are used, whereas the 
difference between the activity of the solutions to which the other columns 
relate is one of degree only. ‘his is very probable, in the light of other con- 
siderations, and will be commented on later. 


100 Mr. E. Ponder. The Hemolytic 


The Protective Action of Histamine. 


It has been shown that histamine possesses the accelerating action on the 
hemolysis produced by sodium glycocholate, being in this respect similar to 
the action of the serum albumin solution dealt with in the beginning of the 
paper. It remains to be shown whether or not it has the protective action of 
the serum albumin solution (Table IX). 

This is a simple matter, in view of the information conveyed in Table XIII. 
The following experiment illustrates this :— 

To 1 ce. of an active standard blood suspension, add 0:1 c.c. of 1 in 500 
histamine. Allow this tube to stand for 5 minutes. 

If 0:2 c.c. of this suspension be added to glycocholate and histamine in a 
tube, it will carry with it a small amount of additional histamine. For instance, 
if 0-4 ¢.c. of glycocholate have added 0:2 c.c. of this suspension, and also 
0:4 c.c. of 1 in 5000 histamine, the final dilution of histamine in the tube will 
be 1 in 8330, instead of 1 in 12,500, which it would have been if instead of 
this suspension containing histamine, a standard suspension had been used. 
Consulting Table XIII, it will be seen that if 1 in 500 glycocholate be 
employed for hemolysis, this shght increase in the histamine concentration is 
of little consequence, altering the hemolytic time only by 1 or 2 seconds. 
Therefore the effect of a dilution of 1 in 500 glycocholate on the standard 
suspension, before and after it has had histamine added in this quantity, will 
decide whether or not the histamine has caused a protection of the blood 
cells. 

To two tubes, then, add 0-4 c.c. of a 0°5 per cent. solution of sodium glyco- 
cholate ; add in the case of one tube, 0°2 c.c. of standard suspension, and in 
the case of the second, 0°2 c.c. of the suspension containing histamine, as 
prepared above ; after a 5 seconds’ interval, add 0°4 c.c. of 1 in 5000 histamine. 
The tube containing the standard suspension hemolyses in 30 seconds, that 
containing the suspension, plus histamine, in 34 minutes. This demonstrates 
that the histamine has a protecting effect on the cells against hemolysis by 
the glycocholate-histamine system. 

If the cells of this suspension plus histamine be washed in saline in the 
centrifuge, the suspension resulting from adding them to the appropriate 
amount of saline is still slow in being heemolysed by these quantities of elyco- 
cholate and histamine. This demonstrates that, as in the case of the 
protection conferred by serum albumin solution, the protection conferred by 
the histamine is not due merely to the presence of the latter in the saline 
surrounding the red cells, but due to some change produced in the cells 
themselves. 


Action of Sodium Glycocholate. 101 


The Action of Histidine. 


Histidine acts in a manner similar to histamine in accelerating hemolysis 
by sodium elycocholate, and, if used in another way, in protecting cells 
against hemolysis by histidine and glycocholate. In general, its action in 
these respects is less marked than that of histamine. 

These properties belonging to histamine and to histidine do not appear to 
be general properties of amino-acids. Glycine and arginin, for instance, do 
not possess them. A full study of this question is being made, and the point 
will not be further dealt with in this paper. . 


Discussion. 

It is a much easier matter to observe the phenomena described in this 
paper than to explain them. A brief discussion of certain points is 
. desirable. 

It appears obvious that the explanation of hemolysis by sodium glyco- 
cholate on the grounds that this salt dissolves the envelope of the corpuscle 
is inadequate, in view of the unusual behaviour of the salt in certain high 
concentrations, and especially in view of the action of non-hemolytic 
substances like histamine when in the presence of a blood suspension and 
sodium glycocholate. A more probable explanation is one which is based on 
changes of surface tension; possibly the solvent action of the salt plays a 
subsequent part. To advance a theory to explain these occurrences is at 
present impossible; the following suggestion, however, is supported by the 
majority of the facts, and may be taken as a working hypothesis, useful for 
the present until further facts are brought to light. 

If we consider first the addition of blood cells to a solution of sodium 
glycocholate, we may say that two occurrences take place: (1) the glyco- 
cholate becomes condensed at the interfaces, and therefore on the surface of 
the red cells; and (2) a solvent action of the glycocholate on the envelope of 
the cell begins. If histamine, which it has been seen, powerfully disturbs 
colloid solutions, be added, the colloidal glycocholate probably undergoes a 
sudden change of physical state, resulting in a sudden variation of the surface 
tension at the red-cell interfaces, where the glycocholate is collected. This 
sudden alteration ruptures the cell wall, all the more so as the glycocholate is 
already attacking the envelope, and therefore is, as it were, continuous with 
the substances composing it. The rapid hemolysis produced by the addition 
of histamine may be thus explained. If, on the other hand, the histamine be 
added to the cells first, it will not be so condensed at the interfaces as the 
colloid would be, and will certainly not dissolve the envelope. On the 

VOL. XCIII.—B, I 


102 Mr. E. Ponder. The Hemolytic 


addition of glycocholate, then, a change of physical state of the latter occurs, 
with a sudden change of surface tension, as before, but more evenly distri- 
buted throughout the fluid, instead of beimg more marked at the cell interfaces. 
Hemolysis will therefore be slower ; the possibility that the glycocholate and 
the histamine may form some species of adsorption compound which has 
scarcely any hemolytic action might further enter into the explanation. 

Such a consideration is further supported by the fact that the occurrences 
are so influenced by small changes in reaction; the process of adsorption and 
similar colloid phenomena being very sensitive in this respect. It may be 
also noted that the curious results obtained by varying the quantities of the 
interacting substances point to changes more complex than simple chemical 
interaction. It appears at times that the phenomena do not occur until 
certain quantities of the interacting substances are present, ¢.g.,in Tables XIII 
and XIV, columns 1 and 2. Here possibly the amount of histamine added 
was insufficient to disturb the glycocholate sufficiently to cause the change of 
surface tension necessary to produce hemolysis. 

Not the least interesting of these occurrences is the change which seems to 
occur in the blood cells themselves, both on standing and under the action of 
histamine. The latter occurrence seems to have no explanation which is even 
probable at present. The fact that blood cells, prepared in such a way that 
coagulation is permitted, are insensitive, may be due to some protective action 
exerted by some product of coagulation. 

The whole subject, which may at first sight seem of little practical conse- 
quence, is of great importance. The facts show that hemolysis by simple 
chemical substances depends on complex factors, and any information which 
can be gained regarding the true manner of action in these relatively simple 
cases is of interest in the consideration of the vastly more complex phenomena 
of hemolysis by heemolysins of animal origin. 


Summary. 


1. Sodium taurocholate and sodium glycocholate are to be considered as 
colloids. They protect colloid gold against precipitation of electrolytes. 

2. Sodium glycocholate is a feebly hemolytic agent. If histamine or 
histidine be added to it in suitable proportions, a highly hemolytic mixture 
results, although histamine and histidine are of themselves non-hemolytic. 
The reaction of the hemolysing fluid influences the speed of heemolysis. 

3. Histamine, if brought in contact with blood cells, renders them 
immune to hemolysis by the histamine glycocholate mixture. Histidine 
acts similarly. This appears to be due to some change in the cells 
themselves, and not merely to the presence of the histamine in the fluid. 


fa 


Action of Sodium Glycocholate. 103 


4, Blood cells which are rapidly heemolysed by glycocholate and histamine 
become insensitive on standing, but to a less degree with an old suspension 
than with a freshly prepared one. 

5. Serum albumin and peptone solutions, and also pituitrin, produce both 
the rapid hemolysis when mixed with sodium glycocholate, and also the 
protective effect when added to blood cells. This latter occurs in vivo as 
well as in vitro. These occurrences may be due to the presence of histamine 
or allied substances. 

6. Several facts suggest that these phenomena are mainly due to dis- 
turbance of surface tension, similar to those which are met with in colloidal 
solutions. They cannot be explained by the theory that the bile salt 
dissolves the corpuscle envelope. 

7. Suspensions of cells which are derived from blood drawn into a fluid 
which prevents coagulation behave differently from suspensions of cells 
which are derived from blood drawn into a fluid which permits coagulation 
to occur. 

8. A protection against hemolysis by the histamine-glycocholate mixture 
is also conferred by blood serum. 

9. The presence of blood serum inhibits the hemolytic action of sodium 
taurocholate and sodium glycocholate. 


REFERENCES. 


Ponder, H., “A Method for investigating the Hemolytic Activity of Chemical 
Substances,” ‘ Roy. Soc. Proc.,’ B, vol. 92 (1921). 

Schafer, ‘ Text-book of Microscopic Anatomy,’ p. 370 (1912). 

Von Noorden, ‘Metabolism and Practical Medicine,’ vol. 2, p. 260 (1907). 

Muller, P., ‘Serodiagnostic Methods’ (Transl. Whitman), p. 20 (1913). 

Abel and Kubota, ‘ Jour. Pharm. and Exp. Ther.,’ vol. 13, p. 248 (1919). 

Bayliss, W. M., ‘Nature of Enzyme Action,’ pp. 22 et seg. (1914). 


a 


CCR LS SSS) 


104 


The Mechansm of Ciliary Movement. 


By J. Gray, M.A., Fellow of King’s College, Cambridge, and Balfour Student, 
Cambridge University. 


(Communicated hy Prof. J. 8S. Gardiner, F.R.S. Received June 1, 1921.) 


The mechanism of ciliary movement has been extensively studied from the 
morphological point of view, and although there is a general consensus of 
opinion as to the structure of the “ciliary apparatus,” there is no adequate 
account of the functions of the various parts of the mechanism. 

The material used for this work has been the gills of Mytilus edulis, and 
has already been described (Orton, 27). It is entirely due to the movement 
of the cilia that an efficient stream of water is kept passing on to the face of 
the gill, and that the food is moved up to the mouth of the animal. By 
means of carmine particles the existence of these currents is easily detected 
by the naked eye. 

The production of a constant current of water in a definite direction implies 
that the cilia are capable of performing work in a remarkably efficient manner. 
If we watch the movement of a single cilium, it is obvious that the beat is 
divisible into two phases: (a) a very rapid forward or effective stroke ; and 
(6) a slower backward or recovery stroke. It is during the rapid effective 
stroke that the cilium performs work on the surrounding medium, and in 
doing so, of course, expends energy. At the conclusion of the effective 
stroke these cilia possess no energy which can be used for work, but by the 
time the recovery stroke is completed a new supply of potential energy is 
available and isin turn converted into kinetic energy during the next effective 
stroke. 

We are, no doubt, entitled to assume that the energy expended by a cilium 
has its origin in some chemical compound, either in the cilium itself or in 
the cell to which it is attached. Our main problem is to throw what light 
we can on the sequence of events which leads to the conversion of chemical 
energy into the kinetic energy of movement. 

The first evidence which will be presented is that gained by an observation 
of the living cells under normal conditions. 


I. Tue STRUCTURE AND BEHAVIOUR OF NORMAL CILIA. 


On the gill filaments are three main groups of ciliated cells—the lateral, 
the latero-frontal, and the frontal cilia (see Gray, 12). These cilia, like all 
other living cilia, appear to be optically homogeneous; they are strongly 


The Mechanism of Cihary Movement. — 105 


refractive and possess a considerable degree of elasticity. In this respect 
Engelmann (8) expressed the view that: “Tous les organes vibratils sont 
résistants, trés flexibles, et dans une large mesure parfaitement élastiques.” 
The cilia of Mytilus are entirely independent of any control by the animal 
and are in constant motion during the life of the cells. 


Lateral Cilia. 


On the sides of each gill filament are three rows of rectangular cells, each 
bearing a brush of cilia. These are the lateral cilia (see figs..1 and 2). The 


: 
Lee 
seb 


ayyoy cee aT Te 
Hee CUT A ni sa awe: 


Wty ie bay ere 
wn ult ‘3 wt Wee A i Ww NY AS 
aye ORNS CKURAUETTLOROOR 


i a 


Fic. 1.—Lateral view of gill filament of Mytilus (moditied from Orton). (a) Terminal 
cilia, (6) frontal cilia, (c) latero-frontal cilia, (d) lateral cilia, (e) vertical water 
current set up by lateral cilia, (f) direction of water current from frontal cilia. 


effective stroke causes a strong current of water to flow on to the gill surface 
at right angles to it (see fig. 2). All the cilia arising from a single cell beat in 
the same phase, as do also the cilia of the three cells comprising each vertical 
row. 

The most distinctive feature of the lateral cilia is their marked rhythm. 
The cilia on adjacent cells beat in succession, so that a continuous wave 
passes along the whole line of lateral cells from one end of the filament to 
the other; the wave passes in opposite directions on the two sides of the 
filament. This metachronial rhythm provides an interesting example of 
co-ordinated movement which is not associated with any visible nervous 
elements. Isolated individual cells from the lateral epithelium continue to 
exhibit active movement. 

Latero-frontal Cilra. 

It will be seen from fig. 2 that on reaching the surface of the gill the water 
columns set in motion by the lateral cilia meet the large latero-frontal cilia. 
During one phase of the beat these cilia rapidly pass from the form of 
straight rods to that of curved hooks, the point being directed towards the 
free surface of the filament. The cilia then flatten out more slowly; the 


106 : Mr. J. Gray. 


flattening begins at the base and proceeds to the point. These cilia also exhibit 
a certain degree of metachronism, but to a much less marked extent than 


Fic. 2.—Transverse vertical section of three gill filaments, showing the deflection of the 
vertical current on to the frontal cilia. (a) Frontal cilia, (6) latero-frontal cilia, 
(c) lateral cilia. 


the lateral cilia. The function of the latero-frontal cilia appears to be two- 
fold: (4) they act as vanes which deflect the water currents on to the surface 
of the filaments; (2) they keep the individual filaments apart, so as to give 
free play to the lateral cilia. 


Frontal Cilia. 


The whole of the flat frontal surface of the gill is covered by the frontal 
cilia, whose effective beat is parallel to this surface and directed towards its 
free edge (see fig. 1).* 

When the movement is very greatly reduced in speed by the addition of 
gum arabic to sea-water, it is seen that, during the effective beat, the cilium 
behaves as a more or less rigid rod, which moves forward on a pivot at its 
base. During the recovery stroke, however, the cilium assumes entirely 
different properties—it is drawn back as a imp non-elastic body in which a 
stress is set up which starts at its base and is transmitted to the free end, 

* At the end of the filaments the frontal cilia are modified so as to deflect the current 


towards the food grove and towards the oral end of the gill. ‘These modified frontal 
cilia are very obvious, and will be referred to as the terminal cilia. 


The Mechanism of Ciliary Movement. 107 


exactly as is the case in a fishing line during the backward movement of a 
cast. As the cilium moves back it loses its limpness, and at the end of the 
recovery stroke possesses a considerable degree of rigidity (fig. 3). 


When movement is taking place fairly quickly the cilium does not appear 


SN 
max 


b =? 
Fie. 3.—Diagram illustrating the form of the terminal cilia of Mytilus during (a) the 
effective and (6) recovery beats. 


to straighten out completely at the end of the recovery stroke, but moves 
forward in the form of a sickle. During the final phase of the effective beat 
the hooked shape is always lost and re-develops during the recovery stroke. 

It should be mentioned that the effective stroke is always quicker than the 
recovery stroke. When movement is very rapid it is impossible to see the 
cilium during the effective heat. 

The change in the consistency of the cilium during the two phases of its 
beat appears to be an observation of considerable significance, but does not 
appear to have been commented on by other observers. The illustrations 
given in Verworn’s (31) text-book of the cilia of Urostyla grandis appear to 
indicate the same phenomenon. The only detailed description of the move- 
ment of large cilia is that by Williams (32) of the cilia of a molluscan larva, 
which clearly indicates a difference in the elastic properties of the cilia 
during the two phases of the beat (fig. 4). 

The effect of stimulation on a muscle fibre has been compared by 
Bayliss (1) to the conversion of a stretched lead spring to a stretched steel 
spring, so that the excited fibre is capable of expending energy in the form 
of work. The cilia of Mytilus, and to a still greater extent the cilia of 
Ctenophores, can be compared, with ‘equal justice, to bent strips of lead and 
steel wire. Jt seems fairly certain that the energy which is expended by the 
ciliwm 1s stored as tension energy. 

Let us now consider the point of origin of the stimulus to which the 


108 Mr. J. Gray. : 


movement of the cilium is the mechanical response. The cilia on adjacent 
cells of the lateral epithelium beat in a definite sequence. If, however, 


Fic. 4.—Diagram showing successive stages in the stroke of a cilium on the velum of a 
gastropod larva (after Williams). A, position of rest; B, position at end of 
recovery beat ; Cand D, stages during effective beat ; E, end of effective beat. 


individual cells are separated experimentally, they continue to exhibit active 
and prolonged movement (Gray, 12). All attempts to detect the operation of 
nervous elements in the epithelium, or in the cells themselves, have failed. 
It may be concluded, therefore, that these ciliated cells provide an example 
of an automatically contractile tissue. The cells are comparable to cardiac 
muscle cells; each cell is capable of independent movement, although, under 
normal circumstances, there is a definite co-ordination between adjacent cells. 

When a piece of living Mytilus gill is teased in sea-water under the 
microscope, portions of the cuticular layer with attached cilia are often 
stripped away from the cells themselves. Such cilia are invariably motion- 
less. It seems certain, then, that an essential part of the mechanism hes in 
the cell itself ; as long as there is a small portion of normal protoplasm at 


~\ 


B A 
Fic. 5.—Diagram of cilia of Pleurobrachia. A is the position of rest ; B is the end of the 
effective stroke, whose direction is shown by the arrows. 


the distal end of the cell near the base of the cilium, the latter continues 
to move.* 


* R.S. Lillie observed that detached but active cilia from the larva of Polygordius 
possessed a knob-like expansion at their proximal ends. 


The Mechanism of Ciliary Movement. 109 


It is interesting to mention a few experiments which were performed on 
the ciliated plates of the Ctenophore Plewrobrachia (see fig. 5). 

Like the lateral cilia of Mytilus there is here definite metachronial 
movement, but the movement of each cell is dependent upon a stimulus 
passing to it from the cell next to it. If the ciliated comb is cut, then all 
those cilia situated on the oral side of the cut cease to beat, and come to rest 
at the beginning of the effective beat. Any cell of this oral portion can be 
thrown into motion by stimulating the cell immediately above it. As far as 
one can see, these cilia resemble skeletal muscle rather than cardiac muscle, 
since some form of external stimulus is necessary to produce a mechanical 
response from the individual cells. 


IJ. THe INFLUENCE OF THE ENVIRONMENT ON CILIARY ACTIVITY. 


It has already been shown (Gray, 12) that a satisfactory medium for 
ciliary activity is provided by a Van’t Hoff’s solution containing NaCl, KCl, 
MgCl, and Cals, in the same proportions as in sea-water, and whose 
hydrogen ion concentration is about Py 78. We can therefore regard the 
other constituents of normal sea-water as unessential. 


(a) The Effect of Varying the Hydrogen-1on Concentration. 


The fact that the cilia on the gill of Mytilus cease to move when the 
hydrogen ion concentration of the surrounding medium reaches a limiting 
value, and that the cilia will resume their movements when the acidity of 
the medium is reduced (Gray, 12), led to the following investigation of 
Bernstein’s well known hypothesis. . 

If the hypothesis be sound, then the inhibiting powers of an acid medium 
should be inversely proportional to the rate at which the acid can enter the 
cell. It has been shown that mineral acids enter living cells very slowly, if 
at all, whereas the fatty acids enter readily. Pieces of living gill, stained 
with neutral red, undergo no change in colour when placed in Van’t Hoft 
solution, to which sufficient hydrochloric acid has been added to produce a 
hydrogen ion concentration of Py 3:4: when placed in a similar solution, 
made acid with acetic or butyric acid, the gill instantly changes from a dull 
brick ved colour to one of brilliant red—indicating that the acid has 
entered the cells. Corresponding facts apply to the alkalies: ammonia 
rapidly enters a living cell, whereas sodium hydroxide does not. 

It is obvious, therefore, that a method is available for deciding whether the 
cessation of movement of cilia in an acid medium is due to an interference 
with the electrical properties of the cell surface, or whether the affected 
elements lie within the cell itself. 


110 My. J. Gray. 


The procedure adopted was to determine the critical concentration of 
hydrogen ions in the external medium, which was just sufficient to cause 
complete stoppage of ciliary movement in 1 minute. A very large number 
of experiments were performed, whose results are summarised in the 
following Table :— 


- | Critical Px | : Critical Px 
aoe | concentration. on concentration. | 
Hiydrochloniciymey.aesesesnees: 3 4 Oxalic! cet esiectane. aeceee een 31 | 
| SHUGH OIG 5. sedn0cavcoannone: | 3:1 IROTMIC Ase seoseaneren ee eoEER tees 4°0 | 
IENGiGisl CoE aes, tee RCS eg he 3:4 A Getic) (.22..dd roan 4°8 
Citricts ahs inten cece Coan 3°4 [ile Buatyricisssceecseeuedecrn cee 52 


It will be observed that the mineral acids are of practically uniform 
efficiency, and the same hydrogen ion concentration of each is required to: 
produce the same physiological effect. The fatty acids, on the other hand, 
-form a series which is more efficient than the mineral acids, and the higher 
member of the series is distinctly more efficient than a lower member. 

It has already been shown (Gray, 12) that, when ciliary activity has. 
ceased in the presence of an acid, recovery takes place when the surrounding 
medium is made alkaline, so that it is possible to compare the effects of the- 
weak and the strong alkalies as restoratives. 

When movement has been stopped by means of a fatty acid, the cells. 
rapidly recover in a solution which is not more alkaline than normal 
sea-water (Py 78): by means of gill fragments stained with neutral red, 
this recovery can be seen to be due to the rapid rate at which the acid is. 
removed from the cell. If, however, the cilia have been stopped by a 
mineral acid, recovery in normal sea-water is relatively slow (Gray, 12), so 
that such fragments form satisfactory material for testing the restorative 
powers of the various alkalies. In the following experiments, fragments of 
the same gill were exposed to a definite strength of mineral acid (HCl, 
Py 3°3) for a definite period (5 minutes). Individual fragments were 
then transferred to normal sea-water, and to sea-water whose Py had 
been raised to the same abnormally high value by NaOH and by NH,OH 
respectively. 


Sea-water. Sea-water. Sea-water. 
+ NaOH. + NH,OH. 
lhe 7 sis Py 8-4. Pu 8-4. 
| 
Movement began ............... 12! la i 
TRU REGO VCAT codon scahoossd6e0030 25! 19! 3! 


The Mechanism of Ciliary Movement. al 


The comparative efficiency of ammonia and sodium hydroxide is also 
seen from the following experiment. Fragments, after previous acid treat- 
ment, were trausferred to sea-water whose alkalinity had been raised to a 
known value by NaOH and by NH,OH. 


Time in minutes for full recovery in 

Pu. =r 

| NH,OH. NaOH. 
9°5 3 5 
9°2 $ 7 
9-0 z 8 
8°7 1 8-10 
8°5 3 10-12 
8 °4 5 12-16 


The above figures all apply to fragments of the same gill after precisely 
the same acid treatment. The experiment was repeated several times with 
identical results. 

It is therefore clear that the weak acids which enter the cell are more 
efficient inhibitors of ciliary movement than are the strong acids which do not 
enter readily, and conversely the weak alkalies are inuch more efficient restoratives 
than the strong alkalies. 

It is impossible to accept the suggestion that the normal activity of the 
ciliated cells is upset by acids owing to a disturbance of the cell surface. 
The physiological effects of both acids and alkalies depend upon the ease 
with which these reagents penetrate to the cell interior. It may be noted 
that another series of experiments showed that the presence of neutral 
electrolytes in external medium has but little effect upon the efficiency 
of either acids or alkalies. 

It is important to note that the cilia come to rest in an acid solution by a 
gradual diminution in the rate of the whole beat, without any reduction in its 
amplitude. Both the effective and the recovery strokes become slower, and 
there is often a marked pause at the beginning and end of each stroke, 
so that a complete beat may take as long as 10 seconds. It is difficult 
to imagine how this could occur where there is actual derangement of the 
contractile elements—since we would expect such to be attended by a 
gradual reduction in the amplitude of the beat. 

Again, the cilia invariably come to rest at the end of the effective stroke ; 
that is, in that position in which the cilium itself possesses no available 
potential energy. 

Interesting evidence is available from a study of spermatozoa which are 


112 Mr. J. Gray. 


known to possess a limited amount of reserve chemical energy. The effect 
of acid on such cells is precisely similar to that on ciliated cells (Gray, 13). 

Cohn (5) has shown that when the movement of such spermatozoa has 
ceased in an acid medium, there is no loss of energy,* the conversion of 
chemical into potential energy has ceased, and can be switched on again 
by removing the acid from the external environment. J¢ seems highly 
probable, therefore, that the movement of cilia 1s stopped in an acid mediwin 
because there 1s no longer a conversion of chemical into potential energy. 


(b) Metallic Ions. 


We have already seen that the cilia of Mytilus beat normally in an 
artificial solution containing NaCl, KCl, CaCl, and MeCls, and whose 
hydrogen ion concentration is the same as sea-water. On the whole, ciliary 
activity is remarkably independent on the absolute concentration of any 
particular ion or upon the exact ratio between different ions. In an 
investigation of the effects of individual ions it is necessary to maintain the 
normal hydrogen ion concentration and also the concentration of other ions 
in the solution. Further, in making a comparison between the effects of an 
ion on ciliary and on muscular activity, comparison must be made to a 
spontaneously contractile muscle (e.g., the auricle of the heart). These facts 
explain the difference in the conclusions arrived at in this paper, and those 
of Lillie (20), Hober (16) and Mayer (23). 

Experiments with Potassium Chloride—Ilt KCl be omitted from the external 
medium and its place taken by NaCl, the /ateral cilia come to rest. The 
time taken for the movement to cease varies considerably in different gills. 
In most cases movement slows down after 5-10 minutes, and in less than 
20 minutes the cilia are stationary. In a few cases in which the lateral 
cilia showed very active movement before the experiment, movement was 
continued in the absence of potassium for as long as 45-60 minutes; move- 
ment recommences vigorously on the subsequent addition of potassium, or 
on the addition of a small amount of alkali sufficient to raise the Py to 
about 8°5. It should be mentioned that in several cases the lateral cilia 
ceased to beat in normal sea-water after about 13 hours, but on addition 
of a slight amount of KCl, vigorous movement took place. This may 
possibly be due to the fact that the blood of the animal contains a higher 
concentration of potassium than does normal sea-water, so that when isolated 


* KE. G. Martin (22) found that, in the presence of alkali, spontaneously beating strips 
of the ventricle of the tortoise gave out a constant total amount of energy for a unit 
mass of tissue. 


Bo. 


The Mechamsm of Ciliary Movement. $5) DUS 


in sea-water the gill is in an environment which may have a sub-normal 
concentration of potassium. 

In contrast to the lateral cilia, the fronto-lateral, the frontal and the 
terminal cilia beat normally for very long periods (more than 48 hours) in a 
solution containing no potassium. 

This contrast is paralleled by the action of such a solution when perfused 
through the heart of different molluses. In the case of Pecten Mines (25) 
showed that potassium could be omitted from the perfusion fluid without any 
derangement of the heart beat: on the other hand, the heart of the Octopus 
gradually stops in such a solution (Kleefeld, 17),and can be revived on adding 
potassium. 

If potassium is present in excess a similar contrast is found in its 
effects on the different cilia. Until the concentration of potassium is raised 
to about ten times the normal value, little or no effect is noticeable upon the 
cilia, although there is a tendency for a rapid secretion of mucus of the 
surface of the gill, which may clog the frontal and terminal cilia. Above this 
concentration the fronto-lateral cilia are affected in a curious way—they pass 
into a state of contraction which persists for a considerable period. At first 
the tips of the cilia remain bent at the end of the recovery phase of the beat, 
then a wave passes along a whole series of the cilia which accentuates this 
bend toa marked degree; this is followed by another wave in which one 
cilium after another remains fixed in a completely contracted position (v¢., at 
the end of the effective beat). In this position the cilia often exhibit a 
curious quivering movement. 

There is thus a regular “staircase” effect—similar to that found in the case 
of the heart. 

Ii the concentration of potassium has not been too strong, the cilia 
recover on transference to normal sea-water after about 15 minutes. LTEven if 
contraction of the fronto-lateral cilia is brought about by a solution in which 
the whole of the NaCl in the Van’t Hoff solution has been replaced by KCI, 
it is noticeable that after about 45 minutes, the cilia begin to recover in the 
original solution, the amplitude of the beat getting gradually larger until the 
complete beat is resumed. The rate of the recovery in such a solution, or in 
normal sea-water, is greatly hastened by the presence of alkali. : 

In all solutions containing excess of KCl, the beat and rhythm of the 
lateral cilia is well maintained and is often more rapid than in the normal 
gill; the frontal and terminal cilia are either unaffected or beat more rapidly 
than normal. Here again the differential action of potassium on different 
tissues is clearly illustrated. 

Lillie and Héber have both emphasized the maintenance of ciliary movement 


114 Mr. J. Gray. 


in the presence of excess of potassium, and have contrasted this with 
the depressant effect of such an excess on skeletal muscle. In view of the 
effect of KCl on the fronto-lateral cilia in preventing the recovery heat, it is 
interesting to note that this is also its effect upon skeletal muscle, viz., the 
latter is thrown into a state of prolonged contraction (Mines); the same 
thing occurs in the vertebrate heart (Burridge, 3). 

The recent work of Kolm and Pick (18) on the effect of potassium on the 
heart brings out clearly three points: (i) the marked quickening effect on the 
automatically contractile auricles and sinus; (11) the differential action on 
different tissues in the same organ, viz., auricles, ventricle ; (iii) the prolonged 
contraction which is caused by high concentration of potassium: the con- 
traction eventually passing off in the presence of the same perfusion fluid. 

As far as I am aware there is no evidence against the view that in the case 
of automatically contractile tissues the effect of low concentrations of 
potassium salts is to increase the rate of movement, while stronger concen- 
trations cause a prolonged contraction which is not, however, permanent. 

The relative immunity of cilia as compared to a muscle cell to potassium is 
probably due to the fact that the latent period of the cilium is very much 
less than that of the average muscle cell, so that an environment which 
throws the latter into tonic contraction by increasing the rate of activity of 
the cell, has much less effect on the cilia. 

The Effects of the Sodiwn Ion—We have already mentioned that the 
absolute concentration of Na can be raised considerably without deranging 
ciliary movement. If the concentration of CaClz, MgClz, and KCl be kept 
constant, and the sodium chloride replaced wholly by isotonic saccharose, 
ciliary movement is well maintained for several. hours. 

It seems reasonable to conclude therefore that the sodium ion plays no 
specific 7éle in activity—although it probably enters into the conditions of 
the general equilibrium within the cell. 

The Effects of the Magnesium Ion—If magnesium be omitted from the 
Van’t Hoff solution, and its place taken by an appropriate amount of calcium, 
ciliary action is well maintained for many hours (more than 48 hours). 
Within wide limits a variation in the concentration of magnesium in the 
medium has little effect upon the form or rate of beat of the terminal 
cilia. 

This fact is in accordance with observations upon the heart of the Octopus 
(Fredericq), the arms of Lepas (Mayer), and the heart of Salpa (Mayer)— 
all of which are insensitive to an absence of magnesium. The sensitivity 
of the heart of Pecten (Mines) is doubtless correlated with its high sensitivity 


to hydrogen ions (Mines). 


The Mechanism of Ciliary Movement. 115 


In a subsequent paper, however, evidence will be presented which indicates 
that magnesium plays an important ré/e in the economy of the cell. 

The Effects of the Caleiwm Ion.—lf calcium be omitted from the external 
medium, and the other conditions be the same as in normal Van’t Hoff 
solution, prolonged ciliary movement takes place. The effect of the absence 
of calcium is, however, seen in the increased sensitivity of the cell to 
hydrogen ions. This is seen in the following experiment :— 


| 
| Duration of movement in 
Pu, | 
Van’t Hoff solution, | Van’t Hoff solution, 
containing calcium. without calcium. 
7°8 More than 48 hours , More than 48 hours. 
7:0 | More than 48 hours 15-45 mins. 


If, after the cessation of movement in the absence of calcium, the alkalinity 
of the solution be raised, rapid recovery takes place. If, on the other hand, 
calcium is added, the amount of recovery, at least fora time, depends upon 
the time which has elapsed between the cessation of movement, and the 
addition of the calcium. If the time is short, rapid and complete recovery 
takes place within 1 minute. If the time be prolonged, the recovery, on 
addition of calcium, is slow: the amplitude of the beat is regained almost 
at once, but the rate of both the effective and recovery strokes is slow, 
and there are often marked pauses at the beginning and end of each stroke. 
The whole phenomenon recalls the effect of acid, and one might conclude 
that the effect of the absence of calcium is possibly due to a change in the 
cell produced by a change in the hydrogen ion concentration. 

The cessation of movement in the absence of calcium, and the recovery 
of movement on the addition of calcium or hydroxyl ions, is paralleled by 
the reaction of such solutions on the heart. 


(c) The Effect of varying the Osmotic Pressure of the Surrounding Medium. 


Although the cilia are not sensitive to slight changes in osmotic pressure, 
yet, if this exceeds a certain value, the cilia are rapidly brought to a com- 
plete standstill. On reducing the osmotic pressure, instant and complete 
recovery takes place. These facts are extremely easy to demonstrate, and 
can be repeated a great many times with the same piece of gill. 

It does not matter whether the increase in osmotic pressure is brought 
about by the addition to sea-water of non-electrolytes or by balanced 
electrolytes (¢g., 24 M. Van’t Hoff solution). In a solution which is not 


116 Mr. J. Gray. 


quite strong enough to cause complete stoppage, it is noticeable that a 
reduced movement is maintained for a very long time (more than 24 hours); 
in such a solution the amplitude of the beat is much reduced, whereas the 
rate of beat is almost unaffected. In a solution which is strong enough 
to cause complete stoppage, the cilia remain in the position shown in the 
accompanying diagram (fig. 6), so that they are unusually conspicuous. 


Fie. 6.—Diagram illustrating the appearance of the terminal cilia of Mytilus when 
brought to rest by high osmotic pressure of the external medium. The arrow shows 
the direction of the normal effective beat. 


It is important to notice that the stoppage of the cilia in hypertonic 
solutions is brought about in an entirely different way to the stoppage in an 
acid solution, and it is therefore not surprising to find that the stoppage in 
hypertonic solutions is not influenced by the presence of hydroxyl] ions, nor 
is the effect of an acid solution altered by reducing its osmotic pressure. 

The effect of hypertonic solutions on muscular activity has not been 
extensively studied, but Demoor and Phillipson(7) have shown that the 
skeletal muscles of a frog lose their excitability to a direct stimulus when 
immersed in hypertonic Ringer solution; the muscles shorten somewhat, 
and the response is gradually abolished. These effects are entirely reversed 
by treatment with normal Ringer solution. Carlson (4) also found that the 
rate of beat of the auricles of the tortoise is unaffected by perfusion with 
hypertonic Ringer solution; the amplitude was, however, much reduced— 
and recovered in normal Ringer. 

The fact that the amplitude of the beat is affected by an inerease wm the 
osmotic pressure of the external mediwm seems to indicate that a loss of water 
JSrom the cell interferes not with the periodic liberation of energy, but with the 
actual contractile mechanisin. 


IV. SUMMARY AND DISCUSSION. 


Let us now review the whole of the available facts, and attempt to form a 
working hypothesis of the nature of the ciliary mechanism. We have seen 
that the ciliated cells of Mytilus provide an example of an automatically 
contractile tissue ; they differ from cardiac or smooth muscle in that their 
latent period is extremely short, and the rate of beat very much quicker than 


vr 
yaa 


The Mechanism of Ciliary Movement. ney, 


corresponding muscle cell. The cilium is essentially an elastic fibre in 
communication with and dependent upon the protoplasm of the cell.* The 
cilium is capable of storing potential energy (supplied to it from the cell) in 
the form of tension, and of liberating this energy in the form of work, The 
amount of tension developed depends on the existence of free water in the 
cell. The rate at which the energy is stored and liberated by the cilium 
depends upon the hydrogen ion concentration of the cell interior. Whereas 
the rate of movement of the cilium depends almost completely upon the 
concentration of hydrogen ions inside the cell, it is largely independent of 
the presence of specific metallic ions (except in certain cases potassium) in 
the external medium. 

A suggestion as to the way in which cheiniecal activity within the cell may 
lead to the development of a tension in a fibrous structure (in the presence 
of water) is provided by the experiments of Fischer and Strietmann (9). 
These authors have shown that, if a piece of catgut, suspended in water, 
comes into contact with an acid, the fibre absorbs water and develops a 
considerable tension. The same phenomenon occurs in the presence of an 
alkali. In this experiment the essential conditions are: (1) the liberation of 
a chemical (acid or alkali) on the surface of a fibre ; (11) the presence of water. 
These facts form the basis of an hypothesis of muscular actions, but they 
can be applied with equal force to the ciliary mechanism. In the case of the 
musele fibre, the production of an acid (lactic acid) during stimulation has 
been demonstrated. If the same fact be assumed to be true in the case of a 
cilium, then many of the facts stated in this paper receive a reasonable 
explanation. The rate at which lactic acid is produced from its carbohydrate 
precursor depends upon the hydrogen ion concentration of the medium 
(Kondo, 19). Hence, the rate at which chemical energy can be converted 
into potential energy will also depend upon the hydrogen ion concentration 
of the cell interior. It is therefore clear why the rate of the recovery stroke 
of the cilia of Mytilus is affected by acids which enter the cell. By our 
hypothesis, at the end of the recovery stroke the cilium possesses potential 
energy, owing to the stress set up in its elastic structure by the tension of 
those fibres at whose surface an acid is situated. This potential energy can 
only be liberated by the relaxation of the fibres; that is, by the removal of 
the acid. The rate at which the acid is removed will depend upon the 
degree of alkalinity of the surrounding cell contents. In other words, an 
explanation is available for the effect of acids and alkalies on the rate of the 
effective stroke of the cilium. 


* The truth of this statement can be seen by reference to most text-books of histology, 
or to the work of Saguchi (29). 


VOL. XCIII.—B. K 


118 Mr. J. Gray. 


The alteration in the length or tension of a fibre exposed to acid depends 
upon the concentration of salts present (Fischer), or on the ease with which 
water can be drawn from the surrounding fluid. Hence, when ciliated cells 
are exposed to a solution whose osmotic pressure is capable of withdrawing 
a considerable amount of water from the cells, the amount of tension, set up 
by a normal amount of acid at the surface of the fibres, will be reduced ; 
consequently, in solutions of high osmotic pressure, the amplitude of the 
beat is affected, and the cilia stop when the amount of free water is zero. 

The above conception of the ciliary mechanism has two advantages: (i) ib 
does not endow the cilium with any hypothetical structure ; (ii) it brings the 
mechanism into line with what is known of other contractile tissues. There 
is, however, one corollary to the hypothesis which applies equally to cilia and 
to muscle cells. If the liberation of an acid at the surface of the ciliary 
fibrils enables the cilium to store potential energy and perform the recovery 
stroke—then, when ciliary activity ceases in the presence of an experimental 
acid, we must assume that the latter acid does not come into contact with 
the contractile fibrils, since the cilia come to rest with the fibrils relaxed. 
When, however, stronger concentrations of acid are used, the cilia stop 
partly (in some cases almost completely) contracted, and occupy a position 
near the end of the normal recovery stroke (see fig. 7). We may well 


Fie. 7.—Diagram illustrating the effect of acid on the terminal cilia of Mytilus. 
(a) With acid just strong enough to stop cilia ; note all the cilia stop at the end of 
the effective beat. (b) Acid of considerably greater strength ; note the cilia come 
to rest between the two phases of the recovery stroke. (c) and (d) Positions 
at beginning and end of the normal effective stroke. 


suppose that in this case the experimental acid has reached the contractile 
fibres. Precisely the same phenomena are found in the heart: weak 


The Mechanism of Ciary Movement. Ekg) 


concentrations of acid stop the heart in diastole, stronger concentrations 
stop it in systole. 

Finally, if the cilia of Plewrobrachia are considered, it will be obvious how 
very closely the known facts agree with the hypothesis of muscular action 
outlined by Hill and Hartree (15). The position of rest of these cilia is 
at the end of the recovery stroke, so that (like a striated muscle fibre) they 
possess a definite amount of potential energy, which can he released at the 
moment of stimulation. The cilium may be regarded as a series of fibres, B,* 
which are in communication with a network, A; the walls of the latter 
are kept stretched by the presence of water in the interstices of the 
network. It is this turgidity which provides the cell with the potential 
energy possessed at the position of rest. At the moment of stimulation, 
some chemical substance is set free at the surface of the fibres, B, which 
promptly take up water from the network, A, so that the cilium flies 
forward, owing to: (i) the liberation of the energy stored in the walls of A; 
and (11) the tension developed in B. At the end of the effective stroke, the 
chemical substance is removed from the fibres, B, and the water flows away 
from the fibres (¢g., by osmosis) into the interstices of the network, A, 
thereby stretching the cilium back to the resting position. 

It should be understood that the above analysis of the ciliary mechanism 
is nothing more than a working hypothesis. At the same time the remark- 
able similarity between the conditions necessary for ciliary and muscular 
activity, coupled with the apparent similarity in the fibrous structure of the 
two types of cell, leads to the conclusion that the two mechanisms may be 
essentially similar. It is also clear that the same scheme might be applied 
to pseudopodial movement. 


Summary of Hxperimental Results. 


1. The cilium is capable of expending potential energy in the form of work 
as long as it is in organic connection with the cell protoplasm. 

2. Kach ciliated cell of Mytilus is capable of independent movement when 
isolated. The cilia of the Ctenophore Plewrobrachia require a definite stimulus 
to induce their beat. Both types of cilia show metachronial rhythm. 

3. The cilium is an elastic fibre or bundle of fibres. In the large majority 
of cases the cilia are in communication with the cell protoplasm by means of 
intracellular fibrille. 

4. The ciliary beat consists of a rapid effective stroke and aslower recovery 
stroke. The form of the recovery stroke often differs markedly from the 
effective stroke. 


* The fibrous nature of these cilia is well seen in preserved specimens. 


KF 


120 Mr. J. Gray. 


During the former the cilium resembles a slack string or fibre, whereas 
during the effective stroke its rigidity is distinctly greater. 

5. When exposed to an acid solution of appropriate strength the cilia of 
Mytilus come to rest by a gradual slowing of the whole beat: the amplitude 
of the beat is not affected. The cilia always come to rest at the end of the 
effective stroke, 7.c.,in the position in which the cilium possesses no convertible 
potential energy. 

The cessation of movement in an acid solution is due to a change which 
takes place inside the cell, and not at its surface. Evidence is advanced 
which suggests that the presence of acid prevents the conversion of chemical 
energy into kinetic energy. 

The effect of acid is entirely reversible by alkalies. The rate of the beat is 
most simply controlled by controlling the hydrogen ion concentration within 
the cell; up to a certain point the higher the internal alkalinity the more 
rapid is the ciliary beat. 

6. Under normal circumstances the activity of the lateral cilia depends on 
the presence of potassium ions. This effect is probably due to the general 
effect of the ion in quickening the beat, which leads in the case of the fronto- 
lateral cilia to a state of prolonged contraction when potassium is in excess. 

7. Ciliary activity is not sensitive to change in the concentration of 
magnesium or sodium in the external medium, although these ions play a part 
in the general equilibrium between the cell and its environment. 

8. The absence of calcium ions may bring about a cessation of ciliary 
movement, which is antagonised by hydroxy] ions. 

9. The reaction of cilia and of muscles to the various chemical constituents 
of their environment is essentially the same. The apparent differences are 
due to: (i) the greater sensitivity of most muscles as compared to cilia ; (ii) the 
cilia have a much shorter latent period than most muscles; (iii) a ciliated cell 
cannot be regarded as directly comparable to a neuro-muscular system. 

10. Cilia are brought to rest if the osmotic pressure of the external 
medium exceeds a certain value. The stoppage is brought about by a gradual 
reduction in the amplitude of the beat. These effects are entirely removed on 
reducing the osmotic pressure. 

11. An hypothesis is put forward that the mechanism of ciliary movement 
is essentially the same as that of muscular movement. 


The expenses of this research were in part met by a grant from the 
Government Grant Committee of the Royal Society. 


The Mechanism of Ciliary Movement. ail 


BIBLIOGRAPHY. 

1. W. M. Bayliss, ‘Text-book of General Physiology,’ p. 439, London (1918). 
2. J. Bernstein, ‘ Electrobiologie,’ Brunswick, 1912. 

3. W. Burridge, ‘Quart. Journ. Exp. Physiology,’ vol. 5, p. 347 (1912). 

4, A. J. Carlson, ‘ Amer. Journ. of Physiol.,’ vol. 15, p. 357 (1912). 

5. E. J. Cohn, ‘ Biological Bulletin,’ vol.-34, p. 167 (1918). 

6. D. Dale, ‘ Journ. Physiol.’ vol. 46, p. 129 (1918). 

7. J. Demoor and M. Phillipson, ‘ Bull. Acad. Méd. Belg.,’ p. 655 (1908-9). 

8. T. Engelmann, “ Cils Vibratils,” ‘ Richet’s Dictionnaire de Physiologie,’ vol. 3, p. 785. 
9. M. Fischer and Strietmann, ‘ Kolloid Zeitschrift, vol. 10, p. 65 (1912). 
10. H. Fredericq, ‘ Bull. de la Cl. des Sci., Acad. Roy. Belgique,’ 1913. 
11. F. W. Frohlich, ‘ Zeit. f. Allgem. Physiol.,’ vol. 5, p. 288 (1905). 
J. Gray, ‘Quart. Journ. Micros. Sci., vol. 64, p. 345 (1920). 
J. Gray, ‘Roy. Soc. Proc., B, vol. 91, p. 147 (1920). 
14. J. Gray, ‘Proc. Camb. Philos. Soc.,’ vol. 20, p. 352 (1921). 

A. V. Hill and W. Hartree, ‘ Phil. Trans.,’ 5, vol. 210, p. 153 (1920). 

16. R. Hober, ‘Biochem. Zeits.,’ vol. 17, p. 518 (1909). 
17. G. Kleefeld, ‘ Bull. de la Cl. des Sci., Acad. Roy. de Belgique,’ p. 91 (1913). 
18. R. Kohn and H, P. Pick, ‘ Pfliigers Archiv,’ vol. 185, p. 235 (1920). 

19. K. Kondo, ‘ Biochem. Zeit.,’ vol. 45, p. 68 (1912). 


20. R. 8. Lillie, ‘ Amer. Journ. Physiol.,’ vol. 7, p. 25 (1902). 

21. R.S. Lillie, ‘Amer. Journ. Physiol.,’ vol. 17, p. 89 (1906). 

22. E. G. Martin, ‘ Amer. Journ. Physiol.’ vol. 15, p. 303 (1905). 

23. A. G. Mayer, ‘ Publ. No. 132,’ Carnegie Instit., Washington, 1910, 
24. A. G. Mayer, ‘ Publ. No. 47, Carnegie Instit., Washington, 1906. 
25. G. R. Mines, ‘Journ. Physiol., vol. 43, p. 467 (1912). 

26. G. R. Mines, ‘Journ. Physiol., vol. 46, pp. 1, 188, 349 (1913). 

27. J. H. Orton, ‘Journ. Mar. Biol. Assoc.,’ vol. 9, p. 444 (1912). 

28. S. Ringer and D. W. Buxton, ‘Journ. Physiol. vol. 8, p. 288 (1887). 
29, S. Saguchi, ‘Journ. Morphol.,’ vol. 29, p. 217 (1917). 

30. T. Sakai, ‘ Zeit. f. Biologie,’ vol. 64, p. 1 (1914). 


31. M. Verworn, ‘ Allgemeine Physiologie,’ p. 302 (1915). 
32. L. W. Williams, ‘ American Naturalist, vol. 41, p. 545 (1907). 


122 


The Mechanism of Ciliary Movement. U.—The Effect of Lons 
on the Cell Membrane. 


By J. Gray, M.A., Fellow of King’s College, Cambridge, and Balfour 
Student in the University of Cambridge. 


(Communicated by Prof. J. 8S. Gardiner, F.R.S. Received October 7, 1921.) 


In 1906 B.S. Lillie (7) published an account of the effects of various 
pure sodium salts upon the ciliated epithelium of Mytilus edulis. He found 
that the ions could be arranged in the following order of “ toxicity,” 


Cl <@NO;< By ol =< sCNe 


Lillie also referred to the effect which various anions have upon the 
amount of water taken up by the cells from their external medium, and 
arranged the ions in the following order of efficiency in causing an absorption 
of water by the cells. 


CH3COO’ < Cl’ < NO;’ < C103’ < Br’ < I’ < SCN’ < BrO;’ < OH’. 


These results form the basis of the following statements found in recent 
text-books, viz., Héber (6) and Bechhold (1) :— 


(i) That the above series represents the effect of anions on ciliary 
movement. 

(ii) That the order in which anions affect ciliary movement is the reverse 
of that in which they atfect muscular movement. 


One of the objects of the present communication is to consider whether 
these important statements are justified. 

It has already been shown (Gray (3)), that if the ciliated epithelium of 
Mytilus edulis is placed in a solution containing NaCl, KCl, CaCl, and 
MeCle, the whole tissue remains normal and in activity for a very prolonged 
period. If we wish, therefore, to determine what specific rdle, if any, is 
being played by the chlorine ion, it is necessary to replace this ion in the 
above solution by other anions. It is not permissible to use a solution 
which does not contain either K’, Ca’’, or Mg’’, nor is it permissible to 
ignore the hydrogen ion concentrations of any experimental solution 
(Gray (3)). 

In the experiments tabulated in Table I, each solution contained the same 
molecular concentration of cations, and the hydrogen ion concentration was 
kept well within the limits to which the tissue is indifferent. 


The Mechanism of Ciliary Movement. 128 


Table I.—The Effect of Mixtures of Na’, K’, Ca’’, and Me™’ upon the Move- 
ment of the Terminal Cilia of Mytilus, 


| Time. | 

Anion. Pu. 
| 30 mins. | 1 hour. 2 hours. 3 hours. 5 hours; exp; 
| discontinued. | 
eee ne We 
Chlorides ...... Normal move- Normal moyement ; no absorption ane See| 
ment; no of water. | | 
absorption of | | 
water. | | 
é | | 
Nitrates ......... Normal movement; no absorption of water He aeek | 
} 
I@eliGkS, Gaoeneaue Very rapid movement ; Normal movement ; no 8-0 | 
no absorption of water. absorption of water. | 

Bromides ...... Normal movement ; no absorption of water 7°8 
Acetates......... Normal movement ; no absorption of water ee | 
Sulphates ...... Very rapid movement ; Normal movement ; no absorption 8:0 | 
no absorption of water. of water. | 
Tartrates ...... Normal Slow Very slow No movement eS | 

movement. movement. movement. 
No absorption of water. 
Citrates ......... No movement ; no absorption of water 78 


Such experiments shew quite clearly that in a solution containing 
balanced cations, the substitution of the normal anion chlorine by NOs’, I’, 
Br’, Ac’, or SO” is not attended by any interference with either the 
activity of the cilia or the amount of water taken up by the cells. It is 
possible that in the case of the iodide and sulphate mixtures there is 
an actual increase in the rate of beat of the cilia, although the observed 
effects may possibly be due to the slightly higher hydroxyl ion concentration _ 
of these solutions. 

In considering the possible significance of the cessation of movement in 
balanced solutions of tartrates and citrates, we are confronted with a purely 
chemical problem. Calcium tartrate and calcium citrate are botl very 
sparingly soluble in water; the same statement applies, though to a less 
degree, to the magnesium salts. When, however, these salts are added to a 
solution of the corresponding sodium salt, they dissolve to a very marked 
extent. The suspicion arises that in making up a physiological mixture 
of tartrates or citrates, the metals magnesium and calcium are not present 
as free ions. This suspicion seems justified by the following facts. When 
sodium phosphate and ammonia are added to a saturated solution of 


124 Mr. J. Gray. 


magnesium citrate in water, a definite precipitate of magnesium phosphate 
is formed. If, however, sodium phosphate and ammonia are added to a 
solution of magnesium citrate in sodium citrate, no precipitate is formed, 
although a considerable amount of magnesium is in solution. We may 
conclude, therefore, that the inability of tartrate or citrate mixtures to 
maintain the normal equilibrium of ciliated cells may prove to be due to 
the absence of free magnesium and calcium ions and not to the direct effect 
of the anions. Confirmation of this view will be given later in this paper. 

As far as I am aware, the only satisfactory investigation of the effects of 
anions on other living processes is that of Sakai(9) on the heart of the 
frog. This author used solutions containing balanced cations, but does not 
refer to their hydrogen ion concentration. His results may be summarised 
as follows :— 


I’, Br’, NO’ Beat well maintained, even faster than normal. 
CUE Renee Normal. 

SOR Grete Beat well maintained, after initial slowing. 
Cie ects Rate steadily falls and finally heart stops. 


There is clearly no fundamental difference between the effect of anions 
on ciliary and on muscular activity. Both types of tissue are remarkably 
indifferent to wide variations in the nature of the anions in the external 
environment. 

(b) Lhe Lffects of Solutions of Pure Sodium Salts. 

Whereas in a solution of a sodium salt containing K’, Ca’, and Mg’’, the 
ciliated epithelium is remarkably indifferent to a variation in the nature 
of the anions present, yet in a solution of a sodium salt, which does not 
contain other cations, the tissue shows a very marked sensitivity to par- 
ticular anions. Table II shows the general course of events. 

It will be seen that the salts can be divided into two main groups: 


A. Those salts which cause the cells to swell up by the absorption of 
water: Ol <<" INO; <<e Bir —<aeuilige 
B. Those salts which’ do not cause the cells to swell. SO.’’, Tartrate, 
Citrate ; 
while the acetate forms an intermediary type. 
It is also clear that these solutions cannot indicate the direct effect of the 
" environment on the contractile mechanism; their effect on the cell is of a 
more general nature, and the contractile mechanism is only secondarily 
involved.* 


* Cilia often remain active when the process of absorbing water is relatively far 
advanced. 


The Mechanism of Ciliary Movement. 125 


Table 11.—The Effect of Pure Sodium Salt Solutions upon Ciliated 


Epithelia. 
1 
Time. 
Anion. Pr greens cotta hese oe, Pu. | 
30 mins. 1 hour. 2 hours. 3 hours. 5 hours. 
| 
Corder ccn..:--- Some cilia | Cells consider- | Tissue disorganised. 78 
active. A little} ably swollen. | Cells much swollen. | 
swelling. | Some cilia Cilia destroyed. 
destroyed. 
UNifrate™ <25..- 23.6: A few cilia Cells much Tissue disorganised. 7°8 
' active. Most swollen. Few Celis much swollen. 
cells begin to | cilia left. Cilia destroyed. 
swell. ; 
IBFOWMNGE = 2s. 5. <.< 0c A few cilia Cells much Tissue disorganised. 78 | 
active. Most | swolien. Few) Celis much swollen. 
cells begin to cilia left. Cilia destroyed. 
swell. 
Wodidea ete. 235-7 | Nearly all Tissue disorganised. 8-0 
| cells markedly Cells much swollen. 
swollen. Cilia destroyed. | 
Neetate! oo ac+.s.c No movement. No movement. Cells slightly swollen ae 
Cells normal. 
Sulphate ......... No movement: cells do not swell. Cilia remain healthy 80 | 
in appearance. | 
Tartrate ......... No movement: cells do not swell. Cilia remain healthy 7°8 
in appearance. 
Citrate <22.....-00 No movement: cells do not swell. Cilia remain healthy 7°8 


| 
in appearance. 


The effects of the various sodium salts upon the water-content of the cells is 
precisely what one would expect from the effects of the same salts on the water- 
content of such non-living colloid gels as fibrin or gelatine. Such gels swell 
readily in the presence of hydroxyl ions, but the uptake of water is affected 
by different salts in precisely the same way as we have found for ciliated 
cells. 

Now, during life the interior of the cell is always more acid than the 
external medium; although at the same time it must be remembered that 
the cell colloids are on the alkaline side of their isoelectric point. Hence, 
if the cell interior is allowed to come into contact with the external medium, 
the amount of water taken up at any particular hydrogen ion concentration 
will depend upon the nature of the anions present in the medium. This 
is seen above actually to be the case, when the cells are put into pure 
solutions of sodium salts. It is not the case when the cells are in a solution 


126 Mr. J. Gray. 


containing balanced cations. In the latter type of solution the hydroxyl 
1ons outside the cell cannot penetrate the cell, nor can the anions outside 
the cell exert any influence upon the uptake of water by the cell. Such a 
condition of affairs is obviously due to the semi-permeable nature of the 
normal cell membrane. The inference to be drawn is, of course, that when 
placed in a solution of a pure sodium salt the observed effects are the direct 
outcome of the loss of semi-permeability by the cell membrane. Direct 
proof of this statement is provided by a determination of the electrical 
conductivity of normal tissues placed in pure sodium salts. Osterhout (8) 
showed that the electrical resistance of Laminaria tissue placed in sodium 
chloride solution fell steadily, until it was one-third of its original value; 
Gray (4) showed that sodium citrate caused a marked fall in the resistance 
of Echinoderm eggs, and quite recently Shearer (11) has shown the similar 
effects of sodium chloride on bacteria. 


(c) The Nature of the Cell Membrane. 

Lillie(7) showed that the toxie effect of pure sodium salts is prevented 
by the presence of the alkaline earth metals. This has been confirmed and 
the statement can be somewhat enlarged. In the first place, either 
magnesium or calcium can prevent the destructive action of a pure sodium 
chloride solution upon the cell-membrane ; or, as is perhaps the more correct 
mode of expression, the presence of magnesium or calcium is necessary 
for the semi-permeable properties of the cell membrane. Since both calcium 
and magnesium occur in sea-water, it is of importance to determine, if 
possible, which of these two metals maintains the normal stability of the 
cell surface. 

In the following experiment the concentration of each salt was the 
concentration in which it normally occurs in sea-water.* 


INGO eonaceoumnaurbess Pir gO ex oeatna Complete disintegration after 3 hours. 

NaCl+CaCl, ...... Brera eee No disintegration after 24 hours. Some cilia 
destroyed, and some filaments separated from | 
| their neighbours. | 
) INEM WROls co ats5 dete 70) Gacccaans Tissue quite healthy, but cilia motionless after | 
| 


24 hours. | 


It is clear that the amount of magnesium in normal sea-water is alone 
capable of maintaining the cell-surface: at the same time the amount of 
calcium is also sufficient to produce a very well-marked stabilising influence. 
In the solution which contains only caleium and sodium in their normal 


* The composition of the sea-water used was :—NaCl, 28°3 grs. per litre ; KCl, 0°76 grs. 
per litre; MgCl,, 5°01 grs. per litre ; CaCl,, 1°22 grs. per litre. 


The Mechamsm of Ciliary Movement. We 


concentrations, the tissue does not remain so healthy as in that containing 
magnesium and sodium; in the former solution there is a distinct tendency 
for the finer cilia to become opaque and detached from the cell: this is 
particularly the case with the small cilia on the ciliated junctions, so that 
individual gill filaments show a distinct tendency to separate from each 
other (see Gray (3)). Interesting facts concerning the action of calcium are, 
however, available from a study of cells whose normal semi-permeability 
has been destroyed by immersion in those sodium salts which prevent the 
uptake of an abnormal amount of water by the cell. When ciliary move- 
ment has ceased in sodium citrate, the cilia and the tissue appear quite 
healthy and translucent. On transference to normal sea-water, however, 
the cilia at once become opaque, and are completely destroyed; at the same 
time there is a rapid uptake of water by the cells which swell up in the 
usual way. If, on the other hand, the tissue (after initial citrate treatment) 
be placed in sea-water containing no calcium, the cells and cilia remain 
quite healthy, and complete recovery of movement takes place, although 
the rate of beat is usually slower than the normal; after a time the rate 
of beat falls off, but can be revived permanently by adding calcium. The 
same recovery from sodium citrate treatment can be effected by treating the 
tissue with any solution containing magnesium but no calcium. The same 
experiments can be performed with tissues previously treated with sodium 
sulphate or sodium tartrate. It is clear from numerous experiments that 
magnesium is the only ion in sea-water which will re-form a semi-permeable 
membrane round a cell which has lost this structure by exposure to a pure 
sodium salt. 

At this point it is interesting to note that calcium has a double action 
on ciliated cells: (a) It is capable of maintaining the cell surface in a normal 
State of semi-permeability ; (b) it is necessary for continuous movement in 
a solution of Py 7-0 (see Gray (5)). In the first of these functions calcium 
can be completely replaced by magnesium, but cannot be so replaced in 
the second. 

The destructive effect of sodium salts upon the cell membrane is shared 
by other monovalent cations, although to a variable extent. In the case of 
the chlorides, there is little difference between Na’, NH,’, and K’, while 
the effect of Li’ is considerably less. In the case of other salts, cg., tartrates 
and citrates, the erosive power of the potassium salts appears to be distinctly 
less than that of the corresponding salts of sodium. Pure solutions of 
magnesium and calcium salts have no erosive action for a considerable time 
(six to eight hours), after which the cell membranes begin to be affected. 

It is curious to notice that magnesium has little or no stabilising action 


128 Mr. J. Gray. 


against the erosive power of potassium, whereas calcium has a well-marked 
action. It is conceivable that this fact may ultimately be correlated with 
the observation that calcium and potassium are mutually inhibitory in the 
case of certain tissues; also, if the cell surface be normally maintained by 
magnesium, then it is possible that potassium may be able to penetrate 
under normal conditions whereas sodium does not. 

Since we can control the destructive effects of the monovalent salts by 
means of magnesium and calcium, it is interesting to note that the direct 
action of the different monovalent ions upon the ciliary mechanism forms a 
well-marked series. 

In the following experiment the monovalent salts were present in the 
molecular concentration of the sodium in normal sea-water; the divalent, 
salts were in the same concentration as they normally occur in sea-water :-— 


{ 
1 


| | 
| Movement of terminal cilia. | 
| Solution. | Py. 7 an] aie Sp. fe 7 =o iy MENA cus 
| De Sie SO 20’. 30’. 60’. 
| 7 | | 

HiCl, MgCl; CaCl... | 0) 0 0 (0) 0) 0 0 
| eNicCl) MeCly, CaGla =... -.2... | 7-2 | se ak | shar sb ae ++ ++ af Fe 
| INTEL COIL, Wiel, CAVCTy So cen one rac) coon ee) amare crenasy) are | ade 
| KCl, MgCl., CaCl, ......... fie? Tactadalijecheh chee a 
| Sea-waterl (Cl) ieee ens 7°2 | ++ ++ +4 ape 


; ++ ++ 
| | 
| 


0 = no movement. 
+ + = normal rate of beat. 
+ + = quicker than normal. 
+++ = very rapid beat. 
Apart, therefore, from their action on the cell-wall, the monovalent ions 
exert a definite effect on the rate of beat of the cilia. The order in which 
the various ions increase the rate of beat is well marked, and is as follows :— 


hie Na <q Nike 


The stoppage of the cilia in the lithium mixture is of the same nature as 
that observed in acids (Gray (5)), and it is not surprising to find that by 
raising the alkalinity of the lithium mixture, a rapid and well-maintained 
beat takes place. 

The fact that the presence of potassium induces a more rapid rate of 
beat than a corresponding amount of sodium enables us to test whether the 
normal semipermeability of the cell membrane is essential to ciliary activity 
or not. The experiment can be performed as follows :—Guill fragments are 
placed in an isotonic solution of sodium citrate until all movement has 
ceased. Individual pieces are now placed in the following solutions and 
their behaviour noted. 


The Mechanism of Ciliary Movement. 129 
aD : . ina i= al 
WI DEG Se octet area! etry facto he eRe No recovery. Cilia remain healthy, but cells 
absorb water rapidly. 
SGAAMGUGP ercneecrer cere are (ooh) jeep mapeee Cilia at once become opaque. Cells rapidly 
absorb water. 
AU? IKON ne egeneeierermenel Sot eal fiat sje eee Rane Marked recovery of beat: recovery is temporary, 


and cells rapidly swell. 


If, whilst the period of recovery in M/2 KCl is well marked, the gills 
are transferred to sea-water or to any solution containing calcium, the cilia 
instantly become opaque, and the cells swell rapidly. It is clear, therefore, 
that although such cilia may beat rapidly in the presence of potassium 
chloride, yet the semipermeability of the cell-wall has been entirely 
destroyed by the previous treatment with sodium citrate. The only metal 
capable of re-stabilising the cell-wall is magnesium. 

A further study of this problem obviously leads to a consideration of the 
nature of the cell-membrane itself, and although our knowledge is very far 
from complete, interesting analogies may be pointed out in the case of non- 
living systems. One of the most characteristic features of cell-membranes 
is their capacity fox allowing weak alkalies and acids to pass into the cell 
and yet exclude the strong alkalies and acids. These facts have, of course, 
led to the suggestion that the cell-membrane is essentially lipoid in nature— 
or that, at least, it contains a lipoid phase. Now Clowes(2) has shown 
that the nature of an oil and water system depends upon the nature of the 
ions present in the system. The truth of this statement may be very 
simply verified as follows :—Into five test-tubes are placed 10 c.c. of olive 
oil containing a little oleic acid, and an equal volume of test solution, 
together with 0°5 cc. N/10 NaOH. The tubes are then thoroughly shaken 
by hand, and examined after about five minutes. 

Clowes maintains that a system of water-drops in a continuous phase 
of oil may be converted into a system of oil-drops in a watery phase by 
means of sodium chloride. It must be pointed out, however, that grave 
difficulties attend the suggestion that the surface of the cell possesses a | 
continuous oily phase; nevertheless, these experiments indicate the possible 
mode of operation of bivalent metals on the cell-surface, if the latter in 
any way resembles an oily emuision. 

In some ways an even closer analogy to the experiments described in 
this paper is provided by the experiments of Schryver(10). This author 
has shown that when a 2 per cent. sodium cholate solution is heated in the 
presence of calcium a gel is formed. This gel is eroded by solutions of the 
salts of monovalent cations, but is completely stable when in contact with 
a solution of balanced monovalent and divalent cations. The erosive power 


130 Mr. J. Gray. 


Table LIL. 


Vest tube. Test solution. Nature of resulting system. 
r 
A 10 ¢.c. distilled water ...... | A uniform milk-white emulsion of oil drops 
in water. 


Discontinuous phase—Orn. 
Continuous phase—W ATER. 


B pe OES NI PAINEN Cleon cn ccse Two phases separate out in equal volumes— 
| (i) Water almost free of oil. 
| (ti) Oil almost free of water. 


© | 10 ec. M/10 MgCl, .......... A uniform greasy emulsion of water drops 
| im oil: 

Discontinuous phase—W ATER. 

| Continuous phase —Orn. 


D 10 e.c. M/10 CaCl, sie | Same as C. 


FE | 10 c.c. sea-water ............. Two phases separate— 

(i) A large volume of water drops in a con- 
tinuous oil phase. 

(ii) A small volume of oil drops in water. 


of the monovalent ions depends upon several factors: (a) their concentra- 
tion, (0) the salts existing in the gel. In concentrations equivalent to those 
used in the present series of experiments (viz., about M/2), the erosive 
power of LiCl is much less than that of Naor K. A study of Schryver’s 
results shows that the analogy between them and the experiments here 
described is strong. It remains, however, to be proved that such a system 
as a cholate gel possesses the same semipermeable properties as a living 


membrane. 
Summary. 


(i) The ciliated cells of Mytilus edulis are insensitive to the following 

anions : 

Cl NOz, Bry lea vAceh wo One 
as long as the normal equilibrium of the cations Na’, K’, Ca”’ and Mg” is 
maintained in the surrounding medium. 

(ii) In solutions containing tartrates and citrates, the bivalent metals 
Mg” and Ca” are probably not present in the ionic state, and the cells 
behave as though these metals were absent. 

(iii) There is no justification for the statement that the order in which 
anions affect ciliary motion is the reverse of the order in which they affect 
muscular movement. 

(iv) Pure solutions of sodium salts destroy the normal semipermeable 
nature of the cell-membrane, and the cell colloids behave as an elastic gel 
in direct contact with the external medium. 


The Mechanism of Cirary Movement. 131 


(v) The action of sodium salts can be inhibited by magnesium or by 
calcium. Probably in normal sea-water the stability of the cell-membrane 
is due to magnesium and not to calcium. 

(vi) In balanced solutions the monovalent cations have a direct effect 
upon the rate of ciliary movement. The rate of movement in solutions 
of the same hydrogen ion concentration is slowest in lithium and fastest 
in potassium. The ions can be arranged in the following well-marked 


series :-— 
Ibi Nay NIG) Ke 


(vii) The normal semipermeability of the cell-wall is not an essential 
condition for ciliary movement. 

(viii) The way in which solutions of the different metals affect the cell- 
surface is in complete agreement with the effect which they produce on the 
electrical conductivity of the cell. 

(ix) There is a marked analogy between the action of salts on the living 
cell-membrane and on a cholate gel, or oil and water emulsion. 


The expenses of this research were in part met by a grant from the 
Government Grant Committee of the Royal Society. 


BIBLIOGRAPHY. 
1. H. Bechhold, ‘ Die Kolloid in Biologie und Medizin,’ p. 273 (1912). 
2. G. H. Clowes, ‘ Journal of Phys. Chemistry,’ vol. 20, p. 407 (1916). 
3. J. Gray, ‘Quart. Journal Micro. Science,’ vol. 64, p. 345 (1920). 
4, J. Gray, ‘Phil, Trans. Roy. Soc.,’ B, vol. 207, p. 481 (1916). 
5. J. Gray, ‘ Proc. Camb. Phil. Soc.,’ vol. 20, p. 352 (1921). 
6. R,. Hober, ‘ Physik. Chem. der Zelle und Gewebe,’ pp. 508, 532 (1914). 
7. B.S. mies ‘Amer. Journ. of Phys.,’ vol. 17, p. 89 (1906). 
8. W. J. Osterhout, ‘Science, vol. 39, p. 544 (1914). 
9. T. Sakai, ‘ Zeit. fiir Biologie,’ vol. 64, p. 1 (1914). 
10, J. B. Schryver and M. Hewlett, ‘ Roy. Soc. Proc.,’ B, vol. 89, p. 361 (1916). 
11. C. Shearer, ‘Journ, of Hygiene,’ vol. 18, p. 337 (1919). 


132 


On the Hypertrophy of the Interstitial Cells in the Testicle of the 
Guinea-Pig under different Experimental Conditions. 


By ALExanper Lipscuiirz, M.D., Professor of Physiology (in collaboration 
with BENNo Ortow, M.D., CHARLES WaGNER, Sc.D., and FELIX BoRMANN). 


(Communicated by F. H. A. Marshall, F.R.S. Received October 11, 1921.) 
(From the Physiological Institute of the University of Dorpat, Esthonia.) 


[PLatEs 1 AND 2.] 


1 


Quantitative problems are, no doubt, of the greatest theoretical and 
practical interest in the study of the internal secretion of the sexual glands. 
We are explaining different physiological and clinical situations by changes 
in the quantity of internal secretion of the sexual glands or of other glands 
of internal secretion connected with the former. It suffices to mention 
normal puberty, menstruation and gravidity, eunuchoidism and pubertas 
precox. (Quantitative problems were already discussed when the first steps 
were made in the study of the question of the site of the internally secretory 
function of the sexual glands(1). Bouin and Ancel(2) tried to cause by 
different experimental means a compensatory hypertrophy of the interstitial 
cells of the testicle; they extirpated the testicle in rabbits on one side and 
ligatured the vas deferens on the other side; they found a proliferation of 
the interstitial cells, whereas the number of the cells of Sertoli remained 
unchanged. Further, they extirpated the normal testicle of pigs with 
unilaterally retained testicle (3). They found in these. experiments that 
the weight of the retained testicle was about twice as much as when the 
normal testicle was present. They found also in these cases a marked hyper- 
trophy of the interstitial cells. Sand (4) confirmed these statemeuts in his 
experiments on rabbits and guinea-pigs using his method of experimental 
eryptorchism. From all these experiments one could conclude that the 
hypertrophy of the interstitial cells takes place as a compensatory hyper- 
trophy of the elements acting as an organ of internal secretion. 

But there are some objections one can make against this conception. 
Ribbert (5) has shown that after unilateral castration the remaining testicle 
is greater than normally, and that there is a marked hypertrophy of the 
seminiferous part of the testicle. From experiments performed in our 
laboratory, we can state (6) that the hypertrophy of the remaining testicle is 
so marked that it weighs twice or thrice as much as a normal testicle of an 


On the Interstitial Cells in Testicle of Guinea-Pig. 133 


animal of the same age. Sand showed that the remaining testicle can 
undergo the same hypertrophy even when the vas deferens is ligatured ; this 
is due to the fact that, even after the vas deferens is ligatured, the 
spermatogenesis can proceed in a normal way, and the degeneration of the 
tubules begins only when the spermatogenesis is more or less completed. So 
one may object that, when the testicular mass is diminished, there is 
hypertrophy not only of the interstitial cells but also of the seminiferous 
part. This is why I said, two years ago(7), that the situation seemed to be 
more complicated than Bouin and Ancel supposed, basing their conclusions 
on ingenious experiments, performed about twenty years previously, when 
nobody could foresee the extraordinary development which the study of the 
internal secretion of the sexual glands has undergone in recent years. 

We again took up the question of the hypertrophy of the interstitial 
cells in the testicle in connection with another problem of internal secretion 
of the sexual glands. We studied the question as to how the development 
of the sexual characters of mammals depends upon the quantity of secretion 
of the sexual glands present in the body. For this purpose we used a 
method consisting of making unilateral castration and of cutting away more 
or less from the second testicle (“ partial castration ”)(8). We made a great 
many of these same experiments on guinea-pigs, and we were able to state— 
in accordance with Pézard (8A)—that even very small particles, representing 
1 or even less than 1 per cent. of the normal testicular weight, are sufficient 
for a normal masculinisation of an animal(9). In some cases we observed 
that the development of the sexual characters was slower than normally ; 
but we have experimental evidence that this phenomenon of retardation 
was caused, not by a simple quantitative deficiency in internal secretion, 
but by a slower development of the sectioned testicle (10). 

In all these experiments we had theoretically to confront a very important 
complication, «4.¢., the possibility of a compensatory hypertrophy of the 
elements, to which we ascribe the function of internal secretion in the 
testicle. And indeed in some cases where the particles were especially 
small, we found an extraordinary development of the interstitial tissue. 
The size and the number of the interstitial cells may sometimes be enormous, 
In such a small segment of the upper pole of the testicle, nourished by the 
arteria spermatica interna, the number of interstitial cells may no doubt 
attain, or even greatly surpass, the number of interstitial cells in two 
normal testicles together, although such a particle represents, as mentioned, 
not more but even less than 1 per cent. of the weight of two normal testicles. 

I never saw such an enormous hypertrophy of interstitial cells as in these 
' small particles in upper partial castration; only one such case of enormous 
VOL, XCIII.—B. L 


134 Dr. A. Lipschiitz and others. 


hypertrophy of interstitial cells is to be found in the literature, in a paper 
recently published by Poll(11), who described the degenerating testicles 
of hybrids of birds. Anyone looking at a microscopical preparation of 
some of our cases, and comparing it with a normal testicle (Plate 1, 
fig. 1), would agree that the hypertrophy of the interstitial cells is here 
excessive—as in some tumours described in the pathology of the testicle 
in man. 

Tn view of this hypertrophy one might object that even in our smallest 
particles there may have been a production of the sex specific secretion 
no smaller than the normal. It is true we have no definite evidence that 
the interstitial cells are the organ of internal secretion in the sexual glands; 
but there are so many facts showing that the interstitial cells have some- 
thing to do with the internal secretion of these glands, that it is impossible 
to avoid this objection when we discuss the quantitative problems in the 
internal secretion of the sexual glands on the basis of experiments with 
partial castration. Whether or not the interstitial cells are really the 
organ of internal secretion in the testicle, the partial castration was, at any 
rate in some cases, counteracted by an hypertrophy of this organ. 

We have some experimental evidence that this hypertrophy of interstitial 
cells is not a compensatory one, 7.e., that this hypertrophy is caused, not by 
an exaggerated function of these cells for the body as a whole, but by local 
conditions. 

aie 

The experimental evidence we have that the hypertrophy of the interstitial 
tissue is not a compensatory one, is of four different orders. 

A. Going through all the cases where small particles of testicular substance 
were sufficient for a masculinisation, in different degrees, of guinea-pigs, 
we saw all transitions between a normal number of interstitial cells and a 
highly augmented number of the latter. But there seemed to be no constant 
relation between the number of interstitial cells and the degree of develop- 
ment of the sexual characters. We will give in another paper a full 
description of all our experiments with partial castration, considering them 
from the point of view of the problem of the site of the function of internal 
secretion in the testicle. Here only the following facts are of importance 
for use 

(1) That there seems to be no constant relation between the number of 
interstitial cells and the degree of masculinisation, although there does not 
exist a case where masculinisation took place without fully developed 
interstitial cells being present in the testicular fragment. 

(2) That a normal masculinisation is possible even when the number of 


On the Interstitial Cells in Testicle of Guinea-Pig. 135 


interstitial cells in a small testicular fragment is not very much augmented, 
so that the number of the interstitial cells is highly diminished in com- 
parison with those in a normal testicle. 

B. In the experiments with partial castration on guinea-pigs, mentioned 
above, we used in reality two different methods. In some of these experi- 
ments we left in the body, as "previously said, a small segment of the upper 
pole of one testicle. In other experiments of this series we left a segment 
of the under pole of the testicle above the cauda epididymidis. In the 
latter we never saw the enormous hypertrophy of interstitial cells observed 
in some cases of “upper” partial castration, although in “under” partial 
castration a marked increase in the number of interstitial cells occurs. But 
the “under” testicular fragment degenerates, in general, so far as to become 
sclerotic, whereas the upper fragment can resist longer against sclero- 
sation (114). We explain this dissimilitude by a difference in the blood 
supply in the two methods. In the “ under” partial castration the testicular 
fragment is supplied with blood by the arteria deferentialis, the artery of 
the vas deferens, which gives off branches from the under part of the 
testicle. These branches, as is known in human anatomy, have an anasto- 
mosis with the branches of the arteria spermatica interna supplying the upper 
half of the testicle. The art. def. is a small one in comparison with the 
art. sp. 1., and we supposed that in our experiments the blood supply of 
an upper fragment was better than the blood supply of an under 
fragment. We found the plexus pampiniformis unchanged, so that it is 
very probable that a small upper testicular fragment received the same 
quantity of blood from the art. sperm. int. as the whole testicle. We think 
it right to conclude from these observations that the good blood supply 
explains, in a sufficient manner, the great development or the hypertrophy 
of the interstitial tissue in upper testicular fragments as related above. 

C. Experimental evidence that the latter conclusion is true, and that the 
hypertrophy of the interstitial cells in the upper testicular fragment is caused 
by local conditions, is shown by the following observations. On six guinea- 
pigs of different ages the one testicle was cut into two fragments, both of 
which were left in the body; the upper one supplied by the art. sp.i, the 
under one supplied by the art. defer. (On the other testicle we made—for 
other experimental purposes—incisions going through about half or more of 
the testicle, but not touching the ductus epididymidis.) All these animals 
showed during four months of observation normal somatic sexual characters. 


A résumé of the six experiments we performed is given in the following 
Table :— 


Dr. A. Lipschiitz and others. 


136 


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On the Interstitial Cells in Testicle of Guinea-Prg. 13%, 


As we see from the weight of the animals used for these experiments, they 
were all at an age when the spermatogenesis in guinea-pigs has attained a 
very high degree, or when the production of spermatozoa begins ; some of 
them were adult animals. 

The result of these experiments is that in all the six cases there was, four 
months after the operation, a very marked degeneration of the under 
fragment of the operated testicle. This degeneration concerned both the 
seminiferous and the interstitial part of the testicular fragment, the latter 
being transformed more or less completely into connective tissue. Having 
observed a great number of testicles under different experimental conditions, 
I should like to mention here that there seem to be different forms of 
degeneration which the testicle may undergo; but I have not enough insight 
into this field of pathological anatomy to judge on this question. 

Unlike the under fragment, the upper fragment was, in four cases, still 
resisting degeneration and sclerosation. No. 69 showed in the upper frag- 
ment, four months after the operation, tubules with spermatozoa which 
were even present in the caput epididymidis. Other tubules in this 
fragment were in the state of desquamation or in the juvenile stage. 
In No. 70 all tubules are in the juvenile stage, corresponding to that of 
an animal about three weeks old. In agreement with Benda (118), we 
mentioned in another paper that it is in reality not justifiable to speak about 
a “degeneration” of the seminiferous tubules occurring after ligature or 
section of the vas deferens, transplantation, radiation, and so on. There is 
in reality only a process which leads up to a juvenile stage, a process which 
occurs in an indefinitely smaller measure also in the normal testicle. To 
understand that there is no other change than a return of seminiferous 
tubules en masse to a juvenile stage, it suffices to compare a preparation of 
No. 70 with one of a normal animal about three weeks old, as given in 
Plate 1, fig 2. I do not think that this “ backward development,” to use a 
notion of Eugen Schulz (11c) is the only possible way of reaction of the 
seminiferous tubules in different experimental conditions, and I do not think 
that a seminiferous tubule which has returned to a juvenile stage will 
always have the same destiny or life-history as a juvenile tubule in a normal 
testicle; on the contrary, in our upper fragments and in other experimental 
cases we several times observed complete degeneration of such tubules. 
Evidently, the same experimental condition which may lead to backward 
development may also lead to complete degeneration of these juvenile 
tubules. 

In No. 64 the upper fragment showed about the same condition as No. 69 
Some tubules showed spermatozoa, others were in the juvenile stage. Beginning 


138 Dr. A. Lipschiitz and others. 


from the level of the incision, connective tissue grows inwards in the 
testicular tissue and a few weeks afterwards this fragment would surely have 
been in the same condition of sclerosation as an under fragment. We found 
this condition in No. 68, where no testicular tissue was to be recognised in 
the mass of connective tissue. A degeneration of the upper fragment took 
place in No. 75, but in a somewhat different way. Another case, No. 76, is 
of the greatest interest for us. It is the fourth of the experiments where 
the upper fragment was still present and in good condition as compared with 
the under fragment already wholly degenerated. In this case (Plate 2, fig. 3) 
the seminiferous tubules have only one stratum of cells; I am not able to 
say whether there are here only cells of Sertoli or some spermatogonia 
also; the first is the more probable. The interstitial tissue was in a 
state of hypertrophy, like that in some cases of “ upper” castration. This 
one case, where we have an enormous number of interstitial cells in an 
upper fragment, although the second testicle is present in the body, is 
sufficient to decide the question whether the hypertrophy of interstitial 
tissue In some cases of upper partial castration is a compensatory one or not; 
this hypertrophy is not a compensatory one, but one caused by local 
conditions. 

D. Further experimental evidence is given by the following observations: 
Instead of sectioning the testicle near the upper pole, as in the foregoing 
experiments, we sectioned the testicle near the under pole and cut away a 
very small fragment of the under pole, together with the cauda epididymidis. 
We made this operation on both sides. In principle, this is the same 
operation as that performed unilaterally in the experiments reported in C, 
but with the difference that, instead of having a small fragment supplied 
by the art. sp. i. on one side, the animals of this series had dig fragments on 
every side supplied by the art. sp. i. If an hypertrophy of the interstitial cells 
occurs under these experimental conditions also, it cannot be compensatory, 
because the quantity of testicnlar mass is not diminished by the operation. 

We made three identical experiments, a réswmé of which is given in the 
Table on page 139. 

As we see, there was in two cases a very marked hypertrophy of the 
interstitial tissue, especially in No. 72, illustrated by fig. 4. The hypertrophy 
is not so striking as in some small upper fragments. But one must take 
into consideration that, on examining a great number of testicles even under 
identical experimental conditions, as already mentioned, all transitions in 
the quantity of interstitial cells exist; even the two testicles of the same 
animal treated in the same manner may show very striking differences as 
concerns the interstitial tissue and the seminiferous tubules. On examining 


On the Interstitial Cells in Testicle of Guinea-Pig. 139 


Weight of the 


Duration animal 
No. of of a se : 
Protocol. | experi- Condition of testicle. 
ment. At 
beginning. ah oat. 
days. erm. grm. 
I 73 54 470 495* | Both testicles grown together. All semi- 
niferous tubules with one stratum only. 
Interstitial cells in a markedly increased 
quantity. Sclerosation at the level 
where the incision was made. 
TL 72 109 516 670 Right testicle: All seminiferous tubules 


in the juvenile stage. Sclerosation 
beginning at the level of the incision. 
Very markedly increased quantity of 
interstitial cells. 

Left testicle: Tubules in full spermato- 
genesis with spermatozoa, tubules in the 
stage of desquamation and with one 
stratum only. Sclerosation as on the 
right. Increased quantity of inter- 
stitial cells. 


Til 60 125 130 420 Both testicles grown together. A great 
number of seminiferous tubules in full 
spermatogenesis ; others in the stage of 
desquamation and with one stratum 
L only; some tubules in the juvenile 
stage. Quantity of interstitial cells 
not increased. 


| 
\ ' | 


* The animal died of illness, and was weighed the last time nine days before its death. 


the two testicles of a normal animal, one will also sometimes observe very 
striking differences in weight and in the development of the seminiferous 
and interstitial apparatus. Great differences are also to be found between 
two animals of the same litter. This is why it is often impossible to have 
real control animals in experiments where conclusions should be based on 
weight or age relations. 

On looking at fig. 4, one will see that there is a striking resemblance 
between the condition of this testicle and the testicle of a young animal 
(fig. 2). The condition of the seminiferous tubules, the condition of the 
interstitial cells, and the distribution of the latter embedded in a granular 
or homogeneous mass, all these remind one in a very striking manner 
of the testicle of a guinea-pig of about 2 or 3 weeks of age, when the 
testicle of this species has entered on its rapid development to spermato- 
genesis and puberty. This juvenile stage of the testicle, which one can 
observe under experimental conditions, is all the more interesting in that our 
animal No. 72 was at the time of the operation fully grown, weighing 
already more than 500 grm. There can be no more striking instance of the 


140 Dr, A. Lipschiitz and others. 


fact that a “backward development” of the testicle is possible under experi- 
mental conditions. 
Ti 

After all, there can be no doubt that the hypertrophy of the interstitial 
cells, as observed under different experimental conditions, has nothing to do 
with the function of the testicle for the organism as a whole, but that this 
hypertrophy is caused only by local conditions in the testicle itself. 

Kyrle (12), who studied the hypertrophy of the interstitial cells under 
different experimental conditions, has suggested that this hypertrophy has 
something to do with a regeneration process which the seminiferous tubules 
are undergoing. This point of view is in connection with another conception 
of the function of the interstitial cells, namely, that these cells represent 
a trophic organ for the seminiferous tubules. The latter conception plays a 
great 7rdle in the attacks, made in the last few months by different German 
authors (13), upon the theory of Bouin and Ancel (which was supported 
and further developed by Tandler, Steimach, Sand and myself) that the 
interstitial cells are an organ of internal secretion. 

This is not the place to discuss the question whether our conception of the 
internally secretory function of the interstitial cells is right or not. Indeed 
this conception will not remain unaltered and it is impossible now to say how 
much of this theory will permanently stand in view of the further develop- 
ment of scientific research in our special field. And, it may be, the results of 
the experiments made in our laboratory and reported by myself in this com- 
munication will be interpreted by some as a withdrawal in some measure 
from the position I held and tried to strengthen in my book on the “ puberty 
gland.” But on the other hand I think that there are not yet sufficient 
data to attribute definitely to the interstitial cells a special local function in 
relation to the seminiferous tubules, although the possibility of such a function 
cannot be denied, even though the interstitial cells should play a véle as an 
organ of internal secretion. It is necessary for us to emphasise this point 
since Stieve in a recent publication(13) has seriously misrepresented our 
views. 

Summary. 

In experiments with partial castration one may observe in small testicular 
fragments enormous hypertrophy of the interstitial tissue, the number and 
the size of the interstitial cells being very markedly augmented. ‘This hyper- 
trophy is not a compensatory one, as is shown by the following experimental 
evidence :— 

A. Hypertrophy of the interstitial cells is not present in all cases of partial 
castration and very small testicular fragments with a not very much 


On the Interstitial Cells in Testicle of Guinea-Prg. 141 


augmented relative number, and consequently with a highly diminished 
absolute number, of interstitial cells may suffice for a normal masculinisation. 

B. On comparing testicular fragments supplied by blood in different ways 
(a fragment from the upper and a fragment from the under pole of the 
testicle) one finds that the fragment which seems to be better supplied by 
blood shows more pronounced tendency to hypertrophy of interstitial cells. 

C. Enormous hypertrophy of interstitial cells may take place in an upper 
testicular fragment even when the other testicle is present in the body. 

D. Marked hypertrophy of interstitial cells may take place when we 
transform both testicles into upper “fragments,” by sectioning the testicle 
near the under pole and by so cutting away only a very small quantity of 
testicular mass. 

In view of all these experiments it seems clear that hypertrophy of the 
interstitial cells, as observed in different experimental conditions, has nothing 
to do with the internally secretory function of the testicle in its relation to 
the organism as a whole. This hypertrophy is caused by local conditions, and 
is not brought about in response to general compensatory requirements. 


REFERENCES. 


) lLipschiitz, ‘Die Pubertitsdriise und ihre Wirkungen, Berne, 1919. See 
Chapter IV. 
(2) Bouin et Ancel, ‘‘ Rech. sur le réle de la gl. interstit. du testic. Hypertrophie com- 
pensatr. expérim.,” ‘C. R. Acad. Sc.,’ vol. 137, p. 1289 (1903) ; Ancel et Bouin, 
“La gl. interst. a seule, dans le test., une action génér. sur l’organisme,” ‘C. R. 
Acad. Sc.,’ vol. 138 (1904). 
(3) Ancel et Bouin, “ De la gl. interst. du testic. des mammif,” ‘Jl. de physiol. et pathol. 
génér.,’ vol. 6 (1904). 
(4) Sand, ‘Experim. Studier over Kgnskarakterer hos Pattedyr.,’ Copenhagen, 1918. 
(5) Ribbert, “Ueber die kompensator. Hypertr. der Geschlechtsdr.,” ‘ Virchow’s 
Archiv,’ vol. 120 (1890). ; 
(6) Lipschiitz et Ottow, “Sur les conséquences de la castr. partielle,” ‘C. R. Soc. Biol.,’ 
vol. 83 (1920). 
(7) Lipschiitz, ‘ Die Pubertiitsdr., etc.’ See p. 135. 
(8) lLipschiitz et Ottow, Joc. cit., sub. 6. 
(84) Pézard, “Castr. aliment. chez les cogs soumis au régime carné exclusif,” ‘C. R. 
Acad. Sc.,’ vol. 169 (1919). 
(9) Lipschiitz, Ottow et Wagner, “Nouvelles observat. sur la castra. part.,” ‘C. R. Soc. 
Biol.,’ vol. 85 (1921). ; 
(10) Lipschiitz, Ottow et Wagner, ‘Sur ie ralentissement de la masculinisation dans la 
castr. part.’ (In print.) 
(11) Poll, “Zwischenzellengeschwiilste d. Hodens bei Vogelmischlingen,” ‘ Beitr. z. 
patholog. Anat.,’ vol. 67 (1920). 
(11a) Lipschiitz, Ottow et Wagner, “Sur des modific. histolog. subies par des restes du péle 
infér. du testic. dans la castr. part.,” ‘C. R. Soc. Biol.,’ vol. 85 (1921) ; Lipschiitz, 
Ottow et Wagner, “Sur des modific. histolog. subies par des restes du pole superieur 
du testic. dans la castr. part.,” ‘C. R. Soc. Biol.,’ vol. 85 (1921). 


— 


( 


142 On the Interstitial Cells in Testicle of Guinea-Prg. 


(11z) Benda, “ Bemerk. z. norm. u. patholog. Histol. der Zwischenzellen d. Menschen 
u. d. Saugetier,” ‘ Archiv f. Frauenkunde,’ vol. 7 (1921). 

(llc) Schulz, Eugen, ‘ Ueber Umkehrbar Entwicklungs-prozesse,’ Leipzig, 1908. 

(12) Kyrle, “Ueber d. Regenerationsvorgange im tier. u. mensch]. Hoden.,” ‘Sitzungsber. 
Akad. d. Wissensch., Wien.,’ vol. 120, III. Abt., 1911. 

(13) Kohn, “Der Bauplan d. Keimdr.,” ‘Arch, f. Entwicklungsmech,’ vol. 47 (1920) ; 
Stieve, “ Entwickl., Bau u. Bedeut. d. Keimdriisenzwischenzellen,” ‘ Ergebn. d. 
Anat. u. Entwicklgesch., vol. 23 (1921); Tiedje, “ Unterbindungsversuche am 
Hoden, unter besonderer Berticksichtigung der Pubertatsdr.-frage,” ‘Deutsche 
Medizin. Wochenschr.,’ 1921, No. 18. 


DESCRIPTION OF PLATES. 
(The figures are drawn by Miss L. Lehbert, Dorpat.) 


PLatE 1. 


Fig. 1.—Two sections through the testicle of a normal animal aged about 44 months 
(Prot. No. 27). On the left: interstitial cells in form of a triangle between 
tubules in full spermatogenesis ; on the right: interstitial cells embedded 
in a granular mass in the neighbourhood of blood-vessels. 

Fig. 2.—Two sections through the testicle of a normal animal about three weeks old 
(Prot. No. 32). The interstitial cells are rich in protoplasm, the nucleus is 
large. In the left half the interstitial cells are embedded in the granular 
mass. 


PLATE 2. 


Fig. 3.—Section through the upper testicular fragment of an animal subjected to the 
“complex” testicular section (Prot. No. 76). The hypertrophy of the inter- 
stitial cells is enormous. The seminiferous tubules with only one stratum of 
cells (cells of Sertoli). 

Fig. 4.—Section through the testicle of an animal subjected to the operation described 
in D (p. 138). (Prot. No. 72). The seminiferous tubules are in the juvenile 
stage, although the animal was fully geown when operated on. The operation 
was performed about four months previously. The number of interstitial 
cells is very markedly increased. 


All the testicles or testicular fragments were fixed in the solution of Helly (solution 
of Miiller with 5 per cent. formol) and stained by eosin and hematoxylin. Only fig. 4 
is made from a fragment fixed in Flemming and stained by Heidenhain’s iron-alum 
hematoxylin. All the preparations were made by Dr. Wagner. 


Lipschutz, &e. Koy, Soc. Proc. B, vol. og, pi. 


| 
Lipschutz, &e. Roy. Soc. Proc. B, vol. 93, pl. 


143 


On the Irritability of the Fronds of Asplenium bulbiferum, with 
Special Reference to Graviperception. 


By T. L. PRANKERD, B.Sc., F.L.S. Lecturer in Botany, University College, 
Reading. 


(Communicated by Prof. W. M. Bayliss, F.R.S. Received March 10,—Revised 
November 11, 1921.) 


[PLATE 3.] 


1. Introduction. 

Our knowledge of the phenomena of irritability in vascular plants is 
largely confined to Angiosperms. Very little work seems to have been done 
on Pteridophytes; and from his few observations on fern fronds, Darwin 
[(1), p. 509] even doubted if they possessed any power of response to gravity. 

In a preliminary account (4) of the distribution of the statolith apparatus in 
plants, I mentioned that it was present in young fern fronds, and I have since 
found that this was partially known to Dehnecke(3). He referred to fern 
fronds as possessing non-assimilating chlorophyll grains resting on their 
physically lower cell walls; but he did not connect these grains with the 
perception of gravity, nor, so far as I am aware, have they ever been photo- 
graphed or indeed noticed since. 

An investigation, therefore, of the physiology and cytology of young fern 
fronds seemed advisable, in order to ascertain if, and how far, any connection 
could be traced between them. The common plant Aspleniwm bulbiferum 
proved a very suitable object for detailed study, though many of the observa- 
tions described have been confirmed by work on other genera. 


2. The Growth and Movements of the Fronds. 


The plants used were grown in pots, generally under bell jars, in rooms 
each of which had only one window; thus subjecting them to one-sided 
illumination the direction of which was known. A few control observations 
were made on plants exposed to all-round illumination, and on others grown 
in the dark. The latter were very little used, because it was desired to study 
the behaviour of plants as far as possible under natural conditions. Many 
series of observations extended over weeks or even months, where prolonged 
darkness would have proved very deleterious, if not fatal. 

The position of a frond under observation was determined by measuring as 
nearly as possible the angle made by the midrib with the horizontal. This 
was done by avery simple, but effective instrument consisting of a transparent 


144 Miss T. L. Prankerd. On the Irritability of the 


protractor (A, fig. 1) mounted between two strips of glass (B) fitted upright 
into a slot in a wooden block (C). The two pieces of glass are held together, 
and keep the protractor in place, by an ordinary letter- 
pee clip (D) at the top. In order to bring the protractor 
on a level with the frond, it can be lowered by simply 
pressing it down from above, or raised by pushing it up 
from below, at the same time opening and holding 
down the clip to keep the supports in place. The 


sea latter are made of glass, so that the whole frond should 
be visible, and also to secure the easy adjustment of 
the 90° line of the protractor against the frame of the 

Php y window—usually quicker than using a plumb line. 
Three phases may conveniently be distinguished in 
aa the life-history of a fern frond; the first or infant 


phase when the apex of the frond is curled, the second 
Fic. 1.—Instrument or adolescent phase, from the time when the first 
ee ee leaflets appear beneath the apical coil, till the frond is - 
Feirrond. quite uncurled, when the third or mature phase is 
reached. Growth cannot be measured as precisely in 

ferns as in angiospermic stems and roots, owing to the circinate vernation and 
the considerable individual variation in the process of uncurling. It frequently 
happens in the middle of the second phase, that the apexis raised very quickly 
(2.e.,1n about 24 hours), so that the recorded height shows a greater increment 
than that due to growth in length. A fern frond, like the organs of angio- 
sperms, shows a grand period of growth. When it first appears above the 
soil it grows slowly: 


05-1 mm.orsoaday. This increases till a young frond 
3-10 cm. in height, i.2., in the second phase, may show a daily increment of 
4-5 mm. In the third phase, growth slackens, and very gradually ceases 
altogether. . 

At least six types of movement are shown by fern fronds :— 

(1) Nutation—This is exhibited most strikingly in the second phase of 
growth, and the results I obtained for Asplenium are very similar to those 
recorded for Nephrodiwm molle by Darwin [(1), p. 257]. 

(2) Rectipetality, by which I mean the tendency in growth for the different 
parts of the mid-rib to be in the same straight line. This is sometimes 
apparently secured in the adolescent stage by an autotropic movement of part 
of the frond. 

(3) Sagging, due to the weight of the developing pinne, is characteristic of 
the end of the adolescent and beginning of the mature stage, and gives the 
frond its final graceful form. 


Fronds of Asplenium bulbiferum. 145 


All these movements are slight; the first almost imperceptible to the 
unaided eye, the second merely occasional, and the third only occurs when 
growth is nearly over. They will, therefore, not concern us further, and we 
are left with the three most important movements, due to the following 
causes :— 

(4) Epinasty, present throughout the first two periods, causes more or less 
striking curvatures towards the end of the adolescent phase. The tip of the 
frond, which may or may not be entirely uncurled, approaches the ab-axial 
side so that the upper part forms a loop, sometimes almost a complete circle, 
a position from which it eventually recovers by slight hyponasty. 

(5) Lvght has a directive effect upon the frond which varies with the position 
of the latter, and does not seem to come into play till it is several centimetres 
in height, 7.2, in the middle of the first phase. Curvature is shown most 
strongly if the frond is placed abaxially to the light, when the apical part 
generally moves through 90° in the direction of the incident rays (see 
fig. 2, A). 


A 
Light Gravity 


(o> 


ey if can 


Fig. 2. Fig. 3. 


Fic, 2.—Explanation in text. (Imagine plane of paper vertical.) 
Fic. 3.—Explanation in text. (Direction of light through plane of paper, which is 
imagined vertical.) 


The effect is much less pronounced, and seems to come into play later, if 
the frond is placed so that the light strikes it laterally (z.c., at right angles to 
the plane of the paper, fig. 2), when a slight positive movement takes place in 
the first period, and diaheliotropism, caused by a torsion of the rachis, is 
shown by the leafiets in the second. If, however, the frond is placed so that 
the incident rays strike the adaxial surface (fig. 2, B) little or no curvature 
takes place, but the frond grows approximately vertically well into the 


adolescent stage, since the effects of light and epinasty balance one another. 


The backward curve produced by the latter in the second period is 
considerably lessened. 

(6) Geotropism.—Irritability to gravity exists from the inception of the 
frond till near the end of the second phase. It is best studied in fronds 
placed horizontally and adaxially to the incident light, when the ergan will 


146 Miss T. L. Prankerd. On the Irritability of the 


curve through 90° to bring itself into the vertical. Placed horizontally but 
abaxially to the light, the frond will move into a vertical plane, at the same 
time curving towards the incident light. If fronds are placed horizontally, 
with their ab- or adaxial surfaces uppermost, the subsequent positions assumed 
are shown in fig.3. In the former case (A) gravity and epinasty act together ; 
in the latter (B) they are balanced against each other, though gravity seems 
to have the greater effect of the two, at least in the first phase, since an 
upward curvature is often produced, though never approaching 90°. 

Although very little exact work has as yet been attempted, it is interesting 
to note that both the latent* and reaction} periods for gravity are probably 
considerably longer for ferns in general than angiosperms. In one experi- 
ment Asplenium bulbiferum was compared with Sidalsia sp. The frond of the 
one and the inflorescence axis of the other, neither unsevered from the parent 
plant, were placed horizontally in the open at about 27°C. In 4 hours a 
distinct geotropic curvature was visible in Sidalsia, and 1} hours more 
proved sufficient to bring the axis back to the vertical, while no trace of 
curvature was to be seen in Asplenium. And while I have made few exact 
measurements{ of the reaction time for ferns, it has never been as low as 
54 hours,except in Marsilia sp. at 18°C. The latent and reaction periods 
vary greatly with temperature and perhaps with the season of the year. 

An attempt to express the relative effect of the last three factors and their 
magnitude in the different phases of the life-history of the fern frond is made 
in fig. 4, though the curves, with the exception of that of growth, are only 
approximate. In the former case, the ordinates are proportional to heights 
reached by the fronds on days represented by the abscisse. The gravity 
curve rises with the growth of the frond, reaches a maximum in the 
adolescent stage, and ceases probably before the frond is entirely uncurled. 
‘It must, however, be understood that this, like other curves, attempts 
to express the average of many observations, and does not necessarily hold 
good exactly for any particular frond. ‘The effect of light is represented by a 
curve which begins after that of gravity, rises in the second phase to the 
same height, and ceases probably soon after the beginning of the third phase, 
and therefore after that of gravity. Finally, epinasty§ starts with the 


* The time elapsing between the first presentation of the stimulus and the beginning 
of the response. 

+ The time elapsing between the first presentation of the stimulus and the end of 
the reaction. 

{ The duration of these periods is now under investigation by my student, Miss 
F. M. O. Waight. 

§ The subsequent relatively slight hyponasty is included here for convenience under 
the term epinasty. 


Fronds of Asplenium bulbiferum. 147 


inception of the frond, and is represented towards the end of the second 
phase higher than gravity or light, since its effect at this time is more pro- 
nounced than either, and seems to dominate the situation. It ceases at a 
period somewhere near the complete uncurling of the frond, but which varies 
greatly with the individual. Indeed, the last curve must be taken as even 
more approximate than either of the others, since epinasty is very variable, 
both in amplitude and the time of appearance and disappearance of its 
remarkable expression towards the end of the second phase. 


| 3 phase 


Fie. 4.—For explanation see text. 


It is important for our purpose that the time at which geotropic irritability 
ceases should be ascertained as nearly as possible, but in actual practice this 
is very difficult, as the hyponastic curve is apt to simulate that due to 
geotropism, and the matter is complicated by the individual variability of 
the frond, which is most apparent in the very unstable adolescent stage. It 
seems at least certain that growth continues after the cessation of geotropic 
irritability, the frond being at this time only 70-80 per cent. of the length 
finally attained. 

The fern frond is in many ways the biological equivalent of the angio- 
spermic shoot, and, like it, will bring itself back into the vertical should it be 
displaced from this position. It accomplishes this by passing the vertical 
and swinging like a pendulum backwards and forwards till it remains upright 
in a manner similar to that well known for flowering plants, and originally, I 
believe, described by Darwin [(2), p. 508]. This takes place if the position 
- of a frond is altered in the first, or in the earlier part of the second phase. 
Should, however, a frond be placed horizontally towards the end of this 


148 Miss T. L. Prankerd. On the Irritability of the 


period, especially if the conditions are such that the reaction period is long, 
it!will probably never reach the vertical, but remain at some angle, continuing 
its growth in the same straight line. In most cases when this occurs, there 
is a certain amount of oscillation before the frond becomes fixed in the oblique 
position. 

Fig. 5 illustrates the life-history of an actual fern-frond moving under 
gravitational stimulus. The dotted line of growth is plotted as before, the 


Pot horizontal Pot upright Pot horizontal 


Fie. 5. 


other curve showing the rise of the frond, where the ordinates represent 
degrees. When the frond was only a centimetre or so in height, the pot was 
placed horizontally, and the frond slowly rose till it was at an angle of 60° 
with the horizontal. Like most fronds under these conditions, it never 
reached the vertical, probably in order to avoid the darkness due to the 
proximity of the soil. When the pot was placed upright, the frond attained 
the vertical position. Again placed horizontally, the frond rose to an angular 
height of 70° and then dropped. This was not due to the cause just 
explained, since the same thing has occurred in experiments when the pot 
was upright (see below). The frond is seen to oscillate for some time, and 
finally to take up a position at an angle of 45°, the recorded deviations from 
this being well within the limits of experimental error. 

_ Text-fig. 6 is from a tracing made on glass of the actual position occupied 
by a fern frond on the recorded dates. A complete movement through 90° 
having been obtained by previously placing the pot horizontally, it was on 


Fronds of Asplenium bulbiferum. 149 


February 12 replaced in the upright position, making the frond therefore 
horizontal. It was slowly rising, till on the 18th it had reached an angular 
height of 45°. Round that position it oscillated, gradually uncurling, till 


Fic. 6. 


this was complete on March 2. Soon after, the sagging effect due to the 
weight of the developing leaflets becomes apparent in the upper part of the 
frond. It will be seen that epinasty is not well marked in this particular 
ease. As I read it, this record illustrates most of the movements described 
above, except that due to light, which was shown in the diaheliotropism of 
the leaflets not represented. 


3. The Statolith Apparatus of Asplenium bulbiferum. 


Most attention has been paid to the frond, though some stems have been 
examined. Starch does not seem to be very abundant in the rhizome of this 
plant, but was always found at the apex. Im one case, in a stem which 
seemed dormant, very little was present, and it was quite unoriented. 
Statoliths are, however, always present beneath the rudiments of a 
developing frond, though some diversity is shown in the cytology of the 
statocyte. In one case the ground tissue was composed of cells with obvious 
statoliths, but possessing in addition starch grains round the nucleus, which 
latter was situated in the middle of the partial statocyte. In another case 
the nuclei were seen among the statoliths, though probably only loosely 

VOL. XCIII.—B. M 


150 Miss T. L. Prankerd. On the Irritability of the — 


attached to them, if at all. This, however, was the only case in which any 
orientation of the nuclei in A. bulbiferum was ever observed. 

The very youngest tissue—an actual meristem—never contains statoliths, 
which is scarcely possible, since little or no sap cavity is as yet developed in 
which they can lie loose; but, as the young frond develops, the chloroplasts 
about the bundle at the base become free, and this gradually extends outwards, 
till practically the whole green part of the ground tissue is composed of 
chloro-statenchyma (photo. 3). This well-characterised tissue (4) follows, so 
to speak, the growth of the petiole upwards, by being freshly formed as 
development proceeds to within a centimetre or so of the apex, and dying 
away behind as the xylem becomes lignified, and, later still, selerenchyma is 
developed. Since more statenchyma is formed apically. than dies away 
towards the base of the petiole, the amount gradually increases, till it 
probably reaches a maximum as the lower pairs of leaflets develop. It never 
seems to be found higher than the third or fourth pair, and dies away 
altogether before the end of the adolescent phase (fig. 7). 


Fic. 7.—Fronds of Asplenium bulbiferum increasing in age from (1)-(4). Note that (4) 
is older morphologically than (3) though smaller. Dotted area indicates amount 
of statenchyma. 

The statolith apparatus is very beautifully shown in the fronds of 
Asplenium bulbiferum. The statoliths are large, bright green bodies full of 
starch, and apparently indistinguishable from chloroplasts, except that they 
are completely free from the protoplasm and nucleus. Nearly every cell of 


Fronds of Asplenium bulbiferum. | 151 


the inner ground tissue (at the right time and place, see above) is a statocyte, 
and practically every chloroplast a statolith, so that we have a very complete, 
if not the highest, type of apparatus. In good transverse sections cut from 
fronds laid horizontally for an hour or two, the little heaps of chloro- 
statoliths may easily be seen with a simple lens lying on the walls which 
have been lowest. The statocytes are relatively large in cross-section, 
though shorter than usual in length, being only about twice as long as they 
are broad. 


4, Summary and Conclusion. 


From the foregoing facts, it will therefore be seen that the life-history of a 
fern frond of Aspleniwm bulbiferum falls naturally into. three periods, charac- 
terised not only by external morphology, but by physiological response and 
cytological differentiation. This may be expressed in tabular form, thus :— 


Stage. | Infant. | Adolescent. | Mature. 
| 
PON eer adeni weicssiseeiicatcen « seletoncaucacsednene Curled Uncurling Uncurled. 
Heliotropic irritability ....| Beginning At a maximum | Ceasing early. 
Geotropic irritability..................++ ....| Increasing os 3 Absent. 


Statocyte tissue .............04 qebenadooou0scaac ” ” ” ” 


Darwin’s failure to recognise apogeotropism for ferns is accounted for by 
the discovery of (1) the much greater reaction time; and (2) the com- 
paratively early disappearance of geotropic irritability in these plants. In 
his experiment [(1), p. 509],* the older frond was probably losing its power 
of response to gravity, and the period of horizontality (46 hours) would not 
always be sufficient to induce curvature in the case of a frond “ with the tip 
still inwardly curled.” Darwin does not give the exact stage of development 
of this frond, nor the temperature of the experiment—points both of which 
have been shown greatly to influence the time of reaction. 

Next in interest to the demonstration of the existence of both gravi- 
perceptive power and the possession of statoliths by fern fronds, comes their 
very close association. This has, of course, been noted for angiospermic 
Shoots (2), but in fern fronds it is more striking. In the first place, a 
considerable part of the growth of the frond takes place after both its 
statoliths and its irritability to gravity have disappeared, but when it is s¢id/ 
able to respond to light ; and, again, these disappearances are, as far as can be 
ascertained, synchronous. 


* I demonstrated apogeotropism in the identical species used by Darwin—Wephrodium 
molle—at Kew Gardens in 1916. 


152 On the Irritability of the Fronds of Asplenium bulbiferum. — 


Graphically, this is shown in fig. 4, where a curve for the rise and fall of 
the statolith apparatus would practically correspond with that of response to 
gravity—certainly more nearly than with any other. The result of this 
work may therefore be held to support the view that the possession of 
statoliths is causally connected with graviperception in plants. 


All acknowledgements will be made on the completion of my work on the 
cytology of the statolith apparatus in plants. 


LITERATURE CITED, 


(1) Darwin, C. and F. (1880). ‘The Power of Movement in Plants.’ 

(2) Darwin, ©. (1904). ‘On the Perception of the Force of Gravity in Plants,” ‘ Brit. 
Ass. Rep.,’ Cambridge. 

(3) Dehnecke, C. (1880). “ Ueb. nichtassimilirende Chlorophyll-Korper,” ‘ Bot. 
Zeitung.’ 

(4) Prankerd, T. L. (1915). “ Preliminary Observations on the Nature and Distribu- 
tion of the Statolith Apparatus in Plants,” ‘ Brit. Ass. Rep.,’ Manchester. 


e 


EXPLANATION OF PLATE. 


Photomicrographs of transverse sections of a frond of Asplenium bulbiferum a little 
younger than (8) in text-fig. 7. 

(1) is taken above the second pair of leaflets, and within the curl. The chloroplasts are 
scattered in the cells, and there is no trace of statoliths. 

(2) cut just above the first pair of leaflets. The ground tissue is transitional between 
(1) and (3) ; statoliths are beginning to be formed. 

(3) from below the first pair of leaflets—nearly all the cells of the ground tissue are 
statocytes. 


Prankerd. IROOM, SOC, J270C., 15, Vol. YS, ple « 


2S Ue te Me 


Ets 


kal 


cdi a 


BIOLOGICAL SCIENCES. 


CONTENTS. 


The Dia-Heliotropic Attitude of Leaves as determined by Transmitted 
Nervous Excitation. By Sir JAGADIS CHUNDER BOSE, F.RS., 
Director, Bose Institute, Calcutta. Assisted by SATYENDRA CHANDRA 
GUHA, MSc., Research Student, Bose Institute, Calcutta 


; Be The Ultra-Violet Absorption Spectra and the Optical Rotation of the Proteins 
a x : of Blood Sera. By S, JUDD LEWIS, D.Sc. (Tibingen), B.Sc. (London), F.L.C. 


The Colouring Matter of Red Roses. By GEOFFREY CURREY 


The Kata-thermometer as a Measure of Ventilation. By LEONARD HILL, 
F.R.S., H. M. VERNON, and D. HARGOOD-ASH 


On the Heating and Cooling of the Body by Local Application of Heat and 
Cold. By LEONARD HILL, M.B., F.R.S., D. HARGOOD-ASH, B.Sc., 
and J. ARGYLL CAMPBELL, M.D. 


M@a the Oxidation Processes of the Echinederm Egg cue Fertilisation. By 
C. SHEARER, F.R:S. 


"The Depressor Nerve of the Rabbit. By B. B. SARKAR. (Plate 4) 


7 


Memorial Fellow 


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a The Craalinine of Protein by Sunlight. By E.G. YOUNG, Ramsay .. 


Page. 


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178 


194 


198 


207 


213 


230 


235 


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153 


The Dia-Heliotropic Attitude of Leaves as determined by 
Transmitted Nervous Hacitation. 


By Sir Jacapis Cuunprr Boss, F.R.S., Director, Bose Institute, Calcutta. 
Assisted by SATYENDRA CHANDRA GUHA, M.Sc., Research Student, 
Bose Institute, Calcutta. 


(Received May 25, 1921.) 


The leaves of plants adjust themselves in various ways in relation to the 
incident light. The heliotropic fixed position is assumed by means of 
curvatures and torsions of the motor organ which may be the pulvinus, 
or the petiole acting as a diffuse pulvinoid. In some cases the motor organ 
alone is both perceptive and responsive; in others, the leaf blade does exert 
a directive action, the perceptive lamina and the motor organ being separated 
by an intervening distance. This directive action of the lamina has been 
found by Vochting in Malva verticillata, and by Haberlandt in Begonia 
discolor, and in several other plants. In connection with this it should 
be borne in mind that this characteristic does not preclude the possibility 
of the motor organ being directly affected by the stimulus. In a nerve-and- 
muscle preparation, the muscle is excited, not merely by indirect but also 
by direct stimulus. As regards the heliotropic adjustment of leaves, the 
stimulus of light acts, in the cases just mentioned, both directly and 
indirectly, the indirect stimulation being due to some transmitted effect 
from the perceptive lamina. We may regard the coarse adjustment to be 
brought about by direct, and the finer adjustment by indirect stimulation. 

Certain leaves thus assume a heliotropic fixed position so that the blades 
are placed at right angles to the direction of light, the directive action 
being due to certain transmitted reaction, yet unknown. No explanation 
has, however, been forthcoming as regards the physiological reaction to 
which this movement must be due. Suggestions have been made that the 
dia-heliotropic position of leaves is of obvious advantage, since this position 
assures for the plant the maximum illumination. But such teleological 
considerations offer no explanation of the definite physiological reaction. 
It is, moreover, not true, as I shall show in the course of this paper, that 
there is anything inherent in the plant-irritability by which the surface of 
the leaf is constrained to place itself perpendicular to the incident light. 

I have for many years been engaged in pursuing investigation on the 
subject, and have recently succeeded in discovering the fundamental reaction 
to which the directive movement is due. I shall be able to show that the 


VOL. XCIII.—B. N 


154 Sir J. C. Bose and Mr. S. C. Guha. 


particular attitude assumed by the leaves is brought about by transmitted 
“nervous impulse,” which impinges on the motor organ, which is not simple 
but highly complex; that there are several distinct impulses which react 
on the corresponding effectors grouped in the motor organ. 

For a full and satisfactory explanation of the phenomena, it will be 
necessary to deal briefly with the characteristics of the motor organ and 
the nervous impulse which actuates it. It will also be necessary to show 
that the physiological reactions of the “sensitive” and “ordinary” plants 
are essentially similar. As a type of the former I shall take Mimosa pudica, 
and for the latter, Helianthus annuus. I propose to deal with the subject 
in the following order :— 

I.—General description of the dia-heliotropic phenomena. 

II.—Characteristics of the motor organ : 

1. Mechanical response due to differential excitability of the pulvinus of 
Mimosa and pulvinoid of Helianthus. 

2. Response to stimulation of adaxial and abaxial halves of the motile 
organ. 

3. The mechanism of heliotropic curvature. 

4, The diurnal movement. 

5. Torsional response to lateral stimulation. 


II].—The nervous mechanism in plants : 
6. Receptor, conductor, and effector. 
7. Localisation of nervous tissue in plants. 


IV.—tThe transmitted nervous impulse: 
8. Definite innervation. 
9. The directive action of propagated impulse in heliotropic leaf adjust- 
ment. 


I.—GENERAL DESCRIPTION OF THE DIA-HELIOTROPIC PHENOMENA. 


Before entering into the experimental investigation of the subject, it is 
desirable to describe the dia-heliotropic phenomena, as typically exemplified by 
Mimosa and Helianthus. A photograph of the former is reproduced in fig. 1, a, 
in which the plant placed in a box had been exposed to the northern sky and 
not to direct sunlight. It will be seen that the leaves which directly front the 
light have been raised, and so placed that the sub-petioles, with their leaflets, 
are at right angles to the strongest illumination. The side or lateral leaves 
have, on the other hand, undergone appropriate torsions—the plane of theleaflets 
being adjusted perpendicular to the light. It will be noticed that in executing 
this, the petioles to the right and the left have undergone opposite torsions. 


The Dia-Heliotropic Attitude of Leaves. 155 


After the assumption of this position, the pot containing the plant was turned 
round through 180°. This brought about a new adjustment in the course of 
twenty minutes, the plane of all the leaflets being once more at right angles 
to the light. The new adjustment necessitated a complete reversal of the 
former movements and torsions. Such perfect adjustment is brought about 
by bright light from the sky, and not so well by direct sunlight, for reasons 
which will be given later. j 

In fig. 1, 6, is seen the heliotropic adjustment of the leaves of sunflower, grown 
near a wall, the plant being exposed to light from the western sky. The 
adjustment is essentially similar to that seen in Mimosa. The lateral leaves, 


Fic. 1.—Dia-heliotropic adjustment of leaves : (a), in Mimosa ; (6), in Helianthus annuus ; and 
(c), in a different species of Helianthus. (From photographs.) 


1 and 3, have undergone appropriate torsions—right-handed or left-handed— 
so that the leaf-blades placed themselves at right angles to the light. The 
leaf numbered 2 has been raised, placing its lamina perpendicular to the light. 
A contributing factor in this is the bending over of the stem, due to positive 
heliotropic curvature, which accentuated the rise of the leaf number 2. The 
same bending often causes an apparent fall of the leaf marked 4. When the 
stem is tied to a stake, the bending over of the stem is prevented; the leaf 
numbered 2 is then found raised by heliotropic action; but there is little or 
no fall of the opposite leaf. 

I reproduce (fig. 1,¢) another photograph of the heliotropic curvature and 
adjustment of a different species of sunflower, which was grown in the open. 
In the morning the plant bent over to the east and all the leaves exhibited 

N 2 


156 Sir J. C. Bose and Mr. S. C. Guha. 


appropriate movements and torsions. In the afternoon the plant bent over 
to the west, all the previous adjustments and torsions being completely 
reversed. The plant continued to exhibit these alternate swings day after 
day till the movement ceased with age. 


I].—CHARACTERISTICS OF THE Motor ORGAN. 


I have shown elsewhere (2) that there is no essential difference between 
the response of “sensitive” and “ordinary” plants. I shall now show that 
all the characteristics of the response of the leaf of Mimosa are also found in 
the leaf of Helianthus. These will be specially demonstrated as regards 
normal response and recovery, the response of adaxial and abaxial halves of 
the organ to stimulus, the effect of direct and indirect stimulus in inducing 
heliotropic curvature, the daily periodic movements of the leaves, and the 
torsional response to lateral stimulation. 


1. Mechanical Response due to Differential Excitability of the Pulvinus of 
Mimosa and Petiole of Helianthus. 


In Mimosa, owing to differential excitability of the upper and lower halves 
of the pulvinus, a diffuse stimulus, such as that of an electric shock, causes a 
responsive fall from which there is a recovery on the cessation of stimulus. 
It has been thought that the upper half of the pulvinus is inexcitable. I have 
shown (3) that this is not the case, since local stimulation by light induces a 
contraction and resulting up-movement of the leaf. The upper part of the 
pulvinus is about eighty times less excitable than the lower half. 

Experiment 1—In Helianthus, the entire petiole acts as a motor organ, of 
which the upper half is relatively less excitable. Diffuse stimulation by 
electric shock induces a responsive fall, followed by a recovery on the cessation 
of stimulus. The response-records thus obtained are very similar to those 
obtained with the leaf of Mimosa. In Helianthus the reaction is relatively 
sluggish and the contraction is not so great as in Mimosa. The difference 
between the two responsive reactions is one of degree and not of kind. 


2. Response to Stimulation of Adaaial and Abaxial Halves of the Organ. 


As stated before, the upper half of the pulvinus of Mimosa responds to 
application of light by local contraction; the leaf is thus erected and the 
movement towards light may be described as positive heliotropism. The 
leaflets attached to the sub-petioles are thus made to face the light. Under 
strong and long.continued sunlight the excitation is transmitted across the 
pulvinus, and causes at first a neutralisation, and finally a reversed or 


The Dia-Heliotropic Attitude of Leaves. 1D 


negative movement by the contraction of the more excitable lower half of 
the organ. This is the reason why the dia-heliotropic adjustment is less 
perfect under strong sunlight. 

We obtain parallel reaction with Helianthus; here the petiole acts as an 


extended pulvinoid. Light applied from above causes an erectile movement ; 


when applied below it causes a more energetic down movement. As the 
transverse conductivity of the petiole is feeble, the positive heliotropic 
response, induced by light acting from above, is rarely reversed into negative. 


3. The Mechanism of Heliotropic Curvature. 


A few words may now be said of the mechanics of curvature by which the 
stem of Helianthus bends towards light (fig.1,0). All forms of stimuli, including 
that of light, induce a diminution of turgor and consequently contraction, 
and retardation of the rate of growth of the directly excited side. But 
this is not the only factor in bringing about the positive curvature. I have 
shown (4) that while the effect of direct stimulus at the proximal side of the 
stem induces diminution of turgor and contraction, its effect on the distal 
side, where it acts indirectly, is the very opposite, namely, an increase of 
turgor and expansion. The positive curvature is thus due to joint effects 
of direct and indirect stimulus at the two opposite sides. I have been able 
to demonstrate the induced increase of turgor at the distal side by experi- 
menting with the stem of Mimosa. The stimulus of light is applied at a 
point directly opposite to -the motile leaf, which by its movement indicates 
the change of turgor, the induced increase of turgor being indicated by an 
erection, and diminution of turgor by a fall of the leaf. Application of light 
at a point on one side of the stem was thus found to induce an increase of 
turgor at its diametrically opposite point. 

Parallel experiments which I have recently carried out with Helianthus 
gave identical results. Are light was continuously applied at a point 
opposite the indicating leaf; this induced an increase of turgor, as exhibited 
by a continuous erection of the leaf. We thus find that while direct stimu- 
lation induces a diminution of turgor at the proximal side, indirect stimulation 
causes an increase of turgor at the distal side. The positive heliotropic 
curvature is thus due to the joint effects of contraction of the proximal and 
expansion of the distal side. 


4, The Diurnal Movement. 


The daily periodic movements of the leaf Mimosa and of Helianthus 
exhibit a further similarity which is remarkable. I have shown elsewhere (5) 


158 Sir J. C. Bose and Mr. S. C. Guha. 


that in plants sensitive to light the operative factors in the diurnal move- 
ment are :-— 

a, The variation of geotropic action with changing temperature. A rise 
of temperature is found to inhibit the geotropic action ; a fall of temperature 
accentuates it. In consequence of this the leaf, subject to geotropie action, 
undergoes a periodic up-and-down movement; the maximum fall of the leaf 
takes place at thermal noon, which is about 2 P.M., the maximum rise is at 
thermal dawn, about 6 A.M. 

b. The action of light is, generally speaking, antagonistic to that of 
temperature. In the forenoon, rise of temperature causes a fall of the leaf, 
but continuous light acting from above tends to raise it. The rapid 
diminution of light towards evening acts virtually like a stimulus, causing 
an abrupt fall of the leaf. 


The diurnal movements of Mimosa and Helianthus exhibit four phases 
which are very similar :— 

(1) The leaf, owing to fall of temperature, erects itself from 2 to 5.30 P.M., 
or thereabouts. 

(2) After 6 P.M. there is a rapid diminution of light, and the leaf undergoes 
a sudden fall, which continues till about 9 P.M. 

(3) After 9 p.m. the leaf begins to erect itself with the fall of temperature, 
the maximum erection being attained at thermal dawn, which is at 6 A.M., 
approximately. 

(4) In the forenoon the leaf is acted on by two antagonistic reactions, 
the effects of rising temperature and of increasing light, the effect of rise 
of temperature being predominant. The leaf thus continues to fall till 
thermal noon, which is about 2 P.M. 


5. Torsional Response to Lateral Stimulus. 


I shall now refer to a very important type of responsive movement 
induced by lateral stimulus. A stimulus is called lateral, when it acts 
either on the right or the left flank of a dorsiventral organ. I shall 
presently show that a dorsiventral organ responds to a lateral stimulus 
by torsion. That this effect is universal will be demonstrated by experi- 
ments on the “sensitive ” Mimosa, and the “ordinary” plant Helianthus. In 
order to eliminate the effect of the weight of the leaf, and also for obtaining 
record of pure torsion, the petiole is enclosed in a hooked support of glass, 
with a smooth internal surface (fig. 2). Friction and the effect of weight 
are thus practically eliminated ; the circular support prevents any up or down 
movement, yet allows freedom for torsional response. I have recently 


The Dia-Helrotroyie Attitude of Leaves. 159 


employed another device which is even more perfect. Instead of the 
tubular support, the petiole is slightly stretched in a horizontal direction 
by an attached thin elastic string of indiarubber tied to arod. The up 
or down movement is thus prevented, whereas the string offers but feeble 
resistance to torsion. The torsion is magnified by an L-shaped piece of 
aluminium wire appropriately tied to the petiole, so that the long arm is 
at right angles to the petiole. The end of the arm is attached by a silk 
thread to the short arm of a recording lever, R; there is thus a compound 


Fie. 2.—Diagrammatic representation for obtaining record of torsional response : 
H, hooked glass rod to secure pure torsional effect ; L, bent piece of metal for 
magnification of torsional movement ; R, recording lever; G, oscillating smoked 
glass plate. Direct stimulation of the right flank, 7, or indirect stimulation of the 
right sub-petiole, 7’, induces right-handed torsion. 


magnification of the torsional movement, a right-handed torsion producing an 
up-curve, and a left-handed torsion a down-curve. The oscillating recorder 
gives successive dots at definite intervals of time, which may be varied, 
according to requirements, from twenty to sixty seconds. Time relations 
of response may thus be obtained from the dotted record. 

Fig. 2 gives a representation of the experimental method for studying the 
torsional response of various “sensitive” and ordinary leaves. Diverse stimuli 
are applied at one flank of the organ, which is the junction of the unequally 
excitable upper and lower halves of the pulvinus or petiole. We shall 


160 Sir J. C. Bose and Mr. 8. C. Guha. 


presently find that the responsive reaction in all dorsiventral organs obeys 
a definite law in regard to the relation between the direction of incident 
stimulus and the resulting torsion. The torsion induced is either right- 
handed or left-handed, clockwise or anti-clockwise. In describing the 
direction of torsion, the position of the observer in regard to the plant 
must be definite; he should stand in front of the responding leaf and look 
at the central stem. When the right flank of the pulvinus or the petiole 
is struck by a horizontal beam of light 7, coming from the right, the induced 
torsion is right-handed. Light acting on the left flank induces a torsion 
which is in the opposite or left-handed direction. On the cessation of 
stimulus the leaf recovers its normal position (fig. 3). 


Fre. 3.—Record of torsional response of pulvinus of Mimosa by lateral light. Stimula- 
tion of the right flank induced right-handed torsion, R, represented by up-curve : 
stimulation of the left flank induced left-handed torsion, L. The two thick dots 
represent the duration of stimulus. Successive dots are at intervals of 20 seconds. 


The response described above takes place when the pulvinus or the 
petiole is exposed to lateral light, the leaflets of the lamina being 
completely shielded from it. The differentiaily excitable organ thus 
undergoes a twist, in consequence of which the less excitable upper half 
of the pulvinus is made to face the stimulus. The leaflets or the lamina 
attached to the petiole are thus carried passively, like so many flags, to face 
the hypothetical source of light. It is obvious that the response is brought 
about by a definite physiological reaction and not for the utilitarian purpose 
of securing maximum illumination of the leatlets or the lamina. Teleo- 


The Dia-Heliotropic Attitude of Leaves. 161 


logical considerations, often adduced, offer no real explanation of the 
phenomena; such arguments are, moreover, highly misleading, for similar 
responsive torsion is induced, not merely by light, but by modes of stimu- 
lation so diverse as electrical, thermal, geotropic, and chemical. 

Response of all anisotropic organs to lateral stimulus is included in the 
following generalisation :— 

An anisotropic organ, when laterally excited by any stimulus, undergoes 
torsion by which the less excitable side is made to face the stimulus. 

In a dorsiventral organ, it is the upper side which is, generally speaking, 
the less excitable. Hence the above generalisation may be expressed in 
the following simpler terms: Lateral stimulation of a dorsiventral organ 
induces a torsion which is right-handed, when the right flank is stimulated. 
‘Left-handed torsion is induced by the stimulation of the left flank. 


Torsional response of petiole of Helvanthus.—The above generalisation finds 
independent support from the response of the petiole of Helianthus to 
various stimuli applied laterally. 

Lxpervment 2.—Two fine pins are thrust about 1 cm. apart on the right 
flank of the petiole of Helianthus, to serve as electrodes for application of 
induction shocks from a secondary coil; a similar pair of electrodes are 
attached to the left flank. On application ofa feeble tetanising shock to 
the right flank, the petiole exhibited a right-handed torsion; stimulation of 
the left flank induced a left-handed torsion. Electric stimulation quickly 
stirs up the internal tissues, hence the latent period is short, and the 
responsive reaction is rapid (fig. 4,a). I next took a different specimen, 
and applied the stimulus of light to the right and the left flanks alternately. 
This gave rise to right- and left-handed torsions as under electric stimulus, 
the only difference being in the slower reaction and prolonged latent period 
(which was 15 minutes) (fig. 4, 6). It must be remembered that in the case 
of light the excitation is gradually transmitted from the outer surface to the 
inner tissues. As regards the direct action of light, the results given above 
show that the responsive reactions of sensitive and ordinary plants are not 
different, but essentially similar. With reference to the heliotropic adjust- 
ment of leaves, we found that, when light strikes symmetrically in front, 
the leaf bends towards it. The growing stem itself is excitable, and its 
induced curvature is a contributory factor in placing the surface of the 
lamina at right angles to the light. Leaves struck laterally by light undergo 
torsion which is definite, being determined by the direction of the incident 
light. The torsion thus induced places the leaflets of the lamina at right 
angles to the light. These effects are produced, as stated before, when the 


162 Sir J. C. Bose and Mr. S. C. Guha. 


responding pulvinus or petiole are directly exposed to light, the leaflets and 
lamina being protected from it. 

The heliotropic adjustment of leaves often takes place, as we have seen, 
when the motor organ is in the shade, or is artificially kept so. There must, 
therefore, be transmitted impulses by which the distant motor apparatus is 
so actuated that the leaflets or the lamina are placed at right angles to the 
light. The transmitted impulse, if single or diffuse, cannot evidently exert 
the necessary directive action. I shall presently show that the transmitted 
impulse is of a nervous character, that the impulses are more than one, and 
distinct from each other, and that they travel by different channels from the 
lamina which perceives light to the distant motor region where movement is 
effected in response to transmitted excitation. 


Fic. 4.—Torsional response of petiole of Helianthus in response to (a), electric stimulus, 
and (6), to stimulus of light. R and L are the opposite responses, due to stimulation 
of the right and left flanks. Successive dots are at intervals of 20 seconds. The 
prolonged latent period under light is not shown in the ‘record. The portion of 
record exhibiting recovery is also omitted. 


The Complex Character of the Motor Organ.—As regards the motor organ 
itself, 1 have stated that it is not simple but very complex. This will be 
understood from the following experiments on the pulvinus of Mimosa. Let 
us imagine the pulvinus to be diagonally divided into four quadrants. When 
the upper quadrant is subjected to light acting from above the responsive 
movement of the leaf is upwards; stimulation of the lower quadrant by light 
acting from below induces a down movement. The stimulation of the right 
quadrant (right flank) by a horizontal beam of light induces, as we have seen, 
a right-handed torsion ; the left quadrant responds by a left-handed torsion. 
The four quadrants may, therefore, be regarded as four independent effectors, 


The Dia-Heliotropic Attitude of Leaves. 163 


the resulting movement being determined by their combined effects. We 
may distinguish these four effectors as the upper, the lower, the right and the 
left effectors. We shall presently find that these different effectors are set in 
action not merely by direct stimulation, but by the transmitted impulses from 
a distance, along definite conducting strands by which the different effectors 
are definitely innervated. 


III. THz Nervous MECHANISM IN PLANTS. 


I shall now describe the “nervous” mechanism by which stimulus received 
at the receptive end gives rise to an excitatory impulse, which is conducted 
along certain definite channels. It is necessary here to justify the use of 
the terms nervous tissue and nervous impulse in regard to plants, since 
the idea has long been prevalent that there is nothing in plants which 
corresponds to the nervous impulse in animals. The transmitted effect of 
stimulus in Mimosa was thus regarded not as a propagated excitation 
but merely a hydro-dynamical disturbance. I have shown, however (7), 
that the transmitted impulse is of an excitatory character, that it may be 
blocked by various physiological blocks, and that, like the nervous impulse 
in animals, the velocity of transmission is enhanced with a rise, and 
depressed or even arrested by a fall of temperature. 


6. Receptor, Conductor, and Effector. 


The nervous system of plants must be regarded as of a comparatively 
simple type. In speaking of the evolution of the nervous system, Parker points 
out that the contractile tissue or muscle appeared first as an independent 
effector, and that the nerve developed secondarily in conjunction with such 
muscles as a means of quickly setting them in action; that a receptor or 
sense organ alone would be of no service to an organism, neither would 
nerve or nerve centres alone; whereas a muscle cell or effector is of use if it 
can be stimulated directly (1). 

In plants we find clear indications of these different stages. Thus in the 
leaf of Hrythrina indica, and in the terminal leaflet of Desmodium gyrans, 
the pulvinus is the independent effector, the connecting nerve link being 
absent or functionally ineffective; heliotropic movement thus takes place 
when the pulvinus is directly stimulated, illumination of lamina having no 
effect. In Mimosa and in Helianthus, on the other hand, the intermediate 
nerve network has, as we shall find, become effective, the leaflets or the 
lamina serving as receptive organs. Haberlandt (9) has shown that in many 
cases the epidermal cells of leaves are of a lenticular shape, for increasing 


~ 164 Sir J. C. Bose and Mr. S. C. Guha. 


the perception of light. He rightly observes that “in zoological nomen- 
clature, organs concerned with the perception of external stimuli have 
always been known as sense organs, even among lower animals and in other 
cases in which it is doubtful if the organs in question are responsible for 
sensation in the psychological sense. It is, therefore, not only permissible, 
but necessary in the interest of consistency to apply the term sense organ 
to the analogous structure in plants.” 

With regard to nerve and nervous impulse, I quote the following from 
Bayliss (1), italicising the important passages :— 

“We find the presence of nerve at a very early stage of evolution. . . ~ 
The effect of anything happening at one end of such a thread is conveyed 
with great rapidity to the other end of the nerve, wherever it may be. Merve 
Jibres have no other function than that of carrying excitation. When set into 
activity by some influence, the disturbances set up disappear spontaneously 
after a very short time if the stimulus ceases to act. . . . Jt 1s usual to 
speak of a ‘propagated disturbance’ passing along the nerve, or sometimes o 
‘nervous impulse. The most sensitive apparatus has been able to detect with 
certainty one kind of change accompanying the passage of the propagated 
disturbance, namely, an electrical effect.” 

All the characteristics of nerve described above are also found in the con- 
ducting tissues of plants. As regards the velocity of transmission of impulse, 
it is not so high as in higher, but not so slow as in lower animals. Thus 
in the frog’s nerve the velocity is about 32 metres per second ; in Eledone it is, 
however, as low as 1 mm. per second. The velocity in Mimosa is about 30 mm. 
per second. Though the propagated disturbance causes nv visible change, 
yet the nervous impulse in plant, as in animal, may be detected by definite 
electric change of galvanometric negativity ; the disturbance set up disappears: 
spontaneously on the cessation of stimulus. If the electric contact be made 
only at one point of the plant nerve, the other being at a distant indifferent. 
region, the electric response is monophasic. But if the contacts are made at, 
two points of the nerve, the proximal is the first to become galvanometrically 
negative; the propagated disturbance then reaches the distal point with con- 
comitant negativity of that point. We thus obtain the characteristic diphasic¢ 
response of the nerve (see below). 

Since the nervous reactions in animals and plants are so essentially similar, 
delay in full recognition of this fact will undoubtedly retard the advance of 
science. I shall in the present paper demonstrate certain striking effects in 
plants, which at first sight would no doubt appear as very astonishing, but 
which in reality result from nervous reaction, usually regarded as the special 
characteristic of the animal. I shall be able to show that in the plant a 


The Dia-Heliotropic Attitude of Leaves. 165 


definite nervous link exists between the receptor and the effector, and that 
there is a well-developed system of innervation, by which the “ attitude” of 
the plant-organ becomes adjusted to the incident stimulus. 


7. Localisation of Nervous Tissues in Plants. 


I have in my previous works shown that in Mimosa stimulus gives rise to 
an excitatory impulse, which is transmitted with a definite velocity, that this 
impulse has all the characteristics of the nervous impulse in animals. The 
most important problem in connection with this subject is the localisation of 
the conducting or nervous tissues. I succeeded in isolating a length of such 
a tissue in ferns and was able to obtain with it many results which are 
regarded as characteristic of nervous tissue in animals. In Mimosa, however, 
it is impossible to isolate the nervous tissues without injury, and I have for 
many years been confronted with the problem of localising im sitw the 
particular tissue which serves as the conductor of excitation. I have recently 
been successful in my efforts, the method employed being that of the 
electric probe (6), by which I was able to localise the geotropic sense organ 
in plants. 

Limitation of space enables me only to give the essential details of the 
method of localisation of nervous tissues and some typical results. A fuller 
account will be given in the forthcoming number of the ‘ Transactions’ of my 
Institute. 

The principle of the method will be understood if we take the somewhat 
analogous case of a cable along which electric messages are being transmitted. 
The conducting strand is here embedded in a non-conducting sheath. We 
can localise the embedded conductor and pick up the transmitted message 
by gradually thrusting in the electric probe, which is insulated except at the 
extreme tip. A galvanometer included in the circuit of the probe will 
begin to pick up messages that are being transmitted from the moment of 
contact of the tip of the probe with the conducting strand. The depth of 
insertion for contact can be read on a suitable scale and the position of the 
conductor may thus be determined. 

We may similarly localise the exact position of the conducting nerve 
embedded in the petiole of Mimosa (fig. 5). Excitation of the sub-petiole will 
give rise to an excitatory impulse which travels in a centrifugal direction 
towards the stem. This excitatory impulse is of galvanometric negativity. 
The conducting nerve will be most intensely excited by the transmitted 
impulse, and the induced electrical change of this particular tissue will be 
maximum. Excitation will no doubt be irradiated to the adjoining tissue, 


166 Sir J. C. Bose and Mr. S. C. Guha. 


but this will undergo a rapid diminution in radial directions outwards. If 
the stimulus be moderate or feeble the irradiation will be slight. 


Fig. 5.—The Electric Probe for localisation of nervous tissue in plants. P, the probe 
in circuit with the galvanometer, G ; S, the screw head, by the rotation of which 
the probe enters the petiole in successive steps ; I, index by which the depth of 
intrusion may be determined. 


The experimental procedure is as follows:—The probe is intruded perpen- 
dicularly along the diameter of the petiole. The intrusion of the probe is by 
steps, say of 0°05 or 0:1 mm. ata time. The slight wound produced by the 
insertion of the tip of the probe causes an excitation, which subsides completely 
in the course of about fifteen minutes. The sub-petioles are now stimulated 
by suitable stimuli, which may be chemical, thermal, mechanical or electric. 
The excitatory impulse is propagated preferentially along certain conducting 
channels in the petiole. The results to be described were obtained with all 
the different modes of stimulation. The electric mode of stimulation has the 
advantage that it can be maintained constant or varied in a graduated manner. 
Special precautions are taken that there should be no disturbance caused by 
leakage of the stimulating current; this is verified by the fact that reversal 
of primary current which actuates the secondary coil causes no change in the 
electric response; the excitatory electric change in different layers of tissue 
is, moreover, definitely related to the character of the tissue. 

I shall anticipate results by describing the characteristic effects. The 
excitatory electric change detectable in different layers as the probe passes 


The Dia-Heliotropic Attitude of Leaves. 167 


from the epidermis to the central pith is found to rise suddenly to a maximum 
in the phloem portion of the fibro-vascular bundle; the xylem shows little 
or no transmitted excitation. Hence we arrive at the conclusion that it is 
the phloem which functions as the nerve of the plant. The characteristic 
electric maximum was not found in experiments where the probe missed the 
phloem ; greater experience now enables me so to direct the passage of the 
probe as not to miss the nerve tract. 

In the diagram of the transverse section of the petiole of Mimosa usually 
given in text-books there is in each bundle a single phloem strand outside 
the xylem. I was, therefore, considerably puzzled by the fact that in 
traversing the bundle I obtained two electric maxima, one before reaching 
the xylem, and the second after passing it. In order to determine the cause 
of this anomaly I made transverse sections of the petiole of Mimosa. 
Differential staining clearly brought out the fact that the phloem strand is not 
single but double, one above and the other below the xylem. The second 
electric maximum coincided with the inner phloem. 


My, Go 

a." 
- tes oman es: 
> oF Om ; 


pu Se SS 


Fig. 6.—Micro-photograph showing a quadrant of the petiole and the fibro-vascular 
bundle. The tissues seen in the section are: the epidermis, the gortex, the bundle 
sheath, the first phloem, the xylem, the second phloem, and the central pith. 


168 Sir J. C. Bose and Mr. S. C. Guha. 


It may be stated here that in petioles provided with four sub-petioles there 
are four distinct bundles with four nerve trunks. But in specimens with 
two sub-petioles we only find two bundles, corresponding to the two sub- 
petioles. Two sub-petioles are found, generally speaking, in younger 
specimens. The micro-photograph (fig. 6) shows one of the bundles. 

Experiment 3.—Electrical excitation in different layers: I shall now give 
detailed results of localisation of the conducting tissue. The probe enters the 
epidermis and is pushed in by steps of, say, 0'05 mm.; it passes in succession 
the cortex, C, the outer phloem, P, the xylem, X, the inner phloem, P’, and 
the central pith, O. The thickness of the different layers is modified by age 
of the specimen. In the records given below (fig. 7) the electric response 


Fie. 7.—Galvanometric record of transmitted excitation in different layers of the petiole: 
the first is the positive response of the epidermis, the second is the feeble negative 
response of the cortex, the third, fourth, and the fifth are the enhanced responses in 
the first phloem, the sixth shows absence of excitation in the xylem, the seventh is 
the enhanced response in the second phloem, the eighth is the diminished response 
in the pith. 


of the epidermis= +12 divisions of the galvanometer. I have shown else- 
where (8) that the epidermis, which protoplasmically is more or less dead, gives 
either a zero or a positive, in contradistinction to the normal negative response 
of living tissues. The probe at a depth of 0°1 mm. encountered the cortex and 
the response there was —17 divisions. The phloem extended through 0:15 mm., 
the average depth being 0'2 mm. The response in this region underwent a 
sudden enhancement, as seen in the three responses —61, —65 and —40 
divisions. The xylem which was at a depth of 0°3 mm. showed no response, 


The Dia-Heliotropic Attitude of Leaves. 


169 


proving that it was a non-conductor; when the probe reached a depth of 
0°35 mm. it encountered the second phloem, where the response underwent a 
second enhancement of —56 divisions. The probe reached the border of 
the pith at a depth of 0-4 mm. and the response underwent a diminution 
to —26 divisions. In cases where the incident stimulus on the sub-petiole 
is feeble the irradiation effects are greatly diminished; the excitatory trans- 
mission is then found onlyin the phloem. I give below a summary of results 


obtained with ten different specimens :— 


Table I—Showing Intensity of Transmitted Excitation in 


in Ten Different Specimens. 


Different Layers 


Transmitted excitation. 
Different 
layers. 

ite II iit, |) YY, VW Wal, }) WOE, |] WD |) IDs< X. | Mean. 
Epidermis....| +1) 0 OFa 0 0 0 0) +4; 0 0 +0°5 
Cortex —2 —3 | —50 0 (0) 0 0 (0) 0 0 —5°5 
Phloem ...... —30 | —30 |—100 | —30 | —36 | —44 | —33 | —18 | —20 | —24 |—36°5 
Xeylem\ eee: -—8 -9 0 (0) 0 —10 0 —4 —8 —8 | -—4°7 
Phloem ...... —30 | —30 | —84 | —10 | —36 | —20 | —12 | —18} —20 | —16 |—26'8 
Pathycscase: (0) —6 | -—29 0) (0) -7 0 0 (8) 0 | —4°2 


It will be seen that in all cases the phloem is invariably found to be the 
best channel for conduction of excitation. The following curve (fig. 8), 


© uo14n} EEOC & PypsunLy 


zE Cc P x P’ 
Layer of cells. 


Fic. 8.—Curve showing the different intensities of transmitted excitation in different 
layers: E, epidermis ; C, cortex; P, first phloem ; X, xylem; P’, second phloem ; 


O, pith. 
VOL. XCIII.—B. 


(0) 


170 Sir J. C. Bose and Mr. S. C. Guha. 


plotted from the mean values given in Table I, illustrates this in a striking 
manner. . 


IV. Tot TRANSMITTED NERVOUS IMPULSE, 


In certain experiments with petioles having four bundles, I allowed the 
probe to pass vertically through the petiole when it encountered the upper 
and lower bundles. I thus obtained maximum transmitted excitations in the 
phloems of the upper fibro-vascular bundle, and a similar maximum in the 
phloems of the lower bundle, the intervening layers of tissue being practically 
non-conducting. From this it follows that excitatory impulse is propagated 
along definite channels through the length of the petiole. 


8. Definite Innervation. 


We shall now follow the nervous strand from the perceptive lamina to the 
motor organ. In Mimosa, the leaflets attached to the sub-petioles form the 
perceptive area for light. The excitation is conducted along the phloem 
strand of the sub-petiole, and thence through the connected phloem in the 
‘petiole. In leaves with four sub-petioles there are, as stated before, four 
main bundles which reach the motile organ, the pulvinus. There the fibro- 
vascular bundles apparently fuse, but very fine section of the pulvinus shows 
lines of separation. In any case, I shall be able to show that nervous strands 
are physiologically distinct. These terminate in the four effectors, of which 
two are lateral, the right and the left effectors; the other two are upper and 
lower effectors. In younger specimens of Mimosa there are two sub-petioles 
instead of four, and the two nerve strands are continued to the right and 
left flanks of the pulvinus, the particular innervation being to the right and 
left effectors respectively. In Helianthus, the right and left nerve pass along 
the right and left flank of the petiole, which, as we have seen, serves as an 
extended motor organ. The following results will show that these strands 
function as distinct nerves :— 

Experiment 4.—One electrode was pricked in so as to make contact with 
the phloem of the right bundle embedded in the petiole; the second contact 
was made with a distant indifferent point. Electric stimulation of the 
right vein of the lamina of Helianthus gave rise to electric response of 
galvanometric negativity, the response being mono-phasic. Application of 
thermal and chemical stimulus produced similar results (fig. 9). 

Experiment 5.—The second electrode was in this case thrust into the 
nerve of the plant about 1 cm. behind the first electrode. The response is 


The Dia-Heliotropic Attitude of Leaves. ifs 


now diphasic, since excitation reached the two points in succession (fig. 9). 
Discontinuity of the nerve stops the transmitted impulse, as will be seen 


below. 


Fig. 9.—Galvanometric record of transmitted excitation in the nerve of Helianthus. 
The first is in response to electric stimulus, the second and the third to thermal and 
chemical stimulus. Note in these multiple responses due to strong stimulation. 
The fourth exhibits diphasic response (see text). 


9. The Directive Action of Propagated Impulse in Heliotropic Leaf-adjustment. 


In Mimosa and in Helianthus I have traced the nervous channels from 
the receptor to the effector, and showed how the nervous impulse is 
propagated along definite channels. The most difficult problem that 
confronts us now is to explain the responsive movement and torsion of the 
motor organ, by which the expanded leaf surface faces the light. I shall now 
describe the motor reaction when different parts of the leaf surface are locally 
stimulated, not only by light, but by diverse modes of stimulation. 


(a) Mimosa pudiea. 

Expervment 6.—For this experiment I first took specimens of Mimosa leaf 
having two sub-petioles. The right sub-petiole was stimulated by feeble 
tetanising electric shock. The response was by right-handed torsion. The 
latent period was 2 seconds, and the torsional movement continued for 
20 seconds, even on cessation of the stimulus, after which there was a 
slow recovery, not shown in the record (fig. 10, a). The propagated impulse 
has thus followed its definite path, and reached the right flank of the 
pulvinus or the right-effector. We saw that the characteristic response 
of this particular effector is by a right-handed torsion. Thus the same 
response takes place, whether the effector is directly stimulated or by 
transmitted excitation. This finds strongest confirmation from the following 


experiment, where the responsive movement is made to undergo reversal. 
0 2 


1 Sir J. C. Bose and Mr. 8. C. Guha. 


Experiment 7.—The left sub-petiole was now stimulated by feeble 
tetanising shock, as in the last experiment. The response was now by a 
left-handed torsion (fig. 10, a); the nervous impulse now reached the left 
flank of the pulvinus, or the left-effector, the characteristic response of 
which is by a left-handed torsion. The leaf may thus be twisted to the 
right or to the left by alternate stimulation of the two sub-petioles. A 
feeble tetanising shock should be used for these experiments, since a strong 
excitation becomes diffused as it reaches the fibro-vascular ring in the 
pulvinus, and the predominant excitation of the entire lower half would 
then mask the characteristic effects of the right or the left effectors. As 


Fic. 10.—Responsive torsion by transmitted excitation in Mimosa under (a), electric 
stimulus ; (0), under stimulus of light. Note right-handed and left-handed torsions 
by stimulation of the right and left sub-petioles. Successive dots in (a) are at 
intervals of 2 seconds, and in (6) 20 seconds. . Note the quick reaction under electric, 
and slower reaction under photic stimulation. 


regards leaves with four sub-petioles, we shall presently find that they 
transmit definite impulses to the four quadrants of the pulvinus, to the right 
and to the left, to the upper and the lower effectors, thus giving rise to 
definite reflexes. 

Expervment 8.—Stimulus of Light.—I next tried the action of stimulus of 
light on the leaflets of the right sub-petiole; here also the transmitted 
excitation imduced a right-handed torsion. The latent period was, for 
reasons explained before, longer than in the case of electric stimulation. 
It should be remembered that the light was applied vertically, and the 
responsive torsion was such that the amount of light absorbed by the 


The Dia-Heliotropie Attitude of Leaves. 173 


leaflets became reduced by the torsion. Hence it is obvious that it is not 
the advantage of the plant, but the inevitable physiological reaction, that 
determines the movement. Stimulation of the leaflets of the left sub-petiole 
induced a left-handed torsion. When the leaflets of both the sub-petioles 
were illuminated by vertical light, the two resulting torsions balanced each 
other. While in this state of dynamic balance, if the intensity of light on 
one of the sides, say the left, be diminished by interposition of a piece of 


Fic. 11.—The upper figure is a diagram of stimulation of nerve-ending of Helianthus. 
The record below shows that stimulation beyond the cut gives (a), no response ; 
while stimulation at b, induces right-handed torsion. 


paper, the balance is at once upset, and we find a right-handed torsion. It 
is thus seen that equilibrium is only possible when the entire leaf-surface 
(consisting of the two rows of the leaflets) is equally illuminated; and that 
would be the case when the surface is perpendicular to the incident light. 
The dia-heliotropic attitudes of leaves is thus brought about by distinct 
nervous impulses, initiated at the perceptive region actuating the different 
effectors. 


In the case of leaves with four sub-petioles, illumination of the extreme 


174 Sir J. C. Bose and Mr. S. C. Guha. 


right induces, as already stated, a right-handed torsion; that of the second 
sub-petiole from the right brings about a movement of erection ; the stimula- 
tion of the third causes a down movement, while that of the extreme left 
causes a left-handed torsion. The leaf is thus adjusted in space by co-ordinated 
action of four distinct reflexes. 


(b) Helianthus annuus. 

Results in every way similar are obtained with leaf of Helianthus. Here 
we can distinguish three main veins or nerves, which collect excitation from 
different regions of the lamina. 

Experiment 9.—I first tried electric stimulation. The insertions of the 
electrodes were made in the manner shown in the diagram (fig. 11). 

Experiment 10.—Effect of Discontinuity.—A cut is made between a and J, 

‘thus interrupting the continuity of the nerve. Electric stimulation at a 
induced no responsive movement; stimulation at 6 induced, however, the 
normal response by right-handed torsion (lower record fig. 11). 

Experiment 11.—Alternate Electric Stimulation——The right and left nerve 
endings in the lamina were stimulated alternately. This gave rise to right- 
handed and left-handed torsions respectively. In fig. 12, a, is given the record 
of right-handed torsion. 

The following experiments will show that photic stimulus induces a reaction 
which is similar to that of electric stimulus :— i 
Experiment 12.—Stimulus of Light.—Sunlight was thrown first on the 
right half and then on the left half of the lamina. The transmitted excita- 
tions induced corresponding torsional responses (fig. 12, 0). A balance was 
produced when the two halves of the lamina were simultaneously exposed to 
equal illumination. Here also, as in Mimosa, the heliotropic adjustment is 

brought about by balanced reactions of the different effectors. 

The movement of a dia-heliotropic lamina has been figuratively compared 
with the movement of the human eye by which it points itself to a luminous 
object. It is strange that there is more truth in this comparison than was 
snspected. In describing the rolling of the eyeball Bayliss says (1): “ When 
there are two sets of muscles acting on a movable organ, such as the eye or a 
part of a limb, in such a way that they antagonise one another, it is clear that 
for effective performance of a particular reflex movement, any contraction of 
the muscles opposing this movement must be inhibited. Further, the inhibi- 
tion of one group must proceed pari passu with the excitation of the other 
group to ensure a well-controlled and steady motion.” 

Now, in the torsional adjustment of the leaf due to unequal stimulation of 
the two receptors—the right and left halves of the lainina—let us take the 


The Dia-Heliotropic Attitude of Leaves. 13) 


extreme case where one half, say the right, is alone stimulated, either by 
light or by electric shock. The two effectors for torsional movement, the 
right and the left, are the responding tissues in the right and left flanks of the 
petiole. These are actuated by the nervous impulses transmitted along the 
two conducting strands. When the right half of the lamina is stimulated 
the transmission of excitation along the conducting strand on the right is 
detected (Experiment 5) by an electric change of galvanometric negativity, 
and the corresponding mechanical response of the right effector is, as shown 
before, by a right-handed torsion. We may next inquire the nature of the 
transmitted impulse along the left flank of the petiole concomitant with the 


Fic, 12.—Torsional response due to transmitted excitation in Helianthus: (a), right- 
handed torsion due to electric stimulation of the nerve-ending in the right half of 
the lamina ; (6), right-handed and left-handed torsions due to transmitted excitations 
caused by alternate illumination of the right and left half of the lamina. Light 
was stopped after the thick dot. 


excitation of the right haif of the lamina. It is obvious that a similar 
excitatory impulse on the left flank (the electric indication of which is 
galvanometric negativity) would oppose and thus neutralise the particular 
directive movement. Hence for-ensuring a steady directive motion, in 
response to stimulation of the right half of the lamina, all excitatory impulse 
to the left flank of the petiole should be inhibited. Further, the directive 
movement induced by the stimulation of the right half of the lamina would 
be actively helped if the motor reaction of the left flank of the petiole be of 
an opposite character to that in the right flank. We found that the right- 
handed torsion is induced by a differential contraction of the right flank 
and for concordant effect the reaction of the left flank should be opposite, 
1¢., a differential expansion. The nervous impulse which actuates the right 


176 Sir J. C. Bose and Mr. S. C. Guha. 


effector when the right half of the lamina is alone stimulated, is indicated by 
galvanometric negativity ; for concordant movement under the above con- 
dition, the impulse which actuates the left effector should be of opposite 
sign, 2.¢., of galvanometric positivity. 

I carried out two sets of experiments on the above lines with an identical 
leaf of Helianthus. First, I carried out the usual experiment of the electric 
detection of transmitted excitatory impulse. In this, one of the contacts 
was made with the right nerve in the petiole, the second being with a distant 
indifferent point. The nerve endings on the right half of the lamina were 
electrically stimulated and the transmitted impulse along the nerve gave the 
usual excitatory reaction of galvanometric negativity. A second pair of 
contacts were made for detection of transmitted impulse in the nerve of the 
left flank of the petiole. Stimulation of the nerve termination of the right 
half of the lamina gave in the left nerve a reaction of galvanometric positivity. 
In practice stimulus was always applied to the right half of the lamina, and 
galvanometric connections were made alternately with the right and left 
nerve. The results were always the same and showed that excitation of a 
nerve gave rise to an opposite reaction in the contiguous nerve. There is no 
doubt that these two nervous impulses of opposite signs reaching the 
antagonistic tissues of the two flanks of the motor organ must be of import- 
ance in the co-ordination of the resulting movements. 


General Summary. 


In certain leaves the heliotropic adjustment is brought about by trans- 
mission of nervous impulse to the motor organ. A continuity is shown to 
exist in the response of “sensitive” and ordinary plants. Mimosa pudica 
is taken as a type of the former, and Helianthus annuus of the latter. 
Mechanical response is brought about in both by the differential excitability 
of the upper and lower halves of the motile organ. The lower half in both is 
the more excitable. Local stimulation of the abaxial half of the organ induces 
an erectile movement, that of adaxial half a more rapid downward movement. 

Heliotropie curvature of a stem is due to the joint effects of contractile 
reaction of the proximal and expansion of the distal side. 

The daily periodic movements of the leaves of Mimosa and of Helianthus 
are essentially similar. The diurnal movement is brought about by the 
variation of the geotropic action with changing temperature, and by the 
varying intensity of light. The leaves erect themselves during the fall of 
temperature from thermal noon at 2 p.M. to about 5.30 P.M. Owing to the 
rapid diminution of light in the evening the leaves undergo an abrupt fall 


| ad 


The Dia-Hehotropic Attitude of Leaves. WET. 


which continues till 9 p.m. After this the leaves erect themselves, till the 
maximum erection is attained at 6 A.M., which is the thermal dawn. The 
movement of the leaves is then reversed and there is a continuous fall till 
the thermal noon at 2 P.M. 

A very important motile reaction in the adjustment of leaves is the torsional 
response to lateral stimulus. The following is the law which determines the 
directive movement: An anisotropic organ when laterally stimulated by any 
stimulus undergoes torsion by which the less excitable side is made to face 
the stimulus. In a dorsi-ventral organ the upper side is, generally speaking, 
the less excitable side, and the response of such an organ to lateral stimulus 
may be expressed in the following simple terms. Lateral stimulation of a 
dorsiventral organ induces a torsion which is right-handed, when the right 
flank is stimulated. Left-handed torsion is induced by the stimulation of the 
left flank. 

The effects described above take place by direct stimulation of light. ‘They 
also take place under transmitted excitation. 

The motor organ may be regarded as consisting of four effectors; the 
response of the right effector is by a right-handed torsion, and of the left 
effector by a left-handed torsion. The upper and lower effectors respond by 
rectilinear up-and-down movements. 

The nervous tissue in plants was localised by means of the Electric Probe 
which was made to pass by successive steps through the petiole. The 
maximum transmitted excitation was localised at the phloem portion of the 
fibro-vascular bundle. Hence the phloem functions as the nerve of the plant. 

Excitation at the receptive region is propagated along a definite conducting 
channel, which is traced from the receptive area in the lamina to the corre- 
sponding effector in the motor region. 

In a petiole of Mimosa, provided with two sub-petioles carrying rows of 
leaflets, stimulation of the right row of leaflets by light gives rise to an 
excitatory impulse which reaches the right effector and induces a right-handed 
torsion. Stimulation of the left row of leaflets induces the opposite, or left- 
handed torsion. When both the sub-petioles are illuminated equilibrium is 
only possible when the entire leaf surface (consisting of the two rows of 
leaflets) is perpendicular to the incident light. The dia-heliotropic attitude 
of leaves is thus brought about by distinct nervous impulses initiated at the 
perceptive region actuating the different effectors. 

In Mimosa with four sub-petioles, illumination of the second sub-petiole 
induces an up-movement; that of the third sub-petiole a down-movement. 
The leaf is thus adjusted in space by the co-ordinated action of four reflexes. 

Results similar to the above were also obtained with Helianthus. 


178 Dr. 8S. J. Lewis. Ultra-Violet Absorption Spectra and 


For the movement of the eye the contraction of the muscle opposing the 
movement has to be inhibited. In the torsional movement of the leaf it is 
found that the stimulation of one nerve causes in a contiguous nerve an 
opposite reaction. The nervous impulses of opposite signs reaching different 
flanks of the motile organ is thus of importance in the co-ordination of the 
resulting movement. 


LITERATURE. 


(1) Bayliss, ‘ Principles of General Physiology,’ 1915, pp. 378, 465, 494. 

(2) Bose, J. C., ‘Plant Response’ (Longmans), p. 40 (1905). 

(3) Bose, J. C., and 8. Das, “ Petiole Pulvinus Preparation of Mimosa pudica,” ‘Roy. 
Soc. Proc.,’ B, vol. 89, p. 213 (1916). 

(4) Bose J.C., and G. P. Das, “ Researches on Growth and Movements in Plants,” ‘ Roy. 
Soc. Proc.,’ B, vol. 90, p. 364 (1918). 

(5) Bose, J. C., ‘Life Movements of Plants,’ vol. 2, p. 597 (1919). 

(6) Bose, J. C., and Guha, ‘ Life Movements of Plants,’ vol. 2, p. 480 (1919). 

(7) Bose, J. C., and G. P. Das, “ An Automatic Method for Investigation of Velocity of 

Deanarnission of Excitation in Mimosa,” ‘ Phil. Trans.,’ B, vol. 204, p. 63 (1913). 
(8) Bose, J. C., ‘Comparative Electrophysiology,’ p. 299 (1907). 
(9) Haberlandt, G., ‘ Physiological Plant Anatomy’ (Englisk Translation), 1914, p. 572. 


The Ultra-Violet Absorption Spectra and the Optical Rotation of 
the Proteins of Blood Sera. 
By 8. Jupp Lewis, D.Sc. (Tiibingen), B.Sc. (London), F-.I.C. 


(Communicated by Prof. J. N. Collie, F.R.S. Received April 29, 1921.) 


The earlier part of this investigation was described in a paper entitled 
“The Ultra-Violet Absorption Spectra of Blood Sera,’ communicated by 
Sir William Ramsay, K.C.B., to the Royal Society in 1916 and ue in 
the ‘ Proceedings’ (series B, Vol. 89, pp. 327 to 335). 

At the close of the paper, attention was directed to the inadequacy of the 
sector spectrophotometers then available, and reference was made to one of 
new design then under construction. In the meantime, a full description of 
this instrument has been published ina paper entitled “ A New Sector Spectro- 
photometer” by the present writer, in the ‘Transactions of the Chemical 
Society’ (1919, vol. 115, pp. 312 to 319), together with figure and diagrams. 
With this instrument completely satisfactory results have been obtained, and 
with it most of the work now to be described has been done. 

The earlier work had reference to serum as a whole; and as foreshadowed 


the Optical Rotation of the Proteins of Blood Sera. 179 


in the paper cited, the later effort has been directed to a study of the proteid 
components of serum and their individual influence on the spectrum. For 
this, the Beit Research Fund Committee of the British Homeopathic 
Association have again given generously the necessary financial aid out of the 
funds placed at their disposal by Mr. Otto Beit for purposes of scientific 
research. 

A search in the literature has failed to reveal any information on the subject, 
so that in all parts of the work it was necessary to break new ground. Two 


‘papers on “ Ultraspectroscopic Studies in Blood Serum,” one by T. Tadokoro 


and the other by T. Tadokoro and Y. Nakayama, appeared in America in the 
‘Journal of Infectious Diseases’ for January, 1920 (vol. 26, pp. 1 to 7, and 8 
to 15), and recall the subject matter of the first paper cited, but they do not 
in any way anticipate the present communication. 

It has already been observed that the absorption band of serum is caused 
entirely or almost entirely by the proteins contained, and it became a matter 
for inquiry as to whether the albumin, pseudo-globulin and eu-globulin were 
similarly or variously absorbent of ultra-violet light. 

The necessary preliminary to a spectroscopic examination of these com- 
ponents was to separate them in a pure state, and to devise means for deter- 
mining the concentration of the solution employed. This proved an 
unexpectedly difficult task, partly because of the confused state of the informa- 
tion available and partly because of the necessity of employing solutions perfectly 
free from preservatives and other substances capable of affecting the spectrum- 
absorbing power of the solutions. In the end there was no alternative to 
relying on one’s own discretion and to devising the details of the processes 
of separation and purification. Great care was exercised with a view to purity 
and constancy of product. 

In formulating the processes, much attention was paid to those published 
by Hardy, Hartley and Haslam, but more particularly to the researches of 
Dr. Harriette Chick on the physical conditions which control the precipitation 
of the proteins of serum, published in 1913 and 1914 in the ‘ Biochemical 
Journal.’ 

Method of Separating the Proteins. 

In the processes of separation ammonium sulphate was practically the only 
reagent employed. The purest qualities obtainable commercially were 
carefully tested for organic matter, as traces of this would affect the absorption 
Spectrum, and the best specimens were selected. 

The manner of procedure was in principle such as that usually followed, 
depending on suitable application of various concentrations of ammonium 
sulphate; but numerous details were carefully studied, and amongst these the 


180 Dr. 8. J. Lewis. Ultra-Violet Absorption Spectra and 


following may be mentioned. It was found desirable to employ fairly large 
quantities of material, such as two litres, of the clear serum and at each stage 
to work at first with solutions of ammonium sulphate, which were as precisely 
as possible of full, one-half or one-third saturation, and then, when required, 
to add small but known excesses. The albumin precipitates were dissolved in 
such a quantity of water as to produce a 14 or 16 per cent. solution, and 
reprecipitated, the first time by the addition of specially recrystallised 
ammonium sulphate, and on subsequent occasions by the aid of ammonium 
sulphate and a very small quantity of acetic acid, and left to stand over for 
some days to allow the precipitate to become micro-crystalline. With horse 
serum the best crystals were obtained after the fourth precipitation. With 
human serum, the particles did not form cystals, but they exhibited a well- 
defined uniformity of shape, suggestive of an approach to a radiate crystalline 
structure. 

The mixed globulins were precipitated three times, and then separated from 
one another by dissolving in sufficient water to produce an approximately 


2 per cent. solution, and precipitating by increasing the ammonium sulphate . 


concentration to one-third saturation. 

The pseudo-globulin was freed from accompanying eu-globulin by slightly 
increasing the ammonium sulphate to nearly 36 per cent. saturation and 
filtermg, and then by adding four small equal quantities of saturated 
ammonium sulphate solution, and allowing to stand after each addition, so 
that the final concentration was nearly but not quite 37 per cent. saturated, 
with a view to removing any remaining eu-globulin. After filtration, the 
pseudo-globulin was reprecipitated by increasing the ammonium sulphate to 
one-half saturation, and collected for use. 

The eu-globulin was reprecipitated by ammonium sulphate four times from 
dilute solutions. With ascitic fluid, a small amount of a brown jelly-like 
substance sometimes accompanied the eu-globulin. When this was the case, 
the impure eu-globulin was dissolved as completely as possible in a small 
quantity of water and then saturated ammonium sulphate solution was added, 
until a small but considerable precipitate was formed, stirred well and filtered. 
The jelly-like substance remained on the filter together with a small quantity 
(probably 20 or 30 per cent.) of the eu-globulin. The filtrate was then quite 
clear, and was treated as an ordinary solution of eu-globulin. Subsequent 
precipitations usually gave no trouble. 


Optical Rotation of the Proteins. 


It was at first assumed that each protein would have a constant rotatory 
power, and that observations of the specific rotations would settle the question 


the Optical Rotation of the Proteins of Blood Sera. 181 


of purity. However, the literature of the subject revealed the widest 
disparity of figures for the same protein, and as reliable data did not appear 
to be available, recourse was had to determining the concentrations of the 
solution by chemical means and to taking advantage of the opportunity which 
the principal work afforded of determining the specific rotations of the several 
proteins. In view of the separations having been carried out with such 
thoroughness, the figures should be fairly accurate and reliable. 

There is probably a normally definite specific rotation for each of the 
globulins, although experimental results do not favour this view with regard 
to the albumins. On the other hand, one must take into consideration such 
eases as that of the pseudo-globulin from specimen No. 291. The specific 
rotation was determined twice on two entirely independent solutions of 
different concentration, the concentrations having to be ascertained separately 
by chemical means. The two results are —43°26° and —43°82°, whereas the 
adopted figure for other pseudo-globulins is —46°. This seems to show that 
if there is a normal value, there are specimens having abnormal values. A 
study of the figures as a whole leads to the conjecture that a given specimen 
may be pure in the chemical sense, but consist of a mixture of optical 
isomers of the protein. A closer examination of the data reveals many 
irregularities not apparent at first sight. For example, the two most recent 
and best specimens of pseudo-globulin from the horse had the specific 
rotations —52:06° and —52°17°, while an earlier specimen gave —49-50°. 
Corresponding specimens of human origin gave —43°0°, —46:97°, —47-66°, 
and —45°35°, the first three having been separated from ascitic fluid and the 
last from normal serum. There is thus exhibited a well-marked differentia- 
tion between the rotation of the human, which may be taken as —46°, and 
that of the horse, which may be taken as —52°. 

Similarly with eu-globulin: the figures for the two best and most recent 
specimens of horse are —43:03° and —43-04°, and for an earlier one —40:98°. 
The human gave —50°24°, —49:12°, —47-13°, and —47-89°, the first three 
referring to the protein separated from ascitic fluid and the last from normal 
serum. 

From these one may adopt —43° for eu-globulin from horse and —48° for 
the human. 

With albumin the results fluctuated considerably. For horse —57-40° is 
the only figure available. For human, —65°36°, —64°43°, —55-05°, —59°14°, 
—50°58°, —54°83°, the first four referring to albumin separated from ascitic 
fluid, and the last two to that from normal serum. 

The rotations were observed with solutions containing a little ammonium 
sulphate. By experiment it ‘was ascertained that the change of rotation 


182 Dr. S. J. Lewis. Ultra-Violet Absorption Spectra and 


on varying the concentration of the ammonium sulphate is so small as not 
appreciably to affect the specific rotations found. The effect under the 
prevailing conditions of experiment is not likely to be greater than +0-2 on 
the specific rotation. 


Spectrophotometry of the Solutions. 


Strong solutions of the proteins were obtained by dissolving the purified 
precipitates described above in water, and their concentrations were ascer- 
tained by determining the total solids and the ammonium sulphate in the 
solution, and taking the difference as protein. This method gave constant 
and apparently satisfactory results. In every case the work was done in 
duplicate, and sometimes in triplicate. 

The strong solution was polarised with the object of determining the 
specific rotation, and suitably diluted with distilled water for use in the 
spectrophotometer. The concentrations found to work best, that is, such as 
to exhibit a well-developed band, have been found to be 0:08 per cent. for the 
albumin, 0°04 per cent. for the pseudo-globulin and for the eu-globulin. 

The strengths of the solutions were estimated approximately by means of 
the polarimeter for immediate use, and corrected later when the chemical 
figures became available. 

The dilution was filled into a 2-cem. observation tube fitted with quartz 
ends, and a second tube was filled with a solution of ammonium sulphate of 
approximately the same strength as was the protein solution with reference 
to this salt: usually this was obtained by diluting saturated ammonium 
sulphate solution 150 times. The latter tube was used as a blank in the one 
path of the spectrophotometer, so that the observations made with the tube 
of protein solution placed in the other path express the spectrum-absorbing 
effect of the protein only. 

The process of spectrophotometry was conducted in the manner indicated 
in the paper first cited, with all the refinements described in the paper dealing 
with the new instrument. The series of photographs for each experiment 
extended over three plates, making a series of fifty-four in all. 

The absorption curve is plotted with extinction coefficients as ordinates 
and wave-lengths as abscisse. The extinction coefficient is calculated on a 
t-cm, layer of a 01 per cent. solution of the protein, which, according to 
Beer’s law, is the same as that on a 0°l-mm. layer of a 10 per cent. solution. 
This corresponds with the protein concentration of serum as nearly as decimal 
figures permit; serum contains about 8 per cent of proteins. Hence the 
curves approximate in their values to those already described with reference 
to serum itself. In order to correlate the two sets of values, either the 


the Optical Rotation of the Proteins of Blood Sera, 183 


extinction coefficients of serum must be multiplied by 1°25 or those of the 
proteins by 0°8. 

Each of the protein curves is in general similar in form and character to 
that of serum (/oc. cit.), which demonstrates that the band produced by serum 
is an expression of its protein-content, especially since serum deprived of its 
* protein gives no such band (loc. cit.). 

It now remains to consider the specimens employed and the curve for each 
protein in detail. In all, eleven specimens of serum were studied, six derived 
from the horse and five human. The primary object in employing horse serum 
was to ascertain the best conditions for separating and purifying the proteins, 
so that the human serum might afterwards be studied with greater confidence 
and certainty. The two or three earlier numbers amongst the horse specimens 
may therefore be regarded as practice numbers, and to that extent the figures 
for these must be held as less reliable. 

The horse serum was as nearly as possible strictly normal, as the first 
three specimens were supplied as such from a physiological laboratory ; and 
the three later specimens were derived from animals slaughtered for use as 
human food. The serum was mixed with an equal volume of saturated 
ammonium sulphate solution within 24 hours of the slaughter of the horse. 

The last of the specimens of human serum (No. 205) was declared to be 
strictly “normal,” and was from a case of cerebral hemorrhage. The other 
four were selected specimens of ascitic fluid. Hach one was quite clear and 
had the appearance of good serum: No. 200 being the least satisfactory, 
although that was good. 

The three later specimens of horse serum and those of ascitic fluid were 
all sufficiently large. That of normal human serum was smaller, namely, 
250 c.c.; but in this case, by careful manipulation, satisfactory separation 
and purification of the three proteins were effected. 

The experimental data are collated in Tables I to VI, in which the specific 
rotations are repeated for the sake of easy reference. The results are 
graphically displayed in the accompanying curves, which have been arranged 
in two groups, namely, the three proteins from horse serum in the one, and 
those of human origin in the other. This is convenient, because the curves 
for the two pseudo-globulins are so very nearly alike that they may be 
regarded as the one a replica of the other. 

The values brought together in the Tables for study and comparison are 
(a) the extinction coefficient at the head of the absorption band at a wave- 
length of about 2800; (0) the extinction coefticient at the point in the band 
where the light-absorbing power is least at a wave-length of about 2500, 
that is, in the depression of the curve; (c) the difference between (a) 


184 Dr. 8. J. Lewis. Ultra-Violet Absorption Spectra and 


and (6), or the “amplitude” of the band, which shows considerable 
regularity, and appears to be significant; (d) the wave-length of the region 
of greatest absorption in the band, that is, at the head; (e) the wave-length 
of the point of least absorption in the band, that is at the foot of the 
curve in the depression. 


Table I.—Pseudo-Giobulin from the Horse. 


Extinction coefficient. Wave-length. 
| Specimen | Specific 
| number. Of head | Of foot | Difference | 47,44 Hoof | ee 
| at 2800. | at 2500. | or amplitude. eae aie 
| 
| 193 112 | 0-48 0-69 | 2790 | 2510 ¥ 
194 1°31 | 0:52 0°79 2800 2510 
| 197 118 | 0-46 0-72 | 2780 | 2518 
| 198 270 |) vdicaal 0°60" | 278079 a eaan —49 -50 
| 199 129) et OL Tame 0°72 | 2800 | 2540 —52°06 
| 203 fa S, a MOAG sy 0°72 | 2790 2500 —52-17 
| Adopted 1°19 0°47 0-72 | 2790 2520 —52 
Table I1.—Pseudo-Globulin (Human). 
| 
Extinction coefficient. Wave-length. 
Specimen Specific 
sumber. Of head | OF foot Difference Head Foot rotation. 
at 2800. | at 2500. | or amplitude. ; : 
| | | 
200 ascitic fluid 1°26 0°61 0-65 2750 2520 
201 5 Fr 1°35 0°63 0°72 2780 2528 (a) —43-26 
| (6) —43 82 
202 5 1°46 0°72 0°74 2790 2520 —46 97 
204 =, 3 1-40 06% | 0°73 2805 2525 —47 66 
205normalserum| 1°28 058 | 0-70 2790 2520 —45 °35 
Adopted 1°35 0°63 | 0°72 2790 2521 —46 


Observations on the Figures for Pseudo-globulin, Tables I and LI. 


Omitting the earlier numbers, 193 and 194, which represent the first 
efforts, and 200, which was not a very good specimen, the figures exhibit a 
remarkable regularity in the amplitude of the absorption curve as expressed 
by the difference in the extinction coefficients. With one exception 
(No. 198), they all lie between 0°70 and 0°74. Very little difference 
between the horse and human specimens is evident. The chief distinction 
is that the corresponding coefficients are a little higher for the human than 
for the horse. 


the Optical Rotation of the Protems of Blood Sera. 185 


One of the most striking features of the work is the discovery that 
pseudo-globulin has probably identically, certainly almost identically, the 
same form of absorption band of the same magnitude, whether it is of horse 
or human origin. This is good evidence of pseudo-globulin being a chemical 
entity. 


Table [1] —Eu-Globulin from the Horse. 


Extinction coefficient. W aye-length. | | 
Specimen | l are | Specific | 
number. - | rotation. 
: | Of head | Of foot | Difference | 
| at 2800. | at 2500. | or amplitude.| "ead. | Foot. 
| ° 
193 1°68 0°48 1°20 2800 | 2539 
194 — | 0-88 _ = 2515 | 
“| ai eee ec | 
198 1°44 | 0 62 0°82 2760 | 25380 —40 ‘98 
199 1°09 | 0°62 0°47 2770 2530 | —43°03 | 
203 gt 42 I 2089 0°53 2780 2535 | —43°04 | 
Adopted | 1°42 | 089 | 058 | 2775 2532 | —48 
| | | | | | 
Table [V.—Eu-Globulin—Human. 
| | 
Extinction coefficient. Wave-length. 
Specimen | Specific 
Ean Es Of head | Of foot Difference Tea Cece. | Folatiou: 
at 2800. | at 2500. | or amplitude. ere a es 
| | = 
200 ascitic fluid | 1°37 0-71 066 | 2750 2680 | —50-24 
ae 1-46 0°78 0-68 | 2770 2510 —49-12 
17 a 1°39. | 0°67 0°72 | 2800 2540 —47 13 
ia, 1°53 0-85 0°68 | 2805 2525 
205normalserum| 1°52 0°8 0-63 2790 2530 —47 “89 
Adopted 151 | 0-85 0-66 | 2795 | 2530 | —48 
| | | 


Observations on the Figures for EHu-globulin, Tables III and IV. 


The curves for the two eu-globulins are so similar in their general form 
and magnitude to those for pseudo-globulin as to leave no doubt of a close 
chemical relationship between the two groups, but the minor quantitative 
distinctions are too great and too well-defined to allow of their being 
regarded as mere varieties of the same substance. 

At the present time, it is a matter of great interest to discover any funda- 
mental differences between pseudo-globulin and eu-globulin. Dr. Harriette 

VOL. XCIII.—B. P 


186 Dr. 8. J. Lewis. Ultra-Violet Absorption Spectra and 


Chick considers the two globulins to be the one a modification of the other.* 
The results of the present inquiry may, on the whole, be held to support this 
view, but they do certainly indicate also that there is some important 
difference between them, which is borne out by the two following con- 
siderations :— 

(a) The amplitude figure is much greater for the pseudo-globulin than it is 
for the eu-globulin. 

For pseudo-globulin this figure is 0°72 for both horse and human; for 
eu-globulin it is 0°53 for horse and 0°66 for human. Therefore, with both 
varieties, there is a marked difference between the two globulins. 

Also, for both horse and human proteins, the curve for pseudo-globulin is 
very slightly broader than that for eu-globulin; this is so for the human 
more than for the horse. 

(6) The intensity of the selective absorption is much greater for the 
eu-globulin than it is for the pseudo-globulin, as shown by the magnitudes of 
the extinction coefficients. 

For horse, the figures for eu-globulin are 1:42 for the head and 0°89 for the 
foot, mean 1:16, against those for pseudo-globulin, which are 1:19 for the 
head and 0:47 for the foot, mean 0°83, giving an excess in favour of © 
the eu-globulin of 0°33, or 39 per cent. 

For human, the corresponding figures for eu-globulin are 1°51 for the head 
and 0°85 for the foot, mean 1:18, against those for pseudo-globulin, which 
are 1:35 for the head and 0°63 for the foot, mean 0°99, giving an excess in 
favour of the eu-globulin of 0°19, or 19 per cent. 

In passing, it may be observed that both horse and human pseudo- 
globulin exhibit the same amplitude in the band, namely, 0°72, and that the 
bands for both horse and human eu-globulin have nearly the same mean 
values for the extinction coefficient, namely, 1:16 and 1:18. 

Although the problem is not yet solved, it may be said that the spectro- 
scopic phenomena are in harmony with the view expressed by Dr. Chick, 
that eu-globulin is a protein-lipoid complex resulting from the interaction of 
pseudo-globulin with a minute proportion of a lecithin. 


Observations on the Figures for Albwmin, Tables V and VI, 


The extinction coefficients of albumin contrast strongly with those for 
either of the two globulins. The amplitude value is only 0:36 for the horse 
or 0:23 for the human, instead of about 0:7, which was found for the two 


globulins. 


* ‘Biochem. Journ.,’ vol. 8, pp. 404-420 (1914). 


the Optical Rotation of the Proteins of Blood Sera. 187 


Table V.—Albumin from the Horse. 


Extinction coefficient. Wave-length. 
Specimen - | Specific 
number. | . rotation. 
Of head | Of foot Difference | 
at 2800. | at 2500. | or amplitude. Lae NOG | 
° 
193 —_— —_— —_ — — 
194 0°55 0°32 0°23 2780 2520 
197 1°60 1:21 0°39 2780 2540 
198 1°42 0°97 0°45 2770 2540 —57 °40 
199 0°89 0°68 0-21 2770 2545 
203 1°19 0°84 0°35 2800 2550 
Adopted. 1°20 0°84 0°36 2785 2545 
Table VI.—Albumin—Human. 
Extinction coefficient. Wave-length. 
Specimen (= a Specific 
number. ‘ rotation. 
Of head Of foot Difference 
at 2800. | at 2500. | or amplitude. Blend. Hoot: 
200 ascitic fluid 1-05 0-86 0°19 2770 2575 —65 °36 
20) Bole 0°67 0°37 030 2765 2540 — 64°43 
Boar evi), 0-70 0°38 0°32 2795 2540 — 55-05 
CL vO ea 0°58 0°33 0°25 2780 2540 —59 14 
205normalserum| *0°70 0-51 0:19 2780 2555 — 50°58 
+0 66 0°45 0°21 2790 2538 —54°83 
Adopted 0°68 0°45 0:23 2783 2540 
* st crop. } 2nd crop. 


The distinction is not in the amplitude alone. The extinction coefficients 
dt the head and fcot of the curve in the horse series are fairly high, while in 
the human series they are exceptionally low, so that the horse albumin is 
well differentiated from the human albumin. 

On tabulating the adopted figures for the amplitude of the band, the 
wave-length of the head and the wave-length of the foot, and extracting 
their means as shown in Table VII, one is impressed with the uniformity in 
the wave-length of the head, which is very nearly the same for the three 
proteins, and yet clearly not identical for pseudo-globulin (2790) and 
albumin (2784), while the two varieties of eu-globulin provide extremes 
at 2775 and 2795. The wave-leneths of the foot for the three proteins are 
also nearly the same, although again clearly differentiated: 2521 for pseudo- 
globulin, 2531 for eu-globulin, and 2543 for albumin. The differences are 


small, but there is no reason for doubting that they arereal. The approximate 
EZ, 


188 Dr. 8. J. Lewis. Ultra-Violet Absorption Spectra and 


Table VII.—Comparative Observations on the Results. 


Amplitude. A for head. A for foot. 
Pseudo-globulin— 
HIOPS@RR en coetneden cen caaveacteumeedeey 0°72 2790 2520 
Elum ane paar ernie 5 Sonic spieteenaecieacees 0°72 2790 2521 
MCAT Pct casens nent dncweeas een 0°72 2790 2521 
Eu-elobulin— 
ELOY SO a ak oanecscaeeseme ane ueseincces meses 0°53 2775 25382 
FELUIM BN Asses nec bee eee yack <naceoeereeeee 0 ‘66 2795 2530 
Mean >. va dameamelsnaembainee teenie 0°60 2785 2581 | 
t 
Albumin— 
| TET OTSE: © sasose cea cance oc sscteeenreat eee 0°36 2785 2545 | 
FERMI. Aes sai cee nanan emacs eee 0°23 2783 2540 
| | 
| Mean: i's estat aerorchens cos 0°30 2784 2543 | 


identity of the wave-lengths signifies the close similarity of chemical 
constitution for the three proteins, while the small differences, which are 
well substantiated so far as the present series of experiments goes, may 
possibly be significant of differences in the nature of the subsidiary groups 
present in the respective molecules. On the other hand, the amplitudes, 
which are respectively 0°72, 0°60 and 0°30, reveal differences which are too ' 
great to be lightly set aside. 

It remains, then, that it is mainly in the magnitude of the extinction 
coefficients that the differences among these three proteins find expression. 
This has already received some attention in the comparison of eu-globulin 
with pseudo-globulin ; but, with albumin, the divergence from the apparently 
well-defined pseudo-globulin is much greater, and not of quite the same 
order; and, further, there is no doubt as to horse albumin being quanti- 
tatively distinct from human albumin. 

If the ratio of the adopted extinction coefficients for the two varieties of - 
albumin be taken, we get 


Head of band. Foot of band. Amplitude. 
WELOUSO)e) eklatsnice ys astels iL 20ihe Be 084. 073.6) aaa 
iainvanaph aeanascieet eae ace ieee 023 qa 


or, if the figures for the two specimens of human albumin from “normal” 
serum alone be taken, we get 
0-84 0:36 


1:20 ; wae 036 _.. 
rie je 9:20 7 1 8 


the Optical Rotation of the Proteins of Blood Sera, 189 


This regular ratio for the several parts of the absorption curve would 
ordinarily signify a corresponding difference in the concentration of the 
substance in solution, but, asmuch as the percentage concentration of the 
albumin was the same for all the solutions, both in the experiment and in 
the ultimate calculations, some other explanation must be found. The work 
of Kober* and others has shown that aromatic amino acids exhibit selective 
absorption, while the absorption spectra of several of the aliphatic amino 
acids and the simpler polypeptides exhibit no selective absorption, and that 
even 40 mm. layers of 0:05 per cent. solutions of some of the latter show no 
general absorption beyond a wave-length of 2500. In view of the way in 
which the proteids are built up of amino-acids and similar groups, some 
exerting selective absorption and some not, and of the difficulty in accounting 
for some of the properties of the proteins when viewed as chemical entities, 
it is not unreasonable to regard them as products resulting from an essentially 
physical association of substances comparable with, but not so chemical as, the 
association of a salt with its water of crystallisation. It may then be assumed 
that the aggregate composing human albumin may result from such a union 
of asubstance comparable with that composing horse albumin with a substance 
or substances possessing no selective absorption. Such an hypothesis of 
physical association gains some support from the fact that five out of six of 
the amplitude values, 0°72, 0°72, 0°53, 0°66, 0°36, 0:20, are approximately 
simple multiples of 0:18, and from the view held by Prof. F. Gowland 
Hopkins} that even the apparently well-defined crystallised egg albumin is 
composed of several proteids, and that much the same may be said of serum 
albumin. It is conceivable that each of the proteins now studied is an 
aggregate resulting from the physical association of a proteid substance 
exhibiting selective absorption with various but definite proportions of such 
simpler bodies as those described above as exerting only general absorption. 
Had the results now recorded been anticipated, it would have been instructive 
to determine in each specimen the yield of phenylalanine, tryptophane and 
similar products exhibiting selective absorption, to see whether the proportion 
was correlated with the extinction coefficient or not. It would have given 
some indication of how far the association of the groups is physical or 
chemical. 

Moreover, if all the curves be re-drawn so as to have the same amplitude, 
say 0°72 or 1:0, very little difference will be observed in their form. How- 
ever, the two albumins would be distinguished from the globulins by the 
general absorption being slightly greater, extending to about 2450 at the 


* “J. Biol. Chem.,’ vol. 22, pp. 433-441 (1915). 
+ ‘J. Physiol.,’ vol. 25, pp. 806-380 (1900). 


190 Dr. 8. J. Lewis. Ultra-Violet Absorption Spectra and 


extinction coefficient of the head of the band against 2420 for any of the 
globulins, and to this one may perhaps attribute the higher figure, 2543, for 
the wave-length of the foot of the curve in the albumins. The inference 
from this is that the general absorption of the simpler body in albumin is 
somewhat greater than it is in the globulins. It isinteresting also to observe 
that the absorption curve of pseudo-globulin which has the greatest amplitude 
proved to be the most constant of all in the course of the experiments now 
described, and was the same whether for horse or man, and would thus appear 
to be a much more definite substance than either eu-globulin or albumin; it 
may even be a chemical entity. 

On the other hand, it is possible that the differences between the absorption 
spectra of the proteins are essentially chemical in their significance, and then 
they are not capable of such simple explanation. 


The Absorption Curves. 


Tt will be seen that at the foot of each column in the Tables I to VI, an 
“adopted” value is given.» This value is not the arithmetic mean of the 
experimental figures, but the value deemed to be the best after considering all 
the circumstances, giving great weight to the most successful experiments and 
little or nothing to those of doubtful value. Hence, the factors for the later 
and better specimens differ only very slightly from the adopted values as 
shown in the Tables. 

With these values a mean curve has been drawn in the following way. 
First, the band for each separate specimen of protein, that is the part of the 
curve covered by the “difference” or “amplitude” values was divided at the 
proper extinction coefficients into ten equal parts and the points of division were 
designated “ position 0” at the extinction coefficient of the head, “ 1st position” 
one-tenth of the way down, “ 2nd position” two-tenths of the way down, and 
so on until the “10th position” is at the extinction coefficient at the foot of 
the depression. All the curves were then re-drawn to the scale expressed by 
the adopted amplitude or “ difference” of extinction coefficient. 

Next, the wave-length at each “position” on the curve was read from the 
curve for each specimen, and a value adopted. The adopted values are set 
out in Table VIII and have been used in plotting the curves (see p. 192). 

It should be observed that although the mean curves show points plotted 
at only about twelve extinction coefficients, and at similar positions for all the 
specimens, absorption spectra were photographed at 50 or more extinction 
coefficients, and hence each original curve has a corresponding number of 
points plotted, similar to those plotted in the curve figured in the first paper. 


the Optical Rotation of the Proteins of Blood Sera. — 191 


Table VIII. 


Position. 


Protein and Section 
variety. z | | | 
0. | Ieee ron nes aun cay Rak |) ge 


Adopted wave-lengths. 
Pseudo-globulin— | | 

ISIG IESG): Segpp gate seeadHeeeee ay 2790 | 2847 | 2860 | 2875 | 2900 | 2980 | 2935 | 2948 | 
t 2790 | 2735 | 2710 | 2670 | 2630 | 2590 | 2562 | 2520 
4 2410 | 2415 | 2420 | 2430 | 2445 | 2460 | 2475 | 2520 | 


Son ScdosnBonRAGEo’ Y 2790 | 2845 | 2858 | 2864 | 2895 | 2929 | 2985 | 2943 | 
€ 2790 | 2744 | 2710 | 2686 | 2685 | 2595 | 2572 | 2521 | 
g 2418 | 2423 | 2428 | 2430 | 2445 | 2461 | 2480 | 2521 | 


HONS OWeaceatck c-ectetisdewsss Y 2775 | 2825 | 2849 | 2865 | 2885 | 
€ 2775 | 2740 | 2718 | 2675 | 2635 | 
(6 2420 | 2435 | 2437 | 2443 | 2460 | 2480 2490 | 20382 | 


lumens hoo. heen sesess y | 2795 | 2830 | 2850 | 2864 | 2880 | 2905 | 2920 | 2930 | 

e | 2795 | 2760 | 2780 | 2680 | 2648 2605 | 2578 | 2530 | 

& | 2422 | 2424 | 2426 | 2487 | 2450 | 2469 | 2480 | 2530 | 

Albumin— | | | | 
LOLOL sates ca iek y | 2785 | 2840 | 2952 | 2863 | 2870 | 2880 | 2885 | 2897 | 

« | 2785 | 2730 | 2720 | 2688 | 2660 | 2620 | 2592 | 2545 | 

& | 2456 | 2457 | 2460 | 2465 2475 2490 | 2508 | 2545 | 

THlivtea hse eae y | 2783 | 2815 | 2825 | 2850 | 2871 | 2880 | 2882 | 28865 | 

« | 2783 | 2755 | 2730 | 2685 | 2645 | 2610 | 2575 | 2540 | 

& | 2450 | 2455 | 2457 | 2465 | 2480 | 2495 | 2510 | 2540 | 


i 


The Characteristics of the Mean Curves. 

The curves are divided into Sections a, B, y, 6, ¢, & as before (loc. cit.). 
Among the more remarkable characteristics are the curious form of the head, 
Section 6, and the step-like prominences or “steps” to which reference was 
made in the first paper. It was hoped that the analysis of the serum into its 
several proteins would have revealed the origin of these steps by showing 
them to be irregularities due to imperfect superposition of the bands of two 
or more proteins, but that expectation has not materialised. On the other 
hand, these characteristics appear in both the globulins with unerring 
regularity, and with but little variation, in either magnitude or position. In 
albumin the “steps” are not so numerous, and they are not much in evidence 
above the tenth position. Below that two large “steps” are evident. 

It is difficult to say whether their positions, that is, their extinction 
coefficients, are precisely the same for all specimens of a given protein or not. 
They repeat themselves with sufficient regularity to suggest that the positions 
may be definite approximately as shown; but on the other hand, the variation 
is too great to attribute it to experimental conditions. The general conclusion 


192 Dr. 8. J. Lewis. Ultra-Violet Absorption Spectra and 


0 
3400 3000 2500 3300 3000 2500 


Al bumin..w...-cessee Eu-globulin_____ Pseudo-globulin 


is that the protein varies, since not only do the positions change more ov less, 
but also sometimes the number of steps in a certain section. 


the Optical Rotation of the Proteins of Blood Sera. 193 


Section @ has been developed in only a few cases. The most notable 
observation is that the absorption in this region is usually very small. The 
curves and especially the spectrum photographs demonstrate in several cases 
that the maximum extinction coefficient is considerably less than 0:01. It is 
certainly very much less with the proteins than it is with serum. 

Section y presents no peculiarities, save only that the sweep is broken by 
four or five “steps,” especially by a prominent one not far from extinction 
coefficient 0:25. 

Section 6 is found to characterise all the proteins more or less, although not 
always in so pronounced a manner as many sera. The mean curve does not 
exhibit this quite so well as the individual curves do, as the construction of 
the mean curve has a smoothing-out effect. With human eu-globulin; the 
head of the curve is narrowed from both sides. 

* Section ¢ is usually the most irregular in form for any given specimen, and 
it is here that there is most disturbance in the wave-length. This is partly, 
but not wholly, accounted for by the “ steps.” 

Section € defines the limit of the general absorption, and shows very little 
variation. Only slight “steps” occur occasionally. The most notable feature 
is that it bends sharply towards the red at the bottom where it joins the 
Section e. 

Summary. 


1. The primary object of the investigation was to ascertain the contribution 
made by each protein constituent of serum to the ultra-violet absorption 
spectrum curve of blood serum. 

2. It has been shown that the absorption curve of pseudo-globulin is 
constant, and is the same for both the horse and human varieties. 

3. The absorption curve for eu-globulin differs considerably from that for 
pseudo-globulin in extinction coefficients, but not in general form. This 
favours the view that the differences between pseudo-globulin and eu-globulin 
do not result from differences in the structure of the chemical molecule. 

4, The absoption curves for the horse and human varieties of albumin have 
been shown to be the same, except for a constant ratio in their magnitudes, 
and this difference may be due to the physical, or possibly chemical, associa- 
tion of an aggregate, possessing little or no selective absorptive power—for 
example, an aliphatic amino-acid or a polypeptide—with the principal or 
absorbing aggregate. 

5. The close similarity in form of all the curves when corrected to a common 
amplitude and the fact that the amplitudes are nearly all simple multiples of 
a common factor, point to similarity of constitution amongst these proteins 
and to a variable “ concentration” of the active group. 


194 Mr. G. Currey. 


6. Comparisons between the absorptions of the proteins of human serum 
reveal absorption bands for the horse somewhat greater in dimensions than 
those for the human. : 

7. The optical properties of the proteins of serum have been investigated 
with fairly satisfactory results. 

8. Processes for the separation and purification of the proteins have been 
elaborated. 


Fi The Colouring Matter of Red Roses. 


By GEOFFREY CURREY. 
(Communicated by Prof. F. Keeble, F.R.S. Received August 12, 1921.) 


An examination of the petals of the red rose “ George Dickson,” has shown 
that the anthocyan pigment contained therein is the cyanidin glucoside, 
cyanin. It is present to the extent of about 9-10 per cent. by weight of 
the dried petals, and exists in the petals as an oxonium salt (7.¢., in combina- 
tion with a plant acid). A yellow glucoside sap-pigment also occurs in the 
same flowers, but beyond the fact that it has been shown to be capable of 
producing an anthocyan, by reduction, and that it is not a glucoside of the 
flavonol myricetin, it has not been further identified, on account of the small 
quantity present. Further work may show it to be a glucoside of quercetin, 
and corroborate the work of Dr. Everest,* on the purple-black viola, in which 
it was shown that an anthocyan (“violanin”) and the flavonol glucoside from 
which it could be produced, by reduction (a glucoside of myricetin), are 
present, side by side, in the same flowers. This would be additional evidence 
in favour of the hypothesis that “ anthocyans are produced, in nature, by the 
reduction of the flavonols.” It is interesting to note that this rose, grown in 
Australia, contains the same colouring matter as was isolated by Willstatter 
and Nolan} from the rose known as “ Rosa Gallica,” grown in Europe, and 
shows how widely these colouring matters are distributed in nature. 

The rose “ George Dickson” was chosen for this investigation on account of 
its deep red colour, which would indicate a fairly large percentage of the 
anthocyan pigment. The flowers from which the petals were gathered were 
grown by Mr. G. Knight, at his nursery, Parramatta Road, Homebush, and 


* “Roy. Soc. Proc.,’ B, vol. 90, p. 255 (1918). 
+ ‘ Annalen,’ vol. 408, p. 1 (1915). 


The Colouring Matter of Red Roses. 95 


his generosity in supplying me with sufficient material enabled the work to 
be successfully accomplished. 

For the isolation of the anthocyan pigment, the methods used by 
Willstitter and Nolan, in their investigation of the “Rosa Gallica,” were 
adopted, while the examination of the flavonol pigment was carried out on 
somewhat similar lines to that used by Dr. Everest in his examination of the 


viola. 
HW#XPERIMENTAL. 


Isolation of the Anthocyan Pigment. 


100 grm. of the petals (which had been first air-dried in the shade, at 
room temperature, and finally over concentrated sulphuric acid), were 
allowed to stand in a closed vessel, with about 300 c.c. of methyl alcohol, 
containing 2 per cent. of concentrated hydrochloric acid (to prevent pseudo- 
base formation), for about twenty-four hours; the mass was then pressed, to 
obtain as much extract as possible, and the residue treated with a further 
quantity of methyl alcoholic hydrochloric acid. After standing some hours, 
this was filtered, with suction, and as the extraction was still incomplete, the 
residue was again extracted with the same solvent. The residue after 
filtering and washing possessed but a pale pink colour, and further extraction 
was deemed unnecessary. The filtrates and washings were united ; they 
_ possessed a fine, deep red colour, with a bluish-violet tinge at the edge of the 
solution. The combined filtrates and washings were poured into about three 
times their volume of ether, well agitated, and allowed to stand for some 
hours. Practically all the anthocyan pigment separated out as a dark brown, 
gummy mass, from which the supernatant solution could be readily decanted. 

The erude anthocyan pigment was redissolved in methyl alcoholic hydro- 
chloric acid, and reprecipitated with ether (about two and a half times 
the volume of the alcoholic solution). After standing some hours, to allow 
precipitation to be as complete as possible, the ether-alcohol solution was 
decanted off and the precipitate allowed to stand for twenty-four hours in 
contact with a mixture of methyl alcohol and glacial acetic acid (to remove 
impurities capable of being hydrolysed or acetylated), and finally collected on 
a filter, washed with a small quantity of methyl alcohol (containing 1 per cent. 
hydrochloric acid), and air-dried. 

The dark brown powder thus obtained was dissolved in boiling water, an 
equal volume of ethyl alcohol (containing 3 per cent. hydrochloric acid) added, 
and the solution allowed to cool; the colouring matter separated out in the 
form of dark brown leaflets, possessing a golden reflex. These were collected 
and air-dried. or identification purposes the anthocyanin chloride thus 


196 Mire Ge Cunrey:. 


obtained was examined for the following properties, viz., crystalline form, 
colour, and reflex; colour changes on the addition of ferric chloride to 
aqueous and alcoholic solutions; colour of solutions in aqueous acid and 
alcohol ; colour changes with alkalies; behaviour with Fehling’s solution (hot 
and cold); colour of precipitate with lead acetate; behaviour with sodium 
bisulphite ; behaviour with zine and dilute acid; solubility in ethyl alcohol 
at 19° C.; solubility in aqueous hydrochloric acid (1 per cent. and 1:5 per 
cent.) at 20° C.; and the distribution of the pigment between amyl alcohol 
and dilute aqueous acid. 

On comparing these properties with those given by Willstatter and Nolan 
for cyanin chloride, they were found to be identical; the anthocyan pigment 
of the red rose “ George Dickson” is, therefore, the di-glucoside eyanin. 


Hydrolysis of the Cyanin Chloride. 


_ The hydrolysis was carried out by boiling a small quantity of the glucoside 

pigment with 20 per cent. hydrochloric acid for three minutes; on standing, 
the sugar-free pigment separated in small needles (not long ones, as obtained 
by Willstatter), possessing a metallic lustre. On examination these were 
found to possess the same properties as described by Willstatter and Everest 
for cyanin chloride. 


Examination of the Yellow Sap-Pigment. 


The ether-alcohol liquor, decanted from the anthocyan precipitate, was 
shaken with powdered calcium carbonate, and, after being allowed to stand 
for a short time, filtered. The filtrate, which possessed a clear, deep yellow 
colour, was concentrated to a small bulk and treated in the following 
manner :—A portion was evaporated to dryness and the residue extracted 
with ether; the ether solution was washed with dilute hydrochloric acid, to 
free it from traces of anthocyan pigment, filtered, and the filtrate shaken 
with dilute sodium carbonate solution, which became yellow in colour. 
Having shown that this alkaline solution contained the pigment in the form 
of a glucoside, the remainder of the concentrate was poured into water and 
boiled, to expel the small quantity of alcohol still present; the aqueous 
solution was then boiled with hydrochloric acid, to hydrolyse the glucoside 
present, allowed to cool, and extracted with ether. The ether extract was 
washed several times with aqueous acid, of different strengths, to ensure the 
removal of traces of anthocyanidin, and finally filtered. The filtrate was 
then shaken with dilute alkali, when the pigment passed into the alkaline 
layer, which assumed a deep yellow colour (the absence of a green colour here 
shows the absence of myricetin), which rapidly became dark brown on 


The Colouring Matter of Red Roses. 197 


exposure to air (a possible explanation of this oxidation is given later). 
The alkaline solution was acidified, extracted with ether, and the ether 
extract evaporated to dryness. The residue, on being dissolved in absolute 
aleohol, to which a small quantity of hydrochloric acid had been added, 
yielded, on the addition of a small piece of magnesium ribbon, a fine red 
coloration, similar to an alcoholic solution of cyanidin chloride. It is 
possible that the yellow pigment present in the petals is a quercetin 
glucoside, but it was not possible to make further tests, as the quantity of 
pigment available was very small. In order to add additional weight to the 
assumption that a glucoside of quercetin is present, the above-mentioned 
tests were carried out with a known glucoside of quercetin, viz., “myrti- 
eolorin.” A small quantity was boiled with acid and hydrolysed, and the 
resulting sugar-free pigment “ quercetin” treated, in alcoholic solution, with 
hydrochloric acid and magnesium ribbon in the same manner as with the 
sugar-free pigment from the rose, exactly similar results were obtained. 
Tests made with both pigments in a glucoside form also agreed. It is 
hoped that further work on this rose will result in sufficient of the yellow 
flavonol sap-pigment being isolated to confirm its identity, by means of 
derivatives. For the sample of “myrticolorin” used in the above tests, 
I am indebted to Mr. H. G. Smith, of the Technological Museum, who very 
kindly supplied me with it; it is a rhammo-glucoside of quercetin, and was 
isolated from eucalyptus leaves. 


~Possible Cause of the Rapid Darkening of the Alkaline Solution of the Yellow 
Sap-pigment from the Rose. 


Although it is the property of the flavonols to undergo oxidation on 
exposure to air in alkaline solution, the very rapid oxygen absorption of the 
solution previously mentioned would appear to be due to some other cause. 
It was thought that this might possibly be due to tannin matter. An 
examination of the petals of the rose revealed the fact that both pyrogallol and 
catechol tannins were present, the former (probably gallo-tannin), being 
present in the greater quantity. The alcoholic extract of the petals would 
contain the tannin matter in solution, and the boiling with acid, to hydrolyse 
the glucosides, as previously mentioned, would also hydrolyse the gallo-tannin, 
with the production of gallic acid; the subsequent extraction of the acid 
solution with ether, to obtain the hydrolysed glucosides, would also extract 
the gallic acid, and this would accompany them into the alkaline solution ; 
the presence of a small quantity of gallic acid would be quite sufficient to 
cause a rapid darkening of the solution, on exposure to air. 


198 


The Kata-thermometer as a Measure of Ventilation. 
By Lronarp Hitt, F.R.S., H. M. Vernon and D. Harcoop-Asu. 


(Received August 20, 1921.) 
(From the National Institute for Medical Research, Hampstead.) 


In two papers previously published, one by Leonard Hill, O. W. Griffiths 
and M. Flack,* and the other by Leonard Hill and D. Hargood-Ash,+ the kata- 
thermometer was described in detail, and formulee were given connecting the 
heat loss with temperature, wind velocity and vapour pressure. Various 
discrepancies were found to occur, however, and seeing that the kata-thermo- 
meter has become recognised as a measure of ventilation the whole matter has 
now been carefully reinvestigated. Large wind tunnels such as those at the 
National Physical Laboratory, which were not available during the war, owing 
to the urgency of aeroplane work, were now at our disposal. 


Experimental Work. 


To obtain known wind velocities for the experimetal work two methods 
were adopted, that of the “wind tunnel” where the air is drawn through a 
long tunnel by means of a propeller at one end, and that of the “ whirling 
arm” where the “kata” is made to move through the air on a revolving arm. 
The wind tunnel work was carried out in the engineering department at the 
National Physical Laboratory by kind permission of Dr. T. E. Stanton, where 
Miss D. Marshall gave us valuable assistance in the determination of wind 
velocities ; also at Hast London College, where Dr. N. A. V. Piercy was good 
enough to allow us the use of the tunnel and to help us in determining the 
wind velocities. 

Observations with the whirling arm method were taken at the Physiological 
Laboratory, Oxford, and at the National Institute for Medical Research, 
Hampstead. The observations at Oxford were made: (1) in a Jarge room, all 
the doors and windows of which were closed, and air currents were further 
reduced by surrounding three sides of the table on which the apparatus stood 
by screens covered with several thicknesses of brown paper. The fourth side 
had to be left open, so as to admit of light, and of a point from which to view 
the “kata.” Air draught from below was prevented by means of horizontal 
screens placed on the table supporting the apparatus. A moving air current 
of known velocity was obtained by clamping the “kata” to a brass rod fixed 


* ¢Phil. Trans.,’ B, vol. 207, pp. 183-220 (1916). 
+ ‘Roy. Soc. Proc.,’ B, vol. 90, pp. 488-447 (1919). 


The Kata-thermometer as a Measure of Ventilation. 199 


at one end to the central pillar of a Sherrington recording drum, and revolving 
it in a horizontal circle; (ii) other observations were made in the respiration 
chamber, which is a closed room of 105x65x4°5 ft. capacity. The 
experiments by the whirling arm method at Hampstead were very similar to 
those carried out at Oxford. A screen of American cloth, 4 ft. high, was 
erected on the floor of a large room, so as to form an enclosure 4 ft. square, 
the top being open. The door and windows of the room were closed, so that 
the air inside the enclosure was still. A transparent celluloid window was 
inserted all round the screen at the height of the “kata” stem, so that 
readings might be taken of the cooling, and the thermometer read. 
In the whirling arm method the actual velocity, v, of the “kata” is 

given by 

_ 2£aTrn 

= 


where r is the radial distance of the “kata” from the axis of revolution, 7 the 
number of revolutions in the time ¢ (estimated by stop-watch). L. V. King* 
and others have shown, however, that this is not the true velocity relative to 
the air, a certain “swirl” being set up by the revolution of the arm. This 
was determined and allowed for in our observations. The procedure by which 
this swirl was allowed for will be described later. 


The Dry “Kata”—Wind Velocity and Temperature. 


As shown in the previous papers, sources of radiant heat being absent, the 
rate at which the “kata” cools in air depends almost entirely upon the wind 
velocity and air temperature. It is shown that the heat loss per second per 
square centimetre of bulb surface is obtained by dividing a “ factor,’ deter- 
mined for each instrument by the time of cooling, expressed in seconds, which 
the alcohol meniscus takes to fall from 100° to 95° F., the unit of heat being 
a millicalorie (1000 millicalories = 1 grm. calorie). This is known as the 
“cooling power,” H,and it was found that a connection between the variables 
is given by an expression of the form 

H = (4+6,/v)0 
where v is the wind velocity and @ the difference between the mean tempera- 
ture of the range of cooling, viz., 365° C. (97-7° F.), and the air temperature 
in degrees Centigrade, whilst a and 6 are numerical constants. 

To obtain the constants a and 6,a number of observations of the cooling 
power were taken, the temperature and wind velocity being accurately deter- 
mined at the same time. The results are shown graphically in figs. 1 and 2. 


* L, V. King, ‘ Phil. Trans.,’ A, vol. 214, p. 373 (1914). 


200 Prof. L. Hill, Messrs. H. M. Vernon, and D. Hargood-Ash. 


Each point in these graphs is the mean value obtained from three to seven 
observations of cooling, taken consecutively. Where points coincide the 
observations were taken at different times. Several kata-thermometers were 
used, the factors in each case being verified. The high velocity observations 
were taken in the wind tunnels and the low velocity ones by the whirling 
arm method. Unfortunately, owing to experimental difficulties, it was not 
found possible to make these two methods overlap, the lowest wind tunnel 
velocity available being 3°05 metres per second, and the highest whirling arm 
velocity for the dry “kata” 1°77 metres per second. 

No equation of the above form will apply for all velocities over the range 


30 


2:8 


2-6 


24 


6 7 8 9 10 II I2 13 14 I5 16 17 
Velocity - Metres per second 


Fig. 1. 


2 a ga anes 


tested, but from the value ,/v = 1 to ,/v = 42, that is, between the velocities 
1 metre per second and 17 metres per second, a curve may be drawn to fit the 
points as shown, the equation of which is 


H = (0134047 ,/v) . 0. (i) 


It should be noted that the above limits include observations in air currents 
obtained by both methods. Below 1 metre per second velocity another curve 
may be drawn, of which the equation is 


H = (0:20+0°40,/2) . 0. (ii) 


The Kata-thermometer as a Measure of Ventilation. 201 


These two lines are shown with the experimental points as the lower curves 
in the diagrams. 


0) Ole 0:2 ar: 3aht 0-4 0:5,,' RO-6—e 4071 730-8 250-9. V0 
Velocity-Metres per second 
Fia. 2. 


The Wet “Kata”—Wind Velocity, Temperature, and Evaporation. 

Wet “kata” observations were made in a similar manner to the dry “kata” 
ones, except that the bulb of the instrument was covered with a muslin 
“finger,” which remained moist during the time of cooling. In this case a 
velocity as high as 2°50 metres per second was obtained by the whirling arm 
method. With the wet “kata” heat is lost by the evaporation which takes 
place in addition to the heat lost by convection and radiation, as in the dry 
“kata,” so that the wet cooling power, H’, is always greater than the dry 
cooling power, H. It is evident, then, that in addition to the temperature 
and wind velocities, some term must be introduced to take account of the 
humidity of the air. In the ‘Phil. Trans.’ paper the formula deduced from 
theory and experiment was of the form 


H’ = (a+b,/v)0+(c+d fv) (F—-f)*, 
VOL. XCIII.—B. Q 


202 Prof. L. Hill, Messrs. H. M. Vernon, and D. Hargood-Ash. 


where H’ was the heat lost per second per square centimetre from the wet 
“kata,” » the wind velocity, F the maximum vapour pressure at 36°5° C., 
J the actual vapour pressure of the air at the time of the observation, @ equal 
to the difference between 36°5° C. and the air temperature, and a, b, ¢, and d 
numerical constants. This was afterwards modified to the form 


H’ = (a4 0',/v) 04+(e' +d’ v3) (F—Sf)#3, 
which was given in the later paper. 
Our new investigations give an equation of the form 
H! = (a +b" 4/0) O+(6" +d" 5/0) (Ff), 
but, just as for the dry “kata,” two equations are found to be necessary, one 
for velocities greater than 1 metre per second and one for velocities less than 
this. These equations are 


H’ = (0134047 ,/v) 6 +(0:035 + 0:098 ,/v) (F—)#8 (iii) 
for velocities greater than one metre per second, and 
H’ = (0:204.0:40 ,/v) 0+ (0:060 + 0:073 x/ v) (F—f)#8 (iv) 


for velocities less than 1 metre per second. 

In each case the first term on the right-hand side of the equation repre- 
sents the dry “kata” heat loss. 

These expressions are cumbersome for practical use, so that when the 
question was re-investigated we proposed to try if a formula in which the 
humidity was represented by the wet bulb temperature would give satis- 
factory results; that is to say, a formula of the form 

H’ = (a+b2*) 6’, 
where @’ is the difference between 36°5° C. (97°7° F.) and the wet bulb tem- 
perature, ¢’, a and b being numerical constants, and z some power to which 
the velocity, v, had to be raised. 

When our experimental values of H’/6’ were plotted against the wind 
velocity values, as shown in the upper curves of figs. 1 and 2, it appeared that 
such a relation existed between the variables, and that, considering the whole 
range of velocities from zero up to 17 metres per second, the value z = 1/3 
gave satisfactory results. 

We may, therefore, write an empirical wet “kata” formula in the form 

H’ = (a+ b,/v) 6, 
where H’ is the heat loss per second per square centimetre, v the wind 


velocity, and 0’ the difference between 365° C. (97°7° F.) and the wet bulb 
temperature, ¢’. As before, two equations are necessary to cover the whole 


The Kata-thermometer as a Measure of Ventilation. 203 


range. The constants a and 6, when vis in metres per second, H’ in milli- 
calories per second, and 0’ in degrees Centigrade, are given by the following 
two equations :— 


H’ = (0:104+110 °/v) & for velocities greater than 1 metre per second, (iii) 
H’ = (0:35 +0°85 @/v) 6’ for velocities less than 1 metre per second. (iva) 


Our results with the wet “kata ” in winds are only over a limited range of 
humidities (the wet bulb temperature varying from 9°5° to 19°8° C.), owing to 
the difficulty of controlling the humidity in the large rooms in which the 
wind tunnels are situated. In the case of still air, however, it is possible to 
obtain more varied conditions, and considering the still air wet “kata” 
formula 

H’ = 0270+ 0-085 (F—/)*, (v) 
given in the original paper, it seems probable that for very dry or very moist 
air, equations (iii) and (iv) are more correct than (ilia) and (iva). It is not 
possible to find an equation for still air of the form 

He ab. 

This can be seen at once by putting in fixed values of the wet bulb tempera- 
ture in (v) and varying the humidity. In this way it is found that @ varies 
from 0°5 to 0°7 as the temperature and humidity vary from 10° C. and 
10 per cent. relative humidity to 20° C. and 100 per cent. relative humidity. 
For any given wet bulb temperature the value of @ increases with the 
humidity, the increase being much more rapid at low relative humidities. 

With respect to the wind velocity index, when considering evaporation, we 
find that observers have come to many different conclusions on the matter. 
John Dalton gives the value as unity. A number of late investigators found 
it to be 0°5.* Recent observations made by Bigelow,+ in which he deter- 
mined the rate of evaporation of water from pans 2 to 6 ft. in diameter in 
wind of various known velocities, gave results agreeing with Dalton’s. It 
may be pointed out that for certain ranges of wind velocity, e.g., between the 
velocities of 0°3 and 2°5 metres per second, our results hold for a value of 1 
for this index, but many more than two equations would be necessary to 
cover the whole range of our experimental work with this value, while for the 
index of 1/3 very close agreement is obtained by the use of two equations 
of the same form. The velocities recorded by Bigelow were mainly low. It 
can hardly be expected that the conditions of evaporation from large vessels 
and from such instruments as the “kata,” where the evaporation surface is 


_ vertical and the water only a thin film, should be identical. 


* Of. Hann, ‘ Lehrbuch der Meteorologie, Hd. 3, 1915. 
+ Bigelow, ‘Monthly Weather Review,’ 1910, p. 307. 


204 Prof. L. Hill, Messrs. H. M. Vernon, and D. Hargood-Ash. 


It will be noticed that the rate of cooling, both of the dry “kata” and the 
wet “kata,” begins to take an abnormal course at the same point, viz., an air, 
velocity of 1 metre per second. There can be little doubt that the abnor- 
malities are due to convection currents. Currents of warm air, heated by the 
cooling “kata,” tend to rise vertically from the sides of the bulb, and this air 
considerably impedes the rate of cooling of the “kata” in still air. It can 
have little or no effect in the presence of horizontal air currents of fair 
velocity, as it will be swept aside, but air currents of low velocity are not 
sufficiently powerful to effect this completely, and in consequence the rate of 
cooling is retarded. 

Determination of Swirl. 


It has already been mentioned that in the whirling arm experiments 
account must be taken of the swirl produced ; that is to say, the true velocity 
of the “kata” relative to the air is less than the actual velocity, because the 
air is carried round with the revolving arm to a certain extent. A method: 
similar to that described by L. V. King* was adopted to determine the amount 
of swirl. In the case of the Oxford experiments, a “ kata” was fixed at one 
end of a horizontal arm and it was revolved at the rate of 1, 2, or 3 metres 
per second, and its bulb passed close to the bulb of another “ kata,” which was 
fixed in a stationary position. Cooling observations of the stationary “kata” 
were made while the moving one revolved at room temperature. When the 
centres of the bulbs were 2 cm. apart there was only 2 mm. spacé between 
the bulbs. 

From the series of data obtained we were able to calculate what the H/@ 
values would have been had it been physically possible for the bulb of the 
stationary “kata” to occupy the place through which the bulb of the moving 
“kata” passed in its revolutions. From them it has been possible to calculate 
the velocity of swirl of air against the stationary “kata” bulb by using an 
approximate formula already determined from observations taken without 
allowance being made for swirl. These swirl velocities vary from 6°5 to 
7:96 per cent. of the velocity of the revolving “kata,” but they are all subject 
to a certain deduction. Now the velocity of swirl found by King when using 
a wire was 6 per cent.; therefore, assuming that this value holds likewise 
with the “kata”—the assumption being warranted by the above experi- 
mental results—then it follows that the values must be reduced by 6 per cent. 
This correction has been applied to all the Oxford results. 

Similar experiments at Hampstead gave a value for the swirl of about 
9 per cent., which was deducted from the calculated velocities before 
recording. 

* L. V. King, ‘ Phil. Trans.,’ A, vol. 214, p. 373 (1914). 


The Kata-thermometer as a Measure of Ventilation. 


The Kata-thermometer in Practical Use as an Anemometer. 


205 


To test the accuracy of the wind formule for practical purposes, series of 
experiments have been carried out at Kew Observatory, where observations 
were taken by kind permission of Dr. Chree; at High Beech, Loughton ; at 
Walberswick, by Mr. P. F. Alexander; and at Eskdalemuir, by Mr. P. N. 


Shelton, under the direction of Mr. L. F. Richardson. 


in the Table. 
“kata” agrees well with that given by other anemometers. 


The results are shown 
It will be seen that in all cases the wind as determined by the 


Place of H 6 = (36 -5—2) Velocity Cup Kew Eskdalemuir| Time of 
observation. o cn ‘| “Kata.” | anemometer.|anemometer.| air meter. | observation. 
mins. 
(| 24:2 175 7-1) 6-4) 6-0) an 8 
| 21°5 18:0 5:1 | 5°7 6:0 | —_ 3 
ew ........2| 21:9 18-0 5'435°8| 5:5'5-7 | 6:3$5°9 = 5 
|| 22-9 17°9 6:0 | 5°5 | 4:9 | = 7 
L| 21:9 17:9 5°6 J 5°7) 6:0) — 7 
Aienrk 220 4:7) 4°97 | ” a= 5 
|| 27-0 22-0 5°5 | 57 | | = = 10 
High Beech 4| 25°5 22-2 4745-4] 5°5$5°6 ae a 10 
L 27 °3 21°5 5°9 | 5 °4 | —_— — 6 
28 6 22-0 6-2) 6°5 J = a 6 
(| 35°5 33-2 4:0 4,0 
35 °5 33°2 4°0 4,°4, 
48 ‘0 37 °3 6'1 5°3 
33 ‘1 87 °6 2°5 2°8 
451 37 °5 5:2 5:0 
Walberswick 55°'5 37 °6 8:3 75 
|| 40-4 eyicil 42 4°6 
32 °5 346 3°0 3°0 
| 29:0 28 °2 3°7 4°) | 
32 °0 30 *4 3°8 4°2 
L| 29°4 29°6 3°3 3°O 
(| 23:2 33 6 1°3 — —_— 2°1 
| 34°6 33 °8 3°6 — — 2°9 
} OM Hl 33 ‘1 2°2 = = 24 
24 °2 33 "1 1°6 — —_— ets 
| 22°5 30°3 io — 0-7 
a 40°1 35 °8 4°4 — 4°7 
pe tdalemuir, 268 32-°8 29 TE 2:3 
35 °8 29°5 5:3 — | — 5:2 
31°5 33 °2 30 —_— | —— 2°5 
26-7 32°7 2-2 ey 2-2 
36 °'8 30 °6 5:2 — | — 3°95 
(| 28:0 31°6 2°6 — — 2°4 
Summary. 


From the above considerations then we consider that we are justified in 
saying that the “kata” is an instrument which shows a definite relation 


206 The Kata-thermometer as a Measure of Ventilation. 


between the heat loss and the surrounding conditions, and that this relation 
may be expressed in the form 
H = (a+b,/v)0 
for the dry “kata,” where H is the heat loss per second per square centimetre 
of “kata” bulb surface, @ being the difference between 36°5° C. and the dry- 
bulb temperature, and v the wind velocity in metres per second. The equation 
takes the following numerical forms for air velocities above and below 1 metre 
per second : 
H = (0134047 4/v)@ (ii) and H = (0:20+ 0°40 ,/v) 6. (aii) 

The wet “kata” formule are more complex (see p. 201), but for ordinary 
atmospheric temperatures and humidities an approximation is yielded by the 
formula 

H’ = (a'+0' 0/0) 6, 

where H’ is the heat loss per second per square centimetre of wet “kata” 
bulb surface, and @’ is the difference between 36°5° C. and the wet bulb 
temperature. 

This equation takes the following numerical forms for air velocities above 
and below 1 metre per second, 

H’ = (0:104110./»)0’ (iv) and HH’ =(03540-85./0)6. (vy) 

(It should be noted that the above equations are not true when v is less 
than 0:04 metre.) By means of equations (ii) and (iii) the “ kata” may be used 
as an anemometer, both for estimating wind velocities out of doors, or the 
velocity of air currents indoors for purposes of ventilation. For the latter 
purpose it has a special value, for it estimates cooling effects of air currents 
whether unidirectional or eddying. 


207 


On the Heating and Cooling of the Body by Local Application 
of Heat and Cold. 


By Leonarp Hitt, M.B., F.R.S., D. Harcoop-Asu, B.Sc., and J. ARGYLL 
CAMPBELL, M.D. 


(Received January 7, 1922.) 


(From the National Institute for Medical Research, Hampstead.) 


The object of this enquiry is to find out how much heat can be gained, or 
cold lost from the body, by the local cooling or warming of a small part, ¢.., 
by cooling the hands in astream of cold water, warming the feet in a hot foot- 
bath, or by a foot-warmer. In order to secure the beneficial effect of open 
windows, the breathing of cool air of low-vapour tension, and stimulation of 
body metabolism by such air ventilating the clothed and naked parts of the 
skin, the general heating of rooms by hot-water coils might be replaced by 
small heaters kept a few degrees above body temperature and locally applied 
to each individual, and each under the individual’s control. Electric heaters 
have been used by aeroplanists placed beneath their outer garments. 

One of us (1) recently published results showing that heating or cooling the 
hands can effectively heat or cool the whole body. We record further experi- 
ments of a like nature. 

In some of these experiments, in which the hands were placed in cold 
water, we estimated the respiratory exchange by the Douglas-Haldane method 
of indirect calorimetry. We obtained in most cases a small rise in body 
‘metabolism after immersion of both hands in cold water at 15° C. for about 
30 minutes. The rise in metabolism was too small to be termed definite. 
The amount of heat lost from the hands to the water was evidently replaced 
by cutting down heat lost from other parts of the body. The actual amount 
of heat lost from the two hands in 30 minutes was, on an average, 20 kilo- 
calories. A greater loss of heat is therefore necessary before metabolism is 
affected. 

The temperature of the skin over the median vein at its bifurcation on the 
front of the elbow was recorded by means of a flat-coiled thermometer insulated 
from the air. The temperature of this portion of skin fell several degrees in 
the above experiments. 

Macleod (2) and others have applied heat and cold to the surface of the 
shaved body of rabbits to study the changes of temperature in underlying 
tissues and in various organs—muscle, liver, kidney and brain. They used 
thermo-electric couples mounted in hypodermic needles. They showed that 


208 Prof. L. Hill, Mr. D. Hargood-Ash, and Dr. J. A. Campbell. 


the changes produced vary with vascularity in different tissues. Heat applied 
over the liver had little effect on the liver, compared with that on muscle, of 
heat applied over the same. Applied over the lung both heat and cold had 
little effect; over the brain there was a prompt change of brain temperature 
but little effect on general body temperature. 

In our experiments to determine the amount of heat lost by the hand to 
the water, the hand was placed in a tin can containing a known volume of 
water; a similar can with a similar volume of water and at approximately the 
same temperature was placed beside the former to act asa control. When 
the temperature of the skin over the median vein was found to be constant, 
the hand was immersed in the water as far as the wrist joint. The tem- 
peratures of the water-bath and the control were read immediately before the 


6) 2 4 6 8 10 12 14 16 18 20 


Time - Minutes 
Fic. 1, A. 


immersion of the hand, and readings were then taken of the skin temperature 
at the elbow, and of the bath and control temperatures, every minute. 

To determine the heat given to the water, the temperature readings of the 
bath and control were plotted. Fig. 1, A shows a typical example. The 
initial temperature of the bath was 9°7° C. and of the control 93°C. The 
bath curve, ABC, shows that for the first 3 minutes the rate of rise of 
temperature was rapid compared with the rate afterwards. From the eighth 
to the twentieth minute the rate of rise of temperature may be taken as 
constant. The curve, EF, shows the rise of temperature of the control bath; 
it will be seen that this rate of rise was constant throughout the experiment. 
GH is drawn parallel to EF, therefore the difference between the lines GC 
and GH gives the rise of temperature due to the immersion of the hand. At 


Heating and Cooling of the Body by Local Heat and Coid. 209 


the point JK, 10 minutes from point G, the difference is 0°38° C., therefore 
the rise for 1 minute was 0:038° C. The water in the can was 6 litres, 


_ therefore the rate of heat loss to the water per minute was 0°228 kilo-calories 


during this time. 
Todetermine the total heat loss from the hand during the 20 minutes’ immer- 
sion; the total rise of temperature of the bath was 11°6° C.—9°7° C.=1:9° C., 


O 
° 
vu 
= 
= 
= 
0 
= 
v 
a 
£ 
2 
= 


Hand a from water and dried 


“Gazi Gu LID IG) alin een ac iam 
Time- Minutes 


Fie. 1, B. 


and that of the control 9:8° C.—9:3° C. = 0°5° C.; therefore the total rise due 
to the hand was 1:9° C.—0:5° C., = 14° C., and the total heat loss from the 
hand 1-4 x 6 = 8-4 kilo-calories. 

Fig. 1, B, shows the changes in temperature of the skin over the median 
vein for the 20 minutes’ immersion and the succeeding 20 minutes. The 
temperature of this portion of skin was quite different from that of the skin 
around it and nearer the hand, showing that the temperature of the blood 


210 Prof. L. Hill, Mr. D, Hargood-Ash, and Dr. J. A. Campbell. 


in the vein was the controlling factor, and not conduction by the skin from 
tissues affected by the changes in the hand. 

Pain in the hand was intense during the first few minutes if the tempera- 
ture of the water was below 14° C. This pain may probably be due, in part, 
to the extreme contraction of the tissues, and disappeared probably because 
the fluids escaped up the arm from the contracted tissues in the hand, thus. 
relieving pressure. In our metabolism experiments already referred to, we 
used water warmer than 14° C., to avoid the influence of pain. Pain is 
known to raise metabolism. 

In our numerous experiments, with the hand in cold water, we were unable 
to obtain from day to day constant results for a given range of temperature, 
probably because so many bodily conditions, ¢.g., vascular, are variable. 

The double fig. 2 gives the results of an experiment in which the hand was 
placed in hot water (43°2° C.), fig. 2, A, recording the change in temperature of 
bath and control, and fig. 2, B, recording the changes in temperature of the 
skin over the median vein at the point of the elbow. In this experiment 
the heat gained by the hand in 20 minutes was 6 kilo-calories, and the rate of 
gain, after this had become constant, 0°120 kilo-calories per minute. 

In these experiments with hot water the fall of temperature in the water 
of the bath was considerably greater during the first minute or so after the 
hand was immersed, and it was difficult to tell how long this effect, which 
was probably due to the heating up of the hand, lasted. If this etfect 
was only temporary, and the heat gained depended only upon the tempera- 
ture of the water, we should expect that different experiments would give 
curves parallel to each other over the same range of temperature. This, 
however, did not occur. 

To obtain more certain information on this point it was decided to ~ 
immerse the hand in hot water as before, but, instead of allowing the 
water to cool, to supply a constant amount of heat equal to that lost by the 
water, so that if the heat absorbed by the hand at a given temperature 
proved to be constant, the experiments could be repeated at different 
temperatures to obtain a curve. However, the heat absorbed by the hand 
at a given temperature was not found to be constant, similar variations 
being obtained as with cold water ; varying bodily conditions were probably 
responsible. 

We also carried out numerous experiments with the flat-coiled thermo- 
meter, when the hand as far as the wrist was heated by the summer sun’s 
rays. The temperature of the skin over the median vein was increased 
several degrees after the hand had been exposed for a few minutes. In these 
cases the blood in the vein may have been heated in the hand by conduction 


Heating and Cooling of the Body by Local Heat and Cold. 211 


Temperature °C 


© Hand removed From water and dried. 


Fig IB 


Temp.of skin over 


median vein 


Time in Minutes 


212 Heating and Cooling of the Body by Local Heat and Cold. 


from the skin or by rays which had penetrated the skin and had been con- 
verted into heat in the blood, as pointed out by Sonne (3). He used thermo- 
electric couples to determine the effect of different rays from the sun and 
found that the visible rays penetrate the skin and locally heat up the blood. 
To this he attributes the value of the sun in heliotherapy. Dark heat rays 
heat up the surface of the skin much more. 


Summary. 


Experiments in which the hands were heated or cooled by water 
showed that the amount of heating or cooling was large, but not constant, for 
a given range of temperature. Some indication of the degree of heating or 
cooling was obtained from the temperature of the skin over the median vein 
at the elbow, the thermometer used being coiled and insulated from the air. 
Loss of 20 to 25 kilo-calories of heat from the hands in 30 minutes, 7.¢., a loss 
almost equal to the basal metabolism, did not appreciably affect the body 
metabolism. 


REFERENCES. 


(1) Hill, Leonard, ‘Journ. Physiol., vol. 54, Proc. exxxvii. 

(2) Macleod, Self, and Taylor, ‘Lancet,’ September 25, 1920, p. 645; Macleod and 
Taylor, zbid., July 9, 1921, p. 70. 

(3) Sonne, ‘Acta Medica Scandinavica,’ vol. 54, p. 394 (1921). 


213 


On the Oxidation Processes of the Echinoderm Egg during 
Fertilisation. 


By C. SHEARER, F.R.S. 


(Received November 15, 1921.) 


I. INTRODUCTION. 


The following paper is concerned with an investigation of the oxidation 
processes of the animal egg-cell during fertilisation. The subject has already 
received considerable attention and the problem has been approached from 
many different aspects. The first to attempt to measure in definitive quantita- 
tive manner the oxygen consumption of the egg on fertilisation, was Warburg(1) 
in 1908. He made use of the sea-urchin Arbacia, and estimated the amount 
of oxygen that had disappeared from the sea-water in which the eggs had 
remained for some time. The Winkler titration method was employed. He 
found that a quantity of eggs that gave a Kjeldahl determination of 28 mgrm. 
of egg nitrogen, which corresponds roughly to about 4 million eggs, 4-5 c.c. of 
oxygen was taken up in the first hour following fertilisation, while the same 
quantity of unfertilised eggs only consumed 0:5-0'7 c.mm. of oxygen in this 
time. The fertilised egg, therefore, took up six to seven times more oxygen 
than the unfertilised egg. Loeb had previously predicted, that the main 
function of the sperm in the process of fertilisation was. that of setting up a 
series of oxidations on its entrance into the cytoplasm of theegg. Warburg’s 
work was a remarkable confirmation, therefore, of Loeb’s prediction. This 
first paper was followed up by a long series of papers which have added 
greatly to our knowledge of the oxidation processes taking place in the egg cn 
fertilisation. In addition, our knowledge has also been greatly extended by the 
numerous papers of Loeb, and especially the papers of Loeb and Wasteneys (2) 
in which quantitative measurements were also carried out. In 1911 appeared 
the large paper of Meyerhof(3) in which the heat liberation was measured 
and correlated with the oxygenconsumption. In all these papers the Winkler 
method was employed; there are, however, many drawbacks to the use of 
this method, and in the recent work of Warburg and Meyerhof it has been 
finally abandoned for the more convenient and accurate manometer. The great 
advantage of the manometer method lies in the fact that it can be used equally 
well for both oxygen and carbon dioxide determinations, and that continuous 
observations can be carried out minute by minute on the respiratory exchange 
of the material under investigation. Warburg (4) (1915), using this instrument, 
has recently reinvestigated the respiratory exchange in the egg of the sea- 


214 De C. Shearer. On the Oxidation Processes of 


urchin Strongylocentrotus during the first 24 hours of development. He found 
thata quantity of unfertilised eggs that contained 20 mgrm. of egg nitrogen, 
which corresponds to about 3 million eggs, consumed in 20 minutes 
10-14 c.mm. of oxygen at a temperature 23° C. and barometer 760 mm. Hg. 
The fertilised egg under the same conditions, 10 minutes after the addition of 
the sperm, consumed 60-84 c¢.mm. That is 10 minutes after fertilisation the 
oxygen consumption of the egg was six times that of the unfertilised egg, and 
that there was already a rise of 500 per cent. in the oxidation rate of the egg 
in this time. In the sixth hour the oxygen consumption was twelve times 
that of the unfertilised egg, at 12 hours it was sixteen times, while at 
24 hours it was twenty-two times the amount of the unfertilised egg. As 
Warburg remarks, it is extraordinary that in one and the same cell substance, 
which receives no addition of fresh material from any external source, we 
should find, as the result of fertilisation in the course of 24 hours, a rise in its 
oxidation rate of something like 2000 per cent. On the whole the mano- 
meter method seemed to show that there was a much closer agreement 
between the increase in the respiratory quotient and the appearance of visible 
structure in the egg, than had been demonstrated in previous work where 
the Winkler titration method had been employed. In all instances the CO, 
output of the eggs followed closely the oxygen uptake, the respiratory quotient 
being in the neighbourhood of 0°9._ The respiration of a single spermatozoon 
was found to be about 1500-2000 times less than that of the egg. 

In the past season, working at Naples, I have been able to carry the 
investigation of the problem a step farther, by the use of a special type of the 
Barcroft differential manometer, in which it was possible to bring about the 
fertilisation of the eggs in the closed chamber of the apparatus, and so for the 
first time the measurement of the respiratory exchange during the period the 
sperm were actually making their way into the egg was rendered possible. 
The eggs and sperm of Hchinus microtuberculatus were used. 


II. Meruops. 


In the previous experiments of Warburg (4) (1915), where the manometer has 
also been employed, readings were only obtained 10 minutes after the addition 
of the sperm. The following experiments will show that, by the end of this 
time, the most important part of the process of the fertilisation of the egg has 
already taken place. There is a great initial inrush of oxygen into the 
ege anda corresponding output of CO, within the first minute after the 
addition of the sperm to the eggs. It is clear that the spermatozoon sets up 
an instantaneous oxidation process in the egg, which is perhaps unparalleled 
in reactions of the animal cell for its sudden character. 


the Echinoderm Egg during Fertilisation. 215 


In the type of manometer employed in the following experiments the 
instrument, when used for ordinary blood-gas measurements is designed to 
allow a small quantity of potassium ferricyanide, held in a small tube in the 
stopper of the manometer chamber, to run down and mix with the blood 
when the chamber itself is slightly rotated, so that a groove in the chamber 
wall comes to lie opposite the opening of the small tube in the chamber 
stopper. The instrument was readily adapted to the purpose of the present 
experiments by replacing the ferricyanide by a drop of fresh sperm. The 
egos could then be fertilised within the closed chamber of the apparatus, 
when both eggs and apparatus were in complete equilibrium with the 
temperature of the water-bath in which the manometer chambers were 
submerged. 

The calibration of the manometers for oxygen was carried out by the 
Hoffmann (5) method, and for CO: by the liberation in the chamber under 
calibration of a known volume of this gas from 2 c.c. of a NagCO3 solution. 
All calibrations were carried out under the conditions holding for the follow- 
ing experiments. The temperature of the water of the thermostat in which 
the chambers of the manometer were submerged was kept constantly at 
145° C. by means of a coil of piping inside the bath, through which cold 
water was kept circulating. This temperature is almost the same as that at 
which the eggs are normally fertilised in the sea. Moreover, it is convenient, 
as the solubility of CO: in sea-water at this temperature and standard 
barometric pressure is almost 1, so that the CO, will distribute itself evenly 
throughout both sea-water and the air space of the apparatus chambers, and 
no correction need be introduced for solubility of the CO: in the sea-water on 
this account, 

Warburg (4), in his measurement of the COs, has taken elaborate means to 
determine the actual amount of CO, given off, by using bicarbonate-free sea- 
water and estimating the total alkali reserve of his eggs; it is probable that 
the use of bicarbonate-free sea-water in itself introduces conditions that 
render respiration far from normal. 

In a number of determinations made on the solubility of CO2 in sea-water 
at the pressures obtaining under the conditions holding in the chambers of 
the manometer in the following experiments, it was found this solubility 
could be neglected, as it was so small in each case as to be well within the 
error of the manometer reading.* It is only at tensions of 0-4 mm. pressure, 


* The change of pressure in the chamber of the apparatus during an experiment was 
very small, being something of the order of 0005 of an atmospheric pressure. The effect 
of this on the solubility of carbon dioxide in the sea-water of the chamber could safely 
be neglected. 


216 Dr. C. Shearer. On the Oxidation Processes of 


or below this, that a great difference is made in the solubility of CO, in sea- 
water. Tensions of this order never obtained in the chambers of the mano- 
meter under the conditions of the following experiments. In the following 
experiments no special importance was attached to the carbon dioxide measure- 
ments beyond determining roughly the relation of the carbon dioxide output 
to the oxygen consumption. No trouble was taken, therefore, to determine 
the CO, output with any degree of accuracy. It is clear that it follows the 
oxygen consumption very closely, the respiratory quotient being always in 
the neighbourhood of 0°9. 

To absorb the carbon dioxide in the oxygen manometer, a drop of KOH 
was placed in the small cup in the bottom of the chamber stopper, and this 
was renewed after each experiment. 

The manometers were made to clamp on a mechanical shaker, such as that 
used when the instrument is employed for making blood-gas determinations. 
The motion of this shaker had to be slowed down very considerably into a 
very gentle to-and-fro motion, or otherwise considerable injury resulted to 


the ege-membrane. Any injury to the ege-membrane always results in an §f 


immediate and abnormal increase of the oxygen consumption of the ege. It 
was also found that the conical pointed type of chamber with which the 
instrument is furnished for blood-gas work was highly unsuitable for egg 
work. The eggs tend to crowd down in the narrow end of the bottle and 
there clump together, and fertilise very badly. The conical chamber was 
therefore replaced in the following experiments by a more or less spherical- 
ended pattern. In making a determination, invariably 2 c.c. of eggs in sea- 
water were placed in one chamber, while the same quantity of plain sea- 
water was placed in the control chamber. The chambers were then attached 
to the manometer and half submerged in the water of the thermostat ; a drop 
of very dilute fresh sperm was then introduced by a fine pipette into the 
tube in the stopper of the chamber containing the eggs, through a small 
opening in the top of the stopper. When the glass plug closing this opening 
had been replaced, the chambers of tle manometer could be completely 
submerged in the water of the thermostat with the stop-cocks open. It was left at 
least 20 minutes with the shaker working, to come into complete equilibrium with 
the temperature of the water of the bath. 

If the eggs are not properly cooled to the bath temperature, or if the sperm, 
when run down on the eggs, are at a different temperature from the eggs, 
faulty manometer readings will be obtained. To avoid any such errors in the 
present experiments, great care was always taken to have the eggs, sperm, 
and manometer, all at exactly the same temperature as the water of the 
thermostat tank before commencing an experiment. The eggs and sperm, 


the Echinoderm Egg during Fertilisation. 217 


after being washed several times in clean sea-water, were placed in large 
open-mouthed bottles suspended in the water of the thermostat. They were 
taken from these bottles as required for an experiment and were therefore 
always at the same temperature as the bath water. The pipettes used to 
measure out the eggs and sperm were always kept in separate bottles of sea~ 
water when not in actual use, these bottles being also suspended in the 
water of the bath. The manometers, finally, were always kept in position on 
the thermostat when not in use, so that'when required for an experiment 
they were already thoroughly cooled to the temperature of the bath. As 
already mentioned, in addition to these precautions 20 minutes was always 
allowed after the eggs and sperm were placed in the manometer chamber 
before the stop-cocks were closed and the first reading taken. It was found 
from preliminary experiments in which plain sea-water was used instead of 
eges and sperm, that when these conditions were observed in performing an 
experiment no cooling effect could be produced, and that, under these 
conditions, the first reading on the manometer was perfectly accurate and 
satisfactory. In revolving the chambers on the manometers the stop-cocks 
of the apparatus were opened and immediately closed a moment later. It 
was possible that, in the operation of revolving the chambers, they might be 
slightly loosened on their respective stoppers and so the oil drawn round, and 
a faulty reading obtained in this way. This was avoided if the stop-cocks of 
the instrument were open while the chambers were revolved. The chambers 
of my manometers were so accurately ground on to their respective instruments 
that I was unable, as a matter of fact, to produce any such error experi- 
mentally when I turned the chambers while the instruments were closed. 
An error of this kind could only be produced by actually forcing the chambers ° 
completely loose from their manometers. Observing the foregoing precautions 
then, at the end of 20 minutes the hands of the manometer stop-cocks could be 
closed and readings commenced on the unfertilised eggs. The same eggs 
could then be fertilised by turning the chambers and allowing the sperm to 
tun down on the eggs. There was a small error in this reading due to the 
presence of the drop of sperm in the small tube in the manometer stopper, 
but care was always taken to use very dilute sperm so that this error could 
be neglected. To rotate the chambers, when they were attached to their 
instruments and the manometers themselves in position submerged in the 
water of the thermostat, wooden handles were wired on the necks of each 
chamber. These were long enough to project well above the surface of the 
water, so that the warm hand or fingers never came in close contact or 
touched the manometer chamber when they were rotated. Separate mano- 
meters were used for the oxygen and CO2 measurements, the output of CO, 
VOL, XCIII.—B. R 


218 Dr. C. Shearer. On the Oxidation Processes of 


55 


50 FERTILIZED 


45 


40 


35 


304 


254 


204 


104 


UNFERTILIZED 


0 if 5 MINUTES. 10 


Fic. 1.—Graph showing the amount of oxygen taken up and the carbon dioxide given 
off during the fertilisation of half a million eggs (4:06 mgrm. of egg N) of 
E. microtuberculatus. The lower line shows the oxygen consumption of the same 
egg before fertilisation. Broken curve carbon dioxide output of eggs. Respiratory 
quotient 0°92. The quantities of oxygen and CO, are given in cubic millimetres 
plotted against time in minutes. 5 


the Echinoderm Egg during Fertilisation. 219 


being arrived at by deducting the readings of the COz manometer from the 
oxygen apparatus. 

It was inevitable that a considerable difference in the amount of oxygen 
consumption on fertilisation should have been observed between different 
batches of eggs and sperm from different females and males. In some 
individuals the generative products were naturally more ripe and in better 
condition than in others. As the eggs and sperm for experiments of this 
kind have to be cut out of the animals and not laid by the animals in the 
normal manner in the sea, there is always some en as to their being 
perfectly mature. 

The eggs can only be roughly tested by adding a little sperm to a few eggs 
in a watch crystal and seeing how evenly and quickly fertilisation takes 
place. As Warburg (4) remarks, it is obviously necessary in determinations of 
this kind that fertilisation should take place in all the eggs at the same 
moment, and that the rate of progress of this process, once it is set up in the 
eggs, should be the same in all the eggs. These conditions are not always 
successfully hit offin an experiment. In a certain small number of experi- 
ments out of some 200 to 300 performed, these conditions were probably as 
favourable as the experimental conditions would allow, and in these the 
readings were remarkably similar. In the following section the figures of 
three typical experiments of this kind are given. A graph of the readings is 
shown in figs. 1, 2, and 3, where the oxygen consumption in cubic millimetres 
is given plotted against the time in minutes. 


III. EXPERIMENTS. 


Unfertilised Eggs. (20 mgrm. egg N.) 

The oxygen consumption of the unfertilised egg is so low that it is quite 
unmeasureable with any degree of accuracy, anlews a much larger number of 
eggs is used than when a determination is made on fertilised eggs in the 
ordinary way. Instead of using one to half a million eggs, 4 to 5 millions 
(20 mgrm. egg nitrogen) were used in arriving at the correct estimation of the 
respiration of the unfertilised eggs. 

The average (15 determinations) amount of oxygen taken up by 4 to 5 
million unfertilised eggs, in 10 minutes, under the conditions of the experi- 
ments was 7 c.mm. oxygen; at this rate, half a million eggs (4 mgrm. of egg 
nitrogen) would consume 1°5 c.mm. in this time. 


RZ 


220 Dr. C. Shearer. On the Oaidation Processes of 


Fertilised Eggs. 
Oxygen Manometer. (4:06 mgrm. egg N.) 


1st minute after addition of sperm 12 c.mm. oxygen consumed. 


2nd 5 i; 17-0 cmm. oxygen consumed. 
3rd 5 : 20:0 - 2 : 
4th % 7 26:0 5 3 

5th 3 “ 32°2 Fe ; 

6th , s 360 i) = 

7th ‘ i, 41-4 99 

8th <3 5 46°8 1 0 

9th ef i; 50:0 es s 

10th 3 i 56:0 5s 


Carbon Dioxide Manometer. (4°08 mgrm. egg N.) 


At end of 2 minutes after addition of sperm difference was 2 c.mm. 


» 5 29 ” 9 4 » 
~ 

” 8 22 9 2) 9) ” 

? 10 PP) 72 39 6 ” 


At the end of the experiment the manometer chambers were opened, when 
it was found that the eggs in both chambers had all formed normal fertilisa- 
tion membranes and consequently all formed regular two-cell segmentation 
stages. A Kjeldahl determination on the eggs showed that in the case 
of the eggs in the oxygen chamber 406 megrm. of egg nitrogen were 
present, and in the case of the CO. chamber 4:08 merm. of egg nitrogen were 
present. 

Tt will be seen from the foregoing Tables and graph, fig. 1, that on addition 
of the sperm to the eggs there is an immediate consumption of oxygen. In 
the course of the first minute the uptake of oxygen is many times that of 
the same eggs one minute before the addition of the sperm, and more is 
usually taken up in the first minute than is taken up in the second and third 
minutes after the addition of the sperm taken together. 

In all instances the CO: output of the eggs follows the oxygen uptake very 
closely, the respiratory quotient being in the neighbourhood of 0°92. 

In fig. 2 is shown a graph of another experiment similar to the former, 
in which half the quantity of eggs were employed (2°08 against 4:06 mgrm. 
egg nitrogen). It will be seen both curves are similar, the later being half 
the value of the former. 

At standard barometric pressure, and temperature of 14:5°C., 4:06 mgrm. 
of egg nitrogen (half a million eggs) which before fertilisation consumed 


the Echinoderm Egg during Fertilisation. 221 


15 c.mm. of oxygen in 10 minutes, after fertilisation consumed 56 ¢.mm. in 
this time ; there was thus an increase in the respiratory quotient of the eggs 
in 10 minutes after fertilisation of something like 37 times that of 
the unfertilised condition. If we consider the increase taking place at the 
end of the first minute after the addition of the sperm to the eggs, we get 


25] 


FERTILIZED 


20 


154 


10; 


0 5 MINUTES. 10 
Fic. 2.—Graph showing the amount of oxygen taken up and the carbon dioxide given 
off during the fertilisation of a quarter of a million (2:08 mgrm. egg N.) eggs of 
E. microtuberculatus. It will be seen that this graph is similar in all respects with 
that shown in fig. 1, except that it is half the size of the former, half the quantity 

of eggs being used. 


even more striking figures. The oxygen consumption of the unfertilised 
egg is, we have seen (15 determinations), about 15 cmm. oxygen for 
4 mgrm. of egg nitrogen in 10 minutes, and in the case of the present 
experiment a reading of this value was actually obtained on the unfertilised 
egg. If we divide this figure by 10 we arrive at the value of the oxygen 


222 Dr. C. Shearer. On the Oxidation Processes of 


consumption for 1 minute = 015¢mm. The same eggs fertilised consumed 
in the first minute after the addition of the sperm 12 c.mm. of oxygen. 
Thus, the addition of the sperm to the eggs causes, within the space of 
1 minute, an increase in their oxygen consumption of something like 
80 times that observed on the same eggs 1 minute previous to the addition 
of the sperm. 

The examination of sections of fixed material of the eggs of #. micro- 
tuberculatus during different stages of the process of fertilisation shows that 
the sperm take at least 10 to 15 minutes to enter the cytoplasm of the 
egg. In material fixed within 2 minutes of the sperm being added to the 
eggs, the sperm are seen only attached to the external surface of the egg 
membrane. They have not penetrated the membrane itself. 

This initial oxygen consumption of the egg immediately on fertilisation 
must be brought about by the first contact of the sperm with the external 
surface of the egg membrane. We arrive then at the remarkable conclusion 
that contact of the spermatozodn with the external surface of the egg is 
capable of increasing its oxygen consumption in 1 minute by something 
more than 8,000 per cent. Im fact, the total oxygen consumption of the 
eggs shown in figs, 1 and 2, represents some change brought about in the 
egg by the spermatozoon before it has entered the egg, and before it has 
formed a male pronucleus in the egg cytoplasm. It is moreover clear, that 
when the fusion of the male and female pronucleus takes place at a later 
stage of the process, it is not accompanied by any fresh rise in the oxygen 
consumption of the ovum, but instead, a slight drop in the curve is often 
observed about this time (see fig. 3). The nuclear features of syngamy 
therefore, seem connected in no direct way with the oxidations taking 
place in the ovum during fertilisation. 

In fig. 3 is shown a graph of an experiment which illustrates these 
points. It represents the amount of oxygen taken up and the carbon 
dioxide given off during the fertilisation of a quantity of eggs that con- 
tained 2°8 mgrm. of egg nitrogen. The curve extends over a period of 
1 hour after the sperm were added to the eggs, and so to a time when the 
first segmentation division has been completed and the egg has attained the 
two-cell stage. The readings are in 2-minute intervals up to the end of 
10 minutes, and after this at 5-minute intervals. At the end of 2 minutes 
10°5 c.mm. of oxygen had been consumed, which compares favourably for this 
quantity of eggs, with the figures obtained in the previous experiments, the 
graphs of which are shown in figs. 1 and 2. 

It will be seen that at the end of 15 minutes the curve begins to 
appreciably flatten, and this flattening increases at the 25th minute when 


ee 


the Echinoderm Egg during Fertilisation. 223 


the fusion of the pronuclei is taking place. At the 40th to 45th minute 
it begins to rise again and this synchronises with the first segmentation 


CMM. 

704 O2 ‘ 6, | 
+ | 

604 Ole 

+ 

50 “~ 

40 ‘ 

304 + 

20 

10 

0 eas 20 "40 MINUTES. 60 


Fie. 3.--Graph showing the oxygen consumption and carbon dioxide output of a quantity 
of L. microtuberculatus eggs that contained 2°8 mgrm. of egg nitrogen during the 
course of the first hour following fertilisation. It will be seen that the steepest 
part of the curve is in the first 15 minutes, while the sperm are in relation with the 
egg membrane. At the 25th minute, when the male and female pronuclei are 
undergoing fusion, there is a slight flattening of the curve. 


division. The steepest part of the curve is that in the first 10 minutes 
interval, which corresponds to the action of the sperm on the external surface 
of the egg membrane. 


224 Dr. C. Shearer. On the Oxidation Processes of 


As the supply of free energy required by the developing ovum is mainly or 
entirely derived from its oxidation processes, it is surprising to find that 
during fertilisation, where a very large amount of energy is immediately 
rendered available, none of it is liberated as the result of the fusion of the 
egg pronuclei, a feature which heretofore has always been considered the most 
essential and important part of the whole process. 

It is of interest in this connection to compare roughly the oxygen con- 
sumption of the ovum on fertilisation with that of the fixed tissues of the 
adult body. If we take liver tissue as one in which the metabolic rate is high, 
according to Barcroft and Shore (6), 1 grm. of fresh liver tissue from a well-fed 
cat consumes 0024 cc. of oxygen per minute, which is equivalent to 
240 cmm. in 10 minutes. If we take Schryver’s(7) figures of the protein 
nitrogen-content of 1 grm. of well-fed cat’s liver as 22-4 merm. nitrogen, we find 
the oxygen uptake of the ovum in the first 10 minutes of the fertilisation 
process, as compared with that of a similar quantity of liver protein in the 
same time, is in about the ratio of 13°8 to 10°7 in round figures. In the 
unfertilised ege this proportion is 0°37 to 10°7 for the same time. 


IV. DISCUSSION. 


There are good reasons for believing, as the result of Loeb’s (8) experiments 
on the fertilisation of the eggs of Strongylocentrotus with the sperm of Asterias, 
and Lillie’s (9) description of the process of fertilisation in Vereis, that the entry 
of the spermatozo6n into the egg consists of two distinct phases. Firstly, an 
external one, in which certain changes are brought about in the cortical 
substance of the egg the moment the sperm make contact with the external 
surface of the egg-membrane; this would seem to be correlated with the 
initial oxidation taking place in the egg, as described above for H. mécro- 
tuberculatus. Secondly, the changes following the actual entry of the sperma- 
tozoon into the egg cytoplasm itself, which, as Lillie has shown in JWerezs, only 
takes place some 30 minutes after the first phase of fertilisation, and in the 
sea-urchin follows some 10 to 15 minutes after the sperm are added to the 
eges. 

By centrifuging the eggs of Nereis before the sperm has actually penetrated 
the ege-membrane, Lillie was able to separate the jelly surrounding the egg 
and containing the spermatozodn from the egg itself. These eggs complete 
meosis, which has been initiated by the spermatozoén, but never segment. A 
typical segmentation nucleus is, however, formed, which breaks down leaving 
the chromosomes free in the egg cytoplasm ; they split longitudinally in the 
normal manner, but never separate. No asters or miotic spindle appear in 
these eggs, as when the complete process of fertilisation is allowed to take 


the Echinoderm Egg during Fertilisation. 225 


place ; and in the absence of these structures the process of cell division makes 
no further progress, and the chromosomes finally degenerate and break down. 


This experiment clearly proves that the sperm bring about profound altera- 


tions in the egg while still external to the egg-membrane. Loeb (8) has shown 
that when the eggs of Strongylocentrotus are fertilised with the sperm of 
Asterias, in hyper-alkaline sea-water, they only form fertilisation membranes : 
no actual segmentation takes place unless the eggs receive further treatment 


‘so that artificial parthenogenesis is induced. 


Meyerhof and Warburg in many of their experiments have shown that 


any injury or cytolysis of the egg-membrane is invariably followed by a great 


increase in the oxygen consumption of these eggs. Meyerhof (10) found that 
this is usually accompanied by an increased liberation of heat. In eggs 
treated with weak solutions of NaCl, in which the normal condition of the cell- 
wall is destroyed in the absence of Ca and K ions, the rise of oxygen con- 


‘sumption was five times that of the same quantity of untreated eggs. The 


heat production was increased from 0:9 grm.-calories per hour to 3°4 grm.- 


calories per hour, after treatment with valerianic acid, by which artificial 


membrane formation had been induced. 
A great many of Loeb’s and Warburg’s experiments point conclusively to 


‘the cortical layer of the egg and the ege-membrane as being the controlling 


factor in the oxidation processes of the ege. Any change brought about in 
these is immediately reflected in the oxygen uptake of the egg. Loeb has, of 


course, based his method of producing artificial parthenogenesis on the fact 
that alteration of the surface layer of the egg renders the commencement of 
development possible. But how can the cytolitic destruction of the surface 


layer of the egg lead to development? Warburg has shown that there are 


good reasons for believing that the oxidations taking place in the egg oceur 


mainly at its surface, for NaOH, which does not diffuse into the egg, raises 
the rate of oxidations more than NH,OH, which readily diffuses into the egg. 
Moreover, he found (11) that the addition of iron* salts to the broken up eggs, 


-or acetone egg powder, was followed by a considerable increase in the oxygen 


consumption of these egg preparations. If the iron-content of the egg 
powder was doubled, the uptake of oxygen was also doubled. He found 
marked traces of iron in the sea-urchin egg. He suggests that the iron 
probably acts the part of a catalyser. If the iron were located in the lipoid 
layer of the egg in a condition in which it was unable to act, some slight 


-alteration in this layer, due to the action of the sperm, might render it active 


or bring both the iron and the oxidisable substrate into a condition in which 


they could quickly interact. We know from Thunberg’s work (12) that lecithin 


* Warburg found 0:03 mgrm. iron per gramme dried egg substance. 


226 Dr. C. Shearer. On the Oxidation Processes of 


in a watery suspension consumes considerable oxygen in the presence of iron 
salts. The egg of the sea-urchin contains considerable quantities of this 
lipoid. 

Warburg (13) has pointed out that there are many points in which the meta- 
bolism of the fertilised egg resembles that of the yeast cell. In each it has. 
been shown that structure plays a very important part, as acetone prepara- 
tions of both the egg and the yeast-cell retain considerable respiratory power. 
Meyerhof (14) finds, however, that if acetone yeast is well washed with water, 
it soon loses its capacity to take up oxygen. If a little watery extract of yeast 
is added to the washed yeast, it immediately regains its lost respiratory 
power. In the water used in washing the yeast Meyerhof found the presence: 
of some compound containing the (SH) group. Hopkins (15) has recently 
isolated from the yeast cell a substance which is undoubtedly closely related, 
if not identical with this respiratory body of Meyerhof. It proves to be a. 
combination of two amino-acids, glutamic acid and cystine, to which Hopkins. 
has given the name of glutathione. This dipeptide possesses most remark- 
able properties in that, in the reduced (SH) form, it can take up molecular 
oxygen, while in the oxidised (S-S) form so produced it can act as a. 
hydrogen acceptor, and can catalyse oxidations of the Wieland type, in 
which no activation of oxygen probably takes place, but an activation of 
hydrogen occurs instead. In the presence of a suitable acceptor the 
hydrogen is removed and the oxidation of the original substance takes place.. 
It can therefore be both reduced and oxidised under the influence of factors 
known to be present in the tissues themselves. Moreover, it possesses: 
precisely those properties which a co-ferment adapted to an oxidase system 
would possess, and at present stands entirely in a class by itself. Hopkins. 
has shown that it is present in most living cells, but he could find no trace of 
it in the hen’s egg, although it was very obviously present in the 30-hour chick. 

I find, however, that in the ripe eggs and sperm of £, miliaris it is invari- 
ably present in an appreciable quantity in the reduced form, but one minute: 
after fertilisation the same eggs give a very deep magenta colour by the nitro- 
prusside test. It is very readily washed out of the eggs by warming them: 
with a trace of acetic acid in sea-water: the washed eggs then no longer give: 
the test. In the unripe egg,in which the nucleus is plainly visible, I could 
find no trace of its presence by the nitro-prusside test. In the ripe eggs it is. 
present in the reduced form in very variable quantities, no two females giving 
the same result, probably depending on varying degrees of ripeness of their 
gonads. In a number of samples of ripe sperm it seemed to be present in 
much less quantity than in the eggs; but here again in two samples of sperm: 
it was present in much greater quantity than in any of the eggs examined. 


the Echinoderm Egg during Fertilisation. 227 


It is, of course, possible that the dipeptide is present in considerable 
quantity in the eggs and sperm in the oxidised (disulphide) form, and that 
during fertilisation it undergoes reduction. It has, of course, long been 
known that the hydrogen ion concentration of the sea-water exercises a 
marked influence on the uptake of oxygen by the egg of the sea-urchin. 
Thus Warburg (13) found that an increase in the HO concentration in the sea- 
water in which the eggs of Strongylocentrotus were placed from 107§ to 107? 
increased the oxygen consumption of the eggs from 14to 81. Hopkins 
finds that glutathione in the oxidised form is rapidly reduced by fresh 
tissues, but that this reduction is greatly accelerated if the reaction or Py of 
the medium is well on the alkaline side of neutrality, while an acid reaction 
greatly retards this reduction. In a similar manner the oxygen uptake of the 
egg-cell is accelerated by alkali and retarded by acids. 

That the glutathione is readily washed out of the eggs is shown by a 
slight pink colour the wash-water gives by the nitro-prusside test. In 
certain experiments in which the unfertilised eggs were treated so that their 
glutathione was washed out, I could find no trace of respiratory power on 
the part of these washed eggs. The same eggs unwashed showed well- 
marked respiration. The oxygen consumption of the unfertilised egg is so low, 
however, that it is perhaps unfair to assume on this ground that glutathione 
is the sole body concerned in the respiration of the ovum. Prof. Hopkins 
has been so kind as to undertake certain experiments with washed 
ege material for me, and he tells me that on the addition of glutathione to 
these ege preparations, the reduction of this in the presence of fresh tissue 
was markedly greater in the case of the fertilised washed eggs than in the 
case of the unfertilised. There seems to be fairly substantial ground then 
for believing that there is an immediate increase in the quantity of this 
remarkable body in the ovum on fertilisation. 

It has been mentioned that cystin is one of the amino-acids entering into 
the composition of glutathione; itis interesting to note that Warburg (16) 
has recently drawn attention to the fact that cystin, absorbed on the surface 
of carbon particles, is capable of considerable respiration, taking up oxygen 
and giving off carbon dioxide. He found that one gram of blood carbon dis- 
solved in a similar weight of a 1/500 N cystin solution, took up the same 
quantity of oxygen asa similar weight of liver tissue. The carbon-cystin 
system, moreover, under the action of oxygen, gives the same end-products 
as the combustion of egg-white, that is carbon dioxide, ammonia and 
sulphuric acid. 

There are a number of other interesting points brought up by the presence 
of glutathione in the germ-cells of the sea-urchin and the possible réle it 


228 Dr. C. Shearer. On the Oxidation Processes of 


may play in the oxidation processes of the ovum. There can hardly be 
much doubt, therefore, that its investigation in the future will reveal many 
new and hitherto unsuspected facts with regard to the oxidations taking place 
in the ovum on fertilisation. - 


V. SUMMARY. 


1. By the use of a special type of the Barcroft differential manometer the 
oxygen consumption and the carbon dioxide output of the egg of Z. micro- 
tuberculatus has been measured during the period the sperm are actually 
making their way into the egg. The eggs were fertilised in the closed 
chambers of the apparatus and their respiration observed before and during 
fertilisation. 

2. It has been shown that during fertilisation the sperm within a minute 
of their being added to the eggs bring about an immediate increase in their 
oxygen consumption. 

3. The study of sections of fixed material of the eggs during the process of 
fertilisation, shows that within 2 minutes of the sperm being added to the ~ 
eggs they have not penetrated the egg membrane. They are only in contact 
with its external surface. 

4, This contact of the spermatozoon with the external surface of the egg 
membrane, however, is capable of increasing in the space of a minute the 
oxidation rate of the ovum by something more than 8000 per cent. 

5. During fertilisation there is more oxygen taken up in the first minute 
of the process than at any subsequent interval of the same time. 

6. The carbon dioxide output of the eggs during fertilisation closely follows 
the oxygen consumption, the respiratory quotient varying from 0:9 to 0°95. 

7. The curve of the oxygen consumption of the ovum during fertilisation 
points conclusively to the stage when the sperm are in contact with the egg 
membrane as the most important part of the process. 

8. The fusion of the male and female pronuclei in the later phases of 
fertilisation is correlated with no additional increase in the oxygen con- 
sumption of the egg-cell. 

9. The oxidation rate of the ovum on fertilisation is probably considerably 
greater than that of any of the adult body tissues, while the oxidation rate of 
the mature unfertilised ovum is very much less than that of any adult body 
tissue. 

10. The ripe sperm and eges of EH. miliaris contain appreciable quantities 
of the dipeptide glutathione in the reduced (SH) form. In the egg 1 minute 
after fertilisation it is found in much greater quantity than in the unfertilised 
condition. It can be washed out of both the fertilised and unfertilised egg. 


the Echinoderm Egg during Fertilisation. 229 


The washed eges no longer give the nitro-prusside test. In the immature 
germ cells it seems absent, at least in the reduced form. 


REFERENCES. 


(1) Warburg, O., “ Beobachtungen tiber die Oxydationsprozesse im Seeigelei,” ‘Zeit. f. 
Physiol. Chem.,’ vol. 57, p. 1 (1908). 

(2) Loeb and Wasteneys, “‘ Die Beeinflussung der Entwicklung und der Oxydations- 
vorginge im Seeigelei,” ‘ Biochem. Zeit.,’ vol. 37, p. 410 (1911). 

(3) Meyerhof, O., ‘ Untersuchungen tiber die Warmetinung der Vitalen Oxydations- 
vorginge in Hiern,” I, II, III, ‘ Biochem. Zeit.,’ vol. 35, p. 246 (1911). 

(4) Warburg, O., ‘‘ Notizen zur Entwicklungsphysiologie des Seeigeleies,” ‘ Arch. f. ges. 
Physiol.,’ vol. 109, p. 324 (1915). 

(5) Hoffmann, “A Simple Method of Calibrating the Differential Blood-gas Apparatus,” 
‘Journ. Physiol.,’ vol. 74, p. 272 (1914). 

(6) Barcroft, J.,and Shore, “The Gaseous Metabolism of the Liver,” ‘Jr. Physiol.,’ 
vol. 45, p. 296 (1912). 

(7) Schryver, 8. B., “Studies in the Chemical Dynamics of Anima] Nutrition,” ‘ Biochem. 
Jr.,’ vol. 1, p. 123 (1906). 

(8) Loeb, J., ‘Artificial Parthenogenesis and Fertilisation,’ Chicago, 1913. 

(9) Lillie, F., ‘Problems of Fertilisation,’ Chicago, 1919. 

(10) Meyerhof, O., “‘Die Atmung der Seeigeleier (S. /vidus) in reinen Chlornatriumlésung,’” 
‘Biochem. Zeit.,’ vol. 33, p. 291 (1911). 

(11) Warburg, O., “Uber die Rolle des Eisens in der Atmung des Seeigeleis, nebst. 
Bemerkungen iiber einige durch Eisen beschleunigten Oxydationen,” ‘ Zeit. 
Physiol. Chem.,’ vol. 92, p. 231 (1914). 

(12) Tunberg, T., “Unterschungen iiber autoxydable Substanzen und autoxydable 
Systeme von physiologischem Interesse,” ‘Skand. Arch. Physiol.,’ vol. 24, p. 90: 
(1911). 

(18) Warburg, O., “ Beitrage zur Physiologie der Zellen,” ‘Ergebnisse der Physiologie,’ 
vol. 14, p. 254 (1914). 

(14) Myerhof, O., ‘“ Unterschungen zur Atmung getiteter Zellen,” ‘ Arch. f. ges. Physiol.,’ 
vol. 170, p. 367 (1918). 

(15) Hopkins, F. G., “On an Autoxidisable Constituent of the Cell,” ‘Biochem. Jr.,’ 
vol. 15, p. 286 (1921). 

(16) Warburg, O., “Physikalische Chemie der Zellatmung,”’ ‘Festschrift d. Kaiser- 
Wilhelms-Gesellschaft,’ Berlin, 1921, p. 224. 


230 


The Depressor Nerve of the Rabbit. 
By B. B. Sarkar. 


(Communicated by Sir E. Sharpey Schafer, F.R.S. Received October 31, 1921.) 


(From the Department of Physiology, Edinburgh University.) 
[PiateE 4.] 


Since the discovery of the depressor nerve,* much work has been done in 
connexion with its important influence on the regulation of blood-pressure, 
but (so far as I am aware) no attempt has been made to determine its 
histological structure. 


Origin and Cowrse of the Depressor. 


Cyont gives the following description of the origin of the nerve. The depressor nerve 
in the animals worked upon usually begins with two branches at the point of departure 
of the superior laryngeal nerve from the vagus, one from each of the two nerves. The 
nerve soon after its origin passes towards the cervical sympathetic, in company with 
which it descends the neck towards the inferior cervical ganglion. With this ganglion 
it is often connected by fine branches: it then turns inward past the subclavian artery, 
and loses itself at the base of the heart, to which it passes from behind between the 
pulmonary artery and the aorta. Just before entering the heart tissue the two 
depressors lie close to one another. 

This is stated by Cyon to represent the course of the nerve in rabbits, cats, and horses, 
and probably in other mammals in which the cervical sympathetic runs separately from 
the vagus. 


My own observations have been made upon rabbits. I have not been 
able to substantiate the statement that a separate depressor is present 
in the cat, and I have had no opportunity of investigating the subject in 
horses. : 

Besides making a number of sporadic observations on animals killed for 
different purposes, I have examined the depressor systematically on both 
sides in seven rabbits. I find that it varies greatly in its mode of origin, 
which is usually, as stated by Ludwig and Cyon, from two branches, one 
from the vagus, the other from the superior laryngeal (fig. 1). But in some 
cases it was a single nerve throughout (fig. 2), while in others the two 
branches of origin ran separately for a greater or less distance, and even to 
their destination (fig. 3).t When single the nerve was generally found to 

* Ludwig and Cyon, Arbeit. physiol. Anstalt, Leipzig, 1866. 

+ ‘Methodik d. physiol. Experimente,’ 1876. 

{ Figs. 1, 2, and 3, are merely diagrams, and are not intended to represent the actual 
size, distance apart, or length of the nerves. 


The Depressor Nerve of the Rabbit. 231 


originate from the angle where the superior laryngeal leaves the vagus. 
When the two branches of origin were separate, one usually left the vagus 
at this point, but sometimes lower down, the other was derived from the 
superior laryngeal a short distance after it had left the vagus. In certain 
cases the whole nerve ran along the superior laryngeal for a little distance 
before emerging as a separate nerve. As Ludwig and Cyon state, the 
depressor lies close to the cervical sympathetic throughout most of its course : 


Superior Cervical Ganglion of the 
Ganglion —— Vagus Trunk 
Superior Cervical —— __ Ganglion of the s 
Ganglion Vagus Trunk Superior Laryngeal: — 
Nerve 
Depressor 
Superior Laryngeal Cervical Sympathetic iar Nerve 
Nerve Nerve ees 
— Vagus Nerve —— Vagus Nerve 


Cervical Sympathetic —— 
Nerve 


Depressor 
Nerve 


(inferior Cervical — 


nalion . ; sues 
Gang Inferior Cervical 


i 
\ eb Gang ion one 
Va 


Fia. 1. Fie. 2. 


Fie. 1.—Diagram of origin and course of depressor nerve in a rabbit, showing the usual 
derivation of the nerve by two threads, one from the superior laryngeal and the 
other from the vagus trunk. 


Fig. 2.—Diagram showing the nerve originating as a single filament from the superior 
laryngeal. Note the (unusual) splitting and rejoining of the nerve in the middle of 
its course. 


in the lower part of the neck one or two short branches unite it with the 
inferior cervical ganglion. I have usually found it to divide at its lower end 
into three filaments, one of which passes to the aorta and the other two to 
the base of the heart. In two instances in which the two branches of origin 
were quite separate throughout, one branch (the vagal) ran to the aorta 
without forming any connexion with the inferior cervical ganglion,* while the 


* This ganglion, which has always been known to physiologists as the “inferior 
cervical,” is now frequently termed by anatomists the “ middle cervical.” 


232 Mr. B. B. Sarkar. 


other (the superior laryngeal) was connected by two filaments with that 
ganglion and then passed to the tissue at the base of the heart (fig. 3). 


Superior Cervical ___ Ganglion of the. 
Ganglion Vagus Trunk 


Superior Laryngeal — 
Nerve 


Vagus Nerve 
Cervical Sympathetic —— 


Nerve 
Depressor 
Nerve 
Inferior Cervical-__— 
Ganglion 
Y Aorta 


Fic. 3.—Diagram showing the depressor passing down as two separate nerves, one of 
which, arising from the superior laryngeal, goes to the aorta; the other, arising 
from the vagus trunk, to the base of the heart. The first one is connected with the 
inferior cervical ganglion by two fine filaments. (These are also shown in figs. I 
and 2.) 


To determine the exact origin of the nerve, serial sections of the vagus 
were made upwards from the angle where the superior laryngeal and the 
depressor leave it. All three nerves are encased in a single epineurium until 
the superior laryngeal separates from the vagus (fig. 6—see Plate 4). On 
examining such aseries of sections a group of ganglion cells is found lying 
between the superior laryngeal and the main vagus trunk just before the 
superior laryngeal separates out (fig. 4). In some animals these cells are 
continued into the beginning of the superior laryngeal nerve as far as the point 
where its depressor branch leaves that nerve. When the whole depressor or 
one of its parts separates from the vagus after the superior laryngeal branch 
has been given off, the group of ganglion cells in question extends down the 
vagus trunk nearly as far as the point where its depressor branch emerges 
(fig. 5). These cells probably give origin to afferent fibres of the 
depressor. In cases in which the depressor emerges high up, the group 
may lie close below the ganglion trunci, although distinctly separate from 
that ganglion. 


The Depressor Nerve of the Rablit. 233 


Microscopie Examination of the Nerve. 

For histological purposes a piece of the nerve was slightly stretched on a 
card, fixed with osmic acid, washed and transferred to alcohol. Sections 
were cut by the paraffin method (figs. 7 and 8). Teased preparations were 
also examined. Microscopically, the nerve is found to consist of both 
myelinated and non-myelinated fibres (fig. 9). The myelinated fibres are 
both medium-sized and very fine. Most are between 4y and 6y (0-004- 
0-006 mm.) in diameter, but in two nerves two or three fibres of about 8u 
(0-008 mm.) were found. ‘The fine myelinated fibres have the character of 
pre-ganglionic autonomic nerves. The non-myelinated resemble post- 
ganglionic autonomic fibres. Both these kinds of fibres are presumably 
efferent. 

The size of the whole nerve varies in different cases. The right nerve is 
usually smaller than the left and contains fewer myelinated fibres. I have 
made an attempt to count the myelinated fibres in six rabbits, but the small 
diameter of the finest fibres renders the task a difficult one. And it is 
impossible to see the non-myelinated fibres clearly enough in section to be 
able to count them. Nor is it easy to obtain sections so exactly transverse 
as to show each myelinated fibre distinctly. The following figures are 
therefore given for what they are worth :— 

The total number of myelinated fibres of all sizes in the nerves of the two 
sides showed an individual difference of from 375 to 496 fibres, the average 
number being 433 in the two.. The average number of myelinated fibres of 
all sizes in the right nerve was 177 and in the left 256. The total number 
oi fibres contained in the branches when these remained separate fell within 
these limits. 


Summary. 


1. The depressor nerve of the rabbit appears*to be connected, at least in 
part, with a special collection of ganglion-cells in the vagus, distinet from the 
ganglion of the trunk. This collection may extend a certain distance into 
the superior laryngeal, or may pass into the vagus trunk some distance below 
the ganglion of the trunk, but in most it lies in close contiguity with and just 
below that ganglion. The cells of the group in question probably give rise 
to the afferent fibres of the depressor. 

The exact point of origin of the nerve is variable. It is usually formed by 
two branches, one from the superior laryngeal and one from the vagus. In 
some cases it is double throughout, in others single. It is connected below 
by fine branches with the inferior cervical ganglion, and can-be traced to the 
root of the aorta and base of the heart. 

VOL. XCIII.—B. S 


234 The Depressor Nerve of the Rabbit. 


2. The size of the nerve and the number of fibres it contains vary in 
different individuals. The left nerve is generally larger and contains more 
fibres than the right. ; 

3. The depressor contains not only medium-sized myelinated fibres, but 
also a considerable number of very fine myelinated fibres, and others which 
are non-myelinated. It is, therefore, probably not wholly formed, as has 
usually been supposed, of afferent fibres, for these fine myelinated and non- 
myelinated fibres closely resemble those belonging to the autonomic nervous 
system and are presumably efferent.* 


The expenses of this investigation have been mainly defrayed by a — 
grant from the Earl of Moray Fund for promoting Research in the 
University of Edinburgh. 


DESCRIPTION OF PLATE. 


Fig. 4.—Section of vagus trunk just before the superior laryngeal branch is given off. 
On the left is seen the vagus ; on the right, the superior laryngeal; between them 
the group of cells from which the afferent fibres of the depressor probably arise. 
Photograph x75 diameters. Osmic preparation. 

Fig. 5.—Section from the same preparation but taken a little lower down. The superior 
laryngeal is now quite separate from the vagus. The group of cells shown in fig. 4 
is still visible, and fibres of the depressor are beginning to accumulate at the side of 
the vagus. Photograph x75 diameters. 

Fig. 6.—Section from the same preparation still lower down. The three nerves now 
appear as entirely distinct bundles, each surrounded with its own perineurium, but 
enclosed in a common epineurium. On the left, the vagus; on the right the 
superior laryngeal; the depressor is the small bundle of fibres between them. 
Photograph x 75 diameters. 

Fig. 7.—Section ofa depressor nerve. Osmic preparation. Photograph x 400 diameters. 

Fig. 8.—Section of the two filaments of origin of a depressor nerve, just before their 
union. Photograph x 400 diameters. (This section is not from tke same animal 
as that from which fig. 7 was obtained.) 

Fig. 9.—From a teased osmic preparation of a depressor nerve. Photograph 
x 300 diameters. The figure shows medium-sized and fine myelinated fibres and a 
few non-myelinated fibres. 


* According, however, to S. W. Ranson (‘ Physiological Review,’ vol. 1, p. 479) small 
myelinated and unmyelinated fibres are included among visceral afferent fibres. This 
statement is based on his own observations recorded in ‘ Journ. Comp. Neurology,’ vol. 29. 
See also the same Journal, vol. 24. (Private communication to E. S. 8.) 


Sarkar. Ror, Soc. Proc., B, vol. 93, Pl. 4. 


255 


The Coagulation of Protein by Sunlight. 


By Etrip Gorpon Younc, Ramsay Memorial Fellow. 


(Communicated by Prof. F. G. Hopkins, F.R.S. Received July 6, 1921.) 
(From the Biochemical Laboratory, Cambridge University.) 


It was first shown by Dreyer and Hanssen (1) in 1917 that ultra-violet light 
produced a change in protein solutions which appeared to be similar to 
coagulation by heat. They exposed various solutions in quartz chambers to 
the light of a Bang lamp with iron and silver electrodes. Vitellin was. found 
most easily coagulated, while globulin, albumin and fibrinogen showed a 
decreasing sensitivity to ultra-violet rays in the order mentioned. These 
investigators also discovered that acids markedly increase the rate of precipita- 
tion. Soret (2) had shown in 1883 that there are absorption bands in the 
extreme ultra-violet region of the spectrum of various proteins, ¢4., casein, 
ovalbumin, mucin and globulin. Tyrosine likewise has this band in the 
ultra-violet and Soret attributed to this constituent of the protein molecule 
its power of absorbing ultra-violet rays. In this connection Harris and 
Hoyt (3) carried out some interesting experiments on the protective power of 
various substances for paramcecium cultures exposed to ultra-violet radiations. 
They found that gelatin peptone, amino-benzoic acid, cystine, leucine and 
especially tyrosine possessed the power of detoxicating ultra-violet rays when 
placed as a thin layer of aqueous solution over paramcecium cultures under a 
quartz-mercury lamp. The toxicity of the radiations for paramecia or 
protoplasm in general can be understood in the light of the discovery of 
Dreyer and Hanssen coupled with that of Soret. 

From a physico-chemical standpoint Bovie (4) has published a study of the 
coagulation of proteins by ultra-violet light. By exposing solutions of 
erystalline ovalbumin, both dialysed and containing electrolytes, to the light 
of a mercury-vapour lamp, he came to the conclusion that there were two 
reactions involved in the coagulation of ovalbumin by ultra-violet light. The 
first is a photochemical one with a low temperature coefficient,—denaturation ; 
and the second is one with a higher temperature coefficient of two and is 
dependent upon the electrolytes present,—coagulation. While using solutions 
dialysed against tap water Bovie made the observation that the protein 
appeared to become sensitive to light of longer wave-length, for his control 
tubes in glass were slowly coagulated. 

Finally, from a medical point of view Schanz(5) has carried out a few 
experiments on this phenomenon with egg-white, blood serum and pig lenses. 

S 2 


236 Mr. E. G. Young. 


From qualitative experiments only he claims to have discovered that certain 
substances, such as glucose, alcohol, lactic acid, urea and acetone, sensitise the 
serum proteins, while he states that certain German mineral waters protect 
them. He uses these observations to explain certain pathological conditions, 
especially old-age cataract. 

When working with solutions of serum albumin which had been several 
times recrystallised, I had noticed that a marked change was produced in the 
solution while in glass vessels, so that the protein was almost quantitatively 
precipitated by mere exposure to sunlight. This precipitation occurred both 
- with material which had been prepared by the alcohol-ether method and that 
obtained by direct crystallisation from serum, as I have described in a previous 
communication (6). A heavy precipitate appeared inside of 2 hours which 
proved soluble only in dilute alkali. The phenomenon seemed of such 
importance that a more thorough examination of the nature of the chemical 
reaction underlying it was undertaken. . 

AsI have been unable to find any record of such a marked change in 
protein solutions by the action of sunlight in the literature, and further as the 
reaction was obtained with serum albumin of a purity such as has never, I 
venture to think, been achieved before, I attributed my observation of light 
instability to the purity of the preparations. 

The following pages of experimental work are designed to show that the 
coagulant action of ultra-violet light on albumin in aqueous solution is also 
brought about by the visible rays of the spectrum when highly purified 
material is used. Experiments are described which show further that the 
nature of the reaction is identical with coagulation by heat in all points tested 
experimentally. That there are two reactions involved is demonstrated and 
changes in some physical constants of the pure protein solutions by the action 
of visible light rays are described as evidence towards an explanation of the 
primary reaction of protein coagulation. 


METHODS. 


Dialysis.—For the complete and rapid removal of contaminating electrolytes 
from solutions of albumin, the dialysing apparatus devised by Sorensen (7) 
has been used in a slightly modified form. It was necessary that the 
apparatus should possess the particular advantage of allowing the operator to 
maintain the solutions at their original concentration or to further concentrate 
them during dialysis. ; 

The Preparation of Collodion Membranes.—Medicinal cotton wool was 
purified by extraction with boiling dilute alkah, followed by repeated changes 
of boiling water. The cotton was then dried and further extracted in 


The Coagulation of Protein by Sunlight. 237 


refluxing absolute alcohol. After drying nitration was carried out as 
recommended by Blitz and von Vegesack (8). The washed and dried cellulose 
nitrate was dissolved in anhydrous alcohol-ether mixture (5:1) to form a 
3 per cent. solution. This was applied by the Sorensen technique to a test- 
tube of # inch diameter. Six or seven applications were given with a drying 
interval of 10 minutes between applications, and a final drying period of 
5 to 7 hours before soaking in water. 

The Dialysing Apparatus.—The general arrangement of the various parts 
was essentially that of Sorensen, although a smaller, slightly modified cell was 
used. The latter had a capacity of 250 c.c., while the membrane sack held 
40 e.c. By the insertion of an extra limb in the upper part of the cell, very 
close to the neck, it was possible to remove the dialysate completely from the 
lower opening by the admission of air through the extra limb before intro- 
ducing fresh water. This, I have found, allows of more rapid removal of the 
electrolytes present with a minimal use of distilled water, and also of the 
ready withdrawal of samples of the dialysate for analysis. The dialysis was 
carried out at room temperature, i.c., 10-15° C., and the negative pressure 
found necessary varied from 5 to 20 em. of mercury. The dialysate was 
replaced with distilled water twice daily. The only satisfactory way of 
determining whether the dialysing apparatus was in proper working order 
was to carry out a series of analyses on the successive dialysates withdrawn. 
Table I shows how efficiently the cell, was functioning when it contained a 
solution of ovalbumin of 11:09 per cent. strength. After the fifth day no 
further positive test for the predominant impurity, (NH,4)2SO,, could be 
obtained. The solution contained 1-440 grm. (NH4)2SOxz at the start and the 
first two days’ dialysis served to remove more than 98 per cent. of the con- 
taminating sulphate. 


Table I.—Dialysis of Ovalbumin Solution, 11-09 per cent. 


| (NH,).SO, content. | 
Day. SO, test. | Protein test. lags , | 
| Grm. | Per cent. total. | 
| 
1 ++4+4+ — 1°2675 88 -02 
Z it Fe 0:1473 10 -23 
; a = 00187 1°30 
= * ee 0 0023 0:16 | 
2 a ay 0 *GO07 0°05 
| 14365 99°76 
' 


238 Mr. E. G. Young. 


The (NH4)2SO,4 was determined by distillation with aeration as in a micro- 
Kjeldahl determination, using N/20 or N/100 H-SO, in the distillate receiver 
and titrating excess acid by N/20 or N/100 Na,S.O3 in the iodide-iodate 
titration. 

The Preparation of Albumin.—Crystalline ovalbumin from one dozen egg 
whites was prepared by means of (NH4)2SO4 and N/3 H2S0Os, erystallising 
and recrystallising at the isoelectric point. Samples of the successive 
recrystallisations were dialysed in the apparatus previously described and the 
remainder after the third crystallisation treated likewise. 

About 800 cc. of horse serum were treated as described in a previous 
comimunication (6), using the direct method, and the crystals obtained 
recrystallised three times. This material was used for several of the experi- 
ments described below. . 

Technique of Light Exposure—Small test tubes of 2 inch diameter of 
ordinary clear glass were used. About 5 cc. of the fluid under examina- 
tion were placed in a test tube or a small glass spectroscope box and exposed 
to sunlight or artificial light from which the infra-red rays had been removed 
by passage through a vessel with parallel sides containing clear water. A 
5-inch lens with a focal length of 6 inches was used to concentrate the light 
rays, and it was placed about 5 inches distant from the light source in the 
case of the arc. The entire solution was exposed to the rays somewhat in 
advance of the focal point. 

It was highly desirable to obtain a sufficiently powerful source of -artificial 
light with some degree of constancy in place of the capricious sun. The light 
from a carbon arc, made by the Firma Carl Zeiss for use with their ultra- 
microscope and mounted on an optical bench, was used for several experi- 
ments. A current of 10-15 amperes was employed. This will bring about 
the same effect as sunlight if exposure be for a sufficient length of time. 
The intensity of the arc is only one-tenth that of mid-day sunlight, and thus an 
approximately similar result by artificial light requires about five times or more 
the length of exposure to sunlight. Another disadvantage in the use of the are 
is the necessity of changing carbons frequently, for with the current strength 
used the carbons are completely burned up in 14 hours, thus necessitating 
changing three or four times in the course of an experiment with ovalbumin. 

Method of Pa Deéerminations.—The concentration of hydrogen ions in the 
various solutions used was determined colorimetrically, using the standard 
buffer mixtures devised by Clark and Lubs (9). The accuracy of the buffer 
mixtures was checked electrometrically by means of the potentiometer and 
the Barendrecht electrode. After some experimentation, the three following 
indicators were selected as most suitable for the purpose. 


The Coagulation of Protein by Sunlight. 239 


Pu, Indicator. 
3:1—4-4 Methyl] orange. 
4:4—6:0 Methyl red. 
6-4—7'8 Neutral red. 


To obviate possible errors due to the presence of proteins and salts, the 
method of dilution was adopted. Thus 1 cc. of fluid was pipetted into a test 
tube, 4 c.c. of CO2-free water added, and a suitable quantity of the indicator. 
A comparator was employed in cases of turbidity or foreign coloration. The 
procedure of dilution was tested and the values obtained by the use of the 
indicators were verified electrometrically on test solutions. 


RESULTS. 

Experiment 1.—Susceptibility of different Crystallisations to Light Change. 

A series of test tubes were prepared containing solutions of the first three 
erystallisations of ovalbumin. These were approximately of 2 per cent. 
strength and had been dialysed free from electrolytes. They were adjusted 
to the same Py by addition of a few drops of N/10 HCl. Another series was 
prepared containing 1 ¢.c. of the different ovalbumin solutions and 2 c.c. of a 
buffer mixture, made up of equal volumes of N/1 CH;COOH and N/1 
CH;COONa, giving a Py of 4°74. The results are shown in Table II. 


Table I1.—Susceptibility of Different Crystallisations to Illumination. 


Test. Mixture. Pu. Result. 
1 3 c.c. solution, Ist crystallisation ...............00000 5:4 - 
1 cc. AS + 2 c.c. buffer 4°6 - 
2 3 C.c. - 2nd Seve uNateuttstsertaa te aihinecmes 5 °4: - 
1 ce. * As +2 c.c. buffer... 4°7 + 
3 3 ¢.c. 3 3rd SiN) “OMMadaeite Mem mseeemence 5 4 — 
it Ge, 5 » +2 c.c. buffer... 4°7 ++ 
| 


Time of exposure to sunlight, 6 hours. 
Albumin concentration of unbuffered solutions, 2 per cent. 


From the results recorded in Table II, where precipitates appeared in the 
buffered solutions of the second and third crystallisations of ovalbumin, it 
became evident that repeated crystallisation tended to render the albumin 
more sensitive to light. It is to be noted that no precipitate appeared in 
the unbuffered solutions. Now, between the unbuffered and buffered 
solutions there existed three differences. The buffered solutions possessed 
a slightly lower concentration of protein, a slightly higher concentration of 


hydrogen ions, and a much greater electrolyte-content than the unbuffered 
solutions. 


240 Mr. E. G. Young. 


The precipitate obtained was soluble in alkali but insoluble in excess 
water or acid. The amount was small, and only a low fraction of the total 
protein in solution. 


Experiment 2.—The Inflyence of Py Variation on the Light Reaction. 


To determine the possible effect of Py variations, a series of tests were 
prepared containing ovalbumin solution of the third crystallisation product, 
to which a few drops of acetic acid of 10 per cent. strength had been added. 
A control tube containing the same albumin solution, with buffer mixture, 
‘was illuminated at the same time. Further controls of tests 1 and 5 were 
prepared, and kept at the same temperature in the dark. The proportions 
and results are recorded in Table III. 


Table I1I.—Effect of Py Variations on Unbuffered Ovalbumin Solutions. 


Test. Mixture. | Pr. | Result. 
| 1 5 c.c. solution + 2 c.c. buffer solution ............... | 4°8 + 
2 5 c.e. 4 +0085 c.c. acetic acid.................. 4°7 -—— 
| 3 Bc, SORLOtC age eee ine. ae |” aeG ee 
| 4 Siecle ela) eeObl Sic: SUM A Fp ae Se | 4°45 = 
| 5 5) (0.(0r = +0°20 c.c. sya bib ast ce those aca: 4°2 - 


Time of exposure to sunlight, 8 hours. 
Concentration of ovalbumin, approximately 2 per cent. 


The Py values were determined at the close of the experiment, and the 
results were only seen in reality when the tubes were all brought to the 
isoelectric point of ovalbumin. On neutralising the acidic solutions with 
dilute NaOH, a marked precipitate appeared, about Py 5:0 (48-5-4), which 
was soluble in excess of alkali, but reprecipitated at the same Py on adding 
acid. This precipitate remained undissolved on further acidification of the 
solution. The volume of the precipitate varied directly with the original 
degree of acidity. The two unilluminated control tubes showed no pre- 
cipitate whatever when adjusted to a Py of 50. This experiment shows 
that acid increases the rate of the light reaction, but that in solutions very 
low in their concentration of electrolytes, the albumin is not precipitated. 
This observation brings the reaction in very close similarity to heat coagula- 
tion. The absence of a precipitate in the unbuffered solutions of Experi- 
ment 1 can now be explained, in the light of the fact brought out by 
Experiment 2. 

A wider range of Py variations was next tried, with the purpose of 
discovering the influence of alkali on the reaction. Phosphate and phthalate 


The Coagulation of Protein by Sunlight. 241 


buffer mixtures were employed, with a solution of ovalbumin three times 
recrystallised. The time of exposure to very bright sunlight was 5 hours. 
Py determinations were made both before and after the exposure. The 
results are recorded in Table [V. Each test consisted of 2 c.c. of buffer and 
1 ce. of ovalbumin solution. The concentration of ovalbumin in the 
original solution was 2 per cent., and in the illuminated tubes was thus 
about 0-7 per cent. Controls of tests 1 and 6 were prepared and kept in 
the dark, with negative results. 


Table LV.—Effect of Py Variations on Buffered Ovalbumin Solutions. 


Pu. 
Test. = = Result after exposure. At Py 4°8. 
Before. After | | 
— ——— —_ ~ = ie oe 
1 3°0 | 3°5 +++ ++4+4 
2 4.°0 | 4.°3 3E ap ++ 
3 4°8 5°1 qF + 
4 54 5°6 opalescent | + 
5 6-0 56 * | ++ 
6 76 6°5 


x | t++4+4 


Time of exposure to sunlight, 5 hours. 
Concentration of ovalbumin, 0°7 per cent. 


At the conclusion of the period of illumination, the tubes on the acid side 
of Py 54 showed precipitates, the degree of which varied directly with the 
hydrion concentration. The tubes on the alkaline side of Py 5:4 were 
merely opalescent. On acidulation to a Py of 48, however, there appeared 
precipitates, the degree of which varied directly with the hydroxyl ion 
concentration of the original solution. The comparison between heat 
coagulation and light coagulation is thus brought even closer. Both acids 
and alkalies increase the rate of the light reaction, and during this change 
there is a removal of H or OH ions from the solution depending upon its 
reaction. 


Experiment 3.— What Physical Changes does the Protein undergo before 
Flocculation ? 


In order to discover whether some physical change in the solution 
which was being subjected to light bombardment might be used as an 
indication of the rate of the light reaction, several properties were investi- 
gated. It was my intention to study the first reaction quite separately, if 
possible, from the second, involving the flocculation of the altered protein. 

(A) Optical Rotation—A 100-mm. tube, filled with the dialysed solution of 


242 Mr. E. G. Young. 


the third erystalline product of ovalbumin, was exposed to the light of the 
are, and readings of the optical rotation were made frequently by removing 
the tube to a Hilger polarimeter, with direct-vision spectroscope, and 
observing the rotation (a;) of the green line of a quartz mercury-vapour 
lamp (A = 546:1 wy). The instrument was accurate to 0°01° The observa- 
tions were as follows :— 


Table V.—Effect of Illumination on Optical Rotation. 


Time of illumination. an. [a Jn. 
| 
minutes. | © = 

0°79 — 36 ‘60 

10 0°81 — 37°60 

30 0°84 —38°99 

45 0°87 —40°38 

105 0°87 —40 38 

| 


Concentration of ovalbumin, 2°16 per cent. 
arn = observed rotation. 
[ale = specific rotation. 


The results in Table V show that one of the effects of radiations of the 
visible spectrum on pure crystalline ovalbumin is an increased power of 
optical rotation. This increase in specific rotation was observed in a 
solution that was entirely free from precipitate. A control tube of the 
same length, and containing the same solution, was read consecutively with 
the illuminated tube. It gave the same reading of 0°79 throughout the 
experiment. 

An experiment to confirm the above observation was tried, using 
erystalline serum albumin which had previously been exposed to sunlight 
for 2 hours. The protein thus exposed was completely coagulated, for the 
solution contained (NH4)2SO, to the extent of 1 per cent. It is to be noted 
that in this experiment the serum albumin of 3:46, per cent. strength was 
completely coagulated inside of 2 hours, whereas ovalbumin in similar 
concentration is only partially coagulated after 6 to 8 hours’ exposure. 
Now, the original unilluminated serum albumin had had a specific rotation 
of —78'60° by the green line of the mercury spectrum (A = 54671 py). 
To the coagulum, with its supernatant liquid of about 100 cc. were added 
fifteen drops of concentrated ammonia, and complete solution was thus 
brought about. This solution was examined in the polarimeter, and the 
specific rotation of the serum albumin was found to have risen to almost 
5° above that of the undenatured substance. Now, in the case of 
unilluminated ovalbumin, I have been able to show that the specific 


a 


The Coagulation of Protein by Sunlight. 243 


rotation is a constant only at the isoelectric point (6). If acid be added, 
the value for [a] is increased. If alkali be added, the value is temporarily 
decreased. In order to discover whether this was also a fact with the 
denatured albumin, two or three drops of HCl (20 per cent.) were added, so 
as to adjust the Py of the solution near to the isoelectric point. The 
specific rotation rose almost 5° to a constant value of —87-92° in several 
hours. The solution was again made alkaline by means of a few drops of 
ainmonia and again the value for the specific rotation fell to —83:0°. The 
same slow rise to the higher value was observed as in the case of unillu- 
minated ovalbumin. It is to be noted that the fluctuations from —83:0° to 
— 89°6° are around a much higher mean value than the one for undenatured 
material, —78°6°. The significance of this observation is discussed in a later 
section. ‘Table VI gives the data in detail. 


Table VI.—Optical Rotation of Denatured Serum Albumin. 


. 
eae Solution. Temperature. Pu. ar. [a}n. 
| ° 
a Original 15 56 —2°‘71 | —78°6 
— Denatured+NH, . 14.°2 7°4 —2°88 | —83°3 
1 hour Se PHC 15 54 —3-01 —87°1 
5 hours 4 15 54 —3°04 —87°9 
12 hours a 14°5 5 °4 —3 04 —87°9 
5 mins. See SNIEL 15 73 — 2°87 —83 ‘0 
2 days 5 15 7°3 —2:°95 —85°3 
3 days _ 14.°5 7°3 —3-09 —89 4 
1 day 5 +NH; 14°53 7°6 —3°10 —89 6 
1 day » 14:2 6 —3:°09 —89 *4 


Concentration of serum albumin, 3°46 per cent. 
Time of previous exposure to sunlight, 2 hours. 

(B) Viscosity and Surface Tension.—A solution of ovalbumin, three times 
recrystallised and dialysed free from sulphate, was used in the following 
experiments. Changes in viscosity were measured by an Ostwald viscosi- 
meter immersed in a thermostat kept at 20° C. and regulated so that the 
temperature was constant to 01°; 5 c.c. were used for a determination, 
measured by means of a calibrated pipette. 

The surface tension was measured by means of a Traube stalagmometer 
(Gerhardt No. III), standardised by distilled water and found to give 
3710 drops. The instrument was accurate to 0°05 of a drop. It should be 
pointed out here that in such a measurement the tension measured is between 
air and the dispersion medium, not between dispersion medium and disperse 
phase. Any change the latter might undergo, however, would very probably 
be shown in the former, though not of necessity. 


244 Mr. E. G. Young. 


The results obtained as shown in Table VII indicate very smalk changes. 
It is well to point out that the amount of albumin undergoing change is very 
small relative to the amount present. This I have repeatedly shown by heat 
coagulation of the remaining solution at its isoelectric point. The are light 
was used in this experiment and the solutions exposed in glass spectroscope 
boxes. 


Table VII.—Effect of Illumination on Viscosity and Surface Tension. 


j | 
Exposure time. Viscosity. y x 108, | Surface tension. 7: 
hours. seconds. | | drops. 
0) 19 -80 11°75 | 39 -00 69°74 
1°5 | 20 60 | 12-23 | 39 10 69 °56 
3-0 20°80 | 12 :34 | 39-60 68°70 
4°5 21°00 12°47 39 -70 68 *52 


Concentration of ovalbumin, 2°10 per cent. 


There is thus a slight decrease in surface tension (vy) and a slight increase 
in viscosity (7), but the changes are so slight that as a means of following — 
the rate of reaction they are unsuitable. It is, however, significant that 
such changes occur, indicating as they do underlying chemical changes while 
the denatured particles remain dispersed. The work is being repeated with 
serum alhumin solutions. 


Experiment 4.—TZhe Effect of various Substances on the Rate of Denatwration. 


For the following tests a solution of serum albumin, twice recrystallised, 
was used. It still contained the mother liquor adherent to the crystals, so 
that there was an appreciable quantity of (NH,4).SO.4 present (about 1 per 
cent.). This solution was quickly affected by sunlight, depositing a massive 
precipitate in an hour if the sunlight were intense. It was denatured by the 
arc light on 2 hours’ exposure. The precipitate was found to be soluble in 
excess water, acid or alkali. The solution in water or acid could only be 
brought about with precipitates formed quickly and freshly deposited. This 
observation is comparable with that of Michaelis and Rona (10) on heat 
coagulation of serum albumin. If the precipitate forms slowly, its solubility 
in both acid and water is lost. J am not able to say whether the resolution 
is an indication of a truly reversible reaction, or whether it merely indicates 
on the part of the colloid the ability to reassume a charge and disperse itself, 
as is the case of pure gelatin, about its isoelectric point. Michaelis and Rona 
state that in the case of heat coagulation it is a true reversion, but adduce 
no evidence for their belief. In this connection it is interesting to recall the 


The Coagulation of Protein by Sunlight. 245 


observation of Corin and Ansiaux (11) that if freshly coagulated egg albumin 


be vigorously shaken it is again dispersed and a clear solution results. This 


point is discussed under the theory of the whole phenomenon. 

The effect of adding various substances was tried on the serum albumin 
solution, and control tubes were prepared in each case and kept in the dark 
at the same temperature. Electrolytes in moderate concentration were found 
to increase the rate of denaturation, e.g., NaCl, (NH4)2SO1, KSCN. Traces of 
alcohol, acetone and toluene did likewise. Glucose and ether had no apparent 
effect. To be in a position to study the effect of various substances, however, 
on the rate of coagulation, a quantitative method would have to be devised. 
From the above experiments it would appear as if any substances with 


, dehydrating power would increase the rate of denaturation. I am at present 


working upon this subject. 


The Nature of the Inght Reaction. 


From the foregoing experiments it will be apparent that the reaction 
brought about by sunlight is very similar to heat coagulation, if not identical 
with it. The main facts associated with the phenomenon of heat coagulation 
may be briefly summarised for purposes of comparison. Chick and Martin (12) 
have conclusively demonstrated that, under certain circumstances, 7.¢., when 
the water present is in large excess and the hydrogen ion concentration is 
kept constant, the reaction can be proved to be of the monomolecular order. 
The reaction is between protein and water and the effect of temperature is 
merely to accelerate it. It has an extraordinarily high temperature coefficient 
and the velocity is influenced by a number of conditions, especially acid and 
alkali. During coagulation, if the reaction be on the acid side of the iso- 
electric point, hydrogen ions are removed from solution; if the reaction be on 
the alkaline side hydroxyl ions are removed. The degree of removal of the 
H or OH ions is dependent on the total concentration of acid or alkali and the 
Py of the solution. Heat coagulation of albumin consists of two processes: 
(1) a reaction between protein and water (denaturation) ; (2) the separation 
of the altered protein in a particulate form (agglutination). From the 
investigation of Sorensen and Jtirgensen (13) on ovalbumin, and of Michaelis 
and co-workers (14) on serum albumin, the maximum flocculation of denatured 
protein has been demonstrated to occur only at its isoelectric point. It is 
interesting to note here that Michaelis and Davidsohn (15) have found that 
the isoelectric point of denatured serum albumin (Py 54) is not the same as 
natural serum albumin (Py 46). Hardy (16) recognised the double nature of 
heat coagulation of egg white as long ago as 1899 and considered the primary 
change as one of an emulsoid colloid to a suspensoid type, which was readily 


246 Mr. E. G. Young. 


precipitated by small quantities of electrolytes. Michaelis endorsed this view 
from his work on serum albumin. Most workers have confined themselves to 
a study of the conditions governing flocculation, and the more fundamental 
primary change from a natural protein to one which is readily precipitated on 
neutralisation of the charge carried by the particles has been neglected. 

The explanation of coagulation generally accepted is due to Hofmeister and 
Pauli. They conceive of the primary change as one of dehydration involving 
the internal neutralisation through the loss of the elements of water of 
terminal NH and COOH groupings. From a study of the coagulation of 
casein by alcohol Robertson comes to the same conclusion. 

Now in the change brought about by sunlight there are two reactions, The 
first change involves a simple chemical one, for which the light is responsible,, 
which causes an increase in optical rotatory power and in viscosity, a decrease 
in surface tension and a decrease in H or OH ions depending on the reaction 
of the nedium. The second stage will only precede if the solution is at or 
near the isoelectric point. It is materially aided by small amounts of electro- 
lytes or dehydrating agents such as alcohol or acetone. Under certain . 
circumstances the second stage is reversible. The explanation would seem to 
lie in the ability of the albumin to reassume charged ions so long as agglutina- 
tion has not proceeded too far. In other words the forces of repulsion of 
similarly charged ions attached to the colloidal particles are sufficient to 
overcome the adhesive forces of the albumin flocculi. It is conceivable that 
in the primary stage not only do we have internal anhydride formation within 
single colloidal aggregates, but that as coagulation proceeds this anhydride 
formation extends to linkages between two or more colloidal aggregates. The 
determination of H or OH ions reveals a diminution during coagulation. 
This can be readily explained on the basis of the amphoteric nature of the 
protein. On the acid side of the isoelectric point the protein is an acid and 
must possess some free COOH groupings. If these become neutralised, as 
in anhydride formation, the acidity of the solution will be diminished. The 
converse will hold true in alkaline solution where NH: groups function as 
basic influences. 

The explanation of the primary reaction as simply involving an internal 
dehydration seems most reasonable on account of the great number of ways 
in which denaturation can be brought about. Light, heat, mechanical shock, 
undue strain as in the surface of expanding air bubbles, or any marked 
double phase such as is produced by the mixing of two immiscible liquids, 
acidity, alcohol, all these agents bring about a change such that the protein 
becomes insoluble. The action of alcohol is interesting in that in this reagent 
we possess one which can bring about both changes, but that if conditions are 


The Coagulation of Protein by Sunlight. 247 


regulated only one will take place. Thus it is possible to remove the water of 
solution or solvate water from the colloidal particles by means of 95 per cent. 
aleohol in the cold. Flocculation is thus brought about, as is the case by 
_ concentrated salt solutions, which is reversible and not accompanied by the 
change called denaturation. If, however, alcohol be added toa protein solution 
in like manner, but at 30° C., then the power of alcohol as a dehydrating 
agent appears so augmented that not only does it remove solvate water, but 
also may be pictured as inducing internal anhydride formation (denaturation). 
The precipitate thrown down is irreversible. In the case of mechanical 
coagulation, the phenomenon fs much more readily produced if a dehydrating 
agent is present in quantity. It would be interesting to know if it would 
occur at all should every trace of electrolyte be removed from the solution. 

From the above discussion, it would appear that the rdle of light as a 
coagulating or denaturating agent is similar to that of heat—a catalyst of 
the primary fundamental chemical reaction. That certain substances can aid 
or hinder its action is very probable from preliminary observations. Certain 
it is, from the results of Experiments 3 and 4, that serum albumin is many 
times more sensitive to light than ovalbumin. In this connection, it is 
interesting to note that the purest serum albumin solutions still contained a 
minute amount of pigment. I am unable to say whether the greater 
sensitivity of serum albumin was or was not due to its influence. 


SUMMARY. 


Serum albumin and ovalbumin which have been several times recrystal- 
lised become sensitive to intense light, either sunlight or strong arc illumina- 
tion, from which the infra-red and ultra-violet rays have been removed. 
Serum albumin is many times more easily affected than ovalbumin. 

The change brought about by light has many of the characteristics of heat 
coagulation. It consists of two separate reactions: (1) denaturation—a 
primary chemical change; (2) flocculation—the precipitation of denatured 
particles. 

_ The primary reaction is accompanied by increase of viscosity and optical 
rotatory power, and decrease of surface tension. The velocity of the primary 
change is increased both by acids and by alkalies. During the reaction, 
H ions are removed if the Py be on the acid side of the isoelectric point ; if 
on the alkaline side, OH ions are removed. 

The secondary reaction does. not follow if the solution be free from 
electrolytes. It is brought about by adjusting the solution to about the 
isoelectric point of the albumin, Py 48-5:4. Under conditions when the 
precipitate is formed readily, it will go back into solution on the addition of 


248 The Coagulation of Protein by Sunlight. 


acid or excess water. If the precipitate is formed slowly, it is only dispersed 
by alkali. 

Certain substances act as accelerators for the reaction, such as alcohol, 
acetone, (NHa4)2SO., NaCl, KSCN. The mechanism of the reaction is 
discussed. 


In conclusion, I wish to take this opportunity of expressing my thanks to 
Prof. F. G. Hopkins for much kind criticism and encouragement. 


7 


REFERENCES. 


(1) Dreyer, G., and Hanssen, O., ‘Compt. Rend.,’ vol. 145, p, 284 (1907). 
(2) Soret, J. L., ‘Compt. Rend.,’ vol. 97, p. 642 (1883). 
(3) Harris, F. I., and Hoyt, H.8., ‘Science,’ vol. 46, p. 318 (1917). 
(4) Bovie, W. T., ‘Science, vol. 37, pp. 24 and 3738 (1918). 
(5) Schanz, F., ‘Biochem. Z.,’ vol. 71, p. 406 (1915) ; ‘ Pfliiger’s Arch.,’ vol. 164, p. 445 
(1916). 
(6) Young, EK. G., ‘Roy. Soc. Proc.,’ B, vol. 93, p. 15. 
(7) Sorensen, 8. P. L., et al. pe Carma Rend. Lab. Carlsberg,’ vol. 12, p. 12 (917). 
(8) Blitz, W., and von Vereen A., ‘Z. physik. Chem.,’ vol. p. 68, 364 (1909). 
(9) Clark, W. M., and Lubs, H. A., ‘ J. Bacteriol.,’ vol. 2, pp. 1 and 109 (1917). 
(10) Michaelis, L., and Rona, P., ‘Biochem. Z.,’ vol. 27, p. 38 (1910) ; vol. 29, p. 494 
(1910). 
(11) Corin, J., and Ansiaux, Q., ‘Bull. Acad. Roy. de Belg.,’ III, vol. 21, p. 355 (1891). 
(12) Chick, H., and Martin, C. J., ‘J. Physiol.,’ vol. 40, p. 404 (1910); vol. 48, p. 1 
(1911) ; vol. 45, pp. 61 and 261 (1912). 
(13) Sérensen, 8. P. L., and Jiirgensen, E., ‘ Biochem. Z.,’ vol. 31, p. 397 (1911). 
(14) Michaelis, L., and Mostynski, B., ‘ Biochem. Z.,’ vol. 24, p. 79 (1911). 
(15) Michaelis, L., and Davidsohn, H., ‘ Biochem. Z.,’ vol. 38, p. 456 (1911). 
(16) Hardy, W. B., ‘ Roy. Soc. Proc.,’ vol. 66, p. 110 (1899-1900). 


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249 


On the Development and Morphology of the Leaves of Palms. 


By Acnes ArBEr, D.Sc., F.L.S., Keddey Fleteher-Warr Student of the 
University of London. 


(Communicated by Prof. F. W. Oliver, F.R.S. Received October 8, 1921.) 


CONTENTS. 

PAGE 

IL. Tinitie@c hbventi@nit seca gconsoadmuue Soo sododobodeonnedoconcouedoe vodosenAondeagonndopedanosseccnoodendds 249 
MepevbelOntoreny, of; the Palm Weak 1.0 ccc... ccecc.+e- ccc ascccecsesccerees seman voeemsries 251 
emeneRElT catedies trio) ce ccncecsacencentennen csctomcselselncanseaeiictnise se « Ssieiaelisnies 251 

9, Nae. Wilesenlorgaine (C(Closbit:) 4), coeoood-huosepscenpacseadeeudoucsessoeccooseapnbubdsoued 254 
Semelilve) Merman elias Clam cl ie aan cen tee hmaetisshia selene ste -tsine sie cteeltdadessaacioetisniene es 255 
Abbey uig ule and WorsalleScaleyencsccscnecssesarecsneesdsepecrsiecctlsedt ene 256 

IIL. The Morphological Nature of the Palm Leaf .................:ccceccseneereeserees 257 
TAP) IDIOTS? dbodentedenandon abosaddooadcadodsbaxsasdhooas dandaqdosSsasnqoeabapodasunedaponeadonlon 260 
PMC ISMO VLCC OTMEMUES MRE t salaieeci cian ecaelaei-ticiatatelamiatlaitatet tie Se clabele telac ssicatlelsinetclalnialas islstsnelsasisielee 261 
estates lemma Olney OiGe desiree haaie-tai cca tite amtinls epleiiavaiecurniie 6 sialslserenietsiisitaninsejcidals si niesjaiis Semeis 261 


I. INTRODUCTION. 


In series of previous memoirs* I have discussed the phyllode theory of 
the Monocotyledonous leaf, both in general and in relation to a number 
of special cases. In the present paper I propose to consider the Palms, 
with a view to determining whether their highly peculiar leaf structure is 
open to interpretation on the lines which have suggested themselves in the 
course of my study of other families, in which the leaves are less obviously 
anomalous. 

The mature Palm leaf consists of a closed basal sheath (sometimes 
continued upwards into an ochrea), a leaf-stalk, and a limb, which may be 
of palmate or pinnate form; the “fan” and “feather” types of limb grade 
into one another, the distinction depending only on the degree of elongation 
of the median rachis. The Fan-palms differ from the Feather-palms in one 
further point on which great stress is sometimes laid—namely, that they 
bear small excrescences at the base of the limb. The outgrowth on the 
ventral side is the so-called ligule; less frequently a corresponding but 
smaller structure, known as the dorsal scale, occurs on the opposite side. 

The morphological value of the various members which constitute the 
Palm leaf will be discussed in a later section of this paper (pp. 257-60). 

It has long been known that the “ compound” appearance of Palm leaves 
| is the result of secondary changes, and that these leaves are thus not truly 
equivalent to the divided or compound leaves of Dicotyledons. Nearly 


* Arber, A. (1921), and earlier papers there cited. 
VOL. XCIII.—B, T 


250 Dr. A. Arber. On the Development and 


a century ago, A. P. de Candolle* pointed out that the young leaves of 
Palms are entire, and that it is only subsequently that they become torn 
into thongs (“ déchirées en lanieres”’); the segments are thus merely pseudo- 
lobes (“ prétendus lobes”). De Candolle emphasises the fact that, though 
the idea of cutting (“découpure”) enters so largely into the technical 
language of leaf-description, it is only to the Palms that such terms can be 
applied with literal exactitude. 

Subsequent workers have carried back our knowledge of Palm leaves to 
the earliest stages in their developmental history. Von Mohil,t Karsten,t 
and Trécul,§ in the middle years of the nineteenth century, followed the 
course of their entogeny, and attempted to trace the stages passed through 
by the leaf-rudiment between its origin and that curiously plicate phase 
which is succeeded by the ultimate subdivision into separate segments. 
About forty years later, the subject received renewed attention, principally 
from Eichler|| and Naumann; the work of Eichler remains, even to the 
present day, the standard account of the development of Palm leaves. 
More recently, Deinega** and Hirmerf} have returned to certain aspects 
of the question which the earlier workers had left in some obscurity. 

A study of the literature shows that, though there is essential unanimity 
as to the manner in which the simple leaf-limb splits into its ultimate 
pseudo-compound form, there are, on the other hand, marked differences 
of opinion about the history of that early developmental period which precedes 
the actual subdivision of the leaf. All workers are agreed that, at an 
extremely early stage, the upper part of the leaf-rudiment shows a series 
of deep furrows alternating with ridges, which give it an apparently plicate 
form. But authorities differ as to the mechanism by which this plication 
is brought about. One school has maintained that there is actual splitting 
of the leaf-tissues (von Mohl, Trécul, Naumann), while others hold that only 
folding takes place (Karsten, Eichler, Deinega, Hirmer). The discussion 
of these conflicting views will be postponed until after I have described my 
own observations on the ontogeny of the Palm leaf. 


* Candolle, A. P. de (1827). 
+ Mohl, H. von (1845). 
t Karsten, H. (1847). 
§ Trécul, A. (1853). 
|| Eichler, A. W. (1885). 
4 Naumann, A. (1887). 
** Veinega, V. (1898). 
++ Hirmer, M. (1919); see this paper for further references. 


7 


q li, 


Morphology of the Leaves of Palms. 251 


IJ. THe ONTOGENY oF THE PALM LEAF. 


1. The “ Plicated” Limb. 


In my study of the Palms I have not confined myself to genera whose 
leaf development has hitherto remained undescribed, but I have examined 
every example of which I could obtain material, because I found that, as 
my standpoint differed somewhat from that of previous workers, the 
existing descriptions did not settle the questions with which I was par- 
ticularly concerned. I have followed the mode of development of the young 
leaves in representatives of four of the five sub-families of the Palms, but 
in the case of the fifth group—the anomalous Phytelephanteze (ipa and 
Phytelephas)—I regret that I have been unable to obtain material. Most 
of my observations relate to the ontogeny of the leaves of seedlings of 
various ages, because it is almost impossible, in this country, to procure 
many apical buds of fully developed Palms. The plumular leaves are 
generally of simpler form than the leaves of the mature plant, but there 
is no reason to suppose that they differ from them in any essential respect 
in the early stages of ontogeny. 

The species which I have examined are distributed as follows among the 
different tribes :— 


I. CoRYPHINE. 


Pheenicee ...... Phema canariensis, Hort., P. dactylifera, L., P. 
Rivieri, Hort. 
SHORES aoe orace Chamerops humilis, L. 


Pritchardia filifera, Lind. 

Lhapis humilis, Blume. 

Thrinax cacelsa, Lodd. 

Trachycarpus (Chamerops) excelsus, H. Wendl., 
T. (C.) Fortune, H. Wendl. 


II. BoRASSINE. 


Borassee ......... Latania Commersonii, J. F. Gmel. 
III. LEPIDOCARYINEA. 


Calamee ......... Demonorops (Calamus) melanochetes, Blume. 
IV. CEROXYLINE. 
ATE CUES tse 5 cee te Areca sapida, Soland. 


Bentinckia nicobarica, Bece. 

Howea Belmoreana, Bece. 

Oreodoxa regia, H. B. et K. 
Cocoinee ......... Cocos Romanzofiana, Cham. 


252 Dr. A. Arber. On the Development and 


I find that in all these species—Fan- and Feather-palms alike—the 
mechanism of development of the plicated or lamellated limb follows 
essentially the same lines. For the sake of brevity, I will limit myself 
to three cases as examples. In the Fan-palm, Trachycarpus Fortunet 
(fig. 1, A-F), the second plumular leaf, examined at a very young stage, 


F 


Fie. 1.—Trachycarpus Fortune, H. Wendl. Series of transverse sections from below 
upwards through the second plumular leaf of seedling (x23). (Sections cut by 
Miss Ethel Sargant.) Fig. 1, A, sheath ; fig. 1, B, petiole ; fig. 1, C-H, process 
of invagination ; fig.1, F, final form. In fig. 1, H, the grooves are numbered in 
the order of their appearance. [Throughout figures—xylem represented black ; 
phloem, white ; fibres, dotted.] In this figure only principal bundles included. 


was found to have at the base a closed sheathing region (fig. 1, A); this 
was succeeded by the petiole (fig. 1, B), an organ of roughly triangular 
outline with a concave upper surface. The petiole retained this char- 
acteristic form for a very short distance only (about 20), and then the 
lamellate structure was rapidly reached by the development of a series 
of grooves penetrating deeply into the leaf-stalk tissue between the 
vascular bundles (fig. 1, C-F). In fig. 1, E, I have numbered the grooves 
to show the order of their origin. The process. of grooving is a very rapid 
one, the stages between fig. 1, B, and fig. 1, E, taking place within a distance 
of about 0°5 mm. 


Fie. 2.—Oocos Romanzofiana, Cham. Series of transverse sections from below 
upwards through second foliage leaf of seedling (x14). Fig. 2, A, sheath; ~ 
fig. 2, B, petiole ; fig. 2, C-H, stages in invagination. 


That development proceeds on the same general principle in both Feather- 
and Fan-palms, is demonstrated by the comparison of fig. 1, A—F, with 
fig. 2, A-E, drawn from the second foliage leaf of Cocos Romanzofiana. This. 


Morphology of the Leaves of Palms. 253 


series does not reach high enough into the leaf to show the final form of 
the limb, but it may be supplemented by reference to figs. 3, A-D, which 
were drawn from a set of sections cut from the third plumular leaf of 
another Feather-palm, Orcodoxa regia, H. B. et K. Fig. 3, A, shows an 
early stage in the grooving of the petiole; this section is interesting for 
comparison with the corresponding stages in the two Palms already 
considered, because the first grooves, in this case, are more or less parallel 
to the upper surface of the petiole, instead of being approximately at right 
angles to it. 


Fie. 3.—Oreodoxa regia, H. B. et K. Series of transverse sections through upper 
part of petiole, and limb, of third plumular leaf (first foliage leaf, which was 
preceded by two scale leaves) of seedling (x14); fig. 3, A, early stage of 
invagination of petiole. 


As we have indicated on p. 250, two alternative explanations have hitherto 
been put forward to account for the resemblance of the young Palm leaf to 
a folded fan: (1) that there is a splitting of the tissues; and (2) that the 
rudimentary organ becomes folded in the course of development. I find 
it impossible to accept the first of these views, since my observations on 
the arrangement of the elements in the neighbourhood of the grooves 
entirely confirm the conclusion—already drawn by Hirmer* from a study 
of the detailed histology in certain genera—that no actual splitting takes 
place. But I am, on the other hand, unable to accept the second view, 
which Hirmer himself supports, namely, that the apparent plication is due 
to the folding of an originally simple leaf-rudiment (“ Faltung der urspriing- 
lich einfachen Blattanlage”). The word folding necessarily implies something 
which is the converse of the eventual unfolding of the blade. But no 
process takes place in the leaf-rudiment which can justly be described by 
such a term. I think it will be recognised that the way in which the 
grooves penetrate between the vascular strands, as shown in figs. 1-3, puts 
the folding hypothesis entirely out of court. My observations thus fail 


to support either the splitting or the folding interpretations ; but they have 


led me to a third view, namely, that the plications originate by invagination, 
or, in other words, by differential growth in the course of which the outer 


* Hirmer, M. (1919). 


t 


254 Dr. A. Arber. On the Development and 


cell-layers develop more rapidly than the inner tissues, and consequently, 
become wrinkled, or folded in, along certain lines. In relation to the 
epidermis alone, the word “folded” might perhaps be admissible, but the 
extension of the term to the leaf as a whole cannot be justified. It may 
possibly be thought that I am exaggerating the value of a slight distinction, 
in laying so much stress upon the difference between invagination and 
folding. But this distinction is by no means so trivial as it may appear at 
a casual glance, and I believe, moreover, that the impression conveyed by 
the description of the young Palm leaf as “ folded” has been just sufficiently 
inexact to prevent botanists realising the true morphological nature of the 
leaves of Palms—a subject to which we shall return in a later section of 
this paper (p. 260). 

When we come to analyse the process of invagination more closely, we see 
that it must depend, in the last resort, on a tendency to disproportionate 
rapidity of growth in the surface layers as compared with those that are more 
deep-seated. And that such disparity should exist in the leaves is not surprising, 
when we realise that the Palms have undoubtedly a general tendency towards 
hypertrophy of the superficial tissues of their various organs. ‘The results of 
this tendency are witnessed in the non-vascular spines which many Palms 
develop on the leaf-rachis, and in the squamiform ovarial hairs, which, in such 
cases as Kaphia Ruffia, Mart., form eventually a woody coat to the fruit. The 
comparatively large size of the root-cap in the Palms may also be a case in 
point, for the calyptrogen, in the Monocotyledonous root, is the only tissue 
which is strictly epidermal. Possibly the integumental outgrowths, which 
ruminate the endosperm in many genera, may be regarded as another 
expression of the same tendency, while a further example is afforded by the 
proliferations producing the “ coiffe” of the young leaf—a structure which 
we shall consider in the following paragraph. 


2. The Membrane (“ Corfe”). 


It has long been known that the lamelle of the very young leaves of certain 
Palms are connected by a kind of ephemeral membrane—the “ Hiille,” “ Haut,” 
“ coiffe,” or “ pellicule”—so that the limb resembles a closed fan sheathed in 
tissue paper. This membrane is ruptured when the leaf unfolds. The view 
taken as to the nature of this envelope necessarily depends upon that held 
regarding the origin of the plications. To Von Mohl, Trécul and Naumann, 
the membrane was a primary constituent of the leaf, and represented the 
survival of the superficial tissues which were not involved in the splitting 
which these authors postulated. By those, on the other hand, who believe 
that the plications originate by folding, the membrane is held to be a secondary 


Morphology of the Leaves of Palms. 255 


product, since it does not penetrate into the grooves. Deinega and Hirmer, 
who applied microtome technique to the problem of the origin of this 
membrane, conclude that it arises through secondary fusions occurring at the 
angles of the folds of the plicate leaf. My observations are ‘entirely in 
harmony with this view, which was also indicated many years ago by Eichler. 
In some serial sections of the plumular leaves of Trachycarpus Fortunei and 
Thrinax excelsa, cut by Miss Ethel Sargant, I have been able to observe 
proliferated epidermal cells occluding the mouths of the invaginations 
(fig. 4, A-C). This occluding tissue no doubt represents the delicate early 


Fic. 4.—Epidermal proliferations in seedling leaves, from sections cut by Miss Hthel 
Sargant. Fig. 4, A-C, Thrinax excelsa, Lodd. Fig. 4, A, transverse section of 
third plumular leaf (x 47); m.b., median bundle. Fig. 4, B, mouth of invagina- 
tion marked x in fig. 4, A (x318). The epidermis and its proliferation, which 
occludes the opening of the groove, are dotted. Fig, 4, C, another occluded 
groove, from lower down in the same leaf (x318). Fig. 4, D, Chamerops 
Fortunei, Wendl. Mouth of invagination occluded by epidermal outgrowth, 
from a, transverse section similar to that shown in fig. 1, F (x 318). 


stage of the membrane which, later on, becomes so conspicuous a feature of 
the young leaf. The condition observed in these two genera thus confirms 
Hirmer’s view, which was based on a study of another genus—Phenix. It is 
much to be desired that some botanist, working at a tropical station where 
the leaves of a number of growing Palms are available, would give usa 
comprehensive account of the structure and history of the “ coiffe.” 


3. The Terminal “ Gland.” 


Attention was drawn by Baillon* in 1895 to the fact that the first leaves 
produced by a Palm seedling are usually entire and parallel-nerved, and end 
in “une sorte de glande terminale, appareil excréteur, dont l’existence est 
passagere.” I have observed this apical structure in the plumular leaves 
of Phenix dactylifera (fig. 5, A-C) and Pritchardia filifera (fig. 5, D-F). In 
these cases the main‘part of the leaf-limb is, as usual, dorsiventral, whereas the 


* Baillon, H. (1895). 


256 Dr. A. Arber. On the Development and 


apex is solid, and cylindrical in section (fig. 5, C and F). I have seen evidence 
of the glandular function which Baillon attributes to this apical region in 


F 

Fic. 5.—Solid tips of plumular leaves, Fig. 5, A-C, Phenix dactylifera, LL. Series 
of transverse sections from below upwards through apex of first foliage leaf to 
show dying out of invaginations (x14); bundles consist chiefly of fibres. 
Fig. 5, D-E, Pritchardia filifera, Lind. Series of transverse sections through 
apex of first foliage leaf (x14) ; fig. 5, E, shows that the apex is slightly hooded ; 
c, cavity. 


the case of Bentinckia nicobarica, where the tip of a plumular leaf bore 
conspicuous stomates and contained a plexus of tracheids. After cutting 
successive sections of the distal region of these leaves, I have come to the 
conclusion that the solid tip cannot be treated as if it were a separate organ, 
for it appears to owe its existence merely to the fact that the invaginations 
die out before they reach the distal end of the leaf. In Pritchardia filifera 
the solid apical part of the leaf is, as it were, slightly undereut by the 
termination of the median invagination, so that a minutely hooded apex is 
produced (fig. 5, E). The solid tips of the seedling leaves of Pritchardia and 
Phenix seem to me exactly comparable with that of such a leaf as Crocus,* 
in which, also, the form of the apex is the result of the failure of the grooves 
to reach to the extreme end. 


(4) The “Ingule” and “ Dorsal Scale.” 


The origin of the outgrowths which occur at the base of the limb in the 
Fan-palms, and are commonly known as the “ligule” and “dorsal scale,” 
cannot be studied satisfactorily in seedlings, since it is only in the later leaves 
that they arrive at their full development. The “dorsal scale” is seldom 
conspicuous, but the “ligule,” though it is very variable in size and shape in 
different species, is often a striking object ; fig. 6 shows its appearance in the 
case of the mature leaf of Zrachycarpus excelsus. In order to follow the 
developmental history of these structures, I cut serial transverse sections 
through the apical bud of a well-grown shoot of Rhapis humilis. Fig. 7, A-D, 
represent the transition from stalk to limb in one of the young leaves of this 
bud. It will be seen that the invaginations, which are responsible for the 


* Arber, A. (1921), fig. 57, B, C, p. 324. 


Morphology of the Leaves of Palms. Pain 


“plication” of the limb, fail, at their proximal limit, to reach the epidermis 
either on the ventral or dorsal side, so that part of the petiolar surface is, in 


Fie. 6.—Trachycarpus excelsus, H. Wendl. Ventral view of junction of leaf-limb 
and petiole to show “ ligule” or ventral crest, v.c. ( x $). 


Fie. 7.—Rhapis humulis, Blame. Series of transverse sections through young leaf 
of mature plant, passing through junction of petiole and leaf-limb from below 
upwards (x9, circa); fig. 7, A, top of petiole ; in fig. 7, B, invagination is just 
beginning and the slightly developed “dorsal scale” or dorsal crest (d.c.) is 
distinguishable ; in fig. 7, C, the “ligule ” or ventral crest (v.c.) is just becoming 
detached, while in fig. 7, D, it is entirely free. The asymmetry of fig. 7, B and C, 
is due to a slight obliquity in the series of sections. 


each case, left overarching the base of the limb. This produces the “ dorsal 
scale” at the back (fig. 7, B, d.c.), and the more prominent “ligule” on the 
ventral side (fig. 7, C, D, wc.). The hooded tip of the plumular leaf of 
Pritchardia filifera (fig. 5, F) is a comparable case on a smaller scale, though 
here it is the distal end of the median invagination which is overarched by 
the ventral surface of the limb. _I think it will be agreed that the almost 
eup-like form produced in Trachycarpus excelsus by the base of the limb and 
the “ligule” together (fig. 6), is consistent with the view that the “ligule” 
merely represents the limiting region of the uninvaginated proximal part of 
the petiole. 

The bearing of the interpretation of the “ligule” and “dorsal scale,” 
which I have just outlined, upon the homologies of the Palm leaf, will be 
considered in the next section of this paper. 


IIL. Lhe Morphological Nature of the Palm Leaf. 


There has never been any question among botanists as to the nature of the 
sheath which forms the extreme base of the Palm leaf; its homology with 


258 Dr. A. Arber. On the Development and 


other leaf-sheaths is obvious. In certain Feather-palms (Desmoncus, Calanwis) 
the apex of the sheath is continued upwards into the so-called ochrea, which 
clearly corresponds to the tubular ochrea of the Polygonacee, etc. 

The sheath is succeeded by a stalk, for which I have throughout this paper 
used the word “petiole,” but of which the homologies, at least in the case of 
the Fan-palms, are a matter of controversy. Domin* takes the view that the 
“ligule” of the Fan-palms is indeed a true ligule, corresponding to that of 
the Graminee, etc., and to the ochrea of the Rotangs; and since ligules are 
always defined as belonging to the leaf-sheath region, he draws the conclusion 
that in the Fan-palms the leaf-stalk, which comes below the “ligule,” cannot 
be a true petiole, but must be of leaf-sheath nature. This view cannot but 
seem strained and artificial when one realises that it consigns the leaf-stalks 
of Fan- and certain Feather-palms to different morphological categories. 
Gliick,f who also regards the ventral outgrowth as ligular, steers a middle 
course by treating it as the free apex of an extremely elongated ligule, which 
is fused with the petiole as far as the point of junction of stalk and limb. 
This speculation is ingenious, but, as I hope to show, unnecessary. 

Both Domin’s and Gliick’s views as to the nature of the “petiole” in the 
Fan-palms, stand or fall with the question of the homologies of the “ ligule ”—a 
problem which we must now consider. I should like to point out, in the first. 
place, that it seems to me that any theory of the “ligule” must also take 
account of the corresponding structure which often occurs at the back of the 
leaf, and is known as the “ dorsal scale.” The latter is more variable than 
the “ligule,” and though in some Fan-palms it is scarcely developed at all, I 
have observed in a Palm, grown under the name of Sabal jfilamentosa, that ib 
may reach much the same degree of conspicuousness as the ventral structure. 
An inspection of the leaves of this Palm certainly suggests that no explana- 
tion of the origin of the “ligule” can be accepted which does not also embrace 
the “dorsal scale.” But it is clear that the “dorsal scale” cannot be ligular, 
since no ligule is ever located behind the leaf to which it belongs, and it seems 
to me highly improbable that the ventral outgrowth should belong to a 
category from which the corresponding dorsal outgrowth is necessarily 
excluded. 

In a preceding section of this paper I have discussed the ontogeny 
of the “ligule” and “ dorsal scale,” and the evidence there adduced has led me 
to the view that these structures have no more claim to be treated as distinct. 
organs than have the solid apice of the plumular leaves described on pp. 255-6. 
The invaginations of the limb, in their proximal region, burrow as it were,. 

* Domin, K. (1911). 
+ Glick, H. (1901). 


Morphology of the Leaves of Palms. 259: 


under the ventral surface of the petiole, and to a much slighter extent, under 
the dorsal surface ; on the ventral side the penthouse thus produced happens 
to be a striking object to the eye, and it may possibly become still more 
noticeable by suffering some further elongation after the invagination of the 
limb is completed, but these are not reasons which can entitle it to rank as a 
morphological entity. The conclusion I have reached is that the“ ligule” and 
“dorsal scale” belong to the petiole, and are merely the outcome of the 
peculiar mode of limb-development characteristic of the Fan-palms. The 
appearance of these structures in the Fan-palms, and their absence in the 
Feather-palms, is due to the fact that in the Fan-palms the whole series of 
invaginations start from the same level, whereas, in the Feather-palms, where 
the plications occur to right and left of the median rachis, there is no crowding 
together of the invaginations at a single point. 

My view of the morphology of the “ligule” and “dorsal scale” leads me 
to suggest that these terms should be dropped, since the structures in 
question have nothing to do either with ligules or with scale-leaves; the 
term ligule will then be left for the tubular ochrea of Desmoncus, etc., to 
which it rightfully belongs. The best substitutes seem to be “ventral crest” 
and “dorsal crest,” expressions based upon the word “ Crista,” already used 
by Drude* for the “ ligule.” If my interpretation of these structures be 
accepted, it leads inevitably to the conclusion that the leaf-stalk, both of the 
Fan- and Feather-palms, is the basal part of a true petiole. 

And now, finally, we have to consider the nature of the leaf-limb. 
Strikingly different in aspect as are the mature blades of typical Fan- and 
Feather-palms, there is not, morphologically, any impassable gulf between the 
two types. If the median rachis is short or non-existent, we get the “fan” 
‘form, while, if there be long-continued growth, the “feather” leaf is produced ; 
the leaves of such a genus as Zicualat show transitional characters. It seems 
possible that the “ fan” type is the older, since the palmate form predominates 
among fossil Palms, while the Feather-palms are in the majority at the 
present day.t But the relative age of the two types must at present be 
treated as an open question, since they were both apparently in existence in 
the Upper Cretaceous. The evolution of the “feather” from the “ fan” (or 
vice versd) must have occurred more than once in the history of the family, 
since both types may be found among Palms which are regarded by system- 
atists as allied. The sub-family Coryphoidee, for instance, includes two 
tribes—the Phoenicee, which are feather-leaved, and the Sabaleze, which are 


* Drude, O. (1889). 
+ Wendland, H. (1879). 
t Unger, in Martius, K. F. P. von (1823-50). 


260 Dr. A. Arber. On the Development and 


fan-leaved ; in the Lepidocaryinez, again, there are two tribes differing in the 
same character—the fan-leaved Mauritez and the feather-leaved Metroxylez. 
These considerations seem to show that the various types of Palm leaf may 
safely be treated as homologous, whether they assume the palmate or the 
pinnate form. 

As I have shown on pp. 251-4, the plicate limb, both in the Fan- and 
Feather-palms, is not, as has hitherto been commonly assumed, the result of 
the folding of a flat leaf-blade; it takes its origin, on the contrary, from the 
upper region of the leaf-stalk, through a series of invaginations which penetrate 
the tissues of this organ, and elaborate its more or less radial structure into 
a flattened and perfectly dorsiventral limb. My observations on the ontogeny 
thus indicate that the Palm leaf is not, as has been generally supposed, 
an organ whose structure is almost without a parallel, but that it falls into 
line with the leaves of other Monocotyledons (e.g., certain Ivids).* 

I thus regard the Palm leaf, as a whole, as a petiolar phyllode, and its 
blade as a pseudo-lamina, analogous to, but not homologous with, the blade 
of a Dicotyledon. The fundamental identity of the leaf of the Palms with 
that of other Monocotyledons is, however, soon masked by secondary fusions, 
and, a little later, by the disintegration and tearing into segments, which the 
leaf undergoes in passing to its peculiar definitive form. 


LV. SUMMARY. 


The evidence from ontogeny and comparative morphology, brought forward 
in the present paper, leads to the following conclusions :— 

1. The leaf-stalk, which succeeds the basal sheath, is, both in the Fan- 
and Feather-palms, the basal or proximal region of the true petiole. 

2. The “fan” or “feather” limb is not, morphologically, a lamina, but 
is a modification of the distal region of the true petiole. The complex 
plication of the limb arises through the development of a series of invagina- 
tions which penetrate into the leaf-stalk tissue between the bundles. 

3. The “ligule” and “dorsal scale” of the Fan-palms are not morpho- 
logical entities, but merely represent the adaxial and abaxial distal margins 
of the uninvaginated proximal region of the petiole. The terms “ ventral 
crest” and “dorsal crest” are proposed as substitutes for the terms “ligule” 
and “dorsal scale.” 

4. The Palm leaf, regarded as a whole, is, on the present interpretation, 
a petiolar phyllode with a pseudo-lamina—a conception which brings it 
into essential relation with the leaves of other Monocotyledons, 


* Arber, A. (1921). 


Morphology of the Leaves of Palms. 261 


* Acknowledgments, 


I am indebted for material to the Director, and to the Keeper of the 
Jodrell Laboratory, the Royal Botanic Gardens, Kew; to the Director and 
to the Superintendent of the Cambridge Botanic Garden ; to the Secretary, 
Royal Botanic Society of London; and to Mr. J. Benbow, of La Mortola, 
Ventimiglia ; Prof. Sauvageau of Bordeaux, and Prof. Gérard of Lyons. In 
preparing the present paper I have also had the advantage of using certain 
specimens and sections from the collection of material, relating to Mono- 
cotyledonous seedlings, formed by the late Miss Ethel Sargant. 


LIST OF MEMOIRS CITED. 


Arber, A. (1921). “The Leaf Structure of the Iridaceze, considered in Relation to the 
Phyllode Theory,” ‘ Ann. Bot.,’ vol. 25, pp. 301-36 (1921), sixty-six text figures. 
Baillon, H. (1895). “ Histoire des Plantes,” ‘Monographie des Palmiers,’ Paris, 1895, 

pp. 245-404, sixty-eight text figures. 

Candolle, A. P. de (1827). ‘Organographie Végétale,’ vol. 1, Paris, 1837, xx +558 pp. 

Deinega V. (1898). “ Beitrage zur Kenntniss der Entwickelungsgeschichte des Blattes 
und der Anlage der Gefassbiindel,” ‘ Flora,’ vol. 85, pp. 439-98 (1898), one plate, 
twenty-two text figures. 

Domin, K. (1911). ‘“ Morphologische und Phylogenetische Studien iiber die Stipular- 
bildungen,” ‘ Ann. du Jardin bot. de Buit.,’ vol. 24 (Ser. 2, vol. 9), pp. 117-326 (1911), 
eleven plates. 

Drude, O. (1889). “Palme,” in ‘Die Natiirlichen Pflanzenfam. (Engler und Prantl), 
Teil I1, Abt. 3, pp. 1-93, sixty-five text figures. 

Eichler, A. W. (1885). ‘Zur Entwickelungsgeschichte der Palmenblatter,” ‘Phys. 
Abhandl. d.k. Akad. d. Wiss.,’ Berlin, 1886 (for 1885), pp. 28, five plates. 

Gliick, H.(1901). “ Die Stipulargebilde der Monokotyledonen,” ‘ Verhandl. d. Naturhist.- 
Med. Vereins zu Heidelberg,’ N.F. Bd. vii, Heft 1, pp. 1-96 (1901), five plates, one 
text figure. 

Hirmer, M. (1919). “ Beitrage zur Morphologie und Entwicklungsgeschichte der 
Blitter einiger Palmen und Cyclanthaceen,” ‘ Flora,’ N.F. Bd. xi (G.R. Bd. cxi), 
1919, pp. 178-89, ten text figures. 

Karsten, H. (1847). “Die Vegetationsorgane der Palmen,” ‘Abhandl. d.k. Akad. d., 
Wiss., Berlin,’ 1849 (for 1847), pp. 73-236, nine plates. 

Martius, K. F. P. von (1823-50). ‘ Historia Naturalis Palmarum,’ 3 vols., 245 plates. 
Munich, 1823-50. 

Mohl, H. von (1845). ‘ Vermischte Schriften,’ Tiibingen, 1845. 

Naumann, A. (1887). “Beitrage zur Entwickelungsgeschichte der Palmenblatter,” 
‘Flora,’ Jahrg. Ixx, pp. 193-202, 209-18, 227-42, 250-7 (1887), two plates. 

'Trécul, A. (1853). “Mémoire sur la Formation des Feuilles,’ ‘Ann. des Sci. Nat.,’ 
Sér. ILI, Bot., vol. 20, pp. 235-314 (1858), six plates. 

Wendland, H. (1879). ‘“‘ Die Habituelle Merkmale der Palmen mit Facherformigem 
Blatt, der sogenannten Sabalartigen Palmen,” ‘ Bot. Zeit.,’ Jahrg. xxxvii, pp. 145-54. 
(1879). 


oJ 


262 


Studies in the Fat Metabolism of the Timothy Grass Bacillus. 


By Marsory STEPHENSON, Beit Memorial Research Fellow, 
and MarGARET DAMPIER WHETHAM. 


(Communicated by Prof. F. G. Hopkins, F.R.S. Received December 14, 1921.) 
(From the Biochemical Laboratory, Cambridge.) 


Studies in fat metabolism have hitherto been chiefly carried out on highly 
specialised vertebrate tissue. By making investigations on a unicellular 
organism, more susceptible of laboratory control, two objects were in view: 
(1) to trace the stages by which the long straight chains of the fatty acid 
molecules are built up from the constituents of the nutritive medium; (2) to 
follow the circumstances and course of their subsequent breakdown. The 
Timothy grass bacillus was the organism selected, on account of (1) its high 
content of fat, and (2) its relationship to the tubercle bacillus, any facts 
substantiated by study of the one probably helping to throw light on the 
chemical habits of the other. 


METHODS OF CULTURE AND ANALYSIS. 


The medium employed contained inorganic salts in the following pro- 


portions :— 
Potassium phosphate (K2HPO,)............... 0-1 grm. per 100 c.c. 
Magnesium sulphate (MgSO4-7H20) ......... 0:07 35 if 
Ammonium phosphate (Amz:HPO,) ......... 0-4 be 
Calcium carborates, sacs .eceee eee eel peee excess. 


together with traces (about 0-006 grm. per 100 cubic centimetres) of sodium 
chloride, introduced when the organism was sown. The calcium carbonate was 
used to maintain the Py of the medium at a constant value, namely, about 
80. The ammonium salt formed the only source of nitrogen. The source of 
carbon varied with different experiments and will be discussed later; nothing 
more complex than glucose was employed. 

Various types of culture vessel were tried, but the only one with which 
we were able to attain any degree of success was the Roux bottle. Attempts 
were made to use flasks or large bottles, with rubber stoppers fitted with 
delivery tubes through which samples of the medium could be withdrawn at 
intervals. Numerous attempts all proved completely unsuccessful for the 
following reasons :—(1) The organism grows mainly in a scum on the surface 
of the medium. Ifa deep vessel is employed this scum is far removed from 


Studies in Fat Metabolism of Timothy Grass Bacillus. 263 


the ,layer of chalk on the bottom, which consequently fails in its function. 
For this or other reasons connected with aeration, the growth of the organism 


slows down and comes to a standstill long before 
the organic constituent of the medium is used up. 
(2) The withdrawal of samples by means of delivery 
tubes plugged with cotton-wool proved completely 
unsuccessful. When the medium was shaken in 
order to distribute the growth evenly before taking 
a sample, the scum formed clots or flakes which 
tended to float, making it impossible to blow out a 
fair sample of medium through the delivery tube. 

The method finally adopted was identical with 
that first employed, viz., culture in Roux bottles. 
The bottles, each containing about 5 grm. of calcium 
carbonate, were plugged in the usual way and baked 
for an hour at 130°-140°. Exactly 150 c.c. of medium 
were then introduced into each bottle, and the set 
of bottles was steam-sterilized on each of three 
successive days. 

The culture was first sown on glycerol potato 
slopes and incubated for four to seven days. A 
number of test-tubes (6 inch by 1 inch) containing a 
small quantity of clean sand, together with about 
5 c.c. of saline, were plugged and autoclaved. Into 
one of these the growth from the potato slopes was 
removed by a platinum loop. The growth from one 
potato slope was sufficient for two or three Roux 
bottles. When sufficient growth had been removed 
to sow the required number of bottles, it was 
emulsified. 

The emulsification of this organism presents 
difficulties. The apparatus shown in fig. 1 was 
employed. A is a solid glass club, the handle of 
which passes through tnbes B and C. These are 
arranged to form a mercury seal. The rubber 
stopper D is fitted into a test-tube of the same size 


WO 

Wa 

GYGN 

Aix4—B 
iar 
BN soak 7 


AS 


BiG 


as those containing the sterile sand and saline, and the whole is wrapped in 


paper and sterilized in an oven at 120°-130° for an hour. 


Stopper D is then 


removed from the empty tube. At the same moment the cotton-wool plug is 


removed from the tube containing the organism, sand and saline, and this tube 


264 Misses M. Stephenson and M. D. Whetham. 


immediately replaces the empty one, and is closed by stopper D. Mercury 
(kept under 5 per cent. formalin) is then introduced by means of a sterile 
curved pipette to form a seal and prevent contamination of the tube whilst 
allowing sufficient play to the glass rod. The pieces of growth can be ground 
in the sand and a fairly even emulsion formed. When this is complete, the 
club and seal are withdrawn with the stopper D and the cotton-wool plug is 
replaced in the test-tube, after about 25 cc. of sterile saline have been 
introduced and mixed with the emulsion. To avoid sowing a contaminated 
culture, the diluted organism was then incubated for 24 hours and sown on 
_to slopes of tryptic agar, which medium favours the rapid growth of con- 
taminating organisms. After 24 hours’ incubation, the growth on these 
slopes was examined by the Ziel-Neelsen method of staining. If satisfactory, 
the original emulsion was sown by means of sterile pipettes, 1 e.c. to each 
Roux bottle. We have employed this method for a large number of experi- 
ments and have only once suffered from contamination, which was detected 
on the slopes before the bottles were sown. 

The bottles thus sown were incubated at 37°, and sample bottles were 
withdrawn at intervals in order to analyse both the medium and the organism 
grown upon it. It will be seen that the success of the experiment depends 
on introducing into each bottle an equal amount of the emulsified culture. 
The analysis of duplicate Roux bottles withdrawn simultaneously shows 
agreement within the limits of accuracy of the analytical methods used, as 
will be shown later. 

Two methods are most frequently used to estimate the amount of growth 
of any organism: (1) plating out and counting colonies; (2) separation of the 
growth by centrifuging, washing, drying, and weighing direct. The former 
was impossible in our experiments owing to the agglutinated character of the 
growth ; the latter was equally unsuitable, since the growth was always mixed 
with the powdered calcium carbonate employed to control the reaction of the 
medium. In our experiments, therefore, we regarded the protein nitrogen 
synthesized asa measure of the growth of the organism. This protein nitrogen 
was precipitated by colloidal iron and estimated by Kjeldahl’s method. The 
other characteristic of the organism to be examined was its fat content. The 
. lipoid material of the bacillus may be conveniently divided into two: 
fraction A is that extracted from the dried organism by purified ether ; this 
fraction contains no phosphorus. From the residue after extraction of A is 
obtained fraction B, by treatment with boiling alcohol and a subsequent 
second extraction with ether; B contains phosphorus. For convenience 
fraction A has been termed the “fat” fraction, B the “ phosphatide ” fraction, 
and the sum of the two “ total lipoid.” 


Studies in Fat Metabolism of Timothy Grass Bacillus. 265 


Estimation of Synthesised Protein. 


The contents of one Roux bottle were washed quantitatively into a beaker 
and made slightly acid with acetic acid; 20 c.c. of “dialysed iron” were 
added and the whole heated on a water-bath, filtered whilst hot, and washed 
with hot water till free from phosphate. The precipitate and paper were 
then transferred to a Kjeldahl incinerating flask and the nitrogen estimated 
in the usual manner. The results are calculated to represent grm. of 


nitrogen in 100 c.c, medium, 7.¢., two-thirds of one Roux bottle. 


Estimation of Inpoids of Bacillus. 


(1) “ Fat.”—The contents of one Roux bottle (150 cc.) were transferred 
quantitatively to a 250-c.c. measuring flask, and made up to volume. They 
were then filtered through a dry pleated filter into a dry flask, and the filtrate 
was set aside for estimation of the constituents of the medium. The 
precipitate, consisting of the insoluble constituents of the medium together 
with the agelutinated bacteria, was transferred on the filter paper to a dish 
and dried in an atmosphere of nitrogen at about 97°. The final stages of the 
drying were carried out 7m vacuo. The filter paper and dried contents were 
then transferred to a Soxhlet extractor and extracted with ether (purified 
from alcohol and aldehydic substances). The extraction flasks employed, of 
about 150 c.c. capacity and about 35 erm. in weight, were fitted to the 
extractor with ground glass joints, in order to get good quantitative results. 
After about 8 hours’ extraction, the ether was distilled off and the flask dried 
in nitrogen and later in vacuo at 97°. The extract thus obtained represents 
the “fat” fraction described above. 

(2) “ Phosphatide.”—The Soxhlet thimble and contents were then trans- 
ferred to a wide-mouthed extraction flask fitted with a condenser. To this 
about 30 c.c. of alcohol (previously distilled from caustic soda) were added ; 
the alcohol was boiled on a water-bath for 20 minutes and then distilled off 
and the flask and contents dried in an atmosphere of nitrogen. The small 
amount of material which had dissolved out of the thimble was then washed 
baek with ether and the thimble and contents were returned to the Soxhlet 
extractor; a second extraction was then made with ether and the flask dried 
and weighed as before. This extract is the “phosphatide ” fraction referred 
to above. 

No detailed chemical examination of these fractions has been made, but it 
has been shown that the “fat” fraction contains no phosphorus, whilst the 
“phosphatide” fraction contains a small but fairly constant quantity 
(estimated by Neuman’s method). Both fractions were tested for nitrogen by 
Kjeldahl’s method : traces (below 1 per cent.) were found in both fractions. 

VOL, XCIII.—B. U 


266 Misses M. Stephenson and M. D. Whetham. 


Phosphorus Estimation. 


| Weight of “ phosphatide ” fraction Merm. of | Percentage of 
| taken ; in grm. phosphorus found. phosphorus. 
0 -0280 | 0346 _ 1°23 

| 0 -0284. | 0 382 1°34 

| 0 0240 | 0270 Lag 

| 0 0241 0-270 1°12 


Nitrogen Estimation. 


| 
Weight of lipoid taken; in grm. siiegeuEen 1 eee os 
| 
0 -1126 (‘fat’) | 0-989 0°78 
0 *1260 (“ phosphatide ”) 0 823 0°73 


Probably both fractions are complex mixtures of lipoid bodies similar to 
those obtained from the tubercle bacillus [Goris, Goris and Liot, 1920 (1)]. 
The two are treated separately in this research on account of the absence of 
phosphorus in fraction A and its presence in fraction B. 


Analysis of the Medium. 


As described above, the contents of one Roux bottle (150 cc. of original 
medium) were made up to 250 c.c., from which the agglutinated bacteria and 
solid mineral matter (chalk, etc.), were then removed by filtration. The 
filtrate was then analysed for glucose or other carbon compound present. 
Glucose estimations were carried out on 5 c.c. of the diluted medium by the 
method of Wood-Ost [Wood and Berry, 1903, (4)]. Ammonia was estimated in 
the first and last samples by the method of Van Slyke [1911, (38)], in order 
to ensure that growth of the bacteria had not ceased through lack of 
nitrogen. 


Experiment 1.—Growth of the Bacillus in respect of Total Nitrogen and Lipoids 
on the usual Inorganic Medium, containing as well 1 per cent. Glucose and 
1 per cent. Acetic Acid, added as Sodium Acetate. 


Twenty-five Roux bottles, prepared and inoculated as described above, 
were incubated at 37° and bottles were withdrawn at intervals in sets of two, 
one for the estimation of total nitrogen, and the other for the estimation of 
lipoids and of the organic constituents of the medium. The disappearance of . 
acetic acid was not followed stage by stage in this first experiment, but it 


Studies in Fat Metabolism of Timothy Grass Bacillus. 267 


was ascertained that by the time the glucose had been utilised the acetic acid 
had also disappeared. 

The reaction of the medium was kept constant by chalk, which was spread 
over the bottom of the Roux bottles; the Py at the beginning, end, and all 
intermediate stages remained 8:0. This was tested by phenol red, and kindly 
checked for us by the hydrogen electrode by Miss Jordan Lloyd. 

The figures given in column 1 of the following table represent the actual 
amounts obtained from single Roux bottles (150 c.c. each), which were of 
necessity the units employed; column II represents the same amounts 
calculated for 100 c.c. medium, from which the points on Curve 1 are taken. 
Where estimations were made on duplicate Roux bottles, the figures are 
bracketed. 


Fat Synthesised. 


| 


| | 
i} - 2” | 
| Fat” fraction. Peeeead Total lipoids. | 
| Day of | Weight in grm. Wei a pons | Weight in grm. | 
) experi- | eight in grm. 
ment. |- 
ee ee ar: 1) ere seet A Sl Rin Te | II. 
| | | 
| | | a BS ae 
i} | } 
| 7 | 00054 | 0-cos6 0 0083 00054 | 00137 | 0-0090 
| | 00198 | 00182 0 -0181 00120 | 0:0379 | 0:0250 
21 | o-opas || 90288 | Gongs || 0018s 0-0 [| 070420 
| 22 | Organic cons\tituents of thle medium co|mpletely utililsed. 
/ 0-01 
58 pel penpals }| 00149 ere } 00160 Pag |) OCEO 
36 | 0-0145 | 00096 | 00230 0 -0152 0:0375 | 0-G124 
0 0089 5 tae 
Boel o.cane. y | £0008! Doles }| 0-010 Gare: fle OREO | 


* The results obtained on the seventh day are too small relatively to the experimental error 
to be important. They merely serve to indicate that growth had begun. 


Protein Nitrogen Synthesised. 


| | 
Grm. per Grm. per 
er Pp . per 
puetiaent Boos bottle, | ee cacia 
. | - 
0-027 | : 

21 0-027 | oe 

28 0-027 | 0-018 | 

36 0-026 | 0-017 | 


268 Misses M. Stephenson and M. D. Whetham. 
Glucose Utilised. 
Day of | Glucose present ; Glucose utilised ; 
experiment. grm. in 100 ¢.c. medium. grm. in 100 c.c, medium. 

0 0:97 0-00 

7 1:00 0-00 

10 0-90 0°07 

14 0°58 0°39 

21 0°14 0°83 

28 0:00 © 0:97 


Norz.—On the 28th day the acetic acid also had completely disappeared. 


It is apparent from Curve 1 that the growth of the bacillus, as measured by 
protein nitrogen and lipoids synthesised, reaches a maximum at the point 
when glucose and acetate disappear from the medium. From this point 
onwards, the bacillus utilises its own lipoid material, which rapidly decreases. 
It is particularly noteworthy that the “fat” fraction decreases at first more 


Disappearance of glucose 


22 ag co 50 55 «O 
Synthesized Nitro 
“Fat 
“Phosphatide «+ -++-+- 
De 100 cc meduurn 


Growth of Bacillus AN 


\ 


25 30 
CorvE 1. 


Studies in Fat Metabolism of Timothy Grass Bacillus. 269 


rapidly than the “phosphatide” fraction, until an equilibrium is established 
in which the “ phosphatide” fraction is maintained at the higher value. 
This suggests that the “fat” fraction represents a form of stored food 
material which is drawn upon when external sources of carbonaceous food 
material fail, whilst the “ phosphatide ” fraction, which decreases less rapidly, 
is probably partially composed of some unit essential to the chemical struc- 
ture of the cell. The duplicate determinations of the “fat” fraction on the 
twenty-eighth day show a wide variation. This is in the period of rapid 
utilisation of stored fat. A slight retardation of the point at which maximum 
growth was attained and utilisation of fat began would result in a large 
observable difference at this point. 

The results of starvation thus show that the fat metabolism of the higher 
animals has its prototype in this micro-organism. 

It also appears from the curve that the protein nitrogen decreases very 
slightly during the first fourteen days of starvation, the drop between the 
twenty-eighth and thirty-sixth day being actually within the experimental 
error. The bacillus thus seems to preserve its protein material intact. It 
must be remembered, however, that throughout the experiment ammonia 
was present in the medium; the bacillus may therefore have broken down 
cell protein as well as lipoid material, and have reconstituted the former 
from the ammonia of the medium and the fatty acid chains of the lipoids. 
Unfortunately no material was available for a final protein nitrogen estimation 
on the sixtieth day. 

It will be noticed that there is a small but definite growth of bacillus 
on the seventh day of the experiment, while the glucose is apparently 
untouched. We have confirmed this observation repeatedly in experiments 
where glucose forms the only organic food material. It may have its ex- 
planation in the small amount of food material carried over from the potato 
slopes, or it may be that in the initial breakdown of the sugar molecule a 
small quantity of a reducing body accumulates which is erroneously 
estimated as glucose; stich a body has been searched for but not as yet 
found. 

During the course of the foregoing experiment, in 100 c.c. of medium, ap- 
proximately 1 grm. of glucose and 1 grm. of acetic acid (as acetate) dis- 
appeared, together with certain unascertained quantities of mineral material 
and ammonia. Their place was taken by 0018 grm. of protein nitrogen, 
corresponding roughly to 0:1 grm. protein, and 0-041 grm. of mixed lipoid 
material. 

In a similar experiment in which glucose was the sole organic constituent 
of the medium, 1 grm. of glucose gave rise to 0:0189 grm. of protein nitrogen 


270 Misses M. Stephenson and M. D. Whetham. 


and 0:028 grm. of lipoid material. In an experiment such as the latter, it 
was hoped that some intermediate products in the change from glucose 
to fat might be isolated, and experiments with that end in view were 
made. 

Experiment 2.—Search for Intermediate Products, 


Five Roux bottles, in all 750 c.c. of medium, containing the usual inorganic 
salts, with calcium carbonate and 1 per cent. glucose, were inoculated in the 
usual way and incubated for fourteen days. The total contents of the five 
bottles were made up to 2,000 cc., and filtered. The glucose was estimated 
in the filtrate and was found to be 0:11 per cent. (calculated on the original 
medium). 0°89 per cent. glucose had thus disappeared from the original 
1 per cent. The medium still had its original reaction, Py 8. Half the 
filtrate (corresponding to 375 c.c. of the original medium) was steam-distilled, 
first from neutral solution. The distillate was collected in cold water in 
fractions and examined for iodoform-producing bodies; a slight iodoform 
reaction was obtained from the distillate collected during the first five minutes, 
but none from subsequent fractions. The iodoform could only be detected by 
the smell; no visible precipitate was obtained. 

The residue in the distilling flask was then acidified with phosphoric acid 
and again steam-distilled, the distillate being collected in standard 0-1N 
baryta (50 cc). At the end of 1} hours the distillation was stopped and 
the baryta back-titrated with hydrochloric acid, using phenol phthalein. 
The difference gave the acidity due to volatile acids and carbonic acid, of 
which a considerable quantity had come over. The carbonate was then 
filtered off, washed, transferred to a flask, mixed with 50 c.c. 0:'1N HCl, and 
boiled under a reflux condenser fitting into the neck of the flask. The residual 
acid was back-titrated with baryta. The results from 375 c.c. of the original 
medium were as follows :— 


Carbonate and volatile acids ............ 362 c¢.c. 0-1N baryta. 
Carponatevalome f jacesec terse eaeeeeaeee DO Ase Ox Nees 
Wolatilemends 2..28.0.ccne ee eee Ge. 5, ONG 


Calculated as acetic acid this is’ equivalent to 0-011 per cent. acetic acid in 
the original medium. The residue from the steam-distillation of the total 
medium (750 c.c.) was evaporated on a water-bath to about 150 c.c., filtered, 
and the filtrate extracted with ether in a continuous extractor for 24 hours. 
The solid matter was dried and extracted in a Soxhlet apparatus. The 
ethereal extract from the liquid extraction contained no solid organic acids. 
The aqueous residue left after the evaporation of the ether was tested for 
lactic acid by Hopkins’ thiophene test ; lactic acid appeared to be present in 


Studies in Fat Metabolism of Timothy Grass Bacillus. 271 


traces, but this could not be established with certainty owing to the rapid 
darkening of the solution by easily charred matter. No solid material was 
obtained from the Soxhlet extractor. 

These results were confirmed by two other experiments, slight variations 
only being obtained in the amount of volatile acids distilled over. It seemed 
therefore that, under the conditions prevailing in these experiments, inter- 
mediate productsin the breakdown of glucose accumulate only in such traces 
that attempts at identification are hopeless. 


Experiment 3.— An Attempt to Obtain Fermentation apart from the Growth of 
the Organisin. 


Six Roux bottles were filled with a medium containing the usual inorganic 
salts and 1 per cent. glucose. From six otherwise exactly similar bottles the 
ammonium phosphate was omitted. These twelve bottles were sown more 
thickly than usual with the emulsion of the bacillus. At the end of fourteeen 
days the sugar in the bottles containing no ammonia was estimated and 
found completely untouched, whilst that in the control bottles had dis- 
appeared in the normal manner. It thus appears that under these conditions 
fermentation does not occur apart from growth, which in its turn is dependent 
on a supply of nitrogen. 

Direct attempts at isolation of intermediate products having failed, 
indirect evidence was sought by growing the organism on possible inter- 
mediate products in the breakdown of the sugar molecule, in order to 
ascertain: (1) whether the organism was capable of utilising and growing 
on the intermediate products; (2) if growth took place, how the forma- 
tion of fat was affected. 

The formation of protein nitrogen (precipitated by colloidal iron) was 
taken as a criterion of growth, the lipoids corresponding to the nitrogen 
synthesised at each period being separately estimated as previously described. 
At the point of maximum lipoid formation (eg., point P, curve 1), the 
growth of the organism was considered to have attained its maximum, and — 
at that point the lipoid formation was compared with the protein nitrogen 
synthesised. 


Experiment 4—Growth on, and Utilisation of, Lactic Acid. 


A series of Roux bottles was started in the usual way containing the 
usual inorganic salts and also 0°68 per cent. lactic acid (dl) added as sodium 
lactate. Sample bottles were withdrawn at intervals and analyses of the 
organism carried out as in Experiment 1. Lactic acid was estimated in 


272 Misses M. Stephenson and M. D. Whetham. 


25 c.c. samples of the diluted medium by the extraction method of Hopkins 
and Fletcher [1907, (2)], and weighed as zinc lactate. 

The disappearance of lactate from the medium and the synthesis of total 
lipoids is summarised below and shown graphically on Curve 2. 


Davee Lactic acid present ; Lactic acid utilised; | Total lipoids formed ; 
ez tay grm. in 100 c.c. of grm. in 100 c.c. of grm. in 100 c.c. of 
P ‘ medium. | medium. | medium. 
0 0°68 | 0-00 0-00 
3 0°66 0-02 Not estimated. 
8 0°13 0°55 0-014 
10 0-01 | 0°67 0 ‘016 
12 0-00 | Total 0 ‘014 
} 


Grm. nitrogen synthesised per 100 c.c. on 10th day, 0 ‘018. 


Disappearance of 
lactic acid 


BOE Synthesis of 


lipoid 


CURVE 2. 


‘It will be seen from the above that the organism readily used lactic acid as 
its sole source of organic food material, and that the course of its growth 
does not differ materially from that on glucose and acetic acid already 
established. Similar results were obtained when the lactic acid was added 
in the form of calcium lactate. The relationship between the total lipoids 


Studies in Fat Metabolism of Timothy Grass Bacillus. 273 


formed and the total protein nitrogen at the point of maximum growth will 
be discussed later. 


Experiment 5.—Growth on, and Utilisation of Acetic Acid. 


Roux bottles containing the usual inorganic salts, with 0°5 per cent. acetic 
acid as sodium acetate, were steam sterilised and sown in the usual way. 

Estimation of the Acetic Acid—The contents of one Roux bottle (150 c.c. 
of original medium) were made up exactly to 250 cc., filtered through a~ 
dry filter into a dry flask, and the acetic acid then estimated by distillation 
of 20 cc. acidified with phosphoric acid. The usual method of estimating 
acetic acid by steam distillation was found unsatisfactory. The amount of 
distillate which must be collected before the acetic acid has completely come 
over is at least 2,000 c.c.; this contains large and variable quantities of 
carbon dioxide, which must be separately estimated by collecting the dis- 
tillate in excess standard baryta, neutralising the excess with standard 
hydrochloric acid, filtering off and separately estimating the barium car- 
bonate. The whole process takes between three and four hours, and gives 
moreover very variable and unreliable results. Instead, a modified procedure 
was adopted. ; 

The distillation of the acetic acid was carried out in vacuo. The apparatus 
used was identical with that described by Van Slyke ((oc. cit.) for the estima- 
tion of ammonia, except that in place of the second smaller distilling flask, 
a thick-walled filtering flask was found more convenient; 10c.c. of diluted 
medium (or an amount containing from 0:06 to 0-1 grm. of acetic acid) was. 
placed in a Claissen flask of 300-400 c.c. capacity, fitted with a capillary 
tube in the first neck, and a tap in the second neck. The liquid was 
acidified with four or five drops of saturated phosphoric acid. An equal 
volume of 95 per cent. alcohol (previously distilled from caustic soda) was 
added. An exact volume (about 25 cc.) of O1N baryta was run from a 
burette into the distilling flask and the filtering flask, and the capillary 
tube was adjusted to dip beneath the surface of the liquid. The contents 
of the Claissen flask were then distilled im vacuo (10-15 mm.), and the 
distillate collected in the baryta in the cooled receiver, any escape of acetic 
acid being prevented by the baryta in the filtering flask. The distillation 
was continued until only about 1 cc. of liquid remained in the Claissen 
flask. The temperature during the last five minutes should be at least 
60°. When the distillation was complete, air was admitted by the capillary 
and by opening the tap in the second neck of the Claissen flask; 20 ce. 
of 50 per cent. alcohol were then poured into the flask and the distillation 
repeated as before, care being taken that the temperature of the water-bath 


274 Misses M. Stephenson and M. D. Whetham. 


reached 60° for at least five minutes. The apparatus was then disconnected, 
the baryta carefully washed into the filtering flask, and the excess titrated 
with standard hydrochloric acid. A small and variable amount of barium 
carbonate was always present and was separately estimated. The neutralised 
liquid was filtered, the solid (barium carbonate) washed twice and then trans- 
ferred into a flask fitted with a small reflux condenser (preferably ground 
in); 10 cc. of 0-LN HCi was then added to the contents of the flask and 
the whole boiled for five minutes under the reflux condenser. The excess 
HCl was back-titrated with baryta. 

The method was tested as follows :— 

10 cc. of a solution of acetic acid were titrated direct against 0°1N 
baryta, using phenol phthalein; 10 e.c. of the same solution of acetic acid 
were distilled into baryta as described above, and the results compared. The 
following are the figures for the distillation :— 


| | | | 
Aveiy beget aeB: ili OF D. BE. | F. 
| | C.c. Ge 
Ge C.c. | O'1N C.c. 0 1N Per cent. 
| C.e. O-1N O-1IN | Ba(OH), O-1N Ba(OH) of acetic 
acetic acid Ba(OH) | HCl used | neutralised Ba(OH), aEeaeen acid in 
taken. fae 2 for back by acetic | neutralised | ae scare solution. 
‘ ; titration. acid and by CO, 5 [D-B] 
| CONMB=CIZ| yy cnlyek Fee ; 
| 
10 25°8 | 2°6 | 23 °2 0-7 | 22°53 1°35 
10 Pe aagaze 1°6 23°8 1 ‘4 | 29-4) 1°34 


10 c.c. of acetic acid titrated direct = 22-1 c.c. 0°1N Ba(OH))s. 
.°. 10 ¢.c. acetic acid contain 0°133 grm. = 1°33 per cent. compared with 1°35 and 1°34 by 
the distillation method. 


The disappearance of acetic acid from the medium is summarized below, 
and shown graphically on Curve 3. 


Day of Acetic acid present ; | Acetic acid utilised ; 
experiment.  grm. in 100 c.c. of medium. | erm. in 100 ¢.c. of medium. | 


4 0-49 0 04, 


28 0-46 | 0:07 


| 
| 
| 0 0°53 | 0-00 
| 
| 


The growth of the organism was too small to admit of accurate estimation. 
From this it appears that, in the conditions holding in this experiment, the 
organism suffers from almost complete inability to use acetic acid as a source 
of organic food material. The slight disappearance of acetic acid is pro- 


Studies in Fat Metabolism of Timothy Grass Bacillus. 275 


bably due to a small amount of carbohydrate carried over from the potato 
slopes (see below). 


0-75 


0:50 


Days O 5 /0 Ve 20 RS 


Curve 3. 


Since it has been shown in earlier experiments that no acetic acid accumu- 
lates during the growth of the organism on glucose, and since acetic acid 
has been shown by many workers to be a frequent intermediate product in 
the breakdown of glucose by bacteria, it seemed worth while to ascertain 
whether the presence of other substances might not confer on the Timothy 
grass bacillus the power of utilising this compound. 


Experiment 5.— Utilisation of Acetic Acid in the presence of Lactic Acid 
or of Glucose. 


Sixteen Roux bottles, containing the usual inorganic medium with 1 per 
cent. lactic acid (added in the form of calcium lactate) and 0:5 per cent. 
acetic acid (added as sodium acetate), were treated as described in Experi- 
ment 1. Acetic acid was estimated on 20 cc. of the diluted medium. It 
was found that a correction had to be introduced owing toa small constant 
amount of lactic acid distilling over with the acetic acid. The amount of 
the correction was ascertained as follows :—20 c.c. of diluted medium, con- 
taining 1-2 per cent. of lactic acid only, were distilled exactly as described 
above for acetic acid. A second distillation of 20 c.c. of the medium, con- 
taining 1:2 per cent. lactic acid and 0°5 per cent. acetic acid was carried out 
under exactly the same conditions. The number of cubic centimetres of 
0-1N baryta neutralised by the lactic acid distilled over in the first experiment 
was then deducted from the number of cubic centimetres of baryta neutral- 
ised by the lactic and acetic acids distilled over in the second experiment. 
The difference gave the number of cubic centimetres of baryta neutralised by 
the acetic acid only. It was found that the amount of lactic acid distilled 
over under the standard conditions was fairly constant (corresponding to 


276 Misses M. Stephenson and M. D. Whetham. 
1:9-2'1 cc. of baryta), and a constant deduction of 2 c.c. of baryta was: 
therefore made. 

The residue left in the Claissen flask after the acetic acid had been 
distilled off was quantitatively removed and the lactic acid estimated as in 
Experiment 4. The result was, of course, too low, owing to the loss of the 
lactic acid which had been distilled over with the acetic acid (z.2., 0:18 erm. 
per 100 c.c. of diluted medium). A small addition had therefore to be made 
to the results obtained by extraction. 


Utilisation of Lactic and Acetic Acids present together. 


Day of | Acetic acid present ;| Acetic acid utilised ; | Lactic acid present ; | Lactic acid utilised ; 
experi- | grm. per 100 c.c. of | grm. per 100 c.c. of | grm. per 100 c.c. of | grm. per 100 c.c. of 
ment. | medium. | medium. medium. medium. 
| 
l 
10) | 0°41 0°00 1°22 0:00 
4 | 0°15 0°26 1°15 0°07 
7 0 055 0 °355 0-94 0°28 
13 0-00 0°41 0°58 0 64 
| 28 _ — 0-32 0-90 


Two similar experiments were carried out with 1 per cent. glucose and. 
1°5 per cent. and 0:7 per cent. respectively of acetic acid (added as potassium 
acetate). 


Utilisation of 1 per cent. Glucose and 1°5 per cent. Acetic Acid present 


together. 
Day of Acetic acid present; | Acetic acid utilised; | Glucose present; | Glucose utilised ; 
| experi- grm. per 100 c.c. of | grm. per 100 c.c. of | grm. per 100 c.c. | grm. per 100 c.c. 
| ment. medium. medium. of medium. of medium. 
| 
| { 
| l 
| 0 1°49 0:00 0:98 0-00 
14 0-18 1°31 0°21 0°77 
| 18 0°14 1°35 0°12 0°86 
Utilisation of 1 per cent. glucose and 0 ‘7 per cent. acetic acid present together. 

0 0-72 0-00 0-95 0-00 
peas 0-08 0-64 0-29 0-66 
[ae 0-09 0°63 0°22 | 0-73 


The above tables and accompanying graphs (Curves 4 and 5) show that, in 
the presence of lactic acid, or of glucose, the organism is capable of utilising 
the lactic acid completely. 


Studies in Fat Metabolism of Timothy Grass Bacillus. 277 


425 


Lacie ae 
ACEC LLL, aicie ae 6a 


70 


O75 


025 


GTQTS per 
100 ¢.c. Of 
mediune 


Days. 7) 


CurvE 4. 
7-50 


Acelit aid ........ 


VRS 1-23 


1-00 7-00 
O75 


OFS 


O30 O50 


O29 025 
(00 6.6 of 700 eh 


0 5 70 15 20 Days 0 
CurvVE 5. 


INFLUENCE OF DIET ON THE CHEMICAL COMPOSITION OF THE ORGANISM. 


Having obtained growth on media with various organic constituents, it is 
of interest to notice what influence these substances have on the proportion 
of lipoids to nitrogenous material in the organism. The graph illustrating 
Experiment 1 shows that the lipoids and synthesised nitrogen rise to a 


278 Misses M. Stephenson and M. D. Whetham. 


maximum at a point closely corresponding in time to the disappearance of the 
organic constituents of the medium, and that after this point the lipoids 
decrease rapidly and the nitrogenous material very slowly. The point of 
maximum lipoid formation is taken as the stage at which the composition of 
the organism shall be estimated for purposes of comparison. The results are 
summarised in the following Table and shown graphically in fig. 2 :— 


A 8” We we = os + 


| b 


| Period of | Gtm- in 100 c.c. of medium. 
Reference | : . IES | $$$ —_— Ratio : 
eo Meee. ipod | Nitrogen Total lipoids Eoeeeipas 
P formation §, 0% peaemces i pocorsn 
fig. 2. | IpRcaey Pas » synthesised | formed by 8 
meays: by organism. _— organism. 
1 , 0°68 p.c. lactic acid... 10 0-019 0-016 | 0 84. | 
2 1°4 p.c. lactic acid .. 11 0-026 0-020 0°78 
1 [io orem beak Cane Ee y 
| 2 { 0-4 p.c. acetic acid ... } a D028 Uae | Be 
| 4 HH J8@; GCOS  aneeouone 12 0 ‘018 0 028 16 
5 {| DROS ah) we taaeoncce i 21 0-018 0-042 234 | 
| Uspienacetic acidiy..-.. 
6 ielprcwolimconemerreeennn 15 0-015 0019 1°3 
7 2 p.c. fe feago04as 15 0-034 0-021 | 0°91 


The results of the two experiments made with lactic acid alone in different 
amounts, approximate closely in the proportion of lipoid to nitrogen 
(fig. 2—1 and 2). When the organism is grown on a mixture of lactic and acetic 
acids, no more nitrogen is synthesised than on lactic acid alone, but the lipoid 
is increased by about 100 per cent. (fig. 2—3). A similar effect appears when 


3 # B 6 7 
6sxlacic 1+#laclic 12xlacte Inglucose 1% glucose 1ngliucose 2% GULCOSE 
acd aca acd 1% Qcelic 
O-440CLUC QE acid 
Fie. 2.—Diagram showing the influence of diet on the chemical composition of the Timothy 
grass bacillus. 


Black blocks represent grams of nitrogen synthesised per 100 c.c. of medium. Shaded blocks 
represent grams of lipoid synthesised per 100 c.c. of medium. 


Studies in Fat Metabolism of Timothy Grass Bacillus. 279 


acetic acid is added to a glucose medium (fig. 2—4 and 5); the added acetic 
acid does not affect the nitrogen synthesised (0-018 grm. in both cases), whilst 
the ratio of lipoids to nitrogen rises from 1°58 to 2°34. 

It seemed possible that this result might not be due specifically to the 
acetic acid, but might be caused by the greater concentration of organic food 
stuff in the medium. Experiments made on 0°68 per cent. and 1:4 per cent. 
of lactic acid (fig. 2—1 and 2), and on 1:0 per cent. and 2:0 per cent. of 
glucose (fig. 2—6 and 7), show that this is not so. Increased concentration 
of food in each case results in an increased production both of synthesised 
protein and of lipoid material, 7.c., increased general growth. The production 
of protein is favoured rather more than that of fat, as is shown by the ratio 
of lipoids to nitrogen. 

The inability to use acetic acid in the absence of carbohydrate or lactic 
acid forms at first sight an analogy to the inability of the higher animals to 
use fat in the absence of a minimum of carbohydrate. From this analogy, 
the authors were led to grow the bacillus on the sodium salts of propionic and 
butyric acids. Growth on both these proceeded readily in a manner com- 
parable to growth on lactate and on glucose. The problem therefore appears 
to be one of some complexity. A detailed study of the growth of this 
organism on various simple straight-chain compounds is in progress, and will 
form the subject of a later communication. 


SUMMARY. 


The growth of the Timothy grass bacillus on a medium consisting of 
inorganic salts, including ammonia as the sole source of nitrogen, glucose, and 
sodium acetate, was followed in detail. The formation of protein nitrogen and 
fat (a phosphatide and a non-phosphatide fraction) were followed stage by 
stage, and correlated with the disappearance of glucose and acetate from the 
medium. Attempts were made to isolate intermediate decomposition products. 
of glucose, but none were found. 

The growth of the organism on possible intermediate products of the break- 
down of glucose was then studied. The growth on lactic acid (as lactate) was 
very similar to that on glucose alone. The lactic acid could be completely 
utilised and the formation of protein and fat resembled that on a glucose 
medium. Growth on acetic acid (present as sodium acetate) was negligible in 
amount, and the acetic acid was not attacked by the organism. A modified 
method of estimating acetic acid is described. Growth on acetic and lactic 
acid (acetate and lactate) showed that the presence of lactic acid enabled 
the organism to utilise the acetic acid. Glucose also enabled the organism 
to utilise the acetic acid. 


280 Studies in Kat Metabolism of Timothy Grass Bacillus. 


A comparison of the composition of the growth on these media showed that 
the acetic acid utilised in the presence of lactic acid or glucose served, not to 
increase the general growth of the organism, but to increase the proportion 
of lipoid material formed. This was shown to be a specific effect of the acetic 

‘acid, and was not merely due to greater concentration of carbonaceous food 
material. 

Experiments were made to ascertain whether the behaviour of the bacillus 
on other straight-chain fatty acids resembled that on lactic acid (and glucose) 
or that on acetic acid. Growth on propionic acid and on butyric acid was 
like that on lactic acid, 1.2. the organism was able to grow on these com- 

‘pounds without the addition of other carbon compounds, and to synthesise 
both nitrogenous and fatty material. 


The authors’ thanks are due to Dr. Graham Smith for kindly providing a 
strain of the Timothy grass bacillus. They gladly take this opportunity of 
expressing their gratitude for the stimulating and encouraging criticism 
afforded by Prof. F. G. Hopkins during the progress of this research. ; 


REFERENCES. 


(1) Goris (Part I), Goris and Liot (Parts II and III), “Composition Chimique du 
Bacille Tuberculeux,” ‘Ann. de l'Institut Pasteur,’ vol. 34, p. 497 (1920). 

(2) Hopkins and Fletcher, “Lactic Acid in Amphibian Muscle,” ‘J. of Physiology,’ 
vol. 35, p. 247 (1907). 

(3) Van Slyke, “ The Analysis of Proteins, etc.,” ‘J. Biol. Chem.,’ vol. 10, p. 15 (1911). 

{4) Wood and Berry, “‘A Rapid Method of Estimating Sugar,” ‘ Proc. of the Cambridge 
Philosophical Society,’ vol. 12, p. 97 (1903). 


281 


Recoil Curves as Shown by the Hot-Wire Microphone. 


By Lieut.-Colonel C. B. Heatp, C.B.E., M.D., and Major W. S. Tucker, 
R.E., D.Sc. 


(Communicated by Prof. C. S. Sherrington, P.R.S. Received November 15, 1921.) 


[PuatEs 5-8. } 


Introductory Remarks. 


The subject matter of this paper deals largely with a description of a 
process which, in its bearing, appertains to physics rather than physiology, 
but as the application is entirely physiological, it has been decided to submit 
it as a physiological contribution. 

In 1916, one of us (W. S. T.), while at work on the perfection of the hot- 
wire microphone, which he had invented for the location of enemy guns, 
realised the possibilities of the hot-wire microphone for obtaining records of 
the pulse, apex beat, etc., and had actually taken records both from the wrist 
and neck, and had shown these to members of the medical profession. 

In the same year, when one of us (C. B. H.) was working upon the 

“examination of cadets for pilots’ certificates in the Royal Flying Corps, a 
converted “ penny-in-the-slot ” weighing machine was used for recording the 
weight. It was noticed that the machine would not permit an absolutely 
steady reading to be taken, as the point of the hand was in constant move- 
ment. Closer observation of this movement showed that it took place in time 
with the heart beat. 

The obvious explanation of the movement of the hand was that the 
propulsion of the blood from the left ventricle of the heart towards the head 
during the first stage of systole was accompanied by a corresponding opposite 
movement of the body towards the feet. If this assumption were correct, 
then, knowing the weight of the body, measurement of the actual distance 
through which it was moved would provide a useful factor in determining the 
efficiency of the heart when regarded as a mechanical pump. 

This simple observation with the weighing machine sufficed, however, to 
attract attention to the importance of employing the recoil of the body for 
the measurement of heart efficiency, the heart being considered as a pump. 

The only previous work along these lines appears to be that undertaken by 
Prof. Yandell Henderson (7), who used a swinging table, in which lateral 
movements were prevented. by an ingenious device; he was thus able to 

VOL. XCIIlL—8, x 


282 Lieut.-Colonel C. B. Heald and Major W. 8. Tucker 


record upon a smoked drum the movements of the table multiplied by 100. 
His remarks on this table are interesting :— 


“With such a table, the heart beat causes not only longitudinal, but 
also lateral movements. The latter have not yet been examined in 
detail. In fact, it is necessary,in order to record the longitudinal move- 
ments with accuracy, that the lateral movements should be prevented. 
It is also necessary, not only that the person under examination should 
he absolutely still, but that he stop breathing during the time the heart 
beats are recorded. Herein, indeed, lies the chief difficulty of the investi- 
gation, for while the total amplitude of the recoil movements is only a 
tenth of a millimetre, and some of the features of the curve amount to 
less than a tenth of this distance, respiration swings the body through a 
distance of many millimetres. Lastly, in order to avoid, so far as 
possible, errors from the table swinging back, after being moved out of 
plumb by the recoil, a pendulum period many times longer than the 
cardiac cycle was found necessary.” 


This author’s comments, even with these precautions, showed that at least 
three great difficulties arose :— 

(1) Periodicity in the recording apparatus. 

(2) Errors due to respiratory movements. 

(3) Errors due to the records having to be taken when the breath is either 
held, or the subject is blowing on a whistle. 

He considers that if these difficulties could be overcome, our knowledge of 
the factors controlling the physical efficiency of the cardio-vascular system 
would be improved. 

It appeared reasonable to think that the hot-wire microphone would give 
faithful records without encountering any of the difficulties above referred to, 
and, at the same time, provide a means of measurement and calibration. 

It was realised that if such measurements and calibration could be accom- 
plished, the results would be of value in examinations for determining physical 
efficiency, especially in the case of aeroplane pilots. 

The recording apparatus (fig. 1) may be divided into three parts :— 

(1) The microphones. 

(2) The galvanometer and timing device. 

(3) The photographic apparatus. 

It may be here stated that some of this apparatus has already been in use 
in sound ranging of guns, but important modifications have been made in 
order to eope with the special difficulties of the method. 


‘ouoydoror Surygverq Sy ‘ ouoydouormt ospnd ‘Fy 
{ rodopaaop o1gvUtogne pus BroULGd. “WH f POOTLA OUT] PUY FYST] JO ooanos “| f LogotMOULVA[BS WOAOYy UHL “OT 
£qmMoalo ospiaq ouoqgsywoyM “CL f pls ouoqdoarormt 4H § aopurjAo Arossooov pur ouoydotorm  f movtydeip puv wap ‘gq § wt0jyeyd ‘yw 


“LDL 


283 


x 


skey 3481] Jo euty 


Recoil Curves as shown by the Hot- Wire Microphone. 


= aaa 


284  Lieut.-Colonel C. B. Heald and Major W. 8. Tucker. 


1. The Microphone. 


The hot-wire instrument is specially capable of dealing with low-frequency 
vibrations such as those imparted to the human body by the heart. It 
consists essentially of two iron drums, connected together by a short piece of 
rubber tubing. 

One of the drums is fixed to the ceiling of the room in which the work is 
done. It consists of a cylinder, the side of which is about half its diameter. 
The platform on which the patient is standing is supported by a hook from 
a diaphragm which forms the circular end of the drum. When, therefore, the 
heart-beats cause motion of the body, the diaphragm responds, thereby altering 
the pressure within the drum in a corresponding way. 

A side tube, let into the wall of the drum, permits the passage of air which 
is set in-motion as a result of these changes. This tube is connected by the 
rubber tube above mentioned toa second cylinder of about twice the capacity, 
and having conical ends, in one of which is a fitting containing the hot-wire 
microphone grid. The air blasts, transmitted along the rubber tube from the 
first drum, are passed into the second drum through a tube opening, and are 
projected past the hot-wire grid, thereby cooling the grid and diminishing to 
a corresponding extent its electrical resistance. 

Records of apex beat or carotid involved the use of a second microphone, 
simply consisting of an open cup whose rim is of ebonite and whose base 
contains the microphone fitting with its grid, and the image of the “ string ” 
recording these effects is thrown on the same recording strip as the recoil 
curve by means of a right-angle prism. The cup is pressed with its rim 
against the chest or neck, and the pulse changes are indicated by air blasts 
passing the grid into the open air (fig. 1). 

The breathing microphone (fig. 1) consists of a single strand of hot-wire, 
mounted on an appropriate fitting attached to a stem resembling that of 
an ordinary tobacco pipe, but with a much wider air channel. The 
mouthpiece is held in the mouth, and the temperature of the grid is 
simply varied by that of the air or of the breath which passes inwards 
or outwards. 

Periodicity of the breathing is thus indicated. A very small electric 
current is used, just sufficient to indicate change in resistance on the 
galvanometer. 

2. The Galvanometer and Timing Device. 


The galvanometer employed is of the Einthoven type. Body movements 
are measured by deflections of a very fine wire mounted between the poles 
of a strong electromagnet. The instrument employed is the Souttar galvano- 


Recoil Curves as shown by the Hot- Wire Microphone. 285 


meter. It is less sensitive than the galvanometer used with the electro- 
eardiograph apparatus, 

The galvanometer used for pulse or breathing records is of the same type 
as the instrument used in the Sound Ranging apparatus. It is less sensitive 
than the Souttar galvanometer. 

All the records obtained, whether of body movement, apex beat or 
breathing, are obtained from one of these galvanometers acting in a 
Wheatstone bridge circuit. ; 

The timing device is a rotating toothed wheel, kept in rotation by a 
synchronous motor, and the current which operates it is supplied from a 
circuit containing a contact opened and closed by a vibrating tuning fork 
electrically maintained. In this way, the time-wheel can only rotate at a 
constant speed satisfying the above conditions, and making the record in 
hundredths and tenths of a second. 


3. The Photographic Apparatus. 


For the production of permanent records, use was made of the automatic 
developing apparatus employed in gun sound ranging. This consists of a 
camera with a roll of sensitised paper which can be fed continuously behind 
a cylindrical lens; the paper thereafter passing automatically through a 
developing and fixing bath. The speed of the sensitised paper can be 
regulated at will, and is capable of being varied from 6 inches per second 


a 1 ~ 
to =), inch per second. 


It will be noted from fig. 1, that the diaphragm which receives the 
body impulses is very highly damped by the rubber ring that supports it. 
The microphone container is purposely made double, with rubber connection, 
since the latter will damp out very efficiently any resonance the air in the 
system might have. 

The vibration galvanometer is heavily shunted so that, although sensitivity 
is sacrificed, the instrument is practically dead-beat. Fig. 2 shows the record 
of a make and break of 1 ohm change in the resistance of the microphone. 
This record also indicates sensitivity, and enables us to standardise all records 
taken from day to day (Plate 5). 

With a standard microphone grid, working with a given electric current, 


~ and with a galvanometer of one definite sensitivity, quantitative comparisons 


of the various records can be made. The recording of minute air currents 
by the microphone is a special feature of our method as distinct from that 
employed by Yandell Henderson, and has been dealt with in a paper by 
one of us (W.S. T.) and Capt. E. T. Paris, on “The Selective Hot-Wire 
Microphone” (15). 


286  Lieut.-Colonel C. B. Heald and Major W. S. Tucker. 


The air, with its small inertia, here takes the place of the mechanism of the 
swinging table. The movements of the air are recorded faithfully and 
without measurable lag by the change in the temperature and electrical 
resistance of the hot-wire microphone. 

The microphone is subject to resistance variation through change in the 
temperature of the room, thus changing the zero. This effect, however, can 
be overcome by simply adjusting the balance of the Wheatstone bridge. 

In order to use the microphone—which has a resistance of about 150 ohm 
when heated—it is inserted in one arm of a Wheatstone bridge, the other 
arm being adjusted by a rheostat to give balance with the string galvano- 
meter. The pulsating currents of air which cool the microphone grid, 
give a corresponding variation of resistance, but it must be noted in this 
investigation that motions of the air, whether positive or negative in direc- 
tion, always produce positive deflections on the galvanometer wire, since all 
air movements produce a fall in temperature and a diminution in resistance 
of the microphone grid. 

Another point to be emphasised is that deflections of the galvanometer 
wire are not proportional to the displacements of the body in a given direc- 
tion, but are proportional to some function of the velocity of the body under 
recoil. What this function is, may be indicated by reference to the previous 
work done on the theory of the hot-wire microphone. 

The deflection of the galvanometer, which may be considered to be pro- 
portional to the change in electrical resistance of the microphone, is . 
dependent not only on the vibratory motions of the air set up by the heart 
‘action, but also on a certain amount of direct air current set up by convec- 
tion, as a result of the disposition of the microphone grid in the orifice 
through which the vibrations are transmitted. Convection effect was 
reduced to a minimum in this investigation, by setting the plane of the 
microphone grid vertical, so that convection currents tend to be perpen- 
dicular to the displacements of the vibrating air. These currents, however, 
could not be completely eliminated, owing to the lack of complete symmetry 
of the enclosure on the opposite sides of the grid. 

It has been shown in the paper above referred to that for a certain type of 
grid similar to that in use and under similar conditions, receiving vibrations 
U sin pt, the total resistance change 


dR = —0:15U?40:15U sin pt+ 0°15 U? cos 2pz, 
where U is the velocity of the air at any moment past the grid, 27/p the 


period of vibration of the air. Other terms may be added to the above 
series, but have been shown to be so small as to be negligible. The con- 


Recoil Curves as shown by the Hot-Wire Microphone. 287 


vection current in direction parallel to the displacements of the vibrating 
particles, is entirely responsible for the term containing U sin pt and from 
the equation it is seen that a simple harmonic vibration in the air is 
recorded by :— 

(i) a displacement of the mean position proportional to the square of the 
velocity ; 

(ii) a periodic term in tune with the air vibration, of amplitude proportional 
to the velocity ; 

(iii) a periodic term an octave above this vibration proportional to the 
square of the velocity. 

Terms (i) and (iii), therefore, are proportional to the kinetic energy in the 
air, while (ii) is proportional to the square root of that energy. 

This relation has been checked by means of an artificial vibrating system 
fixed to the platform referred to above, and consisting of a spiral spring 
supporting a weight which, when displaced, vibrates vertically up and down. 
The spring becomes, for the time, an artificial heart, imparting its vibrations 
through the platform to the diaphragm and to the air in the microphone 
chamber. 

The diaphragm is put under the same tension, etc., by placing weights upon 
the platform equal to the weight of the case under comparison. 

Fig. 34 gives the galvanometer record which closely agrees with the above 
equation in which the maximum velocity of the vibrating air is 2 cm. per 
second, and the convection current about 2 cm. per second (Plate 5). 

One complete vibration corresponds to two peaks, the higher one of which: 
indicates a displacement of the vibrating air in the same direction as the air 
current. 

At any point, the deflection of the galvanometer is proportional to the 
_ funetion of U as quoted in the above equation, but if the function be inte- 
grated with respect to time for a complete cycle, wc, for a time 27/p the 
periodic terms vanish and a quantity proportional to U?, z.¢., to the kinetic 
energy of vibration, is obtained. 

If now the amount of kinetic energy contained in the spring is calculated 
from the mass of the spring, its periodic time and its displacements, we can 
calibrate the records in terms of such kinetic energy. 

The vibrating spring, however, fails to resemble the heart in its action since, 
as seen from the record, the spring loses very little energy per period, 7.¢., it 
is a nearly undamped vibrating system. Had it been heavily damped, the 
spring would rapidly come to rest and the record would show markedly 
decreasing amplitudes. 

With the heart quite a different condition obtains. The projected blood 


288  Lieut.-Colonel C. B, Heald and Major W. 8. Tucker. 


constitutes the mass of the spring and the muscles of the heart the spring 
itself, but the energy which is imparted to the blood, and therefore to the 
body as a whole, is immediately absorbed so that the vibrations appear to be 
dead beat. Itis nevertheless true that the resistance variation, though not 
to be expressed by so simple an equation as that quoted above, can be treated 
in a similar manner, and for a heart cycle, consisting as it does of a number of 
vibrations, superimposed or consecutive, between two so-called heart beats, a 
process of integration can be carried out, and the result will give a quantity 
from which the periodic terms vanish, and which has a value proportional to 
a (velocity), 2.¢., to the kinetic energy imparted to the body during the cycle. 

The process of obtaining the kinetic energy of recoil of the body resolves 
itself, therefore, into the measurement of an area bounded by the heart curve 
and the zero axis, dividing this by the time, and expressing the result in any 
desired units of energy, by comparing with a similar area divided by time, 
given by the vibrating spring of known energy. 

The kinetic energy contained in the spring was found from the relation 
4Mp?d*, where d is the amplitude of the spring and M the effective mass 
which is maintained in vibration with a periodicity of p’/27. This corresponds 
during calibration to an amplitude in the Einthoven string of a. 

The photographic record shows an amplitude of 0, so that the kinetic energy 
of the spring as recorded is :— 

M p?d?b / 2a. 

In the case under consideration this quantity works out to 22 x 10* ergs 
and may be measured by the mean ordinate of the spring curve—say Y. 

Dealing now with the heart curve, examination is made over a complete 
breathing cycle of six heart beats, and the mean ordinate for these is obtained 
(fig.3, B). Calling this y, the kinetic energy exhibited in the body corresponds 


to y/Y x 22 x 104 ergs 


and this is completely absorbed, the time corresponding to it being ¢ seconds. 
Hence the kinetic energy produced averages for a breathing cycle 


y[Ytx 22 x 104 ergs per sec. 
In this determination y/Y = 4 and ¢ = 4:23 secs., so that the heart output 
creates a kinetic energy in the body of 
2-6 x 10* ergs per sec. 
or 0:18 gramme-metres per heart cycle. 


The figures obtained do not represent the total kinetic energy of the heart 
but are, we suggest, proportional toit. They must not, therefore, be compared 
directly with the work done by the heart, which, as given by Starling (12), is 


Recoil Curves as shown by the Hot-Wire Microphone. 289 


in the neighbourhood of 82:3 gramme-metres per beat, of which 81:6 units 
represent work done against arterial pressure, and 0°7 units are measured in 
kinetic energy at the root of the aorta. The latter again is considerably 
greater than the value obtained by our experiments, since our measurements 
are integral of all movements footward and headward. 

One other point should be referred to before proceeding. Yandell 
Henderson obtained records of displacement variation, and these in a given 
mass, that of the patient with the platform that supports him, are also records 
of the potential energy of that mass. 

Thus it will be seen that the measurements made by Yandell Henderson 
and ourselves are complementary, since in any vibrating system the total 
energy at any time is the sum of its potential and kinetic energies. The two 
quantities will be out of phase 7/2, maximum potential energy correspond- 
ing to minimum kinetic energy, and vice versé. 

The peaks of the displacement curve correspond to the minima of the 
records of this paper. Also, while the displacement curves can indicate 
positive and negative values, the curves of our records only give positive 
values. Allowing now for the change of phase of 7/2 on passing from one 
curve to the other, and remembering that the values of the kinetic energy 
curves are always positive, it is possible to show a resemblance between the 
two sets of curves. 

Fig. 4 is a reproduction of a diagram from Yandell Henderson’s paper. 
Taking fig. 3, B, to represent a characteristic heart record obtained by the 
process described in this paper, the curve of fig. 4 can be approximately 
reproduced by reversing alternate peaks to allow for positive and negative 


Carotid 
Pulse 


A. 
AHeadwar?—— 
Centre 

of 
Gravit y 
Feelward 


systole Diastole 


Fie. 4. 


effects. The change of phase would be imposed by moving the curves as a 
whole through the width of half a peak, bearing in mind also that whereas 


290 Lieut.-Colonel C. B. Heald and Major W.S. Tucker. 


Yandell Henderson’s curve reads from left to right, the record of fig. 3 reads 
from right to left. 


Theoretical Considerations. 


The maintenance of life depends ultimately upon the efficiency of the 
circulation. 

Since circulation is maintained by the pumping action of the heart, any 
measurement of its output gives an effective method of testing the controlling 
engine of the body. 

Yandell Henderson used the displacements in the body recoil in an attempt 
to measure the volume discharged from the heart per unit weight of the body 
at each contraction. Thus, a body of weight W would suffer a displacement 
D, corresponding to the propulsion of blood of weight w displacing an amount 
d at each heart contraction, where 


= WiDyiar 


With accurate measurements of body displacement a figure is thus obtained 
proportional to the volume of the discharge per systole. It is obvious, 
however, that the actual work done in producing the movements of the body 
is not the reaction movement of the left ventricle only, but the algebraic 
sums of all the movements of blood or body fluids during the period of 
estimation. 

The difference between our measurements and those of Yandell Henderson 
can now be clearly indicated. His curves are effective indications of the 
potential energy in the body at any moment, and changes in such energy are: 
proportional to systolic discharge. The curves we obtain are indications of 
the complementary kinetic energy of the body, changes in which are propor- 
tional to the changes in potential energy. If Yandell Henderson’s curves, 
therefore, measure systolic discharge, the same claim can be made for the 
curves given by the hot-wire microphone. : 

The advantage of obtaining kinetic energy curves rather than those of dis- 
placement shows itself in one important way. The displacements of the 
body produced by causes other than those due to heart action may be consider- 
able. In the act of breathing, for example, the movements obtained may be 
many times greater than those due to propulsion of the blood. For this 
reason, Yandell Henderson took special precautions, such as holding the 
breath or causing the patient to blow steadily through a whistle. In our 
case, however, we are concerned with velocities rather than displacements, 
and the velocities in the body resulting from breathing are so small as to be 
negligible. The same applies also to slow muscular movements in the body, 
incidental on digestion, ete. Velocities of the air resulting from the 


Recoil Curves as shown by the Hot-Wire Microphone. 291 


diaphragm displacement in the microphone container are here exceedingly 
minute, and we are thus able to assume that they produce no appreciable 
change in the electrical resistance of the microphone. 

We are able to show, however, that breathing does produce a marked effect 
in systolic discharge, as will be clearly indicated in the records described 
later. 

Experimental Work. 

The earliest records were obtained by the simple process of seating the 
patient on a microphone container. It was found that the “spring” of the 
walls sufficed to produce measurable effects. In order to avoid change in zero 
of a balanced electric circuit, the resistance variations were conveyed to the 
galvanometer through the agency of a transformer. 

The next development was that in which the patient was seated in a chair 
which was bolted to a wall of the container at a point beneath its centre of 
gravity. Both of these methods involved careful balance of the body, and 
in any case could give no quantitative results. The chair was then slung by 
chains from the under surface of the diaphragm in the apparatus described 
earlier in the paper, and a foot rest was fitted so that the patient could be 
seated at ease. Here, again, the effect, though more consistent, and though 
to some extent capable of comparable measurement, depended on the attitude 
of the subject, and records showed that marked differences were given when 
the knees were bent to different extents, or when the head was erect or 
bowed. Finally, the subject to be tested was placed on the swinging platform, 
in an absolutely erect position, with a shoulder rest to steady the body. 

A change was also made in the electrical arrangements. It was found 
possible to replace the transformer method by that of the simple Wheatstone 
bridge. This latter arrangement enabled us to get more faithful records of 
the resistance changes of the microphone. Contrary to expectation, the zero 
of the record was found to be quite steady. 


Consideration and Analysis of the Curves. 


The Normal Recoil Curve.—The normal recoil curve is undoubtedly very 
similar to that shown in fig. 5. This curve and the majority of those 
reproduced in this paper have all been obtained from one subject 
(Mr. A. Reading), whose general physical standards are equal to those found 
in the best type of pilot. In addition, the electro-cardiogram and ortho- 
diagrams of this case are normal (Plate 5). 

Very similar records, which need not be reproduced here, were given by 
many other normal subjects. 

As will be seen from the figure, the main features of the normal curve 


292  Lieut.-Colonel C. B. Heald and Major W. S. Tucker. 


consist of (reading from right to left) a small peak, followed immediately by 
two considerably larger peaks, and then by two smaller and somewhat flatter 
peaks. These peaks have been numbered 1 to 5 in the diagram. These five 
peaks, since they are nearly always present in every curve, may be considered 
as the essential elements of the normal recoil curve. In some of the heart 
eycles in the figure other smaller peaks may be observed, and it is generally 
these secondary peaks that tend to become exaggerated, or entirely absent, 
from case to case. : 

Fig. 5 shows also a simultaneous record taken over the carotid by means of 
the special hot-wire microphone previously described. On this record, the 
time relations of the various peaks of the recoil curve in relation to the 
phenomena of a heart cycle have been purposely omitted. This has been done 
to avoid confusion and error, as the peak of the “ hot-wire ” carotid curve, 
which indicates the maximum of velocity, does not occur at the same time 
as that on the ordinary carotid curve. It is, as shown previously, a curve out 
of phase with the ordinary curves which record changes in pressure. When 
the simultaneous records of figs. 5 and 6 were taken we had no comparator at 
our disposal and therefore have not given an exact figure for the time 
interval between the commencement of the first heart sound and the 
commencement of the carotid peak. 

In the heart-sound microphone, the deflections caused by the commence- 
ment of the first and second sounds, corresponding, as they do, to the closure 
of the auriculo-ventricular and semilunar valves, fix very definite points of 
time in the heart cycle for comparative purposes on simultaneous tracings. 

As heart-sound records are free from the difficulty inherent in any 
comparison between potential and kinetic energy curves, a simultaneous record 
_ of recoil and heart sounds has been prepared, and this is shown in fig. 6, while 
the relation of the two sounds has been more clearly defined by lines drawn 
through their commencement to cut the recoil curve. 

As it is undesirable to introduce the recoil curve into the standard composite 
diagram representing changes of pressure in auricles, ventricles, aorta and 
carotid, a special diagram, fig. 7, has been prepared, in which the kinetic 
energy curves of body recoil and carotid are co-ordinated with heart sounds. 
For the time relations of the various events in a heart cycle, the data given 
by Lewis (10) have been used. 

From figs. 5 and 7 the commencement of the carotid curve is seen to 
coincide with the beginning of the first or small peak of the recoil curve. 
This peak (1) is, therefore, probably caused by the feetward movement of the 
body corresponding to the headward movement of blood as it is projected 
from the left ventricle shortly after the beginning of each systole. The 


Recoil Curves as shown by the Hot-Wire Microphone. 293 


interpretation of the two peaks (2 and 3) is, we think, as follows: The first is. 
probably due to the movement of the body headwards as the mass of blood 


[| 10 fossfos| | 


| [VENT SYSTOLE DIASTOLE. 


Auric Excit .Starts. 

Vent Exclt. Starts, 

Aur-Vent Valves Close. 
Avur-Vent Valves Open — 


- Semitunar Open. 
Semilunar Gloee, 


CAROTID 
(HOT WIRE) 


anne ae left to right ) 


passes down the aorta, while the second is caused by the return ’of the 
displaced body to normal. 

The interpretation of the two smaller peaks (4 and 5) is, however, not easy, 
as itis difficult to believe that a recoil of this magnitude can be produced 
entirely by auricular systole. 

In this connection, it should not be forgotten that patients with large and 
heavy hearts, not necessarily dilated post-mortem, frequently shake the whole 
bed. The question, therefore, arises whether, or not, some of the features of 
the recoil curve are due to the actual movement of the heart itself. It has 
been shown by needles passed into the base centre and apex that the base 
moves downward during systole. The elastic recoil of the distended aorta 
also requires taking into consideration. These questions have been left for 
subsequent investigation. 


294  Lieut.-Colonel C. B. Heald and Major W. 8. Tucker. 


The interpretation put forward here of the recoil peaks must be considered, 
at present, as purely tentative. 

In fig. 5 it will be seen that the recoils increase and decrease at- regular 
intervals of time, and the relation of these increases and decreases to respira- 
tion are well shown in tig. 8. In this figure, the shorter parts of the breathing 
record correspond to inspiration, and it will be seen that, in agreement with 
other physiological observations, the maximum recoil occurs just after 
expiration has commenced (Plate 6). 

The effect on the recoil curve, when the breath is held in deep inspiration 
and deep expiration, is well shown in fig. 9. 

The recoils, which are so largely increased in inspiration and diminished 
in expiration, are undoubtedly due to the normal physiological variations 
in output during respiration. During inspiration, the descent of the 
diaphragm increases the positive pressure in the abdomen, thereby tending 
to press blood out of the abdominal veins, and at the same time the negative 
pressure in the thorax is increased, and greater suction exerted. The heart, 
therefore, will be better supplied during inspiration with blood than during 
expiration, and in consequence the output will increase, and enlarged recoils 
be the result. 

The variation in curves given by apparently healthy individuals may be 
seen from fig. 10, where the first example demonstrates a simplified type of 
curve, in which only the primary peaks are recognisable, and from which 
secondary peaks are practically absent (Plate 7). 

The second example shows the type in which the secondary peaks are so 
enlarged that they occasionally make recognition of the primary peaks difficult 
without dividers. 

The third example, taken very shortly after the two upper curves, and 
without any alteration of the apparatus, is from the standard normal subject 
( A. Reading). 

The meaning of these variations is the subject of investigation alone two 
lines, one anatomical, and the other physiological. Small variations in the 
anatomical position of the axis of the heart, or in the disposition of the 
great vessels, may, as referred to above, alter the proportion of the total 
kinetic energy of a contraction acting in the long axis of the body. Or, as 
Krogh and Lindhard (9) have shown, individuals may have a high or low 
coefficient of oxygen utilisation, with a consequent small or big volume 
output from the heart, 2.¢., a small or big recoil curve. 

Variations in Blood Pressure—As soon as it became evident that the 
recoil curves were subject to wide normal variations from subject to 
subject, a certain number of experiments were carried out to determine 


—— 


Recoil Curves as shown by the Hot-Wire Microphone. 295 


the variations which made their appearance in the normal subject under 
different conditions. 

Curves were therefore taken before and after meals over a series of con- 
secutive days at the same hour; and before and after exercise. 

It was thought at first that the variations in the recoil might bear some 
simple relation to the blood pressure. A number of healthy individuals 
were, therefore, examined from this point of view, but yielded no definite 
evidence that small differences of systolic, diastolic or pulse pressure readings 
could be correlated with alterations in the size of the recoil. 

As Tigerstedt (13) had shown, with his Stromuhr, that nitro-glycerine 
increased the minute-volume and lowered the blood pressure, while adrenalin 
tended to the opposite effect, it was decided to try the effect of small doses 
of vaso-constrictors and vaso-dilators, liquor adrenalin and nitro-glycerine 
respectively, taken by the mouth. Accepting Tigerstedt’s results as applic- 
able to the normal human subject, it would seem that enlarged recoils are 
the result of increases in volume output rather than raised blood pressure, 
as is clearly shown by fig. 11. In this figure, the only curve showing a 
definite increase of recoil is (@), v.¢., the one taken five minutes after a small 
dose of nitro-glycerine. Curve (c), taken after a small dose of adrenalin, is 
not appreciably different from any of the control curves. 

It is true that the dose of liquor adrenalin is very small, and was given by 
the mouth, but the effect of the nitro-glycerine is very striking. 

Curves were also obtained after taking by the mouth 0°02 grm. of the vaso- 
constrictor tyramine, a drug which is definitely known to be absorbed, and 
recoils of diminished amplitude were obtained. 

The results are not conclusive, but, taken in conjunction with the 
variations during respiration, are suggestive that alterations in the volume 
output are the chief factors affecting the magnitude of the recoil; this agrees 
with the theory of the action of the microphone dealt with above. 

The result of experiments carried out to show the effect of increased heart 
work on the recoil indicates that all the features of the normal curve 
become enlarged, especially the smaller peaks. An example of a curve taken 
before, immediately after, and some time after exercise, is shown here to 
illustrate this point (fig. 12, Plate 8). 

[t would not, however, be justifiable to assume, at this stage, that a 
curve such as (B) of fig. 10 is produced because the subject’s heart is doing 
more work. 

All the experiments that have been carried out have been directed solely 
towards ascertaining the nature of a normal curve, and the variations to 
which it is subject. As a single control to these experiments, the recoil 


296  Lieut.-Colonel C. B, Heald and Major W. 8. Tucker. 


curve from a case of aortic regurgitation with a high blood pressure, and 
a very large heart, was taken. It is shown here (fig. 13) as an example of 
the extreme deflection caused by the hot-wire microphone, when abnormal 
movements of the body are transmitted to the air in contact with it. 


Conclusion. 


A careful examination of the above records shows that there is an 
additional periodic effect, besides that of the heart cycle and of breathing. 
This reveals itself in the variation in heights of the two highest peaks. At 
certain parts of the record, the first peak is higher, in other parts it is lower 
than the second peak, while we also get the intermediate conditions of 
equality of peaks. One of these effects can be partially explained by the 
difference in sensitivity of the microphone according to the direction of the 
air current; but some physiological explanation is required to account for 
the whole phenomenon, as instrumental variations have been reduced to a 
minimum. This effect will be the subject of further study. 

The apparatus employed for the recoil measurements is now being further 
modified, so that the patient can be examined in a recumbent position. 
Records could then be obtained when the muscles are completely relaxed 
and might easily be taken during sleep. 

This further modification will also permit us to measure the recoil in 
directions perpendicular to the body axis, so that information regarding the 
whole of the body movements should then be available. 


Summary. . 


This paper deals with a new method of measuring body recoil as 
the result of heart action. Attempts have been made to eliminate the dis- 
turbing factors operating against the success of Yandell Henderson’s method. 
To effect this, the hot-wire microphone with suitable galvanometer and 
recording apparatus has been employed, and the records actually made 
measure quantities proportional to the kinetic energy imparted to the body 
by motions of the blood. In this way slow-moving displacements, such as 
those of breathing, fail to be recorded. 

The apparatus is of such form that it can be standardised, giving the same 
responses from day to day for the same body recoils. 

A method is indicated of expressing this kinetic energy of the body in 
C.G.S. units. Attention has been concentrated on the records obtained from 
a favourable subject, and an analysis of these curves shows that the events 
of a heart cycle can be recognised. 


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Heald & Tucker. 


(a) 


(b) 


ec) 


(d) 


(e) 


Fig. 11 


Normal Recoil Curve as 
general Control. 


8 minutes after 1 ounce 
of Peppermint Water (to 
demonstrate presence of 
a psychological effect, if 
any). Showing no psycho- 
logical effect produced. 


5 minutes after 1 ounce 
of Peppermint Water, to 
Which 15 drops of Liquor 
Adrenalin had been added. 


5 minutes after 1 ounce 
of Peppermint Water, to 
which 1/150 grain of 
Nitroglycerin had been 
added. Showing great 
increase in second and 
third peaks. 


Control curve taken half- 
an-hour after (d), show- 
ing complete return to 
normal. 


Roy. Soc. Pizaps 15), Holl, OZ, We 
Fig. 10 


Heald & Tucker. Roy. Soc. Proc. B, vol. 93, pl. & 


Fig. 12 
_ NORMAL HEART RECORD BEFORE EXERCISE 


IMMEDIATELY AFTER EXERCISE 


60 SECONDS AFTER EXERCISE 


Recoil Curves as shown by the Hot-Wire Microphone. 297 


The results so far obtained are consistent with accepted physiological 
data as to the variations in the systolic output of the heart, as affected by 
exercise, respiration or the action of vaso-constrictors and vaso-dilators. 

In conclusion, the authors wish to express their appreciation of the help 
and assistance given to them in carrying out this work by Sir Frederick Sykes, 
the Controller-General of Civil Aviation, and Lieut.-Colonel Cusins, Chief 
Experimental Officer of the Signals Experimental Establishment, where the 
work was carried out. They further desire to express their indebtedness to 
Prof. Yandell Henderson, Dr. A. W. Stott for his kind collaboration during 
the preliminary stages, and to Dr. P. Hamill, when correlation with the 
electro-cardiograph was required, as well as for advice on the physiological 
aspects of the paper. 

The authors particularly wish to recognise the help given by Mr. A. Reading, 
who, besides providing them with a very useful subject for heart recording, 
also manipulated the apparatus most skilfully. 


REFERENCES. 


(1) Bainbridge, F. A., ‘The Physiology of Muscular Exercise,’ London, 1919; ‘‘The 
Influence of Venous Filling upon the Rate of the Heart,” ‘Jnl. of Physiology,’ 
vol. 50, p. 65. 

(2) Bainbridge, F. A.,and Hilton, R., “The Relations between Respiration and the 
Pulse Rate,” ‘ Proc. Physiol. Soc., p. 65 ; ‘Jnl. Physiol.,’ vol. 52. 

(3) Barcroft, J., “Measurement of the Blood Flow through the Chest of the Goat,” 
‘ Proc. Physiol. Soc.,’ London, 1919; “The Measurement of the Work Performed 

by the Heart,” ‘ Proc. Physiol. Soc.,’ London, 1919. 

(4) Cannon, W. B., “Auscultation of the Rhythmic Sounds (Telephone Records) 
produced by Stomach and Intestines,” ‘ American Journal of Physiology,’ 1905. 

(5) Dunn, J. S., “Measurement of Pressure in the R.V.,” ‘Proc. Physiol. Soc.,’ 
London, 1919. 

(6) Golla, F. M. (Croonian Lectures, R.C.P.), “The Objective Study of Neurosis.” 
‘Lancet,’ 16/7/21, 30/7/21, 6/8/21, 20/8/21. 

(7) Henderson, Yandell, “The Mass Movements of the Circulation, as shown by a 
Recoil Curve,” ‘ American Journal of Physiology,’ 1905. 

(8) Hill, A. V., “Electrical Recording of the Pulse,” ‘Lancet,’ 9/10/20 ; “ Hot-Wire 
Microphone,” ‘ Lancet,’ 30/10/20. 

(9) Krogh, August, and Lindhard, J., ‘‘ Measurements of the Blood Flow through the 
Lungs of Man,” ‘Skand. Archv. fiir Physiol.,’ vol. 27, p. 100 (1912); “On the 
Influence of the Venous Supply upon the Output of the Heart,” ‘Skand. Archv. 
fiir Physiol.,’ vol. 27, p. 126 (1912) ; ‘The Regulation of the Supply of Blood to 
the Right Heart,” ‘Skand. Archv. fiir Physiol.,’ vol. 27, p. 227 (1912). 

(10) Lewis, T., ‘The Mechanism and Registration of the Heart Beat,’ London, 1920. 

(11) Mackenzie, J., (a) ‘Diseases of the Heart,’ London, 1913; (6) ‘The Study of the 
Pulse, Arterial, Venous, and Hepatic, and of the Movements of the Heart,’ 
London, 1902. 

(12) Starling, E. H., ‘Principles of Human Physiology,’ London, 1912. 


VOL. XCIII.—-B. Ye 


298 Messrs. J. C. Bramwell and A. V. Hill. 


(13) Tigerstedt, C., “Zur Kenntnis der von dem linken Herzen herausgetriebenen 
Blutmenge in ihrer Abhingigkeit von verschiedenen Variabeln,” ‘Skand. Archy. 
fiir Physiol.,’ vol. 22, p. 173 (1909) et seg. 

(14) Tigerstedt, R., “ Neue Untersuchungen iiber die von dem linken Herzen heraus- 
getriebenen Blutmenge,” ‘Skand. Archv. fiir Physiol.,’ vol. 19, p. 25 (1907) ez seq. 

(15) Tucker, W. S., and Paris, E. T., “A Selective Hot-Wire Microphone,” ‘Phil. 
Trans., A, vol. 221, pp. 389-430. 


The Velocity of the Pulse Wave in Man. 
By J. CRIGHTON BRAMWELL,* M.B., M.R.C.P., and A. V. Hitt, F.RS. 


(Received February 4, 1922.) 


In an investigation now being carried out by us at Manchester observa- 
tions are being made, under various conditions, upon the velocity of the pulse 
wave inman. Asa preliminary to this investigation it was thought advisable 
to study the theory of the transnrission of’ the pulse wave, and the following 
pages contain the results arrived at, together with an account of experiments 
upon the velocity of the pulse wave in an isolated human artery. 

The pulse wave in man travels in the arteries at a speed of 4 to 10 metres 
per second. Its velocity depends, to a small degree, on the velocity of the 
blood in the artery considered, but chiefly upon the elastic condition of the 
arterial wall, which is affected by a variety of factors in health and disease. 
As regards the former, the pulse wave must be considered as travelling, like 
a ripple on moving water, relatively to the fluid in which it occurs. The 
arterial wall merely exerts an elastic constraint upon the surface of the fluid, 
and in the simplified theory of the transmission of the wave (which it is 
necessary for practical purposes to adopt) the inertia of the wall, and of the 
tissues outside it, exerts no influence on the velocity of the wave. Thus any 
experimentally determined value must represent the velocity of the wave 
relatively to the blood, plus the velocity of the blood in the artery. Taking 
0-75 metres per second as an average maximum velocity of the blood in the 
aorta, and 0:25 metres per second as an average maximum in the carotid 
artery (4), we see that the correction for the velocity of the blood itself is small, 
but not negligible, in comparison with the velocity of the wave. Any con- 
siderable increase in the velocity of the blood, caused, ¢.g., by local or general 
exertion, will cause an equal increase in the velocity of the pulse wave. 


* Working for the Medical Research Council. 


The Velocity of the Pulse Wave in Man. 299 


Moreover, the velocity of the blood in the aorta, and to some degree in any 
artery, varies considerably at different moments of the cardiac cycle; such 
differences will cause one part of the pulse wave to be transmitted with a 
greater velocity than another, and so will lead toa certain modification in the 
apparent form of the wave. In the absence of definite knowledge of the 
velocity of the blood in any given case it is not possible to make any 
allowance for it; it is necessary, however, to bear in mind that it may, under 
certain circumstances, appreciably—though not considerably—affect the - 
velocity and modify the form of the transmitted wave. 

Considered in its full complexity the theory of the transmission of the pulse 
wave is difficult. There are, however, two factors which allow us to simplify 
it: (a) the distance over which the wave travels is relatively short ;, (0) the 
wave form, owing to the elastic nature of all the tissues producing it, shows no 
very sharp discontinuities or changes of curvature. In consequence of (6), in 
the analysis of the wave into a system of simple harmonic waves, the shorter 
wave-leneths are relatively unimportant, and it is the transmission of these 
waves which would have required the more complicated treatment. With 
the help of Mr. E. A. Milne, of Trinity College, Cambridge, a fuller theory of 
the wave transmission has been worked out; it is unnecessary to give this 
theory at length, but it may be stated that, with the type of wave occurring 
in arteries, and within the limits of experimental error, the formula given by 
Moens (7) in 1878 is sufficiently accurate for our purpose. We will consider 
the meaning and application of this formula. 

If v be the velocity of the front of the pulse wave, y the radius of the 
artery at the end of diastole, c the thickness of the arterial wall, E the 
modulus of elasticity of the artery for lateral expansion, and p the density of 
the blood, the following relation holds: 


v= / (He/2py). 

Assuming that p is constant, and equal (say) to 1-055, this formula contains 
three variable factors, on which the value of v depends, viz., EK,c and y. In 
this form the expression is of little value, since E,c and y vary from artery 
to artery, and none of them express any easily measurable factor. By a 
simple transformation, however, a formula may be obtained which throws 
much light upon the mechanics of the circulation. A small rise dp in 
pressure may be shown to cause a small increase, dy = y*dp/Kc, in the radius 
y of the artery, or a small increase, 6V = 27y*dp/Kc, in its volume V per 
unit length. Hence 2y/Ece = dV/Vdp, from which 


v= /(Vf[paV /dp]). 


In this equation p is measured in dynes per square centimetre, and v in 


300 Messrs. J. C. Bramwell and A. V. Hill. 


centimetres per second. Expressing p in millimetres of Hg, and v in metres 
per second, and substituting p = 1:055, this equation becomes, 


»v = 0°3574/(V/[dV/dp]). 


But (dV/dp)/V is the relative increase in the volume of the artery, per 
millimetre of Hg increase of pressure. Working in percentages, therefore, the 
equation finally becomes 


v = 3:57/,/ (percentage increase in volume per millimetre of 
Hg increase of pressure). . 


This is the form most intelligible and convenient in use. It requires no 
knowledge of the elastic coefficient as such, nor of the radius and the thick- 
ness of the arterial wall, but only of one simple and directly observable 
function of these, the rate of increase of volume with pressure. Thus an 
observation of the velocity of the pulse wave in any particular vessel tells 
us at once, in absolute units, the degree of extensibility of that vessel. 

The energy expended by the heart, per beat, has been shown by Rhode (2) 
and others, to depend (other things being equal) on the pressure developed by 
it. Thus, if the heart is to work efficiently, the output for a given pressure 
should be as large as possible, which implies a large increase in the volume of 
the arteries per millimetre of pressure developed, and—from the formula—a 
low velocity of the pulse wave. Another sign of an efficient circulation is 
that the flow through the capillaries should remain as high and as constant 
as possible during diastole, which implies a large diminution of volume of 
the arteries for a given fall of pressure, and again a low velocity of the pulse 
wave. Hence a low velocity of the pulse wave is a sign, both of an efficient 
and continuous circulation and of an economical functioning of the heart. 
Thus the velocity of the pulse wave is one important criterion of the general 
efficiency of the circulation. 

In a paper by Roy (3), in 1880, is given a series of curves showing the 
relation between volume and pressure, in the case of arteries and veins, made 
by an ingenious method, commanding every confidence in its accuracy. 
Replotting these curves in rectangular co-ordinates, and measuring their 
slopes at various points, it is possible to deduce the percentage increase in 
volume per millimetre of Hg, and so to calculate the velocity of the 
pulse wave at various pressures. The following results are obtained by 


so doing :-— 


The Velocity of the Pulse Wave in Man. 301 


Table I. 


i | 
PA) whl celui ah id (oul 


Il. Fig. 7—Femoral Artery of Rabbit. 


jhe glee | 20 40 60 80 | 100 | 120 140 | 160 


HID | GEE | SAO | Kes | Was) oO ee 


III. Fig. 9—Carotid of Rabbit, immediately after death. 


| | vil a a | 
60 70 | 80 100 TA MO | GO | 
es | Sal eva Ae Se 1 ox | 17°8 | 


oCO BDH ASE OeSADD 3°4 3°6 3°5 3°6 3°6 3°8 4:3 5°4 


V. Fig. 10.—Carotid of Emaciated Dog, suffering from ill-treatment and 
chronic illness. 


40 60 80 


| 
| | 
Sil | 4-0 fey eal 6:0 


The most striking fact about. these figures is that in a normal healthy 
artery (II, III, and IV) the velocity is constant as the pressure rises from a 
low value up to about 80 mm., after which it increases, at first slowly and 
then more rapidly. At high pressures the velocity is very considerably 
increased. In V the velocity increases considerably throughout. Secondly, 
the velocities in II, III, and IV, at pressures of 80 mm. (about equal to the 
normal diastolic pressure in man), are noticeably less than those observed in 
man. This may be characteristic of the animals investigated, but it seems 
more probable that it is due to the following factor. All living tissues, and 
especially arteries and muscles, show the phenomenon of elastic “ after- 
action,’ continuing to extend for some time if the load or tension be main- 
tained. Roy attempted to avoid errors due to this by making his observations 


302 Messrs. J. C. Bramwell and A. V. Hill. 


very slowly, allowing the tissue a long time to reach its final equilibrium. 
From the point of view of the static effect of the diastolic pressure on the 
arteries, he succeeded; from that, however, of the dynamic effects occurring 
in the rapid cycle of events associated with the pulse, his precautions aggra- 
vated the error, and must have caused the increase of volume per milli- 
metre of Hg to be much larger than that occurring in a rapid change of 
pressure. It is quite conceivable that a pressure, lasting (say) for 0°1 second, 
causes an expansion not greater than half of that resulting from an equal 
pressure maintained for 10 minutes: in this case a calculated velocity based 
on the latter would be only about two-thirds of an observed velocity depend- 
ing on the former. This elastic “after-action” therefore probably causes all 
the velocities in Table I to be too low; the effect is similar in character to 
that caused by adopting the formule for the isothermal expansion of a gas in 
calculating the velocity of sound. The safest thing to do is to measure the 
velocity directly, and so to deduce the constants of the true adiabatie 
expansion. Finally, we see (in I) that in a vein the calculated velocity 
at low pressures is very low, a conclusion which agrees with an observation of 
Morrow (6), and must be borne in mind in comparing the time relations of the 
jugular pulse with those of other events in the heart or circulation. 

The most important point brought out by Table I is the dependence of the 
velocity upon the pressure. In the case of man, the pressure involved is the 
diastolic pressure, that on which the wave is superimposed. This implies a 
decrease in extensibility with increase in length, an effect analogous to that 
occurring in muscle. This is important in various ways, but particularly in 
experimental work, where it shows the necessity of recording the diastolic 
pressure at the same time as the velocity of the pulse wave. Its magnitude 
is emphasised in the experiment described below. 

It is often suggested that in the living animal the velocity of the pulse 
wave may be affected by contraction of the involuntary muscle around the 
arteries. In so far as the contraction of involuntary muscle may affect the 
extensibility of the artery this will be the case, but in no other way. The 
part of the wave whose velocity is measured is the very rapid rise at the 
opening of the aortic valves, a rise which is detectible in a few thousandths 
of a second. It isinconceivable that a contraction of slow involuntary muscle, 
as we ordinarily know it, could affect the rate at which such a sudden rise is 
transmitted. The transmission of the pulse wave, therefore, is a purely 
mechanical phenomenon, its velocity being an indicator of the elasticity of 
the vessels, as modified by any conditions (muscular or otherwise), obtaining 
at the moment. 

The chief difficulty in the observation of the velocity of the pulse wave in 


The Velocity of the Pulse Wave in Man. 303 


an isolated artery lies in the fact that no considerable length of artery can be 
obtained, and the time-interval available for measurement is therefore very 
small. By replacing the blood with mercury, however, this interval can be 
increased 3°58 times, and the utilisation of this fact makes it possible to 
measure the velocity in an isolated artery with fair accuracy, aud then to 
obtain the velocity in an artery containing blood by multiplication by 3°58. 
The reason for this is as follows: the velocity of the pulse wave is inversely _ 
proportiona] to the square root of the density of the fluid in the vessel, so that 

replacing blood (p = 1-055) with mercury (p = 13°5) decreases the velocity in 
the ratio ,/13°5/1:055, 7.2, of 3°58:1. This principle is embodied in the 


= > cow 
Ss ca 
=z = SSE 
OGia  Couvs 
Cc Hee Fa%e 
hw & wreo 
x =a 
— se 


WRITING 


apparatus shown in fig.1. A length of artery is tied firmly at A and B on to 
the copper pipes, which are clamped rigidly to a board. These copper pipes, 
at their other ends, are firmly joined to two long rubber tubes, one of which 
goes to a mercury reservoir capable of being raised and lowered, the other to 
a mercury pressure gauge. Thus the pressure in the artery filled with 
mercury (with bubbles carefully eliminated) can be adjusted to any required 
value. At X and Y in the copper pipes are two small windows, as shown in 
the diagram on the left. Over each window a small thin piece of rubber tube 
is carefully and firmly fixed, and on the rubber, over the edge of the window, 
is glued a minute aluminium angle-piece carrying the finest possible bamboo 


304 Messrs. J. C. Bramwell and A. V. Hill. 


writing lever. The two levers, lying close together, write upon the same” 
revolving drum, and indicate the moments of arrival of a wave in the mercury, 
at X and at Y respectively. The wave is set up by hitting or squeezing the 

left-hand rubber pipe at some distant spot. Its arrival at X causes a sharp 

movement of the writing-point at X, it is then transmitted with very high 

velocity through the almost rigid copper pipes, being delayed, however, in its 

arrival at Y by the slow transmission across the elastic artery between 

A and B. 

After the experiment on the artery is completed, the artery is replaced by 
a rigid copper pipe from A to B, and the observations are repeated, the small 
time interval observed when the rigid tube lies on the path of the wave being 
subtracted from the larger interval when the elastic tube is there. In 
this way any small “zero error” in the instrument is practically eliminated. 
Corresponding points on the records of the two writing levers are com- 
pared, and the time interval between them can be determined with reasonable 
accuracy. Greater accuracy could doubtless be secured by employing a 
photographic method of recording the arrival of the wave at X and Y, but 
for the present purpose this was not necessary. 

The artery employed was the common carotid of a young man (ene had 
died of malignant endocarditis). It measured 6°84 cm. between the ends of 
the copper pipes. At each pressure a series of observations was made of the 
interval between the arrival of the wave at X and Y (fig. 1), and the 
“probable error” of the mean values given below, lay between 0-0005 
and 0°001 secs. The “zero error” to be subtracted, as estimated by the 
interval observed when the artery was replaced by a copper pipe, was 
as follows :— 


| Pressure (mm.) ...... 20 | 40 | 60 80 100 
Zero error (secs.) ...... / 00185 | 0 0142 | 0 0150 0 -0157 0 0165 
i | 
Pressure (mm.)......... 120 140 | 160 180 200 
Zero error (secs.) ...... 0:0174 0:0182 | 0-0190 0-0198 0 -0206 


The following Table shows the results of a series of observations on the 
artery. The values for blood are obtained by multiplying by 3°58. 


Pressure (mm.) ............... 25 57 | 78 92 110 152 200 
Interval observed (secs.) .... 0°079 | 0:086 | 0:0665 | 0-052 | 0-0465 | 00380 | 0°0338 
Zero error (seCs.) .......+-..- 0:014 | 0-015 | 0:0156 | 0-016 | 0:0170 | 0:0187 | 0 -0206 
Difference (secs.) .........-.. 0°065 | 0-071 | 0:0509 | 0-086 | 0:0295 | 0°0193 | 0 -0132 
Velocity (metres p.s.) ...... 1°05 0:96 | 1°34 T9002 -S2Reee5 5:18 | 
Velocity, blood (m.p.s.) ....) 3°76 | 3°45 | 4°81 |. 6°80 |8°3 22 7 18-5 | 
| | 


Shee aegeeeee ek 


Peer NGS 


OF 
_-'THE ROYAL SOCIETY. 
U Series B. Vol 93. —~—‘No B 658 


BIOLOGICAL SCIENCES 


CONTENTS. 
; Page 
_ On a Remarkable Bacteriolytic Element found in Tissues and Secretions. 
By A. FLEMING, M.B., F.R.C.S. (Plate 9) Be ees Wea cme.) 300 
. The Pigmentary Effector System. 1I.—Reaction of Frog's Melanophores 
to Pituitary Extract. By L. T. HOGBEN, M.A., D.Sc. and 
ESE WINTON SNAG der ge ice WLM Gea Pot. | Aga 
Relationships between Antiseptic Action and Chemical Constitution with 
_ special reference to Compounds of the Pyridine, Quinoline, Acridine 
and Phenazine Series. By C. H. BROWNING, J. B. COHEN, F.RSS., - 
R. GAUNT, and R. GULBRANSEN oy é ; : : : . 329 
The tN Gtion of De one ” on Blood and Immunity thereto. By 
J. W. PICKERING, D.Sc., and J. A. HEWITT, Ph.D..B.Sc. .. . ‘ 307 


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The Velocity of the Pulse Wave in Man. 305 


We see here the same effect of pressure as was shown by the calculation 
from Roy’s curves: the velocity increases comparatively slowly as the pres- 
sure rises to about 80 mm., then more rapidly, and finally at high pressures 
very considerable velocities are observed. As regards the absolute value, 
we may compare the velocity given in the above Table with that found, at 
the same (diastolic) pressure, in a normal living subject. According to 
Gallavardin (5) the average normal value of the diastolic pressure in man 
is 70 to 75 mm. Much higher values, however, are given by the use of the 
Pachon oscillometer (80 to 110 mm.). In normal healthy young men our 
observations (to be described elsewhere) have given velocities from 5:8 to 
74 metres per second. Compared with the velocity interpolated in the above 
Table, for a pressure of 70 to 75 mm., these velocities are high: accepting 
the higher estimate of the diastolic pressure in man, the observed velocities 
agree well with those given in the Table; the velocities 5°8 and 74 m.p.s. 
correspond roughly there to pressures of 85 and 102 mm., respectively. On 
the whole, therefore, we may be satisfied that the pulse-wave has a velocity 
in the living man not far different from that in an isolated artery, and that 
its transmission is a mechanical phenomenon depending only on the elastic 
properties of the vessels. 


Summary. 


The theory of the transmission of the pulse-wave in a blood vessel 
is considered, and it is shown that its velocity, in metres per second, is 
given by 


v = 3°57/ / (percentage increase in volume of artery per millimetre 
of Hg increase of pressure). 


This velocity is relative to the blood in the vessel, and must have a small 
correction applied for the velocity of the blood itself. An observation of the 
velocity, therefore, gives directly the degree of extensibility of the vessel, 
and is shown to be one criterion of an efficient circulation. The experiments 
of Roy (1880) on the extensibility of vessels may be used to calculate the 
velocity of the pulse-wave: the calculation shows: (a) that pressure has a 
considerable effect on the velocity, a fact which is confirmed by experiments 
on an isolated human artery, filled with mercury in order to slow the 
transmission of the wave; and (v) that the velocity so calculated is lower 
than observed in man,a fact which is attributed to the phenomenon of elastic 
“atter-action,” which affected Roy’s measurements. The experiments on an 
isolated human artery gave a velocity comparable with that observed in man, 
and it is concluded that the transmission of the pulse-wave is a purely 
VOL. XCIII.—B. Z 


306 Mr. A. Fleming. On a Remarkable 


mechanical effect. its velocity depending on the extensibility of the vessels 
as modified by any condition (muscular or otherwise) obtaining at the 
moment. 

REFERENCES. 


(1) ‘Hermann’s Handbuch,’ vol. 4, p. 229 (1880). 

(2) Rhode, ‘ Arch, £. exp. Path.,’ vol. 68, p. 401 (1912). 

(8) Roy, ‘J. Physiol.,’ vol. 3, p. 125 (1880). 

(4) Luciani, ‘Human Physiology,’ vol. 1, pp. 261-263 (1911). Macmillan. 

(5) Gallavardin, ‘La Tension artérielle en Clinique,’ Paris, 1920, p. 169. Masson. 
(6) Morrow, ‘ Pfliiger’s Arch.,’ vol. 79, p. 442 (1900). 

(7) Moens, ‘ Die Pulskurve,’ Leiden, 1878, p. 90. 


On w Remarkable Bacteriolytic Element found in Tissues and 
Secretions. 
By ALEXANDER FLEMING, M.B., F.R.C.S. 
(Communicated by Sir Almroth Wright, F.R.S. Received February 13, 1922.) 
(From the Laboratory of the Inoculation Department, St. Mary’s Hospital.) 
[Piate 9.] 


In this communication I wish to draw attention to a substance present 
in the tissues and secretions of the body, which is capable of rapidly 
dissolving certain’ bacteria. .As this substance has properties akin to those 
of ferments I have called it a “Lysozyme,” and shall refer to it by this 
name throughout the communication. 

The lysozyme was first noticed during some investigations made on a 
patient suffering from acute coryza. The nasal secretion of this patient was 
cultivated daily on blood agar plates, and for the first three days of the 
infection there was no growth, with the exception of an occasional staphy- 
lococcus colony. The culture made from the nasal mucus on the fourth day 
showed in 24 hours a large number of small colonies which, on examination, 
proved to be large gram-positive cocci arranged irregularly but with a 
tendency to diplococeal and tetrad formation. It is necessary to give here a 
very brief description of this microbe as with it most of the experiments 
described below were done, and it was with it that the phenomena to be 
described were best manifested. The microbe has not been exactly identified, 
but for purposes of this communication it may be alluded toas the Mterococcus 
lysoderkticus. 


Bacteriolytic Element found in Tissues and Secretions. 307 


The fully developed colony of the coccus may be 2 or 3 mm. in diameter ; 
it is round, opaque, raised, and has a bright lemon yellow colour; it grows 
luxuriantly on all the ordinary culture media, and growth takes place well at 
room temperature, or in the incubator at 37° C.; it is aerobic and faculta- 
tively anaerobic: it does not liquefy gelatin or coagulated albumin. 


PRELIMINARY EXPERIMENTS SHOWING THE ACTION OF THE LYSOZYME. 


In the first experiment nasal mucus from the patient, with coryza, was 
shaken up with five times its volume of normal salt solution, and the 
mixture was centrifuged. A drop of the clear supernatant fluid was placed 
on an agar plate, which had previously been thickly planted with 
MM. lysodeikticus, and the plate was incubated at 37° C. for 24 hours, when 
it showed a copious growth of the coccus, except in the region where the 
nasal mucus had been placed. Here there was complete inhibition of 
growth, and this inhibition extended for a distance of about 1 em. beyond 
the limits of the mucus. 

This striking result led to further investigations, and it was noticed that 
one drop of the diluted nasal mucus added to 1 ¢.c. of a thick suspension of 
the cocci caused their complete disappearance in a few minutes at 37° C. 

These two preliminary experiments clearly demonstrate the very powerful 
inhibitory and lytic action which the nasal mucus has upon the JV. lysodeikticus. 
It will be shown later that this power is shared by most of the tissues 
and secretions of the human body, by the tissues of other animals, by vegetable 
tissues, and, to a very marked degree, by ege white. 


FURTHER OBSERVATIONS ON THE EFFECT OF THE LYSOZYME ON BACTERIA. 
1. Inhibitory Action. 

In the preliminary experiments it has been shown that on the surface of 
an agar plate the growth of the nasal coccus is completely inhibited by super- 
added nasal mucus. This inhibitory action can be strikingly demonstrated 
in another manner. 

A small portion of the agar is removed from an ordinary agar plate making 
a cup into which some material rich in lysozyme (tears, nasal mucus, sputum, 
cartilage, egg white, etc.) is placed. A drop of liquid agar, at a temperature 
of about 50° C., is placed on the material in the cup and is allowed to solidify, 
after which the cup is filled with the liquid agar which, in its turn, is allowed 
to set. Liquid agar is then poured all over the plate to make a thin layer 
over the original surface. The whole surface of the medium is now thickly 
planted with the M/. lysodeikticus and the plate is incubated for 24 hours, 
when it will be seen that there is copious growth of the coceus, except in the 

Z 2 


308 Mr. A. Fleming. On a Remarkable 


region of the implanted material. By the method of preparation of the plate, 
in which the material is covered with several distinct layers of agar, there can 
be no mechanical transference of the material to the surface of the plate, but 
the experiment shows that the inhibitory substance is able to penetrate the 
agar and absolutely prevent growth of the coccus for a distance of about 1 em. 
Further, if the plate is kept for a few days, it is found that portions of the 
growth next to the inhibition zone have become almost transparent, and it is 
evident that the lytic substance has continued to diffuse through the agar after 
the microbes have completed their growth, and has dissolved the cocci for a 
distance of 3 or 4mm. The area of inhibition and the partially dissolved 
zone of growth are shown in Plate 9, fig. 1, which is a photograph of a plate 
in which was imbedded 10 c.mm. of tears. 


2. Bactericidal Action. 


If cultures are made from the inhibition zone of a plate, such as has been 
described in the last experiment, no growth results, showing that the bacteria 
implanted on this surface has been destroyed. It can also be shown that if 
lysozyme-containing material be added to a suspension of JZ. lysodeiktieus in a 
test-tube these cocci are destroyed, so that cultures made from the tube remain 
sterile. In one experiment a suspension of JZ, lysodetkiicus, of a strength of 
not less than 1,000 million per cubic centimetre, was exposed to the action of 
1 in 100 nasal mucus and 10 c.mm. volumes were planted out after incubation 
for 1, 2, 5, 10, and 60 minutes. It was found that the cultures remained 
sterile while similar cultures made at the same time from a control tube in 
which the nasal mucus was replaced by normal salt solution, gave copious 
growth up to the end of the experiment, namely, 1 hour’s incubation. 

It was found that after 2 hours’ incubation tears diluted 9,000 times with 
normal salt solution killed the whole of the cocci in a thick suspension of the 
M. lysodeikticus. In the dilutions of tears from 1 in 27,000 to 1 in 245,000 
there was a very marked bactericidal power manifest. 

The bactericidal action of the lysozyme may also be shown with microbes 
other than the nasal coccus. Anexample of this is illustrated in fig. 2, which 
is a photograph of a culture made after incubating a fecal streptococcus for 
2 hours at 45° C., with tears diluted in 1 in 100 and with normal saline 
solution. It will be seen that from the saline tube there resulted a continuous 
sheet of growth, whereas, from the tube containing the tears, there were only 
scattered colonies, showing that the vast majority of the streptococci had been 
destroyed by the tears. 

A similar result was obtained by acting on Streptococcus Jwcalis with the 
inflammatory exudation into a joint cavity. 


Bacterrolytic Element found in Tissues and Secretions. 309 


Lytie Action. 

Naked-eye Changes.—In the second of the preliminary experiments, it was 

shown that, if a drop of nasal mucus be added to a thick suspension of the 

- Micrococcus lysodeikticus in a test-tube, there is, after a short period of incuba- 
tion, a complete clearing of the opaque suspension, so that the fluid becomes 
perfectly clear to the naked eye. It has been noted also that other tissues 
and secretions have the same action. If the material used is rich in 
lysozyme, the action is a very rapid one. Thus, at a temperature of 45° C., 
a 1 in 100 dilution of tears will completely clear the suspension in about 
30 seconds, or a 1 in 5 dilution of egg white in 10 seconds. 

If the bacterial suspension is a very thick one, there is easily to be 
observed a considerable increase in the viscosity of the fluid after lysis of 
the bacteria has been completed, evidenced by the fact that, if the tube is 
shaken, the air bubbles rise much more slowly to the surface of the fluid. 

The lytic action can be strikingly demonstrated by placing on the surface 
of a fully-grown plate culture of M. lysodeikticus a drop of tears, nasal 
mucus, or other material rich in lysozyme. In less than 1 minute at 37° C., 
or in about 10 minutes at room temperature, that portion of the culture on 
which the material was placed will have been completely dissolved, pro- 
ducing a clear space just as if a portion of the culture had been mechanically 
removed. 

Microscopic Changes—When a mixture.of tears and M. lysodetkticus is 
observed with dark ground illumination, it is seen that the cocci rapidly lose 
their sharp outlines, become swollen and gradually disappear. At the same 
time, there appear a very large number of minute granules, somewhat 
similar in appearance to the granules of a polynuclear leucocyte. 

Examined with transmitted light, it is seen that the cocci rapidly swell up 
and become transparent, so that, after 2 minutes at room temperature (when 
the cocci are suspended in undiluted tears), they become quite invisible. 

When the partially dissolved cocci are examined by Burri’s method, they 
are found to be much swollen up, and they are less indistinct, probably 
owing to some of the opaque material used to produce the dark background 
adhering to their glutinous surface (see fig. 3). 

If a similar specimen is coloured with ove of the ordinary bacterial 
stains, the stainable material is found to have diminished in size, giving 
the appearance of small and very irregular cocci. When the lytic action is 
complete, staining fails to reveal any trace of the cocci, 


310 Mr. A. Fleming. Ona Remarkable 


OBSERVATIONS ON THE PROPERTIES OF THE LYSOZYME AND ON THE 
CONDITIONS GOVERNING ITS ACTION. 


The lysozyme is soluble in water or normal salt solution ; it is insoluble 
in chloroform, ether or toluol, and, as these substances do not destroy it or 
inhibit its action, they have been used to preserve lysozyme-containing 
material such as sputum for test purposes ; it retains its potency undi- 
minished after standing at room temperature for several weeks. That 
lysozyme of egg white is not destroyed by desiccation, and that in the 
dried state it can be preserved for long periods, is shown by the fact that it 
is present in large amounts in commercial dried ege albumin. 

From albuminous fluids, protein precipitants such as alcohol, acetone, or 
picric acid, precipitate the whole of the lysozyme with the proteids. : 

Its action takes place most rapidly when a small amount of salt is present 
in the fluid (under 0°1 per cent.), and ceases when more than 5 per cent. of 
salt is present. It acts both on living microbes and on those which have been 
killed with heat. 

Influence of the Reaction of the Fluid. 


It was found that when one drop of sputum extract and one drop of a thick 
suspension of JZ. lysodeikticus were added to 1 c.c. of various dilutions of 
hydrochloric acid or caustic soda, the lytic action was, to some extent, 
delayed in the tubes containing as little as 1/8,000 normal acid or 1/24,000 
normal alkali, and there was complete inhibition of lysis in the tubes 
containing 1/800 normal acid or alkali. These figures are not strictly 
accurate, as alkali-free glass was not used, but they clearly indicate that 
the lysozyme is very sensitive to minute traces of acid or alkali. 


/ 


Resistance of the Lysozyme to Heat. 


Sputum extract, nasal mucus, saliva and subcutaneous fatty tissue, heated 
for 10 minutes at 60° C., had lost but little of their lytic power for bacteria, 
but 5 minutes’ heat at 75° C. destroyed alinost all the lysozyme. Tears, 
diluted 500 times with normal saline solution, were heated for 10 minutes at — 
75° C., and the lysozyme-content was reduced to one quarter. After 
boiling this dilution of tears for 30 minutes, traces of lysozyme remained 
active, but boiling for 1 hour apparently completely destroyed it. 

In saliva, the resistance to heat of the lysozyme and of ptyalin was 
compared. A specimen of saliva was heated to 75° C., and specimens were 
taken at intervals of 1 minute, and their lysozyme and ptyalin-content 
were compared. It was found that these two substances disappeared from 
the saliva at the same time, namely, after heating for 7} minutes. 


Bacteriolytic Element found in Tissues and Secretions. 311 


Influence of Temperature on the Velocity of Lysozymic Action. 

The lytic action takes place slowly in the ice chest and the velocity 
increases up to 60° C. after which it becomes slower again, probably owing to 
the destruction of some of the lysozyme. 

If lysozyme containing material, however, is left in contact with the 
Micrococcus lysodeikticus for 24 hours the cocci are dissolved in the same 
dilution of the lysozyme whether the reaction takes place at room tem- 
perature, 37° C., or 50° C. 


Does the Lysozyme pass through Membranes or Filters ? 


1. Collodion.—One c.c. of a saline extract of sputum was placed in a 
collodion sac and this was suspended in a tube containing a thick suspension 
of M. lysodeikticus and incubated for 6 hours. No lysis of the cocci took place. 
The sac was then punctured and the contents allowed to mix with the 
bacteria when complete lysis oceurred within 2 minutes, showing that the 
sputum extract contained lysozyme, which, however, had been unable to 
pass through a collodion membrane. 

2. Porcelain Filter.—Fifty c.c. of a 1 in 1,000 dilution of tears were passed 
through a Berkfeld filter and it was found that the filtrate was devoid of 
lysozyme action. As it might have been possible that some inhibitory 
substance (¢.g., acid or alkali) had been absorbed from the filter and passed 
into the filtrate a small quantity of the unfiltered tears was added to the 
tubes containing the filtrate and the cocci when lysis promptly occurred 
showing that the lysozyme had actually been retained on the filter and that 
the absence of lysis when the filtrate and cocci were mixed, was not due to 
the presence of any inhibitory substance. 

It was impossible to obtain human secretions, rich in lysozyme, in 
sufficient quantity to filter them in a strong concentration through the 
porcelain filters available. Eee white, however, which is very rich in 
lysozyme, was used for this purpose in a dilution of 1 in 10 in normal saline 
solution. The filtrate was collected in separate portions of about 1 c.c., and 
each portion was tested for the presence of lysozyme. The first 19 cc. 
which passed through the filter had no lytic action on M. lysodeikticus but 
after that the lysozyme passed through and in the 30th c.c. the strength of 
the filtrate was practically the same as that of the unfiltered material. 

These experiments show that a porcelain filter is capable of absorbing a 
considerable quantity of lysozyme, but when that has been absorbed the 
filter offers no barrier to the passage of this substance. We shall see that 
the same thing happens with filters of cotton wool and filter paper. 


312 _ Mr. A. Fleming. Ona Remarkable 


Cotton Wool.—This was tested by two methods which the author had 
previously used to demonstrate the gossypiotropic properties of certain aniline 
dyes. , 

The first method consists in pushing slowly to the bottom of the test-tube con- 
taining a column of about 2 inches of a lysozyme-containing material a tight 
plug of cotton-wool, so that the fluid percolates through the cotton-wool and 
collects above it. Using tears diluted 1 in 1,000 (this sample of tears showed 
lysis up to a dilution of 1 in 5,000,000) it was found that when this 
experiment was carried out the whole of the lysozyme was removed by the 
cotton-wool. 

In the second method a tight plug of cotton-wool was introduced into a 
narrow tube, 1 c.c. of tears (1 in 1,000) placed above this, and with pressure 
exerted with a rubber teat the fluid was driven through the cotton-wool. 
Successive volumes of 1 ¢.c. were driven in this way through the cotton-wool 
and these were separately tested for the presence of lysozyme. It was 
found that a tight plug of cotton-wool 1 cm. long introduced into a piece of 
6-mm. tubing absorbed the whole of the lysozyme from 12 ce. of a 
1 in 1000 dilution of tears. Further volumes of the diluted tears passed 
through this cotton-wool plug all contained lysozyme. 

Filter Paper.—This was tested in the same way as cotton-wool and with 
the same results. Passage through about 0°5 em, of compressed filter paper 
in 6-mm. tubing removed the whole of the lysozyme from 10 ce. of a 
a thousand-fold dilution of tears. 


Ts the Lysozyme Removed from Solution with Substances such as Charcoal ? 


It was found that when a small quantity of blood charcoal was added to a 
thousand-fold dilution of tears and after 2 hours on the bench the mixture 
was centrifuged, the clear supernatant fluid contained no lysozyme. It was 
shown that no inhibitory substance had been absorbed into the fluid from the 
charcoal because, after the supernatant fluid had failed to cause lysis of the 
cocci, a small quantity of the diluted tears was introduced, when lysis promptiy 
occurred. It is evident, therefore, that charcoal removed the lysozyme from 
the fluid. 

Distribution of the Lysozyme in the Body. 

In the first experiments it was found that nasal mucus contained a large 
amount of lysozyme, and it was later found that tears and sputum were 
very potent in their lytic action. It was also found that this property was 
possessed by a very large number of the tissues and organs of the body. The 
lysozyme-content of the tissues was investigated by placing small portions of 
tissue not larger than a split pea in tubes containing 1 cc. of a thick suspen- 


Bacteriolytic Element found in Tissues and Secretions. 313 


sion of the M/. lysodeikticus incubating the tubes at 45° C.,and noting whether 
any lysis took place as evidenced by a clearing of the opacity of the suspension. 
Some of these tissues were obtained from the postmortem room, others from 
laboratory workers or from the operating theatre. The results obtained can 
be summed up by saying that all the tissues and organs possessed some lytic 
power, even a few hairs from the head causing solution of the cocci. While 
in these tests no attempt was made at an exact quantitative estimation, it 
was noticed that lysis proceeded very much more rapidly with some tissues 
than with others. Briefly, it may be said that epidermal structures, the 
lining membrane of the respiratory tract and especially the connective tissues 
(whether fibrous, fatty or cartilaginous) contained large amounts of lysozyme 
affecting MM. lysodeikticus. The rapidity of the lysis with cartilage was so 
striking that an attempt was made to estimate more accurately the amount 
of lysozyme in this tissue. A small portion of cartilage from the patella 
(deep to the articular surface) was weighed and ground up in a mortar with 
a measured volume of salt solution. This was allowed to extract for 6 hours 
when it was centrifuged and the supernatant fluid was added in various 
dilutions to a suspension of the J. lysodeikticus. It was found that with 
an extract corresponding to one part of the original cartilage in 1,300 parts 
of normal salt solution, there was complete lysis of the cocci in 5 minutes 
at 45° C. which shows that cartilage has approximately one-tenth the 
lysozyme-content of tears. 

The presence of lysozyme was sought for in certain physiological and 
pathological fluids, and the results are set forth in Table I. 


Table I. 
| 
Fluids containing lysozyme. Fluids not containing lysozyme. | 

| 
Tears. Normal urine. 
Sputum. Cerebro-spinal fluid. 
Nasal mucus. Sweat (one sample only tested.) | 
Saliva. | 
Blood serum. ies 
Blood plasma. | 


Peritoneal fluid. 

Pleural effusion. 

Hydrocele fluid. 

| Ovarian cyst fluid. 

Sebum. 

Pus from acne pustule. 

Sero pus from a “cold” abscess in the 
popliteal space. 

Urine containing much albumin and 

us. 
Semen (very weak). 


314 Mr. A. Fleming. On a Remarkable 


In connection with the lysozyme-content of the blood, it is to be noted 
that, in addition to its being present in the leucocytes, in the plasma, and in 
the serum, it is also present in rather large amount in the fibrin of the blood 
clot. It is conceivable that this is a protective mechanism for open wounds, 
which rapidly become covered with a layer of fibrin and leucocytes, both of 
which are rich in lysozyme. 

The lysozyme-content of tears, sputum, nasal mucus, saliva, and blood 
serum of the same individval were tested. The specimens were all collected 
at the same time and were tested about 4 hours afterwards. The titrations 
were carried out by making serial dilutions of the various fluids and adding 
to these dilutions a measured quantity of a thick suspension of IZ. lysodeikticus, 
after which the tubes were incubated at 45° C., and readings were made at 
intervals of 15, 30, and 60 minutes. The results are set out in Table II :— 


Table I1—The Lysozyme-Content of various Fluids taken from the same 
Individual at the same Time. 


Time of 
| Material examined. ee Dilution of fluid, 1 in :— 
| 45° C. : 
| 
mins. ios 10a] P30 90 270 810 | 2430 | 
Blood serum......... 15 + + + 6) 0 Oa 
i. 330 + | + es ARE Seema 0 | 
60 2) oiler pati laechoselt? Sl ales oO | 
| | H | 
100 | 300 900 | 2,700 | 
Waste iy 420 aenereebonceccca 16 5 ee en = 0 | O 
| 30 + + ze || 0 | 
60 eS ao 
| | | | 
| | 500 , 1,500 | 4,500 | 13,500) 40,500 | 121,500 
Nasal mucus......... 15 + Ford) pathy el tess + 0 
30 + Bae id Nea seis 3 edie 0 
60 + + + Sey ey oie ee) 
SHOU cononoconane 15 + + + 35 a5 0 
30 + af | + | + 25 0 
60 + + + + yo he 0 
| | | | 
| TISEES) ocqooosanos s5e05¢ | 15 + + | + + 0 | (0) 
30 + + P| Phil penn 0) 
60 ce ert eee aes ae | Pivh ane 
| | { 


+ signifies complete clearing of the fluid. 
+ » partial p 5 
0 » no 2 pb) 


It will be seen from the above Table that tears, sputum, and nasal mucus 
are very rich in lysozyme to the M. lysodeikticus, while saliva and blood serum 
are relatively weak. Fluids from a number of different individuals have 


Bacteriolytic Element found in Tissues and Secretions. 315 


been tested and the relative amounts of lysozyme contained in these have 
been found to be comparatively constant. except in the case of saliva, which 
seems to vary considerably, although it never approaches in lysozyme- 
content tears, sputum, or nasal mucus. 


The Question as to whether Lysozyme exists in Tissues other than Hunan 
Tissuves. 

Only a limited amount of work has been done in this direction, but it is 
sufficient to show that lysozyme is very widespread in nature. Rabbit and 
guinea-pig tissues were examined and it was found that nearly all of these 
contained some lysozyme for the JZ. lysodeikticus, but in general the lysis was 
not nearly so marked asit was with the corresponding human tissues. It may 
be noted that the lachrymal secretion of both these animals contained no 
lysozyme for the MV. lysodeikticus, against which the human tears are so powerful. 
The tissues of a dog were much more lytic than those of the rabbit and 
guinea-pig, but even they were not so active as human tissues. 

It was found that ege-white was very rich in lysozyme for the JL lysodeikticus, 
there being, after incubation for 24 hours, lysis visible to the naked eye when 
a dilution as great as 1 in 50,000,000 was employed. Egg-white also 
contains lytic substances for many other bacteria. It was found also that 
commercial dried egg albumin was very rich in lysozyme. 

In the vegetable kingdom it was found that turnip had a very definite. 
though not very strong lytic action on MW. lysodeikticus. Several of the other 
common table vegetables were tested, but they appeared to be devoid of lytic 
activity. 

Does the Lysozyme act on Bacteria other than the M. lysodeikticus ? 

In the investigation of this problem the method adopted was to make a 
suspension of the bacteria of such a strength that it gave a very decided 
opacity when diluted with an equal amount of saline; } c.c. of this suspen- 
sion was mixed with the same quantity of a 1-in-50 sputum extract ora 
dilution of tears from 1 in 100 to 1 in 1,000. As a control, a twofold dilution 
of the original suspension was made with normal salt solution. The tubes 
were incubated at 45° C.and observations were made at intervals up to 
24 hours, the opacity of the tube containing sputum or tears being compared 
with that of the control tube. 

Three groups of microbes were tested: the first group consisted of 104 
strains of bacteria derived from the air of the laboratory, and of these 
75 per cent. were dissolved, more or less readily, by a 1 in 100 dilution of sputum. 
These air-borne bacteria consisted mainly of cocci of various sorts, but there 
were also bacilli, yeasts, and- two species of mould. 


316 | Mr. A. Fleming. On a Remarkable 


The second group consisted of a series of cultures of bacilli which are 
pathogenic for some animals, but not, so. far as is known, for man. These 
were kindly supplied by Dr. St. John Brooks from the National Collection. 
They were tested with tears (1 in 100) and nasal mucus (1 in 100), in 
addition to the sputum extract, and seven out of eight cultures showed 
some lysis after incubation with one or other of these fluids. These 
cultures included B. abortus of Bang, and B. psewdotuberculosis rodentium, 
to both of which there was some lytic power and which will be referred 
to later. 

The third group consisted of bacteria which had been isolated from the 
human body, and it was found that, whereas most of these were not acted on 
by the lysozyme contained in sputum or tears, some were completely, and 
others partially dissolved. Not one of the various members of the coli 
typhoid group showed the slightest signs of lysis, while sixteen out 
of nineteen strains of intestinal streptococci were dissolved to a greater or 
less extent. 

The results obtained with this group of microbes are set forth in Table III, 
but of necessity, considering the multiplicity of strains involved, this Table 
is incomplete, and it may well be that by altering the conditions of the 
experiment somewhat, a much higher percentage of the bacteria will be 
dissolved. 


Table I11.—Effect of the Lysozyme contained in Sputum or Tears on 
Bacteria isolated from the Human Body, 


{ ow a . a : 
Type of microbe. Number tested. | phaser ea ee isis ae 
SRG MOGOBEE. sneocesedncnoan deo anean 22 16 6 
SHY MOOMOLNEES » snceaatioh onoabootaces 4, 2 2 
IB COM s oem eGR at A ae seit ONAL ER 12 0 12 
JB DPHOOSUIS” sddacanedppomdoscaancass | 1 0 il 
B ParatyPHOSus: ...6..c...e0e ssc 2 0 2 
BS PiOLCUSi aay Amare ota eat ai 2 0 2 
IE, (OOYGLEUS RaSocsaannso chose nae 3 0 3 
EBS PeSbUs essen antenna Setar 1 | 0 1 
Libby, GDI OD SOS 5 .cbcp o6oonn nab 0b o60 0D 1 | 0 1 
Diphtheroid Bacilli ............ 3 0 3 
LEGMTADECED eon 335 snaeonr so vos cones 2 0 2 


Tt was noticed that with different microbes, different fluids in their 
lysozyme-content did not always bear the same ratio to one another. Thus, 
while tears were apparently the most powerfully lytic to the JM. lysoderkticus, 
they had a less powerful lytic effect on some other cocci than had ~ 
sputum or synovial fluid. This may be the explanation of the immunity of 


Fleming. Roy. Soc. Proc., B, vol. 93, Pl. 9. 


Fig. 3. 


Bacteriolytic Element found in Tissues and Secretions. 317 


certain tissues to certain infections, or conversely the well-known predilection 
of certain infections for certain tissues. 

The view has been generally held that the function of tears, saliva and 
sputum, so far as infections are concerned, was to rid the body of microbes 
by mechanically washing them away. Metchnikoff in his treatise on 
“Immunity and Infectious Disease,’ expresses himself very clearly and 
precisely on this point. From the experiments detailed above, however, 
it is quite clear that these secretions, together with most of the tissues of 
the body, have the property of destroying microbes to a very high degree. 

It has not been possible to test extracts of all the different tissues to each 
of many microbes, but it has been shown that human tears and sputum 
can dissolve the majority of the microbes (presumably non-pathogenic) 
recovered from the air of the laboratory. Most of these air-borne bacteria 
are non-pathogenic, and it seems extremely unlikely that they could 
become pathogenic when the human secretions show such a destructive action 
towards them. 

Again, the human secretions showed lytic power to most of the microbes 
tested which, although pathogenic to some animals are harmless to man. 
Notably there was a certain amount of lysis evident with the bacillus abortus 
of Bang and B. pseudotuberculosis rodentium, which are culturally and 
serologically identical with J melitensis and B. pestis respectively, both of 
which latter organisms are very pathogenic for man, and for which there is 
apparently no lysozyme in the human secretions. It may be that it is 
in this sensitiveness to a human lysozyme that the difference between these 
microbes lies. 


DESCRIPTION OF PLATE. 


Fig. 1.—Photograph of agar plate with imbedded tears. 
Pig. 2.—Bactericidal power of tears on streptococci. Upper half—culture from 
streptococci in salt solution. Lower half—culture from same number of 
streptococci in tears (1 in 100). 
Fig. 3—Upper half—Mierococeus lysodeikticus before being acted on by tears. Lower 
half—same partially dissolved by tears. Examined by Burri’s method. 


318 


The Pigmentary Effector System. I.—Reaction of Frog's 
Melanophores to Pituitary Extracts. 
By Lancetor T. Hoacssn, M.A., D.Se., and Frank R. Winton, M.A. 


(Communicated by Prof. E. W. MacBride, LL.D., F.R.S. Received February 2, 
1922.) 


(From the Zoological Laboratory, Imperial College of Science and Technology.) 


CONTENTS. 

PAGE 
Te UNtPOdUcbion.” js cceseeescaens canceeeessctsessms act onsci tensions es cpecs dus eeeet ee ene EReeme 318 
2. Specificity and Localisation of a Pituitary Melanophore Stimulant ...... 320 
3. Sensitiveness of the Melanophores to Pituitary Extracts ...........ce.e0cc0e+ 322 
4, Relation of the Melanophore Stimulant to the other Pituitary Autocoids 323 
5. Relation of the Melanophore Stimulant to Histamine ..............cscseeeees 325 
6. Mode of Action of the Melanophore Stimulant ....................0csscesecaees 326 
Ta) SUMMALY. oo eecienieactlace assis stulesais seen sevice ce sleet veslane se ieee aloe ee eee eee eae eee 328 


1. Lntroduction., 


The ability of certain organisms, including notably the Mollusca and lower 
Vertebrata, to respond to their surroundings by appropriate pigmentary 
changes has long been familiar to biologists; it has been recognised for more 
than half a century that a special type of effector organs (chromatophores, 
melanophores, etc.) are actively instrumental in producing such changes, and 
that, in Vertebrates at least, pigmentary response is partially controlled 
through the nerves by stimuli received from the organs of vision. During 
the past two decades it has been shown that the reactions of the pigment 
cells to stimuli simulate those of other effector organs, especially as regards 
their responses to certain internal secretions. That a fuller understanding of 
their properties might prove of service to practical aspects of physiology, as 
well as the key to a knowledge of “colour adaptation,’ was realised by Lister, 
who concludes his paper on the cutaneous pigmentary system of the Frog 
(1858) with the following comment: “The pigmentary system also promises 
to render good service in toxicological enquiry. Hitherto in experiments 
performed on animals with that object attention has been directed chiefly, if 
not exclusively, to the effects produced upon the actions of the nervous 
centres, the nerves, and muscles. In the pigment cells we have a form of 
tissue with entirely new functions, which, though apparently allied to the 
most recondite processes, yet produce very obvious effects. . . . Such 
experiments are so readily performed, and the effects produced are so 
obviously indicated by the changes in colour of the integument, that I 


The Pigmentary Effector System. 319 


venture to recommend this method of investigation to those who are occupied 
in studying the action of poisons. & 

A considerable literature, which has Feta reviewed too thoroughly and 
recently to merit an extensive epitome here, bears witness to the intricacy of 
the mechanism which underlies pigmental response in Fishes, Amphibia, and 
Reptiles ; and very little work has been done on Molluscs in this connection. 
Tt can be safely asserted that as many distinct types of chromatophores are 
already known as there are different sorts of other effector organs whose 
properties have been studied. Hence it is obvious that, apart from the intrinsic 
interest presented by the phenomenon of colour change, a study of the pro- 
perties of this series of effectors invites consideration in relation to a fuller 


understanding of the rdle played respectively by the character of the tissue 


jy 


and the nature of its nerve supply in the local action of drugs. The research, 
of which this preliminary communication contains an initial account, of the 
response of Amphibian melanophores to pituitary extracts, has been 
approached with both these objectives in view. For present purposes it will 
suffice to recapitulate briefly what has already been achieved in relation to 
the endocrine factors in pigmental response. 

Earlier workers on the pigment effector mechanism concentrated their 
attention on the nervous control. Corona, however, showed as early as 1898 
that a glandular extract could induce pigmental changes in the frog when he 
brought about contraction of the pigment cells with adrenalin. Lieben 
(1906) confirmed this conclusion, and a similar reaction of pigment cells to 
adrenalin has been demonstrated in other Amphibia and in Fishes by various 
workers. Contraction of the melanophores of frog tadpoles was found to 
follow pineal administration by McCurd and F. Allen (1917), whose results 
have been since confirmed by Huxley and Hogben (1921). The latter have 
also shown that melanophore expansion follows pituitary feeding in sala- 
mander larve, an observation which accords with the condition ot generat — 


ge rophore contraction seen to sole He ee expats b ant zt tutige bets 


pituitaries, patalisbiea ee these ee hus, were in p 
workers experimented on Amphibia ; Spaeth (1917), on the other 
that the melanophores of isolated scales in the Teleost fish Bunatahis 
contract in response to both pituitary and adrenal preparations. 

Whatever may be true in the case of Fishes, this is certainly not the mode 
of response among Amphibia, as the present experiments are intended to 
indicate. Pituitary extracts injected into the frog produce a visible 
darkening, with complete and extreme expansion of the melanophores. 
The results recorded show that this is a specific reaction of extracts of 


320 Dr. L. T. Hogben and Mr. F. R. Winton. 


the posterior lobe, is produced by a principle probably identical with the 
uterine stimulant, and is independent of the nerve-endings in the local effect 
which it evokes. 

The normal reactions of melanophores to light in the frog depend on 
several factors besides light, temperature being of predominant significance. 
For the purpose of the experiments, it was necessary to use frogs in which 
the melanophores were fully contracted. Decerebrated animals were 
employed: the room temperature was uniformly 73° to 77° F. At this 
degree of warmth, the frogs placed on a white background under bright 
illumination show complete contraction of the melanophores, which con- 
dition persists after the animals have been decerebrated carefully, so as to 
avoid injury to the pituitary of the animal itself. Throughout this paper, 
the melanophores referred to are in all cases the dermal melanophores. 


2. Specificity and Localisation of the Pitwitary Meclanophore Stumulant. 


The experiments recorded in subsequent sections were based upon 
pituitary extracts prepared commercially for clinical purposes. Such 
preparations are made from the posterior lobe of the gland, which includes 
not only the infundibulum sensw stricto, ue. the pars nervosa, but, in 
addition, the pars intermedia, which is ontogenetically a hypophysial 
structure, though the term hypophysis is sometimes inaccurately applied to 
the whole gland or to the pars anterior (anterior lobe) alone. Hence the term 
infundibulin applied to such extractsis misleading. The following preliminary 
experiments indicate the existence of a specific melanophore stimulant in 
pituitary extracts and its localisation in the pars intermedia and nervosa :— 

(i) An adult female rabbit was decapitated, and its pituitary gland, 
together with pieces of muscle, brain, ovary, pancreas, suprarenals and 
spleen removed at once. The tissues were severally weighed, ground up 
with sand, made up to 1 per cent. in Ringer, and left in the thermostat at 
35° C. for 2 hours. At the end of 2 hours eight pairs of pale frogs were 
taken, being injected in pairs (0°5 c.c. per individual), with the extracts of 
tissues enumerated, another pair being injected with a 0-1 per cent. solution 
of pituitary, and an additional pair with an extract of putrid meat. The 
object of the latter injection was to control the possibility that the effects 
found to follow injections of commercial products were not due to traces of 
the physiologically active (pressor) substance known to occur in putrefying 
tissues. After half an hour, the pairs injected with 1 per cent. and 0-1 per 
cent. pituitary extracts had assumed a coai-black hue, while all the others 
remained pale: microscopic examination of the skin showed that this effect 
was due to the expansion of the melanophores in the former case. 


The Pigmentary Effector System. 321 


(i1) The small size of the pituitary gland in the rabbit as well as the fact 
that the rabbit’s pituitary is remarkably compact, having hardly any cleft 
between the two lobes (which are thus additionally difficult to separate), 
made it impossible to test the effect of extracts from different parts of the 
gland in the foregoing experiment. In the case of the ox pituitary the 
demarcation between the three parts is very striking. It was possible to 
secure ox pituitaries from the slaughter house within little more than an 
hour of killing, and to dissect away portions of the pars nervosa, intermedia 
and anterior separately, for preparing extracts of the three divisions of the 
gland. Each portion was weighed, eround up with sand, and after extraction 
with Ringer at 35° C. for 2 hours made up to a 0:1 per cent. and 0:02 per 
cent. solution. A pair of pale frogs was injected with each of the six 
solutions (0°5 c.c. per individual). After twenty minutes the four frogs 
injected with anterior lobe extract showed no darkening. The pair injected 
with weak (0°02 per cent.) pars nervosa extract likewise displayed no 
darkening, whereas both frogs which had been injected with 0-1 per cent. 
pars nervosa extract and all four animals injected with the pars intermedia 
preparation (0'1 per cent. and 0:02 per cent.) showed intense darkening of the 
skin. Microscopic examination showed that the three pairs injected respec- 
tively with pars anterior, strong and weak extracts, and pars nervosa weak 
extract, had the melanophores in the contracted condition, while the 
remaining six, those injected with pars intermedia and pars nervosa strong 
extract, displayed a state of general melanophore expansion of the 
characteristic type. 

From these experiments it is clear that the pituitary gland contains a 
specific principle which is capable of inducing an extreme type of melano- 
phore expausion in the frog, and that the production of this substance is 
located in the posterior lobe. It will be noted that the extract prepared 
from the pars intermedia was in: the last experiment more potent than that 
prepared from the pars nervosa in corresponding amounts of the fresh - 
glandular substance. The fact that workers like Swingle, who record the 
effect of grafting experiments, claim for the pars intermedia an exclusive 
role in the pigmental control of the pituitary gland, does not necessarily 
conflict with this result, for it is on histological grounds unlikely that the 
pars nervosa actually secretes the autocoid which produces melanophore 
expansion. What is most likely, as has been suggested by other writers, is 
that the pars intermedia secretion rapidly diffuses into the nervosa, in 
which case the term infundibulin applied to such extracts is not merely 
confusing but positively incorrect. The changes which follow injection of 
pituitary extracts in the frog confirm the conclusion that the condition of 

VOL, XCII.—B. 2A 


322 Dr. L. T. Hogben and Mr. F. R. Winton. 


pallor which results from pituitary removal in amphibian larve, as shown by 
Allen and others, is due to endocrine deficiency. 


3. Sensitiveness of the Melanophores to Pituatary Extracts. 

The following tests indicate the mode of response of frogs’ melanophores 
to different concentrations of pituitary extracts. Owing to the extreme 
simplicity of the technique employed, it is not desirable to repeat the 
description where experiments have been repeated for confirmatory purposes. 
The extract employed was in all the remaining experiments Burroughs 
and Wellcome’s liquid sterile posterior lobe extract (“infundin”) in 
0°5 c.c. tubes. 

(1) Solutions 05 per cent, 0:05 per cent., 0:005 per cent., 00005 per cent. 
of the liquid extract in frog’s Ringer were made up, 1 cc. of each solution 
being injected into each of a pair of pale frogs; a fifth pair was injected 
with saline simultaneously as a control. At the conclusion of half an hour 
(by which ‘time maximum reaction is attained with pituitary extracts), the 
two pairs injected respectively with 0°5 per cent. and 0:05 per cent., showed 
the striking pigmentary changes described in the last section; the remainder 
were still pale. On removing a piece of skin from the back of each frog, 
fixing in Bouin’s fluid, dehydrating and mounting in balsam, it was seen 
that in the two pairs that showed visible darkening, the melanophores were 
so fully expanded that their processes appeared to form a continuous web, 
thus rendering the skin almost opaque. The other frogs showed complete 
contraction of the melanophores, except in the 0°005 per cent. pair, in which 
there was perhaps a very slight tendency towards a stellate condition of the 
melanophores. The visible darkening of the skin appears within 10 to 
20 minutes, reaches its maximum in half an hour, and disappears within 
3 hours from the time when injection takes place, as was shown by a further 
injection of the 0°5 per cent. solution into another pair of animals. Pieces 
of skin were also placed in the stronger pituitary solution to test its action 
on the isolated skin. So treated, they displayed on microscopic examination 
half an hour jater, complete expansion of the melanophores, while other 
pieces placed in saline showed the melanophores contracted to fine points. 
It should be explained, however, that if the skin is subjected to a good 
deal of mechanical stimulation, as for example, after being cut into small 
strips 2 mm. square, the melanophores remain contracted and will not respond 
by expansion to pituitary extract. 

(2) A second tube of infundin was used to make up solutions, 0:1 per cent., 
0:075 per cent., 0:05 per cent., 0025 per cent., 0:01 per cent. and 0:005 per 
cent. Six pairs of frogs were taken and injected (0°5 per cent. per individual) 


The Pigmentary Effector System. 323 


severally with the above strengths. The first and fifth doses were, it will 
be noted, quantitatively equivalent to the second and third in the foregoing 
experiment. At the end of half an hour the first two pairs (0°1 per cent. and 
0-075 per cent.) were intensely dark; the others showed but slight, if any, 
perceptible change. Microscopic examination of pieces of the skin showed 
that in the first two pairs the melanophores were expanded, whereas in the 
remainder they were contracted. It may be well to observe parenthetically 
in this connection, that the terms contraction and expansion are used in a 
purely descriptive sense throughout this paper, without prejudice to the 
controversial issue, to be dealt with subsequently it is hoped, whether the 
pigment cell as a whole contracts and expands, or whether on the other hand 
the appearance is due to the independent migration of the pigment granules. 

The above experiments illustrate two significant aspects of the pituitary 
melanophore reaction: (a) there is a fairly definite end point; and (6) it is 
extremely sensitive when the clinical dose is taken as a basis of comparison. 
Since 0°5 to 1 ec. are usually administered clinically (¢g., in parturition), it 
will be seen that less than a thousandth of the clinical dose, or 0:0004 c.c. of 
the commercial (21 per cent.) extract, suffice to evoke the response.* 

To obtain more crucial data it will be necessary to substitute for the crude 
method of injection vid the dorsal lymph sac the intravenous or intraperitoneal 
operation. Considering the extreme technical facility of this method of 
testing the activity of pituitary preparations, the rapidity with which it can 
be carried out, the simplicity and cheapness of the materials employed and 
finally the probable identity—as will be seen later—of the melanophore and 
uterine stimulants, it is not premature to suggest in this place’ that the 
pituitary melanophore reaction merits the fullest consideration as an 
alternative to the very elaborate methods now employed for the standardisa- 
tion of clinical preparations. It would be well moreover to explore the 
possibility of employing strips of frog skin as Hooker was successful in 
culturing the latter. It should then be possible to eliminate the factor of 
individual variation as a source of error. Spaeth emphasised this point in 
relation to the use of fish scales: the frog has the merit of being much more 
easily obtained and reared ; and since excitement and mechanical stimulation 
ordinarily cause the frog’s pigment cells to contract, everything favours the 
success of manipulation. 


4. Relation of the Melanophore Stumulant to other Pituitary Autocoids. 
It is now generally recognised that the diverse responses evoked by extracts 
of the posterior lobe of the pituitary gland are not due to a single autocoid. 


* 021 gr. fresh infundibular substance in 1 e.e. 
2A 2 


324 Dr. L. T. Hogben and Mr. F. R. Winton. 


By extraction of dried and powdered infundibulum with acidified water, 
treatment of the solution with colloidal ferric hydroxide and subsequent 
continuous extraction of the filtrate at reduced pressure with butyl alcohol, 
Dudley (1919) succeeded in separating a crystalline residue containing all the 
uterine stimulant (oxytocic principle) together with a portion of the pressor 
substance. The latter is again differentiated from the uterine stimulant by 
the fact that it is rapidly and completely destroyed by boiling with 0°5 per 
cent. HCl: on the other hand one-fifth of the uterine stimulant remains after 
half-an-hour of acid hydrolysis, and at the end of 6 hours a slight trace—less 
than 1/200ths persists. The rapid destruction of the pressor principle was 
shown by Abel and Nagayama (1920) whose results have been confirmed by 
Dale and Dudley (1921), and extended as indicated above. There are, 
therefore, at least two active principles in the extracts of the infundibulum ; 
and the question thus arises of what relation exists between the melanophore 
stimulant of the infundibulum and the other pituitary autocoids. 

The effect of continued acid hydrolysis was investigated as follows. A 
0°5 per cent. solution of the commercial extract was made up in 0°5 per cent. 
HCl. This mixture was subjected to continuous boiling for 5 hours, a sample 
being removed at the end of 30 minutes. At the conclusion of the experiment 
a sample of the unboiled mixture, the portion which had been subjected to 
only. half-an-hour’s hydrolysis, and the residue were respectively neutralised 
with soda and diluted to a concentration approximately isotonic with Frog’s 
Ringer. From each of the three solutions, A (unboiled), B (boiled half-an- 
hour), C (boiled 5 hours) 0°5 c.c. was injected into a pair of frogs whose 
pigment cells were fully contracted. The macroscopic and microscopic 
examination of the six animals at the conclusion of an hour revealed a marked 
contrast. The A and B pairs were dark and showed the typical pituitary 
reaction. The pair C remained pale. Microscopic preparations of the skin 
showed that in the C pair the melanophores were fully contracted, and in the 
A pair displayed extreme expansion. Those of the B pair were not so 
extremely expanded as those of the A pair. The result of the experiment 
indicates that pituitary extracts retain a considerable potency to induce 
melanophore response after half-an-hour’s boiling with 0°5 per cent. HCl. 
Hence the melanophore stimulant is not identical with the pressor substance, 
and in its slow destruction by acid hydrolysis behaves in a manner identical 
with the uterine or oxytocic principle of infundibular extracts, as far as is 
demonstrable without extensive quantitative estimation of the potency of 


each sample. 


The Pigmentary Effector System. 325 


5. Relation of the Melanophore Stimulant to Histamine. 


The possibility that the melanophore stimulant present in infundibular 
extracts is identical with the oxytocie principle led to experiments on the 


_ effect of histamine, since Abel and Kubota (1919) have raised the question of 


the identity of histamine and the uterine stimulant. Dudley has since 
produced evidence that the effects of pituitary extract on plain muscle are 
not due to the presence of traces of histamine as these workers suggested, 
and more recently Dale and Dudley (1921) have conclusively shown that 
Abel and Kubota were wrong on this point. It can also be stated, with the 
utmost confidence, that the melanophore stimulant is likewise not identical 
with histamine; doses of the latter as considerable as 0:0036 germ. do not 
induce melanophore expansion in the frog. 

That the two substances are not identical is shown further by the action of 
trypsin. Histamine is not acted upon by trypsin,* whereas Dale (1909) and 
Dudley (1919) have shown that both the commonly recognised pituitary 
autocoids are destroyed rapidly by trypsin. In this connection it may be 
noted that Schafer and Herring (1906) claimed that pepsin destroys the 
pressor activity of pituitary extract, leaving intact the diuretic principle ; they 
denied that trypsin destroyed either. The following experiment indicates that 
pepsin does not affect the melanophore stimulant, but that the latter is rapidly 
destroyed by trypsin. 

Six solutions were made up as follows :— 

A. 0°5 per cent. infundibular extract im 0:2 per cent. HCl and 0°5 per cent. ~ 
pepsin. 

B. 0°5 per cent. infundibular extract in 0-2 per cent. HCl, without pepsin. 

C. 0:2 per cent. HCl and 0°5 per cent. pepsin. 

D. 0°5 per cent. infundibular extract in 0°5 per cent. saline trypsin. 

E. 0°5 per cent. saline trypsin alone. 

F. 0°5 per cent. infundibular extract in 0°5 per cent. boiled solution of 
trypsin. 

At the conclusion of 2 hours’ digestion at 34° C. A-C were neutralised, 
boiled, and diluted to a concentration approximately isotonic with Ringer. 
D-F were boiled. After cooling, 0°5 c.c. of each fluid was injected into a pair 
of frogs; and the 12 animals were examined 45 minutes afterwards, a strip 
of skin being removed for microscopic examination, 

Of A-C both the A and B pairs showed macroscopically and microscopically 
the characteristic pituitary reaction. Of D—F, F alone showed expansion of 
the melanophores on being examined by macroscopic and microscopic methods. 


* Dudley (op. cit.). 


326 Dr. L. T. Hogben and Mr. F. R. Winton. 


The result of the experiment indicates that the melanophore stimulant of 
pituitary extracts is not destroyed by pepsin or HCl 0:2 per cent., but that 
it is completely and rapidly destroyed by trypsin. This is in accordance with 
Dale’s work on the uterine and pressor principles. 

As the foregoing experiments show, the melanophore stimulant, like the — 
other pituitary autocoids, is not destroyed by boiling; it is immediately 
destroyed in the presence of a small quantity of H202. One other point 
deserves mention in this connection to correct a possibly erroneous interpreta- 
tion of results previously published by one of the authors. It was stated 
(Huxley and Hogben, 1921) that pituitary feeding induces melanophore 
expansion in salamander larve. In view of the destruction of the melanophore 
stimulant by trypsin, it seems highly probable that the effect was not produced 
as in the case of thyroid feeding by absorption of the autocoid vid the digestive 
system, but by traces in solution in the medium, acting through absorption 
by the skin. 


6. Mode of Action of the Melanophore Stimulant. 


The effect of injection of pituitary extracts on the intact animal might be 
interpreted in at least four different ways :— 


(a) Local effect on the circulation (vasomotor). 
(6) Central stimulation. 

(c) Action on nerve-endings in melanophores. 
(d) Direct action on the melanophores. 


The first two are dismissed in view of the fact that, with appropriate 
preparations, pituitary extracts can be made to exert their characteristic 
reaction upon isolated skin. The second is also excluded by the possibility 
of producing the same reaction after destruction of both spinal cord and 
brain. To discriminate between (c) and (d), two methods may be adopted: 
first, the injection of pituitary extract after the complete degeneration of the 
nerve supply of the skin in a particular area; and, secondly, injection after 
paralysis of the nerve-endings which supply the melanophores by means of 
the usual drug series. The first is the more critical, and it is hoped to carry 
out this method later. The second is open to the objection that the 
evidence is based on the analogy of the operation of drugs on plain muscle. 
As far as the results of experiments on these lines permit legitimate 
inference, it would appear that the action of the infundibular melanophore 
stimulant is direct and independent of any nerve-endings. 

The following re-agents were used for paralysis of different types of 
nerve-endings: cocaine (afferent), curare (spinal efferent), atropine (para- 
sympathetic), and apocodeine (sympathetic). The use of cocaine was 


The Pigmentary Effector System, 327 


suggested by the possibility of an antidromic control. The subsequent 
action of pituitary extract was tested at varying intervals following doses 
of different magnitude, to make certain both as regards time of action and 
amount of drug administered, that the full effect of the latter would be 
exerted. In no case did the normal response fail to make its appearance. 
The indications are therefore that the pituitary melavophore stimulant acts 
directly on the melanophores of the frog and not on the nerve-endings, as is, 
indeed, fully consonant with what is at present known of the mode of action 
of the other pituitary autocoids. 

Neither curare, atropine nor cocaine affect the melanophores of the frog 
in any way. While injection evoked its maximum effect in decerebrated 
frogs which had received just over the normal dose of curare adequate to 
produce general motor paralysis, it should be noted that very large doses of 
curare did prevent the melanophore response to pituitary injection before the 
circulation had actually ceased. Repeated tests, with a wide range of doses, 
showed that atropine does not cause the melanophores of the frog to expand 
as do those of the fish Mundulus when so treated (Spaeth). Laurens (1915) 
has also noted the inefficacy of atropine to induce expansion in the con- 
tracted melanophores of the Axolotl. The different mode of reaction of the 
dermal melanophores of Fishes and Amphibia towards both atropine and the 
pituitary autocoid would suggest that the term “dermal melanophore ” has 
been applied to more than one type of effector organ. It is intended to 
reserve, for fuller discussion at a later stage, the reactions of frog melano- 
phores to drugs; but it may here be mentioned, in connection with the 
antagonism of atropine and pilocarpine in respectively paralysing and 
simulating parasympathetic and secretory uerve-endings, that the latter 
re-agent does not induce expansion of the contracted melanophores in the frog. 
Were these structures innervated by the parasympathetic it would be 
expected that either one or the other would operate in this way. Apocodeine 
which, as Dixon (1904) has shown, effects general sympathetic paralysis, 
brings about a darkening of the skin, not sufficient, however, to mask the 
subsequent effect of pituitary administration. This is fully consonant with 
the reaction of the melanophores to adrenalin, and the direct evidence given 
by Hooker (1912) in regard to the relation of sympathetic stimulation to 
melanophore contraction. 

Finally, the reaction studied in the experiments here recorded, leads to the 
possibility that the nervous system may not be such an all-important factor 
in the mechanism of “colour adaptation” as it has been customary to believe 
in the past. Laurens (1915) concludes from his experiments on the pigment 
responses of blinded and seeing axolotls, that in “colour adaptation” among 


328 The Pigmentary Effector System. 


the Amphibia the stimuli received by the retina and transmitted to the 
central nervous system are “ indisputably” there “transformed and sent out 
along motor nerves to the pigment cells.” That the retina exerts a controlling 
influence is admitted; that the stimuli it receives are transmitted to the 
central nervous system can hardly be questioned; but that the control of the 
pigment cells thereby is of a purely reflex character cannot be conceded till 
the endocrine factor has been eliminated. The existence of an endocrine 
substance capable of producing the reverse response to adrenalin,* raises the 
possibility that the central nervous system may exercise its control over the 
pigment cells partly at least through the secretory activity of the suprarenal 
and pituitary glands. 
7. Summary. 


1. Extracts of the posterior lobe of the pituitary gland have a specitic effect 
on the melanophores of the frog causing them to undergo extreme expansion ; 
an injection, equivalent to less than 1/1000th of the ordinary clinical dose, is 
adequate to produce a conspicuous darkening of the skin visible to the naked 
eye. The effect of pituitary extract on the dermal melanophores of the frog 
is thus the reverse of that which follows administration of adrenalin in the 
frog, and both adrenal and pituitary autocoids in Fundulus, if Spaeth’s 
observations on the latter are correct. 

2. The melanophore stimulant of pituitary extracts is only slowly destroyed 
by boiling with 0° per cent. HCl; it is hence not identical with the pressor 
principle, and in its slow destruction by acid hydrolysis agrees with the 
oxytocic or uterine principle. 

3. That it is not identical with histamine is shown both by the inefficacy of 
this drug to induce expansion of the melanophores, and by the readiness with 
which it is destroyed by tryptic digestion; it is not destroyed in a corre- 
sponding manner by pepsin. 

4. That the pituitary stimulant acts directly on the dermal melanophores 
rather than on the nerve endings is indicated by the failure of apocodeine, 
atropine, curare, and cocaine, to abolish the reaction when administered in 
doses which, on general grounds, would be regarded as sufficient to paralyse 
all nerve endings. 


This research was carried out in Prof. McBride’s laboratory ; acknowledg- 


ment is made to Prof. McBride for his kindness in reading the MS., and to 
Dr. H. H, Dale for generous assistance with reference to the literature. 


* Cf. Introduction. 


Antiseptic Action and Chemical Constitution. 329 


LITERATURE. 


Abel and Kubota, ‘J. Pharm. and Exp. Ther.,’ vol. 13 (1919). 

Abel and Nagayama, ‘J. Pharm. and Exp. Ther.,’ vol. 15 (1920). 

Allen, B., ‘ Biol. Bull.,’ vol. 32 (1917). 

Atwell, ‘ Science,’ vol. 49 (1919). 

Bell Blair, ‘The Pituitary ’ (1919). 

Corona e Moroni, ‘La Riforma Medica ’ (1898). 

Dale, ‘ Biochem. Journ.,’ vol. 4 (1909). 

Dale and Dudley, ‘J. Pharm. and Exp. Ther.,’ vol. 18 (1921). 

Dixon, ‘J. Physiol.’ (1904). 

Dudley, ‘J. Pharm. and Exp. Ther.,’ vol. 14 (1919). 

Hooker, ‘ Zeitschr. Allg. Physiol.,’ vol. 14 (1912). 

Huxley and Hogben, ‘ Roy. Soc. Proc.,’ B (1922). 

Laurens, ‘J. Exp. Zool., vol. 18 (1915). 

Lieben, ‘ Centralblatt f. Physiol.,’ vol. 108 (1906). 

McCord and F. Allen, ‘J. Exp. Zool., vol. 23 (1917). 

Smith, ‘Amer. Anat. Mem.,’ vol. 11 (1920). 

Spaeth, ‘J. Exp. Zool., vol. 15 (1913) and vol. 20 (1916); ‘Am. J. Physiol.,’ vol. 41 
(1916) and vol. 42 (1917) ; ‘J. Pharm. and Exp. Ther.’ (1917). 

Swingle, ‘J. Exp. Zool.,’ vol. 34 (1921). 


Relationships between Antiseptic Action and Chemical Constitution 
with special reference to Compounds of the Pyridine, Quinoline, 
Acridine and Phenazne Series.* i 


By C. H. Brownine, J. B. Couey, F.R.S., R. Gaunt, and R. GULBRANSEN. “ 


(Received October 25, 1921.) 


(From the Organic Chemistry Laboratory, University of Leeds, the Bland-Sutton 
Institute of Pathology, Middlesex Hospital, and the Pathological Department of 
the University and Western Infirmary, Glasgow.) 


BIGLOGICAL SECTION. 

In the course of investigations on the antiseptic properties of certain 
basic benzene derivatives, it was shown that the methochloride of 
diaminoacridine was very highly antiseptic, and that, unlike other powerful 
antiseptics then known, its antibacterial activity was not reduced by the 
presence of blood serum (Browning, Gilmour, Gulbransen, Kennaway and 
Thornton, 2, 7). Diaminoacridine methochloride had been prepared by 


* The work reported in this communication was done with the support of the Medical 
Research Council. 


330 Mr. CU. H. Browning and others. Relationships between 


Benda (1) for Ehrlich, and was named by them trypaflavin on account of its 
powerful therapeutic properties in experimental trypanosome infections. We 
are not aware that its action on bacteria had been investigated prior to the 
work recorded in the above paper (2), although Shiga (10) published almost 
simultaneously results of his investigations on its action on V. cholerw. The 
hydrochloride and sulphate of diaminoacridine were then found to be 
practically equal to the methochloride in antiseptic properties and in efficacy 
of action in serum (8). In the case of these aminoacridine substances the 
power of killing bacteria is exerted slowly ; thus, with the methochloride 
acting on B. coli in serum, the concentration which proves lethal in 2 hours 
(1: 20000) is five to ten times greater than that required to produce sterility 

“after 24 hours; on the other hand, mercuric chloride and carbolic acid pro- 
duce their maximum effect within 2 hours, whether they be tested in a 
solution containing a minute amount of protein (0°7 per cent. of Witte’s 
peptone) or in a rich protein medium such as serum. The acridine com- 
pounds, therefore, may be said to act especially as bacteriostatic agents (7, 8). 
This property, coupled with their relative innocuousness for mammalian 
tissues, as tested by toxicity for the living animal as a whole (4, 7), effect on 
phagocytosis (3, 7), and slight irritating action on the conjunctiva (7), suggested 
that these substances should be specially applicable for the purpose of 
restraining bacterial infections in the tissues. Thus the methochloride, 
under the name of acriflavine, and the sulphate of diaminoacridine base, as 
proflavine, found extensive use in the treatment of infected wounds during the 
late war, and also in the treatment of such relatively accessible infections 
as gonorrheea. The present work records the investigations which have been 
made with the view of tracing the source of the antiseptic property of 
diaminoacridine compounds by examining substances which may be regarded 
as fragments of the acridine molecule. The parent substance of the acridine 
derivatives is a compound of the following formula :— 


4) Oo 
CH CH CH 


3 Ho” \7\/\Non 6 


| | 
MW | 


1) Ones 

Acridine. 
that is, a combination of two benzene and a pyridine ring. If the two side 
wings are removed, a pyridine nucleus remains; if only one wing is detached, 
a quinoline nucleus results. Itseemed, therefore, possible that the antiseptic 
activity of acriflavine (diamino acridine methochloride) might reside either 


Antiseptic Action and Chemical Constitution. 331 


in the pyridine or quinoline nucleus, reinforced by one or more amino groups. 
It is for this reason that substances of this type were first examined. 

In addition, a series of acridine derivatives have been prepared and tested 
for their antiseptic power in order to determine, if possible, whether any 
law could be established relating chemical structure and antiseptic 
action within the group. Also, observations have been made upon 
phenazine compounds, on account of their close chemical relationship to the 
acridine group. 

Methods of Estimating Antiseptic Power. 

The substance to be tested, in a volume not exceeding 01 c.c., was added 
to 1 e.c. of the culture medium, which consisted: (a) of a watery solution 
containing 0°35 per cent. sodium chloride, and 0°7 per cent. bacteriological 
peptone, such as Witte’s, the hydrogen-ion concentration of the mixture 
being adjusted by the addition of caustic soda to yield a Py value between 
72 and 7°8 as indicated by the usual indicators; and (6) sterile ox serum, 
which had been previously heated for several hours at 56° C., in order to 
destroy normal bactericidal power as well as accidental contaminating 
organisms. Seruin, in virtue of its content in protein, has a powerful action 
in reducing the bactericidal effect of most strong antiseptics, at the same 
time it represents the fluid constituent to which antiseptics in contact with 
the tissues are exposed, ¢.y.,in a surgically treated wound ; serum also acts 
as a satisfactory culture medium for the two types of organisms employed, : 
and is extremely constant in composition and reaction. Hence, serum may 
be regarded as a highly suitable medium in which to test antiseptic action. 
The organisms employed in the tests were Staphylococcus awreus and a 
Single strain of B#. coli; the inoculation dose being 01 ¢c. of a 1:1000 
dilution in saline of a 24 hours’ culture in peptone water. Experiments 
have shown that within wide limits the efficiency of the antiseptic, as 
tested by the method described, is practically independent of the size of the 
inoculum (3, 8). But inoculation with very large numbers of organisms should 
be avoided, as these fail to maintain themselves in the medium even in the 
absence of any antiseptic (3). In general, the following series of concentra- 
tions of each substance was tested, 1:1000000, 1:400000, 1: 200000, 
1:100000, 1:40000, 1: 20000, 1:10000, 1:4000, 1:2000, 1:1000. Thus, 
in examining any given compound, the effect of varying concentrations was 
tested at the same time and with the same batch of medium and the same 
cultures of organisms. The mixtures were incubated at 37° C. for 48 hours, 
and then the final readings were made; the occurrence of abundant growth 
was shown by the development of turbidity in the previously clear medium, 
but subcultures frequently yielded growth from tubes which appeared to be 


332 Mr. C. H. Browning and others. Relatronships between 


clear to the naked eye. Accordingly, in all cases the presence or absence 
of living organisms was decided by subculturing each mixture on nutrient 
agar, which was then incubated for 48 hours at 37°C. The results are 
recorded numerically, the highest concentration which permitted vigorous 
growth, and the lowest producing sterility, as tested by subculture of a loop- 
ful of the mixture on agar, being given. In certain cases there is a wide 
zone separating these two concentrations, which indicates that the particular 
compounds are specially bacteriostatic in action, and that concentrations 
considerably less than that required to kill the bacteria still have the effect 
of restraining growth. It is to be noted that more useful information is 
obtained by making subcultures from the mixtures of antiseptic and 
organisms on a solid medium than in a fluid one, as in the latter case it is 
not possible to determine any degree of action of the antiseptic short of 
complete sterilisation. The investigation has been complicated by such 
questions as effects due to differences in solubility and variations in dis- 
sociation, leading possibly to differences in hydrogen-ion concentration of 
the solutions. Thus it has been found, when examining particular com- 
pounds, that comparatively small variations in hydrogen-ion concentration 
may exercise a great influence on the antiseptic potency (Browning, 
Gulbransen and Kennaway, 6). With diaminoacridine methochloride in 
peptone water, the concentration required to sterilise B. coli when the Py 
value of the solution lay between 4 and 5 was 1:2000; within a range from 
6 to 7,1:10000 of the dye sufficed; at 8 to 9, 1:40000 sterilised, while 
at 11 a concentration of 1: 200000 was sufficient. In each ease the 
medium with similar reaction, but without the antiseptic, permitted vigorous 
growth of the organisms. In addition, there is the further factor of 
variability in the behaviour of the bacterial culture. With regard to the 
latter, it appears to be highly probable, if not definitely established, that the 
individuals in a given culture are not all equally susceptible to harmful 
influences ; thus, irregularities are observed when a particular concentration 
of antiseptic is caused to act on duplicate samples of the same infected 
material. This has been drawn attention to by Richet and Cardot (9), and 
has been observed also in our own work. Further, the occurrence of 
variations in the culture from time to time can scarcely be excluded 
although no evidence has been obtained pointing to permanent or to 
reeularly cyclic changes. Repeated series of tests carried out with a view 
to examining the action of diaminoacridine methochloride on a single strain 
of &. coli in ox serum have shown the following variable results. A con- 
centration of 1:1000000 and upwards, sterilised in 8 series, 1:400000 
and upwards, sterilised in 17 series, 1:200000 and upwards in 16 series, 


, 


Antiseptic Action and Chemical Constitution. 333 


1:100000 and upwards, in 24 series, 1: 40000 in 2 series, and 1: 40000 failed 
to sterilise in 1 series. With Staphylococcus awreus, however, the range 
of variation was distinctly less. Accordingly, the numerical values recorded 
here must be interpreted in the light of the above results (5). It should be 
noted, howeve, that in comparing certain closely related compounds in which 
marked alterations in antiseptic power may be produced by relatively slight 
chemical differences, ¢.g., the methochloride and the hydrochloride of the same 
base, the two substances have been tested on the same occasion and with the 
same specimen of medium and culture, thereby reducing as far as possible 
the action of uncontrollable factors. 


Fragments of the Acridine Molecule. 


Table I includes all the compounds tested, and comprises pyridine and 
quinoline derivatives and dinaphthylimine. The striking feature, in general, 
is the low grade of antiseptic power shown by these bodies. Thus, the 
hydrochlorides of quinoline [3], tetrahydroquinoline [15], and the amino- 
quinolines (0[4], m[6], p[8], and @[10]), all failed to sterilise in dilutions 
exceeding 1 : 2000 either in peptone water or in serum. ‘The methochlorides 
of the aminoquinolines [7, 9, 11], except in the case of the ortho-compound [5], 
showed accentuation of antiseptic action in serum, as compared with the 
hydrochlorides of the corresponding bases, a characteristic result which will 
be discussed in more detail when dealing with the acridine group. The 
hydrochlorides of «- [17] and @-naphthoquinoline [19] were slightly more 
active.* No striking difference could be established between these and their 


“ tetrahydro-derivatives [22, 23]. Diamino f-naphthoquinoline [24] also 


showed no enhanced efficiency. The methochlorides (or methosulphates) of 
both naphthoquinolines [18, 20, 21] and of diamino-8-naphthoquinoline [25] 
showed intensified action in serum. The 8-hydroxyquinoline sulphate [12], 
long known as an antiseptic under the name of “chinosol,” is included for 
coniparison ; its activity for Staphylococcus aurews contrasts with the slight 
effect of hydroxyacridine compounds [67] and of the aminoquinoline com- 
pounds as antiseptics, but it is very weakly antiseptic for B. coli. On the 
other hand, it is remarkable that the methochloride [13] and methopicrate [14] 
of the base do not show enhanced action. 1-1-dinaphthyl-2-2-imine [28] 
exhibits great discrepancy between its powerful action on staphylococcus and 
lack of effect on &. coli, which is similar to that exhibited by the triamino- 
triphenylmethane compounds, hexa-methyl and ethyl-violet. But the most 
striking character of this substance is the reduction in action produced by 


* Previous results recorded for a- and 8-naphthoquinoline were obtained with less pure 
preparations (see ‘ Journal of Pathology and Bacteriology,’ vol. 24, p. 127 (1921)). 


Relationships between 


a OOOF : T > é = OOOF : I ae é 
+ QOO0T:T + OOOT : T + QO00T: T sp OOOT:~T |" ae ie agen SO aplLoyTTooyygour amlpourmbourue-» Il 
= d = d = d (41) OOOT * I 
+  Q001'T + 0001‘ T + 0001: T fe eeOOOP ORI [Res ee air ate * epMoryooapéy ompournbourme-» | oT 
= OOOPF * T = ¢ = 0006 * T (-yv2) OOOT * T 
+ Q000F: T + QO0L'T + Q000L‘T + MOOUG IE. hee cae ae apltopyooqyeur eurjourmbourme-d | 6 
= (6 = id = d = ¢ 
+ OO0T : T + O00l: T + OOOT: T + OOOT? LT | aprtoptoorpAy outjoumboutue-d | g 
= OOOF * T = d = OOOF * I (72) 000T : T 
+ QOOOT*T + OOOT : T + QO00T: T ate OOOP:T |" ae a Ga aplco[yooqgeut ourprambourure-w L 
a d = 4 = d = OOOT : T 
+ OOOT: T + NOOT * T ate OOOT : T + 000g? 1 | Pg ibe eee epitopoorpAy outpournbourure-ew 9 
oe d = é = (| = o00c*T | 
ci OOOL: T + OOOT ‘ T = OOOT : T + OOO * T opto (aoTyyeur aurpournbourure-o G 
= d = é = ¢d = 0004 * T 
+  000T'T +  Q00T'T + 0001? I ae OOM a (Gee ee ae eprorqoorpay ourfournboururw-0 | F 
= d = OOOT* I = é =] O00e*T | : 
+ 0001‘ 1 + 0008: f +  000T*T ei WOOO ale I  epoyqooapky ourjoumny | ¢g 
= d = d = d = =000c 1 | ; | 
+ OOOOOT * T an OOOT + T + QOOOT*T | t= OOO0G) i San ee aprporygout eurpraddourmepAygeurp-» | Z 
= d = d = d = d | 
+ Q00TT + OOOL:T | + (00T:T he BOOOK Ge es ae a Me aa aplorqoorpxy ourprcddourure-» | | 
“TAN.LEG “togvm onoqdog “LUNG ‘19jeu ouoydeg 

WNIpsll + 1700 “gr WMIPat + svawny snadvov0phydnj5 ‘oouvySqng ‘ON 

“TUSTURS.LE 


Mr. C. H. Browning and others. 


334 


pus SoaTyVALo(, ouIplAg pue ourjoum?’)—e[nNde[0]J VUIpIIoy oy} Jo syuoMSeay Jo uoNoy odesmuy o4Y,—] e[qeIT, 


Ala 


(‘punoduioos [varmeyo pues tinIpett Jo eAnjxX1uI olf] UL potmmooo sey uoryeqtdrosrd yey, saqevorput ydy 


‘our Ayyydeurcy 


MOLPST[L109S OJOTCUtOd Fo JOYS YYMOAG JO WOTFIGIYUL sojyouop yur {oINGXIW oTL10}s Vv soyvOTpUL — o[IA ‘potoruout sooueysqus 
JO WOTPVAPWOOUOD Of} UL PecINOVO STUSIULOIO OY] JO YJMOIS doAT Yeyy soyvolpur + serqey, quoubosqus pue sty uz) 


" 


335 


‘prow ATSuoTs WOINTOS NOOT? T + 


See | oe aS ee anes 
S| | | 
Se | = d = é | - ie | 
3 + QO00r:T | (74¢)+ oOOOT?T | + wenn | peonone ete 
= oe 3 | ae 2 | & L aA ce PSS a pay cee aulmrg' g-T<yyydeurp T° Zz 
ep = rs + ooos:t | (add)+ — oot T Fe panies meee me 
S a F (ydd)— 008: T Bs U | (an) 000e* T | jopraopyaoapky, ourprrddrp-oysyduurg't | Lz 
ee) O00 Tee Ce) 0007s it (add) + Ovot: T Caan scot 
mS = 0007" = d — oor: | FOOD Fae SOOO USS |e) ceca doprtopyoorpsy ourprakdip-ongydemp T | 9% 
=f © 6000 T ie pee ; Aa } | =— WOOL | TpraAdl 
3 4 c (-yw2) 00OT? T =  Gnohn | 2 ane : : a SHO UPr ona ee outa eae Ouran ia 
OOOT * T + 0002: T : a: | ee } 
5 Ey ; (iD) — ; 2 O00F : I + OOORES Toe ee eee aes “J gutjoumnboyyydeu-g- 
—~ + oooT:1 | (74d) ores (yur) oot? 1 | (7dé)— goog? T | te Reena cee pine 
eek Bae a : Pe 
iS) ‘ d Ca OO0F : T 35 me Lt (pd) Y vee : ! aplcopqooapsy outpournborgydea-g-oapdyeayoy, | 8% 
<S OOOT : T ydd)+ QOool:T S 1: : : 
2 — O00R:T ee ; | ap OOK. (7dd)+ QO00T?T |" apiopqooapay ourpournboygydvu-2-oapAyucyo 
s me Soman Rati ee ‘i cee sil = o0o07:T | : [V4oT | 66 
5 a spp oer ‘T == 000K I =| - 00001 : t a BOONE i en a eee See ae ee 
02 + 000001 : T +  Qoog:T | ee : pce ee 
iS) = é (7dd)— oF T | Giyocnt Ue ace cee OlOURE EE ceraen ae “ epMoryooujeur eurjoumnboyzyden-g | 0% 
Se. | + O0OT*T | + 00001: T | eNO EEO) Ge OO Wa ay 
Ss | x ERO ai | — Q00T:T | — OO00T:T 2 Toor I ae Se eeee ee en cee | lee 
Oia OOOT*T | + Q00F:T | + Q000F: pl ey eet ae ee 
ydd)+ — 000G+T + : 
S 1 a Hite foal aR ecgus ee nt 
Z ee f 5s : bs 000¢ * T + OO00T‘T © apmopypoupsy surjoumboyyydeu-» | ZT 
Ss (4d) +  QO0T:T + : o ‘ A s | 
oooT?T | (7dd)+ O00T'T a Aaa nee nee ears 
= as . EP 1 = ; Opok u aplporyyemt ourpourmboapéyeayes-[AqqeT | OT 
+  000T'T + Q00L'T i 5 Pel 
‘ OOGeEN oe rey te a ; ! + ONE i Ngan gaa eS ae aplopyaoapAy ourpoutrnborpsyeyey, | ST 
000% + T + 00081 + m 
| cian ee oa eee Es 00006 * T + QOOOOT:T | roeesoeessgaproidoygout eurjoumnbéxorpsy-g | FT 
+ 000%‘ T +  QOOOT:T + aoe : a sonoRE a 
ee abit BM ee Pe 006 : Tea 4 OOMOGi7P2 WP ee “+ gprtopqooygeut eutjournb&xoapsy-g | ST 
| + QO00F * I + 00007 *T ae arate t Se az 
OOF? T | +000000¢:T (“°° (,, Josounyo ,,) ezeydyns aurournbAxoapéy-g | ZT 


336 Mr. C. H. Browning and others. Relationships between 


serum; thus 1:2000000 sterilised staphylococci in watery medium, but 
1: 1000 failed to kill these organisms in serum. This is the most extreme 
reduction observed in the case of any substance, being twenty times greater 
than the reduction effected by serum on mercuric chloride. 

So far, therefore, it has not been possible to obtain any fragment of the 
molecule which equals, or even approximates closely to, diaminoacridine in 
antiseptic properties. 

Acridine Group. 

The substances which have been tested are included in Table II. The 
following general conclusions may be drawn from the results :— 

Action of the Amino-Growps.—The introduction of amino-groups enhances 
the antiseptic potency both for Staphylococcus aureus and B. colt, eg., acridine 
[29] and diaminoacridine [35], dimethylacridine [33] and diaminodimethyl- 
acridine [48]. 

Effectiveness in Serwm.—LHfttectiveness in serum is a characteristic of the 
compounds with unsubstituted amino-groups and especially of the metho- 
chlorides of these bases. The further introduction into the diamino compounds 
of a phenyl-group attached to the medial carbon atom (in position 9) has, 
however, a marked effect in diminishing the action in serum ; this is exhibited 
both in the ease of 2-7-diamino-3-6-dimethylacridine [48—51] and 2-amino- 
3-methylnaphthacridine [58—61]. On the other hand, the methochloride of 
9-phenylacridine [32] is more active than that of acridine [30]. 

Comparison of the Antiseptic Power of the Methochloride and the Hydro- 
chloride of the same Base-—The methochloride (or methosulphate or metho- 
nitrate) is never less potent than the hydrochloride in the presence of serum 
and in some cases the increased effectiveness shown by the methochloride is 
very remarkable, ¢y., 2-7-tetraethyldiaminoacridine [55, 56], 2-7-diamino- 
3-6-dimethyl-9-phenylacridine [50, 51], 2-amino-3-methylnaphthacridine [58, 
59], 2-dimethylaminonaphthacridine [62, 63]. In the case of the simplest 
member of the amino series, 2-7-diaminoacridine [35, 36],* and where the 
substituents are directly attached to the outer rings as in 2-7-diamino- 
3-6-dimethylacridine [48, 49], the hydrochloride and the methochloride are 
practically equal in antiseptic power. It is noteworthy, however, that when 
the antiseptic power of diaminoacridine is diminished by substitution of ethyl 
radicals in the amino-groups, the enhanced action of the methochloride [56] 
over the hydrochloride [55] again becomes apparent. So far, no rational 
explanation of the enhanced efficacy of the methochloride has suggested itself. 

e This result has been obtained with carefully purified specimens of these compounds. 


+ The observations of Crum Brown and Fraser on the change produced in the 
pharmacological action of alkaloids when a methyl group is attached to a nitrogen 


Antiseptic Action and Chemical Constitution. 35:7 


The hydrochlorides of certain of the compounds require the presence of a 
slight excess of hydrochloric acid in order to effect solution, e.g., in the case of 
9phenylacridine [31] and 2.amino-3.methylnaphthacridine [58]; but the 
enhanced effect of the methochlorides [32, 59] over the respective hydro- 
chlorides [31, 58] is not to be ascribed to the higher hydrogen-ion concen- 
tration of the solution of the latter, since the addition of hydrochloric 
acid to the methochloride, so as to produce a solution of similar reaction, did 
not reduce the antiseptic power to that of the hydrochloride. The com- 
parative effects of the hydrochloride and the methochloride of the same base 
in peptone water show a much less regular behaviour. 

The Substitution of other Radicals for the Methyl Group in Diaminoacridine 
Methochloride—The following were examined : ethyl [37], propyl [38], n- [39] 
and iso-butyl [40], iso-amyl [41], phenyl [42], benzyl [43], also the chloro- 
acetate [44], chloropropionate [45], and chloroacetanilide [46] derivatives. 
The result was that, within the limits of experimental variation, these com- 
pounds are practically identical with the methochloride [36] in their 
antiseptic power for both organisms. 

The Substitution of Alkyls in the Amino Groups—Tetramethyl- [52] and 
tetraethyl-diaminoacridine [55] hydrochloride and also the methochlorides 
[53,56]and methonitrate [54,57] were investigated. The tetramethyl hydro- 
chloride [52], while practically equal to unsubstituted diaminoacridine [35] 
in its action on Staphylococcus awreus, was distinctly inferior for B. coli, both 
in peptone water and in serum. The tetraethyl compound [55] was still 
weaker ; thus with the latter the sterilising concentration for staphylococcus 
in peptone water was 1: 100000 and in serum 1: 10000, while for B. coli a 
concentration not less than 1: 1000 was required. The methochloride [53] 
and methonitrate [54] of the tetramethyl compound were practically equal to 
the hydrochloride ; also, as in the case of the unsubstituted diaminoacridine, 
the effect in serum with the hydrochloride and methochloride was practically 
equal. On the other hand, the methochloride [56] and methonitrate [57] of 
the tetraethyl compound were much more active in serum than the hydro- 
chloride. 

Groups which interfere with Antiseptic Action—As has been shown above, 
the introduction of methyl and ethyl groups into the amino radicals depresses 
rather than enhances the antiseptic potency [52, 55, as compared with 35], 
thus contrasting with the effect of similar substituents in the diamino- and 
atom, thus converting the compound into a quaternary base, should be recalled in this 
connection (‘Trans. Roy. Soc.,’ Edinburgh, vol. 25, pp. 151, 693 (1868-69) ; ‘ Proc. Roy. 
Soc., Edinburgh, vol. 6, p. 556 (1868-69) ). It has been shown, however, by Lenz that 


diaminoacridine methochloride is totally devoid of curare action, either in cold or warm 
blooded animals (‘ Zeitschr. f. d. gesamt. experim. Med.,’ vol. 12, p. 195 (1921)). 


VOL. XCIII.—B. 2B 


Relationships between 


338 Mr. C. H. Browning and others. 


(7dd) 


— 00000T : T — 00001‘ T — 000002: 1 — 0000F‘T 
+ QO0000F : T + (0002 : I + 000000T : T + 000002: T 
— 000002 * T — 00007: T — 000007 : T — 000002 : I 
+ 0000001 : T + 000002‘ T + 000000T : T + 00000F : T 
— 000007 ‘ T — 00002: 1 — 000002 ‘ T — 000001: 1 
+ 000000T : T + (Q000F:T + 000000T : T + 000002 : T 
— 000002 : T — 00002: 1 — Q000UT : T — 0000F:T 
+ 00000F ‘ T + QO000T :T + 00000F : f + Q0000T : T 
— 00000T : T — 0000T:T — 000007 : T — 00007: T 
+ QO00UF ‘ T + Q0000T : T + 000007 ‘ I + 000002 : T 
— 000002 ‘ 1 — 0000‘ T — 000002: T — 00000T : T 
+ Q0000F : T + QQ000T : T + Q0000F : T + Q0000F : T 
— 000002 ‘ T — 0000F:T — 000002 : T — Q000T:T 
+ 00000% ‘ T + 000001 : T + Q0000F : T + 0000: T 
— 000002 : T — 0000T:T — 000002: T — 0000F:T 
+ (0000F : T + 0Q000F'T + Q0000F : T + QO000T : T 
— 000002 ‘ I — 00001 'T — 000002 : T — 0000: 1 
+ (00000F ‘ T + Q000F ‘1 + Q0000F : T + Q0000T : T 
— 00000T ‘I — 00001: T — 000002 : T — on00F:T 
+ Q0000F : T + Q0000T ‘ T + QO00NF : T + 000002 : I 
— 000001 ‘1 — 00004‘ T — 000002 : T — 000002: 1 
+ 000002 : I + 000%‘ T + Q0000F : T + Q0000F * T 
— Q0000T ‘ T — 00002‘ T — 000002: T — 000002‘ T 
+ 000002 : T + Q000F * T + QO000F : T + Q0000F : T 
— 000F:T — 000%: T — 000P:T — 00002: T 
+ 00001: I + 0001 ‘1 + 0000F : T (72) 000% * 1 
- d = d = d - d 

+ ooor:T | (7¢¢%)+ QOOL?T | (2¢¢)+ oot: t | (-2dd)+ 00T: T 
— 0000T:T — 0002: 1 — 00008: T — 000071 
+ 00002: 1 + 000%: T + 000002 : T + 000000T : T 
- d = 000T:1 - d — 000T:T 
+ O001'T + 000% 'T + QO0T:T + 000%:T 
— 000F:T — 0001'T — 0002'T — 0001 :T 
+ 00002 'T +  Q000T:T + 0000: T + Q000T: T 
- é — 0001: 1 = d — 0002'T 
+  Q00T:T + 0008'T + Q00F'T + 00002: 1 
‘TUn.eg ‘10jeM ou0ydog “uIn.eg *1098M omo4dog 


UINTpoUr : 2700 "gr 


UINIpem : snaimp snaoooophydngy 


“USLUR GIO, 


CRT ES Ey aT KOLO K OW ORK CLE Ee Ze 


OPT[IUBJOdBLO[YO SULpItovouTUTEIp') "Zz | 


eyeuotdoadoxo;yo ourpiovoutmerp') +z 


Don Ooi einai 


97BjooBoOLO[YO ou Iplovournrerp’ LZ 


“ spmoryordzueq autpraovourmerp')'Z 


Bee meee e ress eeeeee 


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epHtop yor Aure-ost ourpltovoururerp:)'Z 
“* eprtoyyoy4qnq-ost ourpiaovouruerp' yg 


epmoyyorAyng-w ourprxovouturerp' yz 


OMe meee ee eee wees oprxortoyAdoad eUIpPILoBvoUIMeIp /°Z 


* eplto[Pooyye ourpmovourmelp' )°Z 


epltopyoorJeut SUrpIxovourUrEIp’ )'Z 
(e38yd[ns puv) oprropyooupy ourpiiovoutureip')'Z 


eplloyqoorj4eut ourprroe,Aqyoup'9'e 
meseerrreereseees  oprtopqoorpAéy ourpitoeyAyjountp’9"¢ 
sree onrropqpooqjeu eurpritoeyéueyd'¢ 


eprxoTqoorpAY ourprxoepAuey dg 


Hreiueservuenecserarerreseeees QNTTOTUDOYIOUL OUIPLOW 


FOUR eee eee ee eee mee eae aes eeesrtons epmtopqoorpAy ouIpMoy 


‘eomeysqug 


‘ONT 


“SOATHBATIO(T OUIPMOW Jo uy ondesyuy— Ty eqeI, 


339 


atution. 


Action and Chemical Const 


aseptic 


Ant 


—0000% : 1 UAT TogIqrqUT FO QUOZ AOPIM v SUM oto] ‘UIVE ‘sqyuoUTTedxo toy40 


ba? 6 
+ o00r't 
+ O00T'T 
a ¢ 
+ 000%'T 
= d 
+ O00 'T 
= d 
+ 0008‘ T 
— 00002: T 
+ Q000F' T 
- d 
(7dd)+ 001: T 
— 000%‘ T 
+ 00002: T 
- d 
(7dd)+ 9990? T 
— 00000T : T 
+ 000000T : T 
(2d@)— O00 1 
(7dd@)+ 000%: T 
— 0000T: I 
+ 000021 
— 00002: 1 
+ 0000: T 
i 
O00T : T 
00002 : T 
QO0OOT : T 
O000F : T 
QOONOT ¢ T 
00002 : T 
0000F : T 
0000T : T 
0000F * T 
é 
OOOT : T 
00000T : T 
00000F : T 
00000T : T 
000002 ! T 
Q000F : I 
000002 : I 


Pelee [|e lt le pe | I 


ge fice | ae [a> [hae f 


(ydd ) — 

+ 
(7dd) — 
(ydd ) + 
(yada ) — 
(7dd) + 


(2dd) — 

+ 
(7da) — 
(7d) + 


Set eee ar ie her! [ce ifict [iieal 4 


G 
OOOT : T 
d 
OOOT * T 
d 
000% * T 
¢ 
OOOT * T 
d 
OOOT * T 
OOOF * T 
00002 * T 
0006 ‘ T 
00002 * T 
OOOT * T 
QOOOT ‘ T 


— G 
(2d) (-yur) 9997 : T 


00002 : T 
OOO00T : T 
OOOOT * T 
00006 ‘ T 
OOOF * T 
OOOOT * T 
OOOOT * T 
OOOO0F * I 
O00T * T 
OOOP * T 
OO0OT * T 
00002 * T 
00002 * T 
OOOOF : T 
OOOF * T 
OOOOT : T 
0002 * T 
OOOPF * T 
d 
OOOT * T 
OOOOT * T 
OOOO0F * T 
G 

OOOT ‘I 
OO000F * T 
OOOOOT ‘ T 


d 
0001‘ T 
— 0002'T 
+ Q008'T 
(yur) OOOT * T 
+  Q00F'T 

= G 
+ 0002: T 
—  000r :T 
+ Q0000F : T 

- 4 
(7dd)+  OO0T‘T 
—  oo00r: I 
(42) 00000T : T 

~ d 
(7dd)+ = 999T? T 
— (00000F : T 
+ QQOOV0T : T 
— 000¢'T 
+ 0000F: T 
— 000002 : T 
+ QOOOOF: T 
— 00000F : T 
+00N0000T : T 
— O000T:T 
+ 00002: T 
— 000002: T 
+ Q0000F : T 
— 000001‘ T 
+ QOQO00F : T 
— 000001 ‘1 
+ 000002‘ T 
— 0C0002 ‘ T 
+ 0000N0T : T 
— O000T:T 
+ 00002‘ T 
— 000002 : T 
+ Q0000F ‘ T 
+— 00000T ' T 
+ 00000 : 1 
— (00000T: T 
+ 0Q00000T : T 


+ OOOT * T 
+ 


— 4 
+ 0008: T 
- F 
+  QO0T'T 
— 0008:1 
+ Q0000T : T 
— 000F:T 
+ Q000F:T 
~ d 
+ O000L'T 
— 00000F : T 
((7%2) 000000T : T 
(7@d)— 00008: T 
+ Q000P' 1 
— 00000T: 1 
+ d 
(ydd)— —-999T ? T 
+ 00gz 
— 000000T 
+ 00000001 : 
— 000002 : 
+ 00000F : 
— 00000F 
+ 0000002 
—000000T 
+ 0000008 
— 00000T 
+ 000002 
— 000002 
+ 000000T 
— 00000F : 
+ 000000T : 
— 00000T 
+ 000002: 
—(00000% 
+ 000000F : T 
— 000002: T 
+ Q0000F : T 
— 00000% : T 
+ 000000T ! T 
— 00008: T 
+ Q0000P : T 
— (Q0000T ‘ T 
+ 000002 : T 


ee i i ee nan! 


UL 4nq ‘89809 peqvodea ut pouregqo sum qinsexr sty 4 


‘xodvd snutyiy 09 prow szyovea MOMNTOS OOOT: T OU x 


oploTpooysout ourpitow [Ayjourp'g'g-LxoapAytp' y's 


[8s wANIpos ourpizoe [AqQoutIp'g'¢-Lxoapyip' pg 
19480 o1,Axoq 


=0@0 ourpitovAuoyd¢-ourmerppAyyouretqo: 1'% 


19980 oI <xoqatvo ourpriovpAuoyd'g-outuerp')'z 


97BJOOvOAOTYO OUIpILovoUTMAEIp[AqeovIp' J" 
“"" oprcopyooyjout ourprcovyyydenourareAqyoutp'z 


“""" gprcopTyooupAyT ourprrovyyydeuourarepAqyemip'g 
eyeydyns 

-oyjom ourlpmovsy4ydeay{uoyd'g-[AqAourg-ourare’g 
ePMOTTO 


-orp£y  outprovyyydvupdueyd g-TAyyourg-vurare'z 


opto qooyjow ourpmovyyydvuyAyyourg-ourme'g 


aplto[qooapAy ourpraoeyyydvupéqyoug-ourure'z 


oPVAGITOYZeM oUIpIMovourmerplAyyouaqoy’ 1 'Z 
epopqooyyour SUIptovourorerppéyqortqog' 1g 


“" gprcopqoorpég ourprtovourmerppéyqourjoy' 1° 


OFVAFIMOT JOU oUTpIIovoUrUreIpyAqyoumns4o9: )°Z 

pa epMolooyyeur ourpiovourmerppAqqourvso}" )'z 
(oSue.t0 ourpri08) 

ouIplovourmerplAyyoures4o4' 2g 
Opmoryo 

-oyjour oeurprouAueyd'g-[AqjoumIp'g9'g-ouroerp' /'Z 
(euravpozueq) eprtopyo 

ourpitovyAneydg-,Ayyourtp'g'g-ourureip")"g 


apiopqooupéy 


-oup&y 


‘“epHoTyoorjeut eurprtow-[Aqyourrp'g'g-ourULBIp' 1°z 
(saoyjof ourprtov) 


epmopyoorpséy — eurptoB-|AqJoutIp'g g-ouTUIEIp' yz 


voeeess TITTED + apo; ooyjout euIpicovouruurp’ pz | 


Lt 


B 


N 


<< 


340 Mr. C. H. Browning and others. Relationships between 


triaminotriphenylmethane dyes (2). The substitution of one hydrogen atom in 
each of the amino groups by acetyl radicals practically abolishes the anti- 
septic action, ¢.g., the sterilising concentration of 2.7. diaminoacridine chloro- 
acetate [44] for Staphylococcus aureus in peptone water was 1: 100000 and in 
serum 1: 200000, and for 5. colc in peptone water 1:20000, and in serum 
1: 400000; on the other hand, with the diacetyl derivative [64] a concentra- 
tion of 1: 2000 failed in each ease to sterilise. 

The carboxylic esters of 2.7. diamino-9.phenylacridine [65] and of 2.7. tetra- 
methyldiamino-9.phenylacridine [66] were so weak as to suggest a marked 
depressing effect of the carboxyl group on the antiseptic property. 

The replacement of the amino groups by hydroxyls also led to practical 
abolition of antiseptic power, as is shown in the case of 2.7.dihydroxy- 
3.6.dimethylacridine, of which both the sodium salt [67] and the metho- 
chloride [68] were tested. 

Comparative Efficiency for Staphylococcus aureus and B. coli.—Antiseptic 
potency for Staphylococcus awreus and B. coli does not invariably run parallel ; 
thus the lethal concentration in serum for staphylococcus is 1: 100000, or 
lower in the case of 2.7.diaminoacridine hydrochloride (or sulphate) [35] and 
methochloride [36], and other analogous derivatives [37-46], 2.7.tetramethyl- 
diaminoacridine hydrochloride [52], methochloride [53] and methonitrate [54], 
2.7.diamino- 3.6.dimethyl acridine hydrochloride [48], and methochloride [49], 
2.7.tetraethyldiaminoacridine methochloride [56] and methonitrate [57], 
2.7.diamino 3.6.dimethyl-9.phenylacridine methochloride [51], 2.amino- 
3.methylnaphthacridine methochloride [59]. But in the case of B. cola only 
the hydrochloride, methochloride, and analogous derivatives of diamino- 
acridine [35-46] and of diaminodimethylacridine [48, 49], and the metho- 
chloride of 2.amino-3. methyl naphthacridine [59] reach this level of 


effectiveness. 
Phenazine Serves. 


The striking feature of this series (see Table III) is the relatively poor 
antiseptic power exhibited by the amino compounds in serum, especially tor 
B. coli. The only compounds exactly comparable with the acridine series are 
those of the phenazine base [69, 70], 2.7.tetramethyl diaminophenazine 
[80, 81], and 2.7.diamino-3.6.dimethylphenazine [86]. The enhanced effect of 
the metho-compounds as compared with the hydrochlorides of the same base » 
is evident in the phenazine series ; but is not so striking as with certain of the 
diaminoacridine derivatives. 

The relatively greater efficiency of the methochloride of 2.dimethyl- 
amino-7.amino-6.methylphenazine [84], as compared with 2.dimethylamino- 
7.aminophenazine [76], and of 2.7.diamino-3.6.dimethylphenazine [86] as 


= "poe4se} 0 OU P[Nod SUOT}BIZNEDTOO AeySIY seouBysqns os044 FO AgtyIquyosul 944 JO gunoo0v UG x 
Se) TOMOVAL OY] FO [OMUOO QUOLOTNSUT JO JANI UO peqqIULO 91B 1098 euojyded ut gg-G9 spunoduloo YIM poeuieqqo sen[eA ot], 
- d = é | 
+ 0009: 1 + 40009‘ T “-"“eprporrpéy oulzeueydourorerplAqjzouresqey 1°% | 18 
ae + 400001 : T + 40000T : T “ eprroTqoorpdy ourzeuoydourmerppAqyouesyoy 1% | 08 
= — 0008: T —  Q000T: T 
= + 0000T * T +  0000F * T “ eprporyjour oulzeueydtAyjour'g-ourmesAqyeutp’s | 6L 
‘Ss +  000T:T + QOOT : T- epioryooupAy ourzeueydyAyjour'g-ourmepAqyeurp’z | 8 
> = d = é (F8 ‘ON Opltopqoouyeur ves) eprxoptpoorpAY 
: S + QOOT'T | +  Q00T‘T ourzeueydpAyyour'g-ourmre’/-ourmepAyzomrp's | LL 
= d — es OO0GE 
© + QOOT:T | + QOO00T : T epllopqooyyem ourzeueydourme'y-ourmelfyjouip'g | OL 
= = d = OOOGaT 
& (2dd)+ QOOT:T + DS QOOR I ale kn ees ee cee terereeeeees QnTIOTpooyyew oulzeueydoutuerp'e'g | SJ, 
= = d ie é 
= + OOOT : T > Se OOO Se s|- Se SS eae | ee epritopqoorpAy ouizeueydourmuip'e'z | FL 
5 — 000%: T — 0007: T 
+ QvOOT:T | EES OOOO Meee ee ees eae ee een ee aplopyoouseut eurzeueydourue'g | 
3 =. See a ara 
SF | + 0001‘ T Oe. OOO GPM ie Pe: ps eel ie epiorqoorpéy ourzeneydourmre'g | Zz, 
8 — 00001: T — 000F‘T 
~ + 0000% : T + 0000T : T ee ere er eaten wee ears ss wee eee aqzeydinsoyyour eulzeueyg TL 
S — 00001: T — 0007‘ T 
= + 00007? T trae (OOOO ae ee ee ee ee epMo[yooujem eulzeueyg | OL 
= d = é | 
x + 0083: 1 A 0008S ae sree PS SS Sue a oak epo[qoorpéy ourzeueyg | 69 
LS po ss at = waa asiaee See ae at eet LT tees Ah Shee 
Q ; ; ; 
2 wInIEg 10jeM ouojdeg unde Tayem ouojdog 
= 
NX UINIpeUl : 2700 “g_ | UWINIpPeUl : snap snooov0jphydnjg ‘eouBysqne ‘ON 
= “UISTURSIC, : | ° 


‘MINIIG Ul seltag ouIzBUEY_ Jo uoOYy oydesyuW—TIT qe, 


‘sonqua oures ey} Aypeomovad oaus oouvgsqns styy Jo punoduoo opidojyo ourz oy, f 
‘poajosstp ATjemaed AjuO 4 


+pe4se9 oq 40U pjnoo suo1yezqUeoMOD JoysIY soouNsqns oseyy JO AyITIQuTOSUT E44 Jo JUNODDG UO 


Ss 
SS) . 
v l | | 
s — 00008: | — 0008‘T — 00008: T — 00000T?T | (punoduroo epixopyo oulz) epLto,qooyyeut | 
D + 00000T*T | + 000F'T + 000002‘ T + QO000P:I  eutzeuoydourme'z -eurjournboapkyeaqoy[q30-N | 
i — 000r‘T | (7d2)— Q00F' 1 Se OU — 000002 ‘ T epllopqooyjeur onrzeueqd 
3 + OQO00L‘T + Q000T‘T + QO00F?T | + Q0000F * T -oy;yden - ourmedqgeurp:z-[Ayjourg-onrme:) | 
aS = d | = é (74d)— QOOT*T | = 000k L 1} epMoryoorpay ourzeneyd — | 
= + 0000P:T | (#4d)+ O00T:T + .QO00T:T | + Q000P?T | -oygyden - oururepAqyourp'Z-[Ayqour g-ourure’) | 
= — 000r'T | — 0002:T — 00001: T — 00000F : I (punoduioo eprto[yo outz) eprtopyooqjeur 
. + 000002:T | + 00001: T + 0000F‘T + 000000T : T eurzeueyd[Aqgoultp'g g-ourme' /-ourmey<z00q"Z 
3 — 0000¢'T | = d | — 00007: 1 — 0000F ‘1 (punoduroo epraoyyo oulz) eprxopqooyjzout 
~S + 0000%:T | + 0008'T + (Q0000T ‘ T + (Q0000F : I eurzeneydAygeunp'g'g-ourme:y-ourmre, <q }aurtp'Z 
Re — 0000Z:T | — 0001: T — Q000F‘T — 000002 * I epMoTqoouyeur 

+ QOO00T ‘TI 4h 0002: T + QOO00F : I + QO0000T : T euizeueydt{yqowip'9'g-ourme' /-ourmeAyjeur'Z 

— 0000F:T = é — 000002 : I — 00000F : I eplo]qooujour 
wo + Qoo0oT:T | (74d)+ 0008‘ T (yu) 00000F : T (72) 000000T : T oulzeueydAyjour'g-ourure’ ,-oygdenourmeZ 
3 — 00008'T | = d — 00000T ‘I — 000002 ‘ T 
as + 00000z:1T | (74¢)+ QO0T'T (72) OOOOOF : 1 (yw) 000000T ?T  ePHopyoorgeut eurzuaeydiAqyeurp g'g-oururerp’ 1'Z 
5 = aon 1 | (72¢)— 0001? I — 00008 + T — 00008: 1 — 
ae) + 00008:T | (74¢)+ 000F' I + (Q0000T ‘ T + QOOOOL?T 7" eploryooujemt ourzenoyd]Ayyour g-ourmerp’ 1°Z 
Sq = 00008: T - é — 000002 : T — 00000T * T fepropqoouyeur 
3 + 000001‘ T +  OQ00T‘T + 00000 *T | + Q0000F * T ourzeuaydtAyyour'g-ourme: 1-ourmeqqeurtp'Z 
an — 00008‘ T — 00071 | 
r= + Q000P:T + QO00r:T | cesses ani coTpooyseut eurzeueydoyyydeaouiure °Z 
a ' 

as i a 
Ss (7dd)+ QO0T‘T (dd) + Gabe 27 | RRC OuE ORG jepmopqoorpAy ourzeaeydoysydeuonime'Z 
2 | | 
Fo Un.eg ‘roqem ouoqydeg “TUNG | royea ou0jydeg 
an | = 
oe) uInIpem :1700 "g | UINIpe :snawmp snovos0jhydnjzg ‘eouBysqng 

| 

ele 
= “UISIUSIO) 
a : é 
= panuyuor— TIT AQeL 


Antiseptic Action and Chemical Constitution. 343 


compared with 2.7.diamino-6.methylphenazine [85], suggests that methyl 
groups attached directly to the benzene rings may play a part in enhancing 
the antiseptic power in this series. For Staphylococcus awreus in serum 
the methochlorides of 2.dimethyl amino-7.amino-6.methylphenazine [84], 
2.7.diamino-3.6.dimethylphenazine [86], and 2.aminonaphtho-7.amino- 
6.methylphenazine [87] are powerful antiseptics, practically equal to the 
most potent of the acridine series; on the other hand, they are markedly 
inferior to the latter in their action on B. coli. 

It cannot be said that the behaviour of the phenazine series throws any 
clear light on the antiseptic properties of the diaminoacridine group. 


CHEMICAL SECTION, 

Amino Pyridine Derivatives |1.2.].—a-Amino pyridine was obtained by the 
method of Tschtschibabin and Leide.* It melted at 57-58° as stated by the 
authors of the process. By heating with a slight excess of acetic anhydride 
and some fused sodium acetate it was converted into the acetyl derivative 
which solidified on pouring the product into water. To purify it, it was 
dissolved in benzene filtered from sodium acetate and the benzene removed 
by distillation. It melted at 72°. The acetyl] derivative was warmed with 
methyl sulphate when the mixture heated spontaneously and on cooling 
solidified to a pasty crystalline mass, which was drained on a porous plate. 
The product, on boiling with cone. hydrochloric acid and evaporating, was not 
the methochloride as it contained no chlorine, but probably undecomposed 
methosulphate. When aminopyridine was heated in methyl alcohol solution 
with 4 molecules of methyl iodide in the water-bath in a sealed tube for 
6—7 hours, it was converted into the dimethylamino pyridine methiodide which 
separated on evaporating the methyl alcohol in brown crystals of the periodide. 
On dissolving the periodide in water iodine separated and the methiodide 
passed into the solution, which is colourless. On evaporation under 
diminished pressure the methiodide separated in fine, colourless needles which 
turned yellow in the air. On analysis the compound gave 50 per cent. of 
iodine (calculated for C;H;N (CHs)2CHsl, I = 48:1 per cent.). The constitu- 
tion is, therefore, represented by one of the following formule :— 


YAN YEN 
\ corn L bsccuecr 
N N 
off, 


* ‘J. S.C. I. Ann. Report,’ 1915, p. 863. 


344 Mr. C. H. Browning and others. Relationships between 


[4-11.] The Aminoquinolines and the Methochlorides.—The 0, m, p and a 
aminoquinolines were prepared from the corresponding nitroquinolines by 
reduction, the latter being obtained from the nitranilines by Knueppel’s 
modification of Skraup’s method* in which anhydrous arsenic acid is employed 
as oxidising agent. The m-nitraniline gives rise to two nitroquinolines, the 
meta and ana derivatives, which were separated by crystallisation from alcohol 
in which the ana-compound, m.p. 65°, dissolves more readily than the meta, 
m.p. 132-4°. In this way the meta-compound was obtained quite pure; but 
the ana-compound after repeated crystallisation melted at 48-50°. 

The reduction of the o- and p-nitroquinolines was effected by a modification 
of the process described by Knueppel with iron and hydrochloric acid. The 
product in the case of the ortho-compound was evaporated to dryness and the 
residue extracted with alcohol in which the hydrochloride dissolves. The 
solution was made alkaline and distilled in steam. The distillate was acidified 
with hydrochloric acid and evaporated to dryness. The base was separated 
by adding caustic soda and extracting with ether. On removing the ether, 
the base separated in almost colourless crystals, which after recrystallisation 
melted at 65°. In the case of the para-compound, the product after 
reduction was made alkaline and extracted with ether. It melted at 108°. 
The m- and a-compounds were reduced with tin and hydrochloric acid. 
Calculated quantities of the materials were introduced into a flask and heated 
for a short time in the water-bath until the reaction began when the flask 
was removed and if necessary cooled. The tin double salt of the meta-com- 
pound, which crystallises on cooling, was separated, decomposed with excess 
of alkali and extracted with ether. The product, crystallised from alcohol, 
melted at 188-190°. In the case of the ana-compound the double salt does 
not separate readily and the solution was, therefore, concentrated on the 
water-bath. The base was extracted with ether after the addition of alkali 
and on crystallisation from alcohol melted at 107°. The acetyl derivatives 
were prepared by boiling gently for } hour 1 grm. of the base with 1 grm. 
of fused sodium acetate and 4 c.c. of acetic anhydride. After cooling, water 
was added and then ammonia gradually until alkaline when the acetyl 
derivatives crystallised. They were recrystallised from dilute alcohol or 
water from which the m-, p- and a-compounds separated in colourless 
plates or flattened prisms; the o-compound had a faint yellow colour. The 
following are the melting points :— 


* ‘Ber.,’ vol. 29, p. 703 (1896) ; ‘ Annalen,’ vol. 310, p. 75 (1900). 


Antiseptic Action and Chemical Constitution. 345 
o-Acetyl aminoquinoline ......... 103° 
m- ” a9 Ff | i Nwimeielees ae 161° 
p- ”? at EIN) elelelote}s{erets 138° 
a- »” he hah ek MDODCCOOOG ice 


By boiling each of the above with a little conc. hydrochloric acid and 
concentrating the solution, the pure hydrochlorides crystallised with a brown 
or yellow colour and were filtered and washed with alcohol. 

The hydrochloride of the m-compound dissolves in water or alcohol with a 
bright green fluorescence. All the hydrochlorides combine with potassium 
cyanate and form orange, crystalline carbamido derivatives. 

The methochlorides were obtained from each of the acetyl derivatives by 
dissolving the substance in three to four times its weight of freshly distilled 
nitrobenzene and heating the solution to 150°. Rather more than the calculated 
quantity of dimethyl sulphate was then added and after a minute the mixture 
was removed from the bath. A portion of the methosulphate crystallised and 
the remainder was precipitated by adding ether. After standing fora time 
the nitrobenzene was removed as far as possible by washing with ether by 
decantation and then evaporating the ether by a current of air. A few cubic 
centimetres of conc. hydrochloric acid were added and the mixture boiled for 


' 4 hour. The solution was concentrated on the water-bath. The o- and a- 


compounds separated on cooling in brown crystalline crusts, the m- and p-com- 


- pounds were crystallised from the hydrochloric acid solution by the addition 


of alcohol and separated in bright yellow needles. The methochloride of the 
meta-compound dissolves in water with a green fluorescence like the hydro- 
chloride. The other three hydrochlorides and methochlorides exhibit no 
fluorescence. 

[14.13] 8. Hydroxyquinoline methopicrate and methochloride.—These sub- 
stances were prepared from the sulphate of the base (Chinosol) as follows :— 

The base was separated from a solution of the sulphate by adding ammonia, 
filtering and washing with water. It was then dried and heated for an hour 
with one part of fused sodium acetate and four parts of acetic anhydride. 
On adding water and a little ammonia until alkaline, the acetyl derivative 
crystallised in long colourless needles, m.p. 75-7° after recrystallisation from 
dilute alcohol. The substance was then dissolved in a little toluene and an 
equal weight of methyl sulphate and boiled for an hour when the yellow 
crystalline methosulphate separated. The toluene was decanted and the dry 
residue boiled with conc. hydrochloric acid. In this way a reddish solution 
was obtained from which the methochloride did not crystallise nor was the 
base precipitated by ammonia. One portion was, therefore, evaporated to 
dryness and formed a greenish-yellow solid which is very soluble in water and 


346 Mr. C. H. Browning and others. Relationships between 


could not be obtained in the crystalline form from other solvents. To a 
second portion picric acid was added which precipitated the picrate in yellow 
clusters of microscopic needles. 


ASIN 


Ge 


SANA 
OH N 


AS 
CH, Cl 
8. Hydroxyquinoline methochloride. 

[15.] Letrahydroquinoline was prepared according to the method of Hoffmann 
and Konigs,* by reduction with tin and hydrochloric acid. The hydrochloride 
crystallised from alcohol in colourless needles, m.p. 181—2°. 

[16.] Methyl tetrahydroquinoline methiodide.—Tetrahydroquinoline obtained 
from the hydrochloride (2 grm.) was mixed with 14 grm. of methyl iodide at 
the ordinary temperature. A clear pale orange solution was obtained, from 
which oily drops separated and gradually solidified toa crystalline mass. The 
product was warmed on the water-bath with reflux for about 1 hour, and left 
overnight at room temperature. The excess of methyl iodide was then driven 
off and the yellowish crystalline mass dissolved in water and filtered. To the 
filtrate ammonia was added until alkaline and the methyl tetrahydroquinoline 
extracted with ether. The aqueous solution was evaporated nearly to dryness 
on the water-bath and cooled. The solid residue was pressed on a porous 
plate and then extracted with alcohol. On the careful addition of ether the 
methiodide was precipitated in colourless needles, m.p. 171-2°. 


Methyl tetrahydroquinoline methiodide. 


[17.] «-Maphthoquinoline was prepared by Knueppel’s method,} applied to 
a-naphthylamine by Claus and Imhoff It was purified by crystallisation 
from petroleum ether and formed colourless needles, m.p. 45°. The metho- 
sulphate was prepared by dissolving the naphthoquinoline in benzene and 
adding an equal quantity of methyl sulphate and heating on the water-bath 
for a short time. Pale yellow needles of the quaternary compound separated 
which were filtered and dried. 

The methochloride was obtained by adding cone. hydrochloric acid and 


* ‘Ber.,’ vol. 16, p. 728 (1883). 
+ ‘Ber.,’ vol. 29, p. 703 (1896). 
t ‘J. prakt. Chem.,’ vol. 57, p. 68 (1898). 


Antiseptic Action and Chemical Constitution. 347 


boiling for a short time; on adding alcohol and cooling the compound 
crystallised in colourless needles 


CH,;0:80,0 1 
spines S/S NON 
ony) on 
bene ee: 
GV NN 
a-Naphthoquinoline a-Naphthoquinoline 
methosulphate. methochloride. 


[20. 21.] B-Naphthoquinoline was obtained by the method described by Claus 
and Besseler,* and purified by crystallisation from petroleum ether. It 
crystallises in pale yellow leaflets, m.p. 93°. It was converted into the metho- 
sulphate and chloride (as described under «-naphthoquinoline); cf. the following 
formule :— 


EN 
c VTE Fs ¢ 
ame <a, ee one 
SNF 
8-Naphthoquinoline 8-Naphthoquinoline 
methosulphate. methochloride. 


[22.] Tetrahydro-a-naphthoquinoline was obtained, as described by 
Bamberger,t by the reduction of «-naphthoquinoline with tin and hydro- 
chloric acid. The base was precipitated from ethereal solution by hydrogen 
chloride as a crystalline mass which was purified by re-crystallisation from 
alcohol acidified with hydrochloric acid. After being decolourised with animal 
charcoal the hydrochloride of the base was obtained in colourless needles, 
m.p. 257°-8°. 

[23.] Letrahydro-8-naphthoquinoline was prepared from the 8-compound, as 
above, in the form of colourless, lustrous leaflets, m.p. 231°. 

[24.] Diamino - 8 - naphthoquinoline.—Five germs. of 8 - naphthoquinoline, 
with 6 germs. potassium nitrate and 20 c.c. conc. sulphuric acid, were heated on 
the water-bath for 3 hours. The nitro-compound was poured on to ice and 
washed with cold water. It was then warmed on the water-bath with dilute 
ammonia, which dissolved a brown coloured substance. The product was 
filtered and dried and crystallised from nitrobenzene. It melted at 230°-245° 
On a second crystallisation it melted’ at 245°-247°. On analysis, 


0:16 grm. gave 21°5 c.c. moist N at 18° and 7475 mm. N = 1511 per cent. 
Calculated for Ci3H;N(NO2)2; N = 15:5 per cent. 


* ‘J. prakt. Chem.,’ vol. 57, p. 49 (1898). 
+ ‘Ber.,’ vol. 24, p. 2475 (1891). 


348 Mr. C. H. Browning and others. Relationships between 


The dinitro-compound was reduced with tin and hydrochloric acid. On 
cooling the double stannic chloride salt crystallised and was filtered, dissolved 
in water, and the tin precipitated by hydrogen sulphide. From the filtrate 
the base was precipitated by sodium hydroxide. It formed pale yellow plates, 
m.p. 250°. It is very unstable and oxidises in the air. 

[26.] 1.4.Naphtho - dipyridine (Benzophenanthroline) was prepared from 
1.4 naphthylene diamine by Karrer’s method.* The diamine was obtained 
by the reduction of benzene-azo-«-naphthylamine with zinc dust and acetic 
acid; 14°35 grm. of «-naphthylamine gave 21°6 grm. of naphthylenediamine 
sulphate, from which the base was obtained, m.p. 118°. The naphtho- 
dipyridine was purified by crystallisation from a mixture of benzene and 
petroleum ether and separated in long, pale yellow needles, m.p. 160°-164°. 


N/ 


| 
TOU 
Wx 


NA 
1.4, Naphtho-dipyridine. 

[27.] 1.5. Naphtho-dipyridine—The 1.5.naphthylenediamine was prepared 
by the reduction of 1.5.dinitro-naphthalene with stannous chloride and 
hydrochloric acid. The naphtho-dipyridine compound was obtained, as 
previously described, and crystallised from benzene in nearly colourless 
needles, m.p. 214°-215°. It combines with methyl sulphate in hot benzene 
solution, from which yellow needles separated on cooling. 


me Ne 
Ve 
1.5. Naphtho-dipyridine. 

[28.] 1.1.Dinaphthyl.2 2-imine was prepared by the method described by 
Meisenheimer and Wittef from 8-naphthylamine. The latter was converted 
into azonaphthalene which was reduced with zine dust and acetic acid. In 
this way 2.2.diamino-1.1.dinaphthyl was obtained, m.p. 191°; 2 grms. of the 
diamino-dinaphthyl hydrochloride were heated in the oil-bath to 240°-250° 


* Marckwald, ‘ Annalen,’ vol. 274, p. 365 (1893). 
+ ‘Ber.,’ vol. 36, p. 4154 (1903). 


Antiseptic Action and Chemical Constitution. 349 


for 5 minutes. The cold fused mass was extracted with alcohol, poured into 
water, and the colourless precipitate filtered. The yield is nearly theoretical, 
and the product melted at 157°. 


per ae Ae OO 
> 
SANG YY VA se oe 
Diamino dinaphthy]l. Dinaphthylimine. 


Acridine Derivatives: [30] Acridine Methochloride.—Acridine base was 
precipitated from pure acridine hydrochloride by the addition of ammonia, 
filtered and dried: 1-5 grms. was dissolved in 7 grms. of nitrobenzene, heated to 
150°, and 1:7 germs. of methyl sulphate added, and, after 1 minute, cooled. 
Yellow prisms of the methosulphate separated and were filtered and washed 
free from nitrobenzene with ether. The methosulphate is soluble in water, 
and from the solution the methoxide was precipitated with ammonia and 
erystallised from alcohol in which it is much less soluble than acridine. It 
erystallises in colourless plates, m.p. 140°. It dissolves readily in hydro- 
chloric acid and the methochloride crystallises on concentration. It may be 
re-crystallised from alcohol and ether. It is very soluble in water, to which 
it imparts a green fluorescence. 

[32.] 9-Phenylacridine methochloride.—9-Phenylacridine was prepared by 
the method of Bernthsen*. 50 grms. of benzoic acid and 70 grms. of diphenyl- 
amine gave 35 grms. of phenylacridine, m.p.183°. To convert it into the 
methochloride the above method was adopted. The methosulphate does not 
separate on cooling, but on adding ether a brown oil was precipitated, which 
soon solidified. The ether solution was decanted and the methosulphate 
washed with ether. Concentrated hydrochloric acid was then added and the 
mixture boiled gently for a short time, when, on cooling, the methochloride 
separated in green leaflets, which readily dissolve in water with a yellow 
colour. 

[33.] 3.6.Dimethylacridine was prepared according to Ullmann’s method.t 
It crystallises from dilute alcohol in yellow needles, m.p. 171°, which fluoresce 
in solution with a blue colour. 

The action of ethyl iodide on acriflavine in ethyl alcohol was examined with 
the object of obtaining the tetraethyl derivative. The mixture heated to 
100° for 6 hours in a sealed tube gave a red crystalline product, which 
appeared to be a periodide. Its antiseptic action was inferior to that of 
acriflavine and it was not further investigated. It is recorded in the Table as 
acriflavine +C2H;I. 


* ¢ Annalen,’ vol. 224, p. 13 (1884). 
+ ‘Ber.,’ vol. 36, p. 1017 (1903). 


350 Mr. C. H. Browning and others. Relationships between 


[36-43.] 2.7.Diaminoacridine Alkyl Chlorides.—In order to study the 
relative antiseptic action of the quaternary alkyl derivatives of diamino- 
acridine in addition to the methochloride (acriflavine), the ethyl, propyl, 
n-butyl, isobutyl, isoamyl, phenyl, and benzyl chlorides were prepared, having 


the following general formula :— 
CH 
NG NTS 


HN A /\ /Sis HC. 


UN 
Alk Cl 


The alkyl chlorides were obtained by the following method: A weighed 
quantity of sodium was dissolved in the alcohol corresponding to the required 
alkyl compound, and the theoretical amount of p-toluene sulphonic chloride 
added. After standing for a time, the product was shaken two or three times 
with water. The washed product was dissolved in ether, dried over calcium 
chloride, the ether distilled, and the residue heated im vacuo on the water- 
bath. In this way the alkyl p-toluene sulphonic esters were obtained in the 
form of pale yellow oils. 

CH3-CgH4-SO2C14+ RONa = CH3-CgH4-SO3R + NaCl. 


About 1 grm. of each ester was allowed to react with an equivalent weight 
of diacetyldiaminoacridine in nitrobenzene solution at 150°-160° The 
product in each case was hydrolysed with cone. hydrochloric acid, whereby 
the alkyl chloride was obtained. 

The benzyl chloride compound, which was prepared in a similar way, was a 
reddish brown crystalline substance, and on analysis 


0:152 grm. gave 0132 grm. AgCl. Cl = 216 per cent. Calculated for 
C2)Hi;N3Cl2; Cl = 19:2 per cent. 


The phenyl chloride compound was obtained by dissolving phenol in the 
calculated quantity of sodium hydroxide solution to form sodium phenate. 
The equivalent amount of p-toluene sulphonic chloride was then added and 
the mixture boiled for about half an hour, and cooled. The phenyl p-toluene 
sulphonic ester which separated was recrystallised from alcohol and melted 
at 95°. It was transformed into the acridine phenochloride in the following 
way: About 10 per cent. more than the theoretical amount of the ester 
was added to the acetyl diaminoacridine dissolved in nitrobenzene and heated 
for 5 minutes to 150°-160°. The product was cooled to 105°, when 5 c.c. of 
cone. hydrochloric acid was added and the mixture maintained at 100° for 
another 5 minutes and cooled to the ordinary temperature. The nitro- 


Antiseptic Action and Chemical Constitution. 351 


benzene was decanted from the phenochloride, which separated, and the 
latter boiled with a little more conc. hydrochloric acid, cooled, and the 
crystalline product filtered, washed with conc. hydrochloric acid, and finally 
with ether. 

[44.] 2.7.Diaminoacridine Chloroacetate—About 1 grm. of diacetyl 
diaminoacridine was dissolved in about 50 c.c. of nitrobenzene and heated in 
a metal bath to 140°-150°. An equivalent amount of chloracetic acid was 
added and maintained at 140°-150° for about 10 minutes. On cooling, 
yellow crystals separated, which were filtered and washed with ether. The 
substance was readily soluble in water. On analysis, 


01485 grm. gave 0:055 grm. AgCl; Cl=9-0 per cent. Calculated for 
Cy9Hy,N30,C1 ; Cl Oy per cent. 


On boiling the diacetyl derivative with conc. hydrochloric acid, red 
erystals were obtained, which were purified by dissolving in water and 
reprecipitating with conc. hydrochloric acid. 

On analysis: 


0-141 grm. gave 0127 grm. AgCl; Cl = 22:2 per cent. Calculated for 
Cy5Hy3N302Cl2; Cl = 20°9 per cent. 


OH CH 
VN GOR 

CH;-CO- ON ae-coce, mal) ee, 
N N 
yo Vio 
COOH-CH, Cl COOH-CH, (1 


On addition of ammonia it was converted into the betaine 
CH 


¥ 
HCC YO 
co 


[46.] 2.7.Diaminoacridine Chloracetanilide was prepared by adding an 
equivalent of chloracetanilide to diacetyl diaminoacridine in nitrobenzene 
(1 grm. of the diacetyl derivative in 50° c.c. of nitrobenzene), heated to 
140°-150° and maintained for 5 to 10 minutes. On cooling, an amorphous 
brown precipitate separated, which was filtered, washed with ether, and 
boiled with conc. hydrochloric acid. Brown crystals were obtained and 
were purified by dissolving them in water, and reprecipitating with conc. 
hydrochloric acid. 


352 Mr. C. H. Browning and others. Relationships between 


On analysis: 


0130 grm. gave 14:5 cc. N at 15° and 757 mm. N= 13 per cent. 
Calculated for CoHigNsOCl,; N = 13:4 per cent. 


The compound has therefore the following formula :— 
CH 
PANY ONIN 


Sa a ia 


JEN 
C,H;NH.CO.CH, Cl 


[45.] 2°7.Diaminoacridine Chloropropionate was prepared like the chlor- 
acetate and was purified by crystallisation from concentrated hydrochloric 
acid. 

[48.] 2.7.Diamino-3.6.dimethylacridine (Acridine yellow) was prepared 
according to the method of Ullmann and Naef* and Ullmann and Marie.+ 
To purify it, it was ground while moist and warmed on the water-bath with 
sufficient sodium hydroxide to render the mixture alkaline ; after cooling it 
was filtered, washed and pressed down. The moist precipitate was then 
dissolved ina small quantity of glacial acetic acid and whilst hot a little 
cone. hydrochloric added gradually until the solution changed to a deep red, 
when water was added till turbid and cooled. The hydrochloride separates in 
fine prismatic, deep orange or red crystals which dissolve in water with a green 
fluorescence. 

[49.] 2.7. Diamino-3.6.dimethylacridine methochloride—The hydrochloride 
prepared as above was precipitated as the base with sodium hydroxide, 
filtered, washed and thoroughly dried in a vacuum desiccator and then 
converted into the diacetyl derivative. 3°6 grms. of the base were mixed with 
13 grms. of acetic anhydride in a small flask furnished with an air condenser 
and boiled gently for } hour. The substance gradually dissolved. It was 
diluted with water, and to the cooled solution just sufficient ammonia was 
added to decompose the anhydride and precipitate the acetyl derivative. 
After filtering, washing and drying it was extracted with a little absolute 
alcohol, which removes a small quantity of a pale yellow substance. 1 grm. 
of the acetyl derivative was dissolved in 10 grms. of nitrobenzene, heated to 
170° and 08 grm. of methyl sulphate added. On cooling, the mixture 
solidifies to a crystalline mass which was filtered and washed free from nitro- 
benzene with ether. The crystalline residue was boiled with conc. hydro- 
chloric acid for 4 hour, when nearly the whole dissolved with a deep red 


* ‘Ber.,’ vol. 33, p. 913 (1900). 
+ ‘Ber.,’ vol. 34, p. 4808 (1908). 


Antiseptic Action and Chemical Constitution. 353 


colour. It was filtered and cooled. The methochloride separates in brown, 
glistening plates, which dissolve in water but are less soluble in dilute hydro- 
chloric acid. It dissolves in alcohol with green fluorescence. 

[67.] 2.7. Dihydroxy-3.6.dimethyl acridine-—The compound was obtained 
from tetramethyl diamino ditolylmethane, by the method of Ullmann and 
Fitzenkam* 


CH 


A bo 
It was converted into the diacetyl derivative by boiling with acetic anhydride 
and fused sodium acetate. 

[68.] 2.7. Dihydroxy-3.6.dimethyl acridine methochloride.—The acetyl deriva- 
tive was dissolved in toluene and to the solution an excess of methyl sulphate 
was added and the mixture boiled. On standing, orange needles separated and 
were filtered and washed with toluene. On heating the substance with cone. 
hydrochloric acid on the water-bath, it first dissolved and then suddenly 
formed a pasty mass of clusters of lemon yellow needles of the methochloride. 
The crystals were filtered and washed with cold water and then with alcohol. 
In the latter it dissolves slightly with green fluorescence. It dissolves to 
some extent also in hot water with a yellow colour, and is readily soluble in 
ammonia and sodium hydroxide solutions. 

[50.] 2.7.Diamino-3.6.dimethyl-9.phenyl acridine (Benzoflavine) was prepared 
according to the method of Meyer and Gross.+ It was purified by pre- 
cipitating the base with ammonia, dissolving the filtered, washed and well- 
pressed base in glacial acetic acid, adding cone. hydrochloric acid and diluting 
until a permanent turbidity was formed. The hydrochloride separates in red 
crystals. 

[51.] 2.7. Diamino-3.6.dimethyl.9-phenyl acridine methochloride was obtained 
by the method employed in the case of acridine yellow by heating the dry 
base with acetic anhydride and methylating the acetyl derivative with methyl 
sulphate. On hydrolysis of the methosulphate with hydrochloric acid the 
methochloride separates in bright red needles. In the absence of hydro- 
chloric acid, it dissolves in water and on cooling gelatinises. 

[52.] 2.7.Tetramethyl dianvino-acridine (Acridine orange) was obtained by 
the method of Behringerf and Ullmann and Marie.§ Tetramethyl-diamino 


* ‘Ber.,’ vol. 38, p. 3787 (1905). 
+ ‘Ber., vol. 32, p. 2356 (1899). 
t ‘J. prakt. Chem.,’ vol. 54, p. 240 (1896). 
S ‘Ber.,’ voi. 34, p. 4307 (1901). 
VOL. XCIII.—B. 


bo 
Q 


354 Mr. C. H. Browning and others. Relationships between 


diphenylmethane, prepared by the action of formaldehyde on dimethyl aniline 
in presence of dilute sulphuric acid was nitrated in the cold with potassium 
nitrate and conc. sulphuric acid and the purified dinitro-derivate, mp. 141°—2°, 
reduced with stannous chloride. After reduction the base m.p. 141°-2° 
was precipitated with caustic soda and extracted from the stannous 
hydroxide with aleohol. It was converted into the acridine compound m.p. 
181°-2° as described under tetraethyldiamino acridine (see below) and the 
latter into the methosulphate. From the solution of the methosulphate 
potassium nitrate precipitates the methonitrate in red needles from which the 
methoxide was obtained as a hygroscopic mass on the addition ot ammonia. 
The methochloride is too soluble and hygroscopic to be separated, and closely 
resembles the corresponding ethyl derivative described below. 

[55.] 2.7. letraethyldiamino-acridine was prepared by the above method 
using diethylaniline in place of dimethylaniline. The tetraethyldiamino 
diphenyl methane is a colourless liquid, which boils at 285°-290° at 10 mm. 
and 305°-310° at 20 mm. pressure. The yield from 100 grms, of diethyl- 
aniline was 50 grm. of the diphenylmethane derivative. On nitration it 
formed a dinitro derivative which crystallised from acetic acid in orange red 
plates m.p. 118°-119°. The latter was reduced with tin and hydrochloric acid 
and the tin partly removed by hydrogen sulphide. The filtered solution on 
evaporation left a colourless, resinous mass of the hydrochloride which 
contained some tin salt. 2 grms. of the hydrochloride were heated with 
4 c.c. of cone. hydrochloric acid diluted with 12 c.c. of water at 140° for 
6 hours in a sealed tube. The red coloured contents of the tube were 
extracted with boiling water in which they dissolved leaving very little 
residue, and to the hot solution ferric chloride solution was added until no 
more precipitate was formed. After the addition of a solution of common 
salt, which throws down a further quantity of the acridine compound, the 
mixture was filtered after cooling and washed with salt solution. The deep 
red solution was made alkaline with ammonia which precipitates the yellow 
base. The latter was filtered and washed and dissolved in hydrochloric acid. 
On concentrating the solution, the hydrochloride crystallises in red crusts 
with a green iridescence. The crystals dissolve in water with a bright orange 
colour. 

[56. 57.] 2.7. Tetraethyldiamino-acridine methochloride and methonitrate.— 
To prepare the methonitrate an excess of dimethyl sulphate was added to the 
finely powdered base in a small basin. The mixture becomes hot. After 
heating for a time on the water-bath, the methosulphate crystallises. Water 
was then added, and heating continued until the excess of methyl sulphate 
was decomposed and a clear red solution obtained. On the addition of a 


Antiseptic Action and Chemical Constitution. 355 


saturated solution of potassium nitrate, the methonitrate crystallised on 
standing in red needles. They were filtered, washed with a little water, and 
dried. On grinding with ammonia solntion the methoxide was obtained as a 
sticky red mass with green iridescence which dissolves in hydrochloric acid 
forming the methochloride as a hygroscopic mass which could not be prepared 
in the crystalline state. 

[58.] 2.Amino - 3.methylnaphthacridine was prepared according to the 
directions of Ullmann and Naef.* The base, after re-crystallisation from 
xylene, melts at 244°. It was acetylated, as described, and the acetyl 
derivative melted at: 320°-321°. 

[59.] 2.Amino - 3.methylnaphthacridine methochloride—06 grm. of the 
acetyl derivative was dissolved in 2 cc. of nitrobenzene heated to 160°, and 
0°3 erm. of dimethyl sulphate added when the mixture crystallised to a semi- 
solid mass, which was filtered and washed with ether. The product, after 
boiling with conc. hydrochloric acid, became dark red, and on concentrating 
the solution on the water-bath and cooling the methochloride crystallised in 
long red needles. 

YIN 
CH 
Glee NaN 


FCLHN ‘A, Dx, 


YON 
CH, Cl 


[60.] 2. Amino - 3.methyl - 9.phenylnaphthacridine. — The substance was 
prepared by adding 5 grms. of tetramino-ditolylphenyl methane (Meyer and 
Gross),f to 8 grms. of 8-naphthol at 150° and heating for an hour to 180°—200°- 
The sticky product was dissolved in 10 c.c. of glacial acetic acid, diluted with 
an equal volume of water, and poured into 10 grm. of caustic soda in 50 e.c. 
of water. The yellow precipitate was filtered and washed with dilute caustic 
soda solution and hot water to remove the naphthol, and the product 
erystallised from alcohol. The undissolved portion was filtered off. It 
erystallises from nitrobenzene and also from benzene in yellowish brown 
needles, m.p. 264°-265°. The alcoholic filtrate was heated to boiling, water 
added till turbid, and allowed to cool. The crystalline product was re- 


crystallised from benzene from which it separates in yellow needles, m.p. 
269°-271°. 


* ‘Ber.,’ vol. 33, pp. 915, 2473 (1900). 
+ ‘ Ber.,’ vol. 32, p. 2356 (1899). 


bo 
Q 
bo 


356 Mr. C. H. Browning and others. Relationships between 


The compound of m.p. 264°-265° on analysis 


0:2026 grm. gave 14:3 ¢.c. moist N at 14° and 725mm. N = 8:37 per 
cent. 

01960 grm. gave 13:7 c.c. moist N at 16° and 753 mm. N = 824 per 
cent. Calculated for Co,HisN2; N = 84 per cent. 


The compound has therefore the formula : 


TEN, 

It was converted into the methosulphate by dissolving in eight times the 
weight of nitrobenzene at 130° and adding half the weight of methylsulphate. 
The crystalline product was filtered and washed with ether. 

[62. 63.] 2.Dimethylaminonaphthacridine and methochloride.— 


JX 
CH 
ale 


It was prepared by the method of Ullmann and Marie,* by fusing tetra- 
methyldiaminodiphenyl methane with @-naphthol at 110°-120°, and raising the 
temperature gradually to 180°-200°. The product was extracted with warm 
alcohol which dissolves the naphthacridine compound, but leaves the hydra- 
eridine. The hydracridine compound was suspended in boiling alcohol, 
acidified with a few drops of hydrochloric acid and ferric chloride added until 
the precipitation of the naphthacridine hydrochloride was complete. The 
precipitate was filtered, dissolved in water, and re-precipitated with cone. 
hydrochloric acid. It was filtered and dried in vacwo over caustic soda. The 
base was precipitated from the dissolved hydrochloride with ammonia, and 
after re-crystallisation from benzene melted at 185°. It was dissolved in 
boiling xylene and the calculated amount of methyl sulphate added. The 
precipitated methosulphate was washed with ether and dried; on dissolving 
it in water and adding sodium chloride solution the methochloride was 
precipitated, filtered, and dried. To remove the sodium chloride the metho- 
chloride was dissolved in alcohol, filtered, and evaporated to dryness. The 
naphthacridine base was obtained from the first alcoholic filtrate by adding a 


* Ber.,’ vol. 34, p. 4318 (1901). 


Antiseptic Action and Chemical Constitution. 357 


boiling alcoholic solution of picric acid. The picrate which crystallised was 

filtered and re-crystallised from boiling aniline. To the picrate suspended in 

alcohol, caustic soda was added and warmed on the water-bath until the 

solution was deep yellow, and then diluted. The precipitated base was 

filtered, washed with water, dried and re-crystallised from benzene (m.p. 185°). 
[65.] 2.7.Diamino-9.phenylacridine carboxylic ester.— 


He 


\ /600G:Hs 


| 
VIN ANON 


LNT 


The substance was prepared by heating with ammonia in sealed tubes 
according to the method of Meyer and Oppelt.* The product was ‘boiled 
with alcohol to remove impurities and the residue suspended in alcohol and 
hydrogen chloride passed in. The alcohol was removed, the hydrochloride 
dissolved in water and precipitated with salt, redissolved and again salted 
out. The product is an orange amorphous powder. 

[66.] 2.7.2etramethyldiamino-9.phenylacridine carboxylic ester. 


| ace 


| 
7 SHINY 


COHN A N/M: 


One molecular proportion of phthalic anhydride was heated with three of 
acetic anhydride and two of m-aminodimethylaniline for 2-3 hours at 
140°-150°. The acetic acid was then distilled off and the residue boiled with 
fifteen to twenty parts of 20 per cent. hydrochloric acid for } hour. From 
the deep red solution the base was precipitated by ammonia, filtered and dried 
in vacuo. It was suspended in ten times its weight of absolute alcohol, 
heated an hour with reflux on the water-bath whilst dry hydrogen chloride 
was passed in. The alcohol was removed, the residue dissolved in hot water, 
filtered and the filtrate salted out. The precipitate was filtered, washed with 
a little cold water and dried. 


* ‘Ber.,’ vol. 21, p. 3376 (1888). 


358 Mr. C. H. Browning and others. Relationships between 


PHENAZINE DERIVATIVES. 


[69-71.] Phenazine was prepared by the method of Kehrmann and Havas* 
by heating a mixture of o-amino- and o-nitro-diphenylamine with fused - 
sodium acetate. It melts at 170°—171°. The methosulphate and metho- 
chloride were obtained in the same manner as the corresponding acridine 
compounds. The phenazine was dissolved in five times its weight of nitro- 
benzene, heated to 120°, and freshly distilled methyl sulphate equal in weight 
to the phenazine was added, the mixture stirred and kept for 5 minutes at 
100°-110° and cooled. The methosulphate separates; ether was added to 
complete the precipitation and the product filtered and washed. It forms 
greenish yellow prisms, The methochloride was prepared by first separating 
the base with ammonia and evaporating the red solution, taking up with 
alcohol to remove ammonium sulphate and then evaporating to dryness. The 
methochloride was prepared by dissolving the base in hydrochloric acid and 
concentrating the solution. It forms greenish crystals. 

[72.] 2.Aminophenazine was prepared by (A) the method described by 
Wohl and Lange,} from o-nitraniline and aniline-hydrochloride in presence 
of fused zinc chloride. The amino-phenazine was separated from the 
product by sublimation and crystallises in brilliant red needles melting at 
283°, which after crystallisation melt at 288°. The acetyl derivative was 
obtained by heating for a short time with an equal weight of fused sodium 
acetate, and twelve to fifteen times its weight of acetic anhydride. When 
poured into water and neutralised with ammonia it formed a buff-coloured 
crystalline powder. The dry acetyl derivative was dissolved in ten times its 
weight of nitrobenzene heated to 120° and methyl sulphate, equal in weight 
to the acetyl derivative, added and the mixture cooled slowly. Ether 
precipitates the methosulphate, which was filtered and washed with ether. 
It was then boiled with water to remove nitrobenzene and filtered. About 
an equal volume of conc. hydrochloric acid was added and evaporated to 
dryness. The methochloride, which is very soluble, was dissolved in alcohol, 
filtered and evaporated. It forms a red, crystalline residue which dissolves 
in water and alcohol with a bright magenta colour. 

(B) A second process for preparing aminophenazine is to pass dry 
ammonia into an alcoholic solution of phenazine methyl sulphate according 
to the method of Kehrmann and Havas.{ The products in the two cases 
appeared to be identical and to have identical bactericidal properties. 

(74.] 2.3.Diaminophenazine was prepared by the method of Ullmann 

* ¢Ber.,’ vol. 46, p. 341 (1913). 
+ ‘Ber.,’ vol. 43, p. 2186 (1910). 
t ‘Ber., vol. 46, p. 481 (1918). 


Re On ae ae ee? ee or 


Antiseptic Action and Chemical Constitution. 359 


and Mauther,* and converted into the acetyl derivative.t It was recrystal- 
lised from nitrobenzene and forms a light brown micro-crystalline powder 
which turns brown at 206° and melts about 270°. The latter was dissolved 
in ten times its weight of nitrobenzene at 150°, and 1 mol. of methyl 
sulphate added. The methosulphate, which was precipitated, was washed with 
ether and the product boiled with cone. hydrochloric acid and evaporated on 
the water-bath. The methochloride crystallised in black needles which were 
filtered and dried. 

[76.] 2.Dimethylamino-7.aminophenazine methochloride. —The compound 
was prepared according to the method described by Karrer,j from a 
mixture of para- and meta-dimethyl phenylene diamine by oxidation 
with potassium dichromate. The phenazine salt was precipitated by zinc 
and sodium chloride and the base separated from the solution in hydro- 
chlorie acid by sodium hydroxide. It was purified by redissolving in acetic 
acid and precipitating with ammonia. It forms a blue-black powder slightly 
soluble in alcohol with a deep violet colour, and readily soluble in dilute 
acids giving a violet solution and forming the hydrochloride 


N 
Y. NT NTN. 


coms Be peat 


TN 
CH, Cl 


[77.] 2.Dimethylamino-T.amino-6.methylphenazine hydrochloride (Toluylene 
red).—The substance was obtained by following the directions of Witt.§ It 
was purified by crystallisation from alcohol. The base was obtained by 
precipitation with sodium hydroxide, and was washed, dried and acetylated in 
the usual way. The acetyl derivative crystallises in brown plates. The 
methiodide was obtained in microscopic black crystals which dissolve in water 
and alcohol with a deep violet colour, by heating the acetyl derivative with 
methyl iodide in a sealed tube for several hours to 100°. 

[84.] 2.Dimethylamino-T-amano-6.methylphenazine  methochloride. — The 
above acetyl derivative was dissolved in five times its weight of nitrobenzene 
heated to 160°, and rather more than the calculated weight of methyl sulphate 
added. After a minute or two the mixture was cooled and the resulting 
black precipitate washed with ether and dried. The product was then 
hydrolysed with cone. hydrochloric acid, when the liquid became deep 

* © Ber.,’ vol. 35, p. 4802 (1902). 
+ Fischer and Hepp, ‘ Ber.,’ vol. 22, p. 358 (1889). 


{ ‘Ber.,’ vol. 49, p. 1643 (1916) ; vol. 50, p. 420 (1917). 
§ ‘Ber.,’ vol. 12, p. 921 (1879). 


360 Mr. C. H. Browning and others. Relationships between 


blue. On adding water or salt solution, the methochloride separated in the 
form of a semi-solid iridescent green mass, which after drying became hard 
and was crystallised from a mixture of benzene and alcohol in the form of a 
micro-crystalline powder with a dark green lustre. It dissolves in water and 
alcohol with a bright magenta colour, similar to toluylene red. On analysis, 
found N = 17-0 per cent.; CH2)N4Cl. requires N = 16°5 per cent. 

The formula is therefore 1: 


. 
UN 


vo 
CH, Ol 


Another method for the preparation is given in D.R. Patent 69188.* It 
consists in heating together 3 mols. p-nitrosodimethylaniline hydrochloride 
and 2 mols. o-aminodimethyl-p-toluidine in 50 per cent. acetic acid solution 
for 6 hours. On dilution, filtration, and precipitation with common salt, a green 
crystalline colouring matter was precipitated, which was filtered and, on account 
of its solubility in water, washed with brine. To the mother liquors, which still 
contained a quantity of the dye zine chloride was added, and the zine chloride 
double salt precipitated which was re-crystallised from dilute hydrochloric 
acid. The substance dissolved with a distinctly magenta colour. There 
appears, therefore, to be a graduation in tint from scarlet to magenta with the 
increase of alkyl radicals in the amino group (compare [86] and [88]). The 
same development of blue in the colour is observed in rosaniline and its 
methyl derivatives which change from magenta to violet. 

Lromination of Acetyl Derivative of Toluylene Red.—The acetyl derivative 
was brominated in chloroform solution, when a dark violet solution was formed, 
which on evaporation gave a dark violet product, which crystallised from 
a mixture of alcohol and ether in microscopic black prisms. The substance 
is, however, a mixture, which can be separated by extracting with ethyl 
acetate. The undissolved portion was dissolved in water and the base preci- 
pitated with ammonia as an oil which solidified on standing. It crystallises 
from alcohol in pale brown transparent prisms, m.p. 205°-207°. It was 
tested qualitatively for bromine, but its further investigation was postponed. 

[78.] 2.Dimethylamino-6.methylphenazine-—Toluylene red was diazotised 
according to Witt’s method (Joc. cit.) in absolute alcohol with sodium nitrite 
and hydrochloric acid. The excess of alcohol was then removed on the 
water-bath, the liquid filtered and poured into a solution of sodium acetate, 


* © Friedlander,’ vol. 3, p. 397. 


Antiseptic Action and Chenucal Constitution. 361 


which throws down the phenazine compound in the form of a crystalline preci- 
pitate having a bronzy iridescence. 

It was recrystallised from dilute alcohol and melted at 168°-169°. The 
ether and benzene solutions are fluorescent, but fluoresce with different colours, 
the former having an orange and the latter a greenish colour. © 

[79.] 2.Dimethylamino-6.methylphenazine methiodide was prepared from the 
phenazine compound by heating with rather more than the calculated 
quantity of methyl iodide in a sealed tube to 100° for 6 hours. A solid dark : 
violet mass resulted, which dissolved in water with a deep violet colour. The 
substance was recrystallised from a mixture of alcohol and ether, from which 
_ it separated in brownish black plates. On analysis, 


(2525 grm. gave 0:1718 grm. AgI; I = 368 per cent. 
0:2540 grm. gave 0:1714 grm. Agl; I= 37-0 per cent. C;HisN3+ CHsl 
requires I = 33:5 per cent. 


The iodine value found is too high, the reason for which is not clear unless, 
as often happens in such cases, a certain amount of periodide is formed at 
the same time. 

[80, 81.] 2.7.Letramethyldiaminophenazine is described by Karrer* as 
being obtained by oxidising a mixture of dimethyl-p-phenylenediamine and 
dimethyl-m-phenylenediamine with potassium dichromate solution in presence 
of hydrochloric acid. The product may have either of the following 
formulee :— 


7 N 
UNAS /\7\/NN(CH,)3 
HC1(GH;)2N\ UK a Jira TELE CML) pb IN 
‘ N 


It was separated in the form of the hydriodide, from which the base was 
precipitated with ammonia, filtered, washed, and extracted with alcohol. The 
residue was dissolved in dilute hydrochloric acid and the solution was 
evaporated in a vacuum desiccator to dryness. The methochloride was 
prepared in the usual way by the action of methyl sulphate on the base, 
dissolved in nitrobenzene and subsequent hydrolysis with conc. hydro- 
chloric acid. It dissolves in water with a bluish red colour. 

[82, 83.] 2. Aminonaphthophenazine was prepared, according to the method 
described by O. Fischer and Hepp, by heating in a sealed tnbe for 5 to 6 hours 
at 160° one molecule o-phenylenediamine and one molecule benzene-azo-a- 
naphthylamine hydrochloride with ten parts of alcohol. On cooling, dark red 


* ©Ber.,’ vol. 49, p. 1643 (1916). 
+ ‘Ber., vol. 23, p. 845 (1890). 


362 Mr. C. H. Browning and others. Relationships between 


crystals separated. They were filtered and crystallised from alcohol contain- 
ing hydrochloric acid. The substance obtained in this way is the hydro- 
chloride of aminonaphthophenazine. It forms dark red crystals, which are 
slightly soluble in water, but on heating are hydrolysed and the base is 
precipitated. ‘The acetyl derivative was prepared by heating the base with 
acetic anhydride and fused sodium acetate. On cooling, the acetyl derivative 
crystallises. It was heated to 160° with ten to twenty times its weight of 
nitrobenzene and the equivalent of one molecule of methyl sulphate added. 
On standing, crystals of the metho-sulphate separated. They were filtered 
aud washed with ether and heated with cone. hydrochloric acid, when the 
hydrochloride of the methochloride slowly crystallised. The substance is 
soluble in water and not so readily hydrolysed as the hydrochloride. 


TaN 
N 
Yn. ESN 


| hema 
Vives Bie 
ZN 
CH; Cl 
2. Amino-naphtho phenazine 
methochloride. 


[85.] 2.7.Diamino-6.methylphenazine methochloride was prepared according 
to D.R. Patent 86608* by heating together and stirring amino-azobenzene 
hydrochloride, amino dimethy]-p-toluidine and glycerol at 110°, according to 
the proportionate amounts given. The mixture froths up during the process, 
aud the reaction is at an end when frothing ceases (about 3 hours). The 
solid product was dissolved in hot water and precipitated as hydrochloride by 
the addition of hydrochloric acid and some common salt. The colouring 
matter was re-crystallised from dilute hydrochloric acid, giving green, glistening 
erystals, which dissolved in water with a scarlet colour. 


N 
/\4A\/\oH, 


EEN NAINA 
EX 
cH, Cl 


NH,HCl. 


[86.] 2.7. Diamino-3.6.dimethylphenazine methochloride was prepared as above, 
using o-amino azotoluene hydrochloride in place of the benzene derivative. 
The product was dissolved in hot water and precipitated with hydrochloric 
acid. On cooling the phenazine compound separated in green prismatic 


* © Wriedlander,’ vol. 4, p. 380. 


Antiseptic Action and Chenical Constitution. 363 
needles, very slightly soluble in cold water, soluble in hot water with a scarlet 
colour. 

N 
cH, 7 VY ‘cH, 


y ms ee ‘HCl. 


ZA NS 

cH, Cl 
[87.] 2.Aminonaphtho-7.amino-6.methylphenazine methochloride—The pre- 
paration was carried out as above, using proportionate quantities of 
benzene-azonaphthylamine hydrochloride and one-and-a-half times the 
amount of glycerol, and heating at 120° for 34 hours. The hot aqueous 
solution of the product was filtered from insoluble matter, and the colouring 
matter precipitated with hydrochloric acid and salt. It was re-crystallised 
from dilute hydrochloric acid from which it separated in small green crystals. 

It dissolves in water with a scarlet colour similar in tint to the other two. 


( ] 
at NYS 0. 
A SS 
CH, Cl 


[88.] 2.Methylamino-7.amino-3.6.dimethylphenazine methochloride-—The sub- 
stance was prepared from nitrosomethylaniline hydrochloride and o-amino- 
p-dimethyl] toluidine dissolved in alcohol according to the quantities given in 
D.R. Patent 80758.* The mixture was boiled for 6 hours. The scarlet 
colour developed in a few minutes and intensified rapidly, until signs of 
precipitation appeared when the liquid was somewhat concentrated and 
cooled. The precipitate was filtered and washed with a little alcohol. The 
phenazine compound consists of a green crystalline powder which dissolves in 
water with a more magenta colour than the previous compound. 


N 
eV Nan, 


CEST NNR 


YAN 

cH, C1 
[93.] N-Methyltetrahydroquinoline-2.aminophenazine methochloride, — The 
tetrahydroquinoline and its N-methyl derivative were prepared by the 


* “Wriedlander,’ vol. 4, p. 376. 


364 Mr. C. H. Browning and others. Relationships between 


method of Hoffman and Kénigs,* and converted into the nitroso compound. 
A mixture of 2°6 grm. nitroso N-methyl tetrahydroquinoline, 15 grm. of 
p-dimethylamino o-toluidine, 15 ¢.c. of glacial acetic acid and 2 c.e. of cone. 
hydrochloric acid were warmed gently on the water-bath. Heat was 
developed and the liquid became a brilliant green which rapidly changed 
through brown black, dull scarlet to magenta. The heating was continued 
from 3 to 4 hours, when the product was dissolved in 50 cc. of hot water, 
filtered and the colouring matter precipitated with zine chloride and brine. 
It forms a green iridescent mass which is at first sticky, but rapidly hardens. 
The substance dissolves in water with a magenta colour similar to that of 
toluylene red methochloride (p. 360). On analysis the zine salt gave 
N=145 per cent.; CsgHaNsgCliZn requires N =141 per cent. The 
formula of the hydrochloride is therefore 


4,07 \/ NAY Ne, 


UC | NH,-HCl. 


NO A WA 
le! WR 
CH, CH, C1 


[89.] 2.Dimethylamino-7.amino-3.6.dimethylphenazine methochloride. — 16 
parts of p-amino-dimethyl o-toluidine and 15 parts of m-dimethylamino 
p-toluidine were dissolved in 300 parts of water, 20 parts of cone. hydro- 
chloric acid and 30 parts of 50 per cent. acetic acid, and cooled to 10° with 
stirring. Thirty parts of sodium dichromate in 400 parts of water were run 
in during 3 hours. A bluish-red coloration develops immediately and 
becomes more intense as the oxidising agent is added. The mixture was 
finally warmed on the water-bath for an hour, filtered and salted out with 
zinc chloride and sodium chloride. In this way the zine chloride salt of 
the colouring matter is obtained. It was purified by solution in water, 
filtration and reprecipitation with salt solution. It could not be recrystal- 
lised from alcohol or dilute hydrochloric acid. The substance by analogy 
with the formation of tetramethyldiamino-phenazine (p. 361) has the 
following structure :— 


| | H,-HCl. 
\ 


* ‘Ber.,’ vol. 16, p. 728 (1883). 
+ Konigs and Freer, ‘ Ber., vol. 18, p. 2388 (1885). 


Antiseptic Action and Chemical Constitution. 365 


[90.] 2.Benzylamino-T.amino-3.6.dimethylphenazine methochloride. — The 
compound was prepared by heating together on the water-bath p-dimethyl- 
amino o-toluidine dissolved in alcohol with nitroso benzyl o-toluidine hydro- 
chloride and hydrochloric acid as described m the preparation of the methyl- 
amino compound (p. 361). The colour developed passing from yellowish red 
to red, with a faintly blue tint. On concentration and cooling the colouring 
matter separated and was filtered and washed with ether. It has probably 
the following formula :— 


N 
cH, 7 ’ cH, 


omcnwal | I BN HCl 
N 


JAS 
CH, Cl 


The zine salt is obtained by precipitating the methochloride in solution 
with zine chloride. . 

[91.] 7.Amino-6.methyl-2.dimethylamino-napthophenazine hydrochloride.— 
The substance was prepared as follows: 2°3 grms. of nitroso dimethyl 
a-naphthylamine hydrochloride were dissolved in 20 c.c. of hot glacial acetic 
acid, and to the solution was added a solution of 1:2 germs. of m-toluylene 
diamine in 20 c.c. of 50 per cent. acetic acid. 1 ce. of cone. hydrochloric 
acid was then added and the mixture heated on the water-bath for 4 hours. 
A magenta colour developed rapidly and lost its bluish tint on continued 
heating, becoming gradually more crimson. It was finally boiled, diluted 
with 100 c.c. of water and the hydrochloride precipitated with brine. For 
purification it was redissolved in water and reprecipitated with brine. It 
has the following formula :— 


() 
Ue 


can | hs H.-HCl. 
VAs 


[92.] 7.Amino-6.methyl-2.dimethylaninenapthophenazine methochloride. — 
The process was carried out as above, but instead of m-toluylene diamine 
the p-dimethylamino o-toluidine was used. 2°4 grms. of nitroso dimethyl 
a-naphthylamine hydrochloride were dissolved in 20 c.c. glacial acetic acid 
and added to 1d grms. of dimethylamino-toluidine in 20 cc. 50 per cent. 
acetic acid, and 1 ¢.c. of cone. hydrochloric acid. The bright crimson colour 
develops rapidly, and after 3 hours’ heating on the water-bath was separated 


366 Antiseptic Action and Chemical Constitution. 


and purified as in the previous case. The hydrochloride of the methochloride 
was even less soluble than the above hydrochloride, but is readily soluble in 
alcohol. The formula is 


oF w 


TA 


N 
WAS ONOE 


iain A | Laas 


REFERENCES. 


. Benda, ‘ Ber. d. Deutsch. Chem. Gesell.’ vol. 44, p. 1787 (1912). 


Browning and Gilmour, ‘Jour. of Path. and Bact.,’ vol. 18, p. 144 (1913); see also 
Browning, Gilmour, and Gulbransen, ‘ Applied Bacteriology, London, p. 65 (1918). 


. Browning and Gulbransen, ‘ Jour. Hyg.,’ vol. 18, p. 33 (1919). 

. Browning and Gulbransen, ‘ Roy. Soc. Proc.,’ B, vol. 90, p. 136 (1918), 

. Browning and Gulbransen, ‘ Brit. Jour. Exper. Path.,’ vol. 2, p, 95 (1921). 

. Browning, Gulbransen, and Kennaway, ‘Jour. of Path. and Bact.,’ vol. 28, p. 106 


(1919). 


. Browning, Gulbransen, Kennaway, and Thornton, ‘Brit. Med. Jour.,’ vol. 1, p. 73 


(1917). 


. Browning, Gulbransen, and Thornton, zbid., vol. 2, p. 70 (1917). - 
. Richet and Cardot, ‘Compt. Rend. Acad. d. Sci.,’ vol. 171, p. 1353 (1920). 
. Shiga, ‘ Zeitschr. f. Immunitiitsforsch., Orig.,’ vol. 18, p. 65 (1918). 


be 
F 


367 


a 
The Action of “ Peptone” on Blood and Immunity thereto. 
By J. W. PickeErineG, D.Sc., and J. A. Hewitt, Ph.D., B.Sc. 


(Communicated by Prof. W. D. Halliburton, F.R.S. Received March 1, 1922.) 
(From the Department of Physiology, University of London, King’s College.) 


The current view on the cause of the non-coagulability of the blood after 
the injection of “peptone” is that the injected material stimulates the liver 
(or the liver plus other organs) to form an excess of a substance named 
antithrombin. The liver is considered further to form antithrombin under 
normal conditions in amount sufficient to maintain the fluidity of the blood. 
The statement that “peptone ” has little or no anticoagulant action on shed 
blood (except in high concentrations) is also generally accepted. Immunity 
to the anticoagulant action of “peptone” which follows its slow injection 1s 
commonly ascribed to hepatic activity. 

The experiments recorded in this paper show that these views can no 
longer be held. 


On the supposed Formation and Secretion of Antithrombin by the Liver. 


Contjean (1), after ligature of the abdominal arterial trunks, and Gley and 
Pachon (2), after ligature of the lymphatics of the liver, found that injected 
“peptone ” had no anticoagulant action. Starling (3) and Delezenne (4) could 
not confirm these observations, while Denny and Minot (5) have shown that 
repeated electrical stimulation of the cceliac plexus or its hepatic branches 
fails to increase the coagulability of the blood. 

The first systematic attempts to prove that the liver secreted an anti- 
thrombic substance appear to have been made by Hédon and Delezenne (6), 
who, after establishing an Hck’s fistula in a dog and, as far as possible, 
removing the liver, failed to reduce the coagulability of the blood by the 
intravascular injection of “peptone.” These investigators concluded that 
something secreted by the liver was the cause of this result. In each of the 
two experiments described, some hours elapsed after the establishment of the 
fistula and before the liver was incompletely excised, and a further hour 
passed before the injection of “ peptone.’ Delezenne (7) reported four similar 
experiments (of which only one is given in detail), and here also there is a 
considerable time-interval, in this case 5 hours, between the end of the 
operation and the injection of the “peptone.” In the 26 minutes immediately 


368 Drs. J. W. Pickering and J. A. Hewitt. 


following the injection of the “peptone” five samples of blood coagulated 
rapidly. 

The common feature of these experiments is the great length of time 
during which the animals were under anesthesia, leading to a decrease of 
oxygen (Buckmaster and Gardner, 8) and a corresponding increase of carbon 
dioxide in the blood. 

Prior to the work of Hédon and Delezenne (Joc. cit.), Lahousse (9) had 
shown that in peptone blood there is an extreme diminution of carbon dioxide, 
while Fano (10) and Wooldridge (11) had demonstrated that the passage of 
carbon dioxide through peptone plasma induces coagulation. Wright (12) 
has also shown that an increase of carbon dioxide in normal circulating blood 
augments its coagulability. 

The following experiments* show that the retarded coagulability of peptone 
blood in animals intact, except for pithing, or in animals in which the liver is 
not acting, may be diminished or annulled by excess of carbon dioxide; the 
general result is that when the vitality of the animal is impaired this 
inevitably occurs with both moderate and larger doses of peptone. 

In these and the subsequent experiments al] animals were anesthetised 
with A.C.E., pithed, and (except in Nos. 14-17) the anesthetic was dis- 
continued for at least a quarter of an hour before observations were made on 
the blood. All blood was withdrawn through evenly paraffined cannule. 
Artificial respiration (except in Nos. 14-17) was employed throughout. The 
animals were kept warm. The “commencement” of clotting was taken to be 
when the first change towards coagulation was observed; “ completion ” of 
clotting when the vessel containing the blood could be inverted without 
spilling. All glass vessels were cleaned with hot hydrochloric acid, caustic 
soda, aleohol and ether. Dust was as far as possible excluded. The percent- 
ages of “peptone” are necessarily approximate, since commercial specimens, 
as used by different investigators, vary enormously in composition. In the 
present work one preparation was employed throughout. 

The results obtained in Experiments 1, 2 and 3 are expressed graphically. 
The points on the upper graph indicate “completion” of clot, those on the 
lower graph “commencement ” of clot. 


* Cats have been used in the experiments now recorded because their susceptibility to 
“peptone” lies midway between that of the dog, which is markedly susceptible, and the 
rabbit which is exceptionally resistant. 


The Action of.“ Peptone” on Blood and Immunity thereto. 369 


i 
Experiment No. 1.—Pithed cat. Peptone injected into right jugular. 


& 
: 70 ~. 
SN 

‘ 8 
NZ iS N NG 
Mae SS ES S 8 
Ry Sy Ss 8 S$ 

RN 'S S 8 'S 
S at 5» = S SS 


2 0 FE 2 25 30 35 40 £45 50 35 60 
Time of observation ,miniutes 


Note-—The clot at 47’ was very loose. From 60’ onwards the heart was 


weak, 


Experiment No, 2.—Pithed cat. Aorta ligatured. 


% 

At) 

N 

‘S 

S A 4 

No aN N 

SS S  wS Sy 

ers ys sy § 

SE Se 53 R 

< NNER ISS = § s 

SZ ws SN Ss 8 . 
% N's S iS s 

» a aN NN 

8 NS So SES 8 
§ 8 E<S ~ 8 S 

&? zw 8 ROSEN 


a 70 IS 20 25 30 IS £0 £5 


Fime of observation, mMinatles. 


Note.—At 30’ and 41', the blood was not completely clotted after 1 hour 


50 minutes. Post mortem examination showed in this and in other experi- 
ments the ligatures intact. 


VOL. XCIII.—B. ; 2D 


Coaguiation time,miniites. 


370 Drs. J. W. Pickering and J. A. Hewitt. 


Experiment No. 3.—Pithed cat. Aorta and inferior vena cava ligatured. 


2410" 24/0" 
G2) 
Tb 
22 
70 
2 
8 | s 
, \ 
6 EK 82S S < 
NL RTE 8 < 
S&S Ad Bs 
4 NES SSs » S 
Ss Sas aw 
RS SAN S S 
Z wS Ss 
2- =8 S88 S S 
PD 
WES 
! 1 4 


& 
S 


19 2025 S3O" 3520 L550. 55. CO. 65 One 
Time Of observation ,miniitles ~ 


Experiment No. 4.—Cat, pithed. Artificial respiration. Aorta and inferior 
vena cava ligatured. 1:625 grm. of “peptone ” was injected rapidly into the 
heart over a period of 4 minutes. Five minutes after the injection of the 
“peptone,” coagulation time showed a retardation of 20 minutes in commence- 
ment of clotting, a complete gelatinous clot appearing 2 minutes 40 seconds 
later. This apparently liquefied, but further clots appeared in the liquid 
during the subsequent 2 hours, and the fluid was completely clotted on the 
following morning. While all other results have been repeated, this is the 
only occasion in these experiments where the phenomenon described in the 
dog by Doyon has been observed. Other samples of blood from this animal 
behaved similarly to those in Experiment No. 3, the increase in concentra- 
tion of “peptone” used not causing any substantially greater inhibition than 
was evident in No. 3. The passage of carbon dioxide through the peptone 
blood from this animal gave typical clotting, as did dilution with distilled 
water. 


Asphyxia lor 40" 


&O 


The total time taken in No.1 was 1 hour 10 minutes, in No. 2 1 hour- 


22 minutes, and, moreover, no anesthetic was being respired. Heédon and 
Delezenne’s experiments occupied “some hours,’ while in Delezenne’s 
observation no less than 5 hours elapsed from the commencement of the 
operation till the observation of the coagulation time of the peptone blood. 
During these protracted periods a volatile anesthetic was being administered 
and no attention was paid to the effect of a possible increase of the concen- 
tration of carbon dioxide in the circulation, under conditions liable to promote 


The Action of “ Peptone” on Blood andLmmumnity thereto. 371 


it. So far as the present writers are aware this criticism applies to all 
experiments in which “ peptone ” has been injected into animals with damaged 
or more or less excised livers. 

Of the facts unexplained by the hypothesis that the secretion by the liver 
of antithrombin is the cause of the fluidity of normal circulating blood and of 
the retarded coagulability of peptone blood, it is noteworthy that the massive 
injection of thrombin does not produce thrombosis, Wooldridge (13). This was 
confirmed by Mellanby (14) who injected, without thrombosis, sufficient 
thrombin into a cat to coagulate 2 litres of blood. After the intravascular 
injection of thrombin, Davis (15) found the coagulation time of shed blood 
to be variable, sometimes slightly lengthened at other times shortened. 
Unfortunately Davis recorded as “coagulation times” only the time of 
completion of the clot. This may be a variable period when the times of 
commencement of clotting are approximately constant (Nos. 1-4). Moreover, 
Davis’ variations in coagulation times are not greater than can be accounted 
for by alterations in the relative concentrations of carbon dioxide and oxygen 
in the blood shed. Apparently this and the corresponding hydrion concen- 
tration did not engage his attention. Davis explains the slightly decreased 
coagulability following the injection of thrombin by suggesting that thrombin 
produces an excessive secretion of antithrombin, while Howell (16) maintains 
that thrombin, or prothrombin is a hormone which stimulates the secretion of 
antithrombin. Davis frankly admits that if antithrombin exists in the 
circulation and is a genuine antibody, secreted as a protection against thrombin, 
then according to all analogies the presence of thrombin should lead to its 
production. 

Nolf (17), however, denies that the intravascular injection of thrombin leads 
to the formation of antithrombin. Further doubt is cast on the accepted 
hypothesis by the recent work of Arthus (18), who observed that delay occurs 
in the coagulation of shed blood after the injection of the venom of Crotalus 
adamunteus into immunised, non-immunised, and even anaphylactic rabbits. 
This not only happened in whole animals, but in those in which the hepatic 
blood supply had been cut off. 

Two views have been advanced in this connection, viz., that of Martin (19), 
and of Barratt (20), where the active fraction of snake venom is considered to 
be a true fibrin-ferment (thrombin), and that of Melianby (21), that it is a 
kinase which, when rapidly injected, causes the rapid formation of thrombin. 
It is evident from the experiments of Arthus, that the “ negative phase” of 
coagulation following either the rapid injection of minimal doses of venom, or 
the slow injection of larger doses, is not produced by the secretion by the 
liver of antithrombin, which combines with thrombin, either existing in, or 

2D 2 


372 Drs. J. W. Pickering and J. A. Hewitt. ; 


produced by, venom. This conclusion falls into line with the recent observa- 
tion of Pickering and Hewitt (22), that the slow addition, drop by drop, of 
tissue extract (nucleo-protein), prepared from kidney, to unsalted bird’s blood 
in vitro, produces a “ negative phase” in its coagulation, and is explicable by 
the suggestion that the “ negative phase” of blood coagulation is a physical 
process involving an adsorptive union of the tissue extract, the substances 
forming the alkali reserve and fibrinogen, and is in its essence closely akin to 
the following in vitro reactions: the Dansyz reaction with the toxins and anti- 
toxins of diphtheria and ricin (Dansyz, 23), the variations in the toxicity 
incidental to the fractional neutralisation of arsenious acid by ferric hydroxide 
(Hewlett, 24), the “ negative phase,” or temporary inhibition of the precipita- 
tion of gelatin by the slow addition of either aleohol or ammonium sulphate, 
and to the similar phenomena following the addition of electrolytes to certain 
inorganic sols (Spring, 25, Hober and Gordon, 26, Paine, 27, Galecki, 28, 
Burton, 29). 

Arthus concluded that in poisoning by proteotoxins, the liver is either not 
the organ which produces anti-thrombin, or is not the only organ. Doyon (30) 
immediately perceived the significance of Arthus’ work, and, adopting his 
methods, injected “ peptone” into dogs in which the hepatic circulation had 
been ligatured. Again, no attention was paid to the probable increase of 
carbon dioxide in the blood during the experiments in which anesthetics 
were employed. Doyon states that, in the cases he observed, the speed of 
clotting of shed blood was normal, but the clots were soft and completely 
dissolved on standing, so that the blood again became fluid. This result 
Doyon ascribed to the action of anti-thrombin formed in parts of the body 
other than the liver, and reverted to Contjean’s views (/oc. cit.), which have 
also been supported by Popielski (31). 

It is, it is thought, clear that Doyon’s clots are not true fibrin clots, as such 
do not re-dissolve i vitro. In a recent paper (Pickering and Hewitt (Joc. cit.) ) 
evidence has been brought forward that the first stage of coagulation of 
mammalian and frog’s blood, surrounded by oil, is the formation of a reversible 
gel, which is soluble in excess of tap water. In the case of frog’s blood this 
stage may, when undisturbed, persist for several hours, but if thromboplastic 
material is added to it a typical clot is formed in a few minutes. It therefore 
appears possible that “ peptone,” as administered by Doyon with the liver out 
of circulation, retarded the coagulation of the blood so that it remained in the 
first stage of the process. Only in one of the series of experiments now 
recorded, in which “ peptone ” was injected into both intact cats and into cats 
deprived of hepatic circulation, has any evidence been noted of the temporary 
formation of a gel which dissolves on standing (No. 4). It has consequently 


The Action of “ Peptone” on Blood and Immunity thereto. 373 


been impossible to investigate any relationship to hydrion concentration of 
this transient but significant phenomenon. 

The earlier observations on the effect of the injection of “ peptone” into 
animals with either impaired or partly excised livers, have formed the basis 
for a vast superstructure of hypotheses. They remain crucial only so long 
as the anticoagulant action of “peptone” has not been observed in animals 
when the liver is not acting. 

Employing a somewhat simpler operative technique to deprive the liver 
of circulating blood with a view of avoiding the prolonged interval before 
the injection of “peptone” (with the consequent accumulation of carbon 
dioxide), the results given in experiments, Nos. 2, 3 and 4 above, 
demonstrate decisively that retardation of coagulation of blood consequent to 
the rapid intravascular injection of moderate amounts of “ peptone” can be 
produced under conditions which preclude the participation of the liver. 

Stress is laid on the importance of working with pithed animals, and in 
allowing at least 15 minutes to elapse after pithing and the discontinuance 
of the anesthetic before the injection of “peptone,” so that any action 
of the anesthetic on the coagulability of the blood may be eliminated. It is 
also advisable to regulate the air supply to the animals so as to maintain, as 
long as possible, normal oxygenation. 


On the action of “ peptone” on blood in vitro. 


The well known observation of Schmidt-Mulheim (32) that very large 
quantities of “peptone” are necessary to retard the coagulation of blood 
im vitro is commonly regarded as showing that “peptone” has no specific 
action on shed blood. Among the earlier observers, Afanassiew (33), 
however, stated that blood shed direct from the artery of a dog, without 
exposure to the air, into 1:225 — 15 per cent. “ peptone,’ and kept at 
40°, remained fluid. Unfortunately the details of the experiment are 
meagre. Pollitzer (34) stated that 2 per cent. of heteroalbumose inhibited 
blood clotting im wtro, while Halliburton (35) recorded that the same 
substance also delayed the coagulation of salted plasma. Working with 
lymph from the thoracic duct which clotted, at room temperature, in from 
10 to 15 minutes, Shore (36) found that when relatively large quantities of 
“peptone” were added in vitro the clotting was as rapid as in pure shed 
lymph, but if smaller amounts of “peptone” were used retardation occurred. 
In one experiment in which the amount of “ peptone” was 0°0377 per cent., 
the lymph remained fluid for 24 hours. 

Camus and Gley (37) reinvestigated the effect of adding “peptone” to 
shed blood, and stated that from eleven to fifteen times more “ peptone” is 


374 Drs. J. W. Pickering and J. A. Hewitt. 


required to produce retardation of clotting in vitro as compared with the 
amount necessary to give this result 7m wivo. Thus the original views 
of the non-specificity of the action of “peptone” on shed blood gained 
general acceptance. 

The present authors find that when blood is shed into “peptone” 
without precautions to preserve it from changes towards coagulation, as in 
the earlier work, then anticoagulant action is absent, except when the 
“peptone” is present in relatively large amount. The following experi- 
ments show that different results are obtained when blood is shed through an 
evenly paraffined cannula from an animal respiring air. 


No. of experiment ......... | 5. | 6. th | 8. 9. 10. 
p.c pc. p.c. p.c. p.c. p.c. 
Percentage of “ peptone” 0°3 0 °325 0°6 1°0 1-0 3°5 
in blood 
Coagulation times of con- | 7 45 5 55 7 45 7 45 5 55 | 7 25 
trol blood ll 45 8 10 ll 45 ll 45 8 10 8 45 
Coagulation times of ‘‘pep-| 13 20 45 20 16 30 14 45 | 21 4 P 
tone” blood 16 40 48 50 20 10 23 40 | 23 40 ? 
Coagulation times of 8 10 Gao 8710 8 10 8 10) Gao 
“water” blood 10 165 7 55 10 15 10: -15 | 10) S15 |e7ias 
Notes. 


1. “ Water” blood is normal blood to which a yolume of water or in No. 6 of 0°9 per cent. 
NaCl equal to that of the peptone has been added. 

2. The upper of the two coagulation times is that of “commencement,” the lower that of 
“completion” of clotting. 

3. The blood was arterial and was withdrawn into glass vessels through paraflined cannule. 
The animals were pithed and were breathing air only. 

4, The ‘‘ peptone” in Experiment No. 6 was dissolved in 0°9 per cent. sodium chloride ; in 
all others in this series in distilled water. 

5. Room temperature was 13-14° C. 

6. The “ peptone ” blood in No. 10 remained fiuid during the next day. 

Experiment No. 5 shows that blood withdrawn from the carotid of a cat 
through a carefully paraffined cannula into a clean glass vessel, and there 
mixed immediately with “peptone” dissolved in distilled water, exhibited 
a distinct retardation of clotting. This delay amounted to 5 minutes 
25 seconds in the time of commencement, and 4 minutes 55 seconds in the 
time of completion of clotting; the amount of “peptone” in the blood was 
only 0:3 per cent. As is seen from experiments 7, 8, 9, and 10 with greater 
concentrations of “peptone,” the inhibition of clotting increased. When 
solution is effected in 0°9 per cent. sodium chloride as in Experiment 
No. 6, the inhibition of coagulation is still more marked. Thus with a 
concentration of 0°325 per cent. of “peptone” in the blood a delay of 
39 minutes 25 seconds in the commencement of clotting was observed, while 


The Action of “ Peptone” on Blood and Immunity thereto. 375 


the delay in completion was 40 minutes 40 seconds. The addition of 
corresponding amounts of 0-9 per cent. sodium chloride to shed blood did 
not produce any appreciable variation in the coagulation time. A com- 
parison of Experiments Nos. 8 and 9 with No. 6 shows that the retarding 
effect of a concentration of 1 per cent. of “peptone” dissolved in distilled 
water was actually less than that of 0°325 per cent. dissolved in normal saline. 
This result falls into line with the observation of Howell (38) that while 
dilution of peptone plasma with water induces coagulation, dilution with 
0-9 per cent. sodium chloride has no such effect. 

In the next three observations, endeavours were made, with success, to 
prolong the anticoagulant action of small amounts of “peptone” i vitro by 
reducing the disturbance to the colloidal complexes of the plasma incidental 
to the shedding of blood, 


Control | Control wee Hee Fie 
bing on | Blood on blood on blood on blood on 
ores a paraffin. paraffin, | paraffin. 
! 
/ “i / “s f “l / “sr f “l 
Time of adding “‘ peptone’”’... OQ. @ Q® ©  @ Q © 0 0 
Time of “commencement”’ 8 55 | 24 20 32 40 84 45 45 20 
of clotting 
Time of “completion” of 12 15 27 40 107 30 | 186 0 135 0 
clotting | 
No: of experiment ............ = _— 11 | 12 13 
Notes. 


In Nos. 12 and 18, after 171 minutes there was still some unclotted blood. 
Many experiments of this nature have been carried out in this connection. Only one is given. 


Hapervments. 

In these experiments, blood from the carotid through a paraffined cannula 
was received into paraffined vessels, as free from dust as possible. With 
0°3 per cent. of “ peptone ” (dissolved in 0°9 per cent. sodium chloride) in the 
blood, again mixed im vitro, the delays in the commencement of clotting 
exceeded 23 minutes, as compared with the coagulation time on glass, a 
result which was not more marked than was obtained with 0°325 per cent. 
of “peptone” dissolved in 0:9 per cent. sodium chloride, mixed in an 
uncoated glass vessel. The distinctive difference between the results on 
glass and paraffined vessels is that, in the former, coagulation, once it has 
commenced, proceeds rapidly, and is completed in a few minutes, while in 
the latter it is a relatively slow process, occupying from 1 to 2 hours, In 
some cases, indeed, the first formed small clots are exhibiting syneresis 
(contraction), while other clots are still forming. Thus, in the less 


376 Drs. J. W. Pickering and J. A. Hewitt. 


disturbed conditions of the paraffined vessels is the continuous inhibitory 
action of the “ peptone ” made evident.. 

A comparison of the coagulation times of pure blood on paraffined 
surfaces with those of blood mixed with “peptone” on similar surfaces 
shows less relative retardation in the time of commencement of clotting 
than is evident when the coagulation of pure and peptone blood in uncoated 
vessels is compared. The relative retardation in the’ time of completion of 
the clots is much more marked when blood mixed with “peptone” is in 
paraffined vessels than when it is contained in glass vessels not so treated. 

In this connection, the partial failure of one experiment is noteworthy. 
The blood contained 0:8 per cent. of “peptone.” It did not commence to 
clot until 31 minutes after the mixing of the blood and “ peptone,” but 
coagulation was complete 2 minutes later, a firm clot being formed which 
could be inverted without spilling. 

This is a marked contrast to Experiments 11, 12 and 13, and cannot be 
ascribed to the higher concentration of “ peptone ” used, as typical slowing of 
clotting has been obtained after the appearance of the first clot in a number 
of cases where this and higher concentrations of “peptone” have been 
employed. In the specific case referred to, examination of the vessel 
containing the peptone blood revealed a small defect in the paraffin lining, 
with adherent fibrils to the exposed glass. Clotting had started from this 
point, and had spread through the liquid. 

Attention is drawn to Experiment 13, where the “peptone” was 
0:6 per cent. of the mixture; here clotting did not commence till 45 minutes 
20 seconds, and was completed only as a semi-gel 24 hours after mixing. 
A small amount of the fluid was withdrawn by a pipette before coagulation 
was complete, and carbon dioxide passed through it. Clotting was observed 
after 10 minutes. This result was also obtained by dilution with distilled 
water. Other observations gave similar results. During the progress of the 
delayed clotting, portions of what remained fluid were, from time to time, 
withdrawn, and mixed with one-fourth of their volume of saturated 
ammonium sulphate. A precipitate of what was probably fibrinogen was 
always obtained (McLean, 39), till, at the end of the coagulation process, 
when the clots appeared to be complete, tests of the residual fluid yielded no 
precipitate. The mixture of “peptone” and blood 7 vitro thus behaved 
precisely as the blood peptonised wm vivo. 

Schmidt-Mulheim (Joc. cit.) gives the dose of “peptone” necessary to 
produce incoagulability of circulating blood of dogs as from 0°3 to 0:6 grm. 
per kilogramme. Pollitzer (Joc. cit.) found cats to be more resistant. Taking 
an animal weighing 3 kgrm., it may be assumed to contain 200 ce. of 


The Action of “ Peptone” on Blood and Immunity thereto. 377 


blood. If 03 grm. per kilogramme of “peptone” is injected, the per- 
centage in the blood is 0°45, or 0'9 if the larger quantity is used. 

If, from a pithed cat which is breathing only air for at least 15 minutes, 
arterial blood is obtained through a paraffined cannula with only smooth 
surfaces exposed to the blood, then less than Schmidt-Mulheim’s minimal dose 
for the dog will produce, in the more resistant cat, an unequivocal anti- 
coagulant action iz vitro, even in uncoated glass vessels. When the concen- 
tration of “ peptone” is increased, but is still 33 per cent. less than Schmidt- 
Mulheim’s larger amount, the results im vitro are parallel to those following 
the injection of “peptone” into the circulation of the living animal. 

These data warrant the following conclusions :— 

1. “ Peptone,” even in small quantities, acts directly on the constituents of 
the blood. 

2. The apparent difference found by other observers between the action of 
“peptone” in vivo and its action in vitro is due to the changes towards 
clotting which take place in blood shed without special precautions to preserve 
its surface conditions. 

3. It is superfluous to assume that the anticoagulant action of “peptone” 
im vivo is due to the secretion of antithrombin either by the liver or by the 
endothelial cells of the vascular system. 


On the Supposed réle of Leucocytes in the Anticoagulant Action of “ Peptone.” 


Delezenne (40) considered that the first action of “peptone” on blood is 
leucolysis yielding two substances: Jewconuclein which hastens coagulation 
and Jewcohistone which retardsit. These both circulate through the liver where 
the former is retained. The latter remains circulating. Several workers 
including Halliburton and Brodie (41), Falloise (42) and Dastre and Studel (43) 
however deny that “peptone” on injection causes leucolysis. Even if this 
process does occur there appear to be no grounds other than pure speculation 
for the supposed liberation of these substances into the blood stream. 

Nolf (44) maintained that under normal conditions an wrknown substance 
is produced by the leucocytes which acts upon the endothelium of the liver 
and stimulates it to secrete antithrombin. The injection of “peptone” 
enhances this process and the excess of antithrombin accounts for the 
retarded coagulability of peptone blood. 

Having demonstrated that the liver is not necessary for the production of 
the anticoagulant action of “peptone,” it becomes of interest to enquire 
whether the leucocytes are concerned in this phenomenon. To this end 
_ Experiments 14-20 were devised. 

Cats were bled through paraffined cannule into paraffined centrifuge tubes 


378 Drs. J. W. Pickering and J. A. Hewitt. 


in ice and without delay these were centrifuged at 4° C., after which the tubes 
were again surrounded by ice till required. By this method a clear fluid 
mammalian plasma was obtained unaltered by foreign substances and which 
had made but little, if any, progress towards coagulation. At the temperature 
mentioned leucocytes do not exhibit amceboid movement and it may be 
assumed that all secretion by them is inhibited, To this plasma “ peptone ” 
was added in concentrations varying from 0°3-1:2 per cent. of the total volume 
of “ peptone” and plasma and the mixture was observed at room temperature 
in paraffined vessels. In all cases except one, the typical anticoagulant action 
of “peptone” was evident. A study of the protocols shows that the results 
are essentially the same as when “ peptone” in similar concentration is either 
added to shed blood at laboratory temperatures or is intravascularly injected 
into whole animals or into animals where the liver is out of circulation. 
Evidence is thus forthcoming that lewcocytes play no part in the inkabition ef 
coagulation which follows the rapid adnuature of blood and “ peptone.” 


| | | | 
| Control | Control | 2’ P-¢: | 06 pc. | 0:9 p.c. | Ce 
plasma | plasma pane peptone | peptone | peptone 
[ae ee Bes plasma | plasma | plasma | plasma 
araffin lass oe as OD. elovieeae 
{a2 ; Cas: paraffin. | paraffin. | paraffin. | paraffin. 
1 Th | 
/ “4 ‘ “i / “ i “s / “ 4 “ 
Time of adding “peptone” ...| O O 0) @) Q @) O10) Oe oye 0) 
Time of “commencement” of | 8 0 5 60 7 40°) 12" 5) | 19) 130s aCe 
clotting | | 
Time of “completion” of | 10 10 8 20 9 10 | 54 50 | Noend | 27 O 
clotting | point 
No. of experiment ............... — — 14 ie elo 17 
Notes. 


Plasma obtained from animal respiring A.C.E. and air. 

1. In No. 16 clotting was not typical; small clots were formed. 

2. In No. 17 the gelatinous clot appeared to liquefy after 29 minutes; 10 minutes later all 
was liquid except one small clot. Syneresis is not excluded. 

3. The residual fluids from 16 and 17 gave, next morning, no precipitate when fifth saturated 
with ammonium sulphate. 

4, No. 14 is the only anomaly in a large number of observations. 


A comparison of Experiments 14-17 with Nos. 18-20 illustrates the 
difference between the action of “ peptone” on plasma from an animal under 
complete anesthesia (A. C. E.) and the action of similar quantities of 
“peptone” on blood from a pithed animal 15 minutes after the discontinuance 
of the anesthetic. 

There is consistently less “peptone” inhibition of clotting when the 
animal is under anesthesia at the time the blood is obtained. Early in these 
experiments it was evident that the shed blood of anzsthetised animals was 


The Action of “ Peptone” on Blood and Immumnaty thereto. 379 
Control 0°6 p.c. 0°9 p.e. 1°2 p.e. 
plasma peptone peptone peptone 

on plasma on | plasma on | plasma on 
paraffin. parafiin. paraflin. paraffin. 
Time of adding peptone ...................65 0 O OREO @ ~@ 0 0 
Time of “ commencement ”’ of clotting 6 ©) 34 45 50 20 24 30 
Time of “ completion” of clotting ......... 23 50 41 45 51 20 30 =O 
Niomole <peniimenty -uccean nescence ne as — | 18 19 20 
Notes. 


1. Repetition of Nos. 18 and 19 gave the same results. 
2. In No, 20, after 20 minutes the gel appeared to liquefy, due probably to syneresis. 
3. Plasma was obtained from animal respiring air. 


more prone to coagulate rapidly than that of pithed animals 15 minutes after 
respiring only air. For this reason, except for the experiments cited above, 
all observations not on pithed animals were discarded. 

In both the last two series of experiments typical syneresis was exhibited 
in the clots formed. The view that blood platelets are necessary for the 


production of syneresis (Bordet and Delange, 45) may be mentioned. 


The Influence of Speed of Injection on the Action and Imnyunity to the Action 
of Peptone on blood, 


Fano (46) found that, if “peptone ” is injected slowly into the circulation, 
it has not any anticoagulant action on the blood, and later Nolf (loc. cit.) 
showed the concentration of “ peptone” as well as the speed of its injection 
to be the determining factors either in the production of anticoagulant action 
or complete or partial immunity thereto. Nolf explained the immunity 
following minimal doses injected rapidly and larger doses injected slowly by 
suggesting that leucocytes are not acted upon by “peptone” if delivered 
sufficiently slowly, and that they become accustomed to increasing doses. The 
leucocytes, he believes, under these conditions fail to produce the wnknown 
substance which stimulates the liver 
fibrinolysin. 


to secrete either antithrombin or 
The experiments described in the preceding section demon- 
strate that leucocytes are not essential for the production of the anti- 
coagulant action of “peptone” im vitro, and there appears to be no valid 
reason for assuming their presence to be necessary for the production of the 
same effect 7m vivo, when the concentrations are strictly comparable. 

In sharp distinction to the generally accepted view is that of Mellanby (47), 
who maintained that fluidity of peptone blood is due to the secretion of an 
excess of alkali by the liver, under the influence of a stimulus of injected 
“peptone,” and the “peptone” immunity is caused by the temporary dis- 


380 Drs. J. W. Pickering and J. A. Hewitt. 


appearance from the liver of the excess of alkali secreted under the toxic 
stimulus. 

The next experiment, No. 21, shows that if relatively large amounts of 
“peptone” are injected slowly, in minimal doses, over a long period of time, 
into a cat in which the circulation through the liver has been prevented, then, 
as in the intact animal, no anticoagulant action is to be obtained. 

Experiment No. 21.—Cat, 31 kilos. Pithed, artificial respiration. Aorta 
ligatured beyond the coronary arteries, inferior vena cava above the 
diaphragm. 

Over period of 1 hour 10 minutes 1°8 erm. of “ peptone” in 09 per cent. 
NaCl was injected in nine equal amounts, each injection being 2°5 cc. in 
volume and being made at approximately equal intervals of time. 

The coagulation times of the blood was taken at frequent intervals during 
the injection of the “ peptone ” and at no time showed any delay. 

Control Blood on Glass.—Commencement of clotting, 9 minutes; comple- 
tion of clotting, 12 minutes 10 seconds. 

The total amount administered was 1°8 grm., and as only about one-fourth 
of the animal’s blood was circulating, the concentration of “ peptone” in the 
blood at the end of the experiment may be estimated at 3°6 per cent., an amount 
much in excess of that required to produce anticoagulant action both in 
whole cats and in cats in which the liver is not acting, provided that such an 
amount is injected slowly. Moreover, reference to Experiment No. 10 shows 
that a concentration of 3:5 per cent. of peptone in the blood im vitro produced 
incoagulability over a period of 24 hours. Assuming in the 7m vivo experi- 
ment mentioned above that the quantity of blood dealt with was 50 cc., a 
concentration of “peptone” exceeding that required to produce prolonged 
incoagulability 7 vitro was injected without anticoagulant effect. 

Some other explanation must therefore be sought for this immunity to the 
slow injection of “ peptone ” than is supplied by the divergent views of Nolf and 
of Mellanby. These hypotheses have one point in common, they assign the 
cause of immunity to hepatic secretion, and it is precisely this point of both 
theories which is shown to be untenable by the experiments given above. 
Whether the alkali reserve or the hydrion concentration of the plasma is 
concerned in these occurrences must be left for future determination. There 
are, however, some indications which point in this direction. Fano (doc. cit.) 
showed that peptone blood could be clotted either by the passage through 
it of carbon dioxide or by neutralisation by acid. Earlier in the present 
paper the influence of the concentration of carbon dioxide in circulating 
blood on the action of “peptone” has been emphasised. It is noteworthy 
that Mills (48) has recorded that a diminution of the alkali reserve occurs 


The Action of “ Peptone” on Blood and Immunity thereto. 381 


part passu with the coagulant action arising from the intravenous injection 
of lung extract. It is well known that the advent of asphyxia will 
determine the appearance of thrombosis after the injection of such amounts 
of kidney nucleoprotein as, in the presence of normal amounts of carbon 
dioxide, tend to produce incoagulability or immunity. In like manner 
Halliburton and Pickering (49) showed that an increase in the concentration 
of the carbon dioxide of the blood will annul the “negative phase ” 
resulting from the injection of minimal doses of these synthesised substances 
which have been shown to be intravascular coagulants (Pickering, 50). 

Still more significant is the correlation of all these “negative phases” 
and temporary immunities to the factors of the speed of action and 
concentration of the toxic substances. In this respect they are similar 
to the “negative phases” which occur in vitro alluded to earlier in 
this paper. 

There is a physical or physico-chemical interpretation indicated. 

An application of the theorem of Le Chatelier (51) may provide a more 
satisfactory explanation than is afforded by current hypotheses. 


On Experiments with perfused Livers. 

It is commonly held that perfusion of the liver affords evidence of the 
secretion of antithrombin by that organ. Delezenne (52) and later Nolf (53), 
who had doubtful results, found that perfusion of the liver with a mixture 
of defibrinated blood and “peptone” gave an incoagulable liquid which, 
when added to shed blood, retarded its coagulation. Apparently these 
workers were unaware of earlier observations of Pavloff (54), who had found 
that the circulation of a mixture of defibrinated and normal blood through 
a heart-lung preparation yielded a fluid which remained uncoagulated for 
several days. Work is now in progress which indicates that certain products 
of autolysis may act as anticoagulants, and in view of Pavloff’s experiments 
it is superfluous to assume that Delezenne’s results were due to hepatic 
activity. Doyon (55) connected the carotid of one dog with the portal 
vein of another, and examined the coagulation times of blood from the 
inferior vena cava. With young fasting dogs the first perfusates coagulated 
normally, but later samples showed delay in coagulation. With adult dogs, 
even after fasting, no retardation of clotting was observed. It is well 
known that the blood of embryos is resistant to clotting and these isolated 
experiments only afford evidence of a similar phenomenon in the blood of 
young fasting dogs. 


382 Drs. J. W. Pickering and J. A. Hewitt. 


Summary and Conclusion. 


1. The retardation of the coagulation of the blood resulting from the rapid 
intravascular injection of “ peptone” into either whole cats or into cats with 
the liver out of circulation is reduced or annulled by increase of carbon 
dioxide in the blood. 

2. The anticoagulant action of “peptone,’ if annulled by an increase 
of carbon dioxide, can be restored by administration of oxygen or of 
excess of air. 

3. In pithed animals respiring air, the retardation of coagulation of shed 
blood following the rapid injection of moderate amounts of “ peptone,” can 
be produced when the liver is out of the circulation. 

4. Previous failures to obtain this result are due to attention not having 
been paid to the increase of carbon dioxide in the blood of animals with 
impaired vitality following operation and prolonged narcosis. 

5. With suitable precautions to preserve the surface conditions of the 
blood, typical “peptone” delays of coagulation can be obtained iw vitvo with 
no greater than are required to produce a similar 


? 


quantities of “peptone ’ 
effect in the living animal. 

6. If special precautions are not taken to preserve the surface conditions of 
shed blood, admixture i vitro with moderate amounts of “ peptone” causes 
no appreciable anticoagulant effect. 

7. Leucocytes play no part in the anticoagulant action of “ peptone” on 
blood. 

8. It is superfluous to assume that the anticoagulant action of “ peptone” on 
blood is due to hepatic secretion of an excess of alkali under the toxic stimulus. 

9. It is unnecessary to assume that the anticoagulant action of “ peptone” 
is due to the secretion of antithrombin, either by the liver or by other cells 
of the body. 

10. Typical immunity to the action of “ peptone ” on blood can be obtained 
by injecting maximal amounts, in repeated small doses, into animals with the 
liver out of circulation. 

11. The current hypotheses on “peptone ” immunity are shortly discussed, 
and reasons given for not accepting them. Immunity to “peptone” appears 
to be a physical process akin to adsorption. 

12. Experiments on the perfusion of the liver do not show that anti- 
thrombin is normally secreted by that organ. 

13. If the foregoing conclusions are correct, it follows that in the inter- 
pretation of the coagulation of the blood it is unnecessary to assume the 
existence of antithrombin, pro-antithrombin, and anti-prothrombin. 


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The Action of “ Peptone” on Blood and Immunity thereto. 383 


14. If antithrombin is not a normal secretion of the liver, some explanation 
of blood coagulation, other than the current “ thrombin theories,” is necessary. 


LITERATURE REFERRED TO IN THE TEXT. 


1. Contjean, ‘ Arch. de Physiol. vol. 7, p. 245 (1895). 

2. Gley and Pachon, ‘Compt. Rend. Acad. Sci.,’ vol. 121, p. 383 (1895) ; ‘ Arch. de 
.  Physiol.,’ vol. 7, p. 711 (1895). : 

3.. Starling, ‘ Jour. Physiol.” vol. 19, p. 15 (1895-6). 

4. Delezenne, ‘Compt. Rend. Acad. Sci.,’ vol. 122, p. 1072 (1896). 

5. Denny and Minot, ‘ Amer. Jour. Physiol., vol. 38, p. 233 (1915). 

6. Hédon and Delezenne, ‘Compt. Rend. Soc. Biol.,’ vol. 3, p. 633 (1896). 
7. Delezenne, ‘ Arch. de Physiol., vol. 8, p. 655 (1896). 

8. Buckmaster and Gardner, ‘ Jour. Physiol.,’ vol. 41, p. 258 (1910-11). 
9. Lahousse, ‘ Archiv Physiol.’ (Leipzig), 1889, p. 77. 

10. Fano, ‘ Arch. Ital. de Biol. vol. 2, p. 146 (1882). 


11. Wooldridge, ‘ Roy. Soc. Proc.,’ vol. 38, p. 69 (1884-5). 

12. Wright, ‘ Roy. Soc. Proc.,’ vol. 55, p.-279 (1894). 

13. Wooldridge, ‘The Chemistry of the Blood, London, 1893, p. 184. 
14. Mellanby, ‘ Jour. Physiol.,” vol. 38, p. 501 (1909). 

15. Davis, ‘Amer. Jour. Physiol.,’ vol. 29, p. 160 (1911-12). 

16. Howell, ‘ Amer. Jour. Physiol.,’ vol. 29, p. 208 (1911-12). 

17. Nolf, ‘ Arch. di Fisiol.,’ vol. 7, p. 1 (1909). 

18. Arthus, ‘Compt. Rend. Soc. Biol., vol. 82, p. 416 (1919). 

19. Martin, ‘ Jour. Physiol.,’ vol. 32, p. 208 (1905). 

20. Barratt, ‘Roy. Soc. Proc., B, vol. 87, p. 177 (1914). 

21. Mellanby, ‘Jour. Physiol.,’ vol. 38, pp. 475 and 501 (1909). 

22. Pickering and Hewitt, ‘ Biochem. Jour.,’ vol. 15, p. 710 (1921). 
23. Dansyz, ‘Ann. Instit. Pasteur, vol. 16, p. 331 (1902). 

24. Hewlett, ‘Manual of Bacteriology, London, 1921, p. 196. 

25. Spring, ‘Rec. Trav. Chim. Pays-bas’ [2], vol. 4, p. 204 (1900). 

26. Hober and Gordon, ‘ Beitrage, vol. 5, p. 432 (1904). 

27. Paine, ‘ Proc. Camb. Phil. Soc.,’ vol. 16, p. 480 (1911). 

28. Galecki, ‘Zeitschr. Anorgan. Chem.,’ vol. 74, p. 179 (1912). 

29. Burton, ‘ Physical Properties of Colloidal Solutions,’ London, 1916, p. 148. 
30. Doyon, ‘Compt. Rend. Soc. Biol.,’ vol. 82, p. 736 (1919). 

31. Popielski, ‘ Zeitschr. f. Immunititsforsch., vol. 18, p. 542 (1913). 
32. Schmidt-Mulheim, ‘Du Bois Reymond’s Arch. f. Physiol.,’ 1880, p. 33. 
33. Afanassiew, ‘Compt. Rend. Acad. Sci.,’ vol. 98, p. 1349 (1884). 
34, Pollitzer, ‘Jour. Physiol.,’ vol. 7, p. 288 (1886). 

35. Halliburton, ‘ Roy. Soc. Proc.,’ vol. 44, p. 264 (1888). 

36. Shore, ‘Jour. Physiol.,’ vol. 11, p. 561 (1890). 

37. Camus and Gley, ‘ Compt. Rend. Soe. Biol.,’ vol. 3, p. 621 (1896). 
38. Howell, ‘ Amer. Jour. Physiol.,’ vol. 31, p. 6 (1912). 

39. Mclean, ‘Johns Hopkins Hosp. Bull.,’ vol. 31, p. 453 (1920). 

40. Delezenne, ‘ Arch. de Physiol.,’ vol. 10, p. 568 (1898). 

41. Halliburton and Brodie, ‘ Jour. Physiol.,’ vol. 17, p. 157 (1894). 
42, Falloise, ‘Bull. Acad. Roy. Belg.,’ vol. 41, p. 521 (1903). 

43. Dastre and Studel, ‘Compt. Rend. Soc. Biol., vol. 55, p. 1847 (1903). 
44, Nolf, ‘Arch. Internat. de Physiol.,’ vol. 2, p. 1 (1904). 


VOL. XCIII.—B. 25 


384 Mr. D. T. Harris. 


45. Bordet and Delange, ‘ Ann. Instit. Pasteur,’ vol. 26, p. 669 (1912). 

46. Fano, ‘Du Bois Reymond’s Arch. f. Physiol.,’ 1881, p. 277. 

47. Mellanby, ‘Jour. Physiol.,’ vol. 38, p. 502 (1909). 

48. Mills, ‘Jour. Biol. Chem.,’ vol. 46, p. 191 (1921). 

49. Halliburton and Pickering, ‘Jour. Physiol.,’ vol. 18, p. 293 (1895). 

50. Pickering, ‘Compt. Rend. Acad. Sci.” vol. 120, p. 1845 (1895) ; ‘Roy. Soc. Proc., 
vol. 60, p. 337 (1896-7). 

51. Le Chatelier, ‘ Recherches sur les équilibres chimiques,’ Paris, 1888. 

52. Delezenne, ‘Compt. Rend. Soc. Biol.,’ vol. 5, p. 354 (1898). 

53. Nolf, ‘ Arch. Internat. de Physiol.,’ vol. 9, p. 407 (1910). 

54. Pavloff, ‘Du Bois Reymond’s Arch. f. Physiol.,’ 1887, p. 452. 

55. Doyon, ‘Compt. Rend. Soc. Biol.,’ vol. 68, p. 752 (1910). 


Active Hyperema. 
By D. T. Harris (Beit Memorial Research Fellow). 


(Communicated by Prof. W. M. Bayliss, F.R.S. Received January 20, 1922.) 


(From the Institute of Physiology, University College, London.) 


CONTENTS. 

PAGE 
Ae Tntro@uction: cosasiies dees ses cee cctecsee none aee saa ceae eae cee eats sav trceee cote eee ene 384 
> Ae Wey) 106 cE SSS EMO BSR nerisaobcanoscone caredoenn ea ccdoncoaskansasnoouccadcest 387 
3. /Vaso-Constrictor Nerves! vescs-ccscseaccsmaeon che cce ee coee rea ree Rete eee eee ERR EeEEEn 389 
4; Vaso-Dilator' Nerves s.osieeesse ns camter seeceecenucewceas tasaeetete eee eee Saree tates eee 390 
5 uMSta bolites:::.cnuncekacaes op dae stes ce cece eae ne teach eee eee aE Ree een 393 
Gatun tional MEby pencemidecnssecesesceeceneceecrn skies ceecbeltcnesteseteck cee eee eeeee 398 
(en @ontrollof Body, Memipenabuneln.necsecens-eeeece es -nesneenecteeee ee reseece reeset 403 
tS ai O09 sey hits} (os: Rn Soe tial denon AAOn AA aaeahtinn A araa Sacecjodadddcouadsbonc 404 


1. INTRODUCTION. 


So conflicting are the statements made by physiologists and pathologists 
alike, relating to the mechanism by which “an increased afflux of blood to a 
part” is brought about, that it was felt desirable to investigate this important 
phenomenon. Let us first consider the present state of knowledge regarding 
the possible factors involved. 

(i) Vaso-constrictor Nerves (neuro-paralytic hypereemia).—The existence of 
vaso-constrictor nerves was first demonstrated in the cervical sympathetic by 
Claude Bernard (1852), and independently by Brown-Séquard. The inter- 
ruption of the normal stream of impulses from the vaso-motor centres‘along 
these nerves gives rise to a “ neuro-paralytic hyperemia,” such as is met with 
surgically in wounds of the neck, or in lesions of the spinal cord. During the 


Actwe Hyperemia. 385 


normal functional activity of a particular organ, reflex excitation of the vaso- 
constrictor nerves of the resting organs liberates a maximum blood supply for 
the working organ; this is a regulating mechanism of the central nervous 
system which ensures a more efficient functional hyperemia of the working 
organ (Lovén, 1866 ; Bayliss, 1920). 

(ii) Vaso-dilator Nerves (neuro-tonic hypereemia).—The first evidence for 
the existence of vaso-dilator nerves was supplied by Claude Bernard (1858), 
who found that stimulation of the chorda tympani caused not only secretion 
by the submaxillary gland, but also dilatation of its blood vessels. Vulpian 
showed the reddening of the tongue upon stimulation of the lingual nerve, 
and proved that the fibres concerned came from the chorda tympani. Goltz 
had noticed that whereas stimulation of the sciatic nerve, when freshly cut, 
caused pallor of the skin of the toes and a lowering of temperature as 
measured by a thermometer placed between the toes, the same stimulation of 
the nerve two days after section caused reddening and a rise of temperature. 
He concluded that vaso-dilator, as well as vaso-constrictor nerves, existed in 
the sciatic, and that they resisted degeneration for a longer time than the 
vaso-constrictors. Bernstein showed that the vaso-dilator fibres in the sciatic 
could be excited independently by means of slow rhythmic shocks, especially 
in a leg which had been previously cooled. 

The remarkable nature of the vaso-dilator supply to the limbs, skin of the 
trunk, and probably of the ears and face, and to the intestine, was not explained 
until 1901, when Bayliss investigated vaso-dilation in these regions produced 
by stimulation of the peripheral cut ends of the corresponding dorsal roots. 
The work of Head and Campbell (1900), on herpes zoster, shows this disease 
to be due to irritative lesions of the dorsal root ganglia, which give rise to 
abnormal impulses in an efferent (anti-dromic) direction along the sensory 
fibres to the skin ; whether ‘stimulation of vaso-dilator nerves can produce 
blisters has not yet been proved. Ninian Bruce (1910) showed that the 
“neurotonic hyperemia ” (Adami), produced in the conjunctiva by an irritant 
like oil of mustard, could be prevented by first paralysing the sensory nerves 
with cocaine, or by section and degeneration ; this was explained as an “axon 
reflex,” since the hyperzemia depends on the integrity of the sensory fibre. 

(iu) Vaso-dilator Substances (a local hyperemia).—It is generally held that 
the hyperzemia of inflammation arises from the action of physical and chemical 
agents directly upon the blood-vessel wall. The great similarity between a 
wheal produced by the lash of a whip, for example, and the effects of a local 
introduction of histamine (@-iminazolyl-ethylamine) into a lightly scarified 
area of skin lends support to this view, since the depressant action of histamine 
locally upon the capillary wall is definitely known (Dale and Richards). 

2E 2 


386 Mr. D. T. Harris. 


What are the possible chemical vaso-dilator substances which may exert 
a “depressor” effect on the plain muscle or endothelium of the blood vessels 
of an organ so as to ensure a greater blood supply during activity ? Two 
possibilities are presented :— 

a. Acid Metabolites: The more or less completely oxidised products, ¢., 
CO, and lactic acid, derived from the energy-producing food-stuffs, 

8. Basie split products resulting from the wear and tear of the cell 
protoplasm. 

(iv) Functional hyperemia.—With this brief survey of the factors which 
produce vaso-dilation, we may embark upon the controversial question of the 
means whereby the functional hyperzmia of an active organ is brought about. 
Leaving out of the discussion the alterations in general blood pressure 
(vide (i)), the issue lies between neuro-muscular vaso-dilation and the 
automatic vaso-dilation produced by metabolites. On the one hand, among 
our best authorities we have some who regard functional vaso-dilation as a 
simple local effect of the metabolites resulting from activity, just as the heat 
evolved results from the chemical changes of activity, and there is, according 
to them, as little need to presume the existence of special vaso-dilator nerves 
as there is to suppose the presence of caloric nerves. 

Barcroft (1914, p. 145) found that during stimulation of the sympathetic 
fibres in the submaxillary gland (cat) by means of adrenalin, the vaso- 
constriction ordinarily produced by adrenalin on blood vessels is more than 
counterbalanced by the action of the vaso-dilator substances produced when 
the gland secretes. 

- On the other hand it is well known that stimulation of the chorda tympani 
in an atropised submaxillary gland causes a vaso-dilation in the total absence 
of secretion ; Barcroft explains this by advancing the possibility of lower 
grades of cellular activity, without external secretion occurring in such 
cases, and he actually found that during stimulation there was an increased 
oxygen intake in the atropised gland. An examination of his figures 
(p. 147) shows, for example, equal metabolic rates for a 4- and 2-fold blood 
flow :-— 

Per cent. increase in oxygen .........+++ 35 ake) 

Per cent. increase in blood flow ....... euos3 22102 


Here there must be, as Bayliss (1920) points out, a factor in addition to 
metabolites called into play; the hyperemia in the last experiment can 
hardly be caused by metabolites arising from activity of such “subliminal 
degree” ; it would appear that the neuro-muscular mechanism is also excited 
to activity. Anrep (1916) has shown that when secretin used to stimulate 
the pancreas is free from the. depressor substance, there is little or no 


Active Hyperenua. 387 


sign of vaso-dilation in the gland associated with secretin; this is also seen 
in some of the experiments of Barcroft and Starling (1904) on the gas 
exchanges in the pancreas. Thus, during the normal rate of activity in 
glandular organs, the metabolites are quickly removed and never reach a 
high concentration. This is not the case with skeletal muscle where a 
prolonged contraction compresses the blood vessels and banks up the 
metabolites. Asher (1910), by means of minute doses of sodium fluoride, 
was able to paralyse the secretory activity of glands without eliminating the 
vaso-dilator response, from which he concluded that vaso-dilation could occur 
independently of metabolites. 

The present investigation is an attempt to clear up these fundamental 
problems :— 

a. To obtain evidence, if possible, for the undoubted existence of vaso- 
dilator nerves. 

b. To determine the relative part played by metabolites and vaso-dilator 
nerves in functional hyperemia. 

e. If vaso-dilator nerves actually exist and are not an essential to functional 
hyperemia, what, then, is their function ? 


2. METHODS. 


1. Measurement of vaso-dilation: a. direct blood flow ; 
b. indirect plethysmograph. 
li. Estimation of metabolites: a. alkali reserve ; 
b. lactie acid. 


The preparation: The tongue, as pointed out by Anrep, possesses unique 
advantages for the examination of these problems: the individuality of 
function of its several nerves, which run separate courses, constituting an 
easy and very certain means of determining the cause of hyperemia. The 
functional activity is almost purely muscular, since the total mass of lingual 
glands is of comparatively insignificant amount. Dogs were found to be the 
most convenient animals for these experiments which involved a good deal 
of operative procedure, the accessibility of the structures in these animals 
reducing injury to the tissues toa minimum. A preliminary dose of morphia 
was given and complete anesthesia maintained by means of chloroform and 
ether; in the few cases in which curare was injected decerebration preceded. 
The lingual and hypoglossal nerves were exposed on both sides, and the blood 
pressure was recorded in the femoral artery. 

Venous outflow: A cannula was placed in the anterior transverse vein 
connecting the two lingual veins, with ligatures and artery forceps so placed 


388 Mr. D. T. Harris. 


as to allow of the collection of samples of blood from the lingual vein while 
permitting the normal flow in the interval. No anti-coagulant was used, 
but all tubes and cannule were paraffined. The rate of flow was noted by a 
drop recorder. : 

For the estimation of lactic acid, 20 c.c. of blood was collected directly 
into 0°5 per cent. KH2PO,; for the determination of the Py and alkali 
reserve, the blood was collected under paraffin, oxalate being used as an anti- 
coagulant. By attaching the hyoid bone to an isometric muscle-lever, the 
tongue muscles could be made to work against a resistance and develop a 
measurable tension (¢.g., fig. 5). 

Plethysmograph of the tongue: The changes in volume of a curarised tongue 
were originally studied by Anrep (senior) and Cybulski (1884), and later 
with the same methods by Piotrowski (1893). For the purposes of the 
present investigation curarisation could not be adopted for maintaining the 
tongue, or rather part of it, placidly in the plethysmograph. In order to 
permit the normal production of metabolites, a technique had to be devised 
which enabled one to take reliable records of an active muscular organ. 
Many failures were encountered before the following arrangement was arrived 
at of accommodating the whole tongue in the plethysmograph and preventing 
its moving in and out during contraction. 

The genio-hyoid muscles were detached from the symphysis of the jaw and 
the mucous membrane of the fioor of the mouth was cut through in close 
contact with the alveolar margin as far back as the anterior pillars of the 
fauces. The tongue was now drawn out ventralwards, all bleeding arrested 
by ligature and cautery, and the cut edges of the mucous membrane 
sewn together round the genio-hyoid muscles, carefully avoiding pressure on 
the lingual vessels and nerves; the whole tongue could thus be placed in 
the plethysmograph. To prevent traction on the root of the tongue from 
without, a tracheal canula was inserted, the trachea being completely cut 
across ; all muscles coming up to the hyoid were cut between ligatures, and 
all branches of XII, other than those to the tongue, were severed; finally, 
to completely immobilise the larynx and hyoid,a steel rod placed through 
the mouth into the larynx, and emerging from the upper end of the 
transected trachea, was firmly clamped to the same stand as the 
plethysmograph. 

A plethysmograph of circular cross-section was selected which just fitted 
the tongue; it was fitted with a cuff of membrane rendered air-tight, yet 
soft, by means of vaseline. When adjusted it was covered with cotton- 
wool and connected to a piston recorder: time was allowed for temperature 


adjustment. 


Active Hyperanua. 389 


3. THE VASO-CONSTRICTOR NERVES. 


The vaso-constrictor fibres are supposed to be derived from the cervical 
sympathetic and run in the hypoglossal nerves: the sympathetic fibres for 
the muscles of the tongue seem to run, not in the hypoglossal, but in the 
chorda tympani (Boeke, 1921). 

The right vago-sympathetic trunk was ligatured in the neck, eut below 
the ligature, and the vagal portion was transected at its emergence from the 
skull. Faradisation of the peripheral end of the R. sympathetic affected the 
venous outflow in the manner shown in fig. 1. 

During stimulation the drops are seen to become less frequent and on 
removal of the stimulus the still further decrease indicates that either the 
vessels immediately relax and accommodate the first outflow of blood, or there 
is a marked “ after effect” following the stimulus. 

Faradisation of the hypoglossal nerve in the curarised animal verifies the 
fact that the vaso-constrictors run’ in this nerve (fig. 2), as was first clearly 
shown by Anrep (senior). 


ms 


TOA TTT 


| Single B shock 10000. e 
Sees... Se ore 


Fie. 3. 


Stimulation of the made-up hypoglossal nerve with only a single shock (to 
avoid any appreciable “metabolite” effect) causes an emptying of the 
collapsible veins and capillaries by simple compression of the contracting 
skeletal muscle. It is here suggested that the arterioles, by the simultaneous 


390 Mr. D. T. Harris. 


stimulation of the vaso-eonstrictor nerves have their lumina rendered less 
liable to extinction: this would prevent a reversal of the blood stream and 
maintain a patent system for the onrush of blood following repeated 
contractions (fig. 4), or a prolonged contraction (fig. 5). It is difficult to 


Stim. 
MsingeB  MsuccessiveB - 


rarpererenrrm neat memset IT ha 


= 


eal 


Fic. 4. 


Lh. lever: 


Fig. 5. 


imagine what function vaso-constrictors, bound up with a motor nerve, 
might serve. Fig. 5 shows the steady diminution of the tension developed 
in the tongue arising from the anemia produced by its own contraction. 


4. THE VASO-DILATOR NERVES. 


Fig. 6, in the opinion of the writer, constitutes the most convincing single 
piece of evidence in favour of the undoubted existence of vaso-dilator nerves. 
Such a huge vaso-dilation in the absence of any muscular activity must be 
independent of metabolites. The effect produced by a similar shock upon the 
motor nerve is seen in fig. 3, where one would expect a greater production of 
metabolites, yet vaso-dilation is absent. 

How very effective a mechanical stimulus is in evoking vaso-dilation 
(Bayliss, 1920), by excitation of the lingual nerve, is seen in the plethysmo- 


a 


graphic record of the tongue in fig. 


Active Hyperamia. 391. 


It must be acknowledged, however, that the vaso-dilator fibres have a 
long latent period (5 seconds), time enough to allow of the production of 
metabolites. To see the drops of venous blood issuing from the lingual 
vein suddenly burst into a continuous stream of bright arterial colour on 
stimulation of the lingual nerve while the tongue remains in a state of 
restful indifference, is a most simple and convincing demonstration of the 
individual existence of vaso-dilator nerves. 


_ Stngle induction shock 4000KU. 
Lingual we eub): o68 aes 


Epa tes 


IBne, th, 


Let us examine further the theory which explains vaso-dilation by the 
action of metabolites. This theory has been based upon the metabolism of 
the submaxillary gland as measured by its oxygen intake, which is found to 
be increased in all conditions in which vaso- dilation accompanies excitation 
of its nerves, and this increased gaseous metabolism only slowly dies down 
(Bareroft and Kato, 1915). The oxygen consumption in the tongue has been 
recently measured by Anrep and Evans (1920), and the mean result of their 
experiments is shown in the following Table. 


392 Mr. D. T. Harris. 


| Flow of blood. Oxygen used. 


c.c./min | ¢.¢./min. 
Pirin gprestie ce leslees aleeeaeee 0°59 | 0-144 | 
| 
Stimulation of lingual nerve...... 2°85 0-142 


From this it will be seen that the stationary oxygen consumption lends no 
support to the metabolite theory for the explanation of the five-fold increase 
of blood flow obtained by these experimenters. 

Measurement of the alkali reserve of the blood from the lingual vein and 
the blocd from the femoral artery during stimulation of the lingual nerve 
shows them to be practically identical. This is seen in the following figures, 
obtained from different dogs under widely varying conditions :— 


ae é 3 
Stimulation of lingual Alkali reserve of plasma. 


nerve. 
| | | 
| dhingnal vein’ -.5..: cee | 49-5 53°5 | 67-0 69 | 70 48°5 
| | | 
| Femoral artery ............... 49°5 | 54:0 675 69...» |noniz td Opes ASO, 


Thus, interrupted faradisation of the lingual nerve produces no measurable 
difference in the alkali reserve (CO2 metabolite), even when the vaso-dilation 
is So great as to increase the blood-flow five to eight times, as in the above 
experiments (of course, the dilution of the metabolite proceeds at the same 
rate). In so far as gaseous metabolites are concerned, there is no denying 
the existence of vaso-dilator nerves. 

Furthermore, no real difference was detected in the lactic acid-content of 
the blood issuing from the tongue during stimulation of the lingual nerve as 
compared with that in the arterial femoral blood, eg., femoral artery, 
64 mgrm. per 100 ¢.c.; lingual vein, 6-0 mgrm. per 100 e.c. 

If vaso-dilator substances are produced when the lingual nerve is excited, 
then they must necessarily be of a non-oxidative character and presumably of 
a very powerful nature; the later stages of this investigation will show that 
such an assumption is unnecessary, since the vaso-dilator nerves of the 
tongue possess independent functions, and are not subservient to the func- 
tional activity of the tongue muscles. 

The glossopharyngeal, the sensory nerve of the posterior third of the 
tongue, responds to stimulation just as the lingual does, but in a milder 
degree (fig. 8); eitherit contains specific vaso-dilator fibres (via tympanic 
branch, Loeb and Eckhard), cr it is capable of conducting anti-dromic vaso- 


Actiwe Hyperemia. 393 


dilator impulses similar to those found by Bayliss (1901) in the sensory nerves 
of the limbs. 
5. METABOLITES. 

Gaskell (1880) was the first to lay stress on the importance of the local 
vaso-dilator action of acid metabolites ; he observed the effect of lactic acid on 
the curarised mylo-hyoid of the frog, and measured with a micrometer eye- 
piece the dilatation of the blood-vessels. About the same time Severini put 
forward the view that “the increased flow of blood through an organ when it 
is in a condition of activity is due to the trophic dilatation of the capillaries, 
and not to relaxation of the vascular muscle.” He stated that oxygen diminishes 
the size of the capillary lumen, because the nucleus of the cells of the capillary 
wall (nucleus of Golubew) becomes more spherical, while conversely with 
the action of CO, it flattens out in the cell, and so the lumen is greater. 

More recently, Krogh (1919) has observed the circulation in the thin 
muscles of frogs and guinea-pigs, chiefly by reflected light, with a binocular 
microscope ; he finds that when muscles contract, either spontaneously or as 
a result of artificial stimulation, many more capillaries spring into view, and 
when the contraction is over they disappear again; capillary dilatation also 
occurs when certain irritants and narcotics are applied locally to the tissue, 
and in these cases the capillary dilatation is not to be explained by dilatation 
of the arterioles. This independent state of contractility of the capillaries 
is seen in the action of histamine, which dilates the capillaries and constricts 
the arterioles (Dale and Richards, 1918). This drug exerts such a powerful 
action on the vessels that the injection into an animal of an amount equal to 
one-millionth of its weight will cause a fall of blood-pressure to one-half 
and a condition indistinguishable from surgical shock (Dale and Laidlaw, 
1919). The possibility of the production in minute quantities of similar 
substances during cellular activity must be kept in mind, though none have 
been isolated up to the present. Still, it must be pointed out that the vaso- 
dilation produced by the action of histamine in cats and dogs is replaced by 
vaso-constriction in the guinea-pig, so that it would be totally unjustifiable to 
assert that this substance plays the part of a general metabolite to bring 
about vascular dilation in active organs. It must also be mentioned that 
adrenalin in very small doses also possesses a dilator action on the capillaries 
(an effect which appears to be independent of the sympathetic), but the 
concurrent action on the arterioles is more pronounced than is the case with 
histamine (Dale and Richards, 1918). 

A similar condition of affairs is seen in chilblains, when the capillaries are 
distended with blood of venous colour, owing to the impoverished oxygen 
supply resulting from the simultaneous arteriolar constriction. 


394 Mr. D. T. Harris. 


Functional Activity. 


A plethysmographic record of the tongue during the application of a single 
induction shock to the hypoglossal nerve (fig. 3) shows a simple compression 
of the collapsible vessels by the contracting muscles and a complete return to 
normal without any appreciable after effect of vaso-dilation. 

When a tongue is made to develop tension against a muscle lever we note 
during the application of a series of.successive induction shocks (fig. 9) a vaso- 
dilation which lasts for more than a minute after stimulation (see also Verzar 
1912). One of the earlier (imperfect) plethysmographic records shows this 
hyperemia very well (fig. 10), the vaso-dilation nate a pulsatile tongue. 


wh vba Aaa 


BaP 


Myogrom 


Stim. 


Secs.” * Wineraalk 7 sec. 30K, 


“Se aaa 


Sates 9. 


eee 


B shocks (20KU) 


Fie. 10. 


During faradisation of the motor nerve the blood flow is almost completely 
arrested: by the compression of the contracting muscles (fig. 11). It is obvious 


Active Hyperemia. 395 


that when a muscle is exerting its maximum effort and there is no alternation 
of contraction between groups of muscle-fibres, to sustain the effort for a 
considerable time involves the working of the muscle in the complete 
absence of fresh blood; in addition to the possible production of metabolites 
of activity we have here to consider the development of asphyxial products. 
This is a point of great importance in physical culture, eg., the maintenance 
of a continued position like “attention.” The phenomenon is seen in its 
most exaggerated form in muscular “ cramp.” 


NERANAAAA Aaa 
STyogram 


JOKL, - 
leago: SYE cal 


Fie. 11. 
Ischemia: The vaso-dilator effect of asphyxial products may be seen by 
placing artery forceps on the lingual arteries (fig. 12); on releasing the 
forceps the tongue dilates to an extent greater than in the resting condition 


and pulsation becomes more marked. This result confirms the findings of 
Anrep (1912) for the fore-limb. 


Fira. 12. 


Venous Congestion: Compression of the lingual vein leads to venous engorge- 
ment of the tongue; on releasing the clip the venules quickly empty but the 


396 Mr. D. T. Harris. 


arterioles and capillaries, probably, remain dilated until the asphyxial products 
are removed (fig. 13). The long upward curve corresponds to the stage of 
“passive hyperemia” while the swelling a few seconds after the release of the 
compression is a true active hyperemia; the former is therapeutically 
regulated in Bier’s treatment, though even here the long application of a 
tourniquet may also involve another doubtful factor—the swelling of the 
body colloids (M. Fischer, 1920). 


On / min. Off 


Ling veins clipped’ * 


Fic. 13. 


Both ischemia and congestion produce like effects and the asphyxial products 
arising from them are additive (fig. 14). A corresponding additive effect is 
obtained with the metabolites of activity and asphyxial products (fig. 15), 
from which a similarity between these substances may be inferred. 

Chemical Vaso-dilator Substances: Schwarz and Lemberger (1911) found 
that the injection of 1 ¢.c. of molar HCl into the central end of the left 
subclavian artery caused obvious dilatation of the blood vessels of the sub- 
maxillary gland. Comparing different acids, their action was not found to 


Lingual art§ and 
veins Clipped 2 mins. 


Fic. 14. 


Active Hyperemia. 397 


correspond to the H-ion concentration. The effect, as they point out, is 
really produced by CO: driven off from the bicarbonate of the blood. Acids 
weaker than CO. were inactive. 


.: is: A Stih® 
Clip on lingual art$ | TT 


ingest inte a 


SEPPCEPPETRETERECT ETC LET POPES TnRTtECEVPCPrrrrryrrreterecc terre. ireeeheort (rensr ener ry eran) 


Fie. 15. 

In order to put this to a more direct and measurable test, 1 c.c. of Ringer’s 
fluid containing the substance was slowly introduced through a fine hypo- 
dermic needle immediately into the lingual artery without occluding it, thus 
minimising the capacity effect. Plain Ringer, sodium bicarbonate, and lactic 
acid were found to produce a vaso-dilation in the order named (so that the 
effect of Ringer alone must be discounted), but the effect produced by lactic 
acid was much the largest and most prolonged (fig. 16) and was in all respects 


i ancl ees is 
we Wont Wy, eA Mec Way \aa/Wn agsrnotratrteety Nays 


\ ag 
Iv WM eh ane aa et 


NYA riyyyaynivie 


| ft 100. 2% ladle acid L 
mpected tito ey: ant? 


SS RI AIR I A A I EN IT 


ine 16. 


‘1% lacticac. 


peal lingent og ae Zee Left ing.artY 


Iie. 17. 


398 Mr. D. T. Harris. 


similar to the vaso-dilation produced by stimulation of the lingual nerve 
(compare figs. 17 and 6) and by the normal metabolites arising from muscular 
activity (compare figs. 17 and 4). Lactic acid, directly or indirectly, is 
therefore a true vaso-dilator substance. 


> 
6. FUNCTIONAL HYPER2MIA. 


We must now arrive at a decision regarding the mechanism by which the 
vaso-dilation accompanying muscular activity is brought about. Let us 
consider the first possibility, viz., that some form of reflex excites the vaso- 
dilator nerves into action at the same time as or soon after the stimulation 
of the motor nerve. 

In glandular organs, the vaso-dilator fibres run in the same bundle as the 
secreto-motor fibres (¢.g., the chorda tympani to the submaxillary gland) and 
do not admit of direct analysis; the attempt to block one or other by means 
of drugs is not as convincing as simple section. 

In our preparation, however, it is the vaso-constrictor fibres which run with 
the motor nerve while the fibres with a vaso-dilator action are bound up in the 
separate sensory nerve; we are thus enabled, by means of section, to interrupt 
any reflex arc (unless it be a peripheral axon reflex) through which vaso- 
dilation might be produced during motor activity. 

An examination of the accompanying plethysmographic records (figs. 18 . 
and 19) shows that as the initial mechanical stimulation of the lingual nerve 
(fig. 7) passes off, the vaso-dilation produced by faradisation of the hypoglossal 
nerve gradually becomes as great after as before section of the lingual nerve. 


Na 


a a aR i 


5secsE. 5secs F 


MM perigh. ~ Mperiph. MI periph. 
(Linguuts cul) aoe lh a 
Fie, 18. 


Division of the motor nerve makes no difference to the form of the curve, 
the same degree of dilation being produced by stimulating the uncut nerve 


Active Hyperemia. 399 


as by stimulating the peripheral end of the divided hypo-glossal (figs. 19 
and 20). 


PA ANN Nai Mi ANN ! 


‘Hh HAN mt INA Nh EAM et NAW 


AAAI AANA 


AD urecut Lig. of Al. 


Fig. 19. Fic. 20. 


A large number of experiments was performed in the attempt to analyse 
the muscular contractions of the tongue during swallowing, elicited by 
. stimulating the central end of the superior laryngeal nerve. The resulting 
ql vaso-dilation was inappreciable, since the long period of rest between successive 


swallowings (5 seconds or more), allowed of the complete removal of meta- 
. bolites; and, on the other hand, section of both lincual nerves made no difference. 
In the absence of any definite evidence for a reflex excitation of the lingual 


nerve, we must examine the metabolite hypothesis as a complete explanation 
for the hyperemia of muscular activity. We have seen that lactic acid 
injected into the lingual artery can produce a typical vaso-dilation. Is lactic 
acid produced in appreciable amount during muscular activity ? The answer 
is in the affirmative, if, in Ryffel’s method (1909), it is really lactic acid which 
is being determined ; estimations by this method gave the following results :— 


Lactic acid in mgrm. per 100 c.c. lingual blood 
during stimulation of 


Lingual nerve. Hypoglossal nerve. 

WD O geste escent ar 6:0 40-8 

| 25 ‘6 (3 mins. later). 
ier slvte eR 6:4 536 

48,0 (1 min. later). 

PGT WBN Cs 16-0* 64-0 
ES ee ee 8-4. 40 °8 
Mean ......... 9-2 49-8 


* After resuscitation from temporary asphyxia. 


bo 
bey 


VOL, XCIII.—B. 


400 Mr. D. T. Harris. 


During stimulation of the lingual nerve there is the same concentration 
within the limits of experimental error, of lactic acid in the blood of the 
lingual vein as there is in the femoral arterial blood (vide infra). Thus one 
finds the lactic acid increased about five-fold in the venous blood of these 
experiments, in which the blood flow was maintained at twice its normal rate 
by interrupted faradisation of the motor nerve; if the normal rate of venous 
outflow had prevailed then the lactic acid concentration would have risen ten- 
fold ; and in tetanus its concentration must be tremendously high. 

The production of lactic acid would lead one to expect a reduction in the 
alkali-reserve according to the equation 


H. C3H;O3 SF NaHCO; = NaC3H;03 + H,0+ COz 


but estimations show a simultaneous increase in the alkali-reserve :— 


Tongue. 
During stimulation of 
Alkali reserve of Resti 
esting. 
plasma. 
Lingual nerve. Hypoglossal nerve. 
Venous. Arterial. Venous. Arterial. Venous. | Arterial. 
| q 
1 Daye LS ie yea aoe 55 55 54°5 55 59 54 
BBG Me cel! athe hea 71 70 71 71 82 "6 
83 74 
55 Un NARA Se eR TR EM cn 64 64 — — 69 64 
ee PUB cosenaeu on emae 52 50 ‘5 — _ 64 54 
SOWA ee CONN Re — | — 49 °5 50 57 50 
at Oger ula lee 61 | 59 60 | 59 73 59 
DAM Sere ay elicit lta — | — 47 | 48 59 43 
Mean v—@......... 1 = 9 


This absolute rise of nine in the alkali-reserve on a mean arterial alkali- 
reserve of 52°5 is equivalent to an average increase of 16 percent. It is 
abundantly clear that both CO, and lactic acid are important metabolites of 
muscular activity. Fletcher (1907) found that an evolution of COs follows a 
production of lactic acid in the frog’s gastrocnemius. 

The carriage of such large increases of acid by the blood became a problem 
of so much interest that the relative part played by plasma and corpuscles 
was investigated. The blood was collected under paraffin, NaF being used 
as an anti-coagulant, since it possesses the double advantage of not interfering 
with the estimation, and also preventing glycolysis (Clogne et Richaud), 
elycolysis having been shown to be associated with the formation of lactic 


Active Hyperama. 401 


acid in shed blood by Evans (personal communication). The results obtained 
are shown here :— 


Stimulation of 
Lingual nerve. Hypoglossal nerve. 
Lactic acid 

(mgrm. in 100 c.c. each of) Plasma. Corpuscles. Plasma. Corpuscles. 
OE ee cisayscritccauraucnce reno tets 16 ‘0 16 ‘0 62-0 68 -0 
SOM ON Lira a ntsetheae telom doles PNoSeRi 12°5 129 18 *4 16-0 
(pi) -| CE ence Ree ERE RROpeE Sane nneeSE aoe 8-0 8°8 16-0 20°8 
Mie ae ye aes ac sense aenrienemese 12-2 126 32-1 349 


Taking into account the hematocrite values of these bloods, the lactic acid 
would appear to be evenly diffused through plasma and corpuscles. 

The corpuscles from exercised muscles seemed to show an acidity of their 
contents markedly higher than the plasma in which they were laked even 
when NaF was used as the anti-coagulant (vide infra). Two dialysing sacs 
were prepared (Dale and Evans, 1920) and half filled with saline and water 
respectively (both of known Py and corrected for) and a comparison made of 
the Py; such results as the following were obtained :— 


Dog 25. Dog 26 
Corpuscles intact (in saline) ............... 735 740 
Ruptured corpuscles (in distilled water) 720 7:25 


It must be mentioned, however, that laked corpuscles (frozen and thawed) 
always exhibit a slightly lower Py than intact corpuscles. 

An increase of lactic acid in the blood is thus seen to be not incompatible 
with a rise of alkali reserve in the plasma, in view of the share played by the 
corpuscles in bearing the onslaught of the acid attack. It must further be 
admitted that the red corpuscle is permeable to the lactic acid ion, and the 
following is advanced as a possible explanation of the events :— 

1°. Diffusion of the lactic acid from the working muscle into the alkaline 
tissue fluid 

Lactic acid + NaHCO; —= Sod. lactate + H2COs. 


The carbonic acid given off diffuses through the vessel wall where it exerts 
its vaso-dilator effect (wide figs. 4 and 17; note in the latter the direct 
astringent effect and the indirect effect of lactic acid due to COz). 

as 


402 Mr. D. T. Harris. 


2°. Diminution of the plasma chlorides and increase of the alkali reserve 
(L. J. Henderson, 1909) 


H2CO3+ NaCl == NaHCO;+ HCl. 


from 1° of plasma alkali reserve. 


3°. Some free lactic acid is formed from part of the sodium lactate in (1) 
and part of the HCl in 2° 


Sod. lactate ++ HCl —~> Lactic acid + NaCl. 


This lactic acid penetrates the corpuscles just as the HCl from 2° does 
(Hamburger, 1904) and the HCO; from 1° (Buckmaster, 1918). 

4°. The oxidative removal of lactic acid continues for some minutes 
(Fletcher and Hopkins) and coincides with the recovery heat production (Hill) 
and the increased oxygen intake (Verzar) following muscular activity. 

It is believed by many, though on insufficient data, that lactic acid and CO, 
act by increasing the H-ion concentration. Calculation of the Py of the blood 
from measurements of the alveolar CO2 may show a difference of 0:02 during 
muscular exercise (Campbell, Douglas, Hobson, 1914); a rise of acidity has 
been inferred from estimations of the affinity of the blood for oxygen 
(Mathison); more recently Barcroft and Parsons (1920) have found that 
muscular work causes a fall in Py of 0:08 in the blood when defibrinated-and 
completely reduced. 

The erythema arising in electro-therapeutics in the area of the kathode 
suggests the possibility of a direct action by the H-ions (or Na-ions?). In 
normal activity, such a direct action of lactic acid is impossible in the 
presence of the bicarbonate of the tissue fluids; the increase of alkali reserve. 
seen to occur above must also tend to keep down the H-ion concentration 
of the plasma, hence the ultimate depressant of the blood- vessel wall must be 
the COs. Patterson and Starling (1914), working on the isolated heart-lung 
preparation, have shown that the heart relaxes more and more as CQz is 
added to the air ventilating the lungs. Evidence is accumulating that CO» 
possesses specific properties in its action upon the tissues which are not 
possessed by other acids (Schwarz and Lemberger, 1914, C. Lovatt-Evans) ;. 
fig. 17 is interesting in this connection. ; 

Finally, we have shown in §5 that CO, and lactic acid, when injected into 
the blood stream, act as powerful vaso-dilator substances, and again in § 6. 
that these acid metabolites are formed in increased amount during muscular 
activity. In so far as functional hyperemia is adequately explained by the 
vaso-dilator action of metabolites, we are in full agreement with Barcroft. 
(1914); but in §4 we have shown the separate existence of vaso-dilator 
nerves which were not, at least in the above experiments, called into play 


Active Hyperemia. 403 


during muscular activity. We must consequently expect to find other 
functions for the vaso-dilator nerves. 


7. CoNTROL OF Bopy TEMPERATURE. 


The fortunate employment of the dog gave the clue to a possible function 
of the vaso-dilator fibres in the lingual nerve. It is a common fact that dogs 
resort to panting and extrusion of the tongue for cooling of the blood. The 
effect upon the blood-flow in the tongue resulting from the stimulation of the 
heat receptors in the skin was therefore studied. 

The fur was cut off, a radiant-heat hemi-cylinder was placed over the trunk 
of the animal, the venous outflow from the lingual vein recorded, and the 
rectal temperature observed throughout the experiment. 

The warming of the skin caused hyperemia and increased lingual blood- 
flow long before the body temperature was appreciably raised (fig. 21, i and ii). 
Tn all cases where this response did not occur the body temperature steadily 
advanced ; Stewart (1913) also emphasises the importance of reduced blood- 
flow in fever. 

To test whether this was an effect of heated blood directly upon the vessels 
or a reflex mechanism, the lingual nerves were cut on both sides and time 
given for the effect of this stimulus (fig. 7) to pass off. The application of 
radiant heat to the skin of the trunk was now not followed by lingual 
hyperemia (fig. 21, ii and iv). 


Lingnvs.cut  -  Lény.nws.cut 
vii DV 


hes, Pil. 


The same results were obtained in another dog, which was immersed in a 
warm bath. It is thus apparent that a nervous reflex of this nature brings 
about a quicker response than could be obtained by the heating up of the 
whole blood; and, further, the nervous reflex ensures vaso-dilation of that 
particular organ which is most favourably disposed for the cooling of the 
blood, whereas heated blood would have a generalised effect on external and 
internal organs alike. It is possible that the flushing of the face in man in 
emotional states and in fever may also be accounted for by similar reflexes 


404 Mr. D. T. Harris. 


involving vaso-dilator nerves. Measurements of the blood-flow, by Stewart’s 
method, in the hands and feet during fever show that the cutaneous vessels 
dilate during fall of temperature by crisis. 

Whether the vaso-dilation of the tongue plays any part in the sensation of 
taste was not investigated. 


CONCLUSIONS. 


1. The lingual nerve contains true vaso-dilator fibres, just as the sympa- 
thetic contains vaso-constrictor fibres; both are equally independent of the 
intervention of metabolites. 

2. No evidence was found that functional hyperemia is due either to 
diminution in vaso-constrictor tone or to increase in vaso-dilator tone. 

3. The experiments show that the increased blood-supply during muscular 
activity is due entirely to the products of metabolism; the absence of a 
simultaneous excitation of the vaso-dilator nerves during voluntary move- 
ment, though probable, is not proved. 


4. Of the metabolites estimated, CO2 and «-OH organic acids were found 
to be increased. 


5. Apart from muscular activity, one function of vaso-dilator nerves 
was found to be concerned with the control of body temperature; active 
hyperemia in the dog’s tongue may be induced by reflex excitation of the 
vaso-dilator nerves through stimulation of heat veceptors in the skin.* 


REFERENCES. 


1884. Anrep, V. (senr.), and Cybulski, N., “On the Physiology of the Vaso-dilators and 
Vaso-constrictors,’ quoted in Hofmann and Schwalbe’s ‘Jahresber.,’ vol. 13, 
pp. 49-55. 

1912. Anrep, G. V., ‘J. Physiol.,’ vol. 45, pp. 318-327. 

1916. Anrep, G. V., ‘J. Physiol.’ vol. 50, pp. 421-433. 

1920. Anrep and Evans, ‘J. Physiol.,’ vol. 54 (‘ Proc.,’ p. 10). 

1910. Asher, L., ‘ Pfliiger’s Archiv,’ vol. 136, pp. 421-428. 

1914. Barcroft, J., ‘The Respiratory Function of the Blood,’ Cam. Univ. Press. 

1915. Bareroft, J., and Kato, ‘ Phil. Trans.,’ B, vol. 206, pp. 149-182. 

1920. Barcroft and Parsons, ‘J. Physiol.,’ vol. 53 (‘ Proc.,’ p. 110). 

1904. Barcroft and Starling, ‘J. Physiol.,’ vol. 31, p. 491. 

1920. Bayliss, W. M., ‘Principles of General Physiology,’ Longmans, 3rd edition, 
pp. 690-707. 

1901. Bayliss, W. M., ‘J. Physiol.,’ vol. 26, pp. 173-209 ; vol. 26 (Proc. 32); vol. 28, 
pp. 276-299. 

1908. Bayliss, W. M., ‘J. Physiol.,’ vol. 37, pp. 264-277. 

1852. Bernard, Claude, ‘Compt. Rend. Soc. Biol.,’ vol. 4, pp. 168-170. 


* Note.—The expenses of this work were defrayed by a grant from the Government 
Grant Committee of the Royal Society. 


1858. 
1921. 
1852. 
1910. 
1914. 
1921. 
1920. 
1910. 
1919. 
1918. 
1921. 
1907. 
1907. 
1920. 
1880. 
1869. 
1874. 
1904. 
1916. 
1900. 
1909. 
1913. 
1870. 
1866. 
1893. 
1914. 
1909. 
1911. 
1878. 
1917. 
1921. 
1913. 
1912. 
1880. 


Active Hyperemia. 405 


Bernard, Claude, ‘Compt. Rend. Soc. Biol.,’ vol. 46, pp. 159-165. 
Boeke, J., ‘ Brain,’ vol. 44, pp. 1, 18. 

Brown-Séquard, ‘ Philadelphia Medical Examiner,’ vol. 8, pp. 481-504. 
Bruce, A. N., ‘Archiv Exper. Pathol.,’ vol. 63, pp. 424-433. 
Campbell, Douglas, Hobson, ‘ Phil. Trans.,’ B, vol. 210, pp. 1-47. 
Clogne et Richaud, ‘ Bull. Soc. Chim. Biol. ITI,’ vol. 2, 66. 

Dale and Evans, ‘J. Physiol.,’ vol. 54, pp. 3, 167. 

Dale and Laidlaw, ‘J. Physiol., vol. 41, pp. 318-344. 

Dale and Laidlaw, ‘ J. Physiol.,’ vol. 52, pp. 355-390. 

Dale and Richards, ‘J. Physiol.,’ vol. 52, pp. 110-165. 

Evans, C. L., ‘J. Physiol.,’ vol. 55, p. 176. 

Fletcher, W., ‘J. Physiol.,’ vol. 35, p. 247. 

Fletcher and Hopkins, ‘J. Physiol.,’ vol. 35, p. 301. 

Fischer, M., ‘Gidema and Nephritis,’ 3rd edition. 

Gaskell, W. H., ‘J. Anat. and Phys.’ vol. 11, p. 720. 

Golubew, ‘Archiv f. mikros. Anat.,’ vol. 5, p. 49. 

Golz, F., ‘ Pfliiger’s Archiv,’ vol. 9, pp. 74-197. 

Hamburger, H. J., ‘Osmotische Druck und Ionenlehre,’ (3 vols.). 
Hamburger, H. J., ‘Wiener Med. Wochenschr.,’ vol. 46, p. 521. 
Head and Campbell, ‘ Brain,’ vol. 23, pp. 353-524. 

Henderson, L. J., ‘ Regulation of Blood Neutrality.’ (Monograph.) 
Hill, A. V., ‘J. Physiol.,’ vol. 46, p. 28. 

Loeb, L., ‘ Beitrage zur Anat. u. Physiol.,’ Giessen, vol. 5, p. 1. 
Lovén, C., ‘ Ber. Sachs Ges. (Leipzig),’ vol. 18, pp. 85-110. 
Piotrowski, G., ‘ Pfliiger’s Archiv,’ vol. 55, pp. 240-302. 

Patterson, Piper and Starling, ‘J. Physiol.,’ vol. 48, pp. 465-513. 
Ryffel, J. H., ‘J. Physiol.’ (Proc., pp. 9 and 31), 

Schwarz and Lemberger, ‘ Pfliiger’s Archiv,’ vol. 141, pp. 149-170. 
Severini, L., ‘ Ricerche sulla Innervazione dei Vasi Sanguinei,’ Perugia. 
Slyk, D. Van, ‘J. of Biol. Chem.,’ vol. 30, pp. 289-368. 

Slyk, D. Van, ‘ Amer. J. Phys. Reviews,’ vol. 1, 1, pp. 141-176. 
Stewart, G. N., ‘J. Exper. Med.,’ vol. 18, p. 372. 

Verzar, ‘J. Physiol., vol. 44, pp. 243 ; vol. 45, p. 39. 

Vulpian and Bouchefontaine, ‘Compt. Rend.,’ vol. 319. 


406 


The Acidity of Muscle during Maintained Contraction. 
By H. E. Roar. 


(Communicated by Sir Charles S. Sherrington, Pres. R.S. Received March 11, 1922.) 


(From the Department of Physiology, London Hospital, Medical College.) 


In 1913, I described a method for recording changes in hydrogen-ion 
concentrations in tissues, by means of a manganese dioxide electrode in 
combination with a calomel electrode (1). By this method it was shown that 
the acidity of muscle probably increased at the same time as, or slightly 
before, the tension increased, and that the acidity decreased as the muscle 
relaxed (2). 

In a paper, which appeared as this note was being prepared for publication, 
Ritchie* states that he has been unable to detect a variation in acidity by the 
use of manganese dioxide electrodes. I am inclined to think that his failure 
is due to the injury to the muscles on insertion of wires into its substance. 
In my own experiments the wires rest on the surface of the muscle. 

The electrical change observed by me is not due to spread of potential from 
the stimulating circuit, because in some records a potential is recorded 
corresponding to the time of stimulation. This potential is quite separate 
from the larger potential ascribed to the acid production, and it may be in the 
same or in an opposite direction from that which accompanies the contraction. 

In my experiments I took precautions to minimise movement of the 
electrode on the surface of the muscle, and any slight movements are probably 
less important with the sartorius than with the gastrocnemius. Change of 
potential, due to movement of the electrode, would probably vary in direction, 
whilst with the manganese dioxide electrode the change is always in the same 
direction. If the electrode did move on the surface of the muscle it is unlikely 
that the potential would return to its former value, as it is seen to do in most 
of my records. 

Other forms of metallic electrodes would give changes of potential depending 
on the chemical reactions that take place in contact with them. 

I do not think that the change shown by a manganese dioxide electrode is 
due to polarisation. If the electrodes are so placed that no difference of 
potential is shown during contraction by non-polarisable electrodes, it is 
difficult to see how any difference of potential will be produced when one 
calomel electrode is substituted by a manganese dioxide electrode and the 


* A.D. Ritchie, ‘ Journ. Physiol.,’ vol. 56, p. 53 (1922). 


The Acidity of Muscle during Maintained Contraction. 407 


contact potential has been properly balanced. If there is no difference of 
potential there cannot be any current, therefore there will be no polarisation. 

If acidity is related to tension in muscle it is necessary to show that 
acidity remains when the muscle tension is maintained, and decreases when 
the tension falls. In order to test the relation of acidity to muscle tension 
two experimental procedures were tried. 

In the first, veratrin was painted on the frog’s muscle after a record of the 
normal contraction had been made. The out-standing feature of the action of 
veratrin is that it does not affect the increase in tension of the muscle, but 
that it delays the relaxation. 

With such a preparation the acidity, as shown by the manganese dioxide 
electrode, remains also. Fig. 1 shows a muscle twitch of a fresh sartorius, 
whilst fig. 2 shows the result with the same muscle after it had been treated 
with veratrin (1: 10°). 

For the second procedure a decerebrate cat was used. This preparation 
shows a marked tension (decerebrate rigidity) in the limb muscles. The 
tension can be abolished by cutting the efferent nerves to the muscles, or by 
refiex inhibition as the result of stimulation of an afferent nerve. 

In decerebrate preparations the rigid muscles show a greater acidity than 
when the muscles are paralysed by cutting their efferent nerve supply, as 
shown by the following results :— 


Table IL—Decerebrate Preparations showing Potential in Volts between a 
Manganese Dioxide Electrode and a Calomel Electrode. 


R. Sartorius. L, Sartorius. 
Date. | > | | 
: if 
Rigid. AUD: CUTIES Difference.| Rigid. AEE cutting _ Difference. 
nerve. nerve. 

21.7.13 | 0 °325 0-004 0-321 0-186 | —0:°169 | 0 355 
14714 ee 0-812 0 :249 0-063 0:°249 «| 0 °242 0 :007 
| 8.3.21 4 0°355 0-206 0-149 GESEGL Oc ral 0-095 
Average ......... 0°178 AMISEIRE 5 cococe 0-152 


Although the absolute values of these are unreliable it is gene Ones paralysing the muscle 
always gives a result corresponding to a decrease in acidity. 


In order to demonstrate further that removal of maintained contraction 
causes decrease in acidity, a reflex vasto-crureus preparation was used (3). 
The sartorius was removed and the electrodes placed on the surface of the 
vasto-crureus. On causing a reflex inhibition by stimulation of the ipsilateral 


408 Mr. H. E. Roaf. The Acidity of 


Fie. 1.—Simple muscle twitch. 


Ieper acess ne AL nwo, Serve 


Fie. 2.—Record of same muscle after treatment with veratrin (1:10°). Records read 
from right to left. ‘Topline, time in 1/5”. Second line, signal for single break-shock 
given to muscle. Bottom line, downward movement shows increased tension by 
muscle. Shadow shows movement of mercury in capillary. Movement upwards 
corresponds to increased acidity at manganese dioxide electrode. 


Muscle during Maintained Contraction. 409 


sciatic nerve, as shown in fig. 3, the end result corresponds to a decrease in 
acidity. The initial result is still doubtful, as the figure reproduced shows a 
movement of the mercury in the opposite direction before the inhibition 
occurs. This result may be due to either a slight contraction before relaxa- 
tion,* or setting free of acid from the muscle preliminary to its removal by 
some other mechanism. 


Fic. 3.—Record of reflex inhibition. Indications as in figs. 1 and 2, but relaxation of 
muscle is shown by a line which starts above time marker and falls across it, instead 
of the record of contraction shown below. Relaxation is accompanied by decrease 
in acidity. 


The results indicate that acidity and tension in muscle are concurrent. 
Thus it may be better to investigate, not how the contraction is maintained, 
but why the acid remains and is not removed as ina simple twitch. The 
results further suggest that as acidity is common to both tetanus and tone 


probably the mechanism for the production of both is the same, «.¢., that there 
is one mechanism in muscle and not two. 


I wish to thank Mr. F. C. Smith for assistance in some of the experiments 
on decerebrate cats. Some of the apparatus for the research was obtained 


* Sir Charles Sherrington informs me that weak reflex inhibition is frequently pre- 
ceded by a slight contraction. This fact is in favour of the view that the slight 
increase in acidity, shown before relaxation occurs, is due to a preliminary increase in 
tension. ' 


410 Dr. C. Shearer. Heat Production and Oxidation 


by grants from the Government Grant Committee of the Royal Society, and 
from the London Hospital Medical College Research Fund, and part of it 
was given by Mr. H. S. Souttar. 


REFERENCES. 


(1) H. E. Roaf, ‘Roy. Soc. Proc.,’ B, vol. 86, p. 215 (1913). 
(2) H. E. Roaf, ‘Journ. Physiol.,’ vol. 48, p. 380 (1914). 
(3) C. 8. Sherrington, ‘‘ Mammalian Physiology,” ‘Clarendon Press,’ Oxford, 1919, p. 117. 


On the Heat Production and Oxidation Processes of the Echino- 
derm Egg during Fertilisation and Early Development. 


By C. Sxeargr, F.R.S. 


(Received March 25, 1922.) 


(From the Biochemical Laboratory, Cambridge.) 


Introduction. 


In the following experiments an attempt is made to measure the heat 
liberation of the ovum on fertilisation and early development, and to 
correlate this with the amount of oxygen consumed and the carbon dioxide 
given off at the same time. New methods hitherto unused for this purpose 
have been employed. The question has already been investigated by 
Meyerhof (1) in an extensive paper published in 1911. He determined the 
heat production and the oxygen consumption of the egg of the sea-urchin 
Strongylocentrotus on fertilisation and early development. The heat production 
was measured directly by means of a finely divided Beckmann thermometer, 
while the eggs were contained in a small closed vacuum flask completely 
submerged in the water of a carefully regulated thermostat. The oxygen 
consumption of the eggs was at the same time determined at intervals of an 
hour, by the titration of the sea-water in which the eggs were kept with 
sodium thiosulphate by the Winkler method. 

The heat given off by a known quantity of eggs expressed in gram 
calories per hour, divided by the amount of oxygen consumed in the same 
time expressed in milligrams, gave him a calorific quotient which he calls 
“Q.” This he found for the early stages of segmentation to be about 2°75, 
but if the heat of solution of carbon dioxide to form bicarbonate with the 
sea-water is taken into consideration this value becomes 2°6. This figure is 


f 


- 


Processes of the Echinoderm Egg during Fertilisation. 411 


so low, however, as to suggest that his data for the heat liberation or the 
oxygen consumption are incorrect, or that the oxidation processes of the egg- 
cell on fertilisation are of a different character from those of adult 
metabolism. It has been shown by Zunst and Schumberg, Rauber, Pfliiger 
and others, that when fat is consumed this figure should be in the vicinity 
of 3°3, when protein 3°2, and carbohydrate 29. Meyerhof could find no carbo- 
hydrate in the egg, and there could be no destruction of protein, but sufficient 
fat was found in the egg to give the quotient observed. In the case of fresh 
sperm, @ was 3:1 or nearly normal. The carbon dioxide production by the 
eggs was not measured. 

The most important fact, however, arising from Meyerhof’s experiments 
was that, whether he took the unfertilised egg, the fertilised, or the fertilised 
egg treated with phenylurethane, so that cell formation was inhibited 
although development proceeded, he found the value of this calorific quotient 
was always the same. If any of the chemical energy liberated in the egg 
as the result of the increased oxygen consumption of the egg on fertilization 
were utilised in producing the visible morphological structure of the egg, 
then the value of this quotient could not be the same in all these instances. 
Warburg (2) had already pointed out, that the oxygen consumption of the 
egg-cell on development always fails to keep pace with the increase in 
morphological structure. In <Arbacia he found the fertilised egg in the 
one cell stage during the first hour of development consumed 4 ce.c. of 
oxygen; in the sixth hour, the same quantity of eggs consumed only 
68 cc., although now the eggs were each composed of thirty-two cells 
instead of one. 

In another experiment where a larger number of eggs were employed, 
13-2 mgrm. of oxygen was consumed by the eggs in the eight-cell stage, 
while in the thirty-two cell stage only 20°5 mgrm. was absorbed. Thus, 
while the oxygen consumption doubled in amount the cellular structure had 
increased four-fold. 

Meyerhof found the heat production of a quantity of unfertilised eggs 
containing 140 mgrm. of nitrogen (about 17 million eggs) to be about 
09 grm. calories per hour, while the same quantity of fertilised eggs, 
liberated 4—4-2 grm. calories in this time. In the second hour, the two-cell 
stage, the heat production rose to 45-5 grm.-calories. In the fourth hour, 
corresponding to the 8-cell stage, it was 6-6°5 grm.-calories. In the sixth 
hour, the thirty-two-cell stage, it was 9°8 grm.-calories, and from this time 
onwards the heat liberation increased rapidly, until in the eighteenth hour, 
when the free swimming stage was reached, it was 17°8 grm.-calories per hour, 
or four times what it was in the first hour of development. Once develop- 


412 Dr. C. Shearer. Heat Production and Oaidation 


ment was initiated the heat production rose steadily without pause or 
interruption. It followed the oxygen consumption closely in all respects, and 
like this, showed no direct relationship to the rate at which morphological 
organisation took place within the egg. No heat production could be 
observed during the formation of the fertilisation membrane or the early 
phases of the fertilisation process itself. 

In all Meyerhof’s experiments great over-crowding of the eggs necessarily 
took place, and it is difficult to believe that under such conditions the heat 
production was normal. In attempting to repeat his experiments, using a 
much larger vacuum flask and a smaller quantity of sea-urchin eggs where 
they were less crowded, I was unable to get them to fertilise in the closed 
flasks. As Loeb first pointed out, an abundant oxygen supply is the 
invariable constant required by the fertilised and developing egg-cell. In 
my own experiments, in order to get my eggs to fertilise and segment 
regularly, I was forced to adopt some means of keeping them aérated during — 
the course of the experiment. If large quantities of eggs were employed 
(300-400 merm. of egg nitrogen), then it was absolutely necessary to carry 
out artificial aération, or otherwise a large number of the eggs quickly died 
and soon cytolysed, and during cytolysis liberated an abnormal amount of 
heat. As I have shown with bacteria (3), the death process and cytolysis of 
all cells is probably accompanied by an abnormally high oxygen consumption 
and heat liberation. On these grounds Meyerhof’s experiments seemed open 
to criticism. It was worth while repeating his experiments, using different 
methods which avoided, as far as possible, this difficulty. Moreover, it 
was of interest to determine if a different method of measuring the heat 
liberation would give figures similar or of the same order as those obtained 
by Meyerhof. 

The Winkler titration method employed by Meyerhof in estimating the 
oxygen consumption of the egg on fertilisation and development is somewhat 
unsatisfactory in that it probably gives too high a figure for the oxygen 
consumption of the egg. The sea-urchin egg on fertilisation discharges a 
certain amount of organic slimy material into the sea-water, which interferes 
to a considerable extent with the accuracy of the titrations carried out by 
this method. The following experiments are for these reasons, to a large 
extent, a repetition of Meyerhof’s work, using different methods for both the 
heat measurement and the oxygen consumption and carbon dioxide output 
of the egg. The eggs and sperm of Hehinus miliaris were employed. This 
species being a shore form it is exceptionally favourable for work of this 
kind. It can be readily reared to the adult stage in small culture jars under 
laboratory conditions. 1 have shown, working in conjunction with 


Processes of the Echinoderm Egg during Fertilisation. 4138 


De Morgan and Fuchs (4), that this species can be readily raised to the 
sexually mature F2 generation in the laboratory if a few simple rules are 
followed in rearing the larve. 


Methods. 


In making the heat measurements the differential calorimetric method has 
been adopted instead of the direct method employed by Meyerhof. It requires 
no expensive fittings or elaborate thermostats, and has the advantage that a 
number of separate determinations can be made at the same time. All the 
following experiments were carried out so that the eggs were efficiently 
aérated. This was carried out so as not to interfere with the accuracy of the 
heat estimations. To test this point many preliminary experiments were 
made.* All final calibrations were carried out under conditions identical with 
those of an actual experiment; the mean of 30 or 40 determinations being 
taken as the final figure. 

The oxygen and carbon dioxide determinations were carried out by the 
employment of a special pattern of the Barcroft (5) differential manometer, 
in which it was possible to fertilise the eggs in the closed chamber of the 
instrument. It was thus possible to record the oxygen consumption and 
earbon dioxide output of the eggs while the sperm were actually making their 
way into the egg. As this instrument and the mode of its use has already 
been described in a previous paper (6), it is unnecessary to give an account of 
it here. 

In the heat measurements, the form of differential calorimetric method 
employed has been that devised by A. V. Hill(7), and has already been clearly 
described by him at some length. The method is based on the fact that, 
within fairly wide limits, a vacuum flask may be given any desired rate of 
conduction of heat to the outside by simply increasing or decreasing the 
volume of its fluid contents. By placing the right quantity of fluid, in this 
ease eggs In sea-water, in one flask, and an appropriate quantity of plain sea- 
water in another flask acting as a control, the two flasks can be given the 
same temperature fall. They can then be used in making a differential 
determination, on being connected with one another by means of a thermo- 
‘couple, with one junction in each flask. The thermocouple being in circuit 
with a delicate galvanometer, any deflection of the mirror gives the difference 
of temperature between the two flasks. A copper-constantan thermocouple 
was used in circuit with a sensitive Ayrton-Mather galvanometer. The 

* Aération was carried out by bubbling a very small volume of water-saturated air 


simultaneously through both fiasks at regular intervals, the eggs in the flask being 
previously well aérated for 20 minutes before the commencement of the experiment. 


414 Dr. C. Shearer. Heat Production and Oxidation 


sensitivity of the galvanometer was such that, at 3:5 metres distance, every 
millimetre of the galvanometer scale represented 0:00139° C. The leads from 
the galvanometer and thermocouples were brought to a specially constructed 
dial box furnished with two keys, by which three or four thermocouples could 
be thrown into circuit with the galvanometer, and also small resistances 
introduced in any of these circuits as desired. All leads and terminals, 
including those of the galvanometer, were made of copper throughout, thus. 
avoiding any possible thermo-electric effects. 

The vacuum flasks were the ordinary narrow-necked silvered Dewar flasks, 
made as “refills” for commercial thermos bottles. They were used in two 
sizes, having a capacity of 400 cc. and 800 c.c. respectively. The larger size- 
have a coefficient of heat loss half that of the smaller, and are therefore more 
accurate to work with where sufficient experimental material can be obtained. 
The selection of the flasks was carried out in the following manner :—Some: 
40 to 50 flasks were obtained for rough testing. These were all filled with 
the same quantity of water at 60° C. They were then closed with plugs of 
cotton wool, and put aside in a corner of the room free from draughts, and, 
allowed to warm up for an hour. Their temperature was then taken with a. 
Beckmann thermometer, after which they were allowed to stand for 
24 hours, when their temperature was again taken with the Beckmann 
thermometer. It was usual to find four or five flasks out of the lot that. 
had very similar rates of temperature fall, and these were selected for 
further calibration. Their coefficients of heat loss were then earefully 
worked out, under conditions as similar as possible to those obtaining in 
experiments by the use of the formula T—T)/A—T)e—kt, a mean of ten or 
twelve determinations being taken. The final calibration was carried out 
under actual conditions of an experiment, with air bubbling through the: 
flask contents, and air tubes and thermocouple junctions in position, and the 
flask itself sunk down in the water of the thermostat. One flask was given 
a slightly higher temperature than the other, which was the exact tem-- 
perature of the bath water; as the temperature of the flask under calibration 
fell slowly to that of the control flask, a series of readings were taken with 
the thermocouple and galvanometer; these, on being plotted out, gave a 
curve from which the value of & could be directly taken. The value given 
by this last method was the one actually employed for experiments. As a 
matter of fact, both methods gave very similar values for & in nearly all 
instances. Prof. Hill was kind enough to place at my service two flasks of 
400 ¢.c. capacity, which were remarkable for having almost the same value 
of k, They could be used differentially by placing the same amount of 
fluid in each. They have been extensively used in the present experiments.. 


Processes of the Echinoderm Egg during Fertilisation. 415 


The flasks during the course of work were recalibrated from time to time, 
and at long intervals were tested by the liberation of a known amount of 
heat in each flask from a small coil of constantan wire. This coil liberated 
21 grm.-calories of heat per hour in the flask under the conditions of the 
test, and the galvanometer scale readings were usually within 3 per cent. of 
this value. To close the mouths of the flasks during an experiment, it was 
found that thick wads of cotton wool were the most effective. When the 
flasks were closed by rubber stoppers and the flask sunk completely in the 
water of the thermostat, it was found that more heat was lost by conduction 
through the stopper than was the case when they were only plugged with 
cotton wool and sunk up to their necks in the water of the bath. The 
flasks were mounted in pairs in open wirework baskets, which were made so 
that they could be clamped on the thermostat, so the flasks were held firmly 
submerged up to within a centimetre of the tops of their necks in the water 
of the bath. The thermostat tank held 50 or 60 litres of water, and was 
kept stirred and in uniform temperature throughout, by having compressed 
air bubbled through it from a number of jets distributed evenly over the 
bottom of the tank. This method of stirring was very effective, for, when it 
was in action, it was seldom possible to distinguish more than a hundredth of 
a degree C. between any two points in the water of the tank. The sides of 
the tank were protected externally by thick layers of felt, and its inner side 
was surrounded by a coil of piping through which cold water could be 
circulated, and the temperature of the tank kept constantly at 145° C. 
The room in which the experiments were conducted was almost entirely 
underground, and underwent little change of temperature between day and 
night, or from one day to another, if the door was kept closed and the 
windows protected. The experiments were carried out in the months of 
July, August and September,-when weather conditions were most favourable 
for work of this kind. It is the special merit of the differential method that 
external temperature conditions can be largely neglected, so long as both 
flasks used in making the differential determinations are affected to the 
same extent by all variations of external temperature. 

In order to get the eggs to fertilise and segment regularly in the flasks, it 
was found necessary to carry out some form of aération. To accomplish this, 
air was slowly bubbled through the contents of both flasks at regular intervals 
during an experiment. This also served to stir up the eggs and prevent their 
settling in a dense mass in the bottom of the flasks. The air used in the 
aération and stirring process was first passed through a large wash-bottle, half- 
filled with sea-water, sunk in the middle of the thermostat tank. The air 
from this bottle was then led into each flask by fine rubber tubes, which 

VOL. XCIII,—B. 26 


416 Dr. C. Shearer. Heat Production and Oxidation 


passed through the cotton plugs used to close the flasks. The air thus 
saturated with moisture and at the same time brought to the temperature of 
the water bath, as Hill has shown, the heat capacity of air being so low, 
produces little or no cooling effect on the contents of the flasks. At the 
commencement of an experiment care was taken to adjust both flasks, but 
especially the control flask, to exactly the same temperature as the thermostat 
water, which, as already mentioned, was kept constantly at 145°C. This 
adjustment in the case of the control flask was always made to within a 
hundredth of a degree C. with a Beckmann thermometer. This adjustment 
was usually carried out several times in succession before an experiment was 
actually commenced. The sperm were suspended in a small bottle in the 
water of the thermostat, so that when finally added to the eggs in the flask 
they were at approximately the same temperature. 

On the addition of the sperm the cotton plugs, with the thermocouple 
junctions and air tubes, were immediately replaced in the necks of the flasks, 
and galvanometer readings commenced. Readings were always taken at 
fairly frequent intervals at the commencement of an experiment, but once 
the experiment was under way, they were usually taken at intervals of 
several hours. The readings obtained, in millimetres on the galvanometer 
scale, were then plotted out on squared paper with respect to time, and a 
curve of observed heat production obtained. As the flasks are meanwhile 
losing heat, a correction for heat loss has to be made. The loss of one flask, 
however, by the conditions of the experiment has been made the same as that 
of the other, so that the rate of temperature-rise in the flask containing the 
egos is immediately given by the formula /(T—T)), where & is the coefficient 
of temperature-loss of the flask, and (T—T;) is the difference of temperature 
between the two flasks, as shown by galvanometer scale readings in milli- 
metres at any instant. The total temperature-rise in the flask is obtained 
by integrating 4(T—T ) with respect to time, and this value is accurately 
given by measuring the area of the curve given by the galvanometer scale 
readings plotted against time. The total heat produced is equal to the 
capacity of the flasks and fluid multiplied by the final temperature difference 
between the flasks plus this value of & (times area of curve), where this last 
expression is equivalent to & [value of the middle ordinate of (T—T))]. 

In the following sections of the paper, a few only of the many experiments 
carried out have been described. In many instances the eggs or sperm, for 
one reason or another, were unsatisfactory and the experiment failed to give 
a result. In other experiments, while the eggs and sperm were perfectly ripe 
and the eggs gave a very high percentage of fertilisation, they failed to 
fertilise in the manometers to the same extent as they did in the flasks, 


Processes of the Echinoderm Egg during Fertilisation. 417 


so rendering comparison of the oxygen intake with the heat production 
impossible. It is essential in experiments of this kind that in both the 
oxygen and COz, and also the heat determinations, all the eggs should fertilise 
at the same time, and that, after fertilisation, they should all develop at the 
same rate, as otherwise no comparisons can be made between different 
portions of the experiment. In all, out of some 500 experiments few were 
satisfactory in all respects. 

To simplify matters the manometer readings in the following experiments 
have all been reduced to standard barometric (760 mm. Hg.) pressure and 
uniform temperature of 145° C. The galvanometer scale readings have 
also been adjusted to start from zero, although it was seldom possible to 
adjust the temperature of the two flasks so closely that the readings should 
actually commence at zero. The galvanometer mirror deflection being always 
either to the right or left the zero was in the middle of the screen. The 
thermocouple was arranged so that the hottest junction should always deflect 
the mirror to the right. 

Experiment 1. 


(a) Heat Determination.—400 c.c. of ripe well washed #. miliaris eggs in 
sea-water were placed in Flask R, and 380 c.c. sea-water in Flask No. 7, under 
the conditions of the experiment both flasks had the same coefficient of heat 
loss. The flasks were sunk down in the bath water, with the thermocouple 
junctions and air tubes in place. The temperature of the flasks was adjusted 
to within a hundredth of a degree of the temperature of the bath, and the 
flasks allowed to remain with air bubbling through them for half-an-hour. 
The flasks were closed with thick wads of cotton-wool. At the end of this 
time the temperature of the flasks was again adjusted as near as possible to 
that of the bath, and after a few minutes a few drops of sperm were added to 
the eggs, and the cotton plugs with air tubes and thermocouple junctions 
replaced in the flasks and galvanometer readings commenced. 


Time 3 P.M. galvanometer scale reading was 0 


A ls, 5) : 5 mm. to R containing eggs. 
BELO, i ; tt 3 5, 
paeleke 5, is 5s Zils 4 en, 3 e 

yh Cas ‘6 rs, 7S ee, ae 


At the end of the experiment 98-100 per cent. of the eggs in the flask 


* In carrying out an experiment the preliminary preparations often took up so much 
time that the actual galvanometer readings could only be started late in the day and had 
sometimes to be carried through to the following morning. 


a 6 2 


418 Dr. C. Shearer. Heat Production and Oxidation 


were in normal morula stages. Kjeldahl determination gave 584 merm.-egg 
nitrogen present. In the first hour after the addition of the sperm the eggs 
liberated 2°9 grm.-calories, 10°5 grm.-calories in the fifth hour, and 22°8 grm.- 


calories in 11 hours (see Curve, fig. 1, a). 


0 
=) 
x= 
a 
- 
60 is 
J 
3 
o 
3b 
E 3) 
i AY 
a 3 
o 40 uN Z 
F 5 E 
oF Ta] 7 
& 46 
= a 452 
a] a Ss 
ea 5 % 4e 
f=) 
ee 2 . 
25 : a 
Ss 3 : 
Ao a =| 
gf v 
20] 3 y 
tb 
on 
a 


ec 


0 5 1Q Time in hrs. 


Fig. 1, a.—Curve of heat production for Experiment I. 58°4 mgrm. egg nitrogen. 


( 4 milligrams egg Nitrogen) 


t) 10 20 30 40 50 60 
Minutes 
Fie. 1, b.—Curve of O, consumption and CO, liberation for same Experiment I. 
4-07 mgrm. egg nitrogen. 


Processes of the Echinoderm Egg during Fertilisation. 419 


(b) Oxygen and Carbon Dioxride Determination.—2 c.c. of the same lot of 
eggs as those placed in the flask in above experiment were put in the chamber 
of oxygen manometer and a drop of KOH put in the cup, 2 cc. of plain sea- 
water being placed in the control chamber. In a second manometer used for 
control the same quantity of eges were placed in the chamber, the KOH 
being left out. 

Temperature of manometer bath 145° C., barometer 760 mm. The mano- 
meters were put in position in the bath and after being brought into complete 
equilibrium with the bath water the cocks of the manometers closed and the 

eggs fertilized. The first manometer showed that :— 


At the end of 15 minutes the eggs had consumed 242 c.mm. oxygen. 
22 > 30 ? 3? ” 33° 0 2? 9 
” ” 60 ” » ; ” 44-0 ” ” 


At the end of the experiment eggs in the chamber showed 100 per cent. 
fertilisation membranes and some commencing two cell stages. Kjeldahl deter- 
mination on the eggs gave 4:07 mgrm. egg nitrogen present. 58°4 merm. of eg 
nitrogen at this rate would take up 631°4 c.mm. oxygen, which is faa 
to 0:'902 mgrm. 

As 2:9 grm.-calories of heat were given off in the first hour following 
fertilisation, for 58-4 merm. N in first part of experiment, 


The heat production in this experiment was 10°5 grm.-calories in 5 hours; 
in 12 hours, 22°8 grm.-calories. 

In the second manometer 4:0 c.mm. of oxygen seemed to be the difference 
between the oxygen intake and CO, output in this experiment, for 
4-07 mgrm. of egg nitrogen. If we assume that 44:0—4.0 gives us the amount 
of carbon dioxide produced in this experiment, we get the respiratory 
quotient of 40/44 = 0-91. 

It will thus be seen the carbon dioxide output of the eggs is almost as 
great as the oxygen consumption. In all the experiments the carbon dioxide 
respiration follows the oxygen consumption very closely, the respiratory 
quotient varying from 0:9 to 0°95 in different experiments. 


Experiment 2. 


(a) Heat Determination—800 c.c. of well washed ripe L. miliaris eggs in 
sea-water placed in Flask No. 3, 763 c.c. sea-water placed in Flask No. 4 
acting as a control. Under the conditions of the experiments with air 
bubbling through both flasks and thermocouple junctions in position, and the 


420 Dr. C. Shearer. Heat Production and Oxidation 


flasks sunk in the water of the thermostat, & for the first flask was 0:0238, 
and for the second k = 0:0236. Temperature adjusted all round to within 
a hundredth of a degree to 145° C. Cooled sperm added and readings 


started. 
Time 3.30 P.M. galvanometer scale reading 0 
Oro Ome ie = 17 mm. R flask containing eggs. 
Or a, A 5 20 m i 33 
, 12.30 AM. . = AN eae is c x 
D0 3, : g. Be) Vy, 6 is 3 


bed 


At end of experiment 95-100 per cent. of eggs were in healthy free- 
swimming early gastrula stages. Kjeldahl determination gave 146-2 merm. 
of egg nitrogen present in flask contents. The heat liberated in first hour 
6°34 grm.-calories, in the fifth hour 28 grm.-calories, and in the eleventh hour 


74-4 grm.-calories (see Curve, fig. 2. a). 


7u.& g.cals.in 11 hrs. 
¥v 


60 


wo 
hy 
x 
w 
r= 
cr 
eo . 
rie n 
ae : 
DE 40 cS) 
AOS - 
G-rt ey 
- 
an } : 
fu iat) 
25 2 pt i 
£3 x yh 
OH i / 
o qi 4 


20 


— 6.35 e.cals. 


0 5 10 Time in hrs. 

Fig. 2, a.—Curve of heat production to Experiment IJ. 1462 mgrm. egg nitrogen. 
(b) Oxygen and CO2 Determination.—2 c.c. of same lot of egg material as 
that put in the flask in the first part of the experiment, placed in a chamber 


Processes of the Echinoderm Egg during Fertilisation. 421 


( 9 milligrams egg Nitrogen } 


0 10 20 30 40 50 60 
Minutes 


Fic. 2, 6.—Curve of O, consumption and CO, liberation for Experiment II, 
9 mgrm. egg nitrogen. 
of oxygen manometer, and the same quantity in a chamber of CO2 apparatus. 
The eggs were fertilised. Those in the oxygen manometer showed that at 


the end of 
1 minute after addition of the sperm eggs had consumed 6°95 c.mm. oxygen. 
2 minutes 5 ‘ 3 : iO - OR aa is 
3 5 5 : :. is RS Oye e 3 
4 3 3 i - pS 2250) es, i 
5 ‘3 ss . - ‘i 23°05 _,, » 
10 . "5 5 a 7 DA Dies. 5; 5 
20 53 , ia if 5 470, es 
30 s if i r Si Disha es 5 
40 33 _ . . .: OG 72g 55 
60 . 5 - i 3S So:0L ey y 


AQ? Dr. C. Shearer. Heat Production and Oxidation 


At end of experiment 100 per cent. of eggs showed fertilisation membranes 
and commencing two-cell stage. Kjeldahl determination gave 9 merm. egg 
nitrogen present. 

At this rate 146-2 mgrm. ege nitrogen would consume 1380 e.mm. oxygen 
in the first hour following fertilisation, 1380 c.mm. oxygen being equivalent 
to 1:97 merm. 

The heat produced by 280 mgrm. egg nitrogen in the first hour following 
fertilisation, as shown in the early part of this experiment, was 635 grm.- 
calories. So that 


The heat production rose in the fifth hour to 28 grm.-calories, and to 
744 grm.-calories in the 11 hours in this experiment. The corresponding 
CO, determination for this experiment gave a respiratory quotient of 0°95. 


Experiment 3. 

800 c.c. of ripe well washed Z. miliaris egos were placed in Flask No. 3, 
763 c.c. plain sea-water being placed in Flask No. 4, acting as a control 
under the conditions of the experiment with flasks sunk in the water of the 
thermostat and air-bubbled through both flasks, & for flask No. 3 was 0:0238 
while that for Flask No. 4 was 0:0236. Temperature was adjusted 
all round to within a hundredth of a degree to 145° C. No sperm were 
added. Galvanometer readings were commenced. At the end of 1 hour 
Flask No. 3 had given off 3:6 grm.-calories of heat. A Kjeldahl determina- 
tion on the flask contents gave 431 mgrm. egg nitrogen present. In the 
same time 8 mgrm. ege nitrogen of the same batch of egg material, consumed 
151 «mm. oxygen. Therefore 431 megrm. of egg nitrogen would consume at 
this rate 812 cmm. oxygen in this time, as 812 cmm. oxygen equal 
1:17 mgrm. O2 we get value of 

3:6 - 
= a a 3:07 
for the unfertilised egg. The value obtained tor the fertilised egg of 
E. miliaris in the two previous experiments was 3-215 and 3:22 respectively. 
Thus the value of Q is somewhat different in the two cases. 

It is doubtful, however, if much significance can be attached to this 
difference. The gonads of so many females have to be used for making 
determination on the heat production of the unfertilised egg, that it is 
impossible that they shotld all be in the same stage of ripeness. Some of the 
gonads are certain to be slightly over ripe, and their eggs will probably 
cytolyse on being placed in the vacuum flask, and will give off an abnormally 


Processes of the Echinoderm Egg during Fertilisation. 423 


large amount of heat; others, again, will be somewhat immature, and will 
consequently give off little heat as compared with the properly mature stage. 
On the whole, from a number of experiments with the unfertilised egg, I am 
inclined to think there is little difference between the value of Q in the 
unfertilised,as compared with the fertilised egg, and that Meyerhof’s conclusion 
that it is the same for both is correct. In three successful experiments with 
the unfertilised egg, the figure for Q obtained in the above described experi- 
ment represents the mean, and it 1s worth noting that this value is slightly 
smaller than in the case of the fertilised egg. 


Discussion. 


The Unfertilised Eqgg.—tIn the foregoing experiments it has been shown that 
the oxygen consumption and the heat liberation of the unfertilised egg of 
HE. miliaris is remarkably small. In 1 hour, 1,000,000 eggs (8 mgrm. egg 
nitrogen) only consumed 15°1 c.mm. of oxygen, and liberated at the same 
time something of the order of 0-067 grm.-calorie heat. If we divide the 
heat liberation in 1 hour’s time, expressed in gramme calories, by the oxygen 
consumption in milligrams, we get a quotient which we may call the calorific 
quotient. In the case of the unfertilised egg this quotient was found to be 
3°07. Meyerhof, in Strongylocentrotus, using the Winkler method for estimating 
the oxygen consumption and the direct method for measuring the heat produc- 
tion, found this egg consumed 9-41 c.mm. of oxygen and liberated 0-038 erm.- 
calorie, under similar conditions. The value of Meyerhof’s calorific quotient 
(Q) varied about 2°8. 

The Fertilised Egg.—On the addition of the sperm to the eggs of #. miliaris, 
there is an immediate oxygen consumption by the egg and a corresponding 
increase in the heat liberated. At the end of the first hour of development 
86:4 c.mm. of oxygen were consumed by 1,000,000 eggs (8 merm. egg N.), and 
0:397 grm.-calorie of heat was liberated. The CO: output of eggs was almost 
the same as the oxygen intake; the respiratory quotient being in the vicinity 
of 0:92. In Strongylocentrotus, Meyerhof found under similar conditions for 
65°38 c.mm. of oxygen consumed a heat liberation of 0:247 erm.-calorie, for a 
similar quantity of eggs. The calorific quotient in this instance again being 
26 to 28. In view of the large number of eggs that have to be used for 
making a heat determination on the unfertilised egg, it is doubtful if much 
significance attaches to the difference found between the calorific quotient of 
307 in the unfertilised egg, as compared with 3:2 in the fertilised condition. 


AQA Dr. C. Shearer. Heat Production and Oxidation 


Summary. 


1. In the present paper an attempt has been made to measure the oxygen 
consumption of the egg of Echinus miliaris on fertilisation and early develop- 
ment and compare it with the amount of heat liberated by the egg at the 
same time. 

2. In making both these estimations new methods have been employed. 

3. The oxygen consumption of the egg has been measured by the use of a 
special pattern of the Barcroft differential manometer, in which the eggs were 
fertilised within the closed chambers of the apparatus. The COz2 output of 
the egg was also measured with the same instrument and the respiratory 
quotient determined, 

4. The heat liberation was measured by the use of the differential micro- 
calorimetric method. 

5. In 1 hour 1 million unfertilised eggs (8 mgrm. egg N.) consumed 
15:1 c.mm. of oxygen and gave off at the same time 0-067 of a gram-calorie of 
heat at standard pressure (760 mm. Hg.) and temperature 14:5° C. 

6. In the same time the same quantity of fertilised eges consumed 
864 cmm. oxygen (with a corresponding output of COs, respiratory 
quotient 0-92) and liberated 0°3976 of a gram-calorie of heat under similar 
conditions. 

7. The fertilised egg in the first hour of development gave off roughly 
6—7 times more heat than the unfertilised egg and consumed at the same 
time 6 or 7 times more oxygen than the unfertilised egg. 

8. In the fertilised egg in one experiment (1) 584 megrm. egg nitrogen 
(about 7°3 million eggs) liberated 2‘9 gram-calories at the end of the first 
hour of development; in the fifth hour, 10°5 gram-calories and 22°8 gram- 
calories of heat in 11 hours. In another experiment (II) 146°2 mgrm. egg 
nitrogen (18°6 million eggs) liberated 6°35 gram-calories in the first hour, 
28 gram-calories in the fifth hour, and in 11 hours 744 gram-calories. (This 
last figure is possibly too high, due to some cytolysis.) On the whole the 
heat liberation of the egg on fertilisation rises steadily, reaching its highest 
point when segmentation has been completed and the free-swimming stage is 
reached. 

9. The heat liberation of the egg during the first hour after the sperm 
have been added to the eggs expressed in gram-calories divided by the 
amount of oxygen consumed in the same time, expressed in milligrammes, 
gives a calorific quotient (Q). 

10. In the case of the unfertilised egg the calorific quotient wasfound to be 
about 3:07, while in the fertilised egg it was found to be 3:22. 


| 


Processes of the Echinoderm Egg during Fertilisation. 425 


11. On fertilisation a greatly increased liberation of chemical energy is 
brought about within the ovum. This is shown by the increased oxygen 
consumption of the fertilised egg-cell combined with its greatly increased 
carbon dioxide and heat liberation. 

12. As, however, the calorific quotient of the unfertilised and the fertilised 
egg-cell is approximately the same in both instances, little or almost a 
negligible quantity of this energy is expended in bringing about the visible 
morphological structure of the developing ovum. It is probably employed 
in keeping the living substance itself intact as a physical system. 


REFERENCES. 


(1) Meyerhof, O., “ Untersuchungen tiber Warmetinung der Vitalen Oxydationsvorginge 
in Eiern,” I, II, III, ‘ Biochem. Zeit.,’ vol. 35, p. 246 (1911). 

(2) Warburg, O., ““Beobachtungen iiber die Oxydationsprozesse im Seeigelei,” ‘ Zeit. f. 
Physiol. Chem.,’ vol. 57, p. 1 (1908). 

(3) Shearer, C., ‘On the Amount of Heat liberated by Bacillus coli when Grown in the 
Presence of Free Amino-Acids,” ‘Jr. Physiol.,’ vol. 55, p. 50 (1921). 

(4) Shearer, De Morgan and Fuchs, “On the Experimental Hybridization of Echinoids,” 
‘Phil. Trans.,’ B, vol. 204, p. 255 (1914). 

(5) Barcroft, J., ‘The Respiratory Function of the Blood,’ Cambridge, 1914. 

(6) Shearer, C., “On the Oxidation Processes of the Echinoderm Egg during Fertilisa- 
tion,” ‘ Roy. Soc. Proc.,’ B, vol. 93, p. 213 (1922). 

(7) Hill, A. V., “ A New Form of Differential Micro-Calorimeter for the Estimation of © 
Heat Production in Physiological, Bacteriological, or Ferment Actions,’ ‘Jr. 
Physiol.,’ vol. 43, p. 261 (1911). 


426 


Further Observations on Cell-wall Structure as seen in Cotton 
Hairs. 
By W. LAWRENCE BALLs, M.A., Sc.D. (Cantab.), and H. A. HANcooK. 
(Communicated by Dr. F. F. Blackman, F.R.S. Received March 3, 1922.) 


[PuatE 10.] 


The present note summarises the results of observations made subsequently 
to the recognition of growth rings in the cell-wall,* of which a photograph in 
transverse section is given in Plate 10, fig. 2. These observations are all 
related to previous physiologica] studies, and most of them have been made 
on material of known origin, 7.e., dated bolls? and pure-line plot crops. 

An excellent memoir on our present knowledge of the cotton cell-wall by 
Mr. H. J. Denham,} now in course of publication, makes it unnecessary for 
us to deal with the historical aspects of the matter. 


Methods. 


(a) The “swelling” technique has been further developed by the use of 
calcium thiocyanate (for which, as well as for the use of naphthamine 
blue as a stain, we are indebted to Mr. H. E. Williams§). Other reagents 
have also been found serviceable when the conditions are adjusted to produce 
an equilibrium state on the verge of actual solution, ¢.g., cuprammonium and 
caustic soda, alone or together, and also sulphuric acid have been largely used. 
The latter is exceptionally interesting as showing specific differences between 
various hairs in respect of the critical concentration. The artifact nature of 
swollen walls has been continuously borne in mind, and all observations have 
been returned to the unswollen state by measurements of the contractions 
and expansions experienced by the hair. 

(2) Our section-cutting technique has been described elsewhere.| 

(c) The junior author devised a simple “ pressure” technique, single hairs 

* W. L. B., “Existence of Daily Growth Rings in the Cell-wall of Cotton Hairs,” 
‘Roy. Soe. Proc.,’ B, vol. 90 (1919). 

+ W. L. B., ‘Raw cotton (Development and Properties of),” chapter 4 (London, 1915). 

{ Denham, H. J., “The Structure of the Cotton Hair and its Botanical Aspects : 
Memoir of the British Cotton Industry Research Association,” ‘Jour. Textile Inst.,’ 
vol. 13, p. 99 (1922). 

§ Williams, H. E., “The Action of Thiocyanates on Cellulose,” ‘ Jour. Soc. Chemical 
Industry,’ vol. 40, p. 221 7 (1921). 


|| Denham, H. J., ‘Nature,’ vol. 107, p. 299 (1921); W. L. B. and H. A. H., ‘ Nature,’ 
vol. 107, p. 361 (1921). 


On Cell-wall Structure as seen in Cotton Hairs. NON 


being stressed enormously, under a cover-slip, by pressing with the blade of 
a knife. This we have found very useful ; it is evidently akin to the breaking 
of aeroplane timber studied by Robinson.* 

(d) The inter-relation of external convolutions and internal wall structures 
has been systematically examined by detailed repeated mapping along the 
length of single hairs. 

(e) Groups of hairs of equal length from single seeds have been similarly 
mapped to obtain the average distribution of convolutions in studying the 
change from day to day in dated samples. A very full re-examination of the 
daily pickings samplest has also been made in connection with this work 
but only slight use will be made of these results at present, as they need 
direct experiment on growing plants for confirmation of our interpretations. 

The evidence on which we base our conclusions is very detailed and 
various, and to particularise every item would be extremely tedious. We 
have, therefore, adopted the plan of summarising our results rather after the 
manner of geological research; and by way of further assistance in keeping 
this note from undue expansion, we shall restrict ourselves to dealing with 
the more debateable features of hair structure as brought out by Mr. Denham’s 
memoir, already referred to. 

The Cuticle. 


This we found to be distinct from the primary wall, though extremely 
tenuous. It possesses a spiral structure, probably showing reversals of the 
spirals, and quite probably identical in frequency, or even in details of 
pattern, with the pit spirals (see below), but the difficulty of correlating the 
two sets of observations is very great; Haller’s method? (SnClz and AuCls) 
was not successful. The spiral lines of weakness and their apparent reversals, 
plus the resistance of the cuticle to solvents, determine the familiar “ beading ” 
of swollen hairs. The miscalled “stomata” of De Mosenthal§ are probably 
primary wall-structures in essence, the cuticle being moulded to them; we 
doubt very much whether the cuticle is actually perforated. A granular 
superficial structure seen after heating (in the thiocyanate process) and 
staining with osmic acid seems to be due to the melting and redistribution of 
the wax film, which varies in amount with varieties and species of cotton 
around 0-4 per cent. of the hair weight. 

* Robinson, W., “The Microscopical Features of Mechanical Strains in Timber and 
the Bearing of these on the Structure of the Cell-wall in 'Plants,” ‘ Phil. Trans.,’ B, 
vol. 210, p. 49 (1920). 

+ W. L. B., “Raw Cotton,” p. 112, doe. cit. 

t Haller, ‘Chem. Zentr.,’ p. 652 (1920). 


§ De Mosenthal, H., “Observations on Cotton and Nitrated Cotton,” ‘Jour. Soe. 
Chem. Ind.,’ vol. 23, p. 292 (1904). 


428 Dr. W. L. Balls and Mr. H. A. Hancock. 


The Primary Wall. 


Various details of our evidence confirm the view that this wall, even when 
adult, is a different cellulose from that of the secondary wall. Further, we 
have seen reason to believe that until growth in length has passed its 
maximum rate, the cellulose (as distinct from the cuticle) has a different 
composition from that which it has assumed when the secondary thickening 
begins. We ourselves, for convenience, called this early stage “ pre-cellulose,” 
and we have since learned* that the general problem is now being investi- 
gated by Priestley. Accidentally, our preparations have shown the secondary 
cellulose completely dissolved, but the primary wall, spirally marked, 
untouched. 

The pit spirals of the secondary wall (see below and figs. 5-7) are con- 
tinuous through the primary wall, and possibly even to the cuticle spirals 
(vide supra). Thus it would seem that the law of Predeterminationt plays 
an important part in the cotton hair, and the bearing of this on our convolu- 
tion maps will be described later. 

The objects discovered and excellently photographed by De Mosenthal{ we 
propose to designate as the “slow spirals.” Their nature is obscure, but they 
are evidently a kind of pitted corrugation in the outer surface of the primary 
wall, to which the cuticle moulds itself. The sides of the trough in which 
the elliptical craters lie often project beyond the surface of the hair, and are 
thus discernible in profile. These slow spirals are particularly easy to see in 
fuzz hairs, but are probably present to some degree along all parts of every 
hair, lint (fig. 3), or fuzz, for they flash out momentarily during swelling with 
critical-strength sulphuric acid, even though not visible previously. The 
spiral shows frequent reversals (fig. 3). The relation of this spiral to the 
pit spirals, in respect of pitch, direction, and reversals, has been studied in 
detail, and we have satisfied ourselves—with some regret for an untenable 
hypothesis—that they are sometimes independent of one another. We have 
no evidence that the slow spiral pattern is pursued into the formation of the 
secondary wall, unlike the pit spirals, nor even that it represents any textural 
modification (as distinct from modification of surface or thickness) in the 
primary wall itself, excepting that when a hair has been “tendered” by acid it 
shows a saw-tooth form of cracking, the long slope coinciding with pit spirals 


* Discussion in Section K on the “Quantitative Analysis of Plant Growth,” ‘ British 
Association,’ 1921. 

+ W. L. B., “ Predetermination of Fluctuation, a Preliminary Note,” ‘Proc. Cam- 
bridge Phil. Soc.,’ May, 1914. 

+ Loc. cit. W.L. B., “ Analyses of Agricultural Yield,” Part III, ‘Phil. Trans.’ B, 
vol. 208, p. 157 (1917-18). 


On Cell-wall Structure as seen in Cotton Hairs. 429 


and the short resembling slow spirals (fig. 4). However, as we have satisfied 
ourselves that these spirals are not invariably opposed in direction, the fact 
may not be relevant, though it does suggest a chemical polarity. 

These slow spirals are thus somewhat mysterious objects, and we must wait 
for direct growth experiments to elucidate their nature. In passing, we may 
note that the distortions produced by swelling make the quick pit spirals 
simulate them very closely (fig. 8), a circumstance which led us to much 
confusion at first. ; 


The Secondary Wall. 


One of us has formerly described the growth rings,* while many observers 
have figured and commented on the existence of spiral markings in the wall 
and on its inner surface especially. The occurrence of somewhat elusive 
simple pits has also been described from fresh material by the senior author, 
and the probable existence of an internal spiral structure has been suggested 
in various forms by non-botanical writers.| We are now in a position to 
co-ordinate all these observations and views. 

By means of the simple “ pressure” method, it is possible to “develop” a 
spiral series of cracks throughout the length of a hair, with little distortion, 
which can then be mapped in detail (fig. 6), and related to a previous charting 
of the external form of the hair (vide infra). Such a pressed hair dissolves 
much more quickly in swelling reagents, presumably from the greatly 
increased free surface, and possibly also (since lower concentrations will 
attack it), because the flank of the patterned and orientated cellulose 
aggregates is, so to speak, exposed. When swollen, only a complex structure 
resembling basket work is produced (fig. 5), and it was the spasmodic 
occurrence of this in slides given us by Mr. Williams§ which started the 
present research. 

By mapping the simple pits in fresh greenhouse material, for which we are 
indebted to Mr. Vernon Bellhouse, and then mapping their spiral cracks, we 
have satisfied ourselves that the pits are simply abnormally wide intervals 
between otherwise contiguous spirals. It is therefore possible that any plant 
cell-wall which shows simple pitting may possess spiral structures similar to 
that of the cotton hair. We would also call attention to the structures in 
wood cell-walls described by Robinson,|| who describes and figures these 


* W.L. B., “The Existence of Daily Growth Rings in the Cell-wall of Cotton Hairs,” 
“Roy. Soc. Proc.,’ B, vol. 90. 

+ W. L. B., “ Raw Cotton,” fig. 12 and p. 78. 

t Vide Denham, Memoir, B.C.I.R.A., loc. cit. 

§ Williams, wide supra. 

{| Robinson, Joc. cit. 


e 


430 Dr. W. L. Balls and Mr. H. A. Hancock. 


so-called slip planes as being interrupted by the fragile middle lamella, which 
seems improbable unless these “slip-planes” are pre-existent. The spasmodic 
occurrence of swollen spirals in thiocyanate preparations merely showed an 
erratic anastomosing series, but with the cuprammonium and soda mixture, 
and better still with critical sulphuric acid (circa 1:540 sp. gr.), the nature 
of these anastomoses was evident; the wall is then seen to consist of about 
a hundred spiral fibrils—a screw of a hundred threads—all approximately 
identical, except in one respect. This exception consists in the frequent 
presence of two spirals, within the series, which stain more deeply than the 
others with naphthamine blue. They do not seem to -be otherwise different 
from their neighbours in any way, and as they seem always to lie at the ends 
of the major axis of the collapsed cell-wall, they might be merely stress- 
produced artifacts. On the other hand, one of them may disappear by 
approaching the other, the interval between them changing. Thus, the 
appearance shown in swollen hairs is altered from a symmetrical double screw 
(figs. 8, 9) to an asymmetric one, and thence to a single-thread screw, as we 
pass along the hair. This is not compatible with artifact origin, and it would 
seem that for some reason unknown two fibrils, lying diametrically opposite 
one another, are somewhat. different from the others. We have noticed 
similar bifurcation and re-union in the spiral thickening of protoxylem 
vessels in other plants. 

The question of the relationship between these radial boundary surfaces 
and the tangential growth-ring boundaries next arises. Numerous attempts 
to demonstrate the matter clearly in transverse section have largely failed ; 
and some considerations relating to free surface, cohesion, and the like, make 
it rather unlikely that these two sets of structures could ever be thus demon- 
strated perfectly and simultaneously in the swollen state. We also suspect 
that the shearing stress of the razor edge may produce molecular disturbances: 
which alter the reactions of the cellulose. Partial demonstrations of the 
existence of these radial boundaries have frequently been obtained, but rarely 
(fig. 1) comparable with the growth-ring demonstration of fig. 2. We thus 
have to depend on optical longitudinal sections, and by this means have 
satisfied ourselves in exceptionally geod preparations that the spirals are 
arranged in layers, each layer constituting a single growth-ring. . 

The cotton hair cell-wall is thus an elaborate structure, laid out on a 
simple plan. In the first instance, a spiral pattern seems to be laid out in 
the primary cell-wall and cuticle ; this, it should be noted, must happen while 
the hair is growing in length. The deposits of secondary cellulose, as growth- 
rings, do not obliterate this pattern, but follow it most strictly. Thus, a radial 
(spiral) structure persists through the concentric deposits. The simple pits 


On Cell-wall Structure as seen in Cotton Hairs. 431 


of any cell, equally with those of cotton hairs, are, after all, merely a special 
case of the same procedure, while an analogy may be found in the medullary 
rays of timber. 

Our partial elucidation of this structure has already thrown light on some 
physical properties of the hair. Abnormal hairs are often found in which, to 
a greater or less extent, the spiral structure is visible without any “ develop- 
ment.” Our colleague, Mr. Slater, in the Physical Section of this Department, 
has, in some preliminary studies, found the flexibility of such hairs to be 
highly abnormal, such hairs standing in the same relation to normal hairs, as 
strands of yarn compared with solid wires of celluloid. We have mentioned 
our opinion that the razor edge may produce molecular disturbances in the 
cellulose. Akin to this is a remarkable phenomenon discovered by the junior 
author, reminiscent of the results described by Griffith* with quartz rods. 
If a hair has been pressure-treated to develop the pit spirals without re-agents 
and then is subjected to stress in longitudinal extension, no alteration is 
noticeable until the hair breaks; after breaking, however, little or no trace 
of the pit spirals is left in any part of the hair. We have failed to obliterate 
the spiral cracks by any tension without actual breakage, and it would seem: 
that, as in Griffith’s work,+ a molecular disturbance is needed, which the 
back-lash of the break provides. 


Dimensions and Constitution of a Pit Spiral Fibril. 

Taking 0:4y as the thickness of a substantial growth-ring, and allowing 100 
spirals to the ring in a hair whose original cell diameter was 15y, and its 
mean wall diameter considerably less, gives us 0-4y square as the approximate 
cross-sectional area of one Pit Spiral Fibril. Its length is apparently that of 
the hair. Without undue speculation it is evident that we are here approaching 
molecular dimensions, the probable size of the cellulose molecules being such 
that some number of them between 1,000 and 100 would constitute the cross- 
sectional area of one such pit spiral. There is even the slight possibility that 
in these pit spiral fibrils we have reached the limits of morphology and are 
examining a chemical (or colloido-chemical) unit. For other reasons, however, 
we rather incline to the view that “cellulose,” even in a pit spiral fibril, is a 
complex of more than one kind of cellulose molecule. 


Origin of the Pit Spiral Structure. 
It seems clear to us that this secondary wall structure is a predetermined 
one, and that, paradoxically, we must therefore look to the period of growth 


* Griffith, A. A., ‘The Phenomena of Rupture and Flow in Solids,” ‘ Phil. Trans.,’ A 
vol. 221, p. 163 (1920). 
+ Griffith, doc. cit. 


VOL. XCIII.—B. 2H 


> 


432 Dr. W. L. Balls and Mr. H. A. Hancock. 


sf 


in length to determine the causation of such abnormalities in it as we have 
mentioned. Physiological work in this direction, under glass, is contemplated. 
But this leaves unsolved the more fundamental problem, as to why even the 
primary wall should be thus patterned and heterogeneous, consisting of 
structures which can sometimes be mechanically broken apart, which are 
possibly only united by a molecular film of water, and which show (when 
enormously swollen and stained with naphthamine blue) granular lines 
alternating with clear zones (fig. 8). 

Until a late stage we were not certain whether the slow spirals were not 
always opposed in direction of rotation to the pit spirals, and a working 
hypothesis was adopted which combined Church’s results* on the fundamental 
geometric structure of the cell with a general idea of protoplasmic cireula- 
tion, and with some earlier studies of growth in a fungus hypha by one of us.+ 
This hypothesis, though now entirely speculative, may yet be of interest. It 
postulated the existence of two “growth centres” mutually exclusive or 
polar in their inter-relations, as a rule; these controlled the longitudinal 
extension of the cell by intussusception, and their micro-bio-chemical opera- 
tions were rhythmic, as in Liesegang ring-formation. It was unlikely that 
such a system would build forward along a straight line, hence revolution 
was postulated, sometimes right-handed, sometimes left-handed (fig. 3), under 
the influence of accident or environment or even of stereo-isomerism. This 
revolution of the builders produced the spiral form, their rhythmic operation 
the successive fibril phases; and molecular predetermination, akin to erystal- 
lisation, produced fibrillar continuity. 

When it became clear that the slow spirals were not the trails of these 
“orowth centres ”—since their direction was not invariably opposed to that 
of the pit spirals—the hypothesis became mere speculation. 

It does, however, remain clear that there is a fundamental geometric 
structure in the cell wall, though our hope of confirming and extending 
Church’s main conclusion has at present failed. 


The Protoplasmic Debris. 
We have only to mention that this is of assistance in swelling technique as 
a rough guide to the amount of longitudinal contraction. 


The Convolutions. 
We now come to a much observed feature of the cotton hair, about which 
nothing definite has been published, whether in respect of their effect on 
* Church, A. H., “ Phyllotaxis in Relation to Mechanical Laws” (Oxford University 


Press, 1904). 
+ W. L. B., “ Temperature and Growth,” ‘Ann. Bot.,’ 1909. 


On Cell-wall Structure as seen in Cotton Hairs. 433 


spinning properties, or on the physical properties of the hair, or of their 
origin. The senior author has regarded them* as a necessary consequence of 
the simple pits in the wall, with modifications caused by wall-thickness 
variations, and we can now extend this to include the pit spiral structure, 
which should completely explain the convoluted form of the collapsed dead 
hair. 

Actually, however, the explanation is not yet complete. Mapping pit 
spiral against convolutions, In any one piece of hair (fig. 10), there is a 
general similarity, but by no means exact identity. It is probable, however, 
that if we could construct scale models of the wall structure (growth rings 
and pit spiral fibrils) in the form of semi-rigid tubes, and then cause them to 
collapse, we should find that local variations of texture, packing, inter- 
fibrillar friction, wall-thickness and hardness, etc., would produce similar 
discrepancies to those we have observed, and we venture to think that the 
existence of convolutions can be explained in this way, if we include the 
reinforced spirals already mentioned, whose presence makes the “arch” 
structure unsymmetrical. 

This, however, leaves a major problem to solve, to wit, the reason why the 
pit spirals, and hence the convolutions, vary their pitch and direction. On 
grounds of convenience, we have studied the convolutions rather than their 
causative spirals, and while no definite conclusions have yet been reached, a 
number of suggestive observations have been made on the daily pickings 
material,t which consists of fruit-capsules (bolls) opening on ninety succes- 
sive days :— 

(a) On counting the number of convolutions in a unit length at the middle 
of the hair (fig. 11, heavy line), we found the average number changing from: 
day to day, as in the case of other hair properties, indicating that environ- 
mental changes affected the convolutions. Similar results were given by 
counting at other Joci (fig. 11), and by taking the percentage distribution as 
between these various /oci (fig. 12). 

(6) Extending these measurements by mapping the convolutions along the 
whole length of the average hair (fig. 13), there were indications that the 
locus of any feature (¢.g., a low number of convolutions) shifted its position 
along the hair on successive days. The earlier the day of boll opening the 
further removed was the particular feature from the base of the hair, thus 
indicating that the environmental determinant of convolution form must have 
operated while the hair was still growing in length. Our evidence is based on 
too slight data to be conclusive in itself, but it will be noticed that it confirms 


* W.L. B., “ Raw Cotton,” pp. 79 and 147, loc. cit. 
+ Vide supra. 


434 Dr. W. L. Balls and Mr. H. A. Hancock. 


O mms. 1 2 4 S & 


1 


( ~\—+—_—_—_—\. Wea 


a 


SSS 7ST 


b 


: Pens Vi 
& 


AAI aT / Kaa He 


VY Ey Va / Dee LTA 
de > <a> RE TE oT DON 


IS2SSS Se Ss SSS" ae 


Ne Aan 4 N ee eae NAC ee oe 
eee oe ee SEO OOO OSES 
é 
eZ a. a NS ee 


Fie. 10—a to f.— Each pair of drawings represents one piece of hair and shows :— 
above—the position of the convolutions with slanting lines drawn to indicate the 
direction at each turn ; below—direction of slope of pit spirals, as determined at. 
various points after pressing. Length corrected to original value. Exceptionally 
discrepant pieces have been selected for reproduction. 


our conclusions already drawn from microscopic work, and thus increases 
their probability. 


On Cell-wall Structure as seen in Cotton Haars. 435 


Sep. 20. @ Oct.10. Oct.20. 


@ 


N 


oO 


No. of Convolutions per |-6mm. field. 


Sep.20. Seps0,__—Occt.10 Ere uOch:20) 


5 8 


Percentage distribution as between the three. 


Fie. 12. 


Figs. 11 and 12.—From capsules opening on successive days in the daily pickings series 


(q.v.) five seeds were taken each day at random, and twenty hairs of similar length 
from each of these five were arranged with their bases in line. The number of 
convolutions included in a microscope field of 1°6 mm. was then determined at three 
loci along the hair, respectively, 5,15 and 25 mm. from the base. The curves as 
shown are the three-point means of the original data, each point thus representing 
the average of 300 hairs. The data are further analysed in fig. 12 in order to 
discriminate between general fluctuation common to the whole length of the hair, 
as indicated in fig. 11, and the existence of differential fluctuation in various parts, 
due to determinations taking place at different times during development. The 
logarithmic plotting shows that the latter fluctuation is quite marked, though its 
amplitude is reduced by a half. 


436 Dr. W. L. Balls and Mr. H. A. Hancock. 


: (cont.) 
191g. .- ai 
October } 
8 19 
2 pe 
. 20 
7 yo” 
Regt 22 
12 Fee 
13 
Q4 
14 
15 : 
: Pe 26 
ater aay 27 
17 


Fie. 13.—Average distribution of convolutions from day to day along the whole length 
of the hair. Data obtained by taking twenty hairs from the same square milli- 
metre of one seed chosen at random each day. These hairs, being roughly of the same 
length, were arranged with their bases in line and the number of convolutions in a 
1°6 mm. field measured at intervals of successive half millimetres. In five cases, 
where only twelve hairs were measured, the scale of the distribution curve has been 
corrected accordingly. The letters a, « and } are placed on some of the curves to 
indicate the general tendency which appears to be shown, though indistinctly, by 
the various modes, in shifting backwards towards the base as the date of opening of 
the capsule becomes later. Base of hair at left hand of each curve. 


On Cell-wall Structure as seen in Cotton Hairs. 437 


The actual data were obtained by selecting usually twenty hairs of equal 
length on each day over a sequence of thirty days, and counting the convolu- 
tions in successive intervals of 1°5 mm. from base to tip; they thus comprise 
some 20,000 measurements, but statistical considerations make it evident that 
they need to be greatly extended in order to be conclusive, and in practice it 
should be found easier finally to attempt the proof by means of direct 
physiological experiment and by observations of the pit spirals. 

A fairly close correspondence was indicated as between the general form of 
the hair-length growth curve (formerly ascertained)* and the shift of any 
convolution-form /ocus from day to day. 

(c) A renewal of a former attempt was also made, in order to see whether 
any forms or markings could be found along the length of the hair which 
would correspond for linear extension to the daily marks of the growth-rings 
in secondary thickening. The system of convolution mapping was extended 
to measure the length and direction of every separate convolution. These 
measurements were then plotted as shown (fig. 14), using rectangles of equal 
area for each one, the bases of which were equal to the convolution length, 
and in this form of plotting it is very evident that the convolution sequence 
along any one hair is at least wave-like. Phases of steep-pitched and short 
convolutions alternate with phases of slow and long convolutions. The 
discrepancies between convolutions and pit spirals seem to happen chiefly in 
the latter phase, which also seems, as might be expected, to contain more 
convolution reversals (fig. 14, 6). The number of peaks (short convolution 
phases), in the curve, seems to tend towards correspondence with the number 
of days (about 25), during which the hairs used in these observations (daily 
pickings samples) were growing in length. 

Here, again, no rigid conclusion can be drawn, but the facts are certainly 


’ very suggestive of a daily environmental effect, acting by predetermination on 


the convolutions through the pit spiral patterning of the primary wall. 

(d) We have not forgotten the probability that mutual pressure inside the 
growing capsule, together with the curling grouping which the hairs thereby 
take up, may influence both pit spiral and convolution, but we anticipate that 
this will be found subsidiary to the other causes indicated. 

(e) A source of error in other observations made on material which had 
been acted upon by softening and swelling re-agents should be noted. When 
the wall cellulose is softened with the hair held in slight tension, the 
convolution spiral may become mechanically unstable and instantaneously 
jump to a new conformation, the hair becoming a cylindrical helix, like an 


* W. L. B., “ Raw Cotton,” p. 76. 


Dr. W. L. Balls and Mr. H. A. Hancock. 


438 


“wu 9g 


/ fi DVI MM _ | VUE NE 


VL WINN /\_ A/D 


pm aca 


“wut O} 


JY /\/ rA\DA 


Win NA\ \ 


‘wuio2 
VSTTISESN IES / YY N/ 


SASS 


0 FT 
pue ‘» FT ur se 
Yjcue] sy. sulMoys 
OY WOVEN OO! = i 
UOTZNTOATIOD yore 
jo woooaitp 943 
894 8OIPUl OUT] BSC 
ey} MOTEq TMeAIp 
seul, sutdots jo 
sotdos 8 ‘9 “FT oy Jo 
O8Bd 9Y} UT “Uol4 
-njoauod = A@aa 
I9AO aie yenba 
JO Sopsueqoor Suy 
-jord Aq yueprtae 
810 OPBUI SI UOTY 
NQI4sIp  IvoeuTT 
ayj jo wuI0y oy, 
“IIV UL OpVUl a19M 
SUOTJVAIASGO 9y2 
‘el “SY Ur ose sw 
‘a “q ‘np ‘sarey 0014 
SUOTe UOLyNTOATOD 
AroA9 Jo woisod 


al} SMOYS FL “Ol 


On Cell-wall Structure as seen in Cotton Hairs. 439 


“ Ayrton spring.” This change is apt to complicate observations on wall 
structure. 
The Fuzz Hours. 

In a previous communication,” one of us has pointed out that fuzz and lint 
appear to be identical in all respects, except that growth in length is inhibited 
in fuzz, while growth in wall thickness is restricted in the lint. None of the 
present observations have revealed any further differences. In many cases 
we have found it convenient to try out new methods, or hypotheses, on the 
fuzz hairs before attempting to apply them to the more delicate lint. 


, 


Conclusions. 


1. A spiral fibrillar radial structure exists in every growth-ring of the cell- 
wall of the cotton hair. 

2. The simple pits of the cell-wall are a special case of this general 
structure. 

3. The pattern of the spiral appears to be predetermined during growth in 
length. 

' 4, This pattern is preserved through all the growth-rings cf the secondary 
wall thickening. 

5. The number of fibrils in cross section of one hair is of the order of 
1,000 upwards. 

6. The pattern (direction, reversal, and pitch) of these spirals seems to be 
the major determinant of the externally visible convolutions of the hair. 

7. There are indications that the unknown cellulose-ageregates, which 
compose any one spiral fibril, have a definite geometric conformation, 
suggestive of stereo-isomerism. 

&. Attempts to elucidate the cellulose-structure further, as by X-rays, will 
probably have to take account of this spiral fibril arrangement. 


While assistance in various ways has been given by our colleagues in the 
Experimental Department of the Fine Cotton Spinners’ and Doublers’ Associa- 
tion, to whose Executive Directors we are indebted for permission to publish 
this account, we would wish especially to acknowledge the interest and 
assistance of Dr. Mary Cunningham. The influence of Dr. H. E. Williams in 
re-energising this inquiry has already been acknowledged, while the inde- 
pendent development of work along similar lines by Dr. S. C. Harland and 
Mr. Deuham, at the Shirley Institute, has been of indirect assistance. 


* W.L. B., “The Existence of Daily Growth Rings in the Cell-wall of Cotton Hairs,” 
Loc. cit. 


440 Dr. J. Hjort. Observations on the Distribution of 


DESCRIPTION OF PLATE. 


Figs. 1 to 7 inclusive, are all photographed with Spencer 4 mm. objective and 10 x 
eyepiece. 


Fig. 1.—Transverse section of Sakel hair, possibly accidentally pressed, and slightly 
swollen with sub-critical strength sulphuric acid ; showing radial (spiral) cracks. 
Fig. 2.—Transverse section of same cotton as fig. 1. Section lightly pressed, then 
swollen with critical strength sulphuric acid ; showing growth rings. 

Fig. 3.—Ordinary Sakel hair mounted in liquid paraffin, untreated. (Denham and 
Harland’s method.) : 

Fig. 4,—Hair of Sakel cotton boiled in dilute HCl, and pressed. 

Fig. 5.—Hair of Sakel cotton pressed heavily in 8 per cent. caustic soda. 

Fig. 6.—Hair pressed in naphthamine blue. Spiral structure entirely due to pressure: 
only. 

Fie. 7.—Hair of Sakel cotton pressed lightly in an adjusted mixture of cuprammonium. 
and soda. 

Fies. 8 and 9 are photographed with Spencer 16 mm. objective only, and 10 x eyepiece. 

Fig. 8.—Hair swollen with calcium thiocyanate, showing double thread spiral. 

Fig. 9.—Hair of Sakel cotton stained with iodine, but not otherwise treated ; showing 
double pit spirals. 


Observations on the Distribution of Fat-Soluble Vitamines %n- 
Marine Animals and Plants. 


By Joan Hoyort, D.Sc., For.Mem.R:S. 
(Received April 27, 1922.) 
(From the Biochemical Laboratory, Cambridge.) 


The Norwegian fishery investigators have for many years been engaged in: 
the study of the growth of fish, mainly the herring and the cod. By means. 
of microscopical study of the scales of the fish it has been possible to determine 
the age of each individual fish, and by means of the assumption, which has. 
been verified within certain limits, that there is a proportion between the 
length of the scale (/,) and the length of the fish (/,) (ae., /.//, = constant). 
it has been possible to calculate the “growth curve” of the fish in different 
years of its life, and in different seasons of the year. The results of this work,. 
which have been summarised up to the year 1914,* proved that the growth 
of the said fish in the Norwegian waters was confined to a few spring—and 
summer—months only, and that the growth of the fish entirely ceases during 


* Johan Hort, “ Fluctuations in the Great Fisheries of Northern Europe,” ‘ Rapports- 
et Procés-verbaux du Conseil International,’ vol. 20, Copenhagen (1914). 


Balls and Hancock. Roy. Soc. Proc., B, vol. 93, Pl. 10. 


— 


4 
; 
; 


Fat-Soluble Vitamines in Marine Animals and Plants. 441 


the winter season. Corresponding to the summer and winter zones, which 
are to be seen on the scales of the fish, we find a periodicity in the increase of 
length and weight of the fish, and a change in the quality of the fish at the 
different seasons of the year. 

The changes in the “quality” of the fish were very early observed to be 
associated with the changes occurring in the content of fat. Chemical 
analyses were made by Mr. H. Bull of the fat contents of herrings and sprats 
at different months of the year,* and from these observations the conclusion 
was drawn that “the supply of fat increases during the summer and is 
consumed during the winter, while water is excreted in the summer and 
assimilated in winter. During the winter, part of the dry matter in the 
system is consumed and replaced by water, so that no great loss in weight is 
apparent. ‘The quality of the fish, however, is considerably affected.” . 

In the herring this is very apparent, the fat being deposited in a special 
peritoneal fat organ (by Norwegian fishermen called the “ister”), which is 
especially developed in summer and in the young year classes which have not: 
yet developed their generative organs. As stated in the paper, the contents 
of the fat organ “varies with the age of the fish, the development of the 
genital organs, in particular, being a factor of great significance. In addition 
to this, the quality of ister varies greatly according to the time of year; this 
applies not only to the ister itself, but also to the contents of fat on the whole 
as shown by chemical analysis.” 

In the cod the changes in quality are most easily demonstrated by the 
inspection of the size of the liver, the contents of which, so far as some 50 per 
cent. is concerned, consist of the “cod-liver oil.” Besides by chemical 
analyses, the liver has been studied by means of weighings. The livers of 
cod of different sizes (in groups of different lengths) have been weighed at 
different times of the year, and it has been found that the liver of the full- 
grown cod during the summer season, when the fish was feeding, weighed no 
less than three times as much as in the winter during the spawning season. 
“The greatest ‘depreciation’ in the quality of the cod takes place during 
their stay on the Lofoten Banks, where the genital organs arrive at full 
maturity and spawning takes place.’+ 

These observations naturally led to a consideration of the problem of the 
conditions which determine the great changes in the growth of the fish 
themselves, and particularly in the organs in which the reserve material (fats) 
are stored. A comparison between the amount of fat in the sprat of the 
west coast and the mean surface temperature of the sea for the different 


* See loc. cit., Chapter V. 
+ Loe. cit., p. 195. 


442 Dr. J. Hjort. Observations on the Distribution of 


months of the year* showed a remarkable correspondence for the spring and 
early summer months, 7.c., the amount of fat increases in close correspondence 
with the rise in temperature in the spring, but this relationship does not 
continue during the late summer and autumn, thus indicating that the 
temperature cannot be considered as the only influence concerned. 

The rise of temperature in the sea during the months of spring is of course 
in northern waters accompanied with a great number of other phenomena, 
viz., the increase of the intensity of light, the increased addition to the sea- 
water of masses of fresh water from the continents carrying with them 
inorganic and organic substances from the land and the seashore. Asa result 
of these events an “eruptive” development of plant life takes place, and 
shortly afterwards plankton animals (i.e, copepods) develop and bottom 
animals begin to increase their growth. 

While the development of the plants and of the small animals which 
directly prey on them may seem easy to understand as a result of the seasonal 
changes of the spring, the increase of the growth of larger animals like the 
cod, the prey of which exist all the year round, seems much more difficult to 
explain. The problem, therefore, seemed worth examination, whether the 
decrease of growth in the autumn and the increase of growth in the spring 
were connected with the availability of certain specific kinds of food, in other 
words if there could be found in nature a variation of chemical qualities in 
the food of the animals. 

The fact that the changes in the quality of the animals during the different 
seasons have been found to be in such a close connection with changes in 
their contents of fats raised the desirability of investigating the distribution 
of the fat-soluble vitamines in organisms of the sea, since these vitamines 
have been found to have such a great influence on the growth of animals. 

During my work at the Biochemical Laboratory of the University of 
Cambridge, Prof. F. G. Hopkins had the great kindness to offer me the 
opportunity of conducting experiments on such vitamine problems, and 
allowed me for this purpose to have the benefit of the organisation for 
experiments on rats which he has established at his laboratory. For the 
purpose of such experiments during the summer 1921 I made a collection of 
different material of plankton and bottom animals from Norwegian waters. 
This material was preserved in alcohol, and it was extracted in the laboratory 
(under anaérobie conditions) with alcohol and ether, in order to extract the 
oils for the experiments. This plan, however, had no success. The fats 
obtained in this way were in the form of waxes, and it was soon realised that 


* Loe, ctt., p. 173. 


Fat-Soluble Vitamines in Marine Animals and Plants. 443 


for some reason they were unsatisfactory for nutritional experiments and gave 
ambiguous results. Other methods had therefore to be applied. 

In the spring of 1922 material was, therefore, collected partly in a fresh or 
sterilized condition, partly in alcohol. In co-operation with my assistant, 
Dr. Axel Palmgren, a different method was developed for the extraction of 
the oils. 

The fresh material was first minced, then dried in thin layers on filter 
paper in the constant-temperature room of the laboratory (at 37°). The 
dried substance proved in all experiments as effective as fresh substance, 
and it could therefore be applied for the purpose of extraction by acetone or 
benzene. ‘The filtrates (from three to four extractions) were evaporated in 
vacuo, the last part of the solvent evaporated off in a water bath at 90°-100° 
in a high vacuum for 15 minutes, till the remaining oil no longer smelt of 
benzene. The oil was then dissolved in olive oil, and the mixture given to 
rats by a counted number of drops, 100 drops making 3 ce. of oil. At. 
a specific gravity of 0-9, one drop would then have a weight of 2°7/100, or 
27 mgrm. If one part of the oil had been dissolved in seven parts of olive 
oil, eight or sixteen drops were given to each rat daily, in order to give the 
rat one or two drops of the oil under experiment. In the following “one 
drop” means one drop of the oil under experiment. The oil was not mixed 
with the food, but administered by means of a pipette kept just over the 
mouth of the rat, which was laid on its back in the left hand. This method 
of feeding was recommended to me by Prof. E. Poulsson, of the University of 
Christiania. 

The experiments are all of a preliminary character, and had mainly the 
purpose of exploring the distribution of fat-soluble vitamines, for future 
more detailed and more quantitative studies. In spite of their preliminary 
character, it has been thought useful to publish the following observations, 
as they may stimulate other workers to investigate this field. 


Marine Plants. 


As a preliminary investigation, it seemed to me of interest to try some 
green alge as representatives of the vegetation along the shore and some 
samples of the first growth of diatoms in the coast waters during spring. 

Green alge (Ulva lactuca and Codiwm tomentosum) were kindly collected for 
me at the Marine Biological Station at Pert Erin by H. H. Thomas and 
S. H. Wadham, of the Botany School of Cambridge. The alge were brought. 
to us in fresh condition, and given partly in fresh and partly in dried 
condition to rats which for some time had been on a diet free from 
fat-soluble vitamines, but containing vitamine B. and other essentials. The 


444 Dr. J. Hjort. Observations on the Distribution of 


three curves (figs. 1 and 2*) all show a marked increase in the weight of the 
rats when given the seaweed. Fig. 1, the lower curve, represents the growth 
on fresh seaweed, kept in cold store. The increase is not so marked as the 
two other curves showing the effect of dried seaweed. This produced an 
enormous increase in the weight. The dried seaweed was added to the food 
as a fine green powder and was thoroughly mixed with the food. 


110 

100 

90 

80 

70 

60 

50 

aa 

5 10 15 20 25 30 3 “5 To 15 20 25 30 35 40 45 
Fic. 1.—Lower curve, fresh ulva, ad lib. Fic. 2.—Dried ulva, 1 grm. per day. 
Upper curve, dried ulva, 1 grm. 
per day. 


In order to see if the growth-promoting factors of the ulva were soluble in 
its fats, dried powder of ulva was extracted with acetone and the oil given 
(1 or 2 drops a day) to the rats. The resulting growth-curves may be seen 


60 a 
20 25 30 35 40 45 5 10" 15% 207 25 S07 35940 


5 10 15 
Fie. 3.—Extract (by acetone) of ulva, Fie. 4.—Point 1, added 1 gr. sterilised 
two drops a day. diatoms per day. Point 2, 15 grm. 


dried diatoms per day. Point 3, 2 drops 
benzene extract of diatoms per day. 


* Tn all the figures the abscissa gives the number of days of experiment, the ordinate 
the weight in grams. 


Fat-Soluble Vitamines in Marine Animals und Plants. 445 


from fig. 3. They demonstrate a marked increase in growth of the rats from 
the moment when the oil was given. 

Samples of Diatoms were collected for me by my friend, Prof. H. H. Gran, 
and Mr. E. Lea on board a Norwegian research vessel. The samples con- 
sisted of a pure “ Diatom-plankton,” as this may be found in the sea in the 
spring before the animals have started their development. The rats fed on 
these diatoms showed first a sharp increase of growth, and then, after a short 
time, a marked decline. The idea suggested itself that the diatoms, which 
contain so much silica, may be very unsuitable as food for a mammal, 
and diarrhoea was also observed in one case (see fig. 4). In some cases, 
two drops a day of green oil, extracted by benzene from the dried mass of 
diatoms, gave at once a marked increase, even in the case of animals which 
had first been fed on the dried material (fig.5). Although these experiments, 
which were hampered by rather scanty material, do not give such a marked 
and striking growth as the curves of the seaweed, I have no doubt that an 
increased growth has been demonstrated as a result of addition to the basal 
diet of the rats of oils extracted from diatoms. 


SemmON MON, (5m 20ka5e:308 35) 40) osu siqimis) 20) 85 50 35 
Fie. 5.—Point 1, ea.0°8 grm. dried diatoms Fie. 6.—Fresh shrimps ad Lib. 

per day. Point 2, 2 drops extract per 

day. 


Shrimps and Prawns. 


Fresh shrimps (Crangon) and fresh prawns (Pandalus borealis) were minced 
and given fresh as an addition to the basal diet of several rats. Figs. 6 and 7 
show a marked increase in the weight of the rats shortly after the addition of 
these substances to their diet. 

Dried powder was extracted by benzene, the extracted oil (two drops a day) 
had in several cases no effect, a fact which I am not able to explain and which 
must be left for future investigation. 


446 Dr. J. Hjort. Observations on the Distribution of 


i aledal eal 
se faa a 
Blind bo, 
Cart 


Se OMISEM 20m 25 SOs : 5 (0 15 20 25 30 35 40 45 50 5s 
Fig. 7.—Fresh prawns ad lib. Fic. 8.—Four drops of oil from roe of herrings. 


eee Cees Ce a 
si le | itl nla 
ie 
Cesta es = aS 
ney Me reo 


130 


120 


5 10 15 20 25 30 35 40 45 50 55 60 65 
Fie. 9.—Fresh cod roe ad lb. 


5) HG) WIS 20, 2530.35) 40445) G0 755 oONiGs 
Fie. 10.—Fresh cod roe, 2 grm. per day. 


| Fat- Soluble Vitamines in Marine Animals and Plants. 447 


Herring and Cod. 


I have mentioned in the introductory remarks above that the fat organ of 

the herring and the liver of the cod both decrease in weight at the same time 

| as the ovaries of the female anima] increase in weight. An examination of 

| the contents of fats and of fat-soluble vitamines of the ovaries (the roe) 

: would therefore seem desirable. Fig. 8 shows the effect of addition of oil 
| extracted from the ovaries of herrings. 

Fresh cod roe (figs, 9 and 10) in a dose of 2-3 grm. per day produced an 

immediate and rapid increase in the weight of rats. Dried roe, prepared in 

Norway for commercial purposes (1 grm. added per day), had — 11 and 12) 


V7 


5 10 15 20 25 30 35 40 45 50 55 
Fie. 11.—Dried cod roe, 1 grm. per day. 


5 10 I5 20 25 30 35 40 45 50 55 


Fic. 12.—Dried cod roe, 1 grm. per day. When first given 
one eye very bad, after some days almost clear. 


just as marked an influence; and this fact must, like the effect of the dried 

ulva, be considered as very important, since it proves that the process of 

drying fish products had no observable destroying effect on the growth- 
VOL. XCIIIL—B, 21 


448 Fat-Soluble Vitanunes in Marine Anamals and Plants. 


promoting factors. Drying of fish and fish products plays a great dle mm the 
fishing industry of different countries, and it is therefore of practical value 
to know that the process does not deprive the products of these valuable 
qualities. 

Extraction of the powdered dry roe by means of benzene produced a clear 
but thick oil, different from the wax, which was obtained by extraction with 
alcohol and ether. The effect of one drop daily of this oil is illustrated by 
the curves on fig. 13. In one rat, which had developed the eye disease 
(keratomalacia) characteristic of cases upon a diet deficient in the fat-soluble 
vitamine, this disease was cured after the addition of the oil of the roe, but 
this rat did not later on show any increase in its growth. 


SR OMISI EOS SO oo tOcto 
Fie. 13.—Benzene extract of dried cod roe, 
1 drop per day. 


The fact that oils extracted from marine plants have been proved to have a 
very strong effect on the growth of rats fed on a diet deficient in fat-soluble 
vitamines seems to indicate the working hypothesis that fat-soluble vitamines 
in the sea arise in plants in a similar way to that proved to be the case in 
plants on land. It seems, further, natural to assume that all marine animals, 
directly or indirectly, obtain these substances from the plants, and that the 
marine animals, like other animals studied, are unable to synthesise these 
substances. An important programme for future work seems then to suggest 
itself, viz., to determine the quantitative distribution of fat-soluble vitamines 
in different types, and in them during different seasons of the year. It may 
be possible by such studies to approach the important problems of the 
influence of the food of animals on that periodicity in their growth which is 
so remarkable in Northern waters. The present investigations seem to 
indicate the problem to ascertain how far the production of fats and fat- 
soluble vitamines in the plants during spring and early summer months will 
have to be regarded as the store on the magnitude of which the growth of 
animals is dependent in Northern latitudes. In Southern latitudes the 


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On Blood-Platelets. re 449 


growth of fish has been found much more uniform throughout the year; the 
changes in the production of plants are there not so marked as in the high 
Northern latitudes. 

Dr. Palmgren and I intend, if circumstances permit, to continue the 
investigations towards the solution of these problems at the Biological 
Laboratory of the University of Christiania. 


On Blood-Platelets: their Behaviour in “ Vitamin A” Deficiency 
and after “ Radiation,’ and their Relation to Bacterial 
Infections. 


By W. Cramer, A. H. Drew, and J. C. Morrram. 
(Communicated by Prof. W. Bulloch, F.R.S. Received March 28, 1922.) 


(From the Laboratories of the Imperial Cancer Research Fund and from the 
Radium Institute.) 


Introductory. General Effects of Fat-soluble Vitamin Deficiency. 


When the fat-soluble vitamin A is withheld from the diet of a rat, the 
general condition of the animal differs from that resulting from a deficiency 
of the water-soluble B vitamin. In the latter case the animal ceases to 
increase in weight almost at once, and then begins to decline. There is a 
progressive fall in the temperature. The animals always die within a 
comparatively short time—two months—and are then found to be in a state 
of profound emaciation, as if they had received no food at all. There is no 
obvious sign of disease or of an infection. For the sake of convenience, we 
will designate briefly this condition by the term “ marasmus.” 

The withholding of the fat-soluble vitamin A alone affects ~ different 
individual rats In a- manner as varied and indefinite as the conditions 
obtained by a deficiency in the water-soluble B are constant and definite. 
When a young rat is kept on a diet from which the fat-soluble vitamin A is 
absent, the increase in weight may cease almost at once, or it may continue 
to increase in weight for many weeks, and almost as rapidly as on a diet 
containing vitamin A, although eventually its growth will come to a 
standstill before the full normal size of an adult rat has been reached. 
We shall, for the sake of convenience, describe these two extremes as the 
“acute” and the “chronic” effect on growth respectively. Eventually the 

VOL. XCIII.—B. 2K 


450 Messrs. W. Cramer, A. H. Drew, and J. C. Mottram. 


rats develop infective conditions, which attack most frequently the eyes, and 
xerophthalmia develops. There may be other organs affected (septic glands or 
pneumonia sometimes develop). 

These infective conditions develop most rapidly in those rats which show 
the “acute” effect on growth, when it may first appear after 6-8 weeks. 
In the rats showing only the “chronic” effect on growth, an infection may 
not develop at all or very much later. We have kept rats on a fat-soluble 
vitamin-free diet for four months without any infection appearing, although 
these rats were kept in the same cage with rats which had developed a very 
intense xerophthalmia. The external appearance of these rats was, in fact, 
such as to make them indistinguishable from normal rats. It will be shown, 
however, that such rats develop the lesion which we consider to be specific 
for the vitamin A deficiency, although not to the same extent as rats 
exhibiting the acute effect on growth. The condition of nutrition of rats on 
a vitamin A-free diet varies, as is to be expected. The rats which show the 
acute condition are ill-nourished, but the extreme condition of emaciation 
seen in the rats suffering from the B deficiency is hardly ever met with. 
Another important difference is that the progressive fall of temperature, 
which is so characteristic of the B deficiency, does not appear in the 
A deficiency. - 

We have been able to control experimentally the conditions under which 
the “acute” and “chronic” effects of fat-soluble vitamin deficiency appear. 
Hitherto, the reason for these differences has not been understood. One 
important factor is the nature of the diet which the rats have received in 
infancy. If the mother during pregnancy and lactation has received a diet 
rich in vitamins, and if this diet be given to the young rats after they have 
been weaned, then the rats are found to be more resistant to a subsequent 
withdrawal of the fat-soluble vitamin. If, on the other hand, the diet has 
not been particularly rich in vitamins, although it may contain an amount 
adequate to maintain the animals in health and to enable them to grow and 
to breed freely, then withdrawal of the fat-soluble vitamin will produce an 
immediate and “acute” effect. It may be added that the diet we refer to 
is not an artificial one, but a natural diet of bread and water, rice and 
maize, which has for years been used in our laboratory as the standard diet 
for our stock of rats. Incidentally, this clearly emphasises the great 
importance of assuring an ample supply of vitamins to the pregnant and 
lactating woman, and to children, and of not being satisfied with the 
comfortable belief that our ordinary food contains sufficient vitamins, 
because we do not suffer from deficiency diseases. . 

Another factor which determines the onset of xerophthalmia is the amount 


On Blood- Platelets. A51 


of the water-soluble vitamin supplied. When a small amount of this 
vitamin is given, xerophthalmia develops much earlier than when a large 
amount is given. This statement refers to experiments in which the water- 
soluble vitamin was supplied in the form of “ marmite.” 

Between the two extremes which we have described, all intermediate 
conditions may be observed. These large variations in reaction to a with- 
drawal of the fat-soluble vitamin make it, of course, very difficult to 
appraise the significance of any changes in tissues and organs which may be 
observed. Many changes which can be seen and have been described must 
be regarded as accidental. For instance, the failure in nutrition which 
may occur is obviously not an essential feature, and is not necessary for the 
_ onset of infective conditions. In connection with our previous work on the 
relation between lymphoid tissue and nutrition, it is of interest to note that 
there may be a considerable lymphopenia when the animals have shown the 
“acute” effect and are in a poor state of nutrition; but when the eye 
condition develops in a well nourished animal, the lymphoid tissue is normal 
and the lymphocyte count shows only a slight diminution. The only general 
feature common to all rats which have been subjected to a fat-soluble 
vitamin deficiency is a greatly diminished resistance to infection. 

We have dealt in considerable detail with the great variety in the general 
conditions of animals on a vitamin A-free diet, because a lesion, if it is to be 
considered specific to this deficiency, must be present in all these animals. 
Further, the severity of such a lesion should be found to vary with the 
extent to which the animal is affected by this vitamin deficiency. And, 
lastly, the lesion should disappear when the deficient vitamin is supplied, 
and the animal recovers as the result. 

We have found such a lesion wn the great reduction in the number of blood- 
platelets. We were led to look for a change in the platelets, because we 
noticed an obvious change in the condition of the blood of rats kept on a diet 
deficient in the fat-soluble vitamin. When the tail was cut for the purpose 
of examining the red and white corpuscles, the blood flowed much more 
freely than in a normal animal, and it was much more difficult to arrest the 
bleeding. When a film was made, it was more difficult to obtain an even 
spreading. There were no constant differences in the number of white 
corpuscles or of the red corpuscles to account for this change, although, as 
will be seen later, there may be, in advanced stages of the deficiency, a 
distinct anemia. But the diminished coagulability of the blood, as it 
manifests itself by the difficulty of arresting the bleeding, sets in long 
before the anemia occurs. The essential importance of the platelets in 
blood coagulation, which the previous observations of Cramer and Pringle (1) 

2K 2 


452 Messrs. W. Cramer, A. H. Drew, and J. C. Mottram. 


and of Bordet (2) had demonstrated, led us to examine the blood for 
platelets. 

Method of Counting Platelets in Rats—The animal is deeply etherised, and 
the tail placed in a watch-glass filled with a solution of 2 per cent. sodium 
citrate in 0°6 per cent. NaCl solution. After cutting the tail and allowing 
the blood to flow until free bleeding is established, the tail is transferred 
quickly to another watch-glass containing about 1 c.c. of the citrate solution 
or of Toisson’s fluid. Blood is allowed to flow so that a mixture of the 
solution and blood convenient for counting the red blood corpuscles and the 
platelets is obtained. After the first few counts it is easy to recognise 
when the mixture is of convenient concentration. The tail is then wiped 
dry and the pipette of the hemocytometer is then filled with blood in the 
usual way, so as to obtain a count of the absolute number of red corpuscles. 
In order to count the number of platelets, the citrate-blood mixture is 
thoroughly stirred, a standard drop placed on a large slide and covered with 
a cover-glass, which is then cemented with melted paraffin. After allowing 
the cells to settle, the proportion of red cells to platelets in each field is 
counted, until about thirty platelets have been counted. From this the 
absolute number of platelets can be calculated. A helpful device for 
counting the platelets, which has proved very useful, is as follows: A coarse 
erating of about 1 mm. squares is ruled on a piece of ground glass with a 
lead pencil. This is mounted in balsam under a cover-glass, and placed 
close up to the source of light at right angles to the beam. The image of 
the grating is then focussed by means of the substage condenser, after the 
preparation has been focussed with the objective, and conveniently divides 
the field for counting. 

Red Cells and Platelets of normal Rats——The following Table gives these 
data for nine normal rats. The counts for the first five rats were made in 
one laboratory, those of the last four in the other. The figures obtained show 


Table I—Normal Rats. 


| Soha? 
Weight in 

erm. Red cells. Platelets. | 

150 10,400,000 960,000 

150 11,840,000 730,000 

150 10,660,000 845,000 

100 10,480,000 720,000 

120 9,960,000 660,000 
50 8,194,000 786,000 
50 10,928,000 1,050,000 
60 10,128,000 912,000 
60 | 9,888,000 1,000,000 


On Blood-Platelets. 453 


that the average red cell count for the normal rat lies approximately between 
9,000,000 and 10,000,000 cells per cubic millimetre, the average platelet 
count lies approximately between 700,000 and 900,000 per cubic millimetre. 
The variations which have been found: include not only individual differences 
of different rats, but also differences due to age, feeding, and those due to the 
personal factor involved in the technique. The various counts made on rats 
showing complete recovery from the vitamin A deficiency (see fig. 4 and 
Table) and from the effects of radium (see fig. 5) give similar figures. 

Effect of Vitamin A Deficiency on Platelets—The rats were kept on a basal 
ration of casein, starch, autoclaved olive oil and the usual salt-mixture, to 
which vitamin B was added in the form of marmite. The olive oil prepared 
as above was known to be free from vitamin A. The casein was freed from 
the fat-soluble vitamin in some experiments by repeated extraction with 
alcohol and ether, in others by heating in shallow trays for 24 hours to 130° 
in air. On such a diet free from the fat-soluble vitamin the platelets show a 
progressive diminution in their number, and this “ thrombopenia”—as it may 
be called—proceeds pari passu with the decline in the general condition of 
the animal. Thus in the one extreme condition, when the weight of the 
animal becomes stationary directly the vitamin A is withheld, and when eye 
symptoms develop within two months, the fall in the number of platelets is 
rapid and pronounced. Taking the opposite extreme, when the animal! 
continues to grow at an almost normal rate for several weeks, and infective 
conditions do not make their appearance until much later or not at all, then 
the fall in the number of platelets is delayed and less pronounced, though 
still distinct. In fact; a slight fall in the number of platelets may sometimes 
be the only sign of the vitamin A deficiency, the rat looking quite normal 
and healthy, and having perhaps only a slightly subnormal weight. Our 
observations show that inféctive conditions (xerophthalmia, etc.) do not 
develop until the platelets have fallen below about 300,000 per cubic milli- 
metre. 

It is important to note that the onset of these infective conditions depends 
on the level to which the platelets have fallen, and not on the length of time 
to which the rats have suffered from a vitamin A deficiency, nor on exposure 
to infection, as will be shown presently. When the deficient vitamin A is 
again supplied after a low platelet count has been established, the number of 
platelets increases. Here again there is a close parallelism between the rate 
of increase in the number of platelets and the degree of improvement in the 
animal. 

These statements are based on and illustrated by the experimental data 
given in the following figures and Tables, which explain themselves and need 


454 Messrs. W. Cramer, A. H. Drew, and J. C. Mottram. 


little further general comment. In the weight curve of each rat the arrow 
indicates a count, and the number attached to the arrow gives the number of 
platelets in thousands. Thus “217” means 217,000 platelets per cubic milli- 
metre. The onset of eye symptoms is indicated by “a.” More advanced 
stages are indicated by “xx” and “zx.” In the figures which illustrate 
the recovery from the vitamin A deficiency, the broken line represents the 
weight curve during the last weeks of the absence of the vitamin, the full line 
gives the weight curve after the addition of this vitamin in the form of cod- 
liver oil. Most of these recovery curves refer to animals dealt with in the 
preceding figures, as will be evident from the rat numbers attached to each 
curve. In these recovery curves the sign (e) stands for the complete disap- 
pearance of the xerophthalmia. 

The figures illustrate all the different varieties of conditions which can be 
observed in rats when kept ona diet deficient in vitamin A. Special attention 
is drawn to fig. 1, which refers to an experiment specially devised to illustrate 
the two extreme conditions and the parallelism between the effect of the 
vitamin deficiency on the general condition of the animals and on the 
platelets. 

In this experiment two groups of three rats derived from two litters, X 
and Y, were taken. The litters were born within three days of each other. 
The mother of the litter X had been kept during pregnancy and lactation on 
the ordinary laboratory diet of bread and water, rice and maize. This diet 
was continued for the young rats after they had been weaned until the actual 
experiment began. The mother of the other litter, Y, had been kept on the 
same diet, to which an ample supply of vitamins A and B had been added in 
the form of cod-liver oil and marmite, and this diet also was continued for the 
young rats. 

When the rats were 7 weeks old, the three heaviest and healthy-looking 
of each litter were selected, and the six rats placed together in one cage and 
fed with the vitamen-free basal ration (purified casein, starch, olive oil, salt 
mixture), to which an ample supply of vitamin B was added in the form ot 
crude marmite. As fig. 1 shows, the rats derived from litter X stopped 
growing almost at once and developed the typical eye infection within 8 or 
9 weeks. Those of litter Y continued to grow fairly well at first. After the 
eleventh week their weight became stationary and remained so for the next 
6 weeks (at the time of writing). Up to that time they had not developed 
any lesion or other infective conditions. They looked perfectly healthy 
normal rats in a good state of nutrition, and formed a striking contrast with 
the small, thin, infected rats of litter X. It may be added that the same 
result was previously obtained in a similar experiment, while in a third such 


On Blood- Platelets. 455 


OL* 


180 


No s8a7 ow 
460 


140 


120 


WEIGHT 


f00 


60 


/ 4 a 72 WEEKS. 
xX = EYE LESION 


Fie. 1.—Weight curves and platelet count in six rats kept in the same cage on the same 
vitamin A-free diet. The figure illustrates the parallelism between the degree of 
thrombopenia and the general condition of the animals. 


Table to Fig. 1. 


Se | kde ce Bale Platelets. | Condition of rat. 
| 
| 
386 4 8,720,000 425,000 No growth. | 
387 4 7,450,000 650,000 Growing rapidly. 
384 5 8,320,000 391,000 No growth. 
388 5 8,400,000 550,000 Growing. 
384 9 6,200,000 180,000 | No growth, thin. 
385 9 9,320,600 390,000 { Xerophthalmia beginning. | 
| | 
386 10 | 8,120,000 217,000 Looks very ill. Advanced 
xerophthalmia. 


387 | 10 | 9,880,000 470,000 ae healthy appear- 


389 | 10 8,800,000 670,000 ance. 


\ 


456 Messrs. W. Cramer, A. H. Drew, and J. C. Mottram. 


experiment the difference, although still present, was not so striking. In the 
present experiment the blood was examined at different times after the with- 
drawal of vitamin A in such a way that the number of platelets in a rat 
of one group could be compared with that of a rat of the other group on 
the same day. The results, which are arranged in this way in the Table 
to fig. 1, show clearly that both groups develop a progressive thrombopenia, 
but that this thrombopenia advances much more rapidly in the severely 
affected litter X than in litter Y. 

Fig. 2 refers to four rats which react in the usual way to the withdrawal of 
the fat soluble vitamin from the diet, and requires no further explanation. 


5 
& 
9 
2. 


120 


/00 
N? 269 &* 

7 | N?274 a 

iS 

RN 

9 N? 2759 
60h 2. 

: ; 
46 

12 16 WEEKS 


r] 4 


& 
«= EYE LESION 


Fic. 2.—Weight curves and platelet count of four rats kept on a vitamin A-free diet. 


Table to Fig. 2. 


Tee! Aes | Red cells. Platelets. Condition of rat. 
269 8 8,720,000 249,000 
16 8,240,000 | 280,000 No eye symptoms. 
273 12 6,720,000 154,000 Intense xerophthalmia. 
Bacteria in blood. 
274 7 10,760,000 | 680,000 
15 10,320,000 380,000 Xerophthalmia. 
275 6 8,000,000 | 313,000 
16 7,800,000 | 160,000 Eye symptoms developed 
| two weeks after this 
count. 


We have stated above that, when A is withheld, the amount of vitamin B 
supplied determines to a certain extent the onset of the typical symptoms. 


On Blood-Platelets. A57 


Fig. 3 refers to an experiment on three rats in which a minimal amount of 
vitamin B was supplied. The amount given was sufficient to prevent a fall 


460 


1/40 


(20 


WEIOCHT 


100 
/ 4 8 WEEKS 
X=EYE LESION 
-----ViTAMINE A PRESENT —— ViITAMINE A ABSENT 


Fie. 3.—Effect of restricting the water-soluble vitamin B to a minimum. Two rats, 
Nos. 398 and 399, received no fat soluble vitamin, and developed the typical eye 
lesion. The third rat, No. 397, received an abundant supply of vitamin A, and 
remained well. 


Table to Fig. 3. 


| Weeks 
+ - . | 
Ne. Op Vitamins Red cells. Platelets. | Condition of rat. 
at. | experi- supplied. 
ment. 
| u ! Bin. rete el 
398 7 A absent, 9,680,000 | 230,000 | Xerophthalmia ; died 
minimal two days later of 
supply of B | pneumonia. 
399 Ul 9,400,000 | 330,000 Xerophthalmia. 
397 7 Ample supply 10,400,000 960,000 Has not grown, but 
Control of A, healthy appearance. 
minimal ; 
supply of B. 


of temperature, but was not sufficient to enable the animals to grow. Two of 
the three animals, Nos. 398 and 399, received no vitamin A, and they rapidly 
developed the typical eye lesion, although their weight was over 100 grm. 
when the experiment began. A third animal, No. 397, received an ample 
supply of the vitamin A,and served ascontrol. It did not grow, but remained 
in good health otherwise. In this rat the number of platelets remained 
normal, while the other two rats developed an intense thrombopenia. Further 
evidence that the absence of vitamin B does not markedly affect the platelets 
will be given below. 

Perhaps the most striking evidence of the relationship between the fat- 


458 Messrs. W. Cramer, A. H. Drew, and J. C. Mottram. 


soluble vitamin and the platelets, is afforded by the behaviour of platelets 
when the animals recover from vitamin A deficiency. This is illustrated in 
fig. 4, When the fat-soluble vitamin is supplied again, the platelets increase 
with extraordinary rapidity. Here, again, there is a close parallelism between 
the recovery from the eye lesion and the increase in the number of platelets. 
Thus, in rat 277, the eye lesion completely cleared up within a week, and the 
number of platelets increased from 380,000 to 870,000. In rat 250, with the 


| N° 251 oS 
G 


780 
&K 
460 10.) 
=I: 
® 
aN ; 4 
7 \o je) 
140 a iB a “a eS a 
ou N° 244 9 
aa, x 
120 LK ‘ (0) 
ees ts S o| N°250 
Id © 
Ps Le) 8 = 
Ts Ses G 
S = i Nien os N° 274 3h 
joo} ay [= SK 6 
2 x Kw NS 5 
~~ 
Q = 
° 
80 ; N°273 9 
; | 
CONS ae 
Stee 
IMME OM co 2 
oes, 
Ls) 
4o Xs} 


+1 WEEK > 
X= EYE LESION =EYE LESION DISAPPEARS 


----ViTAMINE A ABSENT. —— ViTAMINE A PRESENT 


Fic. 4.—Effect of adding vitamin A to the deficient diet. The figure illustrates the 
parallelism between the rate of increase in the number of platelets and the recovery 
from infection. 


low value of 220,000 platelets and the presence of bacteria in the blood as 
demonstrated by a film preparation, the platelets increased to almost 1,000,000 
within three weeks, the eye lesion became completely cured within two weeks, 
and the bacteria disappeared from the blood. The reverse condition is 
represented by rat 386, which was very small and thin and emaciated, and 
which, in addition to having the eye lesion, developed an abscess in the neck. 
When cod-liver oil was given the eye lesion cleared up very slowly, and the 
general nutritive condition of the animal did not improve very much; as the 


On Blood-Platelets. 459 


Table to Fig. 4. 


No. of Defietenoy ror Red cells. Platelets. Condition of rat. 
rat. recovery. 
244 | Deficiency 8,720,000 | 480,000 
Recovery, | 
5 weeks 10,440,000 710,000 
7 weeks 10,000,000 820,000 
250 Deficiency 9,280,000 300,000 Xerophthalmia, bacteria in 
blood. 
Deficiency 10,080,000 220,000 Xerophthalmia worse. 
Recovery, : 
1 week 8,000,000 490,000 Eyes improving. 
3 weeks 9,960,000 997,000 Cured. | 
251 Deficiency 7,560,000 480,000 
Recovery, 
2 weeks 8,360,000 995,000 
273 Deficiency 6,720,000 154,000 Xerophthalmia. 
Recovery, | 
2 weeks 9,720,000 550,000 Hyes cured. 
4, weeks 9,760,000 980,000 
| | 
274 Deficiency 10,320,000 | + ~—-380,000_~—sCX|:-Xerophthalmia. 
Recovery, | 
1 week 10,560,000 | 870,000 Eyes cured. | 
386 Deficiency 8,120,000 217,000 Intense xerophthalmia, | 
abscess in neck. | 
Recovery, hae | 
2 weeks 6,800,000 522,000 Looks thin and ill; xeroph- | 
thalmia had almost cleared | 
| up; abscess still present, | 
| but improving. | 
| | 


weight curve also indicates. The platelet count showed only a slow rise from 
217,000 to 522,000 after two weeks. 

Effect of Vitamin A Deficiency on the Red Cells—The Tables show that, in 
the majority of cases, there is no distinct reduction in the number of red cells, 
even when the platelets are greatly diminished and the animal is in a typical 
condition of A deficiency. Occasionally, however, an anzemia develops. Rats 
Nos. 384 and 273 have a distinct anemia with a red count between 6,000,000 
and 7,000,000. These animals also had the most profound thrombopenia. 
Rat 386 is of interest because it developed an anemia, not while the vitamin A 
was withheld, but later on when it was supplied again. It will be recalled 
that this animal had suffered severely from the deficiency and responded with 
only an incomplete recovery. The anemia cannot, therefore, be regarded as 
the characteristic lesion of vitamin A deficiency. Our observations indicate 
that these occasional anzemias may follow an infection of the blood with 
micro-organisms. 


460 Messrs. W. Cramer, A. H. Drew, and J. C. Mottram. 


Lffect of Vitamin A Deficiency on the Leucocytes—This subject has already 
been dealt with in a previous paper. There are no constant or characteristic 
changes. In the final stage, when infection has supervened, there is usually 
a great increase in the absolute number of polymorphonuclear cells. The 
lymphocytes show, as a rule, no more than a slight diminution, which contrasts. 
sharply with the profound lymphopenia observed when the water soluble 
vitamin is withheld. We have already stated that a lymphopenia may also 
occur in the vitamin A deficiency when the acute effect has been produced 
and the animals are in a very poor state of nutrition. 

There appears to be a change in the number of the polymorphonuclear 
leucocytes, in the sense that the nucleus is less lobulated in the animals 
suffering from the vitamin A deficiency. To establish this fact fully would 
require a very extensive series of observations which we do not propose to 
undertake. We only refer to it here because it may afford an explanation of 
the statement that the so-called “ Arneth index ” (number of lobules of the 
polymorph nucleus) of tuberculous individuals is higher than that of normal 
individuals. The explanation may possibly be found in the fact that the high 
Arneth index of the tuberculous individual is due to his dietary treatment, 
the diet being very rich in the fat-soluble vitamin. 

Effect of Vitamin B Deficiency—Some observations were made on rats kept 
on a diet free from vitamin B, but containing an ample supply of vitamin A. 
The results which are given in the following Table show that when the 
vitamin B is absent and vitamin A present the platelets do not diminish to 
any extent, even at a time when the temperature has become very distinctly 
subnormal, and indicates an advanced stage of the deficiency : 


Effect of B Deficiency. 


| | | | 


No. of Weeks of DEE ONE | 
wae : Temperature. of initial Red cells. Platelets. 
rat. B deficiency. oe 
| weight. 
| | 
| ) 
| aa | 5 35 °7 0g | 11,560,000  ~—-980,000 
| 415 5 35 °2 Og | 9,920,000 | 640,000 
416 5 Below 35 —59 10,000,000 —‘1,200,000 
| | 


In the very last stages of this deficiency we have occasionally obtained low 
ficures for the platelets. But here a technical difficulty arises, because it is 
difficult in that condition to obtain a free flow of blood when the tail is cut. 
This is, however, essential, since the blood platelets tend to stick to the tissues. 
of the wound and disintegrate there, when the blood is oozing out slowly. 


On Blood- Platelets. 461 


Even in a normal animal a low count is obtained if, for some reason, a free 
flow of blood cannot be established. 

Effect of Malnutrition Not Due to Vitamin Deficiency.—In order to study 
this effect young rats of about 50 grm. weight were kept on a protein-free 
diet, consisting of starch, salt mixture, and olive oil, to which the vitamins A 
and B were added in the form of cod-liver oil and marmite. As a result the 
rats decline in weight, but remain otherwise in good health, for three or four 
weeks. The platelets show no diminution at a time when the animals have 
lost 10 grm., 2.¢., 20 per cent. in weight, as the following figures show :— 


Effect of Protein Deficiency in the Presence of Vitamins. 


Weeks of un 
ac. ag protein Temperature. Loe | Red cells. Platelets. 
me deficiency. Pas 
| | 
| 
° | 
420 3 38 *4 109 | 9,776,000 | 1,060,000 
421 3 38 °3 109 | 9,280,000 | 930,000 | 
422 3 38 °3 109 | 10,870,000 | 1,230,000 | 


Effects of Exposure to Radium. 


It is well known that a profound lymphopenia can be produced and main-— 
tained by relatively small doses of @ or y radiation. With larger doses 
additional blood changes occur: first a diminution in the number of poly- 
morphs and with still larger doses a reduction in the number of red cells and 
hemoglobin content. For instance, when rats were continuously exposed to 
radium under constant conditions, a lymphopenia occurred within a few 
hours, a polymorpho-leucopenia in 7 days and an anemia in 13 days. 

Examples of these effects are given in Protocols Nos. 1 and 2. 


Protocol No. 1 showing changes in the polymorphs :— 

Tbree male rats: weights 150, 160, 155 grm., exposed continuously to 
220 merm. RaBre, 2H20, distance 8 inches, screen 0:1 mm. lead, 0°12 mm. 
silver, for 7 days—this is equivalent to 0°55 rads. 

Two control rats: weights 210 and 165 grm. 

Protocol No. 2 showing changes in red cells and hemoglobin. Three female 
rats: weights 80, 85, 75 grm. exposed, continuously to radium, as in Protocol 
No. 1, for 13 days, equivalent to 1:2 rads. 

Three control female rats: weights 80, 85, 75 grm. 


462 Messrs. W. Cramer, A. H. Drew, and J. C. Mottram. 


The following Table gives the results of the blood examinations :— 


Days. 
Rat. | Weight. — 
| | 0. | 2 7 11 16 | 23 : 30 
| 
Radium 160 P| 40} 22] 15 | 19 | 14 | 80 | 4 
Z| 105, | 54) 1:4.) 1:2 | 2-4. | seen 
Control | 210 P 251 76 74 8°3 52) — | — 
| L 13 ‘1 15°0 240 27'1 | 20°2 i 
a he 
0 2 4 8 14, 18 | 25 
Radium | 155 Poa 4:3 | 22 o-9 | 11 || eee 
|) 22056 10 ‘2 31 UPA Bu! 9°6 10°1 
Control 165 P 3°8 3°7 4°6 3°6 5°8 3°2 — 
| L 14°7 15 6 23 °2 20°2 | 7-1 20°3 —_ 
| Oaeree |. 8, 15: | 21. 2 seem | 
| = | | 
Radium | 150 12 | 5°3 | 3°5 Ae) UZE6 2°3 3-7 
L | 151 | 4°9 2°0 7-4 9°6 74 
7 P= polymorphs. L = mono-nuclears in thousands per ¢.mm. 
The following Table gives the results of the blood examinations :— 
| | 
| Days. 
| 
Rat. Weight. = S| 
awe 6. 13 
Radium 80 1 | Oat 8°3 4°] Dead on 17th day. 
H HOZipicl a ovep-c- 53 p.c. 
Control 80 R 75 8-0 al 
H 98 p.c — 105 °5 p.e. 
| | hake: 9. 13. TBs | 
Radium 85 | R 9-2 75 8°0 4:9 Dead on 20th day. 
H 97 p.c. | 915 p.c.| 83 p.c. 69 p.c. 
Control 85 R 8°2 8°5 8°3 10°1 
H |104°5p.c.) 111 p.c. | 1015 p.c.| 115 p.c. 
| 
| | 0. 13 
| 
Radium 75 R 76 21 Dead on 17th day. 
H | 96°5 p.c. = 
Control 75 R 8°5 10°3 
H | 108p.c. | 100 p.c. 


R = red cell content in millions. _H = hemoglobin percentage. 


On Blood-FPlatelets. 463 


At death these animals exhibited signs of a generalised infection with 
micro-organisms accompanied by a bronchopneumonia or an enteritis; in one 
case xerophthalmia was present. If instead of continuing the exposure for 
13 days, shorter exposures be given, then an anemia will either not develop 
or will supervene after varying lengths of time according to the dose and 
the weight of the animal; and it is remarkable that so far whenever an 
animal has developed an anemia it has invariably died within a few days. 
This suggests that the anemia is not directly due to the radiation, but is a 
secondary effect possibly due to the invasion of the blood stream by micro- 
organisms. In view of the similarity between these effects and those 
described above as resulting from withholding vitamin A, an examination of 
the platelet content of the blood was made to discover whether this could be 
the primary change to which the invasion of the blood stream by micro- 
organisms and the anemia was secondary. The findings are given in the 
following protocol. 

Protocol No. 3. 

Five rats: weights 60, 60, 65, 70, 90 aa exposed as in Protocol No. 1 for 
5 days, equivalent to 0°46 rads. 

The red cell and heemoglobin content remained normal in every case. 


1100. 


10 


PLATELETS 


1/8 20 22 24 


Fic. 5.—Effect of radiation on the number of platelets in five rats. Rapid diminution 
during and after exposure to radium, followed by rapid recovery. 


464 Messrs. W. Cramer, A. H. Drew, and J. C. Mottram. 


The changes in the number of the platelets are given in fig. 5, p. 463. 
A profound thrombopenia develops as a result of the exposure to radium. 
This persists for a considerable time after the exposure to radium has ceased. 
It is followed by a rapid spontaneous recovery to the normal number—and 
sometimes exceeding it—within a week, if the dose of radium has not been 
too large, as in the present experiment. With larger exposures an anemia 
develops and the animals die from intercurrent infections. These findings 
have an important clinical bearing upon the cases of pernicious anemia which 
have occurred among radium workers and which have on a few occasions 
resulted in death. In all these cases there has been some more or less 
definite evidence of an infection of the blood stream which has made some 
hesitate to attribute the condition entirely to exposure to radium. These 
experiments probably indicate the particular part which micro-organisms 
play in the manifestation of this type of anemia; and they indicate the 
desirability of examining the platelet content in all cases of pernicious anzemia 
especially of the aplastic varieties. 


The Function of Blood Platelets inthe Mechanism of Resistance to Bacterral 
Infection. 


The observations recorded in this paper demonstrate a striking relationship 
between the resistance of an animal to certain bacterial infections and the 
number of platelets present in the blood. When these latter are diminished 
below a certain level and kept there for some time, either by withholding the 
fat soluble vitamin or by exposure to radium, infective conditions develop. 
When a number of rats are kept in the same cage and on the same vitamin 
A-free diet, only those animals develop infections in which the platelets have 
fallen below the critical level. Those rats in which, for reasons given in the 
paper, the platelets have not been affected to the same extent do not develop 
these infections. When the deficient vitamin is supplied again the infective 
conditions clear up as the number of platelets increase, provided of course 
that these infections have not been allowed to persist for too long a time 
producing secondary changes such as anemia, to which the animal eventually 
succumbs. The infections are as a rule of an avirulent kind. In one case a 
bacteriological examination was made. Blood cultures from the heart blood 
showed the presence of two different gram-positive bacilli belonging to the 
diphtheroid group. 

These facts demonstrate that the platelets fulfil an important function in 
the mechanism of resistance to certain bacterial infections. This conclusion 
links the observations recorded in this paper to the phenomenon described 
previously as “defence rupture” or “kataphylaxia” by one of us in conjunc- 


On Blood-Platelets. 465 


tion with Dr. W. E. Gye. Cramer and Gye (3) found that the injection of 
calcium salts, colloidal silicic acid and other colloids and even distilled water 
produced at the site of injection a diminution in the resistance to infection. 
They showed subsequently (4) that all these different substances have in 
common that they produce the same lesion: a damage to the endothelium of 
the smaller blood-vessels which elicits an agglutination of the platelets 
within the vessel, and the formation of a white thrombus with a resulting 
local disturbance in the circulation (see Plate VI, fig. 3, in VI. Scientific Report 
of Imperial Cancer Research Fund). At the same time lymph and plasma 
pass out into the surrounding connective tissue where they form a gelatinous 
clot. When the washed bacteria of gas gangrene or tetanus are injected at 
the site of this lesion the specific disease (gas gangrene or tetanus as the case 
may be) is elicited in a very virulent form at this site. The same bacteria 
when injected into a different site of the same animal do not elicit the disease 
there but, as in a normal animal, undergo phagocytosis and lysis. At the site 
of the lesion active phagocytosis is still proceeding, but evidence of lysis has 
never been observed. This phenomenon of defence rupture is not restricted 
to the anaérobic bacteria of gas gangrene and tetanus, but holds good also for 
Streptococci, and has recently been shown by Gye and Kettle to be valid also 
for tubercle bacilli. The lesion responsible for this phenomenon is one which 
puts the platelets “out of commission,’ so to speak, locally by agglutinating 
them, and which, by its interference with the circulation, prevents the access 
of new platelets. This leads to a /ocal diminution in the resistance to 
infection. In the thrombopenia of vitamin A deficiency, or after exposure 
to radium, there is a general absence of platelets, and this leads to a general 
diminution in the resistance to infection. 

The literature contains some statements which afford direct evidence that 
the platelets are concerned in the elaboration of bactericidal substances. The 
washed platelets and leucocytes do not contain any bactericidal substances 
when tested against the anthrax bacillus. But when they are mixed with 
tissue fluids which, by themselves, are inactive, they confer upon this fluid an 
intense bactericidal power (Gruber and Futaki(5)). It should be noted that 
these bactericidal substances are not identical with the hemolytic complement. 

More recent work would appear to assign to the platelets a somewhat 
different function. C. G. Bull (6) has shown in a series of papers that 
certain bacteria (staphylococci, colon bacilli, meningococci, typhoid bacilli, 
non-virulent pneumococei and non-virulent influenza bacilli) are rapidly 
agglutinated when injected into the blood of a normal rabbit or dog. 
This is followed by a rapid removal of the bacteria from the circulation 
and by phagocytosis and destruction of the agglutinated bacteria in the 

VOL. XCIII.—B. 21 


466 Messrs. W. Cramer, A. H. Drew, and J. C. Mottram. 


capillary systems of the viscera. Those bacteria which are not agglutinated 
remain in the circulation and produce a progressive septicemia. Generally 
speaking, comparing different types of bacteria, the degree of the agglutination 
of the infecting bacteria in the circulation of the host is a measure of the 
resistance of the host to the particular types of organism. Bull drew special 
attention to the fact that with typhoid bacilli, for instance, the mechanism of 
defence in the living body is very different from that observed im vitro by | 
serum or defibrinated blood. In the latter destruction is caused by bacterio- 
lysis, while in the living animal there is the process of agglutination and 
subsequent phagocytosis in the organs described above. Delrez and 
Govaerts (7, 8) have followed up these observations, and have shown that this. 
process of agglutination in the living animal is brought about by the platelets.. 
They found that a few minutes after the injection of certain bacteria there is 
an agglomeration of the platelets and the bacteria. A few minutes later the 
masses of agglomerated bacteria and platelets can be found in the liver under- 
going phagocytosis. 

The thrombopenia, which is produced in guinea-pigs by the injection of an 
antiplatelet serum (the experimental purpura of Ledingham (9) ), does not 
lead to the development of infective conditions, because the animals either 
die within a few days as the result of the hemorrhages, or when they recover 
the thrombopenia rapidly passes off. The thrombopenia alone is, as Bedson (10) 
has shown, not sufficient to produce hemorrhages. These are the combined 
result of the thrombopenia and of a lesion of the vascular endothelium 
produced by the antiplatelet serum. 


Summary. 

The absence of the fat soluble vitamin from the diet always leads in the 
rat to a progressive diminution in the number of blood platelets. This 
thrombopenia is the only constant lesion which we have found, so far, in every 
case of vitamin A deficiency, and we regard it as the lesion characteristic of 
this deficiency, just as the lymphopenia is characteristic of the vitamin B 
deficiency. A thrombopenia may even be found in rats kept on a vitamin A 
free diet when they do not yet show any obvious signs of ill-health, and are, 
to all external appearances, normal animals. When a profound thrombopenia 
has been established, the addition of the missing vitamin A to the diet is 
followed by a rapid increase in the number of platelets to the normal number, 
provided that the animal has not been allowed to suffer too long and too 
severely from the deficiency. 

Exposure to radium produces not only a lymphopenia, but also with 
sufficiently large doses a thrombopenia. From this the animals recover 


On Blood-FPlatelets. 467 


rapidly, if the application of radium is discontinued, and the dose has not been 
too large. 

If by exposure to radium, or by withholding the fat soluble vitamin, the 
number of platelets has been reduced below a certain critical level—about 
300,000 for the rat—the resistance of the animal to infection is greatly 
diminished and infective conditions develop spontaneously. These may lead 
to a secondary anemia. The infective conditions may clear up again as the 
number of platelets is made to increase. 

The blood platelets fulfil an important function in the mechanism of 
resistance to bacterial infection. Alterations in the local or general resistance 
to infection are associated with local or general changes in the distribution of 
the platelets. 

The radium used in these investigations was a loan from the Medical 
Research Council. 


REFERENCES. 


(1) Cramer and Pringle, ‘Phys. Soc. Proc.,’ July 27, 1912, p. 11; ‘Journ. of Phys.,’ 
vol. 45 ; ‘Quart. Journ. Exp. Physiology, vol. 6, p. 1 (1913). 

(2) Bordet et Delange, ‘ Annales de l'Institut Pasteur,’ vol. 26, p. 657 (1912). 

(3) Bullock and Cramer, ‘ Roy. Soc. Proc.,’ B, vol. 90, p. 513 (1919). 

(4) Bullock and Cramer, ‘VI. Scientific Report Imperial Cancer Research Fund.’ 
Taylor and Francis, 1919. 

(5) Gruber and Futaki, ‘Miinchen. Med. Wochenschr.,’ 1907, p. 249 ; ‘Deutsche Med. 
Wochenschr.,’ 1907, p. 1588. 

(6) Bull, ‘Journ. Exp. Med.,’ vol. 20, p. 236 (1914) ; vol. 22, pp. 475 and 484 (1915) ; 
vol. 23, p. 419 (1916) ; vol. 24, p. 25 (1916). 

(7) Delrez et Govaerts, ‘Compt. Rend. Soc. de Biol.,’ vol. 81, p. 53 (1918). 

(8) Govaerts, ibid., vol. 85, p. 745 (1921). 

(9) Ledingham, ‘The Lancet,’ 1914, p. 1673 ; vol. 1, p. 311 (1915). 

(10) Bedson, ‘Journ. Path. and Bact.,’ vol. 25, p. 94 (1922). 


468 


The Development of the Calcareous Parts of the Lantern of 
‘Aristotle mm Echinus Miliaris. 


By D. W. DEVANESEN, M.A. 
(Communicated by Prof. E. W. MacBride, F.R.S. Received April 12, 1922.) 


[Puates 11-15.] 


CONTENTS. 
PAGE 
Te Entroduction: :2.2<.1.02csece song ces ce teens seers « cause e ote eentons see eee eee ee eee 468 
9© Method’ \.. sac 5/52. ee easaetean bos os soon de Seah SRC ene See eae eee HES 469 
So Whe Saws. ede ideceiwcsds wah tase necpere Gewaen conc v land eR eS eee ee one ee 470 
A. The Wpiphyses).. 4.53. 002 eeeewatciesignss cians te Sons op boosensecheGeee ree’ sonre eee eee 470 
5. The Rottlee yi.cciva.iiicenecaswastnegec sacle sin caves eseeesdeetessadesse=s ese 471 
6. "The Com passes)s.si.o-gn.ccnscsecreementt ore: cmeGients sds asceracc uqties eee e cena te eee eae 471 
%. The Teeth oc baciesssdancecoaace cen anteae ec srnened cs satebaneae na teee steel eto eee 472 
S.. DisCUssSiON © oe. ci fnkse Won ese ce eeemepenalne siseinss ap vide lonesne hades Ose Cac e ee eee eee eee 478 
9. A comparison between Giesbrecht’s account of the development of the 
urchin-tooth as revealed by its root-end in the adult and the construction 
of the same as seen in the young urchin .....2..<....0.c.ccecousere-ceevesoeoeens 481 
LOS SamMAary P.sil54. 1 SAL SRR ee ae oe tow eee ain e bea de enetia Od odo c Gk See ee 483 
1) References:....1.. aii ackeeteatemeetea tea eek iSednedbSeaenTagion. + oskQheeitees ot: eee eeeeeeae 483 
12, Explanation of Plates:2c320) <.dcntdthecncete vn etudeescccecees es deneeeeenacaaeene a eee 485 


1. Introduction. 


This investigation was suggested to me by Prof. E. W. MacBride, F.R.S., to 
whom my thanks are also due for kindly placing at my disposal two meta- 
morphosing plutei and a considerable number of young imagos of Zehinus 
miliaris, all of them fixed in absolute alcohol. 

In the year 1892, Lovén(3) made a few observations on the embryonic 
elements of the calcareous lantern in Goniocidaris canaliculata, a Cidarid with 
a direct development. He also examined the same structures in two ‘young 
urchins which, as he himself admits, were of doubtful identity and in a state 
of bad preservation. In the same year, Théel(7), who was working at the 
early development of the cake-urchin, Echinocyamus pusillus, described certain 
early stages of the lantern in the young urchins. After these researches of 
Lovén and Théel, it appears that no attempts have been made to study the 
development of the lantern ossicles in greater detail and with closer scrutiny. 

It must be stated, however, that in the year 1903, in a near relative of 
Echinus miliaris, viz., Echinus esculentus, though the origin and homology of 
the lantern-ccelom had been investigated by Prof. MacBride (4), the calcareous 
structures it contains remained untouched. One of the obstacles to their 
study was the fact that non-decalcified specimens could not be sectioned 


Caleareous Parts of Lantern of Aristotle in Echinus miliaris. 469 


satisfactorily by ordinary methods; and even if they could be sectioned, there 
was the enormous difficulty of reconstructing, either mentally or otherwise, a 
very complicated system of ossicles. But, happily, these difficulties were 
obviated by following a unique idea very kindly suggested to me by Prof. 
MacBride. He thought that if one could cut away just the top part of the shell, 
then, after some slight maceration, one could see the lantern ossicles below. 
By employing this method were obtained several important facts which are 
recorded in this paper. JI here take the opportunity of expressing my deep 
indebtedness to Prof. MacBride for the many valuable suggestions made and 
for the frequent advice given throughout the course of this investigation, and 
for kindly reading the manuscript. 


2. Method. 


The process of double imbedding im celloidin* and paraffin was employed, 
and the “Jung” sliding microtome was used for sectioning. The Jatter 
instrument exactly suited my purpose, viz., making an opening at the aboral 
surface of the young urchin. Any slight error in orientation can readily be 
corrected in the course of sectioning by the side-to-side and front-to-rear 
adjustments of the object-holder. I must remark, however, that it required 
some skill and judgment to cut away just enough of the aboral part of the 
shell so as not to damage the lantern below. 

When enough of the shell had been sectioned off, the paraffin was dissolved 
out in xylol, and the celloidin was removed by using a mixture of equal parts 
of ether and absolute alcohol. The young urchin was then passed through 
decreasing strengths of alcohol and finally brought into distilled water when 
it was ready to be treated with “ Hau-de-la-barraque.” In this liquid was 
discovered an ideal macerating solution for the young urchin material. 
While it does not act so vigorously as caustic-potash, for instance, it dissolves 
in a given time just enough of the tissues to enable one to see through them 
the contained transparent ossicles. This maceration was watched under a 
low power of the microscope and it lasted never more than a few seconds. 
The young urchin was quickly transferred to distilled water and passed 
through increasing strengths of alcohol, then into xylol, and finally mounted 
in xylol-balsam in the cavity of an excavated slide. Such whole mounts, 
when illuminated with a “ Nernst” electric lamp and examined with a 2mm. 
oil immersion lens, show the ossicles of the lantern very well indeed. 

Entire embryonic teeth were obtained by applying gentle pressure, with a 
mounted-needle under microscopical guidance, to a well-macerated young 


* The imbedding in celluidin was done according to Gilson’s rapid process (Lee’s 
‘“Vade Mecum,’ 8th edition, p. 104). 


470 Mr. D. W. Devanesen. Development of the Calcareous 


urchin. The shell breaks away leaving the lantern in the centre. By further 
manipulations with the mounted-needle the teeth were separated, dried, and 
mounted in xylol-balsam. An examination of whole embryonic teeth 
revealed several facts which were hitherto unknown and which threw fresh 
light on their morphology. 

3. The Jaws. 

In describing the development of the lantern ossicles, it is perhaps well to 
begin with the jaw and proceed upwards. Hach jaw, or a pair of “alveoli,” is 
developed from a pair of tri-radiate spicules deposited in an inter-radius of 
the “echinus-rudiment” of the metamorphosing pluteus; two rays of each 
rudiment of an alveolus are found embracing a tooth, one on the inner side 
and the other on the outer side, while the third ray is seen to stretch aborally 
and towards the rudiment of an “epiphysis ” in the contiguous radius (Plate 11, 
fig. 1, #.A.). The next step in the growth of these spicules consists in the 
branching of the three rays at their ends and in budding new off-shoots over 
their body ; these subsequently join end-to-end. By repetitions of this process, 
there results a perforated ossicle having more or less the shape of an adult 
alveolus. In fact, the rudiments of the alveoli, like all other elements of the 
lantern, with the exception of the teeth, follow the usual method of ossicle 
growth among echinoderms; but, as compared with the growth of the 
epiphyses and the rotul, for instance, these grow mainly along their vertical 
axes. This fact enables one to account for the great height of the lantern in 
urchins generally. This vertical growth of the jaw has caused the ossicles 
above to be pushed farther and farther away from the oral region. In the 
very early stages, as may be expected, one finds all the rudiments of the 
lantern occupying a comparatively low position. 

The upward and radially-directed growth of each alveolus results in a 
junction between it and the rudiment of an epiphysis in the adjoining radius. 
Kach jaw, therefore, bears on the top two epiphyses belonging to different 
radii. 

4. The Epiphyses. 

One finds their earliest traces as pairs of triradiate spicules situated in each 
radius of the “echinus-rudiment,” far below the rotule, and above the level 
of the jaw-rudiments. Each spicule grows by the usual method, and when a 
more or less rectangular plate is formed, each epiphysis gives off from its 
outer corner, in the proximity of a tooth, a process; the two processes 
belonging to a pair of epiphyses diverge from one another (Plate 14, fig. 5, O.Z.). 
One outgrowth meets, over the “foramen magnum ” and on the outer side of a 
tooth, another—I say “another ” advisedly, for it is not its fellow—belonging 
to an epiphysis of a different but adjoining pair (Plate 15, fig. 6, O.#.). Both 


Parts of the Lantern of Aristotle in Echinus miliaris, 471 


processes together form at this stage a comparatively flat bridge, the arch- 
form being attained later. It issignificant that this bridge was not developed 
by the paleozoic Echinocystoida, Perischo-echinoida, Cidaroida, Holectypina, 
and two orders of Centrechinoida. According to Jackson (2, pp. 179 and 183) 
this is an important character found only in the four families of the Temno- 
pleuride, Echinide, Strongylocentrotide, and Echinometride, comprising his 
new sub-order Camerodonta. Evidently the presence of this bridge for the 
attachment of the protractor muscles is a sign of advance belonging to a late 
period in the phylogeny of the sea-urchins. 

The two epiphyses connected by their extensions are further in contact 
below with the same jaw, so thatit is customary in text-books to designate 
them as a pair of epiphyses. But, as far as one could make out from the 
position of their rudiments, it strikes one that the two epiphyses which lie 
one on each side of and below a rotula form a pair. This being so, one may 
say each pair maintains its radial position in the adult, there .being no 
diverging of the epiphyses from a radius. On the contrary, as has been 
mentioned already, the two alveoli of each jaw, growing aborally like the 
limbs of a V, each effect a junction with an epiphysis belonging to an adjacent 
radius. This association, though further strengthened by the formation of a 
bridge or an arch, is to be regarded as only of secondary importance. The 
orientation of the ossicles of the lantern in the adult may, therefore, be as 
follows: compasses, rotule, and epiphyses radial; jaws and teeth inter- 
radial. 

5. The Rotule. 


Each of the five rotule is laid down in a radius of the “ echinus-rudiment ” 
in the metamorphosing larva, as a tri-radiate spicule, always with two rays 
directed towards the cesophagus and one ray turned in the opposite direction 
(Plate 11, figs. 1 and 2, #.R.). By the usual process, a broad, fenestrated, and 
more or less rectangular plate is formed, roofing over the two epiphyses below, 
which are thus concealed from an aboral view of the lantern at this stage 
(Plate 14, fig. 5). In this plate, the boundaries of the primordial tri-radiate 
spicule may still be traced. This stage recalls to one’s mind the broad rotule 
found in Paleodiscus ferox (6). 


6. The Compasses. 


In the absence of any evidence to the contrary, the “compasses” have been 
usually regarded as unpaired elements. The bifurcated ends, however, were 
supposed by Sollas (5) to indicate their paired nature. But their develop- 
ment shows conclusively that, whilst they are duplex structures, the forked 
ends are not indications of a paired nature. 


472 Mr. D. W. Devanesen. Development of the Calcareous 


Each compass can be traced to a pair of tri-radiate spicules placed, not side by 
side, but in a radial line one behind the other (Plate 13, fig. 13, 7.C. and 0.C.). 
One may, therefore, speak of an outer spicule and an inner spicule with reference 
to the central axis of the lantern. Nor are these two spicules deposited at 
the same time as are those of the epiphyses, for instance; the inner first and 
the outer next is the rule. Further, these two spicules, though in the same 
line, occupy different positions and levels in relation to the rotula. The outer 
spicule is situated almost behind the rotula and almost at the same level as 
the latter, if not below it, whereas the inner spicule is placed just above the 
rotula. This fact accounts for the bent condition of the compasses in 
the adult. 

These spicules differ somewhat in the manner of their growth, not only from 
the other members of the lantern, but also between themselves. One of the 
three rays of the inner spicule is absorbed gradually (Plate 12, fig. 3, 7.C.) and 
the remaining two rays stretch end to end, forming a straight rod, more or 
less, directed radially. The free-branching and anastomosing of the original 
rays of the rudiment, found in the development of the rotula for example, 
does not obtain here. The next step in their growth consists in an increase 
in thickness by the appearance and coalescence of branches at the two ends 
of the rod; the middle part of the rod appears to grow in thickness all the 
same, though one does not see as many offshoots there. 

The outer spicule has all the three original rays well developed ; neverthe- 
less they do not branch. Its growth is confined to its size. The three ends 
of the spicule appear in a late stage thick and fenestrated, but the body of the 
spicule is devoid of meshes (Plate 13, fig. 6, COIL). The tri-radiate shape is 
maintained intact and the ray directed centralwards meets the inner piece and 
forms a suture with it, thus constituting the adult compass. It is at this 
suture that the elevator muscles of the compasses are attached, some of the 
fibres to the hinder end of the inner piece and others to the proximal end 
of the outer. This suture between the two pieces is so complete in the 
adult echinoids, both extinct and living, that its double nature has been 
scarcely ever surmised except in one species Strongylocentrotus drdbachiensis 
(Jackson, R. T., p. 179). 

The compasses are the only set of ossicles of the lantern which are absent 
in the “ echinus-rudiment ” of the larva, but appear after the metamorphosis. 


7. The Teeth. 
At the very outset it may be stated that the tooth is, in the lantern 
organisation, an element with a unique structure and a peculiar develop- 
ment. 


Parts of the Lantern of Aristotle mm Echinus miliaris. 473: 


Four stages may be recognised in its construction :— 


(1) The formation of a pair of primordial lamelle ; 

(2) The deposition of secondary pairs of lamelle ; 

(3) The consolidation of all these; and 

(4) The attainment of a relatively stable position by the permanently 
active tooth-germs which will henceforth constitute the root-end.* 


The earliest traces of a tooth, so far as I have made out, are an incipient 
cone, incipient in the sense that, not being completely closed on the outer 
side, it is a cone in making. It will be seen presently that each cone which 
comes into being subsequently arises from a pair of lamelle. One may, 
therefore, reasonably infer that each of these five cones also arises in the same 
way from a pair of lamelle. We shall accordingly call these five cones, the 
“first cones,” and the pair of lamelle of which each first cone is believed to 
be composed, the “pair of primordial lamelle.” Each first cone is situated in 
an inter-radius (Plate 11, fig. 1, #.C.), with its apex turned towards the future 
oral aspect and its base directed towards the future aboral aspect. This cone 
serves as a starting-point for the upbuilding of the entire tooth; for within 
the cavity of this cone in the metamorphosing pluteus are found deposited 
about six pairs of secondary lamelle. The first cone remains as a distinct 
structure, even after a good bit of the embryonic tooth has come into being 
(text-fig. 3, #.C.); it is highly probable that finally it enters into union with 
its successor and forms the tip of the embryonic tooth. 

A study of the numerous pairs of lamelle deposited in succession after 
the first cone has been formed reveals several facts of no small importance 
and interest. Hach lamella is laid down as a round calcareous particle, which, 
as I have evidence to show, is probably the product of a single calciferous 
cell. This particle grows by accretion of lime on one side only in such a way 
that a small triangular plate is first formed (Plate 11, fig. 1, Z, and text-fig. 1). 
At the base of this plate a peripheral growth takes place in one plane, 
resulting in an imperforate transparent lamella. When it has attained its 
maximum vertical growth it has got the contour of an isosceles triangle, with 
its apex directed towards the ventral aspect and its base towards the aboral 
surface. ven before a particular lamella reaches its full size, fresh lamelle 
are deposited at higher levels, one above the other, in quick and regular 
succession, always on the inner side of the preceding lamella. One can, 
therefore, read the several stages passed through by a single lamella from the 
series of lamelle representing different stages in their growth (text-fig. 2). 


* This fact will be elucidated in a paper on the soft parts of the lantern, now in 
preparation. 


474 Mr. D. W. Devanesen. Development of the Calcareous 


-B.L, 
20 acd => ao 


le, oD. B: A 


if 


Text-Fie. 1.—Diagrammatic representation of the formation of a cone from a pair of 
calcareous particles. 1. A pair of calcareous particles. 2,3, 4, 5,6, and 7. Pro- 
gressive stages in the formation of a pair of lamellz ; L.Z., Basal end of a lamella 
where growth takes place by accretion. 8. The pair of lamelle has attained its 
maximum vertical growth, further increase taking place only at the lateral edges ; 
the two lamelle are undergoing slight bending due probably to the pressure of 
lamellae subsequently deposited. 9 and 10. The inner lateral edges of the two 
lamelle have commenced to grow; 4d., The first spot where the latter come into 
contact and fuse. 11 and 12. The inner lateral edges have approximated and are 
gradually fusing ; the outer lateral edges have also begun to join; it will be 
noticed that both the commencement and the completion of the fusion of these 
latter edges are later than those of the inner; this stage in the formation of a 
cone may be called the “incipient cone” stage. 13. The flange /, at the top corner 
of each inner lateral edge, has appeared. 14. The same flanges have joined, 
forming the characteristic beak. 15. A complete cone; BX., Beak. 16. Two 
cones showing their relation to each other; P.P., the spot where the fusion 
between the apices of the two cones takes place, this being the foundation for the 
formation of an axial rod—the “ pars petrosa.” 


The relative position of the two fellow lamellee of a pair deserves notice. 
They are deposited in close proximity to each other, but do not touch, and 
also at different levels so that they alternate. As growth proceeds, however, 


Parts of the Lantern of Aristotle im Echinus miliaris. 475 


the two fellow lamelle are found at the same level, inclined, at first, towards 
each other at an angle which varies with their growth till, finally, as the 
result probably of pressure from succeeding lamelle, their apices are brought 
into contact and fuse together. 

At this stage of our investigation it is, perhaps, well to take into account 
certain fundamental facts revealed to us :— 

(1) We see that a lamella does not pass through a tri-radiate spicule condi- 
tion, unlike, in this respect, the other echinoderm ossicles which do, as far as 
is known at present. 

(2) The growth of a lamella is unusual, being different from the normal 
method of ossicle-formation which obtains among the other component 
elements of the lantern. 

(3) There is a maximum limit to the vertical growth of a lamella, further 
growth being along the two equal edges of each lamella. 

(4) From the fact that numerous pairs of lamelle go to build the adult 
tooth, two conclusions can be drawn, viz., (a) that the urchin-tooth is a paired 
structure, and (0) that a pair of lamelle is the unit of the same. This 
admission of the morphological value of a pair of lamelle as a unit is not, as 
will be seen later, prejudicial to regarding a cone formed by a pair of lamelle 
as an integral structure. 

The process of formation of the tooth from these paired lamelle is not by 
any means simple.* When two lamelle of a pair have attained their 
maximum vertical growth, by the pressure probably of the succeeding lamell 
above, they become bent and concave to an equal extent on their inner 
surfaces, their outer surfaces becoming correspondingly convex. During this 
bending the two lamelle appear to grow along their lateral edges till those 
of one lamella face each to each their two fellows on the other lamella 
(text-fig. 1). This growth is maintained till the confronting edges meet and 
fuse, forming a characteristic cone.t 

While the fusion of the inner edges is taking place, the rudiments of the 
carina are found to arise. An inwardly directed flange appears at the top 
corner of each inner edge (text-fig. 1). At about this time each cone under- 
goes a flattening from the inner to the outer side. Partly as the result of 
this flattening and partly owing to their growth, the above-mentioned flanges 
project well towards the cesophageal side. They ultimately come into contact 

* Lovén’s (3, p. 9) description of the formation of a tooth, in Goniocidaris, from a 
single row of lamellz is extraordinarily simple. He says as follows: “The lamels, 
the general form of which corresponds with that of the tooth, are laid one upon another 
regularly from the top downwards.” Spencer (1904, p. 36), likewise, figures a simple 


arrangement of lamelle in Palwodiscus ferox. 
+ Théel (7) describes also a cone-in-cone arrangement in Hcehinocyamus pusillus. 


476 Mr. D. W. Devanesen. Development of the Calcareous 


and fuse forming a beak-shaped process somewhat like the beak of an ounce- 
glass. It will be seen that in the formation of this beak both lamelle take 
an equal share. When several cones fit into one another, their beaks likewise 
fit together, thus giving rise to a crest which is the precursor of the carina of 
the adult tooth. The lower part of the tooth in the imago appears to be 
devoid of this crest; only the cones arising later develop the beaks in question. 

The fitting into one another of the cones takes place in such a manner that 
the brim of an upper cone always projects a small distance from above the 
brim of the lower cone into which it is enclosed. This fact has a necessary 
bearing, first, on the growth of a carina on the inner side and, second, on the 
formation probably of the middle furrow on the outer side of each tooth 
(Plate 15, fig. 6, C_-A. and text-fig. 1). 

In the aboral end of the embryonic tooth—the root-end in the adult—one 
can see all the intermediate stages between a pair of calcareous particles, 
the simplest condition of a pair of lamelle, and a transparent beaked cone, 
the most advanced stage of the same (text-fig. 2). As has been already 
mentioned, the first point of contact and fusion between every two lamellee of 
a pair is at their apices. The fused apices of one pair unites with those of 
its predecessor and successor even before the pair itself reaches the stage of 
acone. Thus a central rod results to which are attached all the cones and 
the younger pairs of lamelle. Presumably this rod is the “pars petrosa” of 
Giesbrecht (1, p. 90). Curiously enough, the first cone does not appear to 
participate in the formation of this stony part (text-fig. 3). 

It has not been possible to follow closely the next phase in the building of 
the tooth. The coalescence of the cones appears to proceed centripetally from 
the “pars petrosa” outwards. The outlines of the cones gradually disappear 
owing to the fusion of their walls. 

The median furrow of the adult tooth probably marks the place where the 
suture of the two outer edges of a pair of lamellz is formed. Of this line of 
junction a greater portion is bound to disappear in the process of coalescence 
owing to its internal position in the cavity of its predecessor as explained 
already; only the small exposed parts of this suture in the several cones 
probably persist and contribute to the formation of the median furrow. 

On account of the relative position between every two cones, the beaks: 
are, on the inner side, the only exposed parts of the line of junction between: 
the two inner edges of the pairs of lamelle. It must be observed that no 
indication of a suture, however, is to be found on each beak. These beaks, as 
has been already described, are the precursors of the carina which is for this 
reason a composite paired structure. Such is the construction of a tooth so 
far as one could make out from observations of hard structures. 


Parts of the Lantern of Aristotle iv Echinus miliaris. 477 


Res = 


2,.C->=- 


Text-Fia.:2. Trext-Fia. 3. 


‘Text-Fie. 2.—Reconstruction of a fairly advanced tooth as viewed from the inner side. 
R.T., Root-end of the tooth where the lamellze can be seen in all their progressive 
stages; C.P., Calcareous particle, the first beginning of a lamella; /., Flange ; 
L., Lamelle : 22.0’, Two lamelle in the process of cone-formation ; 2Z7.C.", A 
fully formed cone ; BX., The beak of a cone; L.7., Lower part of the tooth where 
the walls of the cones have fused; J/., Mesh-work formed by the coalescence of 
lamelle ; P.P., ‘‘ Pars petrosa,” or the stony part. 


Trext-Fre. 3.—Actual drawing of a tooth less advanced than the one shown in text- 
fig. 2, viewed from the outer side ; the first cone is still intact ; it will be noticed 
that the cones are yet imperfect on the outer side, fusion between the edges 

- thereof being considerably delayed; 2.C., First cone; P.P., the “ Pars petrosa,” 
or the stony part resulting from the union of the apices of the serially fitting 
cones; 22.0’, Two lamelle in the process of cone-formation; JZ., Lamelle ; 
C.P., Calcareous particle ; O0.#., Outer edges not yet approximated. 


478 Mr. D. W. Devanesen. Development of the Calcareous 


8. Discussion. 


In the mouth-frame of recent Asteroidea as well as in most of the fossil 
star-fishes are seen five pairs of generally triangular ossicles situated in the 
inter-radial angles bordering the mouth (Spencer, 1913, p. 26). Originally 
identified by Ludwig as the first pair of modified adambulacrals, they are now 
known as mouth-angle plates. By their position both in the embryo and in 
the adult and by their relation to the mouth, the five pairs of “alveoli” of the 
lantern of Aristotle recall the five pairs of mouth-angle plates in star-fishes, 

In the section on “ epiphyses,” we saw how the two epiphyses each situated 
on an alveolus belonging to adjacent jaws constituted a pair, and not the two 
epiphyses which formed an arch. As the radial canal of the water-vascular 
system runs between the two epiphyses in question,* there is no serious 
obstacle to regarding this pair as corresponding to a pair of ambulacral 
ossicles. 

The interpretation of the rotule offers undoubtedly great difficulties. 
These are not only odd elements as conclusively shown by their origin but. 
also the sole unpaired elements in the lantern of Aristotle. The only odd 
ossicle which gets into touch with the mouth-frame in extinct star-fishes is 
the odontophor. In an account of the family Urasterellide, Spencer (1913, 


p. 135) observes as follows :—“In a private letter tome .... Hudson asks 
me to note (in the mouth-frame of the American species Urasterella pulchella)+ 
‘paired muscle remains . . . . just within the large inter-radial (odontophor).’ 


He goes on to state that there must have been muscles to draw in the mouth- 
angle plates, which in each inter-radius acted as an outer jaw. ‘The origin of 
these adductors may have been on the inner surface of the odontophors rather 
than on the first ambulacralia.” This indicates that in certain asteroids the 
odontophor may get into close relationship with the mouth-angle plates. It 
must be noted, however, that in the lantern of Aristotle the rotula has 
muscular connection with the epiphyses and not with the jaws. 

In certain other fossil star-fishes again, the odontophor has been shown to 
occupy an internal position (Spencer, 1917, p. 180). I quote these two 
features of the odontophor in the extinct asteroidea in order to show how this 
odd ossicle behaves in certain cases, it being far from my purpose to suggest 
thereby any asteroid descent for the echinoids. 

The great impediment to regarding the rotule as modified odontophors is 
the fact that, while the latter are inter-radial, the former are radial. Can it. 
be that, after occupying an internal position in the ancestral urchin, the 


* This fact will be elucidated in a paper now in preparation. 
+ This parenthesis is mine. 


Parts of the Lantern of Aristotle in Echinus miliaris. 479 


odontophors underwent a sinistral or dextral rotation which brought them to 
a radial position? MacBride* with reference to the inter-pyramidal muscles 
says as follows: “These on contraction, approximate the pair of jaws into 
which they are inserted, and it will easily be seen that by the successive 
contraction of the five comminator muscles a rotating movement of the teeth 
would be produced which would cause them to exert an action something like 
that of an auger.” Can this action of the lantern, coupled with the fact that 
the rotule alone of all the ossicles of the lantern are free from muscular 
attachment with the shell, be supposed to have brought about this 
displacement ? 

From its double origin and radial position each compass may be regarded 
as corresponding to a pair of ambulacral ossicles. It is generally known that 
in certain extinct star-fishes, the paired ambulacral plates alternated with 
each other. If the same condition had prevailed among the ancestors of 
urchins, one might conceive of a displacement consisting of one member of a 
pair being pushed in front of his fellow. 

In instituting a comparison between a tooth and other ossicles of the lantern 
or those of the mouth-frame of star-fishes, one should take a pair of primordial 
Jamelle to represent a tooth on the one hand and paired ossicles on the other. 
For I hold an urchin-tooth is not, in the strict sense of the term, an ossicle ; 
it is an aggregate of paired ossicles if the lamelle can be so called. Whether 
the lamelle themselves are ossicles is open to doubt; as we saw in their 
development, they neither pass through a tri-radiate spicule stage nor do they 
grow into fenestrated plates as ossicles of echinoderms do in general by the 
branching and anastomosing of calcareous offshoots. On the contrary a tooth- 
lamella grows by accretion confined in the early stages to one particular side, 
viz., the base of the minute triangular plate. For purposes of comparison, 
therefore, a pair of tooth-lamelle may be, with the above-mentioned reserva- 
tions, taken to represent a pair of ossicles corresponding toa pair of “alveoli” 
or “epiphyses” rudiments. This being so, a whole tooth does not stand in 
the same relation to its rudiments as the other component parts of the lantern 
do; the latter are, par excellence, echinoderm ossicles whereas the former is an 
ageregate of paired structures which are not undoubted ossicles. An epiphysis 
for instance, being the direct outcome of a tri-radiate spicule, is a unit in 
itself; but a tooth is an aggregate, being the product of several paired units, 
the lamellz. A tooth is essentially a double structure lke a pair of “alveoli” 
or “epiphyses,’ the only indication of this in the adult tooth being the 
median furrow which runs longitudinally along its outer side. 

The homologues of the urchin-tooth are to be looked for among the bristles 


* See “ Echinodermata,” ‘Cam. Nat. Hist.,’ p. 526. 


480 Mr. D. W. Devanesen. Development of the Calcareous 


carried by the mouth-angle plates in star-fishes. If two such bristles get 
flattened, assume a cone-shape, and are pushed between the two mouth-angle 
plates thus becoming partly internal, we have the rudiment of an urchin- 
tooth. 

There must have been several physiological forces at work in the 
evolution of the urchin-tooth. First of all, the liability to wear and tear of 
an organ used for browsing purposes could have induced the permanent 
retention of the activity of the embryonic tooth-germ. The ancestor of the 
sea-urchin, whoever that might have been, let us suppose, started with the 
five first cones; these may well have served the primitive animal as organs 
of mastication. If the ancestral animal browsed on things like the brown 
fronds of Laminaria or bored into rocks*—certain sea-urchins are known to 
do both—the conical teeth would be liable to suffer decay. Under such 
circumstances, the power of replacing worn-out teeth would have been of 
immense advantage, and hence the permanent activity in the adult of the 
five embryonic tooth-germs was probably the primary factor in the evolution 
of the urchin-tooth. 

But how can one account for the coming into being of a stout rod as the 
result of fusion of several pairs of lamelle? What could have induced the 
deposition of numerous pairs of lamelle, one above the other, in the imago 
urchin, while even as yet the mouth is not formed? Efficiency is the first 
answer that suggests itself. A short conical tooth, formed by a pair of 
delicate lamelle and renewed frequently, even at its best must have been 
but a weak instrument to the ancestral urchin. If the tooth-germs laid 
down precociously pairs of lamelle, and if by the fusion of these a stout rod 
resulted, that meant efficiency in function and advance in structure. In this 
way, one may account for the formation of a stout rod-shaped tooth through 
the fusion of several cones. 

In this connection, perhaps, it will be well to consider what reaction the 
evolution of the tooth may have had on the jaws. Each tooth in the adult is 
closely and immovably attached to a jaw. This being so, the jaw is bound to 
respond to any adaptations of the tooth. In the “echinus-rudiment,” the 
vertical height of the lantern is at its minimum.t Now, if the tooth 
increases in length in accordance with the causes indicated, it is extremely 
likely that there will be a corresponding response on the part of the jaw to 
adjust itself to the growing tooth. That the tooth may well have been the 


* Vide “The Locomotor Function of the Lantern in Echinus,” by Prof. Gemuinill, 
“ Roy. Soc. Proc.,’ vol. 85, p. 101 (1912). 

+ The height of the lantern in Paleodiscus feror is small when compared with that 
of living urchins (Spencer, 1904). 


Parts of the Lantern of Aristotle in Kchinus miliaris. 481 


leading factor in the evolution of a high lantern is perhaps seen in the lantern 
of the metamorphosing pluteus (Plate 11, fig. 1, Z). Here we find that, even 
while the two jaw-rudiments are in the tri-radiate spicule stage, the tooth is 
in a relatively higher state of development with the first cone and nine 
pairs of secondary lamellee; and it extends aborally beyond the limits of the 
jaw-rudiments. It is perhaps admissible to infer from this ‘that, with the 
growing length of the tooth, the jaw-rudiments kept pace pari passu, and 
the result was a lantern of great height. 

“Summarising the foregoing conclusions, I regard the lantern of Aristotle 
as homoplastie with the buccal armature of star-fishes ; the pyramids are the 
modified first adambulacral plates; the epiphyses have arisen from the first 
ambulacral plates of the Echinoid series; and the teeth represent the 
odontophore, which has acquired a persistent root; the radius and rotula 
remain problematical.” So wrote Prof. Sollas in the year 1899 in his paper 
on “Silurian Echinoidea and Ophiuroidea” (5). I leave the reader to 
compare this statement with what has been indicated on the homologies of 
the lantern of Aristotle in this paper. It will be seen that the idea of the 
homology of the urchin-tooth advanced by me is entirely new, and based on 
indisputable embryological facts. 


9. A Comparison between Gresbrecht’s (1)* Account of the Development of the 
Urchin-tooth as revealed by its Root-end in the Adult and the Construction 
of the Same as scen in the Young Urchin. 


Attempts to probe into the peculiar construction of an urchin-tooth were 
made even as early as the year 1841. Beginning with Valentin,t Meyer, 
Waldeyer, Leuckart, Giesbrecht, Lovén, Théel and MacBride successively 
each gave his attention to this organ. With the exception of Lovén, Théel 
aud MacBride, who studied it in young urchins, these authors employed in 
their researches the root-end of the adult urchin-tooth. The credit of our 
knowledge that the urchin-tooth is built up of lamellee is largely due to 
these workers. Nevertheless, our ideas as to the manner of the origin of 
these lamelle, their growth, arrangement and significance have hitherto been 
rather imperfect. The first attempt, I may say, to interpret the tooth- 
structure in terms of biogenetic laws has been made in this paper (see 
Discussion). 

A very interesting study was made by comparing my own observations, 
based on the embryonic tooth, with those obtained by the examination of the 


* The greater part of this important paper was translated to me by Prof. MacBride. 
+ “Anatomie du genre Hchinus,” 1841, by G. Valentin, the first monograph in 
‘Monogr. d’Kchinodermes viv. et foss.,’ published by L. Agassiz. 


WO, ONT —13, 2 M 


4 


482 Mr. D. W. Devanesen. Development of the Calcareous 


root-end of an adult tooth. From the nature of the case one should expect 
the process in the adult to be similar to that in the young. Hence a 
comparison between the two is inevitable, nay, compulsory. I am bound to 
confess that my own researches do not confirm in their entirety the con- 
clusions drawn by Giesbrecht, the last and most important of the investi- 
gators of the root-end of the adult urchin-tooth. 

According to his observations there are two sets of units for the urehin- 
tooth, one for the wings or the lateral parts and the other for the carina, 
The units of the former are flat structures called * Seales” (Schuppen), which 
are not homogeneous but are made of two lamell separated by a narrow 
interspace. It appears to me that, in this latter inference, Giesbrecht has 
been misled by an artifact, and, likewise also in his other, mentioned in the 
context, that his second set of units, “the prisms,” have an axis-cylinder. 
Each scale is undoubtedly a homogeneous structure of integral value. For 
this reason, and also because it is more appropriate, I have, in the description 
of these plates, retained the use of the term “lamella,” and dropped out the 
word “scale.” His account of the manner of growth of each “scale” is not 
in perfect accord with mine. He says: ‘“ Meanwhile, quite like the shell of a 
mussel, lime is deposited in layers round one ‘ initial point’ (the ‘ calcareous 
particle’ in my description),* though not uniformly in a circle but on one 
side only, so that the initial point always remains at one edge of the plate 
like the umbo of a mussel shell.” Though this description suggests a 
peripheral growth, it differs in two respects: (1) the growth in the pre-cone- 
formation period is by accretion in a straight line along the base of the 
triangular plate (text-fig. 1); and (2) the concentric rows of stripes he 
mentions in the context are non-existent in the lamella. Further, Giesbrecht 
describes the lamelle of one row as alternating with those of the other, and 
has entirely missed out the formation of the cones. 

To me, it would appear, that his second set of units—what he calls 
“prisms,” but, in reality, long needle-shaped structures—is of the nature of 
secondary calcification. In the young imago, the carina is found to arise 
solely by the fusion of inwardly directed flanges (text-fig. 1) of the incipient 
cones, as has been already described in the section on teeth. Giesbrecht 
himself speaks of a certain part of the lamella taking part in the formation 
of the carina. I cannot, therefore, agree to his giving the “prisms” a 
morphological value equal to that of the lamellee. 

On account of the nature of the methods he employed, such as dissecting the 
root-end with a mounted-needle and making ground-sections (Diinnschliffen) 
of the same, I do not know how far one can rely on these doubtful details 


* The parenthesis is mine. 


Parts of the Lantern of Arvstotle in Echinus miliaris. 483 


of his observations. He boiled the root-end with caustie-potash, a treatment 
which undoubtedly would have interfered with the structure of such delicate 
things as the lamella. The wonder is that Giesbrecht accomplished so much 
with the methods at his disposal in those days. 


10. Summary. 

(1) All the calcareous elements of the lantern of Aristotle, with the 
exception of the teeth, are deposited as tri-radiate spicules; in this, as well as 
in their further growth, they resemble the ossicles of echinoderms in general. 

- (2) The two “ epiphyses,” one on each side of and below a rotula, are to be 
regarded as constituting a pair. 

(3) A “compass” arises from two rudimentary spicules. It is the only 
element of the lantern absent in the “ echinus-rudiment.” 

(4) A tooth is a paired structure in consequence of its composition of a 
double row of lamelle. A pair of lamelle is its ultimate unit, although it is 
not inconceivable that originally a pair of lamelle itself, after assuming a 
cone-shape, could have functioned as an integral structure, a primitive kind of 
tooth, in the ancestral urchin. There is a remarkable stage in the consolida- 
tion of these lamellae, viz., the cone-in-cone arrangement. The carina is 
formed by the beaks of the serially-fitting cones. 

(5) Without committing one’s self to the view of a direct descent of the 
sea-urchins from the star-fishes, one may institute a brief comparison between 
the ossicles of the lantern and those of the mouth-frame of a star-fish: a pair 
of “alveoli” corresponds to a pair of mouth-angle plates; a pair of 
“epiphyses,” as understood in this paper, to the first pair of ambulacrals; 
a pair of compasses to the second pair of ambulacrals; the rotula may, 
tentatively, be regarded as.a displaced odontophor; the pair of primordial 
lamelle, the forerunner of the urchin-tooth, may be compared to a pair of 
bristles attached to a pair of mouth-angle plates. 


11. REFERENCES. 
(1) Giesbrecht, W. (1880). “Der feinere Bau der Seeigelziihne,” ‘Morph. Jahrbuch,’ 


vol. 6. 

(2) Jackson, R. T. (1912). ‘Phylogeny of the Echini,” ‘Mem. Bos. Soc. Nat. Hist.,’ 
vol. 7. 

(3) Lovén, S. (1892). “ Echinologica,’ ‘Bihane Kongl. Sven. Veten. Akad. Handl.,’ 
vol. 18. 


(4) MacBride, E. W. (1903). “ The Development of Hehinus esculentus, together with 
some points on the development of #, miliaris and E. acutus,” ‘ Phil. Trans, Roy. 
Soc.,’ B, vol. 195. 


484 Mr. D. W. Devanesen. Development of the Calcareous 


(5) Sollas, W. J. (1899). ‘On Silurian Echinoidea and Ophiuroidea,” ‘Quart. Jour. 
Geol. Soc.,’ vol. 45. : 

(6) Spencer, W. K. (1904). “On the Structure and Affinities of Palewodiscus and 
Agelacrinus,” ‘Roy. Soc. Proc.,’ vol. 74. (1913 and 1917).—‘‘ A Monograph of the 
British Paleeozoic Asterozoa,” Palzeontographical Society. 

(7) Théel, H. (1892). “On the Development of Hehinocyamus pusillus,’ ‘Nova Acta 
Roy. Soc. Scien., Upsala,’ vol. 15. 

(8) Ubisch, L. V. (1918) “Die Entwicklung von Strongylocentrotus lividus,” ‘ Zeit. 
Wissen. Zool., vol. 106, Part III. 


EXPLANATION OF PLATES. 


List oF ABBREVIATIONS USED. 


tils.  Apapsdecanbohodono000 50 Alveolus. 
IB Sa iva ne somrae Sons cae Boss of a spine. 
IB IEG cast han ssodecsoe Beak of a cone. 
BLE vevsievistevsstasten Basal ends of lamelle. 
CA 4 case Seogeebeee Carina of a tooth. 
(GRIN Nocanesonbeccou nose >: Calcareous particle—the starting point of a lamella. 
COME fe) 2 eaten ce neers Compass. 
Ne eac eins neve see Te Calcareous disc of a tube-foot sucker. 
EES, RARE Ee Epiphysis. 
DD ALY sod cesec a ce ec cbs Elevator muscles of a compass. 
VHeraerie «Gece ateveceete ses Flange of a lamella. 
IGI™ \ccosceaceaessicieee ee First cone. 
RG Mars csattacseelceseeseer Inner rudiment of a compass. 
TIN, MISA sects -hecacton 2 Inter-pyramidal muscles. 
Da ect se aeion= ccoete Sees Lamellze of a tooth. 
DINGS Pic vveaktwaecsasce A pair of lamellz in the process of cone-formation. 
DROS” Swenaen tens abcess A fully formed cone. 
OMGS hose. rataveetecnaets Outer rudiment of a compass. 
OTD etl tac «vvadeetiens Csophagus. 
OEE erecta cos se n> see th: Offshoot of an epiphysis. 
OTE ecocnencennnce rece Oral end of a tooth. 
Tee Dik Se hG een PERE A pair of epiphyses. 
TUE ake cao Nab okclicisse 28% « Rotula. 
LEA Wacom tuscacsescusierss Rudiment of an alveolus. 
PCPS acs nes vctncssscaastss Rudiment of an epiphysis. 
UL ios Gdek coeee ee Onna es Root-end of a tooth. 
Tiflis andasaanonis0ebedboee Rudiment of a rotula. 
SBIEY  Sch006 991900080090000 Shell. 
ISPS 9 ven Pocestmanes dew saicet Spine. 
ISSO. eeetene eesti cess Suture between the two rudiments of a compass. 
SEE! secareatsle. taueaee stone Suture between the processes of two epiphyses. 
U eS erect aeerod conte Tooth. 


As the six figures form a progressive series, all of them should be consulted with 
regard to any particular ossicle of the lantern of Aristotle. 


Devanesen. Roy. Soc. Proc., B, vol. 93, Pl. 11. 


c ©) c IR 
Soa Le Ss cao 


‘sp--- OSE 2% ANG 
ZL, O (0) \ e OF) WA 
CZ CRG 2 _GYDBF Xe 
OO 


Devanesen. Roy. Soc. Proc., B, vol. 93, Pl. 12. 


Devanesen. 


toy. Soc. Proc., B, vol. 93, Pl. 13. 


Roy. Soc. Proc., B, vol. 93, Pl. 14. 


Devanesen. 


hoy.S0c. Proc., B, vol. 93, Pl. Up, 


Devanesen. 


; 


| 

| 

{ 

I 

| 
ply 
ar 
ran 
= 


Parts of the Lantern of Aristotle i Echinus miliaris. 485 


DESCRIPTION OF PLATES. 


PuateE 11. 


Fig. 1.—‘ Echinus rudiment” of a metamorphosing pluteus; macerated after all the 
extra-larval parts have been removed by sectioning ; the lantern elements are seen 
from the future aboral surface; they are all, with the exception of the teeth, in 
the stage of tri-radiate spicules ; no trace of compasses. 

Fig. 2.—Aboral view of the lantern elements in a young imago, probably just meta- 
morphosed ; the top part of the shell has been removed and the specimen macerated ; 
the inner rudiment of a compass visible in one radius; branching of the rays has 
commenced in all the spicules. 


PLATE 12. 


Fig. 3.—Aboral view of the same in an imago-urchin slightly more advanced; well- 
marked rudiments of the compasses are laid down in all the radii; epiphyses 
considerably branched ; the teeth are rather diagrammatically represented. 


PLATE 13. 


Fig, 4.—Aboral view of the same in imago still more advanced ; the first branches of 
each rotular rudiment have joined end to end and fresh off-shoots have been put 
forth ; one ray of the inner rudiment of each compass is in process of resorption 
and another ray is being lengthened to meet the outer rudiment ; beaks are not 
formed by the earlier-produced cones; the root-end of a tooth shows the 
characteristic bend of the same seen in the adult; a compass has been omitted 
in one radius ; it will be noticed the specimen is tetramerous in symmetry. 


PLATE 14, 


Fig. 5.—Aboral view of the same in imago more advanced than the one shown in 
fig. 4; the rotule and the epiphyses have grown into fenestrated plates; the 
outline of the original spicule may yet be traced in each rotula ; the two rudiments 
of each compass are still separate; the epiphyses are putting forth processes ; the 
alveoli are not visible ; the compasses in two radii and a rotula in one radius have 
been omitted. ; 


PuateE 15. 


Fig. 6—Aboral view of the same in a fairly grown young urchin; the rotule are 
beginning to sink between the two epiphyses below ; the off-shoots of the latter 
have nearly met across the “foramen magnum”; the two pieces of each compass 
haye met and formed a suture; a carina in each tooth can now be distinctly seen ; 
the jaws are not visible ; a rotula and a compass have together been omitted in 
one radius in order that a pair of epiphyses may be brought into view ; likewise, 
the teeth have been left out in two radii !to show the bridge formed by the 
processes of two epiphyses ; the compass alone has been omitted in one radius to 
bring a rotula into full view. 


2M 2 


486 


Origin and Destiny of Cholesterol in the Animal Organism. 
Part XIII—On the Autolysis of Inver and Spleen. 


By JoHn ADDYMAN GARDNER and Francis WILLIAM Fox (Beit Memorial 
Fellow). 


(Communicated by Sir W. M. Fletcher, F.R.S. Received April 25, 1922.) 


(Report to the Medical Research Council. From the Biochemical Laboratory, St. George’s 
Hospital, and the Physiological Laboratory, South Kensington, London University.) 


In Part XII of this series (1921) the comparison was made of the 
intake and output of cholesterol in normal adults on a known diet and 
over periods of six days, and an average daily loss of 0:3 yrm. of cholesterol 
was noted. The conclusion drawn was that there must be some organ in the 
body capable of synthesising cholesterol. A similar view has been put 
forward by Grigaut (1913), who expressed the opinion that this synthesis is 
the function of the suprarenal glands. It seemed likely that the study of 
the autolysis of various tissues under aseptic conditions might throw some 
light on this question. 

A number of observations bearing on this subject are described in the 
literature, some undertaken with the object of finding evidence of the 
presence of enzymes capable of hydrolysing cholesterol esters, others with the 
object of ascertaining whether destruction or synthesis of cholesterol took 
place on autolysis. The results are very conflicting. 

In 1896, Carbonne, and again Waldvogel, in 1906, claimed to have obtained 
a large increase in cholesterol when lecithin was autolysed with sterile liver 
juice. Craven Moore (1907), however, was unable to confirm this, and 
described one experiment in which 600 grm. of human liver, containing 
0:037 per cent. of cholesterol were autolysed aseptically for 42 days at 37° C., 
and at the end were found to contain 0:038 per cent. Corper (1912) also 
failed to find any marked change in the cholesterol of dog spleen on short 
autolysis. 

Kondo (1910) attempted to decide the question of the presence of enzymes 
capable of hydrolysing cholesterol esters in blood and tissues by the deter- 
mination of the acetyl values of the ether extracts, but his conclusions were 
vitiated by the presence in addition to cholesterol of other acetylisable 
substances, 

In 1912, Schiiltze repeated these experiments, using the digitonin method 
for estimation of cholesterol and its esters. He found that in autolysis of 
human blood and horse blood no hydrolysis of cholesterol estérs took place. 


Origin and Destiny of Cholesterol in the Animal Organism. 487 


Rabbit’s liver gave negative results, but in the case of horse liver a marked 
hydrolysis took place. In experiments with mixtures of blood and liver- 
juice, both in the case of horse and cow, a more or less complete hydrolysis 
took place, but not with serum and liver-juice. Schiiltze made no comment. 
on the constancy or otherwise of the total cholesterol on autolysis, but 
as far as we can ascertain from his protocols, any variation between the 
values of the fresh and autolysed tissues were within the probable errors of 
experiment. 

Cytronberg (1912) continued the work of Schiiltze and showed that the 
cholesterase is contained in the blood cells and not in the plasma. Howard 
Mueller (1916) however was unable to confirm Cytronberg’s work. 

In 1920, T. E. Abelous and L. C. Soula found that spleen pulp on autolysis 
at 37° showed a marked increase in total cholesterol. Some of their results 
are given in the following table :— 


Percentage of Cholesterol in Spleen Pulp. 


; After 24 rs | 
Animal. | Fresh. ease 48 hours. 6 days. | 10 days. 
0-231 0-930 — — | — 
0 500 0 580 | 0-680 0-399 0°150 
0-350 0 °450 0-150 | — = 
0-078 0-459 — = = 
0°317 0-570 0-415 — — 


Initially, it will be noticed, they found a rise followed on prolonged auto- 
lysis by a fall. They also found that these effects increased with rise of 
temperature up to 45° C. 

In one experiment the addition of a minute amount of cholalic acid pro- 
duced a still more marked synthesis of cholesterol. 


| | | 48 hours (with 0°05 
i Hh. soc 3 t grm. 
Animal | Initial content. : 48 hours. cholalic acid added). 


Ralf) 22 pik a 0 500 0-68 | Bag 


They also investigated other organs and concluded generally that nervous 
tissue and liver also have the power of synthesising cholesterol, but in less 
degree than spleen. Other tissues on autolysis show a destruction of 
cholesterol. 

The authors give no details of their methods of extraction and analysis, 
but obviously their results, if correct, are of very great importance. 


488 Messrs. J. A. Gardner and F. W. Fox. Origin and 


We determined, therefore, to study the autolysis of various organs, and in 
this paper give an account of our experiments on liver and spleen. 


Method. 


The whole fresh organ, freed as completely as possible from adhering tissue 
and fat, was finely minced in a mincing machine, pulped in a mortar, and 
divided into the required number of approximately equal portions, which 
were then accurately weighed. One portion was analysed at once and the 
others mixed, in some cases, with sufficient toluene, and in other cases with 
a 2 per cent. solution of sodium fluoride to prevent bacterial growth. They 
were then placed in sterile flasks in the incubator at 37° C. and allowed to 
autolyse for definite periods. The autolysed tissue was not examined 
bacteriologically, but the smell indicated that no appreciable putrefaction 


had taken place. 
Extraction of Fat. 


This is the most important stage of the process, and one in which, in our 
experience, error is most likely to occur. The pulped tissue—fresh or auto- 
lysed—was mixed with excess of alcohol and the fluid portion then drained 
off. The solid matter was placed in a paper thimble and extracted with 
alcohol for several days, the material being taken out at intervals and 
re-ground. Finally the extraction was completed by means of ether. 

The alcoholic fluids were evaporated and the residue thoroughly extracted 
with ether. The ethereal extract was added to the above and made to known 
volume. An aliquot portion was evaporated to obtain the total ether extract, 
and, when desired, to estimate the free cholesterol. 

The extraction was carried out in a flask or matrass with a very long wide 
neck. The paper thimble was placed in an ordinary Soxhlet tube with 
syphon, suspended by a platinum wire in the neck of the flask. Above this 
was suspended a closely wound glass spiral tube through which cold water 
circulated, and which served as a very efficient condenser. By varying the 
height of the Soxhlet tube and the condenser the apparatus could be adapted- 
for use with various solvents, and the temperature of the actual extracting 
solvent could be varied within considerable limits. Another important 
advantage was that all corks and joints were dispensed with, and the 
apparatus was easy to manipulate and clean. 


Hydrolysis of the Fat. 


The fat was hydrolysed in ethereal solution with a large excess of an 
alcoholic solution of sodium ethoxide, as described in previous papers, but as 
the esters of cholesterol are said to be difficult to hydrolyse in the cold by 


Destiny of Cholesterol in the Animal Organism. 489 


this method, the mixture was refluxed for 4 or 5 hours on the water-bath, 
and then allowed to stand 24 hours. The soaps were filtered and well washed 
with ether, or, if too large for this, extracted in the apparatus described 
above. The ethereal solution of unsaponifiable matter was then thoroughly 
washed free from aleohol and traces of soap, made to known volume, and an 
aliquot portion evaporated and weighed. 


Estimation of Sterols Precipitable by Digitonin. 

This estimation was carried out by the method described by Fraser and 
Gardner (1910) on a suitable portion of the solution. 

Most of the experiments were carried out on human liver and spleen, 
obtained from cases of healthy adults killed suddenly by accidents. In these 
cases it was not possible to obtain the material until some 24 hours after 
death, during which time post-mortem changes might have taken place, with 
possible destruction of the enzymes responsible for the changes under investi- 
gation. Control experiments were therefore made on liver and spleen obtained 
from oxen killed at the slaughter-house by the Kosher method. The material 
thus obtained was treated within 2 or, at most, 3 hours after death. In one 
experiment pure cholalic acid was added to the autolysing tissue, to test the 
statement of Abelous and Soula that the addition of a small quantity of this 
substance would markedly increase the synthesis of cholesterol. 

Our results are given in the following Tables—(1) Liver, (II) Spleen. 


Discussion of Results. 


In all these experiments, such aliquot portions were taken for analysis as 
would give between 0:18 and 0°25 erm. of digitonide for weighing. It was 
not possible to use larger quantities than this owing to the scarcity of 
digitonin, but in our experience these quantities are quite sufficient to give 
reliable results, other things being equal. 

When we consider the large and complex series of operations necessary in 
carrying out these experiments the errors must be considerable, however great 
the care taken. We think that errors creep in mainly in the extraction 
process. In the Tables we give the sterol results to the third place of 
decimals, which would be justifiable if we were dealing with pure sterols. 
The relatively minute errors inherent in the digitonin method itself are, of 
course, magnified in calculating the quantities actually weighed to percentage 
of the original material taken for autolysis and extraction. In our opinion, 
second place may be regarded, however, as approximately correct. 

A careful consideration of the figures indicates no evidence whatever of 
either synthesis, or destruction of cholesterol, during autolysis, and as far as 


Origin and 


Messrs. J. A. Gardner and F. W. Fox. 


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492. Origin and Destiny of Cholesterol in the Anamal Organism. 


autolytic experiments afford evidence on the point, these organs are not 
concerned with the synthesis of cholesterol in the organism. 

In column 8, we give the percentages of unsaponifiable matter. These 
figures are probably a little too high, owing to the presence of traces of resins 
which we have shown (1921, Gardner and Fox) are produced by the action of 
alcohol on alkali during hydrolysis, but it will be noticed that the total 
unsaponifiable matter is two to three times the weight of sterol precipitated 
by digitonin. The composition of this unprecipitable portion is under 
investigation. With the Burchardt-Liebermann reagent this substance gives 
a reddish brown colour changing to green, in a inanner similar to that of the 
portion of the unsaponifiable matter of feces not precipitated by digitonin. 

Kstimations were also made of the free and ester cholesterol present in the 
fresh and autolysed tissue, but we do not give the figures at this stage of the 
inquiry, because we think that the question of the presence of ferments capable 
of hydrolysing or synthesising sterol esters, is better attacked by investigating 
the effects of tissue extract on pure cholesterol esters. We may say, however, 
that our results are in general agreement with those of J. H. Schiiltze, 
previously mentioned. 

Conclusion. 

As tar as autolytic experiments are concerned, there is no evidence that 
either liver or spleen are the organs which have to do with the synthesis or 
change of cholesterol, as maintained by Abelous and Soula. 


We take this opportunity of expressing our thanks to the Government 
Grant Committee of the Royal Society, for help towards the expenses of the 
research ; to Dr. Schryver, for kindly supplying a sample of pure cholalic 
acid; and more especially to Dr. Donaldson, of St. George’s Hospital, for the 
care and trouble he has taken in obtaining the post-mortem material; and to 
Dr. Rashleigh, for valued help in some of the earlier experiments. 


REFERENCES. 
1896, Carbonne, ‘ Archiv Ital. di Biol.,’ vol. 26, p. 279. 
1906, Waldvogel, ‘ Miinch. Med. Wochsch.,’ vol. 53, p. 402. 
1907, Craven Moore, ‘ Med. Chron. Manchester,’ p. 230. 
1910, Kenro Kondo, ‘ Biochem. Zeit.,’ vol. 27, p. 426. 
1910, Fraser and Gardner, * Roy. Soc. Proc.,’ B, vol. 82, p. 559. 
1912, Corper, ‘J. Biol. Chem.,’ vol. 11, p. 45. 
1912, Schiiltze, J. H., ‘Biochem. Zeitsch.,’ vol. 42, p. 255. 
1912, Cytronberg, 8., ‘ Biochem. Zeitsch.,’ vol. 45, p. 281. 
1913, Grigant, ‘La cholesterinemie, Paris.’ 
1916, Howard Mueller, ‘J. Biol. Chem.,’ vol. 25, p. 561. 
1920, Abelous, J. E., and Soula, L. C., ‘Compt. Rendu Soc. Biol. 8 Mai. 
1921, Gardner and Fox, ‘ Biochem. Journ.,’ vol. 15, p. 376. 
1921, Gardner and Fox, ‘ Roy. Soc. Proc.,’ B, vol. 92, p. 358. 


OBITUARY NOTICES 


OF 


FELLOWS DECEASED. 


VOL. XCIII.—B. 


CONTENTS. 


PAGE 
WARE ON -<DUCLE! (with pontualh) Macc reseeccesescesee ete eeeereecee Bon CoOCH i 
ADRIAN BROWN \cvceccaccacectstesteemaniesensse retest b ines a aievsecivaciecesiaeeats ena ili 
LowisCOomMPTon, IMEGATL Beevescesmetocbocthes css ceceneecenaseececetneeceneeen cae x 
GrEorGE Stewarpson Bravy (with portrait) .................ceceeee sooner ex 
FRANCIS ARTHUR BAINBRIDGE ..sscoseesscceees echeaes eamedticiamtedsereeamens Xxiv 
Aveustus! Desire WALEER! [ececcccccecesscsess Ancasescod Sancatton ee anatee XXvil 


/ 


EARL OF DUCIE, 1827-1921. 


THE late Earl of Ducie, who died at his seat, Tortworth, Gloucestershire, was 
born on June 25, 1827, and was the “father” of the House of Lords, which he 
entered on the death of his father in 1853. He was also the “father” of the 
Royal Society, to which he was elected in 1855, as well as the oldest Lord 
Lieutenant in England, having been appointed by Palmerston in 1857. 

He was the eldest of ten brothers and four sisters, several of whom survive 
him, but his only son, Lord Moreton, died in 1920, leaving no heir, so that he 
is succeeded by his brother, the Honourable Berkeley Moreton, formerly 
Colonial Secretary for Queensland, where he has resided for many years. 

Lord Ducie was a type of man more common perhaps in the early Victorian 
era, to which he belonged, than at present, who received the honour of our 
Fellowship rather on account of his position and his general interest in 
Science, than for any special study or work that he had done. 

If, however, Arboriculture is a science, and a tree-planter has a claim to 
be elected an F.R.S. in virtue of his lifelong devotion to a pursuit which has 
beautified rural England beyond any other country, then I have no hesitation 
in saying that he would, even as a simple country squire, have deserved it. 
When he inherited the noble domain of Tortworth, 68 years ago, he found 
nothing more to induce him to become a leader in Arboriculture than many 
others have found; for his father, though an eminent agriculturist, had done 
little or nothing to set him an example. Loudon had laid a good foundation 
for more exact knowledge than our ancestors possessed of the great number 
of exotic trees which will thrive in this country, and the introduction by 
Douglas and others of numerous North American conifers, had attracted many 
and created a fashion for planting a “ Pinetum” in many parts of England. 
Some of the most famous of these Pineta were made on soils or in climates 
which later experience has proved to be unsuitable, and in consequence when 
the generation which had planted them died out, they sometimes became 
neglected and many of the species died. But the varied soils and situations 
at Tortworth, coupled with the favourable climate of the vale of Gloucester, 
favoured Lord Ducie’s early experiments so well, that he never rested in his 
labours till he was past ninety, when his bodily and mental powers began to 
fail. But his pleasure and interest in watching the growth of his trees never 
diminished, and he set an example to all planters by the care which he gave 
to his trees for many years; with the result that at Kew alone can a collec- 
tion be found, so well grown, well pruned, and generally well cared for as at 
Tortworth. 

When I first began to collect material for a more up-to-date work on the 
trees of Great Britain, he encouraged and assisted me to a greater extent than 
any other man, and was never so pleased as when he could show me in one or 
other remote corner of the park or plantations, some rare American oak, 

a 2 


il Obituary Notices of Fellows deceased. 


hickory, or other hard-wood which he had planted 40 or 50 years before. 
Visitors were numerous at Tortworth, and though he loved to show them 
round himself, he was such a conscientious man in the performance of the 
numerous public duties which he undertook, that he always seemed to be in 
a hurry to get on the next tree or the next duty, so that one had to repeat 
one’s visits frequently in order to take in the knowledge he possessed. I well 
remember telling him of a tree in Lord Bathurst’s park which I could not 
identify with certainty. He said “ What is the use of your knowledge if you 
cannot name a tree?” and wrote to Sir J. D. Hooker to send down the late 
Mr. Nicholson to name it; who, suspecting, as I did, that it was of hybrid 
origin, identified it with Pyrus utermedia. But it was only after my personal 
visit to Fontainebleau, and a week’s work by Dr. Henry in the Kew Herbarium 
that we came to the conclusion that it must be the same as what Gay and 
other French botanists, many years before, had called the Service Tree of 
Fontainebleau, Pyrus latifolia. 

When Prof. C. 8. Sargent of Boston and the late Dr. Asa Gray visited 
Tortworth about 1880 they were shown three trees natives of California, which 
neither of them had seen alive in their own country and which will not exist 
in the climate of New England. Castanea chrysophylla, the Golden-leaved 
Chestnut, was one of these, and though the Tortworth specimen is rather a 
great bush than a tree, it has supplied seeds for years to all who asked for 
them worthily ; for Lord Ducie was most liberal in distributing young trees 
from his well-managed nursery. 

Though Lord Ducie was for many years a Vice-President of the Royal 
Horticultural Society, and was generally interested in local geology and 
botany, he took no particular interest in other branches of horticulture, 
though Tortworth is celebrated for its fruit. He was an active Volunteer from 
the commencement of the movement, a distinguished marksman with the 
match-rifle, a yachtsman who visited Norway on many occasions, until he lost 
his steam yacht ina fjord there. He was for some years President of the 
National Rifle Association, and a most indefatigable worker in all county 
business. Though Lord Ducie had resisted the request of numerous friends to 
compile or allow to be published a catalogue of his unique collection of living 
trees, it is much to be hoped that such a fitting memorial of his life’s work 
may now be attempted. a Bir an! vs 


rat 


ADRIAN BROWN, 1852—1919. 


AprkIAN Brown was the junior member of the very remarkable amateur 
scientific quartette, Peter Griess, Cornelius O’Sullivan and the brothers 
Brown—Horace and Adrian, once active in the scientific service of brewing at 
Burton-on-Trent. 

The amateur scientific worker is a peculiarly British product—he hates drill 
and grows through force of example, not of precept. He has not matured in 
countries where drill has been in the ascendant. Griess was an outstanding 
example. He spent six or seven years at the University—doing nothing as a 
formal student ; the saying goes, that he wasted his time. Far from this— 
whilst he took his fill of student life, what to-day would be called his sub- 
conscious mind was clearly at work and he suddenly displayed extraordinary 
activity in the laboratory. His great ability was appreciated by Kolbe, his 
teacher, himself a man of the highest intelligence, to some extent trained here, 
under the late Lord Playfair, as fellow worker with the late Sir Edward 
Frankland, the ablest chemist of his time in the laboratory and the author of 
the theory of valency upon which our entire system of structural formule is 
based. Kolbe recommended Griess to Hofmann, then Professor at the Royal 
College of Chemistry, in Oxford Street, London—whence arose the dyestuff 
industry as the outward and visible sign of the great leader's activity and 
example as an original worker. Griess brought with him from Germany his 
discovery of the Diazo-compounds, one of the most remarkable in the history 
of chemistry, as it involved recognition of the fact, that nitrogen, up to that 
time regarded as an inert element, could form compounds of unusual chemical 
activity and extreme instability. He developed his discovery in London, 
until in 1862 he become assistant in Allsopp’s Brewery in Burton-on-Trent. 
Here, until his death in 1888, he occupied an anomalous position, living a life 
all but apart from the brewery, an indefatigable worker, high up in an 
empyrean of constructive organic chemistry. Apparently, Griess did nothing 
in particular for brewing, beyond criticising its products; but he laid the 
foundation of a branch of the dyestuff industry which has since been the 
most remunerative of its many activities. His services were once sought by 
an English dyestuff firm, but the beggarly terms offered him were naturally 
declined and we lost an irrecoverable opportunity. 

In the past scientific workers had their individual patrons who supported 
their inquiries; but the Allsopp firm behaved to their chemist in a way which 
is without parallel in the history of industrial enterprise; they seem to have 
gloried in having so distinguished a man on their staff, without considering 
the direct value of his services. His presence was testimony to their breadth 
of view, as well as to their liberality ; they undoubtedly gained in repute from 
- their action. 


lv Obituary Notices of Fellows deceased. 


Horace Brown entered on his technical career, in 1866, at the time 
when Griess took up the position of chief chemist, at Allsopp’s, vacated by 
Dr. Bottinger, father of the Dr. H. von Bottinger, recently deceased, who is 
noted for the part he played in the development of the German Dyestuff 
Industry. He had been influenced, as a lad, both by Bottinger and by Griess 
but his only didactic training in chemistry was a year spent at the Royal 
College of Chemistry, partly under Hofmann. and partly under Hofmann’s 
successor, Frankland. 


Cornelius O’Sullivan went to Burton in 1867. He too had been a pupil of 
Hofmann, at the Royal College of Chemistry ; he was one of the assistants in 
the laboratory at the time when Horace Brown and the writer entered as 
students. He accompanied Hofmann to Berlin but, after a few months, on 
his recommendation, returned to England to enter the service of Messrs. Bass 
and Co. An accomplished worker, he began the study of the mash-tun by 
investigating the action of the enzymes of malt (diastase) on starch; he may 
be said to have rediscovered maltose in the course of this work and to have 
established its significance as a fundamental unit in the complex starch 
molecule. Although others have followed in his footsteps, to the present day 
we remain ignorant as to the precise nature of the successive changes which 
the starch molecule undergoes on hydrolysis and of the number of enzymes 
concerned in the process. O’Sullivan was also the first to study, in detail, the 
rate at which cane-sugar was hydrolysed by yeast invertase. Finally, he 
undertook an inquiry into the products of the hydrolysis of gum-arabic and 
laid solid foundations which no one yet has built upon. : 


Adrian Brown did not enter the scientific service at Burton until 1873, 
when its foundations had been deeply laid: he then spent several years 
studying for the distinguished part he was to take in the quartette. 

He was born at Burton on April 27,1852. He came of a practical and 
nature-loving stock. His father, Edwin Brown, the son of a small builder, 
left school at an early age, to become clerk at a private bank in Burton, 
ultimately the Burton, Uttoxeter and Ashbourne Union Bank, of which he 
was manager during the last twenty-five years of his life. He died suddenly, 
in 1876, at the age of fifty-seven. He was an ardent naturalist, specially 
known as a coleopterist, with a strong scientific bent and leanings to all the 
sciences, particularly geology. 

He is referred to in the ‘Life and Letters of Charles Darwin’ in a letter 
from Henry Walter Bates, the celebrated Amazonian traveller, to Darwin, 
dated October 17, 1862 :— 


“Mr. Edwin Brown is manager in a large Bank at Burton. I have 
known him twenty-one years; he was my earliest naturalist friend. I 
have always looked on him asa man of extraordinary intellectual ability. 
I have given him my notices on Carabi. He is amassing material 


Adrian Brown. Vv 


(specimens) at very great expense. He has never travelled; this is a 
great deficiency, for the relation of species to closely allied species and 
varieties cannot, I think, be thoroughly understood without personal 
observation in different countries.” 


In the memoir, by Mr. Edward Clodd, prefixed to the reprint of the 
unabridged edition of ‘The Naturalist on the River Amazon,’ published by 
Mr. Murray, in 1892, it is stated that :— 


“Bates was born at Leicester in 1825 and spent his youth in the 
district. Apprenticed to a hosiery business, he left it soon after his 
master died and eventually entered Allsopps’ Brewery as a clerk.” 


Mr. Clodd adds :— 


“ As often as he could he escaped from the desk to the open air, and 
some results of his entomologising are found in a paper on ‘ Coleoptera 
in the neighbourhood of Burton-on-Trent, published in the ‘ Zoologist ’ 
(VI, 1848, 1997). Mr. Edwin Brown, who obtained him the situation 
at Allsopps’, is referred to as the captor of several species scheduled in 
the paper.” 


Edwin Brown appears to have exercised considerable influence upon the 
fortunes of both Bates and A. R. Wallace. Bates became acquainted with 
Wallace at Leicester, where the latter was English master in the Collegiate 
School. Mr. Clodd tells us that :— 


“The two friends often discussed schemes for going abroad to explore 
some unharvested region and at last these took definite shape, mainly 
through the interest excited by a little book, published by John Murray 
in 1847, entitled ‘A Voyage up the River Amazon, including a Residence 
at Para, by Mr. W. H. Edwards, an American tourist.” 


The writer learns from Dr. Horace Brown that this book had greatly 
interested his stepfather, Edwin Brown, who lent it to Bates to read. The 
recovery of such a fragment of history will not be without interest, as giving 
a clue to the mental process whereby the two travellers were eventually led 
to the study of problems of world-wide significance. 

Adrian Brown was therefore nurtured in a scientific atmosphere. He 
attended the local grammar school, but his effective trainmg was at the 
hands of his father and, in chemistry particularly, of his elder brother, 
Horace. He received his special technical training mainly at the Royal 
College of Science, the combination of the Royal College of Chemistry, 
Oxford Street, with the Royal School of Mines, Jermyn Street, then just 
effected at South Kensington. On leaving, he became private assistant to 
Dr. Russell, Lecturer on Chemistry in St. Bartholomew’s Hospital Medical 
School. 

In 1873 he quitted London for Burton, to act as chemist to Messrs. Salt 
and Co., Brewers. 


vl Obituary Notices of Fellows deceased. 


In referring to the Burtonian quartette, which Adrian Brown was the 
last to join, as amateur workers, the writer is not unmindful of the fact that 
all were brewer's chemists and therefore professionally engaged, excepting, 
perhaps, Griess—and it has always been thought that he has not had 
sufficient credit for the work he did in the brewery. None the less, the 
spirit in which they worked was that of the amateur of the past: they 
sought neither gain nor applause: love of their art was their guiding 
light ; they were led solely by desire to explore its fields, to grasp its value, 
to display its beauties. 

Adrian Brown remained twenty-five years in the brewery, leaving in 1899 
to take charge of the newly founded Chair of Brewing and Malting at the 
Mason College, Birmingham. When the University of Birmingham was 
established, he became Professor of the Biology and Chemistry of Fermenta- 
tion and Director of the School of Brewing. He died suddenly on July 2, 
1919, three days after his wife. 

A naturalist from his birth upwards, a man of unobtrusive manner but 
great personal charm, he gained the esteem of all whom he met officialiy 
and the affection of his many friends. Not only was his standard of 
endeavour ever the highest but all he did was characterised by originality, 
great independence of judgment and a consistent logic. 

He was not elected into the Society until 1911. His first work was in 
advance of the time and did not receive the attention it deserved, although 
the subject, the action of oxidising organisms, was one of great interest, 
oxidation playing so determining a part in vital activity. He began by 
studying the action of the well-known Bacterium aceti, used in producing 
vinegar from alcohol; then that of another organism, Bacterium xylinum, 
which he was the first to isolate from “ Mother of Vinegar.” 

The point brought out in the earlier inquiry was the inability of B. aceti to 
condition the oxidation of methylic alcohol, although it grew in presence of 
this compound: then, that it was able to determine the conversion, not only 
of ethylic alcohol but also of propylic, to the corresponding acid; yet was 
without action on isopropylic and isobutylic alcohols; still, it grew in the 
presence of these compounds but was killed by fusel oil and amylic alcohol. 
These remarkable differences in behaviour of compounds so closely related 
remain unexplained to the present day. Assuming that oxidation be deter- 
mined by a catalyst, z.e., at a solid surface, it is most probable that the 
surface is differently affected by the different aleohols: but on any hypo- 
thesis the fact that methylic and isopropylic alcohols are unattacked, 
whilst compounds so close to them in all chemical properties are oxidised, 
is very striking; no other such marked instance of bacterial epicurism is 
known. 

The results of his work on B. aylanwm were of less direct significance, as 
his attention was mainly directed to the membrane which this organism 
produces in sugar solutions ; he thought it was a variety of cellulose, but from 
later work, by Emmerling, it is probable that the product is of a chitinous 


Adrian Brown. vil 


character. Here again, however, a field is opened up for further. inquiry— 
if action take place within the cell, how comes it that such a product is 
secreted without the organism if this be not destroyed in the process ? 

The activities of B. zylinwm were studied, a few years later, by the French 
chemist, Bertrand, who established the fact that it has an entirely remarkable 
discriminative power, as in compounds sensitive to its oxidising influence two 
H.C.OH groups are present, not only in conjunction but so placed that the two 
‘OH groups in these are on the same side of the plane in the formula; thus 
when the two isomerides formed by reducing sorboses are submitted to the 
action of the organism 


H H OH H OH H OH H 
cal oda ical [ae item cf 
CH,OH—C—C—C—C—CH.0OH CH,OH—C—C—C—C—CH,0H 
Vespa ealinee. | lhcaale yal 
OH OH HOH OHH OH 
Sorbitol. Tditol. 


fant 


‘sorbitol alone is oxidised and reconverted into ketose (sorbose). It is in no way 
clear,at present, whether the selective activity displayed by the organism be that 
of an enzyme or traceable merely to a peculiarity in the oxidative process. 

The root ideas underlying our present conception of the nature of enzymic 
hydrolysis are largely traceable to Adrian Brown’s iconoclastic work. 
Beginning with observations on the rate of reproduction of yeast cells, he 
noticed that a constant amount of yeast fermented an approximately 
constant weight of sugar, in unit time, in solutions of varying concentration ; 
the graph of his experiments was a straight line, not a logarithmic curve— 
indicating the change of regularly diminishing amounts in successive unit 
periods—such as was held to be expressive of the simple enzymic change 
conditioned by invertase, on the basis of the experiments made by Cornelius 
O’Sullivan and Tompson, published in 1890. Hence he was led to re-examine 
the evidence adduced by these chemists, in support of their view, that the 
enzymic change was a mass-action effect, strictly comparable with the changes 
taking place in solutions of erystalloids—in other words, that enzymic change 
took place in solution. 

He dealt with this subject in an exhaustive manner. The conclusion 
arrived at was, that during the earlier period of change, as in fermentation, 
the sugar is hydrolysed at a linear rate, the amount converted being 
practically independent of the concentration of the solution, in no way pro- 
portional toit. The rate of hydrolysis is much reduced by the addition of 
invert sugar, that is to say, of the products of change ; lactose, except in very 
large proportion, however, has little effect. 

These conclusions have been fully confirmed by later inquiries. It may 
now be taken as established, that enzymic action is effected at solid surfaces. 
Complete confirmation of this explanation has been given by recent observa- 
tions on the action of a catalyst such as finely-divided metallic nickel in 
determining the hydrogenation of the fatty oils. 


vill Obituary Notices of Fellows deceased. 


Much of Adrian Brown’s work was in criticism of Pasteur’s findings, 
especially the great Frenchman’s conclusion that fermentation was life without 
air; he thought that he had proved the contrary. He studied the effect of 
alcohol and found that it greatly retarded the reproductive growth of yeast ; 
also the effect of carbon dioxide but came to the conclusion that, as it had no: 
greater influence than hydrogen and as there was a much larger increase in 
presence of air, that the repression of growth was due to exclusion of oxygen. 
He was, therefore, led to favour the conclusion that reproductive growth of the 
yeast cell, under ordinary anaérobic conditions, is determined by the amount. 
of oxygen at the disposal of the organism prior to the commencement of 
reproduction. 

Recently, however, Slator, working in the laboratory of Messrs. Bass and Co., 
has adduced proof that not only has carbon dioxide a greater influence: 
than has been supposed on the activity of the yeast organism but also 
that oxygen is not required for reproductive growth of the cell—thereby 
upholding both a common opinion of carbon dioxide and Pasteur’s view as to. 
oxygen being unnecessary. 

It remains to consider what is undoubtedly his most remarkable work— 
that on diffusion into the barley corn, noteworthy both on account of the 
beauty and delicacy of the method he developed and the significance of 
the results. Having eyes to see as well as an inquiring mind, he was led to: 
take special notice of the blue layer just below the skin in certain varieties 
of barley. Desirous of finding out what happened, in the malting process, 
when barley was steeped in water, as it is during the preparation of malt, 
also what would be the influence of impurities in the water, he first studied 
the behaviour of dry barley-corns in water and then in various solutions. 
He saw that in blue barley he had a perfect mechanism for the quantitative 
study of diffusion phenomena. The blue layer furnished the discriminating 
membrane; the finely granular mass of starch within the corn served to- 
attract water into the grain. By placing a set of weighed corns in water 
aud at intervals removing them and determining the increase in weight, at 
various temperatures too, the rate at which water entered was easily ascer- 
tained. The variation in the rate at which it accumulates in the grain, as the- 
temperature is raised, was in agreement with that at which the vapour 
pressure of water rises when this is heated. 

A similar tale is told by solutions of most salts and of substances such as. 
the sugars—these all have vapour pressures lower than that of water and 
water accumulates less rapidly in barley corns placed in these solutions than 
if they were in water, the rate depending on the concentration. 

Solutions of the ordinary strong acids and alkalis also give up water 
to dry barley corns and become concentrated but no acid passes across 
the discriminating membrane; the blue colour remains unchanged in all 
sound corns. 

But weak acids, also weak alkalis, such as ammonia, readily pass through ; 
moreover, the membrane is penetrable by all chemically neutral substances. 


Adrian Brown. 1x 


at all soluble in water—such as acetic acid and its homologues, chloro- 
form, even hydrocarbons such as benzene. ‘These all pass into the corn 
together with water and actually accelerate the passage of water into the 
grain. It is no question of molecular size: such molecules do not penetrate 
the membrane because of their smaller size; on the contrary, butyric acid 
enters more rapidly than acetic and the alcohols of the ethylic series pass 
through the more rapidly the greater their molecular weight, so long as they 
are reasonably soluble in water. 

Eventually an equilibrium is reached within the corn and the concentra- 
tion of the solution may become higher than in the liquid outside; it has 
even been observed, in the case of phenol and aniline, that the internal 
solution is “supersaturated.” To take an example, when the corns are 
saturated in a solution containing 50 per cent. of acetic acid, the solution 
within contains 80 per cent. of the acid—but this is the limit, no more passing 
in from stronger solutions. 

Previous observers on the passage of such substances through living 
tissues have correlated their relative activity and their lethal power with 
the solubility in fats, and have postulated the existence of a lipoid layer 
at the tissue surfaces. Adrian Brown’s observations justify us in putting 
aside all such fancy explanations—the correlation is of consequence only in so 
far as the solubility referred to of substances in oils and fats is usually the 
converse of their solubility in water. 


A full discussion of the work Adrian Brown accomplished and its bearing 
on contemporary inquiries, by the writer, is published in the ‘Journal of 
the Institute of Brewing, 1921, vol. 27, pp. 197-260. From the point of 
view advocated in the present notice, his exemplary career and achieve- 
ments merit most careful attention. His genius lay not on the surface but 
was manifest in a continuity of effort which, in sum, was remarkably 
effective. His work was an expression of himself: it came from within ; 
but that he was induced, if not forced, to display his genius owing to the 
influence of the conducive environment in which he was placed is probably 
a not unwarrantable conclusion. Chamber music such as the Burtonians have 
discoursed so successfully might well and should be more cuitivated, not only 
in industry but in the new Universities, even at Oxford. 

; Eee Er 2Ae 


LOUIS COMPTON MIALL, 1842—1921. 


Louis Compron MIALL was born at Bradford in the year 1842, and was the 
fifth living son of a Congregational Minister, the Rev. James Goodeve Miall. 
His mother was Elizabeth Symonds Mackenzie. The teaching element was 
strong on both sides of his family. The Mialls had been schoolmasters and 
preachers for generations. One of them, Moses Miall, had published a book 
of Practical Remarks on Education. The Mackenzies were also much given 
to schoolmastering, and had come strongly under the influence of the 
Edgeworths, whose educational methods were firmly established in the 
family tradition. Sir Morell Mackenzie and Edward Compton, the actor, 
were first cousins of L. C. Miall on the Mackenzie side, and Edward Miall, 
M.P. for Bradford and Editor of the ‘Non-Conformist, was his father’s 
half-brother. 

The Rev. J. G. Miall was a man of varied attainments, and had a distinct 
gift for teaching. He bestowed much pains on the training of his children, 
for he knew that they would have to fight their way in the world on their 
own resources. He was very particular to train them in self-reliance, to give 
them studious tastes, and the power of expressing themselves well. He had 
himself a beautiful speaking voice, which the children all inherited in 
varying degree, and he taught them to make the best use of it. 

L. C. Miall owed much also to his mother. She, like her husband, had a 
pleasant voice and manner and much charm of personality, and she had 
a power of holding her children’s love and admiration which far exceeded 
his. She was, above all things, deeply religious, but had so courteous and 
sunny a disposition that religion, even the terrible early-Victorian religion, 
could not make her gloomy. 

Louis seems to have been an enterprising and high-spirited child, keen 
about games and mischief, and inclined to wander away from home on 
solitary expeditions. He was sent first to a little school near his home, and, 
later, when he was 9 years old, to Silcoates School, near Wakefield, then, as 
now, a boarding school for the sons of Non-Conformist Ministers. 

At the time that he left school, L. C. Miall’s interests were all classical 
and literary. He had learned little mathematics and no science, but had 
shown himself good at essay writing, and had stored his mind with fine 
passages from Shakespeare and the Latin poets. He said in after life that 
he knew nothing of Natural History as a schoolboy, though he tried to make 
friends with a half-witted boy who could show him, as if by magic, all sorts 
of strange nests and creatures in places where no one else would see 
anything. But even in those days he must have been unusually observant, 
for he tells in the “Natural History of Aquatic Insects” how he and his 
companions watched the dragon-fly escape from its pupa-case, and how they 
saw the larva, “ with its dingy colours, its forbidding shape, and its predatory 
habits . . . stretch out its great paw and secure an unsuspecting victim.” 


Louis Compton Miall. Xl 


In 1857 Louis Miall left school, a boy of fifteen, but looking older, and 
already grave and dignified. He had probably learnt all that Silcoates 
had to teach him, and his father could afford to spend no more upon his 
education, but recognised the lad’s ability, and, being anxious to give him 
further opportunities to study, hit upon a scheme that seemed promising 
from many points of view. This was that Louis should keep a little 
day-school, with his father’s help and direction, prospectuses for which 
were accordingly issued in Louis’s name. A member of the congregation, 
who much admired the minister and knew something of his teaching 
capacity, had already entrusted him with her son’s education, and other 
pupils were soon found to make the nucleus of a small school. The 
time-table was specially arranged so that Louis should have leisure for 
private study; his father apparently took a good deal of the teaching into 
his own hands, and his mother helped with the French, and, altogether, the 
plan seemed to work out very well. It had its drawbacks, however, the 
chief of which was that Louis had to study by himself, for, though his 
father could help him with Latin and Greek, the boy had then no great 
inclination to continue his classical reading, and was more interested in the 
new scientific subjects that were attracting the attention of the younger 
generation. 

His eldest brother was a medical student at Edinburgh and Louis often 
envied his opportunities. Probably it was his example that induced Louis 
later on to take a course of anatomy at the Leeds School of Medicine. It 
meant early rising every day to journey from Bradford to Leeds, but the 
teaching was good and constituted the only training in science that he could 
obtain. 

Meanwhile he was working hard at zoology and geology, and joined a 
Botanical Society at Todmorden, making many friends who were interested in 
Natural History and publishing papers in various periodicals. One of these 
brought him the following letter from Charles Darwin :— 


Down, Bromley, Kent. 
DEAR SIR, January 23, 1860. 

I hope that you will excuse the liberty I take in .writing to you and 
requesting a favour. In the ‘ Annals of Nat. Hist., vol. 15, p. 39, you remark 
“The variations of form in the maxille are of no value among the Phalangida 
in affording generic or specific characters as with the true spiders.” Am I 
to understand from the latter part of sentence that with the individuals of 
the same undoubted species the maxille vary in form? Is not this a very 
surprising fact ? Would you have the great kindness, if the fact be so, to give 
me some details on the amount and kind of variations and in what species. 
And further would you permit me to quote any such facts on your authority ? 

With many apologies for troubling you, I beg to remain, Dear Sir, 


Yours very faithfully, 
CHARLES Darwin. 


xi Olituary Notices of Fellows deceased. 


One wonders what answer was sent, and whether Darwin was aware that 
his correspondent was a boy of eighteen. 

A couple of years later appeared the “ Flora of the West Riding” by Miall 
and Carrington, with an introduction by Louis Miall, which he repented in 
later years ; for, though it has merits, it 1s written in a rather high-flown 
style, and to publish a list of plants and their localities was quite contrary 
to his maturer teaching. 

Besides the difiiculty of studying without teachers, there was another draw- 
back to the life that he was leading at this time. His father was strong-willed 
and autocratic, and Louis’ own strong will was frequently at variance with 
his in the management of the little school. The young man’s scientific 
studies, and the spirit of the age when Darwin and Huxley were fighting for 
freedom of belief, soon brought religious disagreement into the family circle 
and Louis’ change of faith was a great grief to his parents. It hit his father 
on all sides, as parent, schoolmaster and minister, and he felt it very bitterly. 
Altogether the life was neither happy nor hopeful, and the young man decided 
that it could not continue. He would find work elsewhere, and eventually 
he took a post as assistant-master in a school kept by Mr. George Todd at 
Stamford Hill, near London. 

Towards the end of the second year there the situation was changed by 
a letter from his brother Philip, telling him that a Philosophical Society 
was being started in Bradford, and that Philip was commissioned to write 
and offer him the post of Secretary to the Society with a salary of £100 a 
year. This was just what he wanted. He wrote an immediate acceptance 
and gave notice to leave the school at the earliest moment possible. 

This was the turning point in Louis Miall’s career. After six or seven 
years of gradually increasing darkness and discouragement, the horizon 
cleared, and henceforth he advanced without faltering. When he returned 
to Bradford he was very raw and inexperienced and had little idea what to 
make of his new task. The first thing he had to do was to arrange a 
course of lectures, under the guidance of the Committee, who soon left all 
the correspondence in his hands. An interesting course of lectures was 
given between 1865 and 1871, among others by Owen, Huxley and Rolleston, 
who thus came into personal contact with the Secretary of the Bradford 
Philosophical Society. 

Another thing to which the Secretary had to turn his attention was the 
making of a museum from a collection of objects mostly given by people 
who wanted to get rid of them. He finally decided that the only thing he 
could do was to mske a collection of geological specimens for which the 
neighbourhood offered unusual facilities. He prepared a report to the 
Committee, in which he offered to collect what he could from the coalfields 
and limestone districts within reach. For some years it was his delightful 
hobby to explore the district of Craven, to study its geology and to 
collect its fossils. A frequent companion of his on these rambles was 
John Brigg, afterwards Sir John Brigg, M.P. for Keighley, a member of his 


Lows Compton Miall. xill 


‘Committee, who took a great interest in the young Secretary and influenced 
him in many ways. 

In the course of these excursions quite a respectable collection of fossils 
and geological specimens was made for the Bradford Museum. Then a great 
piece of luck befel the Curator. One day there came into his office a coal 
miner bringing some curious bones that he had found in the Low Moor coal 
mine. Miall went to see them himself next day, going down a coal mine for 
the first time in his life. He was shown the bones on the roof of a passage 
in the works, and realised that they would require very careful treatment if 
they were to be removed without injury. So it was decided to apply a 
layer of plaster of Paris to protect the bones and then to have the coal 
carefully worked away, a prop being placed to support the fragments covered 
with plaster. The bones were removed in perfect condition except for those 
that had already been broken off. The block removed was 11 feet long and 
a couple of feet wide. Investigations proved that the bones belonged to a 
Labyrinthodont of a species that was hitherto unknown. On the suggestion 
of the Committee, Miall wrote to Prof. Huxley and offered to take the 
fossil to London and show it to him. Huxley sent an encouraging reply, the 
fossil was carefully packed in a wooden case and taken to London, where it 
was examined with much interest by Prof. Huxley and Prof. Flower. 
Huxley undertook to write a description of it for the Geological Society and 
asked Miall to prepare a short account of its discovery and removal from the 
coal mine. At the next meeting of the Geological Society, Miall read his 
paper and Huxley gave a simple and interesting account of the new 
Labyrinthodont, without notes, explaining it from the specimen as he went 
along. Sir Charles Lyell was present and seemed to be much interested. 

When Miall returned to Bradford and gave the Committee of the 
Philosophical Society a vivid account of what had passed, they asked him to 
repeat the story in the form of a lecture to the Society. It was his first 
public lecture. After spending a good deal of time trying to write it out, 
he resolved to follow Huxley’s example and speak without notes, explaining 
the actual specimen before the audience. There was a good attendance, for 
the matter had aroused interest in Bradford, and the lecture went off very 
well. That was the beginning of Miall’s career as a public lecturer. After 
that we find him giving courses of Lectures in Bradford and Leeds mostly on 
Geology, but also on Botany and the “ Karly History of Domestic Animals.” 

Though very shy and studious, Miall seems to have entered somewhat 
into the social life of Bradford, which happened to be unusually interesting 
at that time. He was fond of music, and indeed had studied it in his usual 
way by sheer force of will and without a teacher, so that he had written 
songs for his sister and could play to some extent on two or three instruments. 
He also had a good deal of talent for painting. He brought back from a cruise 
in the Hebrides in 1868 sketches from which he made some clever little 
water-colour pictures, that still hang beside one or two of his father’s in 
homes of a younger generation. 


XiV Obituary Notices of Fellows deceased. 


At Bradford he met his future wife, Emily Pearce, to whom he was married 
in 1870. Though not scientific, her intellectual and social gifts were, in some 
directions at least, equal to his own. 

In 1871 L. C. Miall was appointed Curator to the Leeds Philosophical and 
Literary Society. He had already delivered a course of lectures on geology to 
the Society and was known to several influential people in Leeds. He must 
have had Huxley’s support, too, in his application, for among his letters of 
congratulation on obtaining the post was one from Huxley, in which he 
characteristically remarks that it would be a matter of great satisfaction to 
him to think that he had in any way contributed “to the putting of an indu- 
bitably square man into the square hole at Leeds.” 

His interests at this time were mainly geological, and he devoted himself to 
the collection of fossils in the Leeds Museum with the same enthusiasm that 
he had given to the geological collection at Bradford. He was helped in its 
re-arrangement by Pengelly, Boyd Dawkins, and others. Later, much help 
and many specimens were given by A. H. Green. When it was re-arranged, 
he wrote a guide to the collection; in the same way he re-arranged the 
different collections of birds, insects, antiquities, and so forth, and wrote a 
guide to each, in which he set forth clearly the general principles of the 
various subjects. 

Since 1869 Miall had been busy with the investigation of the new 
Labyrinthodont that had been found in the Low Moor coal mine. The task 
proved more difficult than he had expected. He was Secretary of the 
Geological Section of the British Association at Edinburgh in 1871, and a 
Committee was then formed, consisting of Phillips, Woodward, John Brigg, 
and three others, with Miall as Secretary, to investigate and compare all the 
known species of Labyrinthodont. It happened that the followimg summer 
John Brigg and his friend, Swire Smith (Sir Swire Smith, whose life has been 
written under the title of ‘The Master Spinner’), decided to go to Germany 
to look into the German system of education and see for themselves how far 
such a system would be possible in industrial England. They invited Miall 
to join them, so that he and John Brigg could combine the investigation of 
Labyrinthodonts with the educational work, all three being in fact interested 
in both subjects. 

They had an instructive tour, and the following year (1873), when the 
British Association met at Bradford, Miall read the report of the Committee 
on Labyrinthodonta. The work had been very thorough: “Some of the 
members have personally examined all the more important Labyrinthodonta 
in European collections, including at least one example of every species 
recorded from the British Isles.” The report created much interest and 
brought Miall into general notice for the first time. 

Miall was now beginning to concentrate his attention on Biology. He 
declined the Professorship of Geology at the newly opened Yorkshire College 
in favour of A. H. Green,a much stronger geologist than he felt himself to be, 
and henceforth his interest in geology began to wane. He never cared greatly 


Lows Compton Miall. ay 


for mere collection and the minute characteristics of the shells in which 
animals had lived. He collaborated with A. H. Green, Thorpe, Riicker, and 
Marshall in a work on ‘ Coal, its History and Uses,’ published in 1878, but 
his serious interest in geology and paleontology ended about the year 1880. 
He never studied petrology, without which much of the recent work cannot 
be appreciated. 

When Miall refused the Professorship of Geology, the Council of the 
Yorkshire College still wished to secure him upon its staff, and appointed him 
the following year (1875) lecturer in Biology, a post which he held concur- 
rently with his curatorship of the Museum. In 1876 he was made Professor 
of Biology. Many of his lectures were given in the library of the 
Philosophical Society, for the Yorkshire College had little accommodation, 
while there was room and a store of material at the Museum. The professors 
of the Yorkshire College frequently gave lectures to the Philosophical 
Society and sat on its Council. Both institutious worked in conjunction with 
the Leeds School of Medicine, which required courses of Botany and Zoology 
for its students. } 

It was in the yard of the Medical School that Miall dissected the Indian 
elephant which chance gave into his hands. A shed was built over the 
animal, and there he worked through the cold winter of 1874-5, helped by 
F. Greenwood, Curator of the Medical School. The memoir on the “ Anatomy 
of the Indian Elephant ” appeared in 1879, and was the second of a series of 
studies in comparative anatomy. The first of the series was the “Skull of 
the Crocodile,” which appeared in 1878, and the third was the “ Structure 
and Life-History of the Cockroach” (1886). There the series ended 
abruptly, for though a short account of Megalichthys, a ganoid fish of the 
Coal Measures, was published in 1885, the fourth book of the series, which 
was to have dealt with that topic, was never written. The author had 
given so much time to the Cockroach, and had become so deeply interested 
in it, that all other research had to give way to the structure and life- 
histories of insects, which occupied him as long as he had vigour and 
eyesight for the work. 

The book on the Cockroach, published in conjunction with Alfred Denny, 
was by far the most important piece of work that Miall had done so far. It 
represented several years of study, begun in the Museum of the Philosophical 
Society, and carried on at the Yorkshire College and at his own home. It 
has since been recognised as marking an epoch in the study of insects in this 
country. In reading up the subject as a preliminary to further research, he 
had become acquainted with the work of the old naturalists, Malpighi, 
Swammerdam, Lyonnet and Straus-Durcheim. He found them so fascinating 
that the first chapter in the “ Cockroach” is devoted to them, and the whole 
book is an exposition of their teaching—a very lucid account of insect 
structure and development. Its value was immediately recognised by 
Prof. Huxley who congratulated Miall on the book. 

The “Cockroach” appeared in 1886. In 1887 we find its author already 

VOL. XCIII.—B. b 


Xvi Obituary Notices of Fellows deceased. 


occupied with another insect, Chironomus, the Harlequin Fly. This was 
chosen because of its abundance nearly all through the year, its trans- 
parency (in contrast to the Cockroach), and the ease with which it can be 
reared. Besides which, he says, Chironomus, in its various stages, has 
a very special biological interest. His attention was concentrated upon it 
for several years. The “Structure and Life-History of the Harlequin Fly,” 
by Miall and A. R. Hammond, did not appear till 1900, though most of the 
work was done by 1892. 

Soon after he began work on Chironomus, Miall visited Leyden to consult 
some books there. Every letter of this period has some reference to 
C@hironomus, and we even find him “reading Dutch for the sake of 
Chironomus”; but, nevertheless, he found time to write on educational 
topics in the ‘Journal of Education’ and to devise “Object Lessons from 
Nature,” which appeared in book form in 1891. 

Nature-study had not at that time become a universal subject of school 
teaching, but object lessons were given habitually by many teachers. The 
“Object Lessons from Nature” were intended to emphasise the value of 
natural history in furnishing object lessons for children. In 1878, a course 
ef nature object lessons to children had been given at the Museum of the 
Philosophical Society, under his direction, so that the idea was not a new 
one to him. 

A natural development of this was the Saturday morning class for teachers 
which was so valuable a feature of the Biological Department for many years. 
The school-masters and mistresses came at first with the idea of getting up a 
few object lessons for their schools, but eventually many of them came year 
after year from love of the work, and were the most enthusiastic students that 
attended the Department. It was a considerable tax on the energy of the 
staff, and Miall was fortunate in having the hearty co-operation of all 
concerned. A further extension of this work with teachers took the form of 
three summer courses in nature study, given in 1901 and the two following 
years, at Berwick, Rothbury, and Hexham. Here, again, he had the help of 
his staff, and all looked back with pleasure on the experience. 

The investigation of Chironomus led to that of aquatic insects in general, 
and, in 1891, Miall gave one of the public lectures to the British Association 
at Cardiff on “Some Difficulties in the Life of Aquatic Insects,” treating 
specially their means of overcoming the surface tension of water. He also 
read a paper on floating leaves in connection with the same difficulty. A 
piece of work on Transformation of Insects, which appeared in “ Nature” in 
1895, was also a product of the Chironomus investigation, and that year, five 
years before the book on the Harlequin Fly was ready, Miall completed the 
“Natural History of Aquatic Insects,” a semi-popular book on the subjects 
that he wasstudying. As in the “Cockroach,” he draws attention to the work 
of “certain old zoologists—Swammerdam, Réaumur, Lyonnet, and De Geer— 
who are at present unjustly neglected.” “Some passages in this book,” he 
says, “if taken alone and read hastily, may appear to disparage systematic 


Lows Compton Miall. XVli 


zoology. This is far from my intention. No one can study the great 
naturalists of the seventeenth and eighteenth centuries without feeling how 
seriously their work is impaired by the defective systems of the time. It is 
not systematic, but aimless work that I deprecate—work that springs from 
no real curiosity about nature and attempts to answer no scientific questions.” 
The book was illustrated by A. R. Hammond, who collaborated with Miall in 
the production of the “Harlequin Fly,’ and made most of the beautiful 
illustrations for that work also. 

In 1892, Miall’s many preoccupations obliged him to give up the Curator- 
ship of the Philosophical Society, though he still continued to serve on its 
council. About this time he left Leeds and went with his wife to live at 
Ilkley, as their children were all scattered for the moment. He subsequently 
took a house at Ben Rhydding, where he wrote “ Round the Year,” a series of 
nature studies, in some respects the most memorable book that has appeared 
from his pen. He was by this time 54, and henceforward undertook no new 
work that involved much close microscopic investigation, such as he had given 
to the Cockroach and the Harlequin Fly, but devoted himself rather to general 
topics of natural history and to educational work. “ Round the Year” may 
almost be regarded as a piece of literature; it has been compared with 
Gilbert White’s Letters and was written in the same spirit, not as work, but 
as a pleasant relaxation in the twilight of a busy day. It led to the study of 
Gilbert White, and to the preparation of a new edition of the Natural History 
of Selborne in conjunction with Dr. W. Warde Fowler. It was followed, in 
1904, by another book of the same kind, “ House, Garden, and Field,” which 
has not quite the freshness of “ Round the Year,” and was meant partly to 
satisfy the teachers who were clamouring for more object lessons. The author 
thought it would be better if they made their own lessons, and that nature 
study could not be taught effectively by those who lacked time or inclination 
to do so, but he was quite willing to suggest topics for those who cared to 
develop them. 

In 1897 appeared “ Thirty Years of Teaching,” which embodies his experience 
in various kinds of teaching, including the education of his own children. A good 
deal of it had been printed in the “ Journal of Education,” and was written in 
the train going to and from Leeds. The most important feature of the book 
is the method of treating University or College students which it advocates— 
a method not indeed new, except as applied to them. 

When the British Association met in Toronto in 1896, Miall was President 
of Section D. His address to the section was an eloquent plea for studying 
life, the modes of growth of individuals and races, the causes of decay and 
extinction, and the adaptation of living organisms to their surroundings. “The 
animals set before the young zoologist are all dead; it is much if they are not 
pickled as well,” he complains, and he asks why we study animals at all, 
giving various answers to the question, but ending “to know more of life is 
an aim as nearly ultimate and self-explanatory as any purpose that man can 
entertain.” Furthermore he urges the historical method of treating various 


b 2 


XVill Obituary Notices of Fellows deceased. 


biological subjects, and shows how much keener interest can be aroused in 
such a topic as the Alternation of Generations by finding out step hy step 
how it was discovered, and sharing the discoverer’s own enthusiasm, than by 
taking it as a mass of cut-and-dried facts. 

After spending half a dozen very pleasant years at Ben Rhydding, Miall 
moved back to Leeds, partly for the convenience of the two sons who were 
then at home again, and he remained in Headingley till he gave up his 
Professorship in 1907. The last years in Leeds were much occupied with 
methods of teaching, and he now attended the new Education Section of the 
British Association when he happened to be present at the meetings. In 
1903 he was chairman of a committee to report on the teaching of Botany. 

In 1902 appeared a volume on “ Injurious and Useful Insects,” an excursion 
into economic entomology, which he felt to be an important field of investiga- 
tion, needing especially complete life-histories of insects to make it valuable. 
No doubt the main idea was right and has since been followed up with good 
results, but Miall was not himself in close enough touch with agriculture to 
make the book altogether a success, from the economic point of view. The 
life-histories of insects that it contains are, however, still useful to economic 
entomologists. 

At the inauguration of the University of Leeds in 1904, Miall was given 
the Honorary Degree of D.Sc., the only academic distinction that he ever 
attained. That year and the year following he had the honour of holding the 
Fullerian Professorship at the Royal Institution. At that time also he was. 
asked to serve on the Council of the Royal Society, but unwillingly declined 
as he had already so much on hand. 

In 1908, after his retirement to Letchworth, he was President of the 
Education Section at the British Association in Dublin, and that was the last. 
meeting that he was able to attend, on account of increasing deafness. 
Many activities had to be given up for the same reason, but he was still able 
to carry on individual teaching. From his wife, who was as keen an 
educationist as himself, he had learnt the direct method of teaching modern 
languages and applied it in a way of his own to the teaching of Latin, writing 
out a series of oral lessons and learning, when over seventy, to speak Latin 
fluently with the modern pronunciation. Since his school days, he had 
never altogether neglected his classical studies and, though he sold most of 
his books when he left Leeds, he had kept such Latin and Greek authors as 
he happened to possess. 

It might be noted here that all his life he loved books and was interested 
in the care and binding of them. He was for many years Hon. Librarian 
of the Yorkshire College. The only half-disparaging remark he was known 
to make about Charles Darwin referred to the ruthless way he treated books. 

Writing was an occupation that he maintained to the end of his life. 
The first book that he wrote at Letchworth was the “ History of Biology,” a 
clear and illuminating résumé of the subject that led to the more important 
work on the “Early Naturalists (1530-1789).” It begins with an intro- 


Louis Compton Miall. X1X 


ductory chapter on Natural History down to the 16th century, and consists 
mostly of biographical sketches of the old naturalists he loved so well, but 
there are also digressions on “The Natural History of Other Lands and the 
Investigation of the Puss Moth and of the Flower.” Of this book Dr. Warde 
Fowler remarks: “He fairly astonished me, after a visit here at Kingham, 
by sending me as a gift the five splendid volumes on insects of Réaumur, 
and later on his own book on the ‘ Early Naturalists, one as great a treasure 
as the other, for his own beautiful English was as clear and enjoyable as 
Réaumur’s French.” 

Miall’s great force lay in his absolute sincerity. Though he could write 
well, and even brilliantly, he never wrote for effect. Everything that he 
published represented all the careful research and investigation that the 
subject demanded. His first attempts at solving a problem were usually 
wrong, he tells us, and in regard to one of his later books he says that 
every time he looked up a fact in the British Museum, he found two 
fresh ones that required investigation. “Fortunately,” he adds, “I am not 
pressed for time.” 

“The Karly Naturalists” was the last book he published. He spent 
some years on “A History of Garden Craft” which was ready for publica- 
tion when the war broke out in 1914, but was then put aside, and after 
that he wrote no more books. He wrote an occasional paper, carried on a 
correspondence (sometimes in French) with one of his brothers, and made 
letter-writing rather a hobby. Gardening had long been a hobby of his 
and he had given a good deal of attention to the laying out of his new garden 
at Letchworth. 

On the death of his wife in 1918, my father came back to his favourite 
haunts in Ben Rhydding and remained there till his last illness. He died on 
February 21st, 1921, at our house in Leeds. By his own wish there was no 
religious service at his funeral, a few words of farewell being spoken by his 
friend, Prof. Smithells, in the presence of a small gathering of relatives, old 
friends and colleagues. Nevertheless, the religious enthusiasm which inspired 
his early manhood had never altogether left him, his attitude to life and the 
unknown was always reverent, and the influence he exerted on those among 
whom he worked was spiritual as well as intellectual. 

W. W. 


xx 


GEORGE STEWARDSON BRADY, 1832—1921. 


G. S. Brapy, M.D., M.R.C.S., D.Sc., LL.D., F.R.S., C.M.Z.S., Professor of 
Natural History, Armstrong College, Newcastle-upon-Tyne, and Consulting 
Physician to the Sunderland Infirmary, was born, he told me, April 18th, 
1832. Presumably also on his authority we learn that the event occurred 
at Gateshead, and that he was the eldest son of Henry Brady, surgeon. 

As his childish education began at the Friends’ School, Ackworth, it is not 
improbable that he owed the name Stewardson to his parents’ acquaintance 
with the Quaker family which gave the popular portrait-painter of that name 
to the early part of the nineteenth century. Certainly the whole tenor of 
Brady’s life seems to have been in tune with the principles of that peace- 
loving community, and even on the scientific side there are many indications 
that triendship was his delight. It has been already explained in ‘ Nature’ 
(January 5th, 1922), among other details, that he became a member of the 
Tyneside Naturalists’ Field Club in 1849. At that early period it is said that 
his interest was “with alge and other plant groups.” Much later on he 
referred to these studies when pointing out in correspondence (November, 
1902), that the organisms which I had described as gland-cells in the amphipod 
genus Urothe, were, in fact, “ parasites, probably alge.” 

With the Natural History Society of Northumberland, Durham, and 
Neweastle-upon-Tyne, of which the Tyneside Field Club was a branch, Brady 
had a long and distinguished connexion, both as a frequent contributor to its 
‘Transactions, and twice President of the Field Club. The respect felt for him 
by fellow-workers in systematic zoology may be partially traced by the use of 
his name in classification. Thus among Copepoda Axel, Boeck names a genus. 
Bradya in 1872, Thomas Scott supplies MWeobradya in 1892, Giesbrecht 
Bradypontius in 1895, and Bradyidius in 1897, Vanhoffen Bradyanus in the 
same year, and G. O. Sars Psewdobradya in 1904. Sars had named a genus 
Bradycinetus in 1865. But this suggests a curious need for caution in that 
many generic names owe the commencing syllables Brady-, not to eminent 
zoologists, but to the Greek @padv, indicating some organic slowness, and 
very inappropriate to the scientific activities of George Brady and his 
brother Henry. For the use of the former’s name in identifying species, his. 
friend A. M. Norman led the way with the Ostracode Cythere Bradw in 1864. 
But this, for technical reasons, gave way to another species, the Marquis de 
Folin’s Cythere Bradii in 1869. Norman, in 1878, named a Copepod 
Cervima Bradyi, Sars in 1884 another of that group Undinopsis Bradyi, and 
Thomas Scott a third in 1892 as Tetragoniceps Bradyi, but this, later on, he 
found reason to place in a new genus with the long-flowing name of 
Phyllopodopsyllus, strictly meaning “a leaf-footed flea,” the species being 
notable for “the large size and leaf-like form of the fifth pair of thoracic feet. 
of the female.” Ina footnote to Tetragoniceps Bradyi, Dr. Thomas Scott 


George Stewardson Brady. XXi 


remarks, “the name is given in compliment to Prof. G. 8. Brady, whe 
instituted the genus, and to whose untiring and disinterested kindness the 
author of these notes owes much of his success in the study of the Entomo- 
straca.” In 1879 Dr. Norman again pays his friend the compliment of using 
his name for a species, this time in the eccentric group of the Sympoda, to 
which he adds the description of Diastylis Brady. 

In the previous year the Ray Society had published the first volume of 
Brady’s “ Monograph of the free and semi-parasitic Copepoda of the British 
Islands.” As the uninitiated may be excused for wondering why men of 
ability should spend a considerable part of their lives in studying creatures 
so Insignificant in size and so generally harmless to mankind, as the Ento- 
mostraca, it may be observed that, as in old Camden’s phrase, “many a 
little makes a mickle,” and as little grains of sand may make a mountain, 
so the stupendous multitudes in which some of the entomostracan species 
oceur make them indirectly yet ultimately important contributors to human 
food and comfort. But, apart from economic values, the true lover of nature 
finds in this seemingly trivial study more than one source of esthetic 
fascination. In the introduction to Brady’s last-mentioned work he says :—- 
“Some of the pleasantest and most profitable hours which I have ever 
spent have been when, after a day’s dredging, I have set out at sunset on 
a quiet boating excursion for the purpose of capturing such prey as could 
be got in the surface net. Many hours of this kind, spent in the company 
of my old friend Mr. David Robertson, amongst the Scilly Islands, on the 
Firth of Clyde, on the sheltered bays of Roundstone and Westport, or on 
the stormier coasts of Northumbria, will long live in my memory, not only 
by their results in the acquisition of valuable specimens, but as times of 
unalloyed delight in the contemplation of nature under a different guise from 
that in which we usually see her.” The David Robertson to whom he here 
alludes, otherwise known as “ the Naturalist of Cumbrae” (see his ‘ Life by 
his Friend, 1891), began a notable career as a penniless herdboy, and ended it 
an Hon. LL.D. of Glasgow University. 

In the bibliography to his luminous work on the Ostracoda of the Bay 
of Napies and the adjacent seas (1894), G. W. Miiller enumerates twenty- 
one contributions by Brady to this branch of Carcinology, together with 
seven others in which his was the leading name in a collaboration. Five 
of these were undertaken with David Robertson, one with Norman, and one 
with Crosskey and Robertson together. When the first volume of the 
“Challenger” Reports on Zoology was published in 1880 under the editorship 
of Sir C. Wyville-Thomson, Brady was already a recognised authority on 
the Ostracoda. He was among those specially consulted as to the disposal 
of the vast “ Challenger ” material, and his was the third memoir to appear. It 
was illustrated by forty-four quarto plates. For the comparative fewness of new 
species he explains that the “ work of the ‘ Challenger’ gave us no collections 
whatever from between tide marks, nor from the laminarian zone, and these 
two zones usually swarm with microzoic life of all kinds.” A later work of much 


oni Obituary Notices of Fellows deceased. 


importance was that which he carried out in partnership with Canon Norman 
on “The Marine and Freshwater Ostracoda of the North Atlantic and of 
North-Western Europe,” the first part appearing in 1889, the second in 1896. 
In this he gives a signal example of his scientific ingenuity which is worthy 
of additional record. He points out (p. 622) that “In consequence of the 
small size of Ostracoda it is extremely difficulty to procure spirit-preserved 
specimens from the deep sea, and although the Myodocopa, being much larger 
than the Podocopa, would be detected by the experienced eye of a Carcino- 
logist who had studied them, yet the Zoologists usually attached to Govern- 
ment Expeditions cannot be expected thus to notice them. Hence it is that 
in a large number of cases the only examples which have come into our 
hands are such as have been picked out of dried material. It struck us that, 
notwitstanding their dried condition, it might yet be possible by maceration 
to get some idea of the withered inmates of the shells. We therefore made 
experiments, and succeeded in restoring the animals beyond our most ardent 
expectations. All the portions of the animals figured [in several genera and 
species mentioned] have been taken from dissections of animals which have 
been preserved in a dried state for very many, in one case, as long as twenty- 
three years, and we are satified that these drawings will be found to be 
almost as exact, so far as they go, as those taken from spirit-preserved 
examples.” 

In 1884, when the editing of the “ Challenger” Reports had passed into the 
vigorous hands of John Murray, the eighth volume of Zoology appeared, having 
as its opening treatise Brady’s Report on the Copepoda illustrated by fifty-five. 
carefully drawn plates. Though the collection thus laboriously discussed 
presented many points of interest, Brady was forced to admit that it was far 
from representative of what the ocean’s resources were likely to contain, and 
that the last word had not been said as to methods of preserving these 
organisms. In his Introduction he makes some remarks which bear on a 
subject previously mentioned :—“ The appearance of these minute creatures at 
the surface depends upon conditions, the nature of which we scarcely at all 
understand. Night, on the whole, seems to be more favourable than daytime, 
but even during the day they sometimes appear in numbers so vast as to 
colour the sea in wide bands for distances of many miles. This appearance 
has been noticed, perhaps, most frequently in the tropics; but even in the 
Arctic seas some species, especially Calanus (Cetochilus) finmarchicus, are at 
times so abundant as to constitute, it is said, a most important item in the 
food of the whale. So far, indeed, as number and size of individuals are 
concerned, it would appear that the cold water of the Arctic and Antarctic 
seas are even more favourable to the growth of Copepoda than the warmer 
seas of the Tropics.” 

With his frequent and arduous contributions to scientific literature Brady 
combined, from 1857 till about 1890, the conscientious exercise of an exacting 
profession, practising as a doctor in Sunderland, “and after that gave up his 
time to his professorship at the Armstrong College, until he resigned in 1906 


George Stewardson Brady. XXIll 


and .... came to live in Sheffield.” His professorship he had held since 
1875. He married in 1859 and had one son and three daughters, losing his 
wife ten years and his son one year before his own death. Two of his 
daughters are married to members of his own profession, one to Dr. Charles 
Atkin of Sheffield and another to Dr. R.S. Hubbersty of Sunderland, the third 
remaining with her father to the close of his days. He died on Christmas 
evening, 1921. Till the last year of what he himself described as his long 
and happy life, he had never realised that he was old. Apart from science 
his amusements had all been of a tranquil kind—gardening, photography, and 
the game of bowls. A friend, who had been reading over many of his 
writings, tells his daughter that: “ Dominating all is the intense love he had 
for nature, religion,and poetry.” Another friend, who often walked with him, 
tells her of the enjoyment derived from the humour, instruction, and high 
tone of his conversation. A long correspondence is in harmony with these 
touches of character. 

A letter from Sheffield, dated June, 1915, shows him at eighty-three, away 
from necessary books, reluctant to undertake fresh work of importance, yet 
unable to be disobliging. He explains that he had declined an invitation to 
describe the Ostracoda and Copepoda collected by the Australasian Antarctic 
Expedition, 1911-1914, under Sir Douglas Mawson, but that the material had 
nevertheless been sent him, with further pressure. Now, the Scientific 
Records of that Expedition show that in Series C the fifth volume contains 
monographs on the Copepoda, the Cladocera, and Halocypride, by G.S. Brady. 


A fine finish! 
DER: 


XX1V 


FRANCIS ARTHUR BAINBRIDGE, 1874-1921. 


In the death of Francis Arthur Bainbridge, at the early age of 47 years, 
Physiology has lost an enthusiastic and successful investigator and a teacher 
of ability and influence. He was elected to the Fellowship of the Royal 
Society in 1919, and it was but a few months later that he showed the first. 
definite signs of the ill-health, which culminated in a brief, acute illness, and 
death on October 27, 1921. 

Bainbridge entered the Leys School with a scholarship in 1888, and 
passed from there in 1893 with an entrance exhibition to Trinity College, 
Cambridge, of which foundation he subsequently became a major scholar. 
His student career was on normal lines for a man of his ability and studious 
habit, but, on finishing the Natural Sciences Tripos, in both parts of which he 
was placed in the first class, he left Cambridge for St. Bartholomew’s 
Hospital, shaping his course with a view to the practice of medicine. After 
qualification, he held several minor appointments in physiology and pathology, 
while waiting for opportunities of advance in the career which he had then 
chosen ; but his natural bent was already obvious, in the devotion of the 
time which he could spare from his official duties to research in physiology, 
which he carried on in the physiological laboratory at University College. 

In this period he made a series of investigations into the mechanism of 
lymph-formation, which brought clearly into view the influence of activity 
in gland cells on the outpouring of lymph from the blood-vessels of the 
gland. These and other phenomena of lymph-formation, which some had 
regarded as indicating a process of active secretion by the capillary endo- 
thelium, he brought into harmony with Starling’s simpler physical con- 
ception, producing ingenious experimental evidence in support of that point 
of view. He became active also in the new field of investigation opened up 
by Bayliss and Starling’s discovery of Secretin, which was made at this time. 
Bainbridge, however, unable as yet to give to physiology an undivided 
allegiance, had neither the time nor the impulse to acquire that full command 
of specialised technique and experience needed for an essentially biochemical 
investigation. When eventually, in 1905, he abandoned the idea of medical 
practice, it was to accept the Gordon Lectureship on Pathology at Guy’s. 
Here, with A. P. Beddard, he began a series of elegant experiments on the 
secretion of urine by the frog’s kidney, which he resumed on his return to: 
physiology in later years. With Beddard also he effected a useful revision of 
then current views as to the meaning of the sequele of partial nephrectomy. 

Bainbridge left Guy’s in 1907, and, wandering for some years yet further 
from physiology, was responsible, at the Lister Institute, for valuable 
contributions to the study and classification of the paratyphoid and food- 
poisoning group of bacilli. This work formed later the basis of his Milroy 
Lectures to the Royal College of Physicians. 


Francs Arthur Bainbridge. XXV 


It would seem that, up to this period, Bainbridge had so divided his aims 
and his interests that onlookers found it difficult to place him. The few who 
knew him intimately felt that his worth and ability had yet to win a full 
and general appreciation. When the intermittent manner of its accomplish- 
ment is remembered, it appears that his output in physiology was already 
remarkable; but he had never yet been in a position to regard it as his 
life-work, and his sound contributions to other branches of medical science 
seemed, with many, to weaken rather than strengthen his claim and his 
promise as a physiologist. 

It was not till 1911 that his election to the Chair of Physiology of Durham 
University, in Newcastle, enabled Bainbridge at length to devote himself 
whole-heartedly to the line of work which most truly held his interest, and 
for which he felt himself best fitted by early training and natural aptitude. 
His department soon attained a high standard of efficiency in the training 
of students, and he resumed, with the late J. A. Menzies, his experiments on 
the frog’s kidney. Later, he entered upon the series of investigations on the 
adjustment of the heart-beat to the demands of muscular exercise, which 
will probably rank as his most important and permanent contribution to 
Science. With Menzies also he wrote what has become one of the most 
popular and useful of the shorter text-books of physiology for medical 
students. 

When war broke out in 1914 Bainbridge took a commission in the 
R.A.M.C., and doubled the duties of his Newcastle Chair with those of 
Medical Officer at a neighbouring military hospital. In 1915 he was 
appointed to the Chair of Physiology at his old hospital, St. Bartholomew’s, 
in London, and combined the duties of this new Chair with those of an 
officer in the Anti-gas Service, experimenting at Millbank, or touring the 
country as a training-officer in defensive measures. Though he was active, 
and even rather athletic by inclination, his constitution was never really 
robust, and was not fitted for this unremitting overwork. The growing 
demands of his teaching necessitated the resignation of his commission, and, 
during the period of moderate health remaining to him, he found time to 
complete a monograph on “The Physiology of Muscular Exercise,’ which 
was published at the end of 1919, and was received with general appreciation 
by physiologists and others interested in its subject. It was, indeed, in 
many ways a model of what such a survey of knowledge should be, and the 
best proof which Bainbridge has left of one aspect of his ability. The 
presentation was clear and logical, and it showed a sound instinct for 
essentials, in a subject of which the main outlines have too often been 
- obscured by controversy concerning details. The note of personal contact 
with the problem was clearly heard, but not unduly emphasised, and the 
monograph was-generally recognised as a sound and scholarly achievement. 

To those who had known Bainbridge long, it seemed that he changed far 
less than most men do. In later years the circle of his friendship widened 
greatly, but the associations formed in the early days always had first place 


XXV1 Obituary Notices of Fellows deceased. 


in his regard. Similarly, his experience and his influence widened; but 
those who had the privilege of intimacy with him found that his tastes and 
convictions, his fundamental attitude to life, changed remarkably little from 
those of his student-days. It may be that a rather prematurely cautious 
and reticent habit of mind had hampered his earlier career. 

His stature was small, his manner quiet and unimpressive, and he had no 
great natural gift of vividness or eloquence in public speaking, though he 
became a clear and effective lecturer to students. These things, with his 
hesitation in committing himself definitely to the work for which he was best 
adapted, rather delayed the recognition which was only beginning to come to 
him in proper measure when hedied. With health and opportunity, he would 
have carried much farther the work that he had begun. 

Bainbridge married in 1905, and, to those who knew him well, the thought 
of his wife’s brave and buoyant comradeship, through times of hesitation and 
disappointment, of success and recognition, and, finally, of stubborn fight 
against ill-health, is inseparable from his memory. His widow, his sisters, 
and his young daughter will have, in their sad loss, the sympathy of all who 
knew him, and especially of that smaller group, who, through years of 
intimacy, had come to know and to prize the steadfast affection, the quiet 
but unwavering loyalty to ideals and convictions, which were outstanding 
features of a fine character. 

babe ela Oe 


XXVii 


AUGUSTUS DESIRE WALLER, 1856-1922. 


Iv was with the greatest surprise and deepest regret that his numerous friends 
heard of the sudden death of Prof. Waller a few weeks ago. He was ill for 
only twelve days; he had a slight stroke, from which his medical attendants 
thought he would fully recover, but other and severer heemorrhages followed 
and he passed quietly away on March 11th. 

He was born on July 12th, 1856, so that at his death he was in his sixty- 
sixth year. He was in full vigour and no one would have expected from his 
energy, both mental and bodily, that the end would come so soon. 

He was the only child of Dr. Augustus Volney Waller, F.R.S., and like 
his father he became famous in physiology. His father’s name in adjectival 
form is familiar throughout the world, and his discovery of what is now 
called “ Wallerian degeneration” stands out as one of the most important 
milestones in physiological history. His son was very jealous of the 
reputation of his father, and I remember one of the few occasions on which I 
have seen him roused to anger was when he thought his father’s work had 
been misrepresented. He dedicated his ‘ Introduction to Human Physiology’ 
to his father’s memory, summarising the latter’s work in the words :— 


Emigration of leucocytes, 1846. 

Degeneration and regenetation of nerve, 1856. 
Cilio-spinal region, 1851. 

Vaso-constrictor action of sympathetic, 1853. 


One of his sons still carries on the physiological tradition, Dr. William 
Waller being one of the junior staff at the University of Liverpool. 

The second Waller, whose loss we have now to deplore, was born in Paris, 
where at the time his father was pursuing his work, and he received his early 
education at the College de Geneve. This early training had considerable 
influence subsequently. He wrote and spoke French fluently, and he usually 
communicated the results of his research work to learned societies in both 
countries. Some of his mannerisms, his expressive and eloquent gestures, 
were doubtless to be traced to the same source. 

In 1870 his father died and he went with his mother to Aberdeen, where, 
after graduating M.B., C.M., in 1878, he finally took his M.D. in 1881. He 
studied also in Edinburgh, but soon migrated to London, and worked at. 
University College under the then Professor of Physiology, Dr. (afterwards 
Sir John) Burdon Sanderson. He received grants from the British Medical 
Association to assist him in his investigations, and in 1884 became Research 
Scholar under the same body. 

His first independent appointment as a teacher was that of Lecturer on 
Physiology at the London School of Medicine for Women, where he met the 
lady who became his wife and life-long companion and helper. He then 


XXVII1 Olituary Notices of Fellows deceased. 


obtained a similar post at St. Mary’s Hospital, and finally, about twenty years 
ago, he was appointed Honorary Director of the Physiological Laboratory at 
the University of London, with the title of Professor. With characteristic 
keenness he had explored the buildings at South Kensington in which the 
University had just been housed, saw the possibilities of using profitably a 
suite of disused rooms, and, with the help of friends, secured them for the 
useful purpose to which, through his efforts, they were ultimately applied. 

Waller’s name became known to the physiologists chiefly through his work 
on the electrical phenomena of the nervous system and of the heart. In this 
work his ingenuity in the devising of experiments and apparatus came to the 
fore. In 1913, he summarised his many years’ work on the heart in the 
Oliver-Sharpey lectures given before the Royal College of Physicians. I 
cannot do better than quote from a letter by Sir Thomas Lewis, F.R.S.,* the 
foremost of present day electro-cardiographers, regarding the value of this 
branch of research. He wrote :— 

“May I add a few words of tribute to the memory of Prof. Waller, whose 
death will be much regretted by both physiologists and physicians in this 
country and in many other lands. He was a man of unusually keen intellect, 
and has been for many years a notable figure in British physiology. His 
brilliant powers of exposition will long render his demonstrations at the 
Physiological Society memorable. His early work on electro-physiology was 
extensive, thorough, and is well known. He was the first to show that the 
currents set up by the beating of the human heart can be recorded; he was 
the first to obtain a human electro-cardiogram; this has been the main 
though by no means his sole contribution to the science of experimental 
medicine. The discovery long preceded the introduction of the string galvano- 
meter, and was the more remarkable in that it was accomplished in the 
eighties.” 

The electrical phenomena in other living structures also attracted his 
attention ; he published numerous papers on the currents found in the retina, 
nerves, muscles, skin, etc., and alsoin plants. They are summarised in his 
book “Sigus of Life from the Electrical Aspect,’ published in 1903. The 
high estimation in which his work was held was shown by his election as 
F.R.S. at the comparatively early age of 36, in 1892. 

During his investigation of nerve and muscle, he made observations on the 
effects of anesthetic vapours and gases on their electrical responses, and thus 
he became interested in clinical anesthesia, and in lectures, demonstrations 
and discussions insisted on the necessity of accurate dosage in the adminis- 
tration of these dangerous means of alleviating suffering, especially in 
reference to chloroform. He invented an apparatus for controlling the 
percentage of the anesthetic in the air a patient breathed, for he was 
convinced that deaths under chloroform could be prevented with proper care. 
In 1901, a Committee was established by the British Medical Association to 
go thoroughly into the matter, and Waller became one of its most earnest 


* ‘Brit. Med. Journ.,’ 1922, vol. i, p. 459. 


Augustus Désiré Waller. Kone Kg 


members, and was at one time its Chairman. The report which was published 
ten years later is a most valuable document, and formed another example of 
how Waller’s academic work was fruitful from the practical standpoint. 

During the last few years of his life, he became interested in three new 
subjects, and threw himself with his usual enthusiasm into all of them. His 
laboratory at one time was, or seemed to be, wholly devoted to one of these ; 
at another time, it was one or other of the remaining two. This was not 
only the case at the South Kensington laboratory, but the same fervour was 
manifested at his private but well known laboratory at his home in Grove 
End Road. The old roomy studio (for the house formerly belonged to a well- 
known artist) was transformed not only into a laboratory, but became the 
principal living room of the professor and his family, where they received 
their friends scientific and otherwise. It opened into a spacious garden which 
also was a great recreation to the workers. The large table in the middle of 
the room was crowded with electrical and other apparatus, when by an 
ingenious arrangement of pulleys, the top was suddenly hoisted ceiling-wards, 
aud a full-sized billiard table was revealed. Waller was as keen on games as he 
was on work, and billiards were not the least of his accomplishments. When 
he first took to driving a motor car, all his energy seemed devoted to mastering 
the intricacies of its mechanism and management. Another of the many 
other interests of his many-sided life was his fondness for animals, and 
especially for bull-dogs. The ancestor of several generations of these was 
Jimmy who became well known as his constant companion in the car and in 
the laboratory. He was the faithful guardian of the car when his master left 
it standing, his fierce countenance being sufficient to repel intruders in spite 
of his gentle nature. Jimmy appeared at several Royal Society Soirées, with 
his paws in basins of salt solution which were connected to a galvanometer 
or electrometer in order to demonstrate the accompanying electrical changes 
of his heart’s activity to an admiring audience. The Home Secretary of that 
day, Mr. Herbert, now Lord Gladstone, had to explain to anti-vivisectionist 
members of the House of Commons that this was not a brutal experiment, 
and that Jimmy suffered as much or as little pain as a child paddling in the 
sea. 

But these are digressions. I had begun to speak of the subjects which 
interested Waller in later years. One of these which only needs a passing 
mention was of a polemical nature, and related to certain movements which 
with the aid of high magnification can be shown to occur in plants. Waller 
attributed these not to growth, but to mere turgescence such as occurs when 
many substances are placed in water. 

The other two topics to which he devoted himself were of a more serious 
nature. One of these was the investigation of the so-called “emotive 
response,” and the other the measurement of the cost of muscular work by 
estimation of the carbonic acid exhaled. 

Both were pursued with characteristic intensity ; his friends inveigled into 
the laboratory had to submit to be put “on the wires” in order that the 


XK Obituary Notices of Fellows deceased. 


change in the resistance of their skin which occurs under various emotions 
might be measured and recorded; one of the many interesting outcomes of 
this work was that most people reacted to the threat of an injury, such as a 
burn, much more strongly than to actual pain. During the air raids he had 
his wife and others “on the wires” and noticed a corresponding effect. The 
investigations on muscular work were carried out on himself, his friends, 
soldiers, colliers, bootmakers, printers and many other classes. He found the 
severity of the work and the output of CO» were parallel. His method has 
been criticised, as no account was taken of the oxygen usage, but Waller 
never claimed absolute accuracy ; he regarded his “short method” for testing 
the cost of work as a practical means to an end which can be accomplished 
only with accuracy by much longer and more complicated methods. It can 
be carried out while the work is in actual progress. It was his intention to 
have compared the two methods in a parallel series of experiments had his life 
been spared, but in criticising his critics showed that their longer methods did 
not always give better results than his own. 

Waller’s contributions to the literature of his subject were numerous, and 
in addition to the books already mentioned, they were mainly published in 
the ‘Proceedings’ and ‘Transactions’ of our Society, and in the ‘Journal of 
Physiology. His academic distinctions were also numerous. In 1889 he 
was made a Lauréat of the Institute of France; he received the Aldini prize 
from the Royal Academy of Science of the Institute of Bologna. He was a 
corresponding member of. many foreign learned societies and academies, and 
an honorary member of the Council of the University of Tomsk. 

Prof. Waller married Alice Mary, daughter of the late Mr. George Palmer, 
M.P. for Reading. It was an ideal union. Mrs. Waller shared in all his. 
work, and he was a devoted husband; her recent illness caused him to 
relinquish his other work; he took a room near the nursing home where she 
was in order to be with her constantly ; later, when she returned home he 
used to carry her to and from her room; his anxiety about her was most 
intense. 

He was an equally devoted father; he had three sons, and two daughters, 
all of whom survive him except the youngest daughter, whose tragic death 
from drowning seemed to leave a permanent mark of sorrow in his character. 
He was no doubt a physiologist first, but in this imperfect survey of his life’s 
work I have endeavoured to show that there were other sides to his 
personality. He passed through many turmoils, of which the last (the 
attempt to close the University laboratory while he was in full vigour 
and it was in full swing of useful work) was by no means the least. He 
won that victory ; he was a good fighter, an ardent and affectionate friend 


and a great man. 
Wi, 1D) 18h. 


INDEX to VOL. XCIIL. (B) 


Address of President at Anniversary, 1921, 1. 

Amphibian metamorphosis and internal secretions (Huxley and Hogben), 36. 
Antiseptic action and chemical constitution (Browning and others), 329. 

Arber (A.) On the Development and Morphology of the Leaves of Palms, 249. 
Asplenium bulbiferum, irritability of fronds of (Prankerd), 143. 


Bacterial variability, studies in (Walker), 54. 

Bacteriolytic element in tissues and secretions (Fleming), 306. 

Bainbridge (F. A.) Obituary notice of, xxiv. 

Balls (W. L.) and Hancock (H. A.) Further Observations on Cell-wall Structure as seen 
in Cotton Hairs, 426. 

Blood-platelets, behaviour in vitamin deficiency and after radiation, etc. (Cramer and 
others), 449. 

Blood sera, absorption spectra and optical rotation of proteins of (Lewis), 178. 

Bose (Sir J. C.) The Dia-heliotropic Attitude of Leaves as determined by Transmitted 
Nervous Excitation, 153. 

Brady (G. 8.) Obituary notice of, xx. 

Bramwell (J. C.) and Hill (A. V.) The Velocity of the Pulse Wave in Man, 298. 

Brown (A.) Obituary notice of, iii. 

Browning (C. H.) and others. Relationships between Antiseptic Action and Chemical 
Constitution, with Special Reference to Compounds of the Pyridine, Quinoline, 
Acridine, and Phenazine Series, 329. 


Campbell (J. A.) See Hill (L.) and others. 

Cell-wall structure in cotton hairs (Balls and Hancock), 426. 

Cholesterol in animal organism ——, autolysis of liver and spleen (Gardner and Fox), 486. 

Ciliary movement, mechanism of (Gray), 104, 122. 

Cohen (J. B.) See Browning and others. 

Cotton hairs, cell-wall structure in (Balls and Hancock), 426. 

Cramer (W.), Drew (A. H.), and Mottram (J. C.) Blood-platelets: their Behaviour in 
“Vitamin A” Deficiency and after “ Radiation,” and their relation to Bacterial 
Infections, 449. 

Currey (G.) The Colouring Matter of Red Roses, 194. 


Depressor nerve of rabbit (Sarkar), 230. 

Devanesen (D. W.) The Development of the Calcareous Parts of the Lantern of Aristotle 
in Hehinus miliaris, 468. 

Dréw (A. H.) See Cramer and others. 

Ducie (Karl of) Obituary notice of, i. 

Echinoderm egg during fertilisation, etc., heat production and oxidation processes of 
(Shearer), 410. 

Echinoderm egg, oxidation during fertilisation (Shearer), 213. 

Echinus miliaris, development of lantern of Aristotle in (Devanesen), 468. 

Fleming (A.) Ona Remarkable Bacteriolytic Element found in Tissues and Secretions, 
306. 

Fox (F. W.) See Gardner and Fox, 

VOL. XCIII.—B, Cc 


XXX 


Gardner (J. A.) and Fox (F. W.) The Origin and Destiny of Cholesterol in the Animal 
Organism. Part XIII.—On the Autolysis of Liver and Spleen, 486. 

Gaunt (R.) See Browning and others. 

Gelatine, titration curve of (Lloyd and Mayes), 69. 

Graviperception of fern fronds (Prankerd), 143. 

Gray (J.) The Mechanism of Ciliary Movement, 104, 122, 

Gulbransen (R.) See Browning and others. 


Hancock (H. A.) See Balls and Hancock. 

Hargood-Ash (D.) See Hill (L.) and others. 

Harris (D. T.) Active Hyperzmia, 384, 

Heald (C. B.) and Tucker (W.S.) Recoil Curves as shown by the Hot-wire Microphone, 
281. 

Heating and cooling of body by local application of heat and cold (Hill and others), 207. 

Hewitt (J. A.) See Pickering and Hewitt. 

Hill (A. V.) See Bramwell and Hill. 

Hill (1.), Hargood-Ash (D.) and Campbell (J. A.) On the Heating and Cooling of the 
Body by Local Application of Heat and Cold, 207. 

Hill (L.), Vernon (H. M.) and Hargood-Ash (D.) The Kata-Thermometer as a Measure 
of Ventilation, 198. 

Hjort (J.) Observations on the Distribution of Fat-soluble Vitamines in Marine Animals 
and Plants, 440. 

Hogben (L. T.) and Winton (F. R.) The Pigmentary Effector System. I.—Reaction of 
Frog’s Melanophores to Pituitary Extracts, 318. 

Hogben (L. T.) See Huxley and Hogben. 

Huxley (J. 8S.) and Hogben (L. T.) Experiments on Amphibian Metamorphosis and 
Pigment Responses in Relation to Internal Secretions, 36. 

Hypereemia, active (Harris), 384. 


Kata-thermometer as measure of ventilation (Hill and others), 198. 


Leaves, dia-heliotropic attitude of, determined by nervous excitation (Bose), 153. 

Lewis (S. J.) The Ultra-violet Absorption Spectra and the Optical Rotation of the 
Proteins of Blood Sera, 178. 

Lipschiitz (A.) On the Hypertrophy of the Interstitial Cells in the Testicle of the 
Guinea-pig under different Experimental Conditions, 132. 

Lloyd (D. J.) and Mayes (C.) The Titration Curve of Gelatine, 69. 


Mayes (C.) See Lloyd and Mayes. 

Miall (L. C.) Obituary notice of, x. 

Microphone, hot-wire, and recoil curves (Heald and Tucker), 281. 
Mottram (J. C.) See Cramer and others. 

Muscle, acidity during maintained contraction (Roaf), 406. 


Obituary Notices :— 


Ducie, Ear] of, i. Brady, G. S., xx. 
Brown, A.., 111. Bainbridge, F. A., xxiv. 
Miall, L. C., x. Waller, A. D., xxvii. 


Ovalbumin and albumin, optical rotatory power of (Young), 15. 


Palms, development and morphology of leaves (Arber), 249. 
Peptone, action on blood and immunity thereto (Pickering and Hewitt), 367. 


XXXIL1 


Pickering (J. W.) and Hewitt (J. A.) The Action of “ Peptone” on Blood and Immunity 
thereto, 367. 

Pigmentary effector system (Hogben and Winton), 318. 

Pituitary extracts, reaction of frog’s melanophores to (Hogben and Winton), :318. 

Ponder (E.) The Hemolytic Action of Sodium Glycocholate, 86. 

Prankerd (T. L.) On the Irritability of the Fronds of Asplenium bulbiferum, with special 
Reference to Graviperception, 143. 

Protein, coagulation of, by sunlight (Young), 235. 

Pulse wave in man, velocity of (Bramwell and Hill), 298. 


Recoil curves shown by hot-wire microphone (Heald and Tucker), 281. 
Roaf (H. E.) The Acidity of Muscle during Maintained Contraction, 406, 
Roses, colouring matter of (Currey), 194. 


Sarkar (B. B.) The Depressor Nerve of the Rabbit, 230. 

Shearer (C.) On the Oxidation Processes of the Echinoderm Egg during Fertilisation, 
213; —— On the Heat Production and Oxidation Processes of the Echinoderm Kee 
during Fertilisation and Early Development, 410. 

Sherrington (Sir C.) Presidential Address, 1921, 1. 

Sodium glycocholate, hemolytic action of (ponder ), 86. 

Stephenson (M.) and Whetham (M. D.) Studies mm the Fat Metabolism of the Timothy 
Grass Bacillus, 262. 


Testicle of Guinea-pig, hypertrophy of interstitial cells under different conditions 
(Lipschiitz), 132. 

Timothy grass bacillus, fat metabolism of (Stephenson and Whetham), 262. 

Tucker (W. 8.) See Heald and Tucker. 


Ventilation, kata-thermometer as measure of (Hill and others), 198. 

Vernon (H. M.) See Hill (L.) and others. 

Vitamines, fat-soluble, distribution in marine animals and plants (Hjort), 440; effect of 
absence on blood-platelets (Cramer and others), 449. 


Walker (KE. W. A.) Studies in Bacterial Variability : On the Occurrence and Develop- 
ment of Dys-, Eu-, and Hyper-agglutinable Forms of Certain Bacteria, 54. 

Waller (A. D.) Obituary notice of, xxvii. 

Whetham (M. D.) See Stephenson and Whetham. 

Winton (F. R.) See Hogben ahd Winton. 


Young (E.G.) On the Optical Rotatory Power of Crystalline Ovalbumin and Serum 
Albumin, 15 ; —— The Coagulation of Protein by Sunlight, 235. 


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ANCE OF THE CAMPBELL TYPE AND SOMES OnY OF IT WITH THE stile ee 


STANDARD. By D. W. Dye, B.Sc.,A.C.G.1.  .. 315 
APPARATUS FOR DETERMINING THE STANDARD "DEVIATION 

MECHANICALLY. By W.L. Batts, M.A., Se. D. (Cantab.) . : 323 
ON THE STRUCTURE OF THE LINE 8G 8 A. OF ‘THE ISOTOPES 

OF LITHIUM. By J. C. McLennan, F.R.S., and D. S. Arnsnin, M.A. (Plate4) .. 342 
INTERFACIAL TENSION AND Ree. TON CONCENTRATION. By 

H. HarrripGeE and R. A. PETERS a 348 


PUBLISHED By Harrison & Sons, Lrp:, 44—47, ST. MaRTin’s Lang, Lonpon, W.C. 2. 


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