wee riaudw * . “ aon a ay ge Oe EN LET mata aly Mh * fe aa “doa® ay 4 4 a een ae LAMA ANN RO sy ee tialte eae aha ae Mh at 4 . "i rn “ Hie | Eee ‘ TW as " ° i LAR e Vert ey b My } wif} ey A A at i uP Ai Johari FN \ SRY 9 Teer iy Me WAS 4g hone Nee eH 4 as FAV EWU DI NR aah MWe (4: i # : as ra : ON t ? y q oy * i ee AY nei Se 4 om Be sts q a He . i M tle Mt uN, st ee i" in i ha fa) ty a Pa re ii Me oe ' ) NA hy ¥} Wk K. ans We i} N re i if wath } ah HWY f co Maur Da fihs hak Lae ite ae oe Mae ies Hace fly peal tare ‘ b ee Hy ae se % y i Day Re y a hin sat y ink 4 HE EN a he Ce Wt: " . 1) A My My iy aS VR OLA Ag " ie " Wh ia a asi hey hid f ‘ i ; she a Hi Pen he: oe Ce Ns i ‘ a tah : , ‘i lh i 1 y ae i mi if A phy ce ioe ge > i as : wi it NW seb ih eSB ae 4h 404 Lan ice nw OM ae SO ea PROCEEDINGS OF THE ROYAL SOCIETY OF LONDON SERIES B CONTAINING PAPERS OF A BIOLOGICAL CHARACTER VOL. LXXX LONDON: PrinteD FOR THE ROYAL SOCIETY anp Sotp By HARRISON AND SONS, ST. MARTIN’S LANE, PRINTERS IN ORDINARY TO HIS MAJESTY. e"Theonlan hs ae mitnsonian Inet. MAR J f) ma % \ UY I9NG a) 40 190g DECEMBER, 1908. LONDON : HEARRISON SONS, PRINTERS IN ORDINARY TO HIS MAJESTY, ST. MARTIN’S LANE. ae oe ae CONTENTS. ——o0F9400— SERIES B. VOL, UXXX. No. B 536.—February 4, 1908. Further Results of the Experimental Treatment of Trypanosomiasis in Rats ; being a Progress Report of a Committee of the Royal Society. By H. G. Plimmer, F.L.S., and J. D. Thomson, M.B., C.M. Communicated by Sir Ray Lankester, K.C.B., F.R.S., Chairman of the Tropical Diseases Committee. (Plate 1) Gee H CBee teseeeereDeFeeeeeetese eee resHFseHFFHFHHSHHHFH HET OCHHETH HSH EO THHHE HHA HOH H HHH HR EH HERE HH RTE OE The Influence of Increased Barometric Pressure on Man. No. 4.—The Relation of Age and Body Weight to Decompression Effects. By Leonard Hill, F.R.S., EMC eenrOCIW OOM, Alley IESE Ce le cgvciswscasdsesteccecacaceecvcesnanVeaaatvenss saesetace On the Distribution of the Different Arteries supplying the Human Brain. By Charles E. Beevor, M.D. Lond., F.R.C.P. Communicated by Professor David Ferrier, F.R.S. (Abstract) Ce ee ee On the Cranial and Facial Characters of the Neandertal Race. By W. J. Sollas, Se.D., LL.D., Professor of Geology in the University of Oxford. (Abstract)... On the Supposed Extracellular Photosynthesis of Carbon Dioxide by Chlorophyll. By Alfred J. Ewart, D.Sc, Ph.D., F.L.S. Communicated by Professor Sees ier ame et MeO E SLES. gh sucaee cee sca ccs vaeneecoshan qr adansunicsonoslesnsadiiesaionevegnstennes On the Occurrence of Post-tetanic Tremor in Several Types of Muscle. By David Fraser Harris, M.D., B.Sc. (Lond.), Lecturer on Physiology and Histology at the University of St. Andrews. Communicated by John G. McKendrick, IABP ANS) ese acea cece ansetenneatst aise sxpalie Seite sblldh sn gon veins vanacecabarata sassy On Reciprocal Innervation of Antagonistic Muscles.. Eleventh Note.—Further Observations on Successive Induction. By C. 8. Sherrington, D.Sc., F.R.S. No. B 537.—March 13, 1908. Address of the President, Lord Rayleigh, O.M., D.C.L., at the Anniversary Meeting on November 30, 1907 ......... ae atari N aioe bCs' iodide axicunnaase deh agaud Seva On the Inheritance of Eye-colour in Man. By C. C. Hurst. Communicated by W. Bateson, F.R.S. eoeeee SbodbbdKKEKHE Hao wBeveeeosnesened boo dbd_abaevoeds babbobecosa CbReeeeeesoneeebseseovene PAGE 30 53 71 85 lV On Some Features in the Hereditary Transmission of the Self-black and the “Trish” Coat Characters in Rats.—Paper IL By Geo. P. Mudge, A.R.C.Sc. Lond, F.Z.S., Lecturer on Biology, London Hospital Medical College (University of London), and the London School of Medicine for Women (University of London). Communicated by A, D. Waller, F.R.S..........0..c006 On the Result of Crossing Round with Wrinkled Peas, with Especial Reference to their Starch-grains. By A. D. Darbishire, Royal College of Science, London. Communicated by J.Bretland Warmer. Wis ncecret sceesec cee cece eee eee eee eee Localisation of Function in the Lemur’s Brain. By F. W. Mott, M.D., F.B.S., and W. D. Halliburton, M.D., FORS. (Plates 9=—4)i i esse ce ceepeenecesene eee ceeenee No. B 588.—April 10, 1908. On the Structure of Sigillaria scutellata, Brongn., and other Eusigillarian Stems, in Comparison with those of other Paleozoic Lycopods. By E. A. Newell Arber, M.A., F.LS., F.G.S., Trinity College, Cambridge ; University Demonstrator in Paleobotany, and Hugh H. Thomas, B.A., formerly Scholar of Downing College, Cambridge. Communicated by D. H. Scott, F.R.S. (Abstract) ......... Dietetics in Tuberculosis : Principles and Economics. By Noel Dean Bardswell, M.D., M.R.C.P., F.R.S. (Edin.), Medical Superintendent, King Edward VII Sanatorium, and John Ellis Chapman, M.R.C.S., L.R.C.P., Medical Super- intendent, Coppin’s Green Sanatorium. Communicated by Sir T. Clifford Allbutt; ReCiBy IRS scacoronsecetecs sen ncive witesic lace nerirs saerevnaate oh astenide eee eae e eee On the Weight of Precipitum obtainable in Precipitin Interactions with Small Weights of Homologous Protein. By Professor D. A. Welsh and Dr. H. G. Chapman. ‘Communicated by Dr.'C: J.oMartin, F.R.S.)) 2.5.00 00.0. sconoeaesns acne Observations upon Phagocytosis carried out by Means of Melanin to Ascertain more particularly whether the Opsonic Index is Identical with the Heemo- phagocytic Index. By 8. G. Shattock and Leonard 8. Dudgeon. Communi- cated by Professor. J. Rose Bradford, PorsSecoR 80%: ivasns-.ccssesenee cease cote A Contribution to the Study of the Mechanism of Respiration, with Especial Reference to the Action of the Vertebral Column and Diaphragm. By J. F. Halls Dally, M.A., M.D. Cantab. Communicated by Professor Sir T. Clifford Allbutty 1K: CB. AEGIS 5.8. dete sees decease dun ahe ins valle sect ein Paee ea senate eek teeta eeeeee No. B 539.—May 14, 1908. The Influence of Temperature on Phagocytosis. By J. C. G. Ledingham, M.B., B.Sc., M.A., Assistant Bacteriologist, Lister Institute, London. Communicated by Dr.’ C. iJ. Martins W. RiSiiiuic wens ohinctscsee sane cee car ebnee saeco eee een ee eee eee Nitrification in Acid Soils. By A. D. Hall, M.A., N. H. J. Miller, Ph.D., and C. T. Gimingham. Communicated by H. E. Armstrong, Ph.D., LL.D., F.RS. The Origin and Destiny of Cholesterol in the Animal Organism. Part I.—On the so-called Hippocoprosterol. By Charles Dorée, Lindley Student of the University of London, and J. A. Gardner, Lecturer in Physiological Chemistry, University of London. Communicated by Dr. A. D. Waller, F.R.S.........0:ce008 PAGE 97 122 136 148 151 161 165 188 196 212 The Origin and Destiny of Cholesterol in the Animal Organism. Part Ij.—The Excretion of Cholesterol by the Dog. By Charles Dorée, Lindley Student of the University of London, and J. A. Gardner, Lecturer on Physiological Chemistry, University of London. Communicated by Dr. A. D. Waller, F.R.S. Bacteria as Agents in the Oxidation of Amorphous Carbon. By M. C. Potter, M.A., F.L.S., Professor of Botany, Armstrong College, in the University of Mirnam.. Communicated by J. B.. Farmer, FRG... ....cccccecceecercesoncnenaqsencnees The Antagonistic Action of Calcium upon the Inhibitory Effect of Magnesium. By S. J. Meltzer and John Auer. Communicated by Professor E. H. Starling, PMT ete any ofolec viendo utocvareleewelcisesiciia calda dueuaaidcame svecijens Sassamnedacsessneuearreniacese Post-tetanic Tremor. (Supplementary Note.) By David Fraser Harris, M.D., MP MCINOMO GOIN)! socnsorastescccacswdsseeacuddecensecensasdtcavessurtsscacseavatiiesccsssnonersncense No. B 540.—June 23, 1908. The Glycogenic Changes in the Placenta and the Foetus of the Pregnant Rabbit: a Contribution to the Chemistry of Growth. By J. Lochhead, M.A., M.D., B.Sc. (Carnegie Scholar), and W. Cramer, Ph.D., D.Sc. (Lecturer on Physiological Chemistry, University of Edinburgh). Communicated by E. A. Schiifer, Pa em anes anreictinti SA satis sands jee ecfeie a hdc eadae co diee tie duet casts duaeate dacderegaauasgess On the Maturation of the Ovum in the Guinea-pig. By J. E. Salvin Moore, A.R.GS., F.L.8., Professor of Experimental and Pathological Cytology, Liverpool, and Miss F. Tozer, B.Sc. London. Communicated by J. Bretland IEP aIMNCIEMR SEU Nao CEH ACCS = A )incis visiaianis nab t,sieloaislewieie's av'utBaaedcigeeawaviek Nas dma ew cunesirnsninne use The Life-history of Vrypanosoma equiperdum. By J. E. Salvin Moore, Professor of Experimental and Pathological Cytology, University of Liverpool, and Anton Breinl, Director of the Runcorn Research Laboratories, Liverpool School of Tropical Medicine. Communicated by Sir Rubert Boyce, F.R.S. (Plates 8 and 9) Pee eserovoeaseeersertereesoseeerectseoeensetasesaseet ese oesseosesseoeeevennaseeeteerasreesasseeeen?e The Alcoholic Ferment of Yeast-juice. Part III.—The Function of Phosphates in the Fermentation of Glucose by Yeast-juice. By Arthur Harden and William John Young (Biochemical Laboratory of the Lister Institate of Preventive iMedieme). Communicated by C. Jt Martin, F.R.S. .1....cccsasdentcccansoserceceesess Studies on Enzyme Action. XJ.—Hydrolysis of Raffinose by Acids and Enzymes. Eoeniatevarmstrons, HOR.S: and,W., H.-Glover, Phi Dii .ccciec.ccscts dustesnannnceres Studies on Enzyme Action. XII.—The Enzymes of Emulsin. By H. E. Arm- strong, F.R.S., BE. F. Armstrong, D.Sc., and E. Horton, B.Sc. ........0.csceeseeeeenee A Further Note on the Nutrition of the Early Embryo: with Special Reference to the Chick. By E. Emrys-Roberts, M.B., Ch.B. Victoria and Liverpool, Sub- curator of the Pathological Museum (Gynecological Section), University of Liverpool. Communicated by Professor C. 8S. Sherrington, F.R.S..........c008e8 : PAGE 227 239 263 288 299 312 32] No. B 541.—August 22, 1908. On Reciprocal Innervation in Vaso-motor Reflexes and the Action of Strychnine and of Chloroform thereon. By W. M. Bayliss, D.Sc., F.R.S. eeceteceresceoceseseectee The Action of Resin and Allied Bodies on a Photographic Plate in the Dark. By William J. Russell, Ph.D., F.R.S. (Plates 10—12) See oeetseonesseeeceecesresseeeeetoeenaees On Some Features in the Hereditary Transmission of the Albino Character and the Black Piebald Coat in Rats.—Paper II. By George Percival Mudge, A.R.C.Se. Lond., Lecturer on Biology at the London Hospital Medical College (University of London) and at the London School of Medicine for Women (University of London). Communicated by Professor A.D. Waller, F.R.S. Have Trypanosomes an Ultra-microscopical Stage in their Life-history? By Colonel David Bruce, C.B., F.R.S., Army Medical Service, and Captain H. R. Bateman, Royal Army Medical Corps CeO ee ose esH SF eesneeeeetooceneseceeseostesesaseeespotes Diphtheria Antitoxin. By John Mellanby, M.D., George Henry Lewes Student. Communicated by Professor J. N. Langley, F.R.S. No. B 542.—September 23, 1908. Croontan Lecture :—The Principles of the Minute Structure of the Nervous System as revealed by Recent Investigations. By Professor Gustaf Retzius, Hor, Mes (RSs tieks gov carszen a sate center se sche cere ce ets seat nae seca: eee 7 et ee ce A Study of the Variations in the Secretion of Hydrochloric Acid in the Gastric Contents of Mice and Rats as compared with the Human Subject, in Cancer. By 8. Monckton Copeman, M.D. Cantab., F.R.S., and H. Wilson Hake, Ph.D., IO) TO Are 1K 02. Sepa AH INANE pr Meena eOm tint Calg CHG EMME AI tin siierceiad so8co07 No. B 543.—November 20, 1908. The Giant Nerve Cells and Fibres of Halla parthenopeca. By J. H. Ashworth, D.Sc., Lecturer in Invertebrate Zoology in the University of Edinburgh. Communicated by Professor J. C. Ewart, M.D., F.R.S. (Abstract) @Coeeceseecseecsen Preliminary Account of the Habits and Structure of the Anaspidide, with Remarks on some other Fresh-water Crustacea from Tasmania. By Geoffrey W. Smith, M.A., Fellow of New College, Oxford. Communicated by Professor E. B. Poulton, F.R.S. (Plate 13) ee Some Experiments made to Test the Action of Extract of Adrenal Cortex. By S. G. Shattock and C. G. Seligmann. Communicated by John Rose Bradford, Bor? See: RS! asaiaase ere Meena ce nt ane aiatley alk ae Reena eee sae eee are Further Results of the Experimental Treatment of Trypanosomiasis: being a Progress Report to a Committee of the Royal Society. By H. G. Plimmer, F.LS., and H. R. Bateman, Captain R.A.M.C. Communicated by J. Rose Bradford; Mi, URS ois aceeeal sient hs s@lh ed ectda s WR ee tients Malt Rete Chl aa eet fe ree eee Complete Survey of the Cell Lamination of the Cerebral Cortex of the Lemur. By Dr. F. W. Mott, M.D., F.R.S., and Agnes M. Kelley. (Plates 14—18) eeereereeres 399 414 444 463 465 473 477 Vil PAGE An Investigation on the Anatomical Structure and Relationships of the Labyrinth in the Reptile, the Bird, and the Mammal. By Albert A. Gray, M.D., F.R.S.E. Communicated by John G. McKendrick, M.D., F.R.S. (Plates 19 and 20)...... 507 No. B 544.—-December 31, 1908. The Natural Mechanism for evoking the Chemical Secretion of the Stomach (Preliminary Communication). By J. 8. Edkins and M. Tweedy. Communi- eaceney lroressor HW; A. Starling, F.R.S 0... cccccesctereewscedecsccsovessusesedeeteceseniees 529 Further Observations on Welwitschia. By H. H. W. Pearson, Sc.D., F.L.S. Com- municaved by Professor A. C. Seward, F.R.S. (Abstract) ...........c.ecscenecenetes 530 On the Presence of Hem-agglutinins, Hem-opsonins, and Heemo-lysins in the Blood obtained from Infectious and Non-Infectious Diseases in Man. (Preliminary Report.) By Leonard S. Dudgeon, F.R.C.P. Lond. Communicated by Pro- Resecatmetion Cr E31 OCLC). Misys evame pn am eee teas sic eien's atve Scieid died ovis Seis cteiseisinwe sewlaca wnnenecsditie vee 531 Preliminary Note on the Occurrence of a New Variety of Trypanosomiasis on the Island of Zanzibar. By Alexander Edington, M.D. Edin., D.P.H. Edin. and Glasgow, F.R.S. Edin. Communicated by J. Rose Bradford, For. Sec. B.S. 545 On Methods for the Continuous (Photographic) and the (uasi-Continuous Registra- tion of the Diurnal Curve of the Temperature of the Animal Body. By Arthur Gamgee, M.D. F.R.C.P., F.R.S., Hon. LL.D. Edin. ; Emeritus Pro- fessor of Physiology in the Owens College, University of Manchester. Reo ie irl Maree ar is ce eh ccc occa dso eof lecicic ate Vow aciseuiga tae tbyelee suacdee obey 550 On Reciprocal Innervation of Antagonistic Muscles. Twelfth Note.—Propriocep- une svehlemes: ) by Cs. Sherrmegton, WiS¢.5 BRS) iicciccis ss cecasedtseasecestesenss 552 Reciprocal Innervation of Antagonistic Muscles. Thirteenth Note.—On the Antagonism between Reflex Inhibition and Reflex Excitation. By C. 8. Be COU SC e 5k GEMS a acc aie'e Si cayjoesiogjr eee siesaaesias sce se cabesiebinges sped beads sawaeidgee'y 565 OBEEUARY NOTICES’OF FELLOWS DECEASED «0.00.0... .ccecccnscsreceeceseed ill RN MEP orice cis nfe oe sos sfe bereits seaie te ctivig uaa da'c ve Mee seis e bales dela len cOpalsantnoleislaite ve Ixxxv te Vote ome ' rg VAY a Myre Sd ot OBITUARY NOTICES OF FELLOWS DECEASED. VOL, LXXX.——B. CONTENTS. PAGE Sir DirtricH BRANDIS... oe Bs Paces oe Be a lil Sir Witit1AM Henry BroapsBent, Bart., K.C.V.O. ... ae a. a val WILLIAM TENNANT GAIRDNER Oa mae ee oy ah ses Ki RoBERT WARINGTON ii iis ee ee ae ae Re eS WALTER FRANK RAPHAEL WELDON Be ney ae ae’ ae xe ll SIR DIETRICH BRANDIS, 1824—1907. By the death of Sir Dietrich Brandis, which occurred at Bonn, on May 28, 1907, a man of world-wide renown has been removed. He was born on April 1, 1824, at Bonn, being the son of Dr. Christian Brandis, Professor of Philosophy in the University of that place. As a boy, he followed his father to Greece, where he spent several years, and thus came into touch at an early age with the life and customs of the nearer East. On his return from Greece he was educated at the Universities of Copenhagen, Gottingen, and Bonn. In 1849 he established himself as “ Privatdocent” on Botany at Bonn. While he thus started life as a Botanist, during his Botanical excursions his attention was soon turned to questions connected with the management of forests. After the occupation of the Province of Pegu, in Burma, Lord Dalhousie offered Brandis the appointment of Superintendent of the Pegu teak forests, an offer which he accepted. He landed in India in 1856, and a year afterwards the rest of the Burma forests was placed under his charge. Brandis proceeded from Calcutta, where he had an interview with Lord Dalhousie, to Rangoon in a separate vessel from that which conveyed his herbarium and botanical library. The latter was wrecked in the Rangoon River, and Brandis thus lost his herbarium and books. He looked upon this almost as a direction to his future line of action. While he never quite abandoned botanical studies, he devoted for years his main energies to. mastering the science and practice of Forestry. It was only thirteen years later that he resumed earnest botanical studies for the space of three or four years, and he then again returned to Forestry for a further period of nine years. After his retirement he occupied himself once more chiefly with botanical work. From 1856 to 1862 Brandis worked indefatigably to bring the forests of Burma under: systematic management. During these years a great conflict raged between the merchants of Burma and the Government, the former maintaining that the supply of teak timber from the forests was inexhaustible, and that, therefore, State interference was unnecessary. Brandis, supported by the Commissioner of Pegu, Major (afterwards Sir Arthur) Phayre, held different views. After a long continued struggle the forests were placed under systematic management, and they, with those of Upper Burma, are now the chief supply of teak timber to the world. If Brandis had done nothing else than.save the Burma teak forests from destruction, he would have deserved well of the British Empire and the world in general. In 1862, he was called to Simla at the suggestion, it is believed, of Dr. Cleghorn, one of the principal pioneers of forest conservancy in India, to advise the Government on forest matters in other parts of the country, and in 1864, he was appointed the first Inspector-General of Forests to the 7 b.2 lv Obituary Notices of Fellows deceased. Government of India. He then set to work to introduce systematic forest management on scientific lines throughout India. A regular department was created, and a forest law was passed which provided for the demarcation and management of the State forests. Brandis travelled from one end of the Bengal Presidency to the other, advising and organising the department. He also visited the Bombay Presidency twice, and he spent two years {1881—83) in Madras on special duty. When he first started operations, he had to do with what staff he could lay his hands on, but he determined to obtain one fit to deal with the requirements of the case. There were already a few military officers in the Department, some of them medical men, and he began by inducing others to join. Some of these did excellent service, and they gave tone to the new Department. In 1866, while on sick leave in England, he obtained the sanction of the late Marquis of Salisbury, the Secretary of State for India, to educate young Englishmen in Continental forest schools, partly in France and partly in Germany. Under this system of training, which lasted until 1886, a number of distinguished Forest officers were supplied to India. About the year 1881, a movement was set on foot to arrange for the education of Indian forest officers in Britain, and this led, in 1885, to the establishment of a School of Forestry in connection with the Royal Indian Engineering College at Coopers Hill. Brandis, who had then retired, was, of course, consulted about this move, and he did not approve of it, considering it to be premature. After some years, however, he agreed to superintend the practical training of the students on the Continent, from 1888 to 1896, when his functions ceased. On the closure of Coopers Hill, the forest branch was, in 1905, transferred to the University of Oxford. But Brandis went a step further. In 1878 he started a forest school at Dehra Dun for the training of natives of India, which now sends annually from forty to fifty trained executive officers into the Service. By these combined means a trained staff of 200 Englishmen and more than 1,000 Indians has been obtained which, assisted by about 10,000 subordinates, manages the Indian State forests, which comprise an area of 239,000 square miles, equal to one-fourth of the area of British India. The results of Brandis’ work in India are very remarkable. The supply of timber, firewood, and a variety of other produce to the teeming millions of India has been placed on a satisfactory footing; the productiveness of the forests 1s increasing year by year; the more important areas are protected against jungle fires, and the net revenue from the forests has risen from £40,000 in 1864 to £660,000 in 1904, although produce valued at a similar sum is given annually free of charge to the people of the country. Brandis was equally interested in the indirect effects of forest vegetation. He started experimental stations at Dehra Dun and in Central India, where observations were made to test the effects of forests on temperature, humidity of the air and the soil, and the preservation of mountain slopes. His interest in the subject is testified by the fact that he was the first to Sir Dietrich Brandis. Vv compile a rainfall map of India, in 1871. It has been improved by subsequent observations, but as regards the main points it holds good to this day. The map served to show clearly the relation between the rainfall and forest vegetation in the several parts of India. Brandis practically relinquished the post of Inspector-General of Forests in India in 1881, when he proceeded on special duty to Madras. He retired finally in 1883. On that occasion the Government of India acknowledged his services in the most complimentary terms, granting him not only a special pension, but a substantial gratuity in recognition of his specially meritorious services. He had been created a Companion of the Indian Empire in 1878, and he was promoted to a Knight Commandership in 1887 for his services in India, as soon as that grade was added to the Order. Apart from British India proper, Brandis did all he could to encourage better forest management in the Native States. He had a considerable share in the development of systematic forest management in many of the Colonies by advising the Government of India to lend competent officers for service in various parts of the Empire, and by advice. After his retirement from India he continued to show uninterrupted interest in his great work by articles published in the ‘Indian Forester, and by letters of advice to his numerous friends in India. As already mentioned, Brandis supervised the practical instruction on the Continent of the Coopers Hill Forest Students from 1888 to 1896. During that time, and afterwards, he also guided the studies of a number of young Americans, who have since established a great Forest Department in the United States dealing with State forests covering more than one hundred millions of acres. His influence in this respect has been so great that President Roosevelt, about a year ago, sent his picture to him with the following inscription: “To Sir Dietrich Brandis, in high appreciation of his services to forestry in the United States. From Theodore Roosevelt.” Thus Brandis has left his mark upon every continent of the earth. As regards this country, his name will go down to posterity as the founder of systematic forest management in the British Empire. Brandis was not only a great administrator, but also a scientific man of a high order. During his career in India he wrote an endless number of reports and papers, and in 1872—74 he interrupted his forest work by writing the “ Forest Flora of North-west and Central India,” a work so highly thought of that he was elected a Fellow of the Royal Society in 1875. The last eight years of his life were devoted by him to the writing of a general Indian forest flora, which he published in 1906 under the title of “Indian Trees,” a monumental work which is likely to be the standard book of reference on the subject for another generation. Of other publications, the following may be mentioned :— 1. “ Vegetation and Country from Narkanda to Pangi.” Simla, 1879. 2. “The Ringal of the North-western Himalaya.” 1885. 3. “ Die Nadelholzer Indiens.” 1886. v1 Obituary Notices of Fellows deceased. 4. “Wall Pictures to Illustrate the Minute Structure of Plants.” Simla, 1886 and 1887. 5. “Three Families of Plants in Engler and Prantl’s ‘Die natiirlichen Pflanzenfamilien.’” 1894. . 6. “Dipterocarpacee.” Brandis and E. Gilg, 1894. 7. “Geographische Verbreitung der Dipterocarpaceen.” 1897. 8. “Ueber die geographische Verbreitung der Bambusen in Ostindien.” 1896 and 1897. 9. “ Biological Notes on Indian Bamboos.” 1899, 10. “Anbau der grossen Bambusen in Deutsch Ostafrika.” 1899. 11. “On Some Martaban Bamboos.” 1906. | 12. “Remarks on the Structure of Bamboo Leaves,” in the ‘ Transactions of the Linnean Society.’ 1906. 13. “The Spruce of Sikkim and the Chumbi Valley.” 1906. Brandis was a Fellow of the Royal Society, the Linnean Society, and the Royal Geographical Society, and LL.D. of the University of Edinburgh ; an Honorary Member of the Royal Scottish Arboricultural Society, the Society of American Foresters, and of the Pharmaceutical Society of Great Britain. He had scarcely completed his great work, “ Indian Trees,” when he fell ill. After a painful illness of several months’ duration he died at the age of 83 years, thus bringing to a close a most remarkable life filled with work from beginning to end, which only his iron constitution enabled him to achieve. He never spared himself, and he was always a warm friend of those associated with him in his work. For the natives of India he was full of sympathy, and he did all he could to advance their education and fit them to partake in the administration of the country, and more particularly of the Forest Department. He married, in 1854, Rachel Shepherd, a daughter of Dr. Marshman, of Bengal. She accompanied him to India, where she died in 1863. In 1867 he married Katherine, daughter of Dr. Rudolph Hasse, of Bonn, who survives him. He left three sons and one daughter. AV. 8. Vi SIR WILLIAM HENRY BROADBENT, Barr., K.C.V.O., 1835—1907. Sm Wittiam Henry BrRoaDBENT, born in 1835, was the son of John Broadbent, of Longwood Edge, Huddersfield. He married, in 1863, Eliza, daughter of Mr. John Harpin, of Birks House, Holmfirth, Yorkshire, by whom he had two sons and three daughters. His two sons, Dr. John Francis Harpin Broadbent, who succeeds his father in the baronetcy, and Dr. Walter Broadbent, are both members of the medical profession. Broadbent had a distinguished career as a student of medicine and after- wards as a great physician. He took a distinguished place in the medical world not only as a practitioner, but as an original investigator of difficult medical questions, and of physiological subjects bearing on the science and practice of medicine. He had the originality of a man who thought for himself; originality shown in finding out new things and not merely in developing a little further the discoveries of others. His professional success was great and well deserved. In 1892 he was appointed physician-in-ordinary to H.R.H. the Prince of Wales, now King Edward VII. Next year a baronetcy was given him, and in 1901 he was made a Knight Commander of the Victorian Order. Nor were honours of this kind limited to those conferred in this country; he was invested with the Grand Cross and Insignia of the Legion of Honour. In 1898 he was appointed physician-extraordinary to Queen Victoria, and, later, physician-in-ordinary to King Edward. Besides these honours he received numerous academic distinctions, including the honorary LL.D. of the Universities of Edinburgh, St. Andrews, and Toronto, and the honorary D.Sc. of the University of Leeds. He was elected a Fellow of the Royal Society in the year 1897. That Broadbent was a man of high intellectual power and of great nobility of character is well known to his friends, and is evidenced by his career and also by the nature of the original medical and physiological work he did. His contributions to medical literature cover a wide and varied field. He was deeply interested in the study of the problems of diseases of the nervous system. One of his earliest publications dealing with this subject was an important paper entitled “The Sensori-Motor Ganglia and Association of Nerve Nuclei,’ in which he enunciated his memorable “hypothesis ” explaining the immunity from paralysis of bilaterally associated muscles in hemiplegia. In 1869 he published a paper on “ The Structure of the Cerebral Hemisphere,” in which he gave a description of the course of the various groups of nerve fibres in the cerebral hemisphere, based on a series of careful dissections which he had been carrying out for some years. In a lecture on “The Theory of Construction of the Nervous System,” delivered at Wakefield in 1876, he referred to these dissections, and gave v1 Obituary Notices of Fellows deceased. a lucid exposition of his views on the mechanism of speech and thought, and the problems of aphasia, a subject in which his interest was maintained throughout his life. To this his papers on “A Case of Amnesia,’ on “A Particular Form of Amnesia,” “Loss of Nouns,” read before the Royal Medical and Chirurgical Society in 1878 and 1884 respectively, and an article in the ‘British Medical Journal’ as late as June 15 this year, on “Some Affections of Speech,’ bear witness. Indeed, at the time of his death he was engaged in writing a treatise on “ Aphasia.” Amongst other publications dealing with diseases of the nervous system, were “ Remarks on the Pathology of Chorea,” published in the ‘ British Medical Journal’ in 1869, the Lettsomian Lecture on “Syphilitic Affections of the Nervous System,” delivered before the Medical Society of London in 1874, and papers on “Ingravescent Apoplexy” and on “ Alcoholic Spinal Paralysis,” read before the Royal Medical and Chirurgical Society in 1874 and 1884. In 1866, the year in which he published his “ Hypothesis,” he contributed an article on “ Prognosis in Heart Disease” to the ‘ British Medical Journal.’ In 1884, before the Harveian Society of London, he gave the Harveian Lectures on “ Prognosis in Valvular Disease of the Heart.” In 1887, at the Royal College of Physicians, he delivered the Croonian Lectures on the “ Pulse,” and in 1891 the Lumleian Lectures on “Structural Diseases of the Heart from the Point of View of Prognosis.” These lectures constituted the foundation of a work on “Heart Disease,” published in 1896, in the preparation of which he was assisted by his son, Dr. John Broadbent, and of which a revised and enlarged edition appeared in 1906. His early interest in the scientific application of therapeutics was shown by a paper entitled, “An Attempt to Apply Chemical Principles in Explana- tion of the Action of Remedies and Poisons,” published in 1869 ; and the line of thought in this was followed out in later years in an address on “The Remote Effects of Remedies,’ read at the annual meeting of the British Medical Association in 1886, and in his Presidential Address delivered before the Clinical Society on “The Relation of Pathology and Therapeutics to Clinical Medicine.” Amongst other notable contributions to the literature on therapeutics is the Cavendish Lecture on “Some Points in the Treatment of Typhoid Fever,” which he delivered before the West London Medico- Chirurgical Society in 1894. Sir Thomas Barlow has observed that, in a remarkable paper submitted several years ago to the Royal Medical and Chirurgical Society, but not published in its ‘Transactions, Broadbent anticipated the development of pharmacology on the lines of chemical affinities of the elements. Broadbent has left a record of splendid work done in the advancement of neurology. The value of what he did towards the elucidation of different problems presented by cases of aphasia is universally acknowledged. This is not the place to give details of any of his researches. I shall limit my remarks to the wide bearings of a great principle he established, to what is known as “ Broadbent’s hypothesis.” This principle has brought method into Sir Wilhkam Henry Broadbent. 1X the analysis of complex symptomatologies of some very different nervous maladies. More than forty years ago (‘British and Foreign Med. Chir. Review,’ April, 1866), he published an article on “the Bilateral Association of Nerve Nuclei to the Higher Centres.” These words, his latest but not his best, deliverance on the subject, are from a lecture he delivered before the Neurological Society, published in ‘ Brain, No. 103, p. 347, 1903. In that lecture he remarked that: “The principle is that when muscles on the two sides of the body always act together, their nuclei, situate in opposite sides of the cord, are so closely associated by commissural fibres as to be practically one nucleus.” Whatever modifications and corrections of details have been required, what is essential in the principle which the hypothesis embodies has not been invalidated. Thus, in a case of ordinary hemiplegia—say right—owing to disease of the left half of the brain there is loss of power of the right limbs, that is, of those parts of the body which are most nearly unilateral, and, as we may say, “most voluntary,” in their actions; but there is no, or very little, disability of the intercostal muscles of either side of the chest. Nevertheless, there is loss of many movements of the intercostal muscles of both sides, that is to say, there is paralysis i the sense of loss of movements of the intercostal muscles of both sides, but without disability of these muscles. The seeming paradox in this statement disappears when we reflect that, as Broadbent told us Iceng ago, in such a case the intercostal muscles of both sides of the chest remain represented in another set of movements in, and are still empowered by, the undamaged right half of the brain; there is almost complete compensation by the right half for the effects of the destruction lesion in the left half. The truth of this hypothesis is demonstrated in two ways. In some cases of what may here be called Rolandic epilepsy (it is sometimes called “ cortical epilepsy ”) there is from a local lesion of the motor region of the cortex cerebri of one half—say, left—of the brain occasionally convulsion of the limbs of the right side of the body and of both sides of the chest. In the facts of hemiplegia contrasted with those of the case of Rolandic epilepsy there is a striking verification of Broadbent’s hypothesis. From a destruction lesion of part of one half of the brain there is no, or very little, obvious disability of the intercostal muscles of either side, whilst from a discharge lesion of a part of the cortex (a part belonging to the same anatomico-physiological system as that part which is the seat of a destruction lesion in hemiplegia) there is sreat spasm of the intercostal muscles of both sides. Another, a third, confirmation of the hypothesis is given by so-called pseudo-bulbar paralysis in cases of this malady there is a double cerebral destruction lesion, causing great disability of bilaterally acting muscles of a certain region of the body—of both sides of the tongue, lips, and palate. A destruction lesion of the left half of the brain only causes very slight, almost no, disability of the bilaterally acting parts mentioned, compeusation being for the effects of that one-sided lesion practically complete. But when that compensation is lost from a lesion of the right half also, there is very great disability of the x Obituary Notices of Fellows deceased. bilaterally acting parts mentioned. Speaking more at large, it follows from Broadbent’s hypothesis that “double hemiplegia” is more than the double of hemiplegia. So much for three applications of the hypothesis. There is another one of great importance to which allusion may be made. The hypothesis “leads (‘ Brain,’ op. cit.) to the conclusion that words are represented in the right as well as in the left hemisphere.” (Broadbent, ‘ Brain,’ loc. cit.) From what has here been said, it will be seen how fundamental and of what wide application Broadbent’s hypothesis is. This basic contribution to neurology has lasted forty years, and is still not only valuable for the explanation of certain neural symptomatologies, but is also fruitful in its indications for further research in medical neurology. Moreover, the writer thinks that it and deductions or inferences from it, will be found of great value in the study of still larger problems than those here dealt with, such as investigations into the physiology of the organism, when that physiology is considered as especially corresponding to psychology, both to the psychology of the sane and of the insane. J. H. J. Nore.—Much of this Obituary is taken from an article, part of which was contributed by the author, in the ‘ British Medical Journal,’ July 20, 1907. Xl. WILLIAM TENNANT GAIRDNER, 1824—1907. WILLIAM TENNANT GAIRDNER was born in Edinburgh on November 8, 1824 and he died in the same city on June 28, 1907. Descended from an Ayrshire stock, he was the son of Dr. John Gairdner, who was for many years a well known medical practitioner in Edinburgh. His mother was Susanna Tennant, a granddaughter of the Rev. Dr. Dalrymple, of Ayr, the “Dalrymple mild” of Robert Burns. Gairdner was educated at the University of Edinburgh, where he graduated in 1845. The teachers who appear to have influenced him most were William Alison, the physiologist, and Robert Christison, the most distinguished pharmacologist of his day. After a short sojourn in Italy, in the company of Lord and Lady Beverley (afterwards the Duke and Duchess of Northumberland), he returned to Edinburgh, was appointed one of the resident assistants in the Royal Infirmary, pathologist to the Infirmary in 1848, obtained wards in 1853, and “became an extra-mural lecturer on Practice of Medicine in the same year. He was elected to the chair of Practice of Medicine in the University of Glasgow in 1862, and this office he held till his retirement in 1902. In 1863, Gairdner was appointed the first “ Medical Officer of Health” for Glasgow, a position he held for nine years. He was President of the British Medical Association in 1888, became a Fellow of the Royal Society in 1893, and, in 1898, Queen Victoria honoured him by creating him a Knight Commander of the Bath. He also represented his University on the General Medical Council from 1893 to 1902. He was President of the College of Physicians of Edinburgh during the years 1893-5. In 1870 he married Miss Helen Bridget Wright, of Norwich, who, with several sons and daughters, survives her husband. In his long and varied career Gairdner engaged in many kinds of work, and the record of his life must, therefore, present various aspects, according as we view him as pathologist, clinical physician, sanitarian, and man of letters. In the early Edinburgh days, while Pathologist to the Royal Infirmary, he devoted special attention to the pathology of the kidney, and, in particular, gave an early description of the waxy kidney. About 1850, he investigated the pathological changes in bronchitis, and more particularly diseases of the lung associated with bronchial obstruction. He enunciated a theory of emphysema, accounting for the changes in the air cells of the lung in that condition by the force of the inspiratory instead of the expiratory act. This theory has not been generally adopted. But Gairdner’s contributions to pathology were not so much in the form of specific investiga- tions as in the general trend of his clinical work. Every case that came before him was subjected to the most rigid scrutiny, not merely in its clinical aspects, but in the verification and correction of these by the facts of the post-mortem theatre. In the case books of the Royal Infirmary of xl Obituary Notices of Fellows deceased. Edinburgh and of the Western Infirmary of Glasgow, there are painstaking records of the most valuable description, monuments of industry, insight, and method. As a clinical physician he belonged to the first rank of workers. In the old days this part of investigation, especially as regards the teaching of students, was carried on in a very loose fashion. The physician often passed from bed to bed in the wards, followed by a crowd of students, who were left to gather wisdom and knowledge as they could from the oracular utterances of the wise man. There was no systematic investigation of cases nor any attempt at methodical teaching of the student. The first great improve- ments were no doubt initiated by John Hughes Bennett, who, fresh from the cliniques of the Parisian hospitals, was the first in this country*to teach the student at the bed side, by causing him to observe the facts of the case, and to discuss, with the teacher, all its features. Gairdner readily took up the same course of Peocedure. More thorough and philosophical than Bennett, he went more deeply into the case, and worked out what may be termed its natural history. He disliked clinical pictures. He disliked the practice of drawing up an imaginary schedule for each disease with a space for each sign and symptom, which had to be filled in whether it happened to be in the case under discussion or not. His method was rather to study each individual case and to closely scrutinise each symptom, to note those belonging to the typical form of the disease that were absent in this particular case, and to draw inferences with care and precision. It was the scientific method that impressed the student and trained him to be an observer. Gairdner always attracted to him the better type of men, and many of his pupils became, in their turn, able pathologists and physicians. In practical therapeutics he also did valuable work. He was one of the first to check the over-stimulating mode of treatment in fevers and pneumonia, and when he did use medicinal substances they were employed in a simple form. He never wrote a “ grape and canister” prescription containing half a dozen ingredients. He had no great faith in drugs, nor in specially vaunted remedies, or modes of treatment. There must be, in his view, a good reason for the employment of a particular remedy and he was not guided by empirical considerations, except to a very limited extent. As a consultant, therefore, he was often somewhat disappointing, as he was more interested apparently in the clinical history of the case and in its pathology than in the treatment. He had a strong view that in many cases natural processes would remove the disease, while, in others, so-called remedies were of little avail. One of Gairdner’s chief claims to distinction is the splendid work he did as the first medical officer of health of Glasgow. During the first half of the nineteenth century, and closely connected with the industrial revolution that followed the introduction of machinery and the factory system, many large towns rapidly increased in population and unsanitary conditions of life were met with everywhere. In no city was this more conspicuous than in William Tennant Gardner. X11 Glasgow. In 1801 the population was 83,805; in 1811, it had risen to 110,400; in 1821, to 147,043; in 1831, to 202,426; in 1841, to 280,692; and in 1851, to 347,001. In 1818, there was a severe epidemic of typhus; in 1832, this was repeated, with, in addition, an epidemic of cholera, and a death-rate of 46 per 1000. In 1837, there was another typhus epidemic ; 1843 brought an epidemic of relapsing fever; 1847 had a typhus epidemic with a death-rate of 56 per 1000; 1848-9 was visited by a second cholera epidemic, in which 3772 deaths occurred; 1851-2 showed more typhus ; and 1853-4 had a third cholera epidemic with 3885 deaths, and a death- rate of 42 per 1000. It is now difficult to realise exactly what those figures mean, and yet this must be attempted if we wish to understand the real state of matters. Thus,in 1837, the population of Glasgow was 253,000, the death rate was 41 per 1000, and the number of deaths from “ fever” was 2180, or 8°6 per 1000. At least 21,800 persons suffered from the disease during that year. In 1847, the number of persons affected by fever must have been about 45,000. If we wish to contrast this with the state of matters in Glasgow at the beginning of the present century, we may take 1901, said to be the worst epidemic year of recent years.* In 1901, the population of Glasgow was 761,712; the general death-rate was 20°6; the number of deaths from fever was 220; the number of cases of fever notifiable, 1385; and the number of deaths from all infectious diseases 3416, or 44 per 1000 living. The total number of cases of infectious diseases registered during the year was 21,145, or less than the number believed to have suffered from “typhus” fever alone in 1837. The cause of the terrible state of matters that prevailed up to the passing of an important municipal act in 1862 was undoubtedly due to overcrowding in insanitary dwellings, to the entire absence in the poorer parts of the city of even the most obvious sanitary apphances, and to the want of hospitals for the segregation of the sick during an epidemic. In January of 1862, (Gairdner was chosen to be the first medical officer of health, and five district surgeons of police were appointed his assistants. At the same time, a “special non-medical inspector ” represented the entire sanitary staff. The first sanitary office was a room measuring fifteen feet by ten feet. These rudiments of a sanitary department soon developed. JDisinfecting and washing houses were established, nuisances were removed, careful inspection was made of specially insanitary districts, committees were formed for special purposes, intra-mural burial grounds were closed, and a hospital for fever was founded at Belvidere, and became in course of time one of the finest fever hospitals in the country. Year by year the sanitary machinery was improved and in particular the danger of overcrowding was combatted * These figures have been obtained from a paper from the late Dr. J. B. Russell, entitled, ‘The Evolution of Sanitary Administration in Glasgow.’ The facts regarding 1901 are given by the present medical officer of health of Glasgow, Dr. A. K. Chalmers, in a footnote to Dr. Russell’s paper, as it appears in a volume entitled, ‘Public Health Administration in Glasgow,’ a memorial volume of the writings of Dr. J. B. Russell, who succeeded Gairdner as medical officer of health. See pp. 1, 4, ete. XIV Obituary Notices of Fellows deceased. by means of “ticketing” houses, that is, specifying on the door of each room the number of inhabitants who were permitted to dwell init. During this period Gairdner wrote many important papers, on sanitary questions, on hospital management, on the training of nurses, on dietetics both as regards the healthy and the sick, and on kindred subjects. In 1871, he resigned the oftice of medical officer of health into the able hands of James B. Russell, one of his own most distinguished pupils, who carried on for many years the work of the Sanitary Department in Glasgow, which is now one of the most complete in the world. Its initiation owed much to Gairdner and he had the satisfaction of watching its progress. Gairdner in many directions showed marked literary gifts. He was a good classical scholar, of a type not common now-a-days in.the medical profession, and he read Virgil and Horace and the New Testament in Greek, as one of his almost daily pastimes. Now and again he published, in addition to works bearing specially on medicine, isolated lectures and essays, such as ‘The Physician as Naturalist, a well-known volume which is a key to his character. Always animated by a thoroughly scientific spirit, he took a wide view of the functions of a physician, as one whose duty it was to investigate the natural phenomena of disease, and uphold the dignity of his calling. In his writings, as in his conversation, one always felt conscious of his lofty ideals and of his transparent sincerity and honesty of purpose. It would not be right not to advert to another marked characteristic. He was a man of sincere piety who ever lived in his daily life under the shadow of the Unseen. Broad and catholic in his religious views, there was always present the spirit of reverence, a generous appreciation of the views and claims of others, a desire to view every question fairly and impersonally, and a high ideal of the nobility of life. J. G. McK. xV ROBERT WARINGTON, 1838—1907. THE name of Robert Warington will ever be associated with one of the most important advances in the agricultural chemistry of the latter half of the nineteenth century, though his classical work on nitrification, which may be regarded as his life-work, bears but a small proportion to the total of that accomplished by him. He, no doubt, owed his chemical proclivities to his father—a Robert Warington also—who was a prominent figure amongst the chemists of earlier days. The elder Warington was one of the first chemical assistants at University College, and was subsequently appointed chemical operator to the Society of Apothecaries. He, also, was a Fellow of the Royal Society, and published several papers on chemical subjects; yet chemistry is more indebted to him for the part which he took in founding the Chemical Society than for the extent of his own original work. It was through his zeal and powers of organisation that this Society was founded in 1841, and the work which he did for it as its secretary during the subsequent ten years helped in no small measure to launch it on its prosperous career. Robert, his eldest son, was born on August 22, 1838, in the parish of Spitalfields. His mother was a daughter of George Jackson, M.R.C.S., to whom science is indebted for several improvements in microscopes which have not yet been superseded, as well as for the invaluable ruled glass micrometer. ‘The original dividing machine made by him for ruling the lines was still being used by a well known optician in 1899, and is probably in use at the present time. Very early in young Warington’s life his parents took up their residence at the Apothecaries’ Hall, and it was here, in the uncongenial atmosphere of the city, that he spent his childhood and youth. His constitution was. naturally feeble, and a life in the heart of London, with but little exercise, and no companions of his own age to assort with, did not tend to strengthen it. All through life he had to contend with a lack of bodily vigour, which rendered his work doubly laborious to him. For his education he seems to. have been chiefly indebted to his parents. While still quite young, he studied chemistry in his father’s laboratory, and had the advantage of attending lectures by Faraday, Brande, and Hofmann. In consequence of the unsatisfactory state of young Warington’s health, his father sought to get him’some employment in the country, and, with that object in view, applied to Mr. Lawes, with whom he was acquainted, and for whom he had done some professional work. The outcome of this was. that in January, 1859, the youth went to work in the Rothamsted Laboratory as Lawes’ unpaid assistant. Here he remained for one year, devoting all his time to ash analyses, of which he had had no previous experience, and examining various methods for obtaining the most. satisfactory results. Dr. Pugh and Mr. F. Rk. Segeleke were also working in. XV1 Obituary Notices of Fellows deceased. the laboratory at that time, and they gave Warington valuable assistance in his work. Of the two series of analyses eventually completed, the first comprised those of the ashes of grass grown under different manurial treat- ment, the results of which were published in Lawes’ and Gilbert’s “ Report of Experiments with Different Manures on Permanent Meadow Land,’* the second series was that of the ash of grain from Broadbalk Field. These latter analyses were never published, their place having been taken by more complete work on the same subject by Richter. Although Warington left the Rothamsted Laboratory in January, 1860, his interest in the work there never ceased, and, until he resumed his connection with Lawes a few years later, he devoted much of his time to studying the Kothamsted results, and was a frequent visitor to the laboratory. His health having been somewhat re-established by his year’s residence in the country, he returned to town, and continued to reside with his parents till 1862, spending his days at South Kensington, where he worked, under Dr. Frankland, as research assistant. But at the end of this period, a further breakdown in health forced him again to seek a country life, and he betook himself to the Royal Agricultural College at Cirencester. Here he remained for four and a-half years, the first nine months of which were spent in doing analyses for Dr. A. Voelcker, and the remainder of the time in fulfilling the duties of teaching assistant under Professor Church. It was during his residence at Cirencester that Warington published the first papers on scientific subjects which appear under his name. ‘These were printed in the ‘Journal of the Chemical Society.’ The earliest of them (1863) dealt with the quantitive determination of phosphoric acid. This was followed by two other short communications on kindred subjects, which preceded and prepared the way for his first work of importance—an investigation into the part played by ferric oxide and alumina in decom- posing soluble phosphates and other salts, and retaining them in the soil. The results of this investigation are embodied in a series of four papers read before the Chemical Society, and are typical examples of the careful work and close reasoning which characterised all Warington’s researches. That ferric oxide acted as a fixing agent for soluble substances applied to a soil, was already known, but the action was attributed to an indefinable physical attraction, which explained nothing. Warington proved, first by experiments with pure ferric oxide, and then with ordinary soil, that the action in the case of calcium phosphate was simply one of chemical decom- position, resulting in the formation of ferric phosphate, whilst, in the case of other salts, such as carbonates, sulphates, nitrates, etc., the chemical character of the action was indicated by the fact that the iron did not retain the salt as a whole, but partially decomposed it, retaining the basic portion in excess over the acid portion. . Warington did not allow his work at Cirencester to sever his connection * “Journ. Roy. Ag. Soc.,’ vol. 20, 1859, p. 407. Robert Warington. Xvi _with Rothamsted, and he offered to analyse three of the most important of the animal ashes which had been prepared there, on the condition that he might make use of the results thus obtained. He consequently received mixed ashes representing the whole bodies of a fat ox, a fat sheep, and a fat pig, and an abstract of the analysis made by him appeared in an article which he wrote for the second supplement to “ Watts’ Dictionary of Chemistry.” The analysis, together with others by Richter, were also published by Lawes and Gilbert in the ‘ Phil. Trans., 1883. In 1864 Warington commenced lecturing to the students at Cirencester on the Rothamsted experiments, and went systematically through all the work which had already been published, together with many additions of as yet unpublished results which had been communicated to him by Lawes and Gilbert. A desire was expressed at Cirencester that these lectures should be published, and negotiations to that end were consequently opened with Lawes and Gilbert. The outcome of these was that Warington was to write a book on the Rothamsted investigations, Lawes guaranteeing him from pecuniary loss, but offering no remuneration. Lawes also reserved to himself the right to supply a preface to the book, on the ground that there would be previously unpublished matter incorporated therein. The writing of this book involved a large amount of labour, especially as, in studying the effect of manures in different seasons, Warington was led to recognise the almost paramount influence of the rainfall on the results, and its action in washing the nitrates out of the soil, an action up to that time unrecognised. For the purpose of examining this action more closely, he compared the results from the plots at Rothamsted with the temperatures and rainfalls supphed to him by Glaisher; at the same time he applied to Gilbert to furnish him with unpublished data respecting the Rothamsted hay crops. Gilbert, however, objected to what now appeared to him in the light of a publication of Rothamsted results by others than Lawes and himself. Dis- cussions ensued, the upshot of which was that the book remained in manuscript, and the seeds of an unfortunate dissension between Gilbert and Warington were sown. Some 120 pages of this book were written (and are still in existence), but Warington declined the pecuniary compensation which Lawes offered to him for his labour. Leaving Cirencester in June, 1867, he became chemist to Lawes’ manure and tartaric and citric acid factories at Millwall, where he remained till 1876. During these years he generally had a long conversation every week with Lawes on those problems in agricultural chemistry which happened to be under investigation at the time, and which were evidently more congenial subjects of discussion to both of them than the problems arising in the factory. Even these, however, were by no means lacking in interest, and at the conclusion of his engagement at Millwall in 1874, Warington remained in the laboratory there for two years longer, working on citric and _ tartaric acids, and ultimately publishing his results in a paper of 70 pages in the ‘Journal of the Chemical Society.’ This paper was published with Lawes’ VOL. LXXX.—B. XV1il Obituary Notices of Fellows deceased. approval, and it is noteworthy for the opinion expressed therein, that “ the large amount of information acquired in the laboratories of our great manufacturing concerns might well be published without any injury to the individual manufacturer.” Eighteen years later, when Warington had for a second time gone to work in Lawes’ tartaric and citric acid factory, he published another paper dealing with these acids, and with the detection of the presence of lead in them. With this solitary exception, all Warington’s subsequent work was on agricultural chemistry, and all of it was done in the Rothamsted laboratory. While still at Millwall, he had been writing a good deal on agricultural subjects—several articles for “ Watts’ Dictionary” and for the Agricultural and Horticultural Co-operation Association—and he had, moreover, as already mentioned, been in continual consultation with Lawes as to the Rothamsted results ; he was naturally, therefore, prepared to receive Lawes’ suggestion that he should go and work in the Rothamsted laboratory. The terms were all settled, and had readily been assented to by Warington; for, although they had involved a reduction of salary to two-thirds of that which he had been receiving at Millwall, he obtained a certain amount of freedom by way of compensation. He was to be at liberty to publish his own work in his own name, provided that it made its appearance as Rothamsted work ; but in cases where the work dealt with subjects which had already occupied the Rothamsted investigators, it was to be published in the joint names of Lawes, Gilbert and Warington. This arrangement, however, owing to some unfore- seen difficulties, was not carried out; and it was not till after a delay of two years that Warington went to Rothamsted (in 1876), under an agreement for a year only, to work simply as Lawes’ private assistant. The engagement was subsequently extended, and all his results were published, either in his own name or in the names of Lawes, Gilbert and Warington. Before removing to Harpenden, he went to work at the laboratory at South Kensington in order to learn water and gas analysis under Frankiand’s assistant, some of the Rothamsted soils being sent to him for practising determinations of nitrogen. While there he devised a method of extracting soils by the vacuum pump, which method has since been largely used at Rothamsted. In the autumn of the same year (1876) he made a short tour among the German experimental stations, and then took up his residence for good at Harpenden. The construction of a gas analysis apparatus (under Frankland’s direction) for the Rothamsted laboratory occupied a considerable time, and, pending its completion, Warington made a study of the indigo method of determining nitric acid. This method, as generally used, he found to be full of sources of error. The principal of these he succeeded in correcting, and, with the method of determination, thus rendered trustworthy, he proceeded to determine regularly the nitrates in the drainage-water from the various wheat plots in Broadbalk field. The chlorides were determined at the same time. No such systematic work had been previously done; whilst the methods of sampling, Robert Warington. X1X which had been adopted when any analysis had been made, had been faulty. Warington now altered these methods, so that the samples analysed should faithfully represent the average composition of the drainage-waters. Having examined the indigo method. for determining nitric acid, he next examined the Crum-Frankland method by agitation with mercury; and, sub- sequently, the method of Schleesing, modified, however, in such a way that the nitric oxide produced was determined by gas analysis. The exhaustive examination of these methods of analysis are described in a series of papers published in his own name in the ‘J ournal of the Chemical Society’ and elsewhere, extending down to 1882. The modified Schloesing method was the one which he finally adopted, and with it he began a long series of determinations of nitrates in soils, and in mangels, swedes, and potatoes. Having satisfied himself as to the methods of nitrogen determination, he next turned his attention to those for the estimation of carbon, and having examined the permanganate and the bichromate methods, and found them wanting, he finally adopted the combustion method, which proved to be thoroughly satisfactory, provided that carbonates were entirely removed by prolonged treatment with sulphurous acid. In this work he was assisted by Mr. W. A. Peake, and the results were brought before the Chemical Society in the names of Warington and Peake. Warington’s results from the examination of the rain and drainage water, together with results previotsly obtained at Rothamsted, formed the subject of a very long report published in the names of the three investigators in the ‘Journal of the Royal Agricultural Society’ for 1882. The subject, however, continued to occupy Warington’s attention long after this date, and we find a report on the subject in the three joint names in 1883, and papers by Warington alone in 1889 and 1887. The last-mentioned paper is an important contribution* to the study of well waters, and deals with the wells in the chalk formation on which Harpenden is situated. In later years (1904) Warington was enabled to give these results a practical bearing on the supposed contamination of the Harpenden water supply, and he saved the community, at any rate, for a time, from adopting an expensive, and, apparently, quite unnecessary system of sewerage. So far, Warington’s work, as here described, consisted largely of examining and perfecting methods of analysis for use in agricultural research. For this work the precision of his nature, and the carefulness of his manipulation pre-eminently fitted him, and most of the methods of analysis which he elaborated have been accepted as standard methods, which promise to remain in use for many years to come. The remainder of his work, however, is that by which he made his name, and, if a strictly chronological © sequence of events had been followed, it should have been mentioned earlier in this notice, for it was in 1877 that he began to study nitrification, and this subject occupied the foremost place in his mind till 1891, when his opportunities for pursuing the subject ceased. During this period he published about ten * “Journ. Chem. Soc.,’ pp. 500—552. . | 6.2 2 Obituary Notices of Fellows deceased. papers on the subject, all in his own name, the principal of which were four communications to the Chemical Society, bearing the title “On Nitrification,” Parts I to IV. That the natural conversion of ammonia into nitric acid was the work of an organism, had been suggested by A. Miiller as early as 1873, but it had been reserved for Schleesing and Miintz to establish definitely that this was the case. In 1877 they showed that, when sewage was allowed to percolate through a column of sand and limestone, the nitrification which occurred during its passage could be prevented by the presence of a sterilising agent such as chloroform vapour, and, after such sterilisation, the activity of the sand could be resuscitated by inoculating it with a few particles of vegetable mould. Questions affecting the problems connected with nitrogen in the soil had naturally been amongst those to which the Rothamsted investigators had, from the first, devoted themselves, and, consequently, they at once set to work to examine such an important observation as that of Schloesing and Mintz. A complete verification of it was obtained by Warington, operating with garden soil only, and a solution of ammonium chloride, instead of sewage; and he was enabled to add the additional information, that nitrification occurred only in the dark. This paper appeared within a year of that of Schloesing and Mintz. Two and a-half years later he published a second paper which added considerably to the facts already established. He showed that the nitrifying organism, besides requiring darkness in order to do its work, must also be supplied with food for its growth—potash, lime and phosphorus—and moreover, that all liberation of free acid must be prevented by the presence of some salifiable base, such as ealcium carbonate. He found, also, that after the introduction of a small quantity of active soil or solution into a liquid capable of nitrification, no action occurred till a certain time had elapsed, this period of incubation being probably due to the organisms having to multiply to a certain extent before they become sufficiently numerous to produce recognisable results. An increase of temperature was found to favour the action up to a certain point, and it was shown that various vegetable moulds and known bacteria were not the organisms to which nitrification could be attributed. Many difficulties, however, still remained to be cleared up, notably the want of uniformity of the action, which resulted in the production of nitrates in some instances, and nitrites in others. We now know that the process is performed by two quite distinct organisms, and that their nutrition is, In some respects, wholly different from that of any other organism hitherto studied; but until this knowledge was gained, work on the subject was singularly difficult, and the results were very perplexing. Warington’s third paper on nitrification added considerably to our knowledge of the circumstances attending the action, and established the fact that the organismsare almost entirely confined to the first nine inches of ordinary soil. The distribution of the organism in the soil was dealt with still more exhaustively in a subsequent communication in 1887. The prize coveted by the workers on this subject was, however, the isolation Robert Warington. | XX8 of one organism itself; and to prepare himself for this task Warington wenf to London for a time, in 1886, to learn bacteriology under Dr. Klein at the - Brown Institution. From Dr. Klein he obtained a large number of pure cultures of various bacteria, and all these, as well as others obtained from his own experiments with soils, he examined as to their behaviour towards ammonia and nitrates, and also as to their mode of growth on skim-milk. The results were brought before the Chemical Society, and proved that none of the bacteria, except the nitrifying organism itself, possessed any appreciable power of nitrification. The majority of the organisms examined, however, were active denitrifiers. Denitrification—whereby nitrates are converted into nitrites, oxides of nitrogen, or even nitrogen gas—was, at this time, a well recognised work of micro-organisms, but was one which, naturally, enhanced to a considerable extent the difficulties met in elucidating the reverse phenomenon of nitrification. Warington’s work added a good deal to our knowledge of the subject, and showed that denitrification is a property actively exhibited by a large number, but by no means by all, micro-organisms, and that in a soil it becomes complete, before the nitrifying organisms begin their task of reversing the reaction. An excellent account of the denitrification of farmyard manure was subsequently written for the ‘Journ. - Roy. Ag. Soc.,’ 1897, vol. 8, Part IV. Warington’s work on nitrification was amply sufficient to establish the fact that the oxidation of ammonia in the soil was the work of an organism, but that organism seems to have been isolated first by Schloesing and Miintz in 1879, though the method which they adopted left, at the time, considerable doubt as to its real identity. But even the isolation of this organism did not solve the whole problem: there was still the independent formation of nitrites and nitrates to be accounted for; and it was here that Warington’s work was most conducive to a solution of the difficulties, for he succeeded in proving that one organism alone could not be held accountable for the various phenomena observed, and that two different organisms must be concerned in the process of nitrification. His success all lay in the chemical aspects of the subject. He was the first to obtain (1879) liquid cultures which converted ammonia into a nitrite, and preserved this power in all sub-cultures, but which was incapable of producing any nitrate ; and shortly afterwards (1881) he obtained cultures which were able to convert nitrites into nitrates, but were unable to oxidise ammonia. This was a practical separation of two distinct organisms, but at the time Warington did not grasp the true meaning of his results, and he associated the change from nitrites into nitrates with a white growth which appeared floating in the liquid, but which really had nothing to do with it. In 1890, after the work of others had resulted in the isolation of the nitrous organism (that which converted ammonia into nitrites), Warington returned to the subject, and found that the white surface organism could not be held accountable for the conversion of the nitrites into nitrates. He eventually succeeded in isolating the organism which really produces this xxii Obituary Notices of Fellows deceased. change, and obtained a nearly pure culture of the nitric organism. At the same time he showed that organic carbon is not necessary for the growth of these organisms, as he had previously imagined, but that they can obtain their carbon from carbonates. These results were published in his fourth paper on nitrification (1891), and were communicated to the Chemical Society only a few days before Winogradski made a similar communication to the French Academy. Winogradski, however, had pushed the matter somewhat further, having obtained the organisms in bodily form, and having shown how they could be cultivated on solid media, a problem which had baffled Warington and other investigators. Warington, therefore, had to share his final hard-won success with another. The practical results of nitrification in the soil were being investigated while the search for the organism was still in progress, and Warington began along series of determinations of nitrates in the Rothamsted soil, the first results of which were published as a lecture given before the Society of Arts, for which he was awarded a silver medal. When, in 1889, Lawes resigned his active control to the present Com- mittee of Management, it was arranged that Warington should leave in the following January. Having, however, in the meantime, reached a very interesting stage in his work on the nitrifying organism, he stayed on at the laboratory till 1891, and succeeded in bringing the work on hand to a successful termination. Although Warington’s original work in agricultural chemistry was brought to a close on his severance from Rothamsted, much useful work remained for him to do. The Committee of Management appointed him American lecturer. under the Lawes Trust, and he, consequently, proceeded to the United States to perform his functions. The six lectures which he there delivered dealt chiefly with the subject of nitrification, illustrated by his own ‘work at Rothamsted. ey, were published by the United States Department of Agriculture. — On his return to England, Sir John Lawes invited him to carry out an investigation at his tartaric and citric acid factory at Millwall on the contamination of these acids by the lead of the vessels used in their preparation. This Warington undertook, and he succeeded in finding a method for obviating the evil. He obtained, in addition, an excellent method for the accurate volumetric determination of lead in the acid. This formed the subject of a communication to the Society of Chemical Industry in 1893, the last communication of any investigation made by him. In 1894, he was appointed one of the examiners in Agriculture under the Science and Art Department, and in the summer of the same year he was elected Sibthorpian Professor of Rural Economy at Oxford for three years. The papers, other than those on original investigations, which Warington wrote, are numerous, and are all characterised by a lucidity of expression and precision of argument which renders them specially valuable. One of the mn, Robert Warington. Xxlll most useful of his writings is, undoubtedly, a little volume entitled “The Chemistry of the Farm.” The amount of appreciation with which it has been received, and the good which it has done, may be measured by the fact that it 1s now in its fifteenth edition, and is accepted as the text book on the subject throughout the world, and as a model of what a text book of that sort should be. Warington continued to reside in Harpenden till the end. His habits and tastes did not predispose him to take any active part in village management, but whenever he thought that his knowledge might be of service to the community he did not hesitate to yive what assistance he could. Educational or charitable work, however, always enlisted his sympathies, and engaged his active support; whilst his strong religious convictions, euided by his clear judgment and absolute sincerity, rendered his church and philanthropic work peculiarly valuable. He certainly had an unusually high sense of public duty, and, persistently throughout life, did what he could to make his fellow-creatures better and happier. Missionary work always held a prominent place in his heart, as also did the training of the young, whether in religious or secular subjects; and, during the last few years of his hfe, much of his time and care was devoted to the Church day-schools. He was greatly interested in all work amongst the poor and needy, and was a liberal supporter of any organised charity which appealed to his judgment. Partly owing to his isolated boyhood and youth, and partly to his lack of robust health, life went harder with him than it otherwise would have done, _ for the characteristics thus developed stood in his way, and often prevented his gaining the sympathy and appreciation which he was so ready to give to others. Warington was elected to the Chemical Society in 1863, and to the Royal Society in 1886. He served for two periods on the Council of the Chemical Society, and for one period as vice-president. For many years he was on the library committee of this Society, and did much useful work for the Fellows during the reorganisation and cataloguing of the books. For this, his extensive acquaintance with chemical literature rendered him specially fitted. Warington was married twice. His first wife was a daughter of G. H. Makins, F.R.C.S., formerly chief Assayer to the Bank of England, and one of the Court of Assistants at the Society of Apothecaries. His second wife was a daughter of Dr. F. R. Spackman, who had for many years been medical practitioner at Harpenden. He has left five daughters by his first wife. In 1906, his health gave way, and he had a serious illness which necessitated a very difficult and dangerous operation. For this he prepared with singular equanimity and courage. The operation was successful; but though he nominally recovered from it, he never regained his strength, and, eleven months afterwards (March 20, 1907), he passed away. With the death of Warington, the first generation of great Rothamsted workers is brought to an end. Their work, whether published in the form XXIV Obituary Notices of Fellows deceased. of independent communications, or as joint productions, constitutes one great whole, of which the various parts are beautifully correlated and interdependent. It has placed agricultural science on an altogether different basis from that which it previously occupied, and the institution which gave birth to it has served as the prototype for similar institutions throughout the whole civilised world. The three men to whom we owe these results—Lawes, Gilbert and Warington—devoted their whole lives and energies to the work, and only those who are acquainted with the difficulties attendant on co-operation in this case can appreciate the devotion to science which was required to master these difficulties. All three workers now lie at rest in the same quiet country churchyard, their combined work in the cause of scientific agriculture forming the most fitting and enduring monument of their labours, for its importance becomes every day more and more evident with the development of the super- structure which is being raised upon it. P48: Baie XXV WALTER FRANK RAPHAEL WELDON. 1860—1906.* WALTER FRANK RAPHAEL WELDON, the elder son of the late Walter Weldon, F.R.S., was born at Suffolk Villa, Highgate. We have no record if he attended a school there. When his parents removed to Puttley he had, as tutor, a neighbouring clergyman. In 1873 he was sent to a boarding- school at Caversham, where he remained not quite three years, and from which, after some months of private study he matriculated, in 1876, in the University of London. In October of that year we find him at University College taking classes in Greek, English, Latin, and French, with two courses of Pure Mathematics. In the summer term of 1877, physics and appled mechanics were studied. During this whole session he also attended Daniel Oliver’s general lectures on botany and Ray Lankester’s on zoology. Later in the Christmas vacation of 1879, after he had gone up to Cambridge, he was for some weeks under Ray Lankester, who set him to work out the structure of the gills of the mollusc “ Trigonia.” In the autumn of 1877 he transferred himself to King’s College, where he stayed for two terms, attending classes in chemistry, mathematics, physics, and mechanics, besides the zoology course of A. H. Garrod and the biology of G. F. Yeo. Divinity under Barry, at that time compulsory, was also taken. At this time Weldon had the medical profession in view. Though only entered on the Register of Medical Students on July 6, 1878, there can be no question that his course, on the whole, was directed towards the Preliminary Scientific Examination of the London M.B. This examination he took in December, 1878, after he had gone up to Cambridge; he was coached for it by T. W. Bridge, now Professor of Zoology in Birmingham, but he had already completed the bulk of the work in his London courses. With the Preliminary Scientific, Weldon’s relation with London University ceased. In 1877 he attended the Plymouth Meeting of the British Associa- tion, and there he was generally to be found in Section D. The presence of a life-long friend, who had already gone to Cambridge, was, at least, one of the causes which led to Weldon’s entering himself as a bye-term student at Cambridge ; and probably his choice of St. John’s College was due to Garrod’s influence. He was admitted on April 6, 1878, as a pupil of the Rev. 8. Parkinson, D.D. At Cambridge Weldon soon found his work more specialised, and he rapidly came under new and marked influences. Under the inspiration of Balfour, Weldon’s thoughts turned more and more to zoology, and the medical profession became less and less attractive. During the years 1879 and 1880, he worked steadily for his Tripos; in the first year he was given * This notice is abstracted (by A. E. S.) from the much longer biography in ‘Biometrika,’ written by Professor Karl Pearson, with some help from Mr. A. E. Shipley. XXVI1 Obituary Notices of Fellows deceased. an exhibition at St. John’s. In the second year a little original investigation on beetles was started; in May he took, for a month, Adam Sedgwick’s place, and demonstrated for Balfour. The Tripos work was continued, in spite of ill-health, till the Easter of 1881, when Weldon was unable to enter for the college scholarship examina- tions. By the influence of Francis Balfour, however, Weldon’s real ability was recognised, and a scholarship was awarded to him. At the very start of his Tripos examination, his only brother, Dante Weldon, who had joined Peterhouse, died suddenly of apoplexy. It says much for Weldon’s self- control that the terrible shock of his brother’s death did not interfere with his place in the first class of the Natural Sciences Tripos. A few weeks later a second bereavement befell him, when his mother passed away. These trials, followed by Balfour’s untimely death in the following year, and by the early death of his own father a few years later, left their indelible impresses upon him. With the Tripos, Weldon’s “ Lehrjahre ” closed, and, as his nature directed, the “ Wanderjahre ” began without any interval of rest. Immediately after his Tripos, Weldon started for Naples to work at the Zoological Station. The charm of Balfour’s personality had aroused the affection of all who attended his classes, and had awakened a keen desire to follow in his foctsteps. In those days the stimulus given by Darwin’s writings to morphological and embryological researches was still the dominating factor amongst zoologists, and Weldon threw himself at first with ardour into the effort to advance our knowledge by morphological methods. In Naples he began his fitst published work, a “Note on the early Development of Lacerta muralis,’ and at the same time did much miscellaneous work on marine organisms. In September he was back in England at the Southampton Meeting of the British Association. Adam Sedgwick, who had succeeded to the teaching work of Francis Balfour, now invited Weldon to demonstrate for him. Thus, the winter found Weldon in Cambridge again, and from Sedgwick’s laboratory was issued the next piece of work: “On the Head-kidney of Bdellostoma, with a suggestion as to the Origin of the Suprarenal Bodies,” and he followed the subject up in the next year by publishing his paper “On the Suprarenal Bodies of Vertebrates.” On March 14, 1883, the anniversary of his parents’ wedding-day, Weldon married Miss Florence Tebb, the eldest daughter of William Tebb, now of Rede Hall, Burstow, Surrey. After the death on January 14, 1883, of W. A. Forbes, a Fellow Johnian, Weldon for four months—June 15 to October 15—acted as locwm tenens for the Prosector at the Zoological Gardens, London, and during that time he read the following papers before the Zoological Society :—“ On some points in the Anatomy of Phcenicopterus and its Allies”; a “ Note on the Placentation of Tetraceros quadricornis” ; and “ Notes on Callithrix gigot.” In the following year (1884) the paper above referred to on the Walter Frank Raphael Weldon. XXVIl development of the suprarenal bodies was published in the Royal Society ‘Proceedings.’ On November 3 of the same year, Weldon was elected toa Fellowship at St. John’s College, and was shortly afterwards appointed University Lecturer in Invertebrate Morphology. About this time he took a permanent home at No. 14, Brookside, which soon became a centre for Cambridge workers on biology. On his return to Cambridge in November, 1884, Weldon had taken up again his invertebrate work. His next Memoir, “On Dinophilus gigas,” dealt with the anatomy and affinities of Dinophilus, at that time a very little known Annelid. The next few years of Weldon’s life were more active than ever. He had now given up coaching, and as he only needed to be in Cambridge two terms of the year, travel and research could occupy the time from the beginning of June to January. On May 8, 1885, he gave his first Friday evening lecture at the Royal Institution on “ Adaptation to surroundings as a factor in Animal Development.” No report of this lecture was published in the ‘ Proceedings, but there are those who still remember the impression caused by the youthful lecturer of twenty-five years of age. Weldon was an adept in lecturing to classes of University students; it brought out all his force and enthusiasm as a teacher. As a writer in the ‘limes’ (April 18, 1906) says: “Seldom is it given to a man to teach as Weldon taught. He lectured almost as one inspired. His extreme earnestness was only equalled by his lucidity. He awoke enthusiasm even in the dullest, and he had the divine gift of compelling interest. In the University Lecture room he always impressed his hearers with the importance of his topic. You could not listen. to him lecturing on a flame-cell or on the variations in the carapace of Pandalus annulicornis without sharing his intense conviction of the importance of the matter in hand. He aroused a consciousness in his students that things were worth studying for their own sake, apart from their examination value.” In July, 1886, Weldon crossed to America, and visited the Bahamas to collect. From his headquarters in the Bahamas, he went with two friends to North Bimini, in the Gulf Stream, and made considerable collections, but his published results were confined to “Haplodiscus piger; a new Pelagic Organism from the Bahamas,” and a “ Preliminary note on a Balanoglossus Larva from the Bahamas.” Working at the Balanoglossus material in 1887, he found that his results differed from those reached by Professor Sprengel. He accordingly went to Giessen at Easter, and finally handed over to Professor Sprengel the whole of his Balanoglossus material. During the Lent and May terms he gave a course of lectures on Economic Entomology to the forestry students at the Royal Indian Engineering College, Coopers Hill. In 1888, the buildings of the Marine Biological Laboratory in Plymouth were nearly completed, and to the Marine Biological Association Weldon gave both time and sympathy during the rest of his life. His annual visits of inspection to their second Laboratory at Lowestoft during the last few XXVlll Obituary Notices of Fellows deceased. years were always a great pleasure to him. Lent and May terms, 1888, were spent as usual in Cambridge, but June to December were given up to Plymouth, with a brief Christmas holiday in Munich. And here we must note the beginning of a new phase in Weldon’s ideas. His thoughts were distinctly turning from morphology to problems in variation and correlation. He has left on record the nature of the problems he was proposing to himself at this time, and they are summed up as follows :— (1) The establishment of a new set of adult characters leading to the evolution of a new family has always been accompanied by the evolution of a new set of larval characters leading to the formation of a larval type peculiar to the newly established family ; the two sets of characters having as yet no demonstrable connection one with the other. (2) The evolution of the adult and that of the larval characters peculiar to a group advance pari passu one with the other, so that a given degree of a specialisation of adult characters on the parts of a given species implies the possession of a larva having a corresponding degree of specialisation and vice Versa. The next year was to place in Weldon’s hands a book—Francis Galton’s “Natural Inheritance,” by which one avenue to the solution of such problems, one quantitative method of attacking organic correlation, was opened out. From this book as their source sprang two notable friendships and the whole of the biometric movement, which so changed the course of his life and work. In 1889, also, another change came. Weldon found that his dredging and collecting work separated him from his books for half his time. Accordingly, he applied for a year’s leave from Cambridge, and he and his wife settled down in a house of their own at Plymouth. This period of hard work lasted through 1890, and was broken only by flying visits to Dresden in September and at Christmas, 1889, and an autumn visit in 1890 to Chartres and Bourges. The intellectual development and the experience and knowledge gained in this period were far more important than the mere published work would indicate. In 1889, Weldon investigated the nature of the curious enlargement of the bladder associated with the green, or excretory, glands in certain Decapod Crustacea, and published in October of the same year his paper of “The Coelom and Nephridia of Palemon servatus.” The result of his investigation was to confirm “the comparison so often made (by Claus, Grobben, and others) between the glomerulus of the vertebrate kidney and the end-sac of the Crustacean green gland.” A little later, June, 1891, he published the results of more extended researches in this field in what proved to be his last strictly morphological paper. It was entitled: “The Renal Organs of certain Decapod Crustacea.” In this he showed that in many Decapods spacious nephro-peritoneal sacs “should be regarded rather as enlarged portions of the tubular system ... than as persistent remnants of a ‘ccelomic’ body cavity, into which the tubular nephridia open.” One further paper of a year later may be best referred to here, Weldon’s Walter Frank Raphael Weldon. XX1X only piece of work on invertebrate embryology, “The formation of the Germ Layers in Crangon vulgaris.” This contains a clear account of the early stages of segmentation, and the building up of the layers of the shrimp, illustrated by excellent figures. And here it may be mentioned that his power with the pencil was not that of the mere draughtsman, accurate in detail but often lifeless; he was an artist by instinct, and he had the keenest pleasure in drawing for its own sake. December, 1890, closed the Cambridge work; Weldon now itiboedaea Ray Lankester in the Jodrell eeu eseone ta at University College, London. In June he had been elected a Fellow of the Royal Society. It has been seen that the years between Weldon’s degree and his first professoriate were years of intense activity. He was teaching many things, studying many things, planning many things. His travels perfected his linguistic powers, and his fluency in French, Italian, and German was soon remarkable. , A word must here be said as to the transition which took place during the “Wanderjahre” in Weldon’s ideas. He had started, as most of the younger men of that day, with an intense enthusiasm for the Darwinian theory of evolution, but he realised to the full that the great scheme of Darwin was onlya working hypothesis, and that it was left to his disciples to complete the proofs, of which the master had only sketched the outline. Naturally he turned first to those methods of proof, morphological and embryological, which were being pursued by the biological leaders of the period, and it was only with time that he came to the conclusion that no great progress could be attained by the old methods. We have already seen that even before the appearance of “Natural Inheritance,’ his thoughts were turning on tbe distribution of variations and the correlation of organic characters. He was being led in the direction of statistical inquiry. The full expression of his ideas is well given in the first part of the “ Editorial,’ with which ‘ Biometrika’ started :— “The starting point of Darwin’s theory of evolution is precisely the existence of those differences between individual members of a race or species which morphologists for the most part rightly neglect. The first condition necessary, in order that any processes of Natural Selection may begin among a race, or species, is the existence of differences among its members ; and the first step in an inquiry into the possible effect of a selective process upon any character of a race must be an estimate of the frequency with which individuals, exhibiting any degree of abnormality with respect to that character, occur. The unit, with which such an enquiry must deal, is not an individual but a race, or a statistically representative sample of a race; and the result must take the form of a numerical statement, showing the relative frequency with which various kinds of individuals composing the race occur.” It was Francis Galton’s “Natural Inheritance” that first indicated to Weldon the manner in which the frequency of deviations from the type could be measured. Kon Obituary Notices of Fellows deceased. In Plymouth, 1890, Weldon started his elaborate measurements on the Decapod Crustacea, and soon succeeded in showing that the distribution of variations was closely like that which Quetelet and Galton had found in the case of man. His paper “The Variations occurring in certain Decapod Crustacea. I. Crangon vulgaris” was, as far as we know, the first to apply the method of Galton to, other zoological types than man. In this paper the author showed that different measurements made on several local races of shrimps give frequency distributions closely following the normal or Gaussian law. In his next paper, “On certain correlated Variations in Crangon vulgaris,” he calculated the first coefficients of organic correlation, 2.¢., the numerical measures of the degree of interrelation between two organs or characters in the same individual. It is quite true that the complete modern methods were not adopted in either of these papers, but we have for the first time organic correlation coefticients—although not yet called by that name— tabled for four local races. These two papers are epoch-making in the history of the science, afterwards called Biometry. It is right to state that Weldon’s mathematical knowledge at this period was far more limited than it afterwards became. The first paper was sent to Francis Galton as referee, and was the commencement of a life-long friendship between the two men. With Galton’s aid the statistical treatment was remodelled, and considerable modifications made in the conclusions. The defect in mathematical grasp, which Weldon had realised in his first paper, led him at once to seek to eliminate it. He set about increasing his mathematical knowledge by a thorough study of the great French writers on the calculus of probability. He did not turn to elementary text-books, but, with his characteristic thoroughness went to the fountain head, and he thus attained a great power of following mathematical reasoning, and this power developed with the years. He had, moreover, a touch with observation and experiment rare in mathematicians. In problems of probability he would start experimentally and often reach results of great complexity by induction. From 1890 onwards, his knowledge, theoretical and experimental, of the theory of chances increased by bounds. Weldon’s work at University College commenced in 1891. The house in Wimpole Street was taken and, if possible, life became more intense. In October came the college inaugural lecture for the session, on the subject of the statistical treatinent of variation. This year and the next were strenuous years in calculating. The Weldons toiled away at masses of figures, doing all in duplicate. At Easter, 1892, they went to Malta and Naples, and the summer was spent over crab-measurements at the Zoological Station in the latter city, and the first biometric crab paper “On certain correlated Variations in Carcinus menas” was issued later in the year. This paper confirms on the shore crab the results already obtained on the common shrimp. The distributions of characters are closely Gaussian, with the exception of the relative frontal breadth, which the author considered Walter Frank Raphael Weldon. XXX dimorphic in Naples. He does not refer to this fact in his memoir. As for shrimps the correlations again came out closely alike for the Plymouth and Naples races. Weldon was not dogmatic on the point; he considered the constancy as at least an “ empirical working rule.” To the biometrician, perhaps, the most interesting committee with which Weldon was associated in his later years was that which came to be called the Royal Society Evolution (Animals and Plants) Committee. His papers ou variation and correlation in shrimps and crabs had brought him closely into touch with Francis Galton, and both were keenly interested in the discovery of further dimorphic forms such as had been suggested by the frontal breadths of the Naples crabs. Weldon was full also of other ideas ripe for investigation. He had started his great attempt at the measurement of a selective death-rate in the crabs of Plymouth Sound; experiments on repeated selection of infusoria were going on in his laboratory; he was gathering an ardent band of workers about him, and much seemed possible with proper assistance and that friendly sympathy which was ever essential to him. As a result of an informal conference held at the Savile Club towards the end of 1893, it was decided to ask the Royal Society to establish a Committee “for the purpose of Conducting Statistical Enquiry into the Variability of Organisms,” and such a Committee was early in 1894 constituted by the Council, with Francis Galton as Chairman, and Weldon as Secretary, the Committee being entitled; “Committee for conducting Statistical Inquiries into the Measurable Characteristics of Plants and Animals.” The use of the words statistical and measurable, somewhat narrowly, but accurately, defined the proposed researches of the Committee. It went on until 1897, with the same members, the same title and scope. Looking back on the matter now, one realises how much Weldon’s work was hampered by the Committee. It is generally best that a man’s work should be published on his own responsibility, and when he is a man of well-known ability and established reputation, grants in aid can always be procured. In this case Weldon had a sympathetic committee, but the members were naturally anxious on the one hand for the prestige of the Society with whose name they were associated, and secondly, they were desirous of showing that they were achieving something. Both conditions were incompatible with tentative researches such as Biometry then demanded. Trial and experiment were peculiarly needful in 1893; the statistical calculus itself was not then even partially completed ; biometric computations were not reduced to routine methods, and the mere work of collecting, observing, experimenting, and measuring was more than enough for one man. Weldon with his “volume of life” was eager to do all these things, and run a laboratory with perhaps sixty students as well. The “ Attempt to Measure the Death-rate due to the Selective Destruction of Carcinus menas, with respect to a Particular Dimension,” formed the first report of the Committee, and was presented to the Royal Society in November, 1894. Weldon’s general project in this case was novel at the time, it XXX Obituary Notices of Fellows deceased. consisted in determining whether the death-rate is correlated with measur- able characters of the organism, or, as he himself puts it, “in comparing the frequency of abnormalities in young individuals at various stages of growth with the frequency of the same abnormalities in adult life, so as to determine whether any evidence of selective destruction during growth could be discovered or not.” | Looking back now on Weldon’s paper of 1894, one realises its great merits : it formulated the whole range of problems which must be dealt with biometrically before the principle of selection can be raised from hypothesis to law. Almost each step of it suggests a mathematical problem of vital ~ importance in evolution, which has since been developed at length, or still awaits the labour of the ardent biometrician. Unfortunately the paper, as well as the suggestive “ Remarks on Variation in Animals and Plants” with its memorable words: “The questions raised by the Darwinian hypothesis are purely statistical, and the statistical method is the only one at present obvious by which that hypothesis can be experi- mentally checked,” fell on barren soil. A further instructive report on the growth at two moults of a considerable number of crabs was made to the Committee in 1897, but appears never to have been published. Later, an account of work on Natural Selection in crabs was given by Weldon in his Presidential Address to the Zoological Section of the British Association, Bristol, 1898. In the paper just mentioned, after several years of discourage- ment and much hard labour, he succeeded in demonstrating that natural selection was really at work, and further that it was at work at a very sensible rate. The labour involved was excessive. One “crabbery ” consisted of 500 wide-mouthed bottles, each with two syphons for a constant flow of sea water. Each crab had to be fed daily and its bottle cleaned. But in the autumn a rest came. The British Association Address was written and Weldon thoroughly enjoyed his presidency of Section D at Bristol. It may not be out of place here to note the great aid Weldon’s artistic instinct and literary training gave to his scientific expression. His papers are models of clear exposition, his facts are well marshalled, his phraseology apt, his arguments concise, and his conclusions tersely and definitely expressed. The result, however, was not reached without much labour. There was never any artificial briluancy introduced in the process; rhetoric in the service of science was intolerable to Weldon. His was simply an attempt to choose the suitable form and the right words for a given purpose. It was comparable with his sense of sound, with his extraordinary gift of appreciating and reproducing the exact intonation of a foreign tongue. Considerable changes were soon to take place in his environment and scheme of work. Lankester had been appointed Director to the British Museum (Natural History), and in February, 1899, Weldon succeeded him in the Linacre Professorship at Oxford. In the February of 1897 the Royal Society Evolution Committee received a large increase of membership; it ceased henceforth to “conduct statistical mquiries into the measurable Walter Frank Raphael Weldon. XXXll _ characteristics of plants and animals,” and became transformed into an Evolution (Plants and Animals) Committee and Weldon and the biometric members ultimately withdrew from it. During the eight years of his London professoriate Weldon’s development was great; he became step by step a sound mathematician, and gained largely in his power of clear and luminous exposition. His laboratory was always full of enthusiastic workers, and over forty memoirs were published by his students. His removal from the London field of work, while an incalculable loss to his colleagues, was not without its compensation to his nearest friends. They knew that the life of the last few years had been one of great tension, that Weldon’s time had been too much encroached upon by committee work, that the separation between the locus of his teaching and of his research work was very undesirable; that even the social life of London involved too much expense of energy. He was a child of the’ open air and the breezes, and it was hoped that he might have more of them, if not in lowland Oxford, at least on the hills around. There was space and air too for the experimental work that had been so cramped in Gower Street. The Daphnia studies, which had oceupied so much energy under unfavourable conditions in London, were at once resumed on broader lines in the ponds and ditches round Oxford. With a basket of bottles attached to his cycle handle, and a fishing creel, filled with more bottles, on his back, the Linacre Professor might be met even as far as the Chiltern Hills, collecting not only Daphnia, but samples of the water in which they lived. His University College work had shown him how widely Daphnia are modified by their chemical and physical environment, and how this modification is largely due to selection. There exist elaborate drawings of the Daphnia from the Oxfordshire ponds, indicating their differentiation into local races, with notes on the peculiarity of their habitat and the chemical constitution of the water. A study of Kobelt’s “Studien zur Zoogeographie,” 1897-9, led him later to take up the same problem with regard to land-snails. What is the meaning of the slight but perfectly sensible differences in type to be found in shells from adjacent valleys or even from different heights of the same mountain ? Weldon attacked the problem in his usual manner; he spent two Christmas vacations collecting Sicilian snails of the same species, from habitats extend- ing over a wide area, the local environments were described, and the snails were often photographed with their immediate surroundings. Innumerable shells were brought back to Oxford, and the Professor delighted to discourse on the significant differences in local type, and yet the gradual change of type to type from one spot to another. No rapidly made measurement on the outside of the shell would satisfy him; the shell must be carefully ground down through the axis, and the measurements must be made on the section thus exposed. Perhaps four or five snail shells could be ground and measured in a day, and at the time of his death not more than a few hundred of the Sicilian thousands had even been ground. But these attempts to get to the kernel of selection in its action on local VOL. LXXX.—B. ad XXXIV Obituary Notices of Fellows deceased. races were far from occupying the whole of Weldon’s thoughts in these early days. In conjunction with his assistant, Dr. E. Warren, he had commenced at University College his first big experimental investigation into heredity. The characters dealt with consisted of the number of scales in particular colour patches upon certain pedigree moths, and the work of counting these was very laborious. In the course of three years, many hundreds of pedigree moths were dealt with, and the observations were reduced. But no definite inheritance at all of the character selected for consideration was discovered. Weldon, apparently thought that there had been some fatal mistake in the selection of pairings, and undoubtedly, in some cases, parents of opposite deviation had been mated, so that a rather influential negative assortative mating resulted. But from other series of pedigree moth data, it seems probable that there is some special feature in heredity in moths, or possibly in those that breed twice in the year, and that the vast piece of work which Weldon and Warren undertook in 1898—1901 may still have its lesson to teach us. | In these three first years at Oxford, Weldon’s intellectual activity was intense. To the pedigree moth experiments was added, in the summer of 1900, an elaborate series of Shirley Poppy growings, 1250 pedigree individuals being grown and tended in separate pots; Weldon’s records were the most perfect of those of any of the co-operators, and his energy and suggestions gave a new impetus to the whole investigation. They were ultimately published in ‘ Biometrika’ under the title, “ Co-operative Investigations on Plants, I. On Inheritance in the Shirley Poppy.” As Weldon himself expressed it, the moths and poppies meant “a solid eight hours daily of stable-boy work through the whole summer and through the Easter vacation, with decent statistical work between.” After the Shirley Poppies were out of hand in the summer of 1900, the Weldons went to Hamburg and thence to Plon. The object of this visit was to collect Clawsilia at Plon and Gremsmiihlen for comparison with the race at Risborough. The same aim—the comparison of local races—led Weldon at Christmas to collect land-snails in Madeira. Thus he slowly built up a magnificent biometric collection of snail shells, z.¢., one sufficiently large to show, in the case of many local races of a number of species, the type and variability by statis- tically ample samples. Of this part of his work only two fragments have been published, “ A First Study of Natural Selection in Clausilia laminata” (Montagu) and “ Note on a Race of Clausilia itala” (von Martens). In the first of these memoirs he shows that two races of (. laminata exist, in localities so widely separated as Gremsmiihlen and Risborough, with sensibly identical spirals, although no crossing between their ancestors can have existed for an immense period of time, and although there are comparatively few common environmental conditions. At the same time, while no differ- ential secular selection of the spiral appears to have taken place during this period, there yet seems to be a periodic selection of the younger individuals in each generation, the variability of the spirals of the young cells being Walter Frank Raphael Weldon. XXXV sensibly greater than of the corresponding whorls of adults. In other words, _ stability to the type is preserved by selection in each new generation. In the second memoir, Weldon sought for demonstration of a like periodic selection in the ©. itala he had collected from the public walks round the Citadel of Brescia. He failed, however, to trace it, and was forced to conclude that C. itala is either not now subject to selective elimination for this character or is multiplying at present under specially favourable conditions at Brescia, or again, as both young and old were gathered in early spring, after their winter sleep, that elimination takes place largely during the winter, and “that individuals of the same length, collected in the autumn, at the close of their period of growth, might be more variable than those which survive the winter.” The problem of growth, to be studied only under conditions of captivity, possibly modifying the natural growth immensely, has made the crab investigation an extremely complex one. Weldon solved the difficulty by the brilliant idea that the snail carries with it practically a record of its youth. If the wear and tear of the outside of the shell to some extent confuses the record there, a carefully ground axial section will reveal by the lower whorls the infancy of the organism. Hence the days given to experi- mental grinding, the training in manipulation and the final success, and then the steady work, grinding and measuring a few specimens a day, till the necessary hundreds were put together; the laborious calculations not in the least indicated in the papers; and the illustration of how shells may be used— by those who will give the needful toil—to test the truth of the Darwinian theory. ? On November 16, Weldon wrote :— “Do you think it would be too hopelessly expensive to start a journal ot some-kind?.... “Tf one printed five hundred copies of a royal ,8vo. once a quarter, . sternly repressing anything by way of illustration except process draw- ings and curves, what would the annual loss be, taking any practical price per number? .... If no English publisher would undertake it at a cheap rate, the cost of going to Fischer, of Jena, or even Engelmann _ would not be very great.” This was the first definite suggestion of the establishment of ‘ Biometrika.’ On November 29, the draft circular, corresponding fairly closely to the first editorial of the first number, reached his co-editor from Oxford with the words: “Get a better title for this would-be journal than I can think of!” The circular went back to Oxford with the suggestion that the science in future should be called Biometry, and its official organ be ‘ Biometrika.’ And on December 2, 1900, Weldon wrote ;— “J did not see your letter yesterday until it was too late for you to have an answer last night. I like ‘ Biometrika’ and the sub-title.” EXXVI Obituary Notices of Fellows deceased. Thus was ‘ Biometrika’ born and christened. The reply to circulars issued during December was sufficiently favourable to warrant further proceedings. By June of 1901 its publication through the Cambridge University Press had been arranged for, and the sympathetic help of the Syndics and the care given by the University Printers enabled us to start well and surmount many difficulties peculiar to a new branch of science. During the years in which Weldon was co-editor with Karl Pearson he contributed much, directly and indirectly, to its pages. He was referee for all essentially biological papers; and his judgment in this matter was of the utmost value. He revised and almost re-wrote special articles. He was ever ready with encouragement and aid when real difficulties arose. Starting on October 16, 1900, and extending throughout the early ‘Biometrika’ letters to his co-editor, runs a flood of information with regard to Mendel and his hypothesis. “ About pleasanter things: I have heard of and read a paper by one Mendel, on the results of crossing peas, which I think you would like to read. It is in the ‘Abhandlungen des naturforschenden Vereines in Brunn’ for 1865. I have the R.S. copy here, but I will send it to you if you want it.’—(October 16, 1900.) Then follows a résumé of the first of Mendel’s memoirs, and for montbs the letters—always treatises—are equally devoted to snails, Mendelism, and the basal things of life. , The earlier part of 1901 was chiefly occupied by snails, but a new factor had come into Weldon’s many-sided occupations. It was settled that ‘Biometrika’ should have in an early number a critical bibliography of papers dealing with statistical biology. Weldon undertook the task of preparing it, as his study of Mendel had led him to a very great number of such papers dealing with inheritance, and the section on Heredity was to be published first. Like all his projects, it was to be done in so thorough and comprehensive a manner that years were required for its completion. A very full list of titles was formed, especially in the Inheritance section, and many of the papers therein were thoroughly studied and abstracted. But such study meant with him not only grasping the writer’s conclusions, but testing his arithmetic and weighing his logic. Thus Weldon’s note on “Change in Organic Correlation .of &icaria ranunculoides during the , Flowering Season,” arose from this bibliographical work and the erroneous manner in which he found Verschaffelt and MacLeod dealing with correla- tion. A further result of this work was that his confidence in the generality of the Mendelian hypotheses was much shaken. He found that Mendel’s views were not consonant with the results formulated in a number of papers he had been led to abstract, and that the definite categories used by some Mendelian writers did not correspond to really well-defined classes in the characters themselves. To those who accept the biometric standpoint, that, in the main, Walter Frank Raphael Weldon. XXXVII evolution has not taken place by leaps, but by continuous selection of the favourable variation from the distribution of the offspring round the ancestrally fixed type, each selection modifying pro rata that type, there must be a manifest want in Mendelian theories of inheritance. Reproduc- tion from this standpoint can only shake the kaleidoscope of existing alternatives; it can bring nothing new into the field) To complete a Mendelian theory we must apparently associate it for the purposes of evolution with some hypothesis of “mutations.” The chief upholder of such an hypothesis has been de Vries, and Weldon’s article on “ Professor de Vries on the Origin of Species” was the outcome of his consideration of this matter. During the years 1902 and 1903 an elaborate attempt was made to grow the numerous sub-races of Draba verna, with the idea that they might throw light on mutations. .The project failed, largely owing to difficulties in the artificial cultivation of some of the species. But for a time all other interests paled before Draba verna. A study of the work of von Guaita had convinced Weldon, early in 1901, that the cross between the European albino mouse and the Japanese waltzing mouse was not one which admitted of simple Mendelian description. In May, 1901, his letters contain inquiries as to Japanese mice dealers. During the summer and autumn the collection of Japanese mice was in progress. These mice were to be bred to test the purity of the stock ; during December about forty does had litters, and pure breeding went on until the autumn of 1902, when hybridization commenced. The work on these mice was for two years entrusted to Mr. A. D. Darbishire, but the whole plan of the experi- ments, the preparation of the correlation tables, and the elaborate calcula- _ tions were in the main due to Weldon. On Mr. Darbishire’s leaving Oxford, Weldon again resumed personal control of the actual breeding arrangements, and from some second hybrid matings carried on the work to the sixth hybrids’ offspring. The work was nearing completion at his death, and through the energy of Mr. Frank Sherlock, the skins of the 600 pedigree mice forming the stud at that time have been dressed and added to those of the earlier generations. Weldon had this work much at heart, and his letters during 1904 and 1905 give many indications of the points he considered demonstrated. The experimental part of the work would have been nearly coinpleted had not his whole thought and energy been directed from November, 1905, into another channel. In the summer ‘ Biometrika’ was edited from Bainbridge in Wensleydale, and the co-editors cycled to the churchyards of the Yorkshire daies, collecting material for their joimt paper “On Assoriative Mating in Man” (34). From Bainbridge, the Weldons went to the British Association meeting at Belfast, where an evening lecture on Inheritance was given. At Christmas came one of the above-mentioned visits to Palermo to collect Sicilian snails. : In the spring of 1903, Weldon was busy, as were the whole available members of the biometric school, in studying the influence of environment d 2 XXXVIil Obituary Notices of Fellows deceased. and of period of season on the variation and correlation of the floral parts of Lesser:Celandine. & “Give my love to the Brethren who are co-operating in the matter of Celandines, and beseech them to make a better map of their country than the enclosed.”—(Oxford, February 23, 1903.) Weldon threw his whole energy and love of minute exactitude into the task, and his letters are filled with an account of the almost daily changes in the type and variability of the Celandine flowers from his selected stations. The result of this enquiry was the collection of an immense amount of data showing that environment and period in the flowering season affected the flower characters to an extent comparable with the differences attributed to local races. At Easter of 1903 a series of mishaps prevented the common holiday, but this was more than compensated for by the summer vacation. The Weldons started with a sea trip to Marseilles and back. They then returned to Oxford, in order that work on the article “Crustacea”-for the Cambridge Natural History might be carried on, and that an eye might be kept on the mice. The data on assortative mating in man collected in the previous year were reduced and a joint paper sent to press; the immense amount of calculation and reduction involved in the mouse-paper was got throuvh ; a joint criticism of Johannsen’s “ Ueber Erblichkeit in Populationen und in reinen Linien” was written by the co-editors, under the title “ Tnheritance in Phaseolus vulgaris,” and a joint study was made, at Weldon’s suggestion, of the relationship between Mendelian formule and the theory of ancestral heredity. It was shown that there was no essential antagonism between the two methods of approaching the subject, and the results were published ultimately at Part XII of the “ Mathematical Contributions to the Theory of Evolution,” Weldon persistently declining to allow it to appear as a joint memoir, because he had taken no part in certain portions of the more complicated algebraic analysis. Christmas found the Weldons in Palermo on the snail quest. His letters thence to his co-editor teem with the fresh- ness of the sky and the joy of open-air work. 3 “Out between five and six, in the dark, without any breakfast, sunrise up in the hills, a day’s tramp on a piece of bread and a handful of olives, and home at seven, laden with snails. Then after dinner to clean the beasts. That is not work, and it makes one very fit, but one gets tired enough to sleep when the snails are cleaned.” At the beginning of 1904 the work on the Brescia Clauwsilia was in progress, the mice were multiplying after their kind, and Weldon’s thoughts were turning more and more to a determinal theory of inheritance, which should give a simple Mendelism at one end of the range and blended inheritance at the other. The summer found the Pearsons twelve miles from Oxford, at Cogges, near Witney, and the Galtons, twenty miles further, at Bibury; there was much cycling to and fro, and the plan of a new book Walter Frank Raphael Weldon. XXXI1X by Weldon on Inheritance was drafted, and some of the early chapters were written. ; The book on Inheritance occupied most of the remainder of the year, and to aid it forward and help those of us who were not biologists to clearer notions, Pearson suggested to Weldon a course of lectures in London to his own group of biometric workers. The project grew, other departments of the College desired to attend, and ultimately the lectures were thrown open to all members of the University and even to the outside public. The lecturer had a good audience of more than a hundred, and enjoyed the return to his old environment. The letters of Weldon to both Francis Galton and Pearson during the years 1904 and 1905 are full of inheritance work, the details of the great mice-breeding experiment, the statement and the solution, or it might be the suggested solution, of nuclear problems leading to detrimental theories ot inheritance. Occasionally, there would be a touch of conscience, and the drawings for the Crustacea would be pressed forward :— “T ought to give my whole time to the ‘Cambridge Natural History ’ for a while. They had been very good to me, and I have treated them more than a little badly. I am rather anxious to get them off my conscience.’—(Oxford, February 15, 1905.) But only the chapter on “ Phyllopods ” was completed, figures and all, and was set up in type. Many figures were prepared for other parts; beautiful things, which gave Weldon not only scientific but artistic pleasure, he had made, but the text remains a mere fragment. In the same way but little was absolutely completed of the article on “ Heliozoa” for Lankester’s “Natural History.” It was not Weldon’s biometric friends who kept him from these tasks, but solely his own intense keenness in the pursuit of new knowledge. The fascination of inheritance problems kept him, however, for months at a time at the Heredity book. At Exeter, 1905, he went to Ferrara because that place had a university, and as such must have a library, where work could be done. The contents of the library were perfectly medieval, a characteristic appropriate in the castle, but hardly helpful in heredity. Still, portions of the manuscript came to England for comment and criticisms, and we were hopeful that the end of the year would see the book completed. It must not be thought for a moment that Weldon was desultory in his work. As Sir Ray Lankester says in a letter: “His absolute thoroughness and unstinting devotion to any work he took up were leading features in his character.” He pursued science, however, for sheer love of it, and he would have continued to do so had he been Alexander Selkirk on the island with no opportunity of publication and nobody to communicate his results to. He never slackened in the energy he gave to scientific work, but having satisfied himself in one quest he did not stay to fill in the page for others to x] Obituary Notices of Fellows deceased. read; his keen eye found a new problem where the ordinary man saw a cow-pasture, or a dusty hedgerow, and he started again with unremitted ardour to what had for himself the greater interest. In the summer the Pearsons were at Hast Ilsley, some seventeen miles from Oxford, and there was cycling out several times a week; there was steady joint work on the determinantal theory of inheritance as suited by Weldon, which, it is to be hoped, is sufficiently advanced to be completed and published. He had in August, 1905, given to the Summer Meeting of the University Extension in Oxford a lecture on “ Inheritance in Animals and Plants,” and this had taken up some of his energy during the summer vacation. Qn the whole, however, he worked persistently at the Inherit- ance book. It cannot be denied that those who were often with Weldon during the last two years were occasionally anxious on his account. The pace at which he worked had been too great—but at no time was it definitely realised that there was cause for immediate alarm. His intellectual activity was never ‘apparently diminished, and his long cycling rides were maintained to the end; though an occasional, but never long persistent, lack of the old joyous- ness of life was noticeable. In November, 1905, Weldon was unfortunately taken off from the work on his Inheritance book by the presentation to the Royal Society of a paper by Captain C. C. Hurst, “On the Inheritance of Coat-Colour in Horses.” He had had no proper summer holiday, but he threw himself nine hours a day into the study of “The General Studbook.” “T can do nothing else until I have found out what it means . The question between Mendel and Galton’s theory of Reversion ought to be answered out of these. Thank God, I have not finished that book. There must be a chapter on Race Horses!” He promised to communicate a note to the Society involving details of his inquiry. This was done on January 18, 1906, in a “Note on the Offspring of Thoroughbred Chestnut Mares.” “The object of the present note is partly to fulfil my promise and partly to call attention to certain facts which must be considered in the attempt to apply any Mendelian formula whatever to the inheritance of coat-colour in race horses.” Here it can only be said that he took up the subject with his usual vigour and thoroughness. But he was overworked and overwrought, and a holiday was absolutely needful. He went to Rome, but the volumes of the Studbook went with him. His letters are filled with Studbook detail till Easter, with hardly a reference to anything else. Re-reading them now, one sees how this drudgery, with no proper holiday, told on him. Hundreds of pedigrees were formed, and a vast amount of material was reduced. At Easter, he and his wife went to the little inn at Woolstone, at the foot of the White Horse Walter Frank Raphael Weldon. xli Hill, and his co-editor came down later to Longcot, a mile away, for the joint vacation. Weldon, still hard at work on the Studbooks, was intellectually as keenly active as of old; and was planning the lines of his big memoir on coat-colour in horses, and showing how they illustrated the points he had already found in the mice. This extraordinary mental activity was now telling upon a constitution never very robust, but the end came with startling suddenness. A day or two of slight illness at Woolstone, which, as usual, he made nothing of, was followed by a visit alone to London on Wednesday, April 11. Here he was taken seriously il, and within a few hours he died of pneumonia, on Good Friday, April 13, 1906. So passed away, not unfitly—for it was without any long disabling illness and in full intellectual vigour—a man of unusual personality, one of the most inspiring and loveable of teachers, the least self-regarding and the most helpful of friends, and the most generous of opponents. And lastly, as to Science, What will his place be? The time to judge is not yet. Much of his work has still to be published, and this is not the occasion to indicate what Biometry has already achieved. The movement he aided in starting is but in its infancy. It has to fight not for this theory or that, but for a new method and a greater standard of logical exactness in the science of life. To those who condemn it out of hand, or emphasise its slightest slip, we can boldly reply, “ You simply cannot judge, for you have not the requisite knowledge.” To the biometrician, Weldon will remain as the first biologist who, able to make his name by following the old tracks, chose to strike out a new path—and one which carried him far away from his earlier coileagues. It is scarcely to be wondered at if those he joined should wish to see some monument to his memory; for he fell, the volume of life exhausted, fighting for the new learning. CON TE Na Se IDENRY “BAKER TRISTRAM Soca. coemegeeenee ake Bawee cco os ven iene sie s teat: ee neaee PNDERED: NEWTON See ctece cas ctcchvacesseeecacneeeenene catenins uiien tecca se eteaener Sir JoHn Evans, K.C.B............ uals eaten slowaiBirale seletetovetie alartemrer cleat ten smote ELEN Rive CLIFTON: SORBY «.ccrcente souserecccseeceman eens Weegee Casitas begat SIR JOSEPH FAYRER ......... «Gib aTSTSIET w nis eters ak 4'dln arS'ase SACRED Ge Ea Ri oe Walter Frank Raphael Weldon. xh _ Hill, and his co-editor came down later to Longcot, a mile away, for the joint vacation. Weldon, still hard at work on the Studbooks, was intellectually as keenly active as of old; and was planning the lines of his big memoir on coat-colour in horses, and showing how they illustrated the points he had already found in the mice. This extraordinary mental activity was now telling upon a constitution never very robust, but the end came with startling suddenness. A day or two of slight illness at Woolstone, which, as usual, he made nothing of, was followed by a visit alone to London on Wednesday, April 11. Here he was taken seriously ill, and within a few hours he died of pneumonia, on Good Friday, April 13, 1906. So passed away, not unfitly—for it was without any long disabling illness and in full intellectual vigour—a man of unusual personality, one of the most inspiring and loveable of teachers, the least self-regarding and the most helpful of friends, and the most generous of opponents. And lastly, as to Science, What will his place be? The time to judge is not yet. Much of his work has still to be published, and this is not: the occasion to indicate what Biometry has already achieved. The movement he - aided in starting is but in its infancy. It has to fight not for this theory or that, but for a new theory and a greater standard of logical exactness in the science of life. To those who condemn it out of hand, or emphasise its slightest slip, we can boldly reply, “ You simply cannot judge, for you have not the requisite knowledge.” To the biometrician, Weldon will remain as the first biologist who, able to make his name by following the old tracks, chose to strike out a new path—and one which carried him far away from his earlier colleagues. It is scarcely to be wondered at if those he joined should wish to see some monument to his memory; for he fell, the volume of life exhausted, fighting for the new learning. VOL, LXXX.—B. Y xlu HENRY BAKER TRISTRAM, 1822—1906. THe Rev. Henry Baker Tristram, long familiarly known to naturalists all over the world as Canon Tristram, was born on May 11, 1822, at Eglingham, near Alnwick, of which large parish his father was vicar. He received his early education at Durham School, and passed to Lincoln College, Oxford, where he graduated in 1844, taking a second class in classics. In the following year he was ordained Deacon, and shortly afterwards became Curate of Morton Bishop. He had not been long engaged in his clerical duties when he developed such signs of a weak chest that it was judged expedient to send him abroad. Accordingly, in 1847, he received the appointment of Acting Naval and Military Chaplain at Bermuda, and held it for two years. That he had been from early boyhood an ardent lover of nature and a keen collector of plants and animals cannot be doubted. But it was probably during his residence in Bermuda that his future career as a naturalist took a definite beginning. Among the officers of the 42nd Highland Regiment, quartered there at the time, was Henry Maurice Drummond (brother of Drummond of Megginch), who had been stimulated into active natural history pursuits by coming under the influence of Hugh Edwin Strickland, until he made himself more than a mere amateur ornithologist. Tristram caught from him the same spirit of scientific observation, and took up the study of shells and birds in the serious way which he never afterwards abandoned. On his return to England, in 1849, he was presented to the living of Castle Eden, in Durham, and in 1850 married a daughter of P. Bowlby, an officer who had served in the Peninsular and Waterloo campaigns. Hight children were born of this marriage, consisting of one son and seven daughters. To the duties of a country clergyman he for some years added those of tutor, and took pupils (with whom he made occasional excursions to the Continent, travelling one year along the West Coast of Norway as far as the Arctic Circle). The lung affection, however, which had necessitated his seeking the warmer climate of Bermuda, again returned upon him. He was advised to spend a winter in Algeria. From this change, which he took in the winter of 1855—56, he received so much benefit that he repeated his visit next winter, and he used to refer to these two sojourns in Africa as the prime cause of his being able to throw off the ailment which had threatened him. Having formed a friendship with the French Governor-General, he was enabled to push his explorations to the furthest French outposts, and beyond these far into the desert, living almost all the time under canvas. In the year 1857 he was joined there by Mr. W. H. Hudleston and the late Mr. Osbert Salvin. This party succeeded in making large ornithological collections, which proved to be of great interest. During the following year (1858—59) Tristram travelled widely in the Eastern Mediterranean basin, including Palestine and Egypt. Henry Baker Tristram. xii In 1860 he became Master of Greatham Hospital and Rector of Greatham, and held these appointments until 1873 when, having obtained a Canonry in Durham Cathedral, he removed to Durham, which thereafter became his home until the close of his life. But his love of travel led him to return again and again to the East in order to gather fresh material illustrative of its geology and natural history. He renewed his acquaintance with Palestine in 1863—64, and again in 1872. In 1881 he travelled through Mesopotamia and Armenia. In 1891 he visited China, Japan, and the North-West of North America. In 1894 he was again in Palestine, and once more in 1897, at the age of seventy-five. On his last visit he had his leg broken by a kick from a horse when riding near Jerusalem, but such was his irrepressible vitality. that, after a few weeks in hospital, he reappeared as hale and hearty as ever. Throughout all these extensive wanderings Tristram showed the true instincts of a born naturalist, cultivated and enlarged by wide and constant experience. To him we are mainly indebted for our knowledge of the plants and animals of Palestine and the surrounding countries. His papers on the ornithology of Northern Africa, which appeared in the ‘Ibis’ for 1859 and following years, were important additions to what had previously been known on the subject. His frequent journeys through Palestine allowed him to acquire an unrivalled acquaintance with the geology, topography, and natural history of that country, and he gathered together an admirable account of his observations in his great work on the ‘Fauna and Flora of Palestine, which was published by the Palestine Exploration Fund in 1884. His scientific labours and his descriptive powers, however, were made more widely known by the separate volumes which appeared from his facile pen in successive years. The first of these, ‘The Great Sahara,’ published in 1860, at once established his place as an accomplished traveller and observant naturalist. It was followed by a series of attractive narratives of his wanderings through Palestine. His friend, the late Professor Alfred Newton, remarked that “ Tristram’s study of the ‘desert forms’ of the birds induced him to declare in the ‘Ibis’ for 1859 (p. 429) his conviction ‘of the truth of the views set forth by Messrs. Darwin and Wallace in their communication to the Linnean Society,’ adding that ‘it is hardly possible, I should think, to illustrate this theory better than by the larks and chats of North Africa.’ Three or four pages follow in which special examples are cited in illustration, and these were written, if not published, before the appearance of ‘ The Origin of Species,’ so that Tristram appears to have been the first zoologist to accept publicly the principles of Darwinism.” ‘He had to modify his expressions some time after, when the ‘ orthodox’ tide was flowing, just as Galileo was obliged to do, but he held them all the same until the end, and great credit is due to him for this.”* * From MS. notes supplied to the writer by Professor Newton, who also, in ‘ Nature,’ for March 16, 1906, called attention to Tristram’s early Darwinian pronouncement. eng xliv Obituary Notices of Fellows deceased. “In all his voyages and journeys, ornithology received Tristram’s chief attention. Among his discoveries may be especially mentioned that of a starling-like bird, named after him by Mr. Sclater, Amydrus Tristram, — peculiar to the gorge of the Kedron, and belonging to a genus previously thought to be purely Ethiopian. But his collection was not at all confined to specimens obtained by himself or his companions on his travels, extensive as these were; but comprehended the birds of the whole world, and formed one of the largest ever brought together by any private person. It was sold in his lifetime to the Free Public Museum of Liverpool.”* It was described in the Report of the Committee of that institution for 1896 as containing “ 20,000 specimens, referable to 6000 species, of which 150 are types.” ‘Tristram likewise amassed a large and valuable collection of birds’ eggs, which he sold to Mr. Philip Crowley, at whose death it passed by will into the Natural History Museum, South Kensington. Canon Tristram endeared himself to a wide circle of friends by the singular modesty and geniality of his nature, by his keen sense of humour, the great range of his acquirements in natural history, and the delightful flow of his conversation, in which he would draw upon his wide and varied experience in so many different countries. He celebrated his golden wedding in the spring of the year 1901. Two years later his wife died, and he himself, retaining his faculties to the end, passed away on March 8, 1906, at the ripe age of eighty-four. A Joe * MS. of Professor Newton. xlv ALFRED NEWTON, 1829—1907. By the death of Professor Alfred Newton, the ranks of British zoologists have lost one of their most venerable and distinguished ornaments, and Ornithology in particular has been deprived of its most learned and accomplished British representative. Born at Geneva on June 11, 1829, he spent his boyhood with his numerous brothers and sisters at Elveden, an estate on the borders of Suffolk and Norfolk, which belonged to his father. His undergraduate life, which began at Cambridge in 1848, does not appear to have been marked by any conspicuous success in the usual subjects of study, though he is said to have gained a considerable reputation in his college for his English essays. Certainly his literary style gave proof of his having cultivated the humanities. His natural bent, however, was already strongly pronounced towards natural history pursuits, which at that time met with but little encouragement at the university, nor were his tastes favoured by his own family, as they did not seem likely to lead to any kind of successful career. In 1853, however, after having taken his B.A. degree, he was elected at Magdalene College to the Drury Travelling Fellowship, which is open to the sons of Norfolk gentlemen. He was thus enabled to throw himself heart and soul into the active prosecution of science. He went abroad during several years, and made various journeys through Arctic latitudes, studying the abundant bird-life of these regions. Lapland, Iceland, and Spitzbergen were successively visited by him in the course of these wanderings, and, not improbably, he then imbibed that affection for northern forms which distinguished him. He likewise took occasion to cross the Atlantic more than once. In 1857 he was in the West Indies, and went thence to confer with the naturalists of the United States in Philadelphia and Washington. In 1862 he spent some time in Madeira. During those fruitful years of active experience, his ready pen was busy in the description of the facts which he had observed at home and abroad. He communicated his notes to the pages of the ‘ Zoologist’ and ‘ Ibis,’ of which latter journal he was one of the original founders. For a long succession of years his numerous papers in these publications, and in the ‘ Proceedings of the Zoological Society, on the occurrence, distribution, structure, and habits of birds, formed notable contributions to Ornithology. They so fully estab- lished his reputation as an experienced naturalist that in 1866 he was appointed to the newly-created Chair of Zoology and Comparative Anatomy in his own university. Afterwards his college elected him to a Foundation Fellowship. For more than thirty years, up to the time of his death, he lived in the picturesque Old Lodge of Magdalene, surrounded with his books and papers, always busy with important and useful work, delighted to welcome his friends to his den, and constantly on the outlook for oppor- xlvi Obituary Notices of Fellows deceased. tunities of doing a kindness to younger men, especially to those who had tastes akin to his own. His Sunday evening receptions were an important — feature in the scientific life of the University. Not a few of the naturalists who have risen into prominent positions in this country can look back to the stimulus they received from those meetings, where the advantages to be derived from personal intercourse among the younger workers were enhanced by the ever ready sympathy and encouragement of the genial _ professor. Newton was all his life a keen collector. His chief interests lay, of course, among birds, but he had the instincts of a true naturalist, and was always on the watch for specimens in all provinces of the animal kingdom which would help to enlarge and enrich the Museum at Cambridge. He was likewise a lover of books, and his rooms, with their well-filled shelves, showed the wide range of his literary tastes, and the success with which he had pursued the quest after rare and valuable works in natural history. He was, above all, a philosophical naturalist, intent rather on the higher and broader questions than on details of species or of structure. He was endowed, too, with a highly critical faculty, and could express his criticisms with pungent clearness. He could never be satisfied with anything less than the completest accuracy attainable, while his literary instinct led him to cultivate great simplicity of style, in which every word was well chosen, and none was redundant. These characteristic features of the Cambridge professor are specially to be noted in the numerous essays which he wrote on questions of large biological interest, such, for example, as the series of articles from his pen which appeared in the ninth edition of the ‘ Encyclopedia Britannica, and which formed the basis of his greatest work, the ‘Dictionary of Birds.’ This ornithological classic, issued in successive parts from 1893 to 1896, was prefixed with a Latin inscription to his youngest brother, Edward, who “ for more than fifty years had been his most assiduous fellow-student in orni- thological pursuits at home, abroad, under the open sky, and in caves.” It shows his critical acumen alike in what he selected for treatment and in what he omitted. His habitual caution is well illustrated by his choice of an alphabetical rather than a taxonomic arrangement of his subject, while the occasionally caustic force of his language is displayed in his preface, where he denounces some attempts at systematic arrangement as “among the most fallacious, and a good deal worse than those they are intended to supersede.” “JT have no wish,” he adds, “ to mislead others by an assertion of knowledge which I know no one to possess.” Alfred Newton was one of the first naturalists in this country to give in his adhesion to the views propounded by Charles Darwin as to the origin of species. A few years after the publication of these views he contributed to the ‘ Proceedings of the Zoological Society ’ (1863) an interesting confirmation and illustration of Darwin’s remarks on the way in which seeds may be dispersed by birds, describing the case of a partridge which had been found Alfred Newton. xlvi with its foot firmly imbedded in a lump of hardened earth. In the address which he gave to the Department of Botany and Zoology at the meeting of the British Association in 1876, while praising the then recently published volume by Alfred Russell Wallace on “The Geographical Distribution of Animals,” he emphatically refers to the modern theory of evolution as worthy of “the chief glory in giving a real and lasting value to the interpretation of the facts of animal distribution.” The subject of the distribution of plants and animals over the surface of the globe was one to which Newton devoted much thought, and on which he wrote with his characteristic breadth, caution, and critical discernment. His treatment of the “ Geographical Distribution of Birds ” in the ninth edition of the ‘Encyclopedia Britannica’ may be referred to as an excellent example of the way in which he looked at such questions of wide biological bearing. He naturally took a deep interest in everything connected with the extinction of species. In the address to the British Association above referred to, he drew a vivid and humorous picture of the effects of human interference with the economy of nature, picturing the consequences of man’s occupation of an island, as seen in the destruction of its indigenous fauna and flora, and their replacement by the animals and plants introduced by him—pigs, goats, rats, rabbits, ferrets, sparrows, and starlings. He entered an eloquent plea for an endeavour to protect and preserve the native forms, and he claimed that the naturalist alone had the knowledge that should guide the efforts to promote the use and prevent the abuse of the animal world. Unfortunately, though something has since been done in the direction pointed out by him, the indiscriminate slaughter, which he so feelingly deprecated, still goes on in various parts of the world. That this subject Jay near to Newton’s heart was shown by his returning to it in his admirable article on “Extermination ” in the ‘ Dictionary of Birds.’ One who contemplated with such keen regret the approaching extermina- tion of many remarkable forms of life could not but feel a saddened interest in those which have disappeared within the times of human experience. Newton was a diligent collector of all the information that could be obtained regarding the Dodo. He wrote a number of papers on this subject, and his article on it in his ‘ Dictionary’ may be cited as an illustration of the learning and the exhaustive treatment with which he could discuss a matter that strongly appealed to him. In the same way he devoted himself to tracing out all that could be ascertained regarding the haunts of the Great Auk or Gare Fowl, so recently exterminated. He published several papers on the subject, and one of the objects of his last yachting cruise was to visit the ledge among the Orkney Islands, where the bird had its latest British home. For many years during the later part of his life Newton had an annual opportunity of enjoying pleasant and easy travel, and of visiting some of the most crowded haunts of bird life in these islands. His friend, Henry Evans, of Derby, also an accomplished ornithologist, gladly welcomed him on board his steam yacht and directed her course to any coast or island that the xlvii Obituary Notices of Fellows deceased. Professor wished to see. Year after year “ Alfred the Great,” as Evans used playfully to call him, was received with open arms not only by his host, but by every member of the crew. And no one could look forward with keener zest to these holidays than Newton, when for some weeks he could escape from the cares of University life to the firths and sounds of the west and north of Scotland, where no letters could reach him, even if he had left an address behind him, which he was generally careful not to do. Nowhere could he be seen to be more completely in his element than on board of the “ Aster.’ He loved the sea and its associations with such a sturdy affection that inclemencies of weather, by no means infrequent in those regions, never drew from him the least sign of impatience, or seemed in any degree to disturb his habitual cheeriness and his enjoyment of the cruise. Clad in the light-grey tweed suit which did duty on these voyages, but without top-coat or waterproof, he would sit for hours on some exposed part of the vessel, smoking innumerable pipes and watching for every variety of sea-fowl that might show itself either in the air or on the water. In the course of a few days sun, wind, rain and salt spray told on his complexion, which then assumed a ruddiness that would have astonished the inmates of Magdalene College. The sharpness of his eyesight in the detection of birds on the wing, even when he had nearly reached the age of seventy years, was always an astonish- ment to his companions. And the enthusiasm with which each fresh form was greeted by him as it flew overhead became infectious to all on board. Most of the crew reappeared year after year from their winter employments to take their places in the annual cruises, and some of them became almost as cunning in bird-life as their master. In successive seasons Newton was in this way enabled to visit almost every bay and sea-loch from the Mull of Cantyre to the furthest promontory of the Shetlands. He repeatedly anchored at St. Kilda, and had excellent opportunities of seeing there at the height of the nesting season the most marvellous and varied crowds of sea-fowl anywhere to be found among the British Islands. Nor were the voyages confined to the Scottish coast. He one year sailed round the whole of Ireland, and was thus enabled to compare the bird-haunts of the Irish cliffs with those of Scotland. Twice the yacht carried him round the Faroe Islands, and afforded him a further display of that boreal bird-life which from his young days had such charms for him. These cruises formed an important element in Newton’s life during his later years. He looked forward to them with almost boyish exuberance and delighted afterwards to recount their varied incidents. They not. only provided a healthful and delightful holiday, but kept him still in close personal touch with birds, which had been the main interest and study of his life. In spite of the lameness which was understood to have been the result of an accident during infancy, he was often the first to enter the boat which had been got ready for a landing on some surf-beaten rock, or for a closer inspection of the caves and stacks at the foot of a bird-haunted precipice. Alfred Newton. xlix On such occasions, so self-dependent was he, he would gently repel offers of the assistance which was always at his service. It was only when the increasing feebleness of his limbs would have made such assistance indis- pensable that he reluctantly gave up the annual cruise. Continuing to hold the zoological professorship for the long space of forty- one years, taking also an active part in the conduct of general business, Alfred Newton became a distinct living force in the University. To him should be ascribed no small share in fostering the rise and progress of the natural sciences towards a recognised place in the scheme of studies of Cambridge. His scientific reputation in the world outside was sustained within the walls of the University by the stimulating and suggestive form of his teaching, by his enthusiastic devotion to the development of the Museum of Zoology, and by his untiring but not obtrusive advocacy of the claims of science. But his wide and beneficent influence in Cambridge sprang also in large measure from his strongly-marked personality, wherein kindness, courtesy, and fidelity, were combined with a fearless independence, an impatient antagonism to untruthfulness in every shape and degree, and a habit of frankly and forcibly expressing his convictions. A, G: SIR JOHN EVANS, K.C.B. 1823—1908. THE death of Sir John Evans has removed from the Royal Society one who for forty years has been among its most conspicuous members, who for half of that long period filled the office of Treasurer, and who from first to last has taken an active and useful part in the general business of the Society. His eminent capacity in the conduct of affairs, the unremitting devotion with which he employed that talent in the Society’s interest, and the genial courtesy which marked his intercourse with the Fellows have given him a strong claim on their grateful remembrance. He came of a stock wherein both science and literature had been cultivated. His grandfather, Lewis Evans (1755—1827), the first mathematical master in the Royal Military Academy, Woolwich, studied astronomy and was elected into the Royal Society in 1823. His father, the Rev. Dr. Arthur Benoni Evans (1781—1854) was headmaster of the Grammar School at Market Bosworth and a prolific writer, who published many poems and theological works, together with a book on ‘ Leicestershire Words, Phrases, and Proverbs’ (1848). His maternal grandmother belonged to a Huguenot branch of an old French family, and from her he perhaps inherited his lightness of heart. He was born on 17th November, 1823, at Britwell Court, Burnham, Buckinghamshire, and was educated under his father at Market Bosworth. Although entered for matriculation at Brasenose College, Oxford, he did not. eventually proceed to the University. His education, however, under the paternal roof had been excellent. He had acquired such a knowledge of Latin and such an acquaintance with classic authors as remained a life-long possession to him. Every now and then, in the course of conversation, some happy phrase or line from a Roman poet would occur to him, with which he would light up and enforce the remarks he was making. His archeological writings indicate how diligently he sought in ancient literature such references as might illustrate the early history of mankind. His father’s care in his upbringing was further shown by his being sent for a short time to Germany, in order to gain some facility in speaking the language. ; Instead of entering the University, he, in 1840, at the age of seventeen, embarked on a commercial career. His maternal uncle, John Dickinson, the head and founder of the well-known firm of paper-makers of that name, and a man of scientific tastes, who became a Fellow of the Royal Society, invited him to join the staff at the paper-making works of Nash Mills, near Hemel Hempstead. John Evans found there the settled home in which he lived almost up to the end of his life and which became more widely known as the abode of the active and enthusiastic antiquary, numismatist, and geologist than as the headquarters of a commercial company. Having married his cousin, the daughter of the head of the firm, he was, in 1851, admitted as one Sir John Evans, K.C.B. hi of the partners, and in course of time he in turn became the senior member of the firm. To those who met him only in his business relations he wa# an active and enlightened paper-maker, keenly alive to every modern improve- ment in machinery and in the processes of manufacture, gifted with great clearness of judgment and remarkable capacity for mastering the most complicated details of business. His energy and initiative largely con- tributed to the success of the various enterprises of the firm. For many years he was President of the Paper Makers’ Association, and took a leading part in the conduct of its affairs. But while thus sedulously attentive to commerce he found leisure to gratify his strong bent towards the study and collection of antiquities and the prosecution of several branches of scientific enquiry. His taste for geology seems to have been developed even in boyhood, for he is said, when nine years old, to have hammered a collection of fossils out of the Wenlock limestone quarries at Dudley. But his geological proclivities were eventually drawn in two main directions, partly by the requirements of his business and partly by his love of antiquities. In paper-making an ample water supply is essential, and in Hertfordshire the subject of water-rights has long been keenly discussed. Evans, in the interest of his firm, studied the question of water-supply, both from the geological and the meteorological side, and he became on these matters a recognised authority, whose advice was often sought and always valued. No one stood up more stoutly and successfully than he for the conservation of the water-supply of his county, which was again and again threatened by the great metropolitan water companies. This important question being thus forced on his attention by pressing practical considerations, he devoted much time to its study. He explored the super- ficial deposits in all parts of his district as well as the water-bearing strata that lie deeper underground. In the course of these enquiries he was led to investigate the relations between rainfall and evaporation, and the percolation of rain through soil—subjects regarding which little information was available at the time when he began his researches. From the year 1853 he had under his own immediate care the rain-gauges and percolation-gauges which had been erected at Nash Mills in 1836 by his uncle. He was drawn into the geological field by another and different pathway. In the first decade of the latter half of last century the discovery of what were alleged to be implements of human fabrication in the old river gravels of the north of France gave rise to a keen discussion among men of science. The conclusion, which some of the early observers drew from the evidence, that man must have lived on the earth for a far longer period than had generally been supposed, naturally aroused much interest among the general public. As far back as 1841 Boucher de Perthes had obtained from the old gravel terraces of the valley of the Somme, at Abbeville, numerous chipped flints which he recognised to be the handiwork of man. In 1847 he began to publish his observations, but they met with little or no support among his fellow countrymen. On the contrary, they were either ignored or hi Obituary Notices of Fellows deceased. denied and even derided, though one or two competent French geologists were convinced of their probable truth. It was not until the autumn of 1858 that Hugh Falconer, who then saw the collection made by Boucher de Perthes at Abbeville and was satisfied that the shaped flints were truly human implements, urged Joseph Prestwich to undertake an examination of the geology of the valley of the Somme, with the view of determining the precise position of these implements and of ascertaining whether or not there was evidence to prove their high antiquity. This task was accomplished in the spring of 1859 by Prestwich, who took Evans with him to assist in the investigation. The conjoint labours of these two observers, which completely demonstrated the accuracy of the French discoverer’s observations and conclusions, formed the first important step in winning general acceptance to the opinion, which had been so stoutly contested, that the human race, together with various tribes of animals that have been long extinct, must have inhabited Western Europe for a long succession of ages, wherein the rivers cut their way deeply into the valleys which they traverse. Prestwich communicated his results to the Royal Society, while Evans submitted a statement on the subject to the Society of Antiquaries, which had elected him one of its number in 1852. This paper appeared in the ‘ Archeologia ’ (vol. 38, 1860, p. 280), under the title of “Flint Implements in the Drift; being an Account of their Discovery on the Continent and in England.” The journey with Prestwich formed the turning-point in Sir John’s scientific career. From that time onwards he specially devoted himself to the investigation of the earliest traces of man which have been preserved in river-gravels, brick-earths, cavern deposits, or elsewhere. He became one of the most enthusiastic and successful collectors of flint implements. His singularly good powers of observation enabled him to detect them even on ground that had been already searched for them, and in any company of hunters for these objects he was generally the most fortunate. Even on a surface so long inhabited as that of Egypt his trained eyes enabled him to pick them up. Both abroad and at home he purchased freely every illustrative type which he could procure, until in the end he had amassed such a series of these objects as is probably possessed by no other private collector. Throughout his life he continued to publish from time to time notices of the progress of discovery in regard to the occurrence and distribution of flint implements. So recently as December, 1907, he communicated to the Geological Society what proved to be his last paper, on “Some Recent Discoveries of Flint Implements,” wherein he expressed his matured opinions regarding the probable origin of the high-level gravels in which these relics of primitive man have been found. But besides writing these scattered papers, Evans rendered a great service to the progress of archeology by his published’ volumes, in which he gathered together all the evidence which had been accumulating in different countries as to the types and distribution of the various relics of early human work- manship. The first of these separate works appeared in the summer of Sir John Evans, K.C.B. laa 1872 with the title of ‘The Ancient Stone Implements, Weapons, and Ornaments of Great Britain. It at once took its place as the chief authority on the subjects of which it treats. The learning displayed in its earlier chapters, the careful arrangement of its material, its detailed yet interesting descriptions, and the importance attached throughout its pages to the stratigraphical position in which the relics had been found showed it to be no mere antiquarian enquiry but a treatise conceived and executed on thoroughly scientific lines. It had an important influence in connecting the pursuits of archeology and geology, by the way in which it marshalled the evidence for a chronological sequence in the relics of early man, and showed that the conclusions derived from a consideration of varieties in types of workmanship were supported by the geological evidence derivable from the positions in which these several types were found. In the midst of the numerous and multifarious duties which claimed his constant attention he brought out a second edition of the work in 1897, greatly revised, and incorporating a large amount of new material. Although Sir John Evans chiefly occupied himself with the archeological side of geology, he occasionally ventured into other parts of the geological domain, and his incursions of this kind were always marked by the same quickness of insight and shrewdness of inference. It was he, for instance, who first detected that the toothed jaw which lay detached on the same slab of stone that contained the original specimen of 0 “shop pp burary O-% < |” 098%. | “88 < a ‘4Nsor eatyedout YIIA ‘MOLIVUL PUB PULTS YIIM poye[Noour o10M ; SYBl OMY PUL ‘wa7L0m sod PUNO, 910M OUONT *(FUOUTZBETY JO 4SIy 2h pus) osvastp 014 Jo Avp YTOF OY} BOUTS Tees UeEq PRY soumosouvd £14 : 8 ON. “Perry sea pte ‘Trey pus Boy- -910F S}I FO grad uoyee PRY IBY 0 “ep WILE TO pod rk CLT. 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H. G. Plimmer and J. D. Thomson. [Oct. 28, Sodium Arsenyl Tartrate or Sodiwm Tartrarsenite—This compound was investigated by Henderson and Ewing* in 1895, who gave it the formula AsONaC,H,0¢.24H20. It was prepared for these experiments by dissolving one equivalent of arsenious oxide in two equivalents of acid sodium tartrate, filtering, evaporating to a small volume, and adding alcohol till crystallisation commenced ; on cooling, the substance crystallised out. This substance does not seem to be anything like so effective as the antimony compound. Of five rats which have been treated with it, four died between the 12th and 24th days, and three of these had a recurrence; one is still living on the 21st day, but we think that this is probably due to the sodium antimony] tartrate which was given after a recurrence, in the same way as if from the beginning of the disease. A mixture of equal parts of a 1 per cent. solution of sodium antimonyl tartrate and of sodium arsenyl tartrate has been tried on six rats. One died on the 14th day and the five others are still living on the 21st day without any recurrence. Immunity. With a view of ascertaining what amount of immunity, if any, had been conferred on an animal which we considered to be cured, a Nagana rat was taken which was inoculated on May 13, and had been afterwards successfully treated with atoxyl and succinimide of mercury, and in which no trypano- somes had been found since it had its first dose on May 16, when the trypanosomes were very plentiful in the blood. On October 7, the 147th day, the rat was re-inoculated from another Nagana rat, and on the 11th trypanosomes were present in numbers in the blood; a dose of sodium antimony! tartrate was given and no trypanosomes have been seen since the 12th. This seems to point to the fact that no immunity is conferred. DESCRIPTION OF PLATE. The microphotographs were made from rough blood-preparations, with a low-power objective (Zeiss 8 mm.), in order to demonstrate the rapid disappearance of the trypano- somes from the blood after administration of sodium antimonyl tartrate. Fie. 1 shows the blood of a Nagana rat, 4 days after inoculation, before treatment. Fic. 2 shows the blood from the same rat half an hour after the injection of 0°35 c.c. of a 1 per cent. solution of sodium antimony]! tartrate. Fic. 3 shows the blood from the same rat one hour after administration of the above dose * Henderson and Ewing, ‘Chem. Soc. Trans.,’ 1895, vol. 67, p. 103. and Thomson. Roy. Soc. Proc., B. vol. 80, Plate 1. Fig. 1. Fic. 3. 1907:] Experimental Treatment of Trypanosomiasis in Rats. 11 Note upon Sodium Antimonyl Tartrate, By R. H. Apers Putmer, D.Sc. Lond. Sodium antimony] tartrate was described in 1842 by Dumas and Piria,* who gave it the constitution CsHs0,)Na0,Sb203H20,+ but did not state how they had prepared it. Clarke and Evans{ obtained a compound of the composition 3Na2C,H.0.¢+ 2Sb(OH)3+3H20, in 1883, by saturating tartaric acid with antimony trioxide and neutralising the solution with sodium carbonate. The first compound does not seem to have been prepared again since 1842. Sodium antimonyl tartrate was prepared according to the methods usually given for preparing tartar emetic, by boiling a solution of acid sodium tartrate (13 grammes) with a little more than the calculated quantity (10 grammes) of antimony ‘trioxide until the latter had almost completely passed into solution. On filtering and concentrating the solution to a small volume no crystallisation occurred, but on adding a little alcohol the whole became solid. This was then dissolved in about twice its volume of hot water, and alcohol was added until precipitation commenced, when, on cooling, the sodium antimony] tartrate crystallised out. This compound at the ordinary tempera- ture dries very slowly and has a moist appearance, but when dried i vacuo over sulphuric acid it becomes anhydrous and loses 24 molecules of water of erystallisation, resembling sodium tartrarsenite in this respect. The substance is very easily soluble in water and its solution reacts faintly acid to litmus. 0°7228 gramme air-dried substance lost in vacuo over sulphuric acid 0°0920 gramme H,O = 12°73 per cent. 1:0538 o 3 <5 e 0°1338 gramme H,O = 12°70 per cent. Calculated for C,H,0;NaSb.25H,O. H,O = 12°8 per cent. I. 0°4058 gramme substance dried tm vacuo over sulphuric acid gave 0°2332 gramme Sb,S, and 0°0974 gramme Na,SO,. Ii. 0°3780 gramme substance dried 7m vacuo over sulphuric acid gave 0°2072 gramme Sb,8, and 00912 gramme Na,SO,. Calculated for C,H,0,NaSb. Sb = 39-09 per cent. ; Na = 7°49 per cent. Found :—I. Sb = 39-28 Na 4578 he If. Sb = 39°14 ms Na = 7°81 re * Dumas and Piria, ‘ Liebig’s Annalen,’ 1842, vol. 44, p. 89. + Old notation. ; Clarke and Evans, ‘ Berichte,’ 1883, vol. 16, p. 2385. 12 Messrs. L. Hill and M. Greenwood, Jun. [Oct. 1, When dried at 105° C., the substance loses only two molecules of water of crystal- lisation :— | 06120 gramme air-dried substance lost at 105° C. 0:0640 gramme H,O = 10°46 per cent. Calculated for C,H,O,NaSb.2H,O. H,O = 10°49 per cent. The remaining half molecule is subsequently lost in vacuo over sulphuric acid. 0°6120 gramme substance dried at 105° C. then lost 7m vacuo over sulphuric acid 00110 gramme H,O. Total loss = 12°25 per cent. On exposure to air, the two and a half molecules of water of crystallisation are again taken up, but the salt does not deliquesce. 0°5370 gramme substance dried <2 vacuo over sulphuric acid, on exposure to air ncreased in weight to 0°6066 gramme. The Influence of Increased Barometric Pressure on Man. No. 4.—The Relation of Age and Body Weight to Decom- pression Lffects. By Leonarp Hitt, F.R.S., and M. GREENwooD, Jun., M.R.CS. (Received October 1,—Read December 5, 1907.) Statistics of caissons and diving works tend to suggest that the percentage number of men affected injuriously by exposure to compressed air increases with age. Pol and Watelle (1) record that men between 18 and 26 stood the work best, and that of the 25 men dismissed for illness from the works under their inspection, 19 were over 40 years old, 5 over 30, and 1 over 28 years. Catsaras(2) investigated 62 instances of paralysis among sponge divers, and we find that, of these, 33 were over 30 years old, 17 over 25, 11 over 20, and 1 over 19. These men dived about 140 feet, spent about 10 minutes below, and were decompressed in about one minute. _ Evidently this variation might depend on—(i) the actual age difference ; (ii) on an increase in mean body weight with age; (111) on a combination of (i) and (ii); (iv) it might be purely random. We cannot absolutely exclude (iv) in the instance of caisson works unless we know the total number of men at each age employed, figures which do not seem to be available. Snell (3) gives the following table (Table I) of his observations, made at the Blackwall Tunnel works. Unfortunately, column 2 gives not the total number employed, but only those who submitted themselves to medical inspection, which was not, at first, compulsory. . | 1907.] Influence of Increased Barometric Pressure on Man. 13 _ Table I. Number of men taken - ill whose ages are Number of men examined Proportion of illnesses to Ages. and passed. necawded: every 100 men passed. 15—20 55 0 0 20—25 145 15 10°3 25—30 152 37 24 °3 30—35 91 19 20 °9 35—40 61 14 22 °9 40—45 38 10 26 °3 45—50 3 5 166-0 Totals... 545 100 Supposing, however, that the men examined by Snell were a fair sample of - the workers, they can be used as a, measure of the age distribution in the whole class of employés. If there be no special liability to illness at any particular age, the number of men of a given age who suffer should be simply n’ PINS total number of cases, where N = 545, n’ the number of men within the specified age limits as recorded above. In this way we obtain the subjoined table :— | Table II. | Ages. Actuai number affected. Theoretical number, Applying the usual test for goodness of fit (4) to this distribution, y? is found to be 56°44, so that the odds are more than a million to one against a worse agreement between theory and observation if the deviations are merely a result of random sampling. In the same way, if we group the 143 cases with recorded ages given by v. Schrotter (2), from the Niissdorf Works, and assume Snell’s age distribution to hold for these works (a not very improbable assumption, since we know that many caissoniers travel from place to place through England and Kurope), we have :— | 14 Messrs. L. Hill and M. Greenwood, Jun. — [Oct. 1, Table III. Ages. Actual number affected. Theoretical number. 15—20 if 14°43 20—25 35 38°04 25—30 43 39 °88 30—35 32 23 °87 35—40 19 16 40—45 8 9 ‘97 | 45—50 5 0°79 and xy? is 39:13, or the odds are about three in a million. It will be seen, therefore, that there is some evidence in favour of an age bias, but the statistics are not sufficiently detailed to give much information. Returning to (i), (11), and (11), it is clear that we must have considerable difficulty in isolating (1) and (i1) under experimental conditions. Thus, in dealing with such animals as rats, it is not easy to obtain a large number of the same age but markedly different body weights. If we attempt to overcome the difficulty by comparing animals of different species, a new factor is involved, the importance of which cannot at present be estimated. It must, therefore, be admitted that in the results about to be detailed the observed differences cannot be ascribed wholly to body mass; other changes associated with growth and decay may be influential, and we must certainly not compare directly animals of different species. With these reservations, regarding which more will be said later, we shall demonstrate an appreciable difference in liability to caisson disease when animais of different weights are employed. | It is known that the velocity of the circulation and rate of respiratory exchange in small mammalia is greater than in large animals, the relatively larger surface exposure in the former necessitating a higher rate of meta- bolism. Since the saturation of the tissues with gas, together with its removal from them, are functions of the blood, it follows that the processes should require less time in small than large animals. If, then, we expose large and small animals to the influence of compressed air for so long a time that both will contain large quantities of dissolved gas, a decompression rate dangerous for the former should be safe for the latter. Our experiments have been carried out on rats, mice, cats, rabbits, and guinea-pigs. (a) Lats. In all experiments the animals were exposed to a pressure of +105 lbs. for periods varying from a-half to two hours; in most cases the exposure was - 1907.] Influence of Increased Barometric Pressure on Man. 15 for one hour. Decompression was effected in four and a-half to seven seconds. In all, 85 rats have been used—54 small, 31 large; of the small rats, 14, or 25°9 per cent., died; of the large animals, 24, 77-4 per cent., died. Unfortunately, all the rats were not weighed, but we have records of the weight in 65 cases, and although this is not a great many from the statistician’s point of view, it is sufficient to make an analysis interesting. The following attempt has been made to measure the relationship between decompression effects and body weight. As we have no quantitative scale of decompression effects, the most satisfactory method to employ would seem to be that of fourfold division, obtaining the coefficient of correlation in the ordinary way(d5). We accordingly made the following table :-— Table IV. Weight in grammes. Recoveries. | Deaths. | Totals. Below 124 ........ui0 28 13 41 ABOVE 124: 00.0. cusen 6 18 24 : Totals ......... 34 | 31 | 65 | From this 7 was calculated to be 0°625 + 0°095. At first sight this would suggest a very close relationship between low weight and immunity from illness, but it is more than doubtful whether the fourfold method is applicable. For this process to be valid it is necessary that the distribution should be normal; thus, the modal severity of caisson illness should be neither among the very slight nor the very severe cases. As a matter of fact, human statistics show that “bends,” slight decompression effects, are vastly more frequent than severe or moderately severe paralysis, so that the severity distribution is, perhaps, skew, and 7, as found above, ceases to be a satis- factory measure of association. _We have, however, two more tests of statistical relationship, the correlation ratio (6) ( __ Standard Deviation of Means of aor . Standard Deviation of the Character and the coefficient of mean square contingency (C,)(7). We can calculate m readily ; the mean weight of all the rats was 122°87 grammes, the standard deviation 67:08; the mean of survivors was 91°62 grammes, of victims 15715, and » = 0°489. As a matter of fact this value differs from 7 by less than three times 16 Messrs. L. Hill and M. Greenwood, Jun. [Oct. 1, the latter’s probable error, but this does not prove linearity of regression,* nor can we apply Blakeman’s test for linearity (8), since the scale of caisson illness is not quantitative. | Finally, the mean square contingency was determined by means of the grouping shown in Table V, and proved to be 0°544 + 0:078.+ : While, therefore, the exact value of the relationship between body weight and decompression effects is hardly expressible in terms of normal correla- tion, it can scarcely be less close than that implied by a contingency coefficient of at least 0°3; in other words, the two variables are quite definitely correlated. We have at present no means of extending this analysis to any other group of animals. Table V. Weight in grammes. Survived. | =) = @ Qu Kj io) —s = mn 40— 60 | 60— 80 | 80-—100 ee DH WH DIAN MAOAE SSSSSSASAUASA 100—120 120—140 140—160 . 160—180 | 180—200 200—220 220—240 300—320 340—360 COCHKHHNPHNONaA SSSSSSSSSASH DWH AOHARHANARO SSSdSSONSUSSS oy or S Totalsy.c-cderscrs 34 °0 ive) pat ral Observations falling at a class limit (e.g., 60) were reckoned as 0°5 in each class. (b) Rabbits. Our experiments have not been numerous, but the following are suggestive :-— Exp. 5.11.06.—Three rabbits, one full grown, another of [medium size, a * In any case, but little weight can be attached to the value obtained for 7, since there are only two arrays available. It seemed best, however, to record the value for comparison. + Deduced by multiplying the probable error obtained from the ordinary formula by 4/3. The complete formula of Blakeman and Pearson (9) gave a lower value, perhaps owing to an undetected slip in the rather involved arithmetical computation. The exact significance of the coefficient may be realised by comparing its value with those obtained by Pearson (7) for the contingency between undoubtedly related variables :— Stature in father and son ... eee oh . C,=0513 Hair colour in brothers ae, Na se .. COC, =0°614 Occupation in fathers and sons _... ies ». CO, = 06275 See pp. 21, 22 (7). 1907.| Influence of Increased Barometric Pressure on Man. 17 third smaller one, were exposed to a pressure of 105 to 110 lbs. for 51 minutes, and decompressed in four and a-half seconds. The biggest rabbit went into convulsions in three and a-half minutes, and the medium-sized one was affected shortly after. On unscrewing the chamber both were found dead, exhibiting the usual post-mortem appearances. The smallest rabbit seemed normal.* (c) Cats. Exp. 27.7.06.—A pregnant cat and half-grown kitten were exposed to 4+ 100 Ibs. for 30 minutes, and decompressed in six seconds. The cat died in 20 minutes. Autopsy.—Veins and arteries full of bubbles and froth. Lungs emphyse- matous and congested in patches, owing to air embolism. Fotuses—Air bubbles in amniotic fluid, which frothed on pouring into a beaker; air in foetal lungs and liquid contents of stomachs, none visible in the blood. The kitten, which appeared normal, was killed for examina- tion within a minute of opening the chamber. No bubbles were seen. The bladder was full of urine, which frothed like champagne when poured into a beaker. 25.1.07.—An old cat and a half-grown cat were exposed to + 110 lbs. for 85 minutes, and decompressed in seven seconds. The old cat was found to be dead on opening the chamber, the young cat survived 20 minutes. 26.7.07.—An old cat and two kittens (half grown) exposed to + 100 lbs. for 30 minutes and decompressed in 10 seconds. The cat was found dead on opening the chamber, the two kittens survived. Collecting our results on rabbits and cats, we have :— Fable Vi Animals. Died. Survived. nee be Ly with probable errors. Young rabbits ......... 5 g 35°7 + 8°64 Old rabbits <2. .0060 006 2 iE 66 67 +18 °36 Wourg, cats:..0<9. 1.24.45. 6 6 50°0 + 9°735 OTA CALS ica sats oaesiecoe des 6 0 100°0 + 8-0(?) The difference in the first group is hardly sensible (30°97 + 20:3), in the second, perhaps, significant (50 + 12:6). * Although this animal had no decompression symptoms, it died four days later from _ pneumonia, produced probably by the rupture of the lung tissue, which occurs in many cases on sudden decompression, followed by infection. VOL. LXXX.—B, C 18 Messrs. L. Hill and M. Greenwood, Jun. [Oct. 1, If young animals have an advantage here also, it is, to say the least, not very marked, and we may reasonably conclude that we have to deal with a group in which the absolute body mass of all members is so great that the decompression period (four to seven seconds) is too short even for the least bulky subjects. In addition to this question of absolute body mass, there appear to be quite marked variations in different species. Thus, it by no means follows that animals of species A will stand decompression better than much heavier animals of species B. So far as our observations go, the guinea-pig is a peculiarly unfavourable subject, even when young and light animals are used ; in 10 experiments we have had eight fatalities. 12.7.07.—Nine guinea-pigs were exposed to + 105 lbs. for one hour, and decompressed in four seconds. Eight (weights, 144, 72, 77, 164:5, 75, 114°5, 108-5, 81 grammes) were dead, and the ninth (108 grammes) paralysed, but recovered completely in a few days. We shall next give details of comparative experiments, which suggest the same conclusion. Rabbits and Cats or Kittens. 26.7.06.—Four rabbits (about one month old), three kittens (about three weeks old) were exposed to + 100 lbs. for 80 minutes, and decompressed in seven seconds. Two rabbits died and one kitten. 21.11.06.—Half-grown cat (3 lbs. 2 ozs.) and large rabbit (3 lbs. 8 ozs.) were exposed to + 105 lbs. for 35 minutes. Decompression time, four seconds. The cat was dead on removal, the rabbit unaffected. Rabbits and Gurinea-prq. oe 10.5.07.—Two young rabbits (3 lbs. 6 ozs.; 3 lbs. 2 ozs.), one guinea-pig (1 lb. 8 ozs.) exposed to + 100 lbs. for 30 minutes, and decompressed in seven seconds. The rabbits died within 15 minutes, and the guinea-pig died in the night, showing signs of pulmonary hemorrhage. Rats and Rabbits. 10.7.07.—Two rats (435 grammes, 87 grammes), one rabbit (1304 grammes) exposed to + 105 lbs. for 14 hours, and decompressed in four seconds. All were dead on removal. Evidently these results are indecisive. Rats and Mice. 7.12.06.—Six rats and six mice were exposed to + 115 lbs. for 25 minutes, and decompressed in four seconds. Four rats died (162, 104, 120, 152 J 1907.] Influence of Increased Barometric Pressure on Man. 19 grammes), two (107, 87 grammes) survived. All the mice survived (18, 18, 12, 10, 11, 10 grammes). 24.1.07.—Fifteen mice and five half-grown rats were exposed to + 115 lbs. for two hours, and decompressed in five seconds. Three rats and 15 mice were unaffected. One rat was dead (froth in heart and veins), one paraplegic. In all our experiments on rats and mice, we find :—: | . . Percentage Died. Survived. mortality. cna IEpeuCS oR ser y Bees 0 6 10 . 37 548-16 54816 | Wiee so. .2. ec: 3 27 10 +3°69 a, ee | Difference ......... —_ = 27 :5+8°96 | | The evidence in favour of the view that mice can stand rapid decom- pression better than rats is reasonably strong. Here body weight is apparently more important than age, full-grown mice being better subjects than young rats. Rats and Kittens. 22.5.07.—Two full-grown rats (11 and 12$ ozs.), and three young kittens (11, 11, 10 ozs.) were exposed to + 120 lbs. for 55 minutes. Decompression time, 54 seconds. One rat was dead on removal; two kittens and the other rat died in a few minutes; one kitten survived. P.M.: The air emboli usually observed were present in all three animals. This experiment shows little advantage on the side of the young animals. The results we have described may be compared with the observations of earlier workers which we have collected and tabulated. These seem to show a relative immunity of the smaller animals to rapid decompression, and a death rate of almost 100 per cent. in the case of cats and dogs at pressures above six to seven atmuspheres. 20) Messrs. L. Hill and M. Greenwood, Jun. [ Oct. 1, Table VII. ae Pressure in atmospheres. eee Result. Sparrows (Bert) (10). (ROP Mame aence Maca: A few seconds Death. Sa(for onmins))yueeeeseeee cee 5 Nil. 8 (for:2 mins) eivecceece ee: _ Nil. Sa(tor 2yhrs:) eee heen a Death. OF (i thr.do Minss) cesses 3 Nil. 10 (some minutes) ............ ct Nil. 12 Arta tude Weaicee we tics " Death. 14 se Malu etter once ci No immediate result; died . next day. | 14 aries see e Death. 15 ABP al es capone an ‘ Death. Mice (Phillipon) (11). 5 7(20 ais) jars sacenessen oer 1 min. Nil. 4 1 (1B, rh UR ere Sead day 1—2 secs. Nii. sy (Ql sts) RGB aBoobonEdHouSccadsb: Momentary Death. Rats (Bert) (10). 5a bred> mims.)o soccer: A few seconds Nil. 6% (1 br. 45 mins.) ......... 3 Nil. GEE hy) ih ica eee ssp ecastees a Nil. | fe RoBribe.g bon sh0oda0n ab ona AbSsoede: 2 mins Death (2 animals). Table VIII. Period of in atmospheres. : | _ Pressure p decompression. Result. Rabbits (Bert) (10). | 63 (1 hr. 45 mins.) ......... 43 mins. ( Nil. 7 (afew minutes) ......... 2 OF iss Nil (2 animals). 8 J (Simin!) Meee eae = 3hiaae Nil. | Ba. Waialua ents Meena ee seer at 2—3 _ ,, Nil (2 animals). Rabbits (Phillipon) (11). Aa! CL WE ye vsntsdaasceia: cies’ Momentary Death. 54 WG Messier Sesotte tethers ae Pa Death. B62 ely este atewte cone eeeene: ye Death. TCD, MINS?) ie onsen scence 3 Nil. 7) CLUE) isaaes Seaseaseuenen: 15 mins. Slight symptoms ; recovered. Cats (Bert) (10). 8. 7(5 mins.) 2.35.2 waees. 2—3 mins Paralysis and death. NOW (O min ss) cece tamales 2—3 ,, Death. | MQ or ccasebenines vac banteaiae neater 2—3 ,, Paralysis. | —1907.] Influence of Increased Barometric Pressure on Man. 21 Table VIl1—continued. * | Pressure in atmospheres. d SEEN 3 Result. ecompression. | Dogs (Bert) (10). inc coche (PoE R Arne ee eae 1—2 mins Nil. Ae LO WAITS.) cin cowadedes stn ox. 2-3 __s,, Nil. Ie fa ead sha wiltiitera i hoes 2-3 , Nil. CP AUR MUING Ve accise beans spe ne- 2—3 _,, Nil. Sem AUS) taehiuce das cena) «sch 20 secs. Nil. OM (OO URNS) sees. es cee sh aoc 20" 5) Nil. Sa Co eee 200% Nil. 6 (afew minutes) ......... 20 Slight paralysis. OMG SR MEE, ) Socncekesnvinweeae 20015 Death. 63 (a few minutes) ......... 4 mins. Nil. Bed NOEs Beats ee eas Paraplegia and death. Ud; TALES, i 8%. aoe cue W153 Paraplegia and death. (dee CRO: UininSe) "5.524 soem oe wae 22, Paralysis and death. MAGUS MITTS Vp. ccctee kee eee lle Game Paralysis and death. 7 (afew minutes) ......... 2. Paralysis and death. 7i Fen es Lares i Paralysis and death. 4 Fe Behr at Paralysis and death. 4 Pred ae LM Maas steak 2s es Paralysis and death. $ A 1 dy Ae er One or A oe Slight paralysis. a 2 hme Coe eee PAP pera Paralysis and death. 73 Po th te he CLs EF aa ts, Nil. & Asam ig eae Set er at oy Paralysis. 8 Seep Sa Caley ier s—4 Ci, Paralysis and death. + One a A ee oe PS: Paralysis and death. a Se | eM: Orn mas Paralysis and death. z Pe Tid peoseaaset Ze 43 Paralysis and death. Table [X.—Catsaras’ Observations on Dogs (12). Atmos compressi Water depth. alee Exposure. oe ee cee Result. metres. secs. 34:0 45 16 mins. 40 Nil. 34:0 43 Loe 50 Nil. 38 ‘0 4,3 7G is 60 Nil. 40 -O 5 Gals 60 Nil. 43 *O 5s 4 ,, 60 Nil. 340 Ay 2% hrs. 40 Temporary ‘paralysis. 36 ‘0 43 Bi fins 50 i * 38 °0 43 Le es 60 Death. 40 °O 5 fee 60 Paralysis and death. 43 °O 55 Pats 60 Dyspnea ; recovered. ; 43 °7 53 Dis 60 Nil. : 43 °7 5s Teas 60 Paralysis. AT °2 3 ‘Deedes 50 - | Temporary paralysis. 53 °2 3 Page 40 Paraplegia. 55 °4 4 25 mins. 60 Death. 60 -O0 7 BOUIN: 60 Death. 45 °O 53 . 30°, ; 30 Paraplegia. 22 Messrs. L. Hill and M. Greenwood, Jun. [ Oct. 1, Further, the observations of L. Hill and J. J. R. Macleod led them to conclude that all adult dogs and cats died when decompressed in a few seconds after an hour’s exposure (or more) to eight atmospheres. If the relative immunity of small animals be due to the greater velocity of the circulation in them, it follows that any agent which damages or slows the circulation would deprive them of their safeguard. We have tested this by exposing small animals to air pressure and then pumping chloroform into the chamber, or upsetting a bottle containing the anesthetic placed inside. 8.5.07.—Guinea-pig (1 lb. 4 ozs.) placed in chamber with CHCl At 11.30 A.M., pressure raised to + 100 lbs.; 11.35, beginning to show signs of ansesthesia; 12.12, decompressed from + 75 lbs. in five seconds. Dead on removal. P.M.: Heart auricles contained an enormous quantity of froth. Every vessel examined full of bubbles. Lungs almost bloodless in appear- ance. Renal vessels frothed on incision. Gall bladder, greatly distended, bile frothed almost lke soda water. Control 10.5.07.—Guinea-pig (1 lb. 8 ozs.) exposed to +100 Ibs. for 30 minutes, and decompressed in seven seconds. The animal seemed normal on decompression, but was found dead next day (Saturday). P.M. (Monday): There were signs of pulmonary hemorrhage, but the body was decomposing. 15.5.07.—Three young rats (4 ozs., 3 ozs., 2 ozs.) and a kitten were com- pressed at 12.50 p.m. to + 110 lbs. At 2.8, chloroform was pumped into the chamber, and at 2.11, when they were all lightly anzesthetised, decompres- sion was effected in five and a-half seconds. On opening the chamber the kitten and one rat were found dead. The other two rats died in a few minutes. P.M.: All exhibited an enormous amount of gaseous embolism except the rat which survived longest, and even in this case many emboli were seen, especially in the vena cava inferior. | These experiments, especially the last, are in favour of the view that immunity does depend on a rapid circulation. In considering the practical bearing of our results, 1t is to be remembered that the conditions which enable a small aniinal rapidly to discharge an excess of dissolved gas also lead to its more rapid saturation. Hence, short exposures are relatively more dangerous for small than large animals. The advantages of small body mass and youth should accordingly be more apparent among caisson workers than in diving operations; we are not acquainted with any statistics bearing on this point. It is interesting to note that we have no reason to think that small animals are more susceptible to oxygen poisoning than large ones. The following observations of L. Hill, J. J. R. Macleod, and C. Ham are suggestive of this :— here. 1907.] Influence of Increased Barometric Pressure on Man. 23 Table X (13). Animal oe Re aa Sidirca cnn occ vasnas oes 8 Lue fe eee Peete 11 Cat and 4 kittens ............... 8—6 Ete, hie s lie holds bdedeiee sds 8 Atmospheres of O, (about 90 per cent.). LL Ue eee 6 oD Se 6 MMs ctiss icadasccsesnscesntsese 6 Li ree 6 CMR Sted dy dornididewe:scaicdsaes 5 MMI feats sic den 1a ene svdvonca snes 5—6 UM Pasa do eo icin et va 59.00%, tng! an 64 HEROES St eres Ses ace wedececesecee’ 6 MIAN 05/4 pobistdn < d guinlna ase videolan sca 6 MRR des dalsiw so. sore isd. ae Hebe ase 6 4—5 MMe iltes Vatdids cseexdyrgeayeds seeqos { - MME EERG Osten 2d Models sioner ois ue stone ne 5 oe ee a a 43 MMM A So ckaccatisenesencde ve 22 PGES FAD DIG... sn sceeccssccecess ) MGUEES THOUS... ...00s0ccecrscccees | 2 Small mouse (16 grammes) ...| > 42 Large mouse (26 grammes) ...| | Small rat (50 grammes)......... J Period of exposure. Guan 1 hr. 20 mins. 1 hr. 40 mins. 15—20 mins. Result. Pneumonia. Death. Pneumonia. Gasping respirations ; re- covered. Pneumonia. »? +B) ” Convulsions. Nil. Convulsions. Nil. All the animals convulsed. In the case of the lungs the action of the oxygen is direct, and there is no reason to suppose that the size of the animals should have any influence As to the convulsions, Lorrain Smith (14) has shown that these are caused not by the amount of oxygen combined, but by the tension of oxygen dissolved in the blood. normal state. Convulsions are as readily produced in an animal whose blood is half saturated with carbon monoxide as in the The immediate factor in their causation is unknown... Conclusions. 1. Small mammals are relatively immune from decompression effects. 2. This immunity depends on rapidity of circulation, and may be destroyed by damaging the latter with chloroform. 3. Age is probably important per se, but of far less importance than body weight. We have no convincing proof that two animals of the same weight but different ages would exhibit unequal resisting powers. 4, There is no evidence that small animals are more quickly poisoned by high pressures of oxygen than large ones. 24 Influence of Increased Barometric Pressure on Man. The practical outcome of this research is that young men of small body weight and possessing a vigorous circulation should be selected for compressed air works. REFERENCES. (1) Quoted by Paul Bert, ‘La Pression Barométrique, Paris, 1878, pp. 379—383. (2) Quoted by Heller, Mager, and v. Seda, ‘Luftdruckerkrankungen, etc.,’ Wien, 1900;-voll, p27: (3) Snell, ‘Compressed Air Illness,’ Trenton 1896. (4) W. P. Elderton, “ Tables for Testing the Goodness of Fit of Theory to Observation,” ‘Biometrika,’ vol. 1, p. 155, 1903. (5) K. Pearson, “On the Correlation of Characters not Quantitatively Measurable,” ‘Phil. Trans.,’ A, 1900, vol. 195, pp. 1—47. (6) K. Pearson, “On the Theory of Skew Correlation and Non-Linear Regression,” ‘Drapers’ Company Research Memoirs,’ Biometric Series, No. 2. . (7) K. Pearson, “On the Theory of Contingency and its Relation to Association and Normal Correlation,” ‘ Drapers’ Company Research Memoirs,’ Biometric Series, Nowl: (8) J. Blakeman, “On Tests for Linearity of Regression in Frequency Distributions,” ‘Biometrika,’ 1905, vol. 4, pp. 3832—350. (9) J. Blakeman and R. Pearson, “On the Probable Error of Mean Square Contingency,” ‘Biometrika,’ 1906, vol. 5, pp. 191—-197. (10) P. Bert, ‘ La Pression Barométrique,’ Table 18, p. 954. (11) Phillipon, ‘Comptes Rendus de l’Acad. d. Sciences,’ 1892, vol. 115. (12) Quoted by Heller, Mager, and v. Schrotter, op. czt., p. 760. (13) Hill and Macleod, ‘Journ. of Hygiene,’ 1903, vol. 3, p. 401. (14) J. Lorrain Smith, “ Pathological Effects due to Increased Oxygen Tension,” ‘Journ. of Physiol.,’ 1899, vol. 24, p. 19. 295 On the Distribution of the Different Arteries supplying the Human Brain. By Cuartes E. Beevor, M.D. Lond., F.R.C.P. (Communicated by Professor David Ferrier, F.R.S. Received May 8,— Read December 5, 1907.) (Abstract. ) The present work was undertaken to ascertain the area of distribution of the different arteries of the brain, when they were injected simultaneously under the same pressure with gelatine containing soluble colours. The number of brains injected is 87. The arteries injected were the posterior communicating, the anterior choroid, the anterior cerebral, the middle cerebral, and the posterior cerebral. The previous observations of Duret, Heubner and Kolisko and other authors are described. The method of investigation consisted in injecting simultaneously by means of pressure bottles three, four, or five of these arteries with different soluble colours. The injection mass used was gelatine, coloured with soluble carmine, Nicholson’s blue, naphthol green, acridine yellow, and Bismarck brown. Twelve different classes of experiments are described. The brains were hardened in formalin, and subsequently cut and examined in the sagittal, horizontal, or coronal planes. The most frequent distribution of the basal branches to. the different parts is by the following arteries :— Regio subthalamica, including the corpus subthalamicum and Forel’s field, by the posterior communicating. Corpus manmulare, by the posterior cerebral. Pes peduncult, anterior one-third by the posterior communicating or anterior choroid, posterior two-thirds by the posterior cerebral. Internal capsuie, anterior division, by the anterior cerebral in the inferior half and by the middle cerebral in its superior half. Posterior division, the inferior part, the anterior one-third by the posterior communicating, posterior wo-thirds by the anterior choroid, which also supplied the retro-lenticular fibres ; the superior part by the middle cerebral. Caudate nucleus, caput, inferior half by the anterior cerebral, superior half by the middle cerebral. Superior horizontal part by the middle cerebral, posteriorly by the posterior cerebral. Surcingle by the anterior horoid. 26 Dr. C. E. Beevor. On the Distribution of the [May 8, Lenticular nucleus, external segment, anterior inferior part by the anterior cerebral, the rest by the middle cerebral. Middle segment by the middle cerebral. Internal segment by the anterior choroid. Optic thalamus, anterior nucleus, by the posterior cerebral. Haternal nucleus anteriorly by the posterior communicating, posteriorly by the posterior cerebral. Internal nucleus by the posterior cerebral, anterior half sometimes by the posterior communicating. Pulvinar by the posterior cerebral. Choroid plexus in the descending and posterior cornua by the anterior choroid, in the central part of the lateral ventricle by the posterior cerebral. Choroid membrane in the descending cornu, anterior part by the anterior choroid, the rest of the membrane by the posterior cerebral. Optic tract by the anterior choroid. Anterior commissure in the median part by the anterior cerebral, in the outer part by the middle cerebral, and in the postero-external part by the anterior choroid. On the cortex the areas of supply of the chief arteries differ in their extent from the description given by Duret, and are most commonly as follows :— Anterior cerebral artery, on median surface of cortex, to half way along the quadrate lobule; on external surface, posteriorly to half way along the parietal lobule, inferiorly to the sulcus frontalis superior. | Middle cerebral artery, on outer surface, posteriorly to the occipital pole or half an inch anterior to it, superiorly to the middle line along the posterior half of the parietal lobule, inferiorly to the middle of the inferior temporal gyrus ; on median surface to include the hippocampal lobule. Posterior cerebral artery, on median surface, anteriorly and superiorly to the posterior half of quadrate lobule ; on external surface at the median line anteriorly to the external parieto-occipital fissure, posteriorly to the occipital pole or to half an inch anterior to this, and inferiorly to the middle or superior border of the inferior temporal gyrus. The optic radiations in the occipital lobe are supplied in the upper (vertical) three-fourths by the middle cerebral and in the inferior one-fourth by the posterior cerebral artery, except in the posterior inch, where all by the posterior cerebral. The following parts are also supplied by the cortical arteries :— The corpus callosum, the genu, rostrum and body by the anterior cerebral ; the tapetum by the anterior, middle or posterior cerebral; the splenium and forceps major by the posterior cerebral artery. The wneus by the anterior choroid artery. The cornu ammons by the anterior. choroid artery or the posterior cerebral artery. 1907.| Different Arteries supplying the Human Brain. 27 The centrum ovale by the anterior, middle, and posterior cerebral arteries. The simultaneous injection of the five arteries supplying the brain, by the same pressure, with soluble colours in gelatine has (it is believed) not been accomplished before, and it is considered that this method gives the most accurate representation of the distribution of each artery, including that of the finest capillaries. The parts of the brain, the arterial supply of which hitherto has not been described, or which are found to be different from other observers, are :— The regio subthalamica, with the corpus subthalamicum and Forel’s field, the supply to which has not been described; the relative supply of the arteries to the pes pedunculi; the corpus mammillare (by the posterior cerebral in place of the posterior communicating); the exact supply to the anterior limb of the internal capsule; the absence of anastomoses of the three arteries supplying the posterior limb of the internal capsule; the exact supply to the caudate nucleus; the absence of anastomosis in the head of the caudate nucleus between the anterior and posterior cerebral arteries; the exact supply of the lenticular nucleus; the supply to the different nuclei of the optic thalamus, which has not been described before (the thalamus is not supplied by the lenticulo-optic arteries of Duret); the anterior part of the choroid membrane, which is supplied by the anterior choroid artery ; the supply to the fornix and the anterior commissure. In the cortex the observations differ from those of Duret in that, though there are great varieties, the anterior cerebral area extends most frequently on the outer surface along the median line posteriorly to midway between the Rolandic fissure and the external parieto-occipital fissure, instead of to the Rolandic fissure ; and inferiorly to the sulcus frontalis superior, instead of the sulcus frontalis inferior. The middle cerebral area on the outer surface reaches the middle line for the posterior half of the parietal lobe, in place of along the whole extent of the lobe; and posteriorly the posterior pole, or half an inch in front of it, in place of the upturned end of the parallel sulcus; and inferiorly the middle of the third temporal gyrus in place of the middle of the second temporal gyrus; this diminution of the extent of the posterior cerebral area on to the outer surface corresponds to the increase of the middle cerebral area. In the supply to the pedunculus cunei and the optic radiations the results agree with Henschen rather than with Monakow, though they differ from both in the case of the supply to the optic radiations ; the supply to the fasciculus longitudinalis inferior is different to that given by Monakow, and corresponds to that of the optic radiations. The exact arterial supply to the corpus callosum, uncus and cornu ammonis 28 Prof. W. J. Sollas. On the Cramal and [May 15, is given. And also that to the centrum ovale as seen in the sagittal, horizontal, and coronal planes. The knowledge of the exact part of the brain which is supplied by any artery is of great importance in the diagnosis of the parts of the brain which undergo softening when this particular artery is blocked by a blood clot. On the Cranial and Facial Characters of the Neandertal Race. By W. J. Souas, Se.D., LL.D., Professor of Geology in the University of Oxford. (Received May 15,—Read November 14, 1907.) (Abstract.) As a result of a detailed comparison of the calvarium of the Neandertal race with that of the aborigines of South Australia, it is shown that a much closer resemblance exists between them than has hitherto been supposed, especially as regards the calottal height, calottal index, Schwalbe’s (“bregma”) angle, the bregma index, the frontal and orbito-frontal angles, the superior (“lambda”) and inferior inion angles, and the fronto-parietal index. The chief differences are to be found in the magnitude of the cephalic index, the continuity of the frontal torus, and the deeply impressed character of the frontal fossa. Comparisons based on the glabella-inion line are misleading, owing to the inconstancy in position of the inion. In the absence of any fixed point in the skull, the centre of figure of the median longitudinal section is chosen for a point of reference, and a radius drawn from this to the basion as an initial line for the measurement of polar co-ordinates. The exterior foramino-basal angle owes its perplexing anomalies to the fact that its magnitude is determined by five independent variables, one of which is connected with the cranial height, so that in depressed forms of skull it acquires a higher value than might otherwise be expected. The Gibraltar skull, preserved in the Royal College of Surgeons, is the only example of the Neandertal race which presents the bones of the face and the basi-cranial axis in undisturbed connection with the calvarium. Its characters, apart from the cranial vault, are unique: no other known skull possesses so long a face, such a large and broad nasal aperture, or such pro- jecting nasal walls. In profile, the nasal curve flows into that of the glabella, p 1907.] Facial Characters of the Neandertal Race. 29 eal without any sudden change of flexure, that is, there is no nasal notch, such as occursin the Australians. | The orbit, as in all skulls of the Neandertal race, is distinguished by its exces- sive height above a line drawn from the nasion to the middle of the fronto- ‘zygomatic suture: in a low South Australian skull this height amounts to between 8 and 10 mm., in the Gibraltar skull to between 12 and 14 mm., and in the Neandertal calotte to between 19 and 20 mm. A further character of the orbits is the absence of a well-defined lower margin. The absence of a canine fossa has been remarked upon by Huxley. In the absence of the prosphenion and ephippium, the sphenethmoidal angle has been measured from the limbus sphenoidalis by a line drawn to the erista galli on the one hand and the basion on the other: it exceeds the corresponding angle of the lowest known South Australian skull, similarly measured, by 16° 30’. The palate is parallel-sided and very dolicho-uranic. The thickness of the frontal bone, measured on one side of the crista gall, is 24 mm. The prognathism of the upper jaw, in whatever way it is measured, is extremely small, so that according to existing nomenclature the skull would be classed as orthognathous. The cranial capacity is estimated as 1250 c.c., and thus makes a close approach to that of the Neandertal calotte. The average capacity of South Australian skulls is very similar, but ranges from 1460 to 1100 cc. If the calotte of Pithecanthropus represents the mean of a similarly variable race, then the extreme forms of such a race would almost completely bridge over the hiatus between man and the higher apes. 30 On the Supposed Extracellular Photosynthesis of Carbon Dioxide by Chlorophyll. By ALFRED J. Ewart, D.Sc., Ph.D., F.LS. (Communicated by Professor J. Bretland Farmer, F.R.S. Received October 11,— Read December 5, 1907.) In two recent papers, Usher and Priestley have brought forward evidence to show that chlorophyll is able to assimilate carbon dioxide and produce formaldehyde outside the plant, and that hydroxyl is the other product and is decomposed into water and free oxygen in the presence of a particular ferment. It is, unfortunately, necessary to point out that their work is to some extent vitiated by certain oversights and by one serious inaccuracy. In their first paper* they confirm Bach’s statement that formaldehyde is produced when light acts on a solution of uranium in the presence of carbon dioxide and water, although formic acid is much more abundant. In a later papert they contradict this result, stating that formic acid alone is present and no formaldehyde, thus confirming Euler’s} criticism of Bach’s results. Usher and Priestley consider that formaldehyde is an intermediate product, but bring forward no satisfactory proof. Their most striking experiment on extracellular photosynthesis is that made by painting a solution of chloro- phyll on gelatine films and exposing them to light in the presence of carbon dioxide. The chlorophyll was bleached, which Usher and Priestley consider to be due to the formation of hydrogen peroxide, being apparently unaware of the fact that chlorophyll is bleached by sunlight in the presence of ordinary free oxygen or in air deprived of all carbon dioxide, and that the absence of hydrogen peroxide from living cells has been definitely established in a number of cases.§ " In regard to the apparent detection of formaldehyde in the gelatine films, this is not surprising, because they contained in all probability an “ aldehyde ” substance before the chlorophyll was painted over them, and further show the same amount whether the films are exposed to light in the presence or absence of carbon dioxide. All the forms of French leaf gelatine (Coignet’s) of cooking (transparent and opaque), Nelson’s, and of impure commercial gelatine turn pink in the presence of a decolorised solution of rosaniline, * ‘Roy. Soc. Proc.,’ B, vol. 77, 1906, p. 369. t+ Loe. cit., B, vol. 78, p. 368. t ‘Ber. d. Deutsch. Chem. Ges.,’ 1904, vol. 37, p. 3415. § Cf. Pfeffer’s ‘Physiology,’ Eng. edit., vol. 1, pp. 545—546. ‘Supposed Extracellular Photosynthesis of Carbon Diowide, etc. 31 with or without gentle warming, and it is on this reaction that Usher and Priestley mainly rely. The best mode of obtaining the reaction is to soak the gelatine in water, melt, cool, and then pour on a little of the decolorised rosaniline, when a pink colour appears near the surface of the gelatine, even if an excess of sulphur dioxide is present. It must, however, be remembered that the same reaction is given by acetaldehyde, and possibly by other aldehydes also. Hence, possibly, may arise the fact observed by Usher and Priestley that the methyleneaniline obtained from the distillate from the gelatine had a slightly different melting point to that of the pure substance.* Even prolonged soaking and washing in water does not remove from the gelatine its power of assuming a pink or violet-pink colour with decolorised rosaniline, and it is quite evident that this oversight renders Usher and Priestley’s method valueless. Repeating their experiments, however, there appeared to be a slight but distinct increase in the amount of “aldehyde” after the chloro- phyll on the film had been bleached by exposure to sunlight, but quantitative estimation was not possible, and the same increase appeared to be shown by films exposed to light in the absence of carbon dioxide. Usher and Priestley state that chlorophyll films on water also produce formaldehyde when exposed to light in the presence of carbon dioxide. I find this method extremely difficult to apply, and have not been able to obtain more than a suggestion of the possibility of the presence of formaldehyde in the subnatant water by the magenta test, and the presence or absence of carbon dioxide appeared to be immaterial. Hence a trace of formaldehyde might have been present in the original chlorophyll, the green colour of which masked the test when first applied. Conclusive results were, however, obtained by using the paler bases of Vallisneria leaves. These showed no trace of formaldehyde when freshly examined, whether killed by heat, chloroform, or ether, but developed a strong pink coloration when placed in decolorised magenta after eight hours’ exposure to sunlight. All green parts gave similar results, although wherever a tissue is dark green it is difficult to be sure of the absence of a trace of formaldehyde in the original material. Owing to the poisonous character of this substance, however, the amount originally present could not be large. After exposure to light, leaves of Vallisnerta give the reaction rather better than leaves of Hlodea and shoots of Chara. In the case of grass and other leaves, they must either be crushed or chopped after bleaching in sunlight, or the epidermis removed in order to allow the reagent to penetrate, otherwise the pink colour is only produced at the cut edges of the leaf. * Usher and Priestley, ¢bzd., p. 320. 32 Dr. A. J. Ewart. Supposed Extracellular [Oct. 11, The chloroplastids in the cells, after exposure and testing, show mostly a strong violet tinge when examined, but a slight tinge can usually be distin- guished in the protoplasm, so that apparently the formaldehyde diffuses somewhat from the chloroplastids to the protoplasm, and, further, is not formed in all the chloroplastids even of one and the same cell. Furthermore, precisely the same production of “formaldehyde” is shown when the green tissues are exposed to light in an atmosphere free from carbon dioxide. It might be suggested that possibly a post-mortem production of carbon dioxide took place. Accordingly, leaves of Vallisneria, Hlodea, and grasses killed by chloroform, ether, and heat were enclosed in vessels on clean strips of glass and kept in darkness for two and for 12 hours. The vessel contained a solution of caustic soda, and half of the inner wall was lined by a sheet of asbestos soaked in caustic soda. On then exposing to light until the leaves were nearly or completely bleached, all of them without exception developed a pink coloration with decolorised magenta, and the colour was as strong as that developed in the presence of carbon dioxide. In similar fresh leaves no formaldehyde whatever could be detected. It is evident, therefore, that Usher and Priestley have misinterpreted the facts, and that chlorophyll, when exposed to light in the presence or absence of carbon dioxide, yields an aldehyde, presumably formaldehyde, merely as a decomposition product. Interesting and suggestive as this fact may be, it affords no proof that formaldehyde is a product of photosynthesis, or that the latter can take place outside the living cell. The idea has more than once been put forward that chlorophyll itself is a product of photosynthesis, and that carbohydrates are formed by molecular changes and rearrangements in it taking place under the action and with the aid of light energy. On examining parts of leaves growing in nature, but which had become bleached or discoloured, traces of formaldehyde appeared to be present in many cases, whether examined in the early morning or evening, and pre- sumably in these cases also the aldehyde results from the decomposition of the chlorophyll. | Usher and Priestley state that on floating the white petals of Saxi/raga Wallacei on water in the presence of carbon dioxide and sunlight, and after painting the petals with chlorophyll, the plastids in the cells developed starch grains, and they consider this to prove an extracellular decomposition of carbon dioxide outside the petals. In any case it would only prove that the traces of formaldehyde produced by the decomposition of the chlorophyll can be synthesised by the plastids to carbohydrate, and the experiment is open to two very serious objections. Firstly, the petals are cuticularised, and formal- dehyde diffuses only very slowly through cuticle, even when comparatively 1907.] Photosynthesis of Carbon Dioade by Chlorophyll. 33 thin. Secondly, except in the case of Composite the plastids of most white petals have faint but distinct traces of chlorophyll, either along the veins or in all parts. By Saxifraga Wallacei, Usher and Priestley presumably mean S. Campost Boiss, living petals of which plant are not procurable in Melbourne, but they would find it worth while to examine the petals for traces of chlorophyll or for a feeble power of independent photosynthesis in parts at least. That this explanation is the correct one is shown by the fact that the petals formed starch in a 0-001-per-cent. solution of formaldehyde when exposed to light, but not in darkness, whereas if the carbohydrate had been derived directly from the formaldehyde, without any photosynthesis of carbon dioxide, it should have been formed both in light and darkness. An equally strong objection applies to the experiment with Hlodea, which formed starch and gave off oxygen in a 0°02 solution of formic acid in light, but not in darkness, the explanation being that the formic acid decomposed and the carbon dioxide produced was assimilated. Hydroxyl and Peroxidase or “ Catalase” Enzymes. No conclusive proof has been brought forward to show that living plant cells even contain any appreciable amount of peroxide of hydrogen, and Pfeffer has conclusively shown in many cases, by the study of cells containing oxidisable pigments or chromogens, that no trace of hydroxyl is formed in them at any time. Russell* explains the action of plant tissues on photographic plates in the dark by assuming that they contain minute traces of hydroxyl, although it is not easy to see how this explanation could apply to a leaf which had been kept dried for 18 months to 3 years and still retained its power of affecting a photographic plate in darkness. There are many other possible explanations of the action, and hence by itself it affords no proof of the presence of hydroxyl. The oxidation of certain organic compounds yields hydrogen peroxide as a by-product, but in the living plant, as Loewt has shown, a catalase enzyme is generally present whose functions may possibly be to prevent the possibility of the accumulation of any hydroxyl, and the existence of such an enzyme was first noted by Schoenheinf. The presence of a “catalase” enzyme is, however, no proof that a plant contains or produces hydrogen peroxide, for ferments have been isolated from many plants to which they cannot be of any value (rennet and diastase ferments in fungi), or have a very doubtful one (peptic enzymes in latex). Usher and Priestley do not mention Loew’s work, and possibly were * © Roy. Soc. Proc.,’ B, vol. 78, p. 385. t Loew, ‘U.S. Dept. Agric. Report,’ 68, 1901. t ‘Journ. Prakt. Chem.,’ vol. 89, 1863. VOL. LXXX.—B. D 34 Dr. A. J. Ewart. Supposed Eatracellular [Oct. 11, not aware of its existence, for Loew discusses the properties, forms, and distribution of his catalase very fully. The Evolution of Oxygen. By smearing gelatine chlorophyll films with a catalase enzyme, Usher and Priestley obtained an evolution of oxygen in the presence of carbon dioxide and light. I have not been able to obtain any satisfactory proof of this fact, and must point out that gelatine films smeared over with an oily film retain oxygen for some time and only give it off slowly in an atmosphere of nitrogen. After covering with chlorophyll and a catalase juice from Vadllisneria, the films were kept in an atmosphere of nitrogen over mercury, a little carbon dioxide added and, after exposure to light, the gas was analysed in a Bonnier-Mangin apparatus. Barely measurable traces of oxygen were present in some cases, but hardly more than can be explained on the above assumption, and the amount of carbon dioxide underwent no appreciable diminution, but if anything a slght increase. Further, if the films were kept over night in an atmosphere of nitrogen in the presence of a piece of pyrogallol-potash paper subsequently drawn out by a thread, on adding carbon dioxide next day and exposing to hght no trace of oxygen was found, although the films at the commencement of the exposure appeared to be green and normal. Apparently, therefore, the decomposition of chlorophyll on exposure to light and the resultant production of aldehyde is not accompanied by any appreciable evolution of free oxygen. Further, no hydroxyl could be detected in any of the films by testing with coloured cell sap and cyanin. Nor was the pink coloration produced on testing the films with decolorised magenta lessened by boiling the gelatine previously to testing, which should have been the case had any hydroxyl been present. Dead cells of Hlodea and Vallisneria were also tested by the bacterium methods for an evolution of oxygen, but without effect, using pure aerobic forms. If the green cells had been exposed to strong light for some hours until bleached and were then tested, it was found that the bacteria ceased to move or only moved very slowly in their immediate neighbourhood, even when the surrounding medium was fully supplied with oxygen. After the leaves had been boiled in dilute hydroxyl and soaked in cold water for a day, they showed the usual chemotropic attraction to aerobic bacteria in the presence of oxygen, due to the exudation of chemotropic substances. It is evident, therefore, that the aldehyde produced by the decomposition of the chlorophyll is able to prevent the detection of the presence of oxygen by the bacterium method, a danger which has not previously occurred to myself or 1907.| Photosynthesis of Carbon Dioxide by Chlorophyll. 35 to any previous worker with this method. This fact takes away all decisive value from the negative observations of Kny* and of Czapekt in regard to the absence of any power of evolving oxygen from isolated droplets of chlorophyll or from droplets imbedded in foreign protoplasm when tested by the bacterium method. The bacterium method must, therefore, be applied with great caution to chlorophyllous cells which have been exposed to strong light when in an abnormal, depressed, or dying condition. The only observations which seem to support those of Usher and Priestley, and show that an evolution of oxygen is possible when chlorophyll is exposed to sunlight apart from living protoplasm, are those of Molisch,t who found by the aid of luminous bacteria that chloroplastids from dried dead cells were able to evolve oxygen. Since, however, Molisch with the same means was unable under any circumstances to detect any evolution of oxygen from healthy cells rich in etiolin, but free from chlorophyll, it is evident that this method of Beyerinck’s is not always reliable. The fact that chloroplastids may remain capable of photosynthesis outside the cell says nothing, for they do not retain this power long, whether kept in light or darkness, and have a photoplasmic basis. Nevertheless, it is quite possible that some of the cases of partial or complete assimilatory inhibition} observed by me may result from an interference with the later processes of polymerisation, such interference rapidly reacting on the primary stages of photosynthesis. This is very probably the case when the accumulation of the products of photosynthesis increasingly retards further production. Photosynthesis is, however, certainly not the simple physico-chemical process that Usher and Priestley imagine it to be, but involves vital activity and control in all its stages. It is, in fact, difficult to see how a simultaneous production of formaldehyde and hydroxyl could be possible in films exposed to sunlight, for as Geisoull has shown, hydroxyl reacts with neutral or acid solutions of formaldehyde, forming carbon dioxide, water, and hydrogen. This reaction is only shown in a test-tube on warming, but if a Vallisneria leaf which has accumulated formaldehyde is soaked in hydroxyl and exposed to strong sunlight or warmed, the formaldehyde disappears. Boiling with hydroxyl does not, however, remove from gelatine its power of producing a pink colour with decolorised magenta. Hence its “aldehyde” constituent is not formaldehyde, a fact which Dr. Rothero has confirmed by distillation tests. * Kny, ‘ Ber. d. Bot. Ges.,’ 1897, vol. 15, p. 388. + Czapek, ‘Ber. d. Bot. Ges.,’ 1902, vol. 20, p. (44). t ‘Bot. Zeit.,’ 1904, vol. 62, I, p. 1. § ‘Linn. Soc. Journ.,’ 1896, vol. 31, pp. 429, 367. || ‘Ber. d. D. Chem. Ges.,’ 1904, vol. 37, p. 515. 36 Supposed Katracellular Photosynthesis of Carbon Dioarde, ete. Usher and Priestley have, therefore, not founded the formaldehyde hypothesis upon any surer basis than had been already established for it by Pollacci* and by Curtius and Reinke,t the only established facts being that chlorophyll decomposes when exposed to light in the presence of oxygen and in the presence or absence of carbon dioxide, and that one of the products of its decomposition is formaldehyde. This production of formaldehyde does not, however, necessarily represent the primary stage in photosynthesis, but is either one of the later ones or a more or less accidental phenomenon shown either in abnormal or dead chlorophyllous cells and tissues or by extracted chlorophyll. In any case we have as yet no satisfactory proof that the production of formaldehyde in dead cells or extracted chlorophyll when exposed to light is accompanied either by a decomposition of carbon dioxide or by a production of oxygen or hydroxyl. * © Atti d. Instit. Bot. Pavia,’ 1900, p. 45; 1902, p. 8; and 1904. + ‘ Ber. d. Bot. Ges.,’ 1897, vol. 15, p. 201. 37 On the Occurrence of Post-tetanie Tremor mm Several Types of Muscle. By Davip Fraser Harris, M.D., B.Sc. (Lond.), Lecturer on Physiology and Histology at the University of St. Andrews. (Communicated by John G. McKendrick, M.D., F.R.S. Received February 15,— Read June 27, 1907.) PART In 1901* I discovered that a frog’s gastrocnemius which had been kept in complete tetanus, either by direct or indirect stimulation, would, if the stimulation were continued until fatigue had begun to manifest itself, fall into a state of obvious tremor. This tremor, whose average periodicity in TAA WEN NAAT OTL MM SA UTS TA LT AS ANN NA MTN TN AN LTTE MMT TAGS Fie. 1.—Gastrocnemius, frog. Indirect stimulation. 5 grammes close to axle of lever giving amplification of 14. Upper tracing, “complete tetanus” for about 25 seconds ; lower tracing shows point of commencement of post-tetanic tremor. Periodicity at first 6 a second. Stimulation (Neef’s hammer) is recorded above time in half- seconds. (Reduced to 3.) * D. F. Harris, ‘Phys. Soc. Proc.,’ March 1, 1903. 38 Dr. D. F. Harris. On the Occurrence of —[Feb. 15, the frog’s gastrocnemius is four to six a second, can be maintained unaltered as to its time-relations, but of slowly declining amplitude, for a relatively long time (half an hour). Fig. 1 shows the smooth line of complete tetanus, breaking off into the post-tetanic tremor in the frog’s gastrocnemius stimulated indirectly.* On investigating as many animal types as I could procure, I found that a post-tetanic tremor could be demonstrated in man, cat, kitten, rabbit, pigeon, and frog, both with and without intact circulation, the mean periodicity of the tremor in all types being between two and six or eight per second. An analogous tremor is elicitable in muscles of fish and lobster. Fig. 3 is a facsimile of the tracing obtained by stimulating the human flexor sublimis digitorum by rapid induction-shocks. After the time of complete tetanus is over, the muscle falls into a state of irregular tremor which in my own arm I caused to be maintained for half an hour, during which time it did not alter either in intensity or in average periodicity. Sponge-electrodes were placed on two spots, an upper and a lower, on the fore-arm over the flexor sublimis digitorum. The middle finger had a ring slipped over its terminal phalanx, and from this a thread passed over a pulley to the spring of an ergograph.t The interruptions here were 60 per second (380 makes and 30 breaks); and as there was previous independent evidence to show that the makes were subliminal, I may take it that 30 break shocks per second constituted the “ stimuli.” | The responses occurred with an average periodicity of four per second. The ratio of stimuli to responses is then 30:4 or 7°5:1, 7e., 1 in 7°5 may be said to be the figure of physiological insusceptibility in this case. Obviously the muscle had its circulation intact. [I may say that at the end of half an hour I experienced no sensation of fatigue in the muscle, but in the above and in all similar experiments on my own muscles the sensations of “the muscular sense ” were quite distinct. A thrill corresponding to the rate of stimulation was perceptible. ] The next type of muscle I investigated was that in the cat. In one experiment, a young cat was pithed under a dose of A.C.E. mixture and artificial respiration done. The Tendo Achillis was fastened to a bell-crank lever, a 50-gramme weight being attached as near the axle as possible. Direct stimulation of a muscle with intact circulation was kept up for * The current in the primary circuit that was usually employed for tetanic stimulation was one of about 3°8 amperes (voltage 4:2). t+ The “ period ” of this spring was 40 to 42 per second. 1907.| Post-tetanic Tremor in Several Types of Muscle. 39 half an hour, at the end of which time, while the muscle was still irritable, it was so feeble that a weight of 20 grammes had to be substituted. The average periodicity of the tremor is three to five per second. Stimuli from 100 a second tuning-fork in Onset of coarse tremor, 8 seconds from commencement (With a lens a tremor can be seen during the complete 10 grammes, same lever as in fig. 1. Periodicity, 8; later, 5 to 6 a second. primary circuit, recorded below time in half-seconds. tetanus, of periodicity 16 to 18 a second.) of tetanus. Fie. 24.—Gastrocnemius, kitten. In only one case, in mammalian muscle, did I obtain a rate slower than three to four per second, viz., one to two a second (fig. 4). Here the 40 _ "Dr. D. F. Harris. On the Occurrence of — [Feb. 15, gastrocnemius was attached in the usual way to a bell-crank lever which carried a 10-gramme weight, TENET Fic. 28.—Portion taken from middle of record of post-tetanic tremor several minutes after commencement. (Facsimile.) un ECU enn TATA EMV TYTNNT Taree ; M SoS Au i N NTN Sao OSS Fic. 3.—Flexor sublimis digitorum (homo) ; sponge-electrodes. Neef’s hammer. Helmholtz side- wire in primary coil. Porter’s ergograph-spring as recorder. Stimulation recorded below time in half-seconds. Tremor maintained for 30 minutes. Average periodicity 5 to 6a second. (Facsimile.) TATA ALT ATAU LTS RET Gao TA TEN A ST Fic. 4.—Gastrocnemius, kitten (pithed, artificial respiration). Complete tetanus (on direct stimulation) suddenly breaking off into post-tetanic tremor. The animal was pithed, artificial respiration was used, and very little blood lost in preparing the muscle. With indirect stimulation, the muscle exhibited 1907.] Post-tetanice Tremor in Several Types of Muscle. 41 complete tetanus for an unusually long time; several minutes after the stimulation had been made direct (owing to the onset of end-plate fatigue) Stimulation direct. Tremor recorded for 20 minutes. Lever as in fig. 1. 20 grammes near axle. Stimulation by Neef’s hammer (side-wire}, recorded above time in half-seconds. Fie. 5.—Extensor longus digitorum, pigeon, pithed. Periodicity, 3 to 5 a second. the tetanus was still complete, but after a few seconds more the muscle quite suddenly broke off into a tremor with a periodicity of about one or two per 42 Dr. D. F. Harris. On the Occurrence of — [Feb. 15, second which, becoming rhythmic, was maintained of singularly constant intensity. To investigate the post-tetanic tremor in the bird, I used the pigeon (pithed). The extensor of the toes was the muscle chosen: a cord from the tendon for the toe passed to a hinged lever weighted with 20 grammes quite close to theaxle. Direct stimulation was maintained for 20 minutes; portions of the tremor during this time are reproduced in fig. 5. The average — periodicity of the tremor is three to five a second. In the case of the muscles of the fish, in some experiments I used those of the tail of the plaice or “spotted flounder” (Plewronectes platessa), the fish being pithed. Considerable difficulty was encountered in fixing the very. soft-fibred muscles to the recorder. Using the hammer as interruptor, and direct stimulation, I obtained a tremor of about one per second, which could not, however, be maintained indefinitely ; fatigue seemed to supervene with considerable rapidity ; I found great difficulty in bringing out smooth complete tetanus at all: the tendency to tremor was present from the first. When we used the muscles that close the jaw we obtained a tremor of a very slow periodicity, a shortening lasting about 1 second and occurring at irregular intervals of 3, 4, 5, or 6 seconds each. : In the case of the lobster (Homarus vulgaris), we used one of the flexors of the abdominal somites. The preparation was fixed by clamping a portion of the carapace in muscle-forceps and attaching the fibres of the muscle to a horizontal lever with a weight (10 grammes) very near the axle. The stimulation was, of course, in all cases direct, fine wires being led from the short-circuiting key in the secondary circuit directly into the muscle substance. On submitting the muscle of the lobster to stimulation similar to that employed in most of the other types of muscle (viz., stimuli given by Neet’s hammer, with side-wire), I failed entirely to produce complete tetanus; in other words, the muscle fell into a state of tremor from the very beginning of the stimulation. This is very well seen in fig. 7, where, using a quite fresh flexor of abdominal somite, a violent tremor is elicited without any preliminary period of complete tetanus. The periodicity of this tremor in the first second was nine a second, but, as fatigue set in, it fell to three a second. Thus, although we cannot use the term “post-tetanic” of the tremor which appears in the muscle of the fish or of the lobster in consequence of stimulation by rapidly recurring (tetanic) stimuli (30 to 70 a second), never- 1907.] Post-tetanie Tremor in Several Types of Muscle. 43 statatatatatatatatatatctntaletntstalatnlalanlalalacavaaalaenlotatataenlel nnn FULL Fie. 6.—Tail-muscle of Pleuronectes platessa. 5 grammes near axle. Lever as in fig. 1. Neef’s hammer, side-wire. Stimulation direct. Time in half-seconds. No complete tetanus. Tremor of periodicity, 1°5 to 2 a second. Patty TT Se a a ee ra SENEGAL INS SINT TET SESE SS TN TENN TTT TA TT TET NTT SG NH ETT ASNT otto uence Fig. 7.—Muscle of abdominal somite, Homarus vulgaris. Lever as in fig. 1. Stimulation direct. Break-shocks only. 10 grammes near axle. Stimulation, given by Neef’s hammer (side-wire), recorded below time in half-seconds. Tremor from commence- ment. Periodicity at first 9 a second, soon becoming 3a second. (Tracing reduced to one-half.) Fic. 8.—Tremor in a not quite so fresh muscle of lobster, average periodicity 4 to 5a second. Stimuli at least 70 a second recorded above the time in half-seconds. Conditions as in fig. 7. (Facsimile.) 44 Dr. D. F. Harris. On the Occurrence of — [Feb, 15, theless the time-relations of this tremor are comparable with those of the post-tetanic tremor of the higher muscular types. Crustacean muscle is no exception to the universality of this phenomenon of slow tremor appearing in muscles subjected to “tetanising” stimuli; i possesses, InN common with mammalian, avian, and amphibian oe protoplasm, that property of functional insusceptibility in virtue of which it avoids exhaustion by falling into a state of comparatively slow “rhythmic ” contractions. Part JJ.—ANALYSIS OF RESULTS AND THEORETICAL. (1) The first point that may be noticed here is that the tremor of post- tetanic onset is in its average periodicity not that of the tremor of the same muscle about to pass from incomplete to complete tetanus. H.g., in frog’s gastrocnemius the maximal rhythm of the incomplete tetanus (just before fusion to complete) is something between 25 and 28 per second,* while the “rhythm” of the tremor that follows spontaneously on a period of complete tetanus is about four to six a second. Instead of 25 to 28 responses per second, the muscle can now exhibit no more than five to six, or about one-fifth of the former. Obviously, fatigue is the cause of this physiological insusceptibility ; fatigue which, by preventing responses at anything like the rhythm of the stimuli (30 to 100 per second), prevents the early onset of complete exhaustion. This physiological insusceptibility may be taken as a protective mechanism against the fatal effects of full fatigue. (2) The post-tetanic tremor is a myogenic phenomenon. It occurs in muscle directly stimulated, whether curarised or not, and a tremor indistin- guishable from it is given by, eg., the dying diaphragm (fig. 10). For tremors of directly stimulated muscles, see figs. 28 and 3. For tremor of curarised muscle (toad), see fig. 9. The so-called “spontaneous” tremor I was able to record for three quarters of an hour in the dying diaphragm of a pithed rabbit which had its phrenics cut. It is a tremor of about six per second average periodicity and, therefore, quite similar in time-relations to the post-tetanic.t (3) While the periodicity of the post-tetanic tremor is rarely, during long periods, more than six per second, it is not of the same periodicity in all the muscles even of the same animal. * T. G. Brodie, ‘Elements of Experimental Physiology, pp. 62, 63. London: Longmans, Green, and Co., 1898. + D. F. Harris, “On the Time-relations of the Spontaneous Tremor of the Diaphragm,” ‘Phys. Soc. Proc.,’ January 26, 1907. 1907.| Post-tetane Tremor in Several Types of Muscle. 45 Thus, while in the frog’s gastrocnemius it is about six per second, in the hyoglossus it is only 2 to 2°5 a second; ratio 3:1. Fig. 9.—Gastrocnemius, toad, curarised. 5 grammes. FBell-crank lever. Stimuli by Neef’s hammer (side-wire). Time in half-seconds. Tremor maintained for 15 minutes. Average periodicity, 2°5 to 3.asecond. (Facsimile.) ae esiieelssieleis delsedels ed Malus seliseisieie stsielsisicl Fie. 10.—Diaphragm, rabbit (pithed, phrenics cut). “ Spontaneous” tremor recorded for # hour. Time in half-seconds. Lever as in fig. 1; no weight. Periodicity at first 10 a second; later, 5asecond. (Facsimile.) HTT TT NY ANI | TSA PHT FTTH TOTES ATT TT TIT UAT YHAMTRH NANNY ANT THAT AN TACT! SONY TY HANTS TT IT, STAT Ly Fic. 11.—Hyoglossus, frog. Direct stimulation ; no weight. Neef’s hammer (side-wire). Time in half-seconds. Tremor. Periodicity, 2 to 3 a second. This difference is interesting in the light of the well-known difference in the number of stimuli necessary to cause complete tetanus in the gastro- enemius and in the hyoglossus respectively, viz., 30 and 10 respectively, ratio 3:1, The post-tetanic tremor, no less than the genesis of their tetanus, proclaims 46 Dr. D. F. Harris. On the Occurrence of — [Feb. 15, the differences in molecular mobility between the gastrocnemius and the hyoglossus muscles. Similarly, a poison like curare, which diminishes protoplasmic mobility, brings down the periodicity of the post-tetanic tremor from the six per second of frog’s normal gastrocnemius to 2°5 to 3 per second in the curarised gastrocnemius (cf. fig. 9). In other words, the physiological insusceptibility is now about three times as great as in the normal frog muscle; or, again, the curarised gastrocnemius becomes reduced, as regards molecular mobility, to the level of the less mobile hyoglossus. (4) The post-tetanic tremor is characteristically irregular in intensity from moment to moment. : Previously to my publishing the first note on the periodicity of this tremor, Dr. Bayliss was good enough to suggest, in a private communication, that the irregularity in the record of the tremor was due to irregular variations in the intensity of the various members of the series of stimuli given by Neet’s hammer, that is to say, that the stimuli varied from submaximal to maximal and vice versd. I had eliminated this possibility, by noticing that post-tetanic tremor could be produced at all distances between the primary and secondary coils, both when the stimuli were submaximal and when they were maximal. As I had already used the Helmholtz side-wire equaliser, and did not consider that the vibrating reed gave stimuli of sufficiently uniform intensity, I decided to test the point by (1) using a 100 a second electro-magnetic tuning-fork as interruptor in the primary, and (2) by using as interruptor, in the primary circuit, a metallic rotating wheel provided with teeth which dipped into a pool of mercury, and was driven at varying speeds by a small air-engine. By neither of these methods was the irregularity abolished; and in the case of certain muscles was not in any obvious manner diminished (notably fig. 2, muscle of kitten, where the 100 a second fork was used). While freely admitting it possible that a series of stimuli of absolutely equal physical intensity would give the least irregular form of post-tetanic tremor, yet I am compelled, from a study of a large number of these tremors through long periods during which the stimuli were as constant in energy as possible, to believe that the irregularity of the post-tetanic tremor is inherently characteristic of it. It seems to me that the irregularities can be explained by :— 1. Non-simultaneous onset of fatigue in the several fasciculi or fibres of the muscle ; so that when we use even physically equal stimuli, a stimulus which is effective for one fibre or fasciculus may not be so for another, or if effective 1907.] Post-tetanic Tremor in Several Types of Muscle. 47 for one fibre, etc., at one moment, may not be so for that same fibre at the next moment. All the fibres are not equally fatigued after the same duration of stimula- tion, and, through their irresponsiveness, certain fatigued fibres rest, to recover their irritability later on, so that a stimulus sub-liminal for a given fibre at one moment may be liminal or supra-liminal for it after a short interval. 2. In the next place, it is not probable that all the fibres composing the depths of a muscular mass receive either in indirect or in direct stimulation their stimuli at absolutely the same instant through the entire muscle—the bi so-called “non-instantaneous innervation ;” and, further, it is known that certain fasiculi have more component fibres than others, more fibres to be innervated. It rarely happens that all the fibres contract simultaneously, but when they do, we obtain a result as in fig. 4—a series of large waves of quite slow rhythm, 1°5 to 2 a second—reminding one strongly in some respects of a cardiac rhythm as seen in fig. 12, which is a record of the so-called “ heat-tetanus ” of the ventricle of the heart of a rabbit. Fig. 12.—Ventricle of heart (rabbit), immersed in continuously heated 1-per-cent.’£ NaCl. Lever same as in fig. 1. 5 grammes weight. Time in half-seconds. Maximal periodicity, 6 to 7 per second. In this case the gastrocnemius muscle had its circulation intact (kitten pithed, artificial respiration); the stimuli were from a tuning-fork in the primary, 7.¢., as physically uniform as possible; stimulation was direct. Here I conclude that the onset of fatigue was simultaneous throughout, practically, all the fibres of the muscle, a condition, for several reasons, very rarely met with (fig. 4). Here, if anywhere, the conditions were such as to favour the simultaneous onset of fatigue in the fibres, for the stimuli were as equal in intensity as was possible, and any conditions due to varying degrees of neural fatigue were eliminated by the previous fatigue of the motor end-plates; all the fibres were still supplied with oxygenated blood, i.e, were under the same conditions as regards nourishment and removal of waste-products, so that there was maximum uniformity of both the physical and metabolic conditions. Except for a second or two, I have seen none but irregular tracings of 48 Dr. D. F. Harris. On the Occurrence of — [Feb. 15, post-tetanic tremors, and this irregularity also characterises even those tremors which are produced by the so-called “constant” stimuli and also certain tremors of “ spontaneous” origin. Fig. 13.—Gastrocnemius, frog. Sciatic nerve drying. Bell-crank lever; no weight. Time in half-seconds. Periodicity of tremor, 4 to 6 a second. (Facsimile.) Fie. 14.—Gastrocnemius, frog. Solid NaCl placed on sciatic nerve. Bell-crank lever ; no weight. Time in half-seconds. Periodicity of tremor, 5 to 7 a second. (Facsimile.) Fic. 15.—Gastrocnemius, frog. Sciatic nerve pinched. LBell-crank lever ; no weight. Time in half-seconds. Periodicity of tremor, 6 a second. (Facsimile.) With regard to tremors due to “constant” or “single” stimuli, we have the tremors from (1) drying of the nerve; (2) chemical stimulation of the nerve, ¢.g., by NaCl; (3) pinching the nerve; (4) heat suddenly applied to the nerve; and (5) the disappearance of anelectrotonus from the nerve: while the tremor of the dying diaphragm is an example of the “spontaneous ” kind (cf. fig. 10). 1907.| Post-tetanice Tremor in Several Types of Muscle. 49 Fie. 16.—Gastrocnemius, frog. Sciatic nerve suddenly heated by hot NaCl 75-per-cent. solution. Bell-crank lever. Time in half-seconds. Periodicity of tremor, 4 per second. (Facsimile.) ee as Seconds. LLL... 1. Fie. 17.—Gastrocnemius, frog. Ritter’s “opening tetanus.” LBell-crank lever; no weight. lcm. of sciatic nerve had “constant” current (5°4 1075 ampere) ascending for 5 minutes through non-polarisable electrodes. To be read from left to right. Time in seconds. Periodicity at beginning of tremor, 5 to 6 a second. (Facsimile.) Sohe Sonn Fic. 18.—Sartorius, frog, completely immersed in Biedermann’s fluid. To be read from left to right. Time in half-seconds. Average periodicity, 4 per second. (Facsimile.) ) All of these tremors are more or less irregular. Now all these tremors are, as to average periodicity, of the same order as VOL. LXXX.—B. | E 50 Dr. D. F. Harris. On the Occurrence of — [Feb. 15, the post-tetanic (three or four to six a second), and as the coarse tremor brought on by fatigue in, eg., the human deltoid (fig. 19). Fic. 19.—Deltoid, human, coarse fatigue tremor of. Arm held out horizontally with 1 kilo. in hand; cord from middle finger to button of cardiograph (Marey’s). Tracing by a recording tambour. Time in half-seconds. Periodicity of tremor, 5 per second. To be read from left to right. (Facsimile.) In these cases we may say that inco-ordination of fibres or of fasciculi is the element common to all, whether this has been brought about by “non- simultaneous innervation ” or by repeatedly varying degrees of the excitability of the fibres brought on either by fatigue or by some hitherto unexplained state of inaccessibility to certain stimuli. I take it that this inco-ordination or non-simultaneousness of innervation is what Dr. Haycraft alludes to when, discussing normal voluntary tetanus, he writes . . . “ muscles exhibit fascicular or other local movements.”* If this be true as regards normal voluntary tetanus, the record of which is very regular compared with the tremors under study at present, it must apply with increased force to those tremors of muscles in which fatigue has set in. While fatigue, inducing fascicular inco-ordination, can be held to explain the irregularity of the post-tetanic tremor and the spontaneous tremors of dying muscle, it is difficult to see how fatigue can be the main factor producing the irregularity of such tremors as those from chemical, mechanical, or thermal stimulation or from anelectrotonus, seeing that the irregular contractions are present from the very first when fatigue obviously cannot have set in. The probability is that those stimuli called “ single ” or “ constant” are, as regards nerve-molecules, anything but constant in their stimulating power. * J. B. Haycraft, “Voluntary and Reflex Muscular Contraction,” ‘Phys. Journ.,’ vol. 11, 1890, p. 366. 1907.] Post-tetanie Tremor in Several Types of Muscle. 51 Drying of nerve in air, abstraction of water by NaCl, increase of temperature, even pinching the nerve cannot be assumed to be respectively uniform in their modes of being stimuli; in fact, physically they represent stimuli varying considerably in intensity from moment to moment. They introduce intra-molecular disturbances of various potentials and of varying disruptive powers as regards the biogens of the nerve. These disturbances “act” like discrete stimuli of varying intensities. Theoretical Explanation of the Meaning of the Post-tetanic Tremor* and Tremors of Similar Perrodicity. The property of functional inertia of muscular protoplasm expressed here as a physiological insusceptibility to certain stimuli seems to me to furnish the key to the meaning of the post-tetanic tremor. I regard it as a protective mechanism which, by permitting the establishment of this fatigue-rhythm of low periodicity, averts for a time the physiological calamity of full exhaustion. If there could be responses to such high rates of stimulation as 30, 40, or 100 per second, the muscle would very soon be utterly tired out and incapacitated for a long time from activity. But, owing to the possession of the property of physiological irresponsiveness (functional inertia), the muscle subjected to continued stimulation responds only to some of the stimuli in the rapid series (every fifth, seventh, or tenth), or responds at a rhythm very much lower than that of the stimuli, thus substituting chronic fatigue for acute exhaustion. Here, in virtue of functional inertia, the living matter preserves itself from that destruction which would overtake it were it possessed of affectability alone. The two properties are co-existent in the living matter: resting muscle has much affectability, but little functional inertia; as activity proceeds, the affectability diminishes and the functional inertia Increases towards the continued stimulation, until a point is at last reached when the ratio between the two properties is such that only every 1 in 7 or 10 stimuli is responded to, the others being functionally disregarded. This state of matters can be exhibited for a long time, biologically speaking, viz., half an hour, as in the post-tetanic tremor of Ene human flexor sublimis digitorum with intact circulation. The biotonic state at any moment depends on the ratio of the degrees of possession of these two properties; were affectability alone present we * D. F. Harris, “Functional Inertia, a Property of Protoplasm,” ‘Roy. Soc. Edin. Proc.,’ vol. 24, p. 196; and D. F. Harris, “ Affectability and Functional Inertia as the two Properties of Protoplasm,” ‘Scot. Micros. Soc. Proc.,’ vol. 4. E 2 52 Post-tetanic Tremor in Several Types of Muscle. would have, as a result of perfect response to all stimuli, a maximum uncontrollable activity, and the rapid end of things in death; were functional inertia alone present, we would have complete irresponsiveness to all stimuli. But a condition of functional compromise between the two biotonic extremes is set up, and thus a mean condition, known as “ fatigue,” established. I take the existence of the post-tetanic tremor as one more expression of the physiological significance of fatigue and of rhythm. In those tremors arising from a single stimulus (figs. 15, 16, and 17), or from continued stimulation (figs. 13 and 14), or spontaneously (fig. 10), we have the property of functional inertia of the muscular substance con- tributing to the explanation of the feature common to them all, the want of correspondence between the character of the responses and the nature and mode of application of the stimuli. Tremors of the same average periodicity, 3 to 6 a second, are elicited by stimuli physico-chemically the most varied, and may be seen charac- terising cases so far apart physiologically as the dying diaphragm and the maximal rate of beat of the heart ventricle. _ This non-correspondence or lack of parallelism between stimulation and response seems to be due to the stimulus-disregarding property of functional inertia in the muscle-substance, the same property which, in the case of Iong-continued rhythmic stimulation, expresses itself in the post-tetanic tremor. Through affectability muscle responds to any stimulus by contracting, but through functional inertia the nature, the mode of application, and even the rhythm of the stimulation is disregarded, and to the most varied kinds of stimulation the response becomes the same—a tremor whose component events recur with the same slow average periodicity. “Tt seems,” writes Professor Biedermann,* “to be an almost universal property of muscular substance to fall, under certain conditions, with all prolonged stimuli into a state of visible rhythmical excitation.” The above data, I submit, are an experimental and statistical verification of this statement. [The expenses involved in the foregoing work have been met by a grant from the Carnegie Trust for the Universities of Scotland, of which I here desire to express my grateful acknowledgment. | * ¢Klectro-physiology,’ vol. 1, p. 107. London: Macmillan, 1896. D3 On Reciprocal Innervation of Antagonistic Muscles. Eleventh Note.—Further Observations on Successive Induction. By C. 8. SHERRINGTON, D.Sc., F.R.S. (Received November 4,—Read December 5, 1907.) (Physiology Laboratory, University of Liverpool.) ? It was shown in previous notes* of this series that in the “ flexion-reflex< ’ of the limb the reflex inhibition of the extensor muscles is frequently followed by contraction of them. This contraction ensues immediately on cessation of the inhibitory stimulus, and is often more accentuated than was the contraction originally inhibited. The phenomenon was ascribed to a process of rebound in the central part of the nervous are inhibited, and this process, on account of its seeming analogy with certain visual phenomena often called “ inductive,’ was termed “successive induction.” The occurrence of this ‘‘ successive induction ” made it seem probable that under favourable conditions the reflex evoked in the limb by a single stimulus would be diphasic with a first phase of flexion followed by a subse- quent phase of extension. The following observations show that this is in fact the case, and under appropriate conditions regularly so. Among conditions requisite for the reflex’s exhibition of this .diphasic character is freedom of the preparation as far as possible from such depressing influences as “shock,” deep narcosis, fatigue, etc. In purely spinal reflexes successive induction does occur; thus, it causes exaltation of activity of the extensor arcs to ensue after their inhibition during a flexion reflex; that was established by observations previously reported.t In those observations the reflex preparation used had been preserved over long periods subsequent to spinal transection, the depression due to spinal shock in the ordinary meaning of the term had had prolonged opportunity to subside. In the present observations the examination of the reflex has been proceeded to within a few hours of the transection, since it was found that the successive induction could be observed without recourse to long periods of recovery if the transection instead of being spinal was pontine, vc, through the gnterior part of the hindbrain. The transection and * * Roy. Soc. Proc.,’ B, vol. 76, p. 160; vol. 77, p. 478; vol. 79, p. 347; also ‘ Inte- grative Action of the Nervous System,’ London and New York, pp. 21, 206, 1906. + Sherrington, ‘Roy. Soc. Proc.,’ B, vol. 76, p. 161; vol. 77, p. 478; ‘Integrative Action of the Nervous System,’ p. 19 ; also article “Spinal Cord,” Schafer’s ‘ Text-book of Physiology,’ vol. 2, p. 841, 1900. Prof. C. S. Sherrington. On Reciprocal [Nov. 4, 1907. | Innervation of Antagonistic Muscles. 55 operation preparatory to that was carried out under deep chloroform narcosis. The brain anterior to the transection was removed. After this removal the chloroform narcosis was lightened. The “ flexion-reflex,” elicited from an afferent nerve or from some appropriate skin-point, is then found to be diphasic. The movement of active flexion is followed by a movement of active extension. During the active flexion the extensor muscles are relaxed by central inhibition; during the active extension the extensor muscles contract. The contraction of the extensor muscle, ze. the discharge of the extensor motoneurones, never in my experience sets in during the actual delivery of the external stimulus used to excite the “ flexion-reflex.” So long as that stimulus continues the extensors remain inhibited (fig. 1).* It is only after withdrawal of that stimulus that the discharge of the extensor motoneurones occurs. The commencement of this discharge, judged by the muscle’s contraction, follows withdrawal of the external stimulus by a time interval longer than that between commencement of external stimulus and onset of the inhibitory relaxation of the muscle. In other words, the latency of the contraction-phase is longer than that of the inhibition-phase (fig. 1, A, and fig. 8, A). That the contraction of extensor muscle, 2.¢., discharge of extensor moto- neurone, never occurs during the continuance of the external stimulus, distinguishes the reaction from a rhythmic reflex such as the “scratch- reflex.” In the scratch-reflex the phases of absence of discharge and of occurrence of discharge succeed each on the other in the motoneurone, ¢.7., of extensor of knee, during unabated continuance of the external stimulus,t and with a period of alternation practically independent of the external stimulus. In the case of the “ flexion-reflex” of diphasic character, the second phase, the phase of discharge of the extensor motoneurone, cannot apparently break through the state of inhibition characterising the first phase so long as the external stimulus is unremitted. The first phase may be termed, if desired, a refractory phase; but if so it is a refractory phase the duration of which depends on the external stimulus, whereas the duration of the refractory phase of the scratch-reflex is independent of the external stimulus. * The time-marker records in fifths of seconds throughout all the figures. The signal marking excitation is below. All read from left to right. In all the records (figs. 1—9 inclusive), descent of the myograph line means relaxation of the muscle, and conversely its ascent means contraction of the muscle. t Sherrington, ‘Journ. of Physiology,’ vol. 29, p. 58, 1903 ; vol. 31, p. xvii ; vol. 34, p. 1; ‘ Integrative Action of the Nervous System,’ p. 45. 26 Prof. C. 8. Sherrington. On Reciprocal [ Nov. 4, Fia. 2. For the contraction-phase to ensue, not only must the external stimulus have ceased, but that stimulus must have possessed an intensity of above a certain minimal degree. In fig. 1, the lower reaction B, showing no atter-phase of contraction, was given by a weaker repetition of the faradic stimulus which in the same reflex preparation had shortly before caused the upper reaction A, showing marked after-phase of contraction. This influ- ence of intensity is well shown when the stimulus is very brief, 7.2, a single make or break induction shock. When the shock is a weak one, inhibitory relaxation of the muscle is its only effect obvious in the preparation (fig. 2). When the shock is more intense, a phase of contraction follows the phase of relaxation (fig 2), and the contraction-phase becomes more marked as the intensity of the shock is made greater (fig. 2). With a quite strong induction shock, eg., a break shock unpleasant to the observer's tongue, the contraction-phase ensuing on the relaxation may reach a height much exceeding that of the contraction pre-existent to the inhibitory relaxation (fig. 2). Also, the contraction-phase may last several seconds, and be much longer than the phase of inhibitory relaxation which preceded it (fig. 2). With longer and more complex stimuli, such as series of double shocks at an intermittence of 30 per second, a similar relaxation is even better evident (fig. 3). When the stimulus is quite weak, the reflex effect in the extensor muscle, ¢.g., vasto crureus, is simply an inhibitory relaxation (fig. 3, A). As the stimulus is repeated with greater intensity, a phase of contraction follows on the phase of inhibition, and the contraction-phase increases with in» crease of the intensity of the stimulus (fig. 3, B, C, D). But the phase of contraction never 1907. | Innervation of Antagomstic Muscles. 57 encroaches on the phase of inhibitory relaxation during the period of actual delivery of the external stimulus. Further, with these stimuli other conditions governing the appearance and intensity of the contraction-phase become apparent. Of these, one is that a TOOT oO TOTTOIM wy weak stimulus which, when brief, does not occasion a contraction-phase, does induce a contraction-phase if its duration be lengthened (fig. 4). Also, a stimulus which, when brief, occasions but slight contraction-phase, occasions IEtGs) a: more and more marked contraction-phase when its duration is more and more lengthened, up to certain limits. That it is less easy to evoke the diphasic 58 Prof. C. 8. Sherrington. On Reciprocal [ Nov. 4, reflex by a single induction shock as stimulus than by a faradic stimulus, is explicable by the influence of mere duration of the stimulus upon the efficacy of a stimulus to induce the contraction-phase. The rule holds generally in reflex reactions that frequently-repeated small stimuli sum to a total effect equal to that of a single stimulus of much greater individual intensity. The above results amount, therefore, simply to this, that the phase of inhibition has to exceed a certain minimal value in order that a phase of contraction ensue from it. In fig. 3 it will be noticed that the amount of lengthening of the muscle in the inhibitory phase of the reflex increases with the strength of the stimulus, as does the ensuing contraction-phase itself. Yet itis not at all necessary, in order to obtain the contraction-phase, or to obtain a marked contraction-phase, that the lengthening of the muscle in the precurrent inhibition-phase should have been extensive. The intensity of contraction- phase bears no close relation to the amplitude of the lengthening in the inhibitory phase. The amount of contraction-phase is independent in great measure of the extent of the fore-running inhibitory lengthening of the muscle. Thus, in figs. 4, 5, 6, and 7, the amplitude of the lengthening registered in the inhibition-phase was very small, yet the ensuing contraction- phase is marked in fig. 4, and is very great in figs. 5 and 6. The amplitude of the lengthening of the muscle, which takes place during the inhibition- phase, depends much on the degree of contraction present at the moment when the inhibitory stimulus is applied. If the muscle be then already fairly at relaxation length, the amount of further lengthening which ensues during the inhibition-phase may be small. The stimulus will then, nevertheless, if it have sufficient intensity or duration, be followed by large contraction-phase (figs. 5, 6, and 7). This seems important, because it indicates that the main predisposing factor for the after-coming contraction is not the peripheral relaxation of the muscle, but the central process of inhibition, and this the muscle only fully expresses under suitable mechanical conditions. We may suppose that the central change underlying this central inhibition increases with increasing intensity of stimulus— ‘intensity including, as was argued above, duration (within limits) of stimulus. The amount of the after-coming contraction-phase seems, therefore, proportioned to the amount of the precurrent inhibition which takes place centrally. This argues in favour of regarding the contraction-phase as due to a central rebound from inhibition to excitation.* It was said above that a stimulus, too weak when brief to evoke an after- phase of contraction, becomes effective when prolonged (fig. 4). The range of * Of. “Integrative Action, &c.,” p. 212 ; ‘Journ.-of Physiology,’ vol. 36, p. 191. 1907. | Innervation of Antagonstic Muscles. BELT SEAT OTT TINS ETE EIS PO TEES EIT A 60 Prof. C. 8S. Sherrington. On Reciprocal [Nov. 4, Fic. 6. duration over which this holds good is wide, but it may be overstepped. - An effective stimulus may, if much prolonged, pass unfollowed by contraction- A ae Innervation of Antagomstic Muscles. 61 phase (fig. 4, H). There seems for each intensity of stimulus a duration which best favours the occurrence and intensity of contraction-phase (fig. 4). The length of this period varies with the condition of the reflex preparation as well as with the intensity of the external stimulus. In fig. 4 the reflex preparation was of relatively low activity. The relation existing between the period of the stimulus and the development of contraction-phase recalls the observations of Bowditch and Warren* on the time-factor in the influence of precurrent stimuli from various sources on the amplitude of the knee-jerk ; also the relation observed by Yerkest between precurrent stimuli and a reflex elicitable from the frog. The contraction of the contraction-phase varies from a brief spasm (fig. 3) to a prolonged tetanus lasting many seconds (fig. 8, A and B). The con- ied traction may be of great amplitude and power. When intense it reaches its maximum with considerable speed (figs. 1 A, 6,7, 8, A and B). Its decline may be rapid (figs. 3, 4), but more usually is quite gradual (figs. 1, 2,7). Its decline is always, in my experience, more gradual than its ascent, and usually very greatly so (figs. 1, 2, 7). This is a point of difference between it and certain forms of strychnine reflex as exhibited by the same extensor muscles, and it constitutes a distinction of some importance for determining the relation of strychnine reflexes exhibited by these muscles to the normal after- phase of contraction which they also show.t * ‘Journ. of Physiology,’ vol. 11, p. 25, 1890. t Pfliiger’s ‘ Archiv,’ vol. 107, p. 207, 1905. { Sherrington, ‘Journ. of Physiol.,’ vol. 36, p. 191, 1907. V ‘8 ‘eld 4, Son ae ee ate | SE ee ey ee be Gig) rr ae a = $e tics nal = eo UuL27]0" | ~ pr uayyy * \ [ Nov 3 i) S ~ Q SS) D RS Ss S ington. of. C. S. Sherri Pr MU NIAVURETNUENLULURENECODSAVALEUEASETIUCUEUTNLUNUTEUSELASLILCELREVUSTEWAESENNSEEU SEALED A ILUSAITNSTLTLIILONIE VENTED SID STEAVNTSINURIVIORTANIVTRLED NIE STOTIAYOLSERTYEVISTIIN ALibesoceosi deer far rss ceo sus bepesepereroe Ces ee ess TL AAU “GL UID % X Oo ay = 2 Pr 2 = a s ~~ > a x = = 2 se S Ss nt) < = py LHLCLILLONPE LE POR OAL LEI ALLL ELI PELE L OETA EEL EL ORL LER PIRI PL ER ACARI SELES RED OLP ALCP ELEELOUL EERE E Ee Eee I OPEL OPTI I ESI ILER ISI RE IAL E DELILE PPLRE LL ARI OPPIPLILLILIIPIRDI OE OECPILLIL LP POO PP Retire reper per Pr reeset rss RELEA AL £022 1907.] 64 Prof. C. 8. Sherrington. On Reciprocal [ Nov. 4, The contraction of the after-phase is extremely easily and rapidly cut short by a repetition of the stimuius upon which it followed. That stimulus inflicts on it with speed and certainty an inhibitory relaxation (figs. 7 and 8, A and B). Even the briefest repetition of the stimulus suffices. I have seen a repetition lasting only one-twentieth of a second cut itdown. In fig.7 is seen the effect of renewal of the stimulus for one-tenth of a second (fig. 7, third application of stimulus). The stimulus cuts it down by inhibitory relaxation, much as it cuts down a crossed extensicn reflex.* On repetition of the original stimulus, the contraction of the contraction- phase is lessened or suppressed according to. the intensity of the stimulus or the duration of its reapplication. On cessation of the reapplication the contraction-phase sets in again (figs. 7 and 8, A, B); subject only to the general conditions already mentioned as governing the contraction and to the condition that the reapplication has not been strongly repeated many times. Thecontraction-phase tires out more quickly than does the inhibition- phase. The contraction-phase that appears after a repetition of the stimulus does not seem tc be simply a continuation or reappearance of the old con- traction-phase that had been inhibited, for it often exceeds in intensity the intensity which the foregoing contraction-phase had at the moment when the inhibitory stimulus was renewed (fig. 7, second stimulation; and fig. 8, B). Nevertheless, the new contraction-phase has not so great intensity as the previous contraction-phase showed at outset (figs. 7 and 8, A). The fresh contraction-phase following each repetition of the inhibitory stimulus is on the whole somewhat less than the one preceding it (figs. 7 and 8, A, B), and the series of contraction-phases show progressive decline (figs. 7 and 8, A, B). Fig. 8, A, B, exhibits the first four and the last two of a series of 15 diphasic “ flexion-reflexes ” provoked in the extensor muscle (vasto-crureus) without pause between them. The external stimulus was not intense, and its intensity remained the same throughout this series, although the duration of its several applications was not exactly the same for each member of the series, since the short-circuiting key opened and shut by hand instead of automatically. The contraction-phase dwindled progressively in its repetitions, and on (fig. 8, B) withdrawal of the sixteenth stimulus no con- traction-phase followed. The faradic stimulus was then (fig. 8, B) repeated with increased intensity, although with somewhat shorter duration. On cessation of this (seventeenth) stimulation, although little or no further lengthening of the already fairly-relaxed muscle had been obvious during the stimulation, a contraction-phase of considerable intensity at once followed (fig. 8, B). On repetition of the stimulus this was at once cut * See fig. 3, in eighth Note of this series, ‘ Roy. Soc. Proc.,’ B, vol. 76, p. 277. EDO s. | Innervation of Antagonstic Muscles. 65 down. On withdrawing the stimulus contraction-phase again appeared almost as intense as on the next previous withdrawal (fig. 8, B). By repeating and withdrawing the stimulus a new series was obtained. This declined more rapidly than the former one, and when its contraction-phases had died out a further strengthening of the stimulus induced a new and still more rapidly-declining series. It is clear, therefore, that in the first series the contraction-phase had been exhausted only relatively to the intensity of the external stimulus used in that series. A stronger stimulus was still able to evoke more contraction-phase. I have shown a similar relation to exist between fatigue of reaction and intensity of stimulus in the scratch- reflex of the spinal dog.* On the whole, in my experience the contraction- phase is relatively little resistant to fatigue. It will often disappear for a given stimulus in three or four severe repetitions of that stimulus. It seems to wear out more rapidly than does the inhibition-phase of the same reflex. It wears out especially rapidly when the reflex excitability of the preparation is low, ¢g., under shock, general exhaustion, etc. It was said above that the contraction-phase is easily suppressed by chloroform narcosis. In the bulbospinal animal exhibiting extensor rigidity, a very considerable depth of chloroform or ether narcosis is required to abolish the rigidity. A depth of narcosis less than that required to abolish the rigidity of the muscles abolishes the contraction-phase of the “ flexion- reflex’ in them. When under deepening narcosis the stimulus for the “ flexion-reflex” is repeated at suitable intervals, the contraction-phase following each inhibitory relaxation of the extensor muscle becomes less, and finally unobtainable before extinction of the decerebrate rigidity, and while the inhibitory relaxation of the muscle is still obtainable. Conversely, after chloroform narcosis has been pushed to the extreme of abolishing all reflex reaction, on lightening the narcosis the decerebrate rigidity reappears, and the reflex inhibitory relaxation of the extensors becomes demonstrable at:a time when there is still no obtaining of the contraction-phase withdrawal of the inhibitory stimulus, occasioning no rebound contraction of the muscle. Asphyxial conditions tend, as is well known, to augment some reflex reactions before finally paralysing them. The “ flexion-reflex” is, as I have frequently seen in working with the spinal mammal, one of the reactions thus increased by impending asphyxia. It might, therefore, be thought that the appearance of the contraction-phase of the extensors in this reflex was attributable to a super-excitability, due to insufficient aeration of the blood from imperfect respiratory ventilation in the paralysed animal. This is not so; on the contrary, defective pulmonary ventilation and experimental —* ‘Journ. of Physiology,’ vol. 34, p. 42, 1906. VOL. LXXX.—B, ¥ “s* . 4 ¥ 66 Prof. C. 8. Sherrington. On Reciprocal [Nov. 4, conditions tending to asphyxia depress the contraction-phase, and, if allowed to continue, soon abolish it. The possible importance of “successive induction” as a coordinator of reflex actions seems obvious. If a reflex A not only temporarily inhibits a contraction B antagonistic to it, but also induces in the are of B, as an immediately subsequent result, an excitation, we have a process qualified to link together simpler reflexes, so as to form from them reflex cycles of action such as characterise the reflex play of the limbs in locomotion. In a previous Note it was remarked that a difficulty in thus applying the experimental results then obtained lay “in the intensity and long duration ” of the stimuli which have to be employed to produce the “successive induction.”* “Such intensity and duration certainly do not occur in the course of the alternating reflexes ’t as they occur naturally. The observa- tions brought forward in the present communication remove that difficulty. Rebound contraction from “successive induction” is shown to be elicitable by stimuli of even momentary duration, eg., a single make or break shock ; and still more readily by stimuli lasting even so brief a time as a twentieth of a second. The intensity of electric stimuli that evoke it need not be more than just perceptible to the tongue. Mechanical stimuli as well as electric also readily produce it. The experiments afford, I think, convincing proof that in both fore limb and hind limb the ordinary “ flexion-reflex,” as expressed by the extensor muscles, is a diphasic reaction. The first phase is relaxation due to central inhibition, the second phase contraction due to central discharge. The first endures throughout the application of the external stimulus, the second immediately follows the withdrawal of that stimulus, and is due to a central rebound from the state of inhibition to a state of excitation. In the first phase of the reflex the extensor muscles abandon the maintenance of a posture, or the execution of a movement in which they were engaged; in the second phase they restore that posture to the limb, or reinstitute movement in the abandoned direction. The series of reflexes of fig. 8, A and B, is a series of “steps” executed by the limb under the sole action of a main extensor muscle of the knee (vasto-crureus). It has been shownj that in each reflex of such a series, the flexors§ contract during the application of the stimulus, that is, at and during time of inhibitory relaxation of the extensors.|| There- * Sherrington, ‘Roy. Soc. Proc.,’ B, vol. 77, p. 495.° + Ibid. : { Sherrington, ‘ Roy. Soc. Proc.,’ vol. 52, 1893. § For list, see ‘ Roy. Soc. Proc.,’ B, vol, 79, p. 341. || For list, of. cbid., p. 342, 1907. | Innervation of Antagonistic Muscles. 67 fore, in a series of reflexes such as that of which fig. 8, A and B, shows the extensor muscle’s reactions, the active movements of flexion and extension, alternating one with another, do not require alternation of two external stimuli, one evoking flexion, the other extension. Such observations show that one and the same stimulus, intermittently applied, suffices fully for the double phases of the reflex movement. Contraction-phase ensues in the muscle antagonistic to the flexor in each pause of application of the external stimulus. In other words, a neural discharge, due to successive induction, produces after each reflex contraction of the flexor a compensatory contraction of the extensor, bringing the limb back to extension and keeping it there until renewal of the external stimulus again inhibits the extensor, and contracts the flexor. In this case, unlike the “ scratch-reflex,” the rapidity of succession of the phases of the cyclic reflex is in the hands of the external stimulus. If the rate of intermission of that stimulus is slow, the alternation of the phases of the reflex is likewise slow ; if the repetitions of the stimulus follow quickly, the cycle of the reflex proceeds quickly. That is to say, the rate of stepping of the limb depends on the rate of recurrence of the applications of a single uniform stimulus which excites, during each of its applications, one phase of the reflex act, and is followed by the other phase. Addendum.—December 3, 1907. (With HerBerT E. Roar, M.D. Toronto.) (Received December 4, 1907.) Mention was made in the foregoing* of the possibility that not only complete withdrawal of the inhibitory stimulus, but likewise mere lessening of its intensity, might induce from the inhibited centre fresh motor discharge and consequent contraction of the muscles previously kept relaxed by reflex inhibition. This proves to be actually the case. To obtain this effect under the experimental condition, the reduction of intensity of the external stimulus has had to be considerable, and the reduction has been abrupt. The method adopted has been that followed in studying the influence of the intensity of the external stimulus on the power of the stimulus to break through reflex fatigue The resistance of the primary circuit has-been suddenly altered by introduction of further resistance of known amount. With the current and inductorium used, no rebound contraction was obtained under our conditions of experiment when the resistance was increased by less than 30 ohms. Fig. 9 shows the effect of abruptly reducing the current * Cf. also ‘ Journ. of Physiology,’ vol. 36, p. 191. +t Ibid., vol. 34, p. 42. 68 Prof. C. 8. Sherrington. On Reciprocal [ Nov. 4, YYW WY YAY WWW VV in the primary circuit by introducing 40 ohms additional resistance. The upper record (A) in the figure shows the reflex effect of faradisa- tion of the afferent nerve with the secondary coil at 40 units of the Kronecker scale, and with the 40 additional ohms in the primary © circuit. The effect is seen to be reflex inhibition, as is evidenced by a slight yet distinct relaxation of the vasto-crureus muscle. The stimulus was, therefore, above threshold value for producing reflex inhibition of the extensor arc. The lower figure opens with the effect of faradisation of the same nerve a minute later with the secondary coil unaltered in position (ze, 40 Kronecker units), but without the inclusion of the 40 additional ohms in the primary circuit. This stronger stimulus is seen to produce smart reflex relaxation of the extensor muscle. When that has reached its full amplitude and endured for something over a second, the extra 40 ohms are abruptly introduced (by un-short- circuiting them) into the primary circuit, and the faradic stimulus is thus reduced to the intensity it had in the upper trace of the figure, 4¢., to an intensity near, but distinctly above, threshold value under the experimental conditions of a minute before. The record shows that those conditions have now under- gone a change, for now, instead of a weak inhibition, a strong extensor contraction sets in. This is the rebound contraction, and the change that has altered the conditions has been “successive induction” in the reflex preparation. The rebound contraction exceeds in intensity that obtaining prior to the inset of the original inhibition. The duration of the less intense faradisation, marked by the upper signal line, is continued for about a second, and then by short-circuiting the 1907. | Innervation of Antagonistic Muscles. 69 additional 40 ohms from the primary circuit, the more intense faradic stimulus is recontinued. The strong inhibitory relaxation of the extensor muscle at once returns. Finally, on discontinuing this, there occurs once more a rebound contraction of the extensor muscle, and this rebound is more intense still. Such observations show that the central rebound from inhibition to super-activity on the part of the reflex arc of the extensor takes place in the face of a weak inhibitory stimulus applied to the arc at the time of the rebound. 7 The result seems to have bearings of some theoretical importance. Of these one appears as follows. When reflex contraction has proceeded, even for a short while, under the application of a full exciting stimulus a weak excitatory stimulus, which prior to the strong reaction was able to excite the reflex contraction though weakly, loses some of its efficacy and becomes less able to provoke its reflex; in other words, tends to fall below the threshold value and may become subliminal.* Such a change in excitatory reflexes is ascribed to, or rather described under, fatigue effects. In the case of the inhibitory reflex just mentioned, an inhibitory stimulus which, prior to the action of a stronger inhibitory, was able to produce reflex inhibition, fails to do so when repeated after the stronger stimulus has been producing inhibition for a short time. If in the case of reflex excitation, causing contraction, the phenomenon is permitted to rank as fatigue, the similar result observed in reflex inhibition indicates that a similar wearing out or blunting of inhibitory effect occurs, and can, under like terminology, be called fatigue. There is thus emphasised one more of several significant likenesses between the converse processes of reflex excitation and reflex inhibition. Another bearing of theoretical importance which the observation allows is the following. The flexion reflex is in reality the reflex stepping of the limb, and such stepping is a rhythmic alternating reflex. The observations in the present Note indicate that instead of having to look for two different alternately-acting stimuli wherewith to explain the two successive phases of flexion and extension characterising the reflex, one stimulus (namely, that exciting flexion) is all that may be needed. But that stimulus has to be intermittently applied, extension by rebound contraction occurring in the intermissions of its application. The additional observations in this addendum show that it is not necessary for the stimulus to be actually intermittent; it will suffice if it merely suffer periodic decrements of intensity—provided the decrements exceed a certain amount. In other * Good instances are seen with the scratch-reflex, cf. ‘Journ. of Physiology,’ vol. 34, p. 1. 70 On Reciprocal Innervation of Antagonistic Muscles. words, in this reflex there is developed in the extensor neurones a slight refractory phase, slight because though effectively blocking a weak inhibitory stimulus, it fails before the insistence of a strong one. Thus analogy is at once revealed between this reflex and one superficially very different from it, namely, the “ scratch-reflex.” In the latter a feature of co-ordina- tion is the occurrence of marked refractory phase. During the progress of the reflex the external stimulus which excites the contraction of the flexor muscles fails to excite them at rhythmically recurring periods owing to supervention of a refractory phase in the spinal centre. And the analogy the extensors of the limb as well as the flexors contract rhythmically,* and presumably their rhythmic 33 is the closer since in the “scratch-reflex contractions alternate with those of their antagonists. The recurrent refractory phase would therefore not only block the flexor excitatory stimulus, but at the same time block the extensor inhibitory stimulus. This is what it seems to be doing in the “step-reflex.” The difference between the two reflexes becomes ~one of quantity and period rather than any essentially qualitative one. The facts indicate that the reflex movement of stepping, with its two opposite phases of flexion and extension, can be excited by one single form of stimulus intermittently or even merely unequally applied, that stimulus being the one which directly excites flexion. This suggests an explanation for the striking inequality with which flexion and extension respectively are represented in the receptive field, both deep and superficial, of the limb itself. The direct stimulation, electrical or mechanical (2.e., ligation), of any afferent limb-nerve, whether cutaneous or deep, excites as its immediate result flexion of the limb itself, not extension.t Somewhat similarly, in the motor cortex (especially of the hind lmb) the primary representation of flexion greatly preponderates over that of extension. The explanation of * By appropriate isolation the following muscles in the cat are seen to exhibit the characteristic rhythmic contraction constituting the scratch-reflex—tensor fascie femoris, psoas magnus, gluteus minimus, front portion of gluteus maximus, pectineus, sartorius, semt- tendinosus, tibialis anticus, posterior portion of biceps femoris, flexor longus digitorum, adductor parvus, vasto-crureus, semimembranosus and anterior part of biceps femoris. Although most of these are flexor muscles, some, eg., the three last mentioned, are extensors. These three can be seen to contract simultaneously together in the rhythm of the reflex. For the rhythmic abduction which accompanies the rhythmic flexion of thigh in the reflex gluteus minimus seems chiefly responsible, since that rhythmic movement persists when all muscles of limb except gl. min. have been paralysed by severance of their nerves. This list probably does not exhaust the whole set of muscles exhibiting the rhythmic contraction of the reflex.—C. 8. S. + ‘Integrative Action of the Nervous System,’ p. 291, 1906; ‘Roy. Soc. Proc.,’ B, vol. 76, p. 293, and B, vol. 79, p. 337. P £ th i * aie T a On i Innervation of Antagonistic Muscles. 71 Ati $ curious inequality may lie in the circumstance that reflex flexion of the 4 limb of itself induces as a sequence to itself extension, so that no local stimulus is, in so far, required for extension. The same argument may also apply to the analogous inequality of jaw opening and jaw closing, both as locally elicitable reflexes and in their primary representation in the “motor” cortex.* At root of the inequality with which movements of opposite direction, forming complemental pairs, are represented, both in the fields of local reflex action and in the “motor” cortex cerebri, may lie ‘successive induction.” * Ibid., and ‘Journ. of Physiology,’ vol. 34, p. 315. Anmversary Address by Lord Rayleigh. 71 this curious inequality may lie in the circumstance that reflex flexion of the limb of itself induces as a sequence to itself extension, so that no local stimulus is, in so far, required for extension. The same argument may also apply to the analogous inequality of jaw opening and jaw closing, both as locally elicitable reflexes and in their primary representation in the “motor” cortex.* At root of the inequality with which movements of opposite direction, forming complemental pairs, are represented, both in the fields of local reflex action and in the “motor” cortec cerebri, may lie “successive induction.” Address of the President, Lord Rayleigh, O.M., D.C.L., at the Anniversary Meeting on November 30, 1907. Since the last Anniversary the Society has sustained the loss of twenty- five Fellows and three Foreign Members. The deceased Fellows are :— Thomas Andrews, Rev. John Kerr, Sir Benjamin Baker, Sir Leopold McClintock, Sir Dietrich Brandis, Dr. Maxwell Tylden Masters, Sir William Henry Broadbent, Prof. Alfred Newton, Dr. Alexander Buchan, Cornelius O’Sullivan, Lord Davey, Sir Wilham Henry Perkin, Dr. August Dupré, | Dr. Wiliam Henry Ransom, Sir Joseph Fayrer, Sir Edward James Reed, Sir Michael Foster, Dr. Edward John Routh, Sir William Tennant Gairdner, Henry Chamberlaine Russell, Lord Goschen, Prof. Charles Stewart, Sir James Hector, Robert Warington. _ Prof. Alexander Stewart Herschel, The Foreign Members are :— Marcellin Berthelot, Dmitri Ivanovitch Mendeleeft, Henri Moissan. * Jbid., and ‘Journ. of Physiology,’ vol. 34, p. 315. VOL. LXXX.—B. | G 72 Annwersary Address by Lord Rayleigh. [Nov. 30, From the length of this list it will be seen that death has been unusually busy during the year. It includes many names of great distinction, of whom I can refer only to a few. : Sir B. Baker was a Councillor at the time of his death, and was well known among us as a frequent attendant at our meetings and as combining scientific interests with the highest degree of successful practice in his profession. The recent catastrophe in America will, perhaps, even enhance his reputation as the designer of the Forth Bridge. In Sir Michael Foster we have lost one, to whom probably more than to anyone else the present position of our Society is due. For 22 years he held the office of Secretary, during about half of which time I was his colleague. It would not be too much to say that the interests of the Society and a desire to extend its usefulness were never out of his mind. It was inevitable that his pronounced views and his energy in giving effect to them should occasionally arouse opposition, but he was impelled always by public spirit, sometimes to the detriment of his own private interests. His work for Cambridge, for the Government in commissions of inquiry, as well as for. the Society, constitute a lasting claim upon the gratitude of our generation. The name of Kerr will go down to posterity as the discoverer of two remarkable phenomena in Electro-Optics. His success is a good example of what may be accomplished under no small difficulties by courage and perseverance. The claims of Sir W. Perkin as the pioneer in the aniline industry and as a distinguished worker in scientific chemistry have recently been celebrated, and are recognised all over the world. It is satisfactory to reflect that, unlike many inventors, he met with full appreciation during his lifetime. Dr. Routh’s name is one that I cannot allow to pass without a word. I was indebted to him for mathematical instruction and stimulus at Cambridge, and I can still vividly recall the amazement with which, as a freshman, I observed the extent and precision of his knowledge, and of the rapidity with which he could deal with any problem presented to him. His book on Dynamics is well known. In its earlier editions it illustrated, perhaps, rather the vices than the virtues of the Cambridge School, but it developed later into a work of first-rate importance. I have always been under the impression that Routh’s scientific merits were underrated. It was erroneously assumed that so,much devotion to tuition could leave scope for little else. On the foreign list we have to lament three chemists of high distinction, Berthelot and Mendeleeff, though still active, had attained old age; but in Moissan we lose one from whom much more might have been expected. All three have been recipients of our medals. 1907. | Anmversary Address by Lord Rayleigh. 73 An important feature in the work of the Royal Society consists of various enquiries, undertaken for different Departments of Government, in regard to diseases which affect the tropical portions of our foreign possessions and dependencies. Among these diseases the attention of the civilised world has been for some years directed to the malady known as Sleeping Sickness. The first concerted action for the study and combating of this apalling scourge arose out of a representation made by the Royal Society to the Foreign Office in the spring of 1902, in consequence of which, at the request of the Treasury, the Society’s Malaria Committee organised and despatched a small scientific commission to Uganda. In the course of a short time the source of the disease was traced by this Commission to the presence of a trypanosome in the blood and cerebro-spinal fluid of the victims, and the further discovery was also made by the same Commission that the trypanosomes are carried by a species of biting tsetse-fly. These important revelations were followed up by detailed studies of the character and distribution both of the disease and of the fly. Besides sending out a succession of observers to prosecute the investigations of its Commission at Entebbe, the Royal Society urged upon the Colonial Office the necessity of organising, and under an increased medical staff, a more comprehensive enquiry into the local conditions under which the disease is propagated. This recommendation was carried out and some valuable information on the subject has been obtained. Meanwhile, though various drugs had been tried with at best only temporary success, no lasting remedy had been found for the malady, which has continued to be fatal and to spread steadily over Central and East Africa. The various European Governments which have possessions in those regions have at last determined to make a united effort to cope with Sleeping Sickness through the instrumentality of an International Conference having a separate bureau in each country concerned and a central bureau in London. The object of this co-operation will be to collect information bearing on the disease, to devise and carry out such scientific researches as may seem to be necessary and to concert measures for dealing with the disease and the populations affected or likely to be affected by it. The Royal Society, having led the way in this subject, has been invited to give the proposed combined inter- national action its support. The Society welcomes the proposal and will be prepared to render every assistance in its power. In the meantime our ‘Tropical Diseases Committee is continuously and actively engaged in the endeavour to discover a drug that may prove effective in the treatment of the ‘disease. Their investigations have been directed to the study of trypano- -somiasis in rats and the latest results obtained are such as to encourage the hope that at least in this direction their labours have been successful. . G 2 74 Anniversary Address by Lord Rayleigh. —[Nov. 30, During the present year three parts of the Reports of the Society’s Mediterranean Fever Commission have been published, embodying the final observations and conclusions in this important enquiry, which was undertaken at the joint request of the Admiralty, War Office, and Colonial Office. It is not often that a difficult investigation of this kind can be brought to a successful conclusion in so short a time as two years and a-half, and the various members of the Commission are deserving of the warmest com- mendation for the skill, zeal, and promptitude with which they have solved the problem submitted to them. They have shown how the scourge of fever, which has been so long rife in Malta, and has so seriously reduced the strength of our garrison there, may be eventually banished from the island. Already their recommendations, so far as they have been followed, have reduced the amount of fever to trifling proportions. It now remains for the authorities to adopt the further precautions pointed out to them, which will probably banish the disease altogether. I have continued to preside at the Meetings of the Executive Committee of the National Physical Laboratory. The work of the Laboratory has grown greatly during the year. The addition to the Engineering Building and the new building for Metallurgical Chemistry are completed and are now occupied, while the building for Metrology is very nearly finished. A new 100-ton testing machine, one of Messrs. Buckton’s latest patterns, has been installed, and the increased accommodation in the Engineering Laboratory enables the work there to proceed more easily and rapidly. Great progress has been made in the equipment of the Electro-technical Laboratory, and research and test-work can now go on there in a rapid and systematic manner. The question of the Commercial Testing undertaken by the Laboratory has been the subject of investigation by a Treasury Committee, before which I was summoned to give evidence. It is understood that the report of this. Committee may be expected shortly. Progress has been made with the buildings at Eskdale Muir, some of which are now ready for occupation. It was hoped that the work might. have begun this summer, and the Treasury have provided a sum of £750 for the expenses during three-quarters of the current financial year. Owing to. the bad weather in the early summer this anticipation has not been realised, but a start will be made very shortly. The buildings are admirably adapted for their purpose, and will render possible the study of terrestrial magnetism under the undisturbed conditions which used to exist at Kew. A list of the more important researches is published in the Report of the 1907. | Annwersary Address by Lord Rayleigh. 75 Laboratory. Among these may be mentioned those by Dr. Harker on the Kew Scale of Temperature and its relation to the International Hydrogen Scale; Mr. Paterson’s paper, read before the Institution of Electrical Engineers—“ Investigations of Flame Standards and the Present Performance ‘of High-voltage Lamps”; and the eighth report of the Alloys Research Committee, by Dr. Carpenter and Mr. Edwards, on the Properties of Alloys of Aluminium and Copper. Professor Ayrton, Mr. Mather, and Mr. Smith have finished their work on the Ampere Balance, and the paper is now being published by the Royal Society in the ‘ Philosophical Transactions, while papers on the Silver Voltameter and the Weston Cell are also in the press. Dr. Stanton and Mr. Bairstow have completed a further research on the measurement of wind pressure, and are well advanced with the investigation into methods of impact-testing. Other researches in progress are those on the measurement of small inductances and capacities, with a view to the standardisation of the wave- lengths used in wireless telegraphy, on alloys of copper, aluminium, and manganese, for the Alloys Research Committee, and on the properties of eutectics. The completion of the work on the electrical units will be satisfactory to those who have been interested in this question. At the time of my own researches about twenty-five years ago, the ohm and the ampere were uncertain to 2 or 3 per cent., and I then scarcely hoped to get nearer than one part in a thousand. The recent work carried on at Bushey would seem to indicate that an accuracy of one part in ten thousand may have been attained. The possibility of such a refinement depends largely upon the use in the instruments of coils composed of a single layer of wire, the position of every turn of which is open to exact determination. The importance of this feature was insisted upon by the late Professor Jones. Accuracy of measurement appeals less to the lay and scientific public than discoveries promising to open up new fields; but though its importance at any particular stage may be overrated, it promotes a much needed consolidation and security in the scientific edifice. A remarkable example of enhanced accuracy is afforded by modern measurements of luminous wave-lengths, for which we are mainly indebted to our Copley medallist. Not only did he introduce the vacuum: tube charged with mercury or cadmium as the best source of homogeneous light, but by a most able use of an ingenious method he determined, with the highest precision, the values of the cadmium red, green, and blue wave-lengths in terms of one another, and of the metre. His work has been skilfully followed up by Fabry and Perot, and numerous wave-lengths are now known with a relative accuracy of one- 76 Anniversary Address by Lord Rayleigh. [Nov. 30, millionth part. When we reflect upon the almost ultra microscopic magni- tude of a wave-length of light, the possibility of such an achievement may well excite our astonishment. For the advancement of science the main requirement is, of course, original work of a high standard, adequately explained and published. But this is not enough. The advances so made must be secured, and this can hardly be, unless they are appreciated by the scientific public. In some branches of pure Mathematics it is said that readers are scarcer than writers. At any rate the history of science shows that important original work is liable to be overlooked and is, perhaps the more hable, the higher the degree of originality. The names of T. Young, Mayer, Carnot, Waterston, and B. Stewart, will suggest themselves to the physicist ; and in other branches, doubtless, similar lists might be made of workers whose labours remained neglected for a shorter or a longer time. In looking into the more recent progress of Geometrical Optics, I have been astonished to find how little correlation there has been between the more important writings. That Coddington should have remained unknown in Germany and von Seidel in England need not greatly surprise us; but in this subject it would appear that a man cannot succeed in making even his own countrymen attend to him. Coddington seems to have heard nothing of Cotes and Smith, and Hamilton nothing of Airy and Coddington. It is true that no two writers on theoretical subjects could differ more in taste and style than do Hamilton and Coddington. The latter addressed himself to special problems, the solution of which seemed to have practical importance. Among his achievements was the rule relating to the curvature of images, generally known as Petzval’s, although Petzval’s work was of much later date. Hamilton, on the other hand, allowed his love of generality and of analytical developments to run away with him. In his Memoir on Systems of Rays, with its elaborate and rambling supplements, there is little to interest the practical optician, though the mark of genius is throughout apparent. It was only in two or three pages of a later paper that he applied his powerful methods to the real problem of Optics. As Finsterwalder has remarked, his “ six radical constants of aberration,” expressing the general properties of a symmetrical instrument, are at once an anticipation and a generalisation of von Seidel’s theorems. But the published work is the barest possible summary. If Hamilton had been endowed with any instinct for Optics proper, he could nave developed these results into a treatise of first-class importance. In more recent times Hamilton’s footsteps have been followed by Maxwell as well as by Thiesen and Bruns, of whom the two latter do not seein to have realised that Hamilton (or even Maxwell) had concerned himself 1907.| Anniversary Address by Lord Rayleigh. 77 with the subject at all, The natural development of Hamilton’s ideas will be found in an able memoir by Schwarzschild (1905). I have spoken of English work that lay neglected, but a scarcely less notable instance is the splendid discovery of the microscopic limit by Fraunhofer, a man who combined in the highest degree practical skill with scientific insight. Thanks to the researches of Abbe and Helmholtz, it is now well known that there is a world that lies for ever hidden from our vision, however optically aided ; but neither of these eminent men realised that the discovery had been anticipated by’ Fraunhofer. Some, perhaps, may doubt whether Fraunhofer’s argument, founded upon the disappearance of spectra from gratings of extreme fineness, is of adequate cogency. To this I may reply that I was myself convinced by it in 1870, before either Abbe or Helmholtz had written a word upon the subject. Enough has probably been said to illustrate my contention that much loss has ensued from ignorance and neglect of work already done. But is there any remedy? I think there ought to be. In all the principal countries of the world we have now a body of men professionally connected with science in its various departments. No doubt the attention of many of these is so engrossed by teaching that it would be hard to expect much more from them, though we must remember that teaching itself takes on a new life when touched with the spirit of original enquiry. But in the older Universities, at any rate, the advancement of science is one of the first duties of Professors. Actual additions to knowledge occupy here the first place. But there must be many who, from advancing years or for other reasons, find themselves unable to do much more work of this kind. It is these I would exhort that they may fulfil their function in another way. If each man would mark out for himself a field—it need not be more than a small one—and make it his business to be thoroughly con- versant with all things new and old that fall within it, the danger of which I have spoken would be largely obviated. A short paper, a letter to a scientific newspaper, or even conversation with friends and pupils, would rescue from oblivion writings that had been temporarily overlooked, thereby advancing knowledge generally and sometimes saving from discouragement an unknown worker capable of further achievements. Another service such _ experts might render would be to furnish advice to younger men desirous of pursuing their special subject. The readers of whom I have been speaking are experts capable of advancing science themselves and of helping others to do so. But there is another class of possible readers of scientific books on behalf of whom I wish to make an appeal. We who are dependent upon sight in almost 78 Annwersary Address by Lord Rayleigh. [Nov. 30, everything that we do must specially sympathise with those unfortunates who are deprived of this most precious gift. A movement is on foot, and has already received valuable support, to promote the publication of standard scientific works in embossed type suitable for the use of the blind. Such publication is costly and can hardly be undertaken upon an adequate scale without external aid. My friend, Mr. H. M. Taylor, a Fellow of this Society, tells me that in the course of the last 12 months he has written out the whole of Mr. C. Smith’s Elementary Algebra in Braille type, has afterwards read the copy with his fingers and again, later, read the whole in proof. There can be no doubt that books in embossed type on such subjects as Mechanics, Physics, Astronomy, Geology, not to mention the various Biological Sciences, would be an immense boon to many blind readers. I commend the proposal heartily to your notice. Another remedy for the confusion into which scientific literature is liable to fall may he in the direction of restricting the amount of unessential detail that is sometimes prevalent in the publication of scientific results. In comparing the outputs of the present time, and of, say, 30 years ago, the most striking feature that appears is doubtless the increase of bulk, in recent years coming especially from young workers stimulated by the healthy encouragement of direct research as a part of scientific education. But I think it may also be observed, and not alone in the case of such early dissertations, that there is, on the whole, less care taken for the concise presentation of results, and that the main principles are often submerged under a flood of experimental detail. When the author himself has not taken the trouble to digest his material or to prepare it properly for the press, the reader may be tempted to judge of the care taken in the work from the pains taken in its presentation. The tendency in some subjects to submit for immediate publication the undigested contents of note-books is one that we hear much of at the present time. It is a matter that is difficult for publishing bodies to deal with, except by simple refusal of imperfectly prepared material, with its danger of giving offence to authors of recognised standing, but it seems not unlikely that at present public scientific opinion would endorse such a course of action. A related difficulty and one that contributes to this trouble, is the tendency, noticeable in some public scientific organisations, to imagine that their activity is estimated by the number of pages of printed matter they can produce in the year. Probably no consideration is further removed than this from the minds of the educated public, whose judgment is alone worth considering. ) #907. | Anniversary Address by Lord Raylezgh. 79 MEDALLISTS, 1907. CopLEY MEDAL. The Copley Medal is awarded to Professor Albert Abraham Michelson, For. Mem. R.S., on the ground of his experimental investigations in Optics. In 1879, Michelson brought out a determination of the velocity of light by an improved method, based on Foucault’s, which gave 299,980 kilometres per second. Three years later, by means of a modification of the method, capable of even greater precision, he found for this constant, of fundamental importance for-electric as well as optical science, the value of 299,853 kilometres. Michelson has been a pioneer in the construction of interferometers, which are now indispensable in Optics and Metrology. With his new instrument, at Paris, he determined the absolute wave-lengths of the red, green, and blue lines of cadmium by counting the number of fringes (twice the number of wave-lengths) corresponding to the length of the standard metre of the Bureau International des Poids et Mesures. He found the metre to be 1,553,164 times the wave-length of the red line of cadmium, a result which is almost in exact agreement with the redetermination last year by Perot and Fabry. Michelson thus proved the feasibility of an absolute standard of length, in wave-lengths, of such accuracy, that if the standard metre were lost or destroyed, it could be replaced by duplicates which could not be distinguished from the original. He had the greatest share in the elaboration of precise experiments on the relative motion of ether and matter. He repeated in an improved form Fresnel’s experiment of the speed of light in moving media, using water and sulphide of carbon. He found that the fraction of the velocity of the water by which the velocity of light is’ increased is 0-434, with a possible error of + 0°02. The fact that the speed is less in water than in air shows experimentally that the corpuscular theory is erroneous; but his results, moreover, established the correctness of Fresnel’s formula for the effect, the theory of which has since become well understood. In conjunction with E. W. Morley, he devised and carried out a very remarkable method by which, on the assumption of ether at rest, an effect depending on quantities of the order (v/V)? would appear to be appreciable. No displacement of the fringes was found. Of this result the simplest explanation would be that the ether near the earth partakes fully in its orbital motion ; but modern electric and optical science appears to demand a quiescent ether, and the existence of this and similar null results is funda- mental for its theory. | 80 Annwersary Address by Lord Rayleigh. [Nov. 30, He has shown the possible application of the Interferometer method to Astronomy, by himself measuring the diameters of the four satellites of Jupiter, which are only about one second of arc. He suggests the further application of the instrument to such of the fixed stars as may not subtend less than one-hundredth of a second of are. In 1898, Michelson constructed a spectroscope which enables us to make use of the great resolving powers of the very high orders of spectra which are absent in the use of the ordinary grating, and with the added advantage of having most of the hight in one spectrum. The echelon consists of a pile of glass plates of precisely equal thickness, which overlap by an equal amount ; with it spectral lines which appear single with the most powerful eratings can be resolved into components. This instrument has been espe- cially useful for the direct observation of the important, because definite, influence of magnetism on light, discovered by Zeeman. With 30 plates, and using the 25,000th spectrum, the echelon has a resolving power of 750,000, while the most powerful gratings do not exceed 100,000. In connection with the analysis of radiations, he has constructed and used various machines for the analysis of periodic motions. For example, in con- junction with Stratton, he perfected a remarkable machine which is based on the equilibrium of a rigid body under the action of springs. Professor Michelson has also investigated by his Interferometer the important subject, both theoretically and practically, of the breadth and the structure of spectral lines, including the effect of a magnetic field, and in various other ways his genius has opened up new ground in experimental Optics. RoyvaL MEDALS. One of the Royal Medals has been awarded, with the approval of His Majesty, to Dr. Ernest William Hobson, F.R.S. During the last 20 years Dr. E. W. Hobson has been distinguished for the fundamental character of his contributions to Mathematics and Mathematical Physics. His earlier published work, from 1888 onwards, deals largely with the so-called Harmonic Analysis, which embraces many topics having for their common aim the solution of the Potential Equation in forms suitable for application to the problems of Physics. The exhaustive examination of the general types of Harmonic Functions contained in his paper in the ‘Phil. Trans.” 1896, has been found to be of high utility for this application. He was led by these researches, and particularly by the difficulty of describing in general terms the characteristics of a function capable of being represented by Fourier’s series, to take part in the revision of the logical basis of 1907.] Annwersary Address by Lord Rayleigh. 81 ‘Differential and Integral Calculus which is now in progress; his Presidential Address to the London Mathematical Society, in 1902, on the questions here arising, aroused general interest among mathematicians; and he has recently (1907) published an extensive volume, dealing with the whole matter and its applications to the theory of Fourier’s series, which is of great importance for the history and development of Mathematics. His Majesty has also approved the award of a Royal Medal to Dr. Ramsay H. Traquair, F.R.S. . Dr. Traquair is honoured on the ground of his long continued researches on the fossil fishes of Paleozoic strata, which have culminated, within the past 10 years, in his discovery of new groups of Silurian and Devonian fishes, and in his complete exposition of the structure of Drepanaspis, Phlyctenaspis, and other remarkable forms. | For nearly forty years Dr. Traquair has been busy with the description of fossil fishes, mostly from the Paleozoic rocks of Scotland, and he is deservedly held to be one of the most eminent paleontologists of the day. He has been highly successful in the interpretation of the often very obscure and frag- mentary remains which he has had to elucidate, and his restorations of fishes _ have won such credit as to appear in all modern text-books of Paleontology. It may be said that his work, notwithstanding the great difficulties of the subject, has well stood the test of time. Dr. Traquair has done much to advance our knowledge of the osteology of fishes generally. His earliest memoirs on the asymmetrical skull of flat- fishes and on the skull of Polypterus remain models of exactness. His acquaintance with osteology enabled him to show how former superficial examination of the Paleozoic fishes had led to wrong interpretations. He demonstrated that Chirolepis was not an Acanthodian, as previously supposed, but a true Paleoniscid. In 1877 he satisfactorily defined the Paleeoniscidée and their genera for the first time, and conclusively proved them to be more nearly related to the Sturgeons than to any of the other modern Ganoids with which they had been associated. He thus made an entirely new departure in the interpretation of extinct fishes, replacing an artificial classification by one based on phylogenetic relationship. His later memoir on the Platysomidee was equally fundamental and of the same nature. All subsequent discoveries, many made by Traquair himself, have confirmed these conclusions, which are now universally accepted. In 1878, Dr. Traquair demonstrated the Dipneustan nature of the Devonian Dipterus, and somewhat later he began the detailed study of the Devonian fishes. His latest researches on the Upper Silurian fishes of Scotland are equally important, and provide a mass of new knowledge for which we are 82 Anmversary Address by Lord Rayleigh. [| Nov. 30, indebted to his exceptional skill and judgment in unravelling the mysteries of early Vertebrate life. Davy MEDAL. The Davy Medal is awarded to Professor Edward Willams Morley. Professor Edward W. Morley is well known both to chemists and to physicists for his work in the application of optical interferences and other physical phenomena to increase the accuracy of measurement. Numerous valuable papers have appeared, either in collaboration with Professor Michelson and others, or in his own name, on such subjects. Special reference may be made to his experiments, in conjunction with Professor Michelson, on the fundamental question of the absence of effect of translatory motion of material bodies on luminous phenomena. His claim to the Davy Medal rests on grounds closely related to these researches; for he has combined thorough mastery of accurate measurement with an intimate knowledge of modern chemistry, and has utilised them in his attempt to solve one of the most difficult and fundamental problems of chemical science. The special problem to which he has consecrated many years of his life is the determination of the relative atomic weights of hydrogen and oxygen; it has been attacked by him with rare insight and skill, and with indomitable perseverance, and he seems to have settled it for many years to come, if not permanently. All the recent work devoted to this problem, and there has been much, has tended to establish more firmly the ratio arrived at by Professor Morley. His determinations of the absolute weights of a litre of hydrogen and of oxygen, and his investigations of the amounts of moisture retained by gases dried by various desiccating agents, are of the very greatest importance for scientific progress. : SYLVESTER MEDAL. Professor Wilhelm Wirtinger, of Vienna, is the recipient of the Sylvester Medal. | He is distinguished for the importance and wide scope of his contributions to the general Theory of Functions. Our knowledge of the general properties and characteristics of functions of any number of independent variables, and our ideas for the further investigation of such functions are, for the most part, at present bound up with the Theory of Multiply- periodic Functions, and this Theory is of as great importance for general Solid Geometry as the ideas of Abel have proved to be for the Theory of Plane Curves. Professor Wirtinger has applied himself for many years to the study of the general problems here involved. A general summary 1907. | Anmversary Address by Lord Raylegh. 83 of his researches is given by him in the Abel Centenary volume (xxvi, 1902) of the ‘ Acta Mathematica. Two of his papers may be particularly referred to, both of 1895. One of these deals with the reduction of the Theory and General Multiply-periodic Functions to the Theory of Algebraic Functions, with a view to their expression by Theta functions; this was one of the life problems of Weierstrass, who did not, however, during his lifetime, publish anything more than several brief indications of a method of solution. Professor Wirtinger’s memoir obtains a_ solution, and is, moreover, characterised throughout by most stimulating depth and grasp of general principles. This paper was followed by two others, one continuing the matter in detail, the other making an application of its principles to the general Theory of Automorphic Functions. Another extensive paper, which obtained the Beneke Prize of the Royal Society of Gottingen, deals with the general Theory of Theta Functions. In it he obtained results of far-reaching importance, for Geometry as well as for the Theory of Functions, the full development of which will require many years of work. HucuHes MEDAD, The Hughes Medal is awarded to Principal Ernest Howard Griffiths. Principal Griffiths has conferred great benefit on physical science by his series of measurements of fundamental constants, mainly in the domain of thermal and electric energy. At a time when the equivalent of the thermal unit in mechanical energy stood urgently in need of revision, he devoted himself to the problem with all the refinements and patient manipulation that could be devised, the result being a value for Joule’s equivalent which at once acquired authority in the lght of the evidence produced, and largely confirmed the corrections already advanced by Rowland and others. A main cause of discrepancy had been found to be the variation of the thermal capacity of water with the temperature; and by an investigation in which this variation was determined, Griffiths elucidated and correlated fundamentally the work of previous observers, from Joule onward. Of special importance also, in the domain of chemical physics, was an investiga- tion of the depression of the freezing point of water by very dilute admixture of dissolved substances, wherein he verified, with all the refinement of absolute physical determinations, that the change of freezing point ran exactly parallel to the electric conductivity when the dilution of the electrolysable salt was comparable to that of gases, being twice as much per molecule as the standard value of the depression for non-electrolytes. 84 Anniversary Address by Lord Rayleigh. BUCHANAN MEDAL. The Buchanan Medal is awarded to Mr. William Henry Power, C.B., F.R.S. Mr. Power’s services to Hygienic Science and Practice have extended over a period of more than thirty years, and have been of the most distin- guished kind. He has himself personally conducted successful enquiries into the causes of the spread of various diseases, and has obtained results which have proved of the greatest benefit to mankind. Moreover, in his lone con- nection with the medical department of the Local Government Board, he has planned and directed numerous general and local investigations whereby our knowledge of disease, and of the methods of coping with it, have been greatly increased. The medical reports issued by the Local Government Board, which are universally regarded as among the most important contributions of our time to this subject, have for many years past been either written by him or owe much to his editorial criticism and supervision. . It is not too much to say that no living man in this country has advanced the cause of scientific hygiene more than Mr. Power, or is more worthy of the distinction of the Buchanan Medal. 895 On the Inheritance of Eye-colour wn Man. By C. C. Hurst. (Communicated by W. Bateson, F.R.S. Received May 7,—Read November 14, 1907.) The following notes on the inheritance of eye-colcur in man are based on material examined by the writer in the village of Burbage, Leicestershire. During the past three years, 139 pairs of parents and 683 of their offspring have been examined, with the following results :— Eye-colour in man depends almost wholly on the colour of the iris. The colour of the iris varies considerably in different families and often in different individuals of the same family. This variability is due partly to the presence or absence of different pigments on the anterior and posterior surfaces of the iris, and partly, in all probability, to actual differences in the structure of the iris. In the Report of the Anthropometric Committee of the British Association for 1880, the following statement concerning the nature of eye-colour in man is given by Mr. Charles Roberts (pp. 154 to 136) :-— “The iris, on which the colour of the eye depends, is a thin membranous structure, composed of unstriped muscular fibres, nerves, and blood-vessels, held together by a delicate network of fibrous tissue. On the inner surface of this membrane there is a layer of dark purple pigment called the wvea ... and in brown eyes there is an additional layer of yellow (and, perhaps, brown-red) pigment on its outer surface also, and in some instances there is a deposit of pigment amongst the fibrous structures. In the albino where the pigment is entirely absent from both surfaces of the iris, the bright red blood is seen through the semi-transparent fibrous tissues of a pink colour; and in blue eyes, where the outer layer of pigment is wanting, the various shades are due to the dark inner layer of pigment—the weea— showing through fibrous structures of different densities or degrees of opacity. “The eyes of new-born infants . . . are dark blue, in consequence of the greater delicacy and transparency of the fibrous portion of the iris; and as these tissues become thickened by use, and by advancing age, the lighter shades of blue and, finally, grey are produced, the grey, indeed, being chiefly due to the colour of the fibrous tissues themselves. In grey eyes, moreover, we see the first appearance of the superficial layer of yellow pigment in the 86 Mr. C. C. Hurst. [May 7, form of isolated patches situated around the margin of the pupil, or in rays running across the iris. “Tn the various shades of green eyes the yellow pigment is more uniformly diffused over the surface of the iris, and the green colour is due to the blending of the superficial yellow pigment with the blue and grey of the deeper structures. In the hazel and brown eyes the wvea and the fibrous tissues are hidden by increasing deposits of yellow and brown pigment on the anterior surface of the iris, and when this is very dense, black eyes are the result.” The above statement agrees well with my own observations, though I would add that the presence of the superficial layer of yellow pigment, when only slightly developed, is to be seen in blue eyes as well as in grey eyes. No albinos were met with in the material examined by me, and my observations, therefore, relate to pigmented eyes only. It has generally been supposed that the various types of eye-colours grade into each other without sensible breaks of continuity. = z cB an S | S : — s . e ‘ re ° ©. 5 ° ° . ake 3 val . é 2a © aia aS EMS BoE ONS eee OHO a Bia | Bs A ‘a 2 Pe Rie Takara. le Sess ieee ie UME a rier et ean a ity ie pees b ° kK 53 Bae eM eCoea edo En Coe o Cee ceedocHeo se < oo E Sg teeddcdde = aes Ties Mel io fenton) Sen Galen 5 US = UD 69 99 OP rl GO 1D Be PAD BH CO mt 99 10 60 Bw 99 > 1D 11D et OD Py et 10 OD Ps 1D DO STS SSA OOOm ar Cle aa aan ge Seen Seles ea Sera cet ea SAAR RAAAAAAAS ° i | 90 simplex. Number and initials of d parent. Oe ee ee a. ee ee a | ' i Ctr AIAN & OV © aha) halt E-E-1-1ai takai b wT Oe Am Pong nOg mPa Wie We & Ob Ob | | | 239 H. 245 J.T. Q AlwownnFrwan or) Duplex. | | | rae j | | fi | J - On the Inheritance of Eye-colour in Man. Duplex-Simplex Matings. Table I1I1.—Duplex x Simplex. Simplex. (a) Giving all Duplex. Sh OOS Soyo) OOo SIOVSVve) The 69 matings of duplex and simplex parents also gave two kinds of results: (a) 17 families produced 66 offspring, all of the duplex type; (0) 52 families produced 258 offspring, of which 121 were duplex and 137 were Table III (a) and (6) give the numbers found in each family. Total offspring. WWNNEBWONNEWEWON ON [May 7, Mr. C. C. Hurst. 92 Table I1]—continued. Total offspring. Simplex. Duplex. Number and initials of 6 parent (b) Giving Duplex and Simplex. MOOMAMMDMWONWMW MAO ANRIOT NMI FP MOANMANOMAMAMANARNDANRHMHAHMRONNONNO regi sacl es MACH dP ain ciel, : SOR donna died abide SUBBR Kid ddan HO HHRERRORBROME REE EERROEMAOUBK dK ORK HOD POD EAA ABO SM OAAAL AAI ADIL AWS ABO HE WMAP ADS AANAN A OO ip 10 DOr RODDOBDODOONNS HH DE alta De Lag ON a a are ey ar ee NYCI pr yee a SE 24, Leal N rs a -1907.] On the Inheritance of Eye-colour in Man. 93 _ With regard to the heredity of the duplex and simplex types, the above tables show that :— _ (1) Simplex parents mated together give all simplex offspring. (2) Duplex parents mated together give either (a) all duplex offspring, or (6) duplex and simplex offspring in the proportion of about 3: 1. (3) Duplex parents mated with simplex parents give either (a) all duplex offspring, or (0) duplex and simplex offspring in the proportion of about 1: 1. It is evident, therefore, that the simplex type, in heredity, behaves as a Mendelian recessive to the duplex type, which is dominant. We have already seen that the duplex type differs from the simplex type by the presence of anterior pigment in the iris. The unit-characters concerned in the heredity of the duplex and simplex types of eyes are, therefore, presence (duplex) and absence (simplex) of anterior pigment in the iris, presence being dominant over absence, which is recessive, The duplex parents in Tables II (0) and III (0) are all obviously heterozygous, carrying the simplex character in a recessive state. The duplex parents in Tables II (a) and III (a) may be either homozygous or heterozygous. The duplex parents in Table ITI (a) with large families are almost certainly homozygous, but in those with small families the numbers of offspring are insufficient to test adequately the gametic constitution of the duplex parents. Similarly in the case of the large families in Table II (a), one of the duplex parents is almost certainly homozygous, while the other parent may be either homozygous or heterozygous. | In the small families the numbers of offspring are again too small to test adequately the gametic constitution of the duplex parents. In proof of this we have the fact that in Table III (a) each of the three duplex parents of families 53 D. A.,125 E. R., and 209 J. H. is known to have a simplex parent and consequently must be heterozygous, though none of them has yet produced simplex offspring. Similarly in Table II (a) both duplex parents of family 13 J. C. are known to have a simplex parent, though they have not yet produced any simplex offspring. As might be expected in a mixed population, some of the simplex parents observed are known to have been extracted from a duplex parent, or to have had duplex brethren. For instance, in Table I, at least one of the simplex parents of families 15 A. F.,97 H.C., 231 W.K., 255 J. H., and 257 J. C. is known to have a duplex parent, while of the families 31 D. N. and 243 J. F. one parent at least has duplex brethren. In accordance with the Mendelian principles, the extracted simplex type 94 Mr. C. C. Hurst. [May 7, breeds true to the simplex character without reversion to its duplex ancestors. In view of recent Mendelian experiments with plants and animals, it did not seem impossible that some simplex individuals at least might be carrying factors which on meeting, in the process of fertilisation, with other complementary factors, might give rise to reversions of the duplex type, but, so far, no such cases have been found. For the present, therefore, while remembering such a possibility, we must take it that the duplex and simplex types of eye-colour in man constitute a simple Mendelian case of presence and absence of a certain pigment. With regard to the different patterns of duplex eyes and their various shades, my facts, so far, do not enable me to determine positively their genetic relations. The chief difficulty is due to the fact that the anterior pigment present in children tends to increase with age, though to what extent, or to what age,is not yet known, owing to the limited period of observation. In the few families observed, with all adult offspring, the evidence suggests that the ringed pattern is recessive to the self-coloured pattern, which is dominant ; but with regard to the genetic relations of the spotted pattern to the ringed and self-coloured patterns there is practically no evidence available. Previous Work. Large numbers of records of eye-colours have been compiled and discussed by anthropologists and biometricians at home and abroad. In most cases, however, the data relate to certain sections of the population, such as school children and conscripts, not analysed or grouped according to their families. So far, I have found only two memoirs which approach the question of the inheritance of eye-colour in man by a comparison of parents with their offspring. The first is that of Alphonse de Candolle.* De Candolle, with the assistance of some 28 experienced observers, collected a number of records of the inheritance of eye-colours in Switzer- land, Germany, and Sweden. De Candolle made two classes of eye-colours, “brown” and “ blue,’ omitting all doubtful shades. The “brown” class included “black,” “brown,” “ yellow-brown,” and “green-brown.” The “blue” class included “blue,” “blue-grey,” “grey,” “green-blue,” and “green-grey.” Comparing de Candolle’s classes and shades with mine, it is evident that all the “ brown” class belong to the duplex type with anterior pigment, but that only part of the “blue ” class belong to the simplex type, without anterior pigment. * “ Wrérédité de la Couleur des Yeux dans |’Espéce Humaine,” ‘ Archives des Sciences, Geneve (3¢me période, vol. 12, 1884, pp. 97—119). 1907. | On the Inheritance of Eye-colour in Man. 95 The second memoir is that of Mr. Francis Galton.* Mr. Galton collected a number of records of family eye-colours in the British Isles, among the “ Records of Family Faculties ”—known as the R.F.F. data. These family records were obtained through the offer of prizes to the public.t From the family records sent in, Mr. Galton made three classes of eye- colours, “ light,” “hazel,” and “dark.” The “light” class included the shades recorded as “light blue,” “blue,” “dark blue,” “ grey,’ and “ blue-green.” The “ hazel” class included “dark grey” and “hazel.” The “dark” class included “black,” “ very dark brown,” “ dark brown,” “ brown,” and “ light brown.” Com- paring the shades of colour sent in to Mr. Galton by his correspondents with mine, it is evident that all of Mr. Galton’s “dark” class belong to the duplex type with anterior pigment. With regard to the “hazel ” class, part of these would probably represent my ringed duplex pattern, while the remainder might belong either to the duplex or simplex type, according to the inter- pretations of the colours by different observers. Mr. Galton apparently regards “dark grey” and “ hazel” as bicolour eyes,f which would make them practically equivalent to my ringed duplex eyes. In view, however, of my experience with popular descriptions of eye-colours, it is highly probable that many of Mr. Galton’s correspondents would record certain forms of self- coloured duplex eyes as “hazel,” and certain forms of “simplex” eyes as “dark grey.” Mr. Galton’s “light” class would apparently consist partly of the simplex type without anterior pigment, and partly of the low-grade forms of the duplex type with some anterior pigment. In the nature of the circum- stances in which the R.F.F. data were recorded, it cannot, of course, be expected that the observations were critical in regard to the presence or absence of anterior pigment in the iris. On the whole, therefore, it does not seem possible to express either de Candolle’s or Mr. Galton’s classes and shades of eye-colours in terms of duplex and simplex pigmentation. It was on Mr. Galton’s R.F.F. data that Professor Karl Pearson based his memoir “On the Inheritance of Eye-colour in Man,’§ and afterwards concluded that nothing corresponding to Mendel’s principles appeared in the characters for eye-colour in man.| * “Family Likeness in Eye-colour,” ‘Roy. Soc. Proc.,’ 1886, vol. 40, No. 245, pp. 402—416. t See ‘ Natural Inheritance,’ 1889, pp. 72—78. t Loe. cit., pp. 142, 144. § ‘ Phil. Trans.,’ A, 1900, vol. 195, p. 102. || ‘ Biometrika,’ 1903, vol. 2, pp. 213, 214. 96 On the Inheritance of Eye-colour in Man. Summary. An examination of the eye-colours of a number of parents and their offspring in a Leicestershire village shows that there are at least two dis- continuous types of iris In man :— (1) The duplex type, with both anterior and posterior pigments, as in ordinary brown eyes. (2) The simplex type, with posterior pigment only, the anterior pigment being absent, as in clear blue eyes. In heredity the simplex type behaves as a Mendelian recessive to the duplex type, which is dominant. The unit characters concerned are evidently presence (duplex) and absence (simplex) of anterior pigment on a basis of posterior pigment, presence being dominant. , The duplex and simplex types can be distinguished at any age. Various pigmental and structural changes take place in the iris during childhood and youth, the extent of which is not yet known. Few families with living parents and offspring, all adult, are to be found in one village. Consequently, it has not yet been possible to determine the genetic relations between the various shades of the duplex type. Note——I desire to acknowledge my indebtedness to Mr. W. Bateson for some valuable criticisms and suggestions in regard to the preparation of this paper, and also to Mr. R. C. Punnett, who came down and examined a number of the simplex eyes recorded above. (Note added 11th January, 1908.) Since the above paper was presented, an article on “ Heredity of Eye- colour in Man ” has appeared*in ‘Science,’ 1907, vel. 26, pp. 589—592 (dated Ist November), in which Professor C. B. Davenport independently arrives at similar conclusions, pointing out the Mendelian inheritance of eye-colour in man. a ol = 97 On Some Features in the Hereditary Transmission of the Self- black and the “ Irish” Coat Characters in Rats.—Paper I. By Geo. P. Mupcez, A.R.C.8ce. Lond., F.Z.S., Lecturer on Biology, London Hospital Medical College (University of London), and the London School of Medicine for Women (University of London), (Communicated by A. D. Waller, F.R.S. Received May 8,—Read November 14, 1907.) CONTENTS. PAGE PAPAL OCHINCEOIY) we. Sion) cna wae seed tine aaeenbae Met aad Ss noacusessa ye Vanutacks 97 B. System of Interpretation of Results..............ccceccecsceeeseeees 99 €. The “ Irish” Character : Nature and Origin .................0005 100 i yeeeperimental Results 1.21.15. gsi deonsccerecisestnencestisrasearesdace cuss 102 Cie trish sb Olbish OG GO CEG. b) saci seckscenccsacess 102 Ge AMIN GE PAR CMCAQ Es cece. ti stv ences: davcaracceevessadubissaves 102 CDOeEte bale Pare med ee 500.5 j. cieetedsacslvvedewete steeds ies 104 (2) “Irish” 6x Piebald black-white (Cr 6 bx Cr 5) ......... 105 (3) i Mp lmian(Or 6 Doc Cr 4.6 oot lswentecea cess 106 (4) is x Grey with White (Cr6 6x Cr 2) | .....cccess. 108 (oP Ayecovic, pattern” albtnOes v2.20: 2. 1907.| Transmission of certain Coat Characters in Rats. 99 The cost of these experiments has been defrayed by a grant from the Government Grant Committee of the Royal Society. B. THE SYSTEM OF INTERPRETATION OF RESULTS. It is possible to adopt at least three ways of representing the allelo- morphic pairs, but so far as the results which I have obtained with rats are concerned, the predictions are identical in each case. One of these “schemes” differs fundamentally from the other two in that it regards greyness as not being allelomorphic to blackness, while the other two do. There are reasons for believing that greyness and blackness may not be allelomorphic. And since this “scheme,” which embodies Bateson and Punnett’s “ Presence and Absence” theory, has successfully interpreted other results of a diverse nature, it will be adopted in this paper. The essential feature in their “scheme” is that the allelomorphic pairs consist of a determinant (factor) for the presence, and of one for the absence of the character under consideration. On the basis of gametic purity, there- fore, any one gamete cannot carry both the positive and negative factor of the same character. But it can carry the positive factor for each of two characters, such as blackness and greyness. This “scheme” predicts accurately the types of rat which result in any cross, in so far that types not predicted never appear. In some cases, however, predicted types have not appeared, but the evidence is clear that this is due only to want of numbers, and that with more litters the missing types will duly appear. Indeed, in several instances, when I first tabulated my results, a certain type which was predicted was absent, but in most of the cases it has since appeared in a later litter. With regard to the expected proportions, the prediction is usually approximately, and in some cases exactly, fulfilled. | In interpreting my results by means of Bateson and Punnett’s “ Presence and Absence” hypothesis, I have conjoined to their conception, that of Cuénot, in which he postulates the idea that colour is due to the interaction of two bodies, one a ferment and the other a chromogen. This idea has received confirmation from Miss Durham’s work. Below Ll append the various symbols employed :— C = colour producer.* c = absence of colour producer. G = grey determiner. = 3 erey determiner. B = blackt .. \C L black ‘4 = self-pattern determiner. We % self-pattern determiner. P = piebald-pattern determiner. p= " piebald-pattern determiner. * Tn this paper the colour producer is regarded as a ferment. + In this paper the grey and black determiners are regarded as chromogenous bodies which develope colour when acted upon by the ferment C. 100 Mr. G. P. Mudge. On the Hereditary — [May 8, The different types of rats will therefore be symbolically represented as follows :— Wild self-grey rat...... CGBSP. Piebald sh hpi eee CGBsP. Wild self-black rat .... CgBSP. Piebald Pe ee ae bsie A lbintostiabee. oe ertee ce cGBSP, cGBsP, cgBSp, cGbSp, ceGbSP, ceBSP, cGbsP,.cgBsP, cGBSp. By various combinations of these forms the number of possible albinoes can be increased. Thus, if cgBSP x cGBSp, a form cGegBSPp will result. It is very doubtful whether an albino not carrying at least one positive colour and one positive pattern member of each allelomorphic pair, exists. In accordance with the indications given by Miss Durham’s work (17), I shall assume that the albino carries the chromogen determiners (G and aD and not the ferment = tyrosinase = colour producer (C). In applying this “scheme” to my results, it will be necessary to assume that every gamete must carry one member of each allelomorphic pair. C. THE “IRISH” CHARACTER = CRAMPE’S Cr 6.* The form known in the “ fancy ” as the “Irish” rat is a nearly wholly black individual with patches of white on the ventral surface. With regard to its visible zygotic characters there are apparently two types which Doncaster (15) has proposed to call the “Irish” @ and the “ Irish” 6. With this subdivision of the “ Irish ” type (Cr 6) I am inclined to agree, and certainly for purposes of description it is a very necessary one. Doncaster’s definition of the zygotic characters of the “%” form is, however, unsatisfactory, because it is not sufficiently precise. According to him this form has more white on the ventral surface than the “a” form. My experience suggests that this is not the chief difference, and that we should define the “ Irish” } as being the form with more or less ventral white and with carpal and metatarsal white bands. The “Irish” @ possesses the ventral white alone and always very little of it, but has no carpal or meta- tarsal bands; the ventral white is usually merely a fleck on the chest or abdomen. Some of my “ Irish” 6 forms in respect of the excessive reduction of the ventral white approach the a form, but are at once distinguished by the presence of the white carpal and metatarsal bands. * The symbols Cr 1—Cr 7, which are used in this paper, indicate the seven varieties into which Crampe divided rats. These varieties are described on p. 119, and also by Bateson (5), and by Doncaster (15). 1907.] Transmission of certain Coat Characters in Rats. 101 As a rule, the ventral white in the “ Irish” 6 form assumes the character of a longitudinal band extending from the chest at the level of the arms back to the umbilicus or farther. In general form, this band is narrow-triangular with its base in the pectoral region. In most cases, the two angles of the base extend off along the ventral surface of the arms and reach the carpal bands. In some instances, however, this extension is absent, and 'in others broken. The longitudinal band itself is often broken, or expanded in some parts, usually in the abdomina! region, and constricted in others, usually just anterior to the umbilical region. In the pattern thus produced there is often symmetry, but usually more or less departure from a strict symmetrical figure exists. In the “ Irish” a form, the ventral white in most cases assumes the shape of a very small triangular patch in the pectoral region, with its apex directed posteriorly. It suggests a reduced pattern of the “Irish” 6 form. Some- times an additional spot of white is present on the abdomen. Origin of the “ Irish” 6. The “Irish” 6 is a heterozygote and in my experiments has been produced in the following 10 different kinds of crosses :— (1) Black var. of wild grey (Cr 7) x Cr 4.* (2) Extracted black (Cr 7) x Cr 4. (Or 2 x Cr 2.7 (4) Cr 2x Cr 4. (5) Cr 2x Cr 5 (carrying albinism recessive). (6) Cr 5x Cr 4, (7) “ Irish” 6x “Trish” 0 (Cr 6 0). (8) “Irish” 6x Cr 5 (homozygote), (9) “Irish” 6 x Cr 5 (carrying albinism recessive), (10) “Trish” 0x Cr 4. Of these, Doncaster has previously obtained “ Irish” 6 from Nos. 6 and 10, and also probably from crosses Nos. 2, 3, 4,5, and 7. In these latter crosses Doncaster had not, at the time they were made, distinguished between “ Irish ” b and a. But in the light of my experiments there can be no doubt his “Trish” forms from these crosses were of the sub-type 0. The origin of “Trish” 6 forms from crosses Nos. 1, 8, and 9, are, therefore, new. In addition to these, Doncaster also obtained this form from crosses of Cr 6 ax Cr 6 3, Cr 7x Cr 5,and Cr7xCr606. It is almost certain from his records, that * See footnote, p. 100. + The Cr 2 forms are similar in their visible zygotic characters to the “Irish” 6, but are grey in colour. 102 Mr. G. P. Mudge. On the Hereditary [May 8, the Cr 7 in the two latter experiments (53 and 34) was not homozygote and was, perhaps, an “ Irish” @ (see Summary of Results, No. 5, p. 115). The Cr 2* types used in my experiments, and also the “ Irish” 0, were of two different origins, one form in each type resulting from a cross of a self-grey or a self-black respectively with a Cr 4, and in the other with a corresponding cross with a Cr 5. The possibility that the Cr 4 in the two former cases carried the piebald pattern determiner must not be overlooked. The homo- zygote nature of the Cr 5 in the eighth line of the column of matings above was tested by crossing it with a Cr 5 known to be carrying albinism recessive. No albinoes appeared in its offspring. D. EXPERIMENTAL RESULTS. (1) Trish” 6x Trish” b= Ci-6 0 GCr Gab: (a) Albino Parentage-—Seven matings of this kind have been made. Of the seven pairs of “Irish” 6 individuals used, two pairs (experiments 49, 50) were derived from a cross of a black var.: wild grey x albino, three (51, 52, 53) from a cross of an extracted black (Cr 7) x Cr 4, and two (experiments 54, 55) from a cross of “ [rish” 6 x Cr 5 carrying albinism recessive. The black variety of the wild grey rat may be regarded as a pure black and as equivalent to an extracted Cr 7. There is no possibility of its carrying the grey chromogen determiner, since grey is dominant to black. We may, therefore, consider experiments 49,50, 51, 52, and 53 together and regard them all as crosses of a Cr 7 x Cr 4. The parental albinoes of these “ Irish” 0 individuals were bred by me from albino grandparents, which were in turn obtained from ordinary dealers. Rats obtained from this source are nearly sure to be of piebald origin, though one of mine thus obtained for another experiment carried the self character, and another the grey chromogen. We may, therefore, assume that these albinoes were carrying the piebald pattern and black chromogen determiners. On this assumption we may represent the “Irish” 6 individuals used by the symbol CegBSsP. It will, perhaps, be as well in this particular cross to show how the predic- tions which are deduced are arrived at, and I will therefore give a table of the possible gametic combinations. Each “ Irish” 0 individual of the zygotic composition CegBSsP will, at the segregation of the characters, form gametes which will carry the combination of characters shown in the following symbols: CgBSP, CgBsP, cgBSP, cgBsP. There are thus 16 possible combinations between the four kinds of gametes. The combinations are shown in the subjoined Table [. * See footnote, p. 100. x 1907.| Transmission of certain Coat Characters in Rats. 103 Table I.—Predicted Offspring in a Cross of “Irish” 0 x“ Irish” 6. Gametes formed by | i Be ae by aygou ee ae ig Pee cea eee j iis,” 6. | ris : of the offspring. offspring. CgBSP x CgBSP == CgBSP = Cr 7 CgBsP x CgBSP = CgBSsP = Cr 6 6 egBSP x CgBSP = CegBSP = Cr 6 a egBsP x CgBSP = CegBSsP = Cr 6b CgBSP x CgBsP = CgBSsP = Cr 6b CgBsP x CgBsP = CgBsP = Cr 5 cgBSP x CgBsP = CegBSsP = Cr66 cgBsP x CgBsP = CegBsP = Cr 5 CgBSP x cgBSP = CcegBSP = Cr 6a CgBsP x cgBSP = CegBSsP == Cr 6 5 egBSP x egBSP = egBSP = Cr 4 cgBsP x cgBSP = egBSsP = Cr 4 CgBSP x ceBsP = CegBSsP = Cr 6 6 CgBsP x cgBsP = CegBsP = Cr 5 cgBSP x cgBsP = cegBSsP = Cr 4 egBsP x egBsP = egBsP = Cr 4 Total offspring = 4 Cr 44+3 Cr 5+2 Cr6a+6 Cr6 6+1 Cr 7. X= gee os aw 4 alb. ; 12 pigmented. =1 : 3 Turning now to the actual results of experiments 49, 50, 51, 52, and 53, there is produced a total offspring of :— OF CrA! 4Or s-423 Cr 6 a-+- 10 Cr 6 644 Cr 7. et Ae aeie pdt e's) ay, oo alee == wheliioy ; 21 pigmented. Thus the experimental result departs from prediction in the expected proportions only. Dividing the actual result throughout by two, we have 454+2415+5+2. This is a very close approximation to the prediction. The actual result is only one Cr5 and Cr 60 too few, and one Cr 7 too many. It is instructive to plot out the prediction and the actual result in the form of two curves (fig. 1). It can be seen at once how close is the resemblance between the two.: It is obvious that the general slope and form is the same, and that the difference is not greater than that between a theoretical normal curve of error, and one of that form obtained from an actual series of measurements. It is to be noted that all the types which are predicted have appeared, and that none which are not expected have arisen. | VOL. LXXX.—B. ’ I as Ts 104 Mr. G. P. Mudge. On the Hereditary [May 8, We may, therefore, conclude that the “ Irish” } form in these experiments may have the gametic composition which I have assumed for it, z.c., CegBSsP. ba b6 7 Fig. 1.-—Curve showing the ie ec oo corr eee, between prediction and actual result in a cross of “Irish” 6 x “Irish” 6. The line indicating prediction is the continuous one, that showing result is the broken one. The abscissee represent the different types of rats, and the ordinates the proportions in which they occur. (b) Piebald Parentage—In experiments 54 and 55 the “ Irish” 5 parents used were derived from a cross of Cr 6 6x Cr 5 (carrying albinism recessive). The Cr 66 grandparent was in both experiments derived from a cross of black variety of wild grey x Cr 4 (see experiments 47, 57, and 59). From such a cross of Cr 66xCr 5, there are expected among the offspring, in addition to the Cr 4 and Cr 5 types, two forms of “ Irish” 0, v.e., CgBSsP and CegBSsP. If we suppose that in both of the experiments (54 and 55) one of the “Trish” b parents was a CgBSsP and the other a CcegBSsP, then the predicted results will differ from that where both are CcegbBSsP in the absence of albinoes. The expectation is :— 2 Cr5+1 Cr6a+4 Cr 6 641 Cr 7. The actual result of the two experiments combined is :-— 3 Cr 5+8 Cr 6 b+1 Cr 7. The special feature of the prediction, 7.¢., absence of albinoes, is thus far confirmed. Considering the smallness of the number, the approximation to prediction, with regard to proportion of the different types, is sufficiently close to be significant. .The proportion of Cr 5 is exact, that of the Cr 7 is as near as it can be with the number of individuals present, while that of the Cr 6 61s two too many. The predicted Cr 6 a is absent. In connection with the missing type, the remark made on pp. 99 and 106 should be borne in mind. Expected types do not always appear in the first litter, and some not until the third litter, even though the early litters may be of large numbers (eight or more). 1907.| Transmission of certain Coat Characters in Rats. 105 We may, therefore, regard these two experiments as affording initial evidence that this particular type of “ Irish” 0, 2.¢., CgBSsP as contrasted to CeeBSsP has been actually produced. (2) “ Irish” b x Prebald Black-White = Cr 66x Cr 5 Five matings of this nature have been made (experiments 56, 57, 58, 59, and 59a). In experiment 56 the Cr 5 parent was known by two previous testings to be homozygote. In experiments 57 and 59 the Cr 5 parent was known from previous testings to be carrying albinism recessive. In experiment 58 the Cr 5 parent was not tested, but since both its parents carried albinism recessive, the chances are equal that it also carried albinism. In experiment 59a the chances are also equal that the Cr 5 parent is either homozygous or carries albinism recessive. The “ Irish” } partners were in four experiments (56—59) members of the same parents, and were derived from a cross between a black variety of wild erey x Cr 4 (experiment 47). The albino parent was derived from albino grandparents purchased from ordinary dealers, and it was therefore, in all probability, carrying both black chromogen and piebald pattern determiners. The “Irish” 0 partners in these four experiments may be, therefore, represented by the symbol CegBSsP. The piebald parents in experiments 57, 58, and 59 will be represented by the symbol CegBsP. The predicted offspring in such a cross will be :— 2 Cr 4+3 Cr 5+53 Cr 6 0. We 4a =) Zale Or pigmented, (== 1 73) The actual result for experiments 57 and 59 combined is :-— i Cr ee Cr 5+20 Cr 6 642 pig. eyes.” 12 alb. 34 pigmented. = 1: : 3 nearly. In these two experiments (57 and 59) where the actual gametic factors are known with some degree of certainty, the prediction of proportions of Cr 4 to the pigmented types and the actual result are practically identical. The proportion, however, of Cr 6 0 is a little too large. In the case of experiment 57 there were five litters, and since the details are significant, I append them :— * At birth the pigmented individuals can be immediately distinguished: from the albinoes by the pigmented eyes, which show through the skin. These two young ones died before their coat colour and pattern had developed. At birth the skin of- all sans is colourless and hairless. 2 106 Mr. G. P. Mudge. On the Hereditary — [May 8, Ist. 2 Or 4 +6 Cr 6 8. 2nd. 2 Cr4+3 Cr5+2 Cr 6 0. srd. 3 Cr4+2 Cr 5+2 Cr 6 6+1 pigmented ae 4th. 3 Cr 442 Cr 5+2 Cr 6 0. oth. 2 Cr4+1 Cr 5+3 Cr 6 041 pigmented eyes. —_— -—_— 12 8 15 2 It is to be noted in the first litter, notwithstanding that it is large, that the predicted Cr 5 type is not present. That in the first, second, and third litters the proportion of albinoes to pigmented individuals is as near as it possibly can be to expectation, with the odd numbers involved. That in the third and fourth litters the albinoes are in excess. These considerations show us, what is also shown in others of my experi- ments, that predicted types will not necessarily appear in any one litter, and that large numbers must be reared before expected Prope ee in all cases will be quite accurately realised by actual results. Considering next, experiment 56, where the Cr 5 parent is known to be homozygote, the prediction is :— 2 Cr 542 Cr 6 0. The actual result is :— A Cro 3 ©r, 0) 0: With the odd numbers concerned this is the nearest approach possible to prediction. The chief feature to notice is that while the predicted types have appeared, unpredicted ones have not. Experiments 58 and 59a may now be considered. The absence of albinoes in their offspring suggests that the Cr 5 parent was homozygous. The total offspring in these two experiments is 4 Cr 5+8 Cr606. Added to experi- ment 56, it gives a total of 8:11 respectively. There is thus, roundly, only one Cr 5 too few, and one Cr 6 0 too many. (3) “Trish” b x | Albino = Cr:'6b6 x Cr'4, Seven crosses of this nature have been made, 7.¢., experiments 60 to 66. All the “Irish” 6 parents used were descended from two individuals of the black variety wild grey rat (experiments 47 and 48). Five of the seven were from mating 47, and the remaining two from mating 48. Three of the albinoes used (experiments 60, 62, and 63) were also derived from the same albino parents, which were obtained from ordinary dealers. In experiment 61 the albino parent was also obtained from the same source, but from a different parentage. Their zygotic composition may therefore be represented by cgBsP. In experiments 64, 65, and 66 the albinoes used were 1907.] Transmission of certain Coat Characters in Rats. 107 extracted ones. In 64 it was extracted from a mating of two “Irish” 6 parents (experiment 50). In experiments 65 and 66 the two albinoes used were brothers, and were extracted from a cross between a Cr 2xCr 5 (experiment 38). I will therefore deal with experiments 60, 61, 62, and 63 together, since all the four albino parents are probably of the same gametic constitution. The expectation 1s :— 2 Cr 441 Cri+1 Cr6 6 = 1 alb.: 1 pigmented. The actual result of the four combined experiments is :-— 14 Cr 447 Cr 5+3 Cr 6 6+6 pigmented eyes.* Ss : oe ae te 14 alb. 16 pigmented. ee Uae gl aa Result and prediction are thus almost identical with regard to the pro- _ portion of albinoes and pigmented individuals; they are so with regard to the nature of the types predicted, and doubtless had the six individuals with pigmented eyes lived, there would have been a near approach to expectation of the Cr 6 b type. Lxpervments 65 and 66. As already stated, the albino parents in this cross were derived from a Cr 2 x Cr 5 (experiment 38). The Cr 2 was derived from wild grey x Cr 4 (of ordinary dealers) (experiment 20).+ The Cr 5 was derived from Cr 6 6x Cr 4 (of ordinary dealers) (experiment 62). The Cr 6 0 from black variety greyxCr4 (of ordimary dealers) (experiment 48). (See Descendants’ Table, Charts II and IIT.) From an ancestry of this nature the albino parent used in experiments 65 and 66 may have one of four zygotic compositions, «., cgBsP, cgBSsP, cGgBsP, cGgBSsP. The “ Irish” 6 parents will be CegBSsP. If the albinoes are of the nature indicated by the last two symbols, and are carrying the grey determiner, then Cr 2 individuals are expected in the offspring. They are absent. Albinoes and the Cr 5 type are also expected in any case, but the Cr 4 is absent in experiment 65 and the Cr 5 in experiment 66. The numbers, however, are small, and it has been shown that expected types do not always appear in the first litter. The results of these two experiments do not coincide in their proportions with prediction, and they are being repeated on a larger scale. _* These died before coat colour had developed. + This experiment will be described in a later paper. 108 Mr. G. P. Mudge. On the Hereditary [May 8, Hxpervment 64. In this experiment the albino was extracted from a cross of Cr 6 bx Cr 6 6 (experiment 50). Reference to the table of gametic combinations on p. 103 will show that an albino thus extracted may be either ceBsP, egBSP, or cgBSsP. If it be egBSP, then a Cr 6 a is expected in its offspring when crossed with a Or 6 6; if it be cgBSsP, then a Cr 5 is additionally expected. Assuming it to be egBSsP, the full expectation is A Cra Cr 5- i) Cr o-22 Gr 60) The actual result of this mating in two litters is 3 Crd+2 Cr6a+8 Cr60. The expected albinoes are therefore missing, and there is a preponderance of the Cr 66 type. But since the Cr 5 and Cr 6a types were both absent in the first litter, it is possible that the expected Cr 4 types would have appeared had the parents hved to produce a third litter. The appearance, however, of the Cr 6 a type among the offspring can only occur if the parental albino is carrying the self-pattern determiner, and the presence of the Cr 5 type can only occur if it carries an allelomorph, 2.2. , which, being recessive to it, will therefore allow P to be manifested. The presence of Cr 6 a together with that of the Cr5 type, must therefore be regarded as initial evidence that this type of albino has been actually produced. And, since it is predicted (see table of gametic combinations, p. 103) that some of the albinoes produced in a cross of Cr6 6x Cr6 6 will be of the two types cgBSP and cgBSsP, it thus far constitutes a fulfilment of the prediction of the existence of this latter type in contrast to the former. (4) “Lrish” bx Grey with White = Cr 6 bx Cr 2. One cross only (experiment 66a) of this nature has been made, but the result is of exceptional interest, and affords a cogent test of the doctrine of gametic purity and of dominance. To understand the case, it is necessary to consider the origin of the Cr2 member of the pair. Its parents were a black variety of the wild grey rat (experiment 48) x with an albino purchased of ordinary dealers. In this mating there were seven young, six being of the expected “Irish” 6 type and one exceptional one, @.¢., a Cr 2. In my other crosses of this nature there were 13 young, all of the “ Irish” 6 type. To explain the production of this Cr 2, it is necessary to assume that the albino rat which was mated to the black variety of grey was carrying the erey determiner.* For reasons which will appear later, I will further * The same result would be obtained by supposing that the black variety of wild grey was carrying the grey chromogen. But since grey is dominant to black this cannot ) - the case. b d : 1907.| Transmission of certain Coat Characters in Rats. 109 assume that the albino also carried the black and piebald pattern determiners. Its composition will then be represented by cGgBsP. Such an albino, mated with a homozygote black, will produce, according to the subjoined Table II, equal numbers of “ Irish” 6 and Cr 2. Table I].—Predicted Offspring in a Cross of Cr 7 (extracted) xCr 4 (extracted). The albino is carrying the grey determiner and may be either cGgBsP or cGgBSsP. Gametes formed by | Gametes formed by Zygotic composition ie uy 2y aa self black. albino. of the offspring. pide insite lec | offspring. CgBSP x cgBsP = CegBSsP = Cr 6 CgBSP x cGBsP = CcGgBSsP = Cr 2 CeBSP % _cGBSP = CeGgBSP = Cr 2 CgBSP * egBSP = CcgBSP = Cr 6a Total Offspring. If albino = cGgBsP = 1Cr2+1(Cr6 4. ie = cGgBSsP = 2 Cr2+1Cr66+1Cr6a. If the assumption that the albino parent is carrying the grey determiner be correct, then the exceptional Cr 2 member among the offspring, in experiment 48, may be assumed to be of the zygotic composition CeGgBSsP. If we ascertain the possible gametic combinations when such a Cr 2 indi- vidual is crossed with an “Irish” 4 of CegBSsP composition, it will be such that the F 1 offspring predicted will be as follows :— Se@m4 ede Or b-28 Cr 24 3.Cr3+3 Cr5+2 Cr 6a+6 Cr 6641 Cr 7 = 1 Cr 4:3 pigmented. Now, the special feature in this progeny is the presence of the black forms, especially the homozygote Cr 7. Grey being dominant to black, black individuals could not appear in the F 1 generation of a cross between grey (Cr 2) and black (“Irish” 6), unless the grey member carried black recessive. Therefore, if black members of any coat pattern actually appeared in the F 1 generation of this cross of “Irish” 6 x Cr 2, it must be accepted as evidence that the Cr 2 carried black recessive. And, since on no other hypothesis, as far as I can see, could these black members appear—bearing in mind the unquestioned dominance of grey—unless gametic purity or segrega- tion of unit characters really occurs, it must also be accepted as a cogent proof of the truth of this doctrine. 110 Mr. G. P. Mudge. On the Hereditary [May 8, The actual offspring (experiment 66a) consisted of :— 1 Cr 4+ Cri+2 Cr 2+1 Cr 541 Cr 7. Thus, two types of black individuals have appeared, z.c., Cr 5 and Cr 7. The predicted “ Irish” form, together with the grey Cr 3 type, is absent. But if we bear in mind that, at the lowest limit, eight individuals at least must be produced in order to represent all types expected, and that actually only a total of six individuals were produced, it is obvious that two types at least. must be absent. Unfortunately, though I kept these two rats mated for a year and a-half after this first litter, they never gave rise to a second one, and are now dead. If the hypothesis above made be correct, then the Cr 7 member of the: offspring should be homozygote. It was tested by mating with three albinoes. _ (experiments 43, 44, 45) in succession, the last of which carried the grey determiner. With the first two albinoes, it had a single litter in each case, of eight individuals, making a total of 16, all of the “Irish” 6 type. No albinoes appeared. The “ Irish” 0 individuals of this offspring were crossed inter se (experiments 51, 52, 53), and gave a progeny wholly confirmative of the homozygote nature of the Cr 7. When crossed (experiment 45) with the third albino (carrying the grey determiner), it gave a wholly pigmented offspring, five in number (infra, p. 114). Thus, in a total offspring of 21, from three albinoes mated with it, not a single albino appeared. There can, then, be no doubt as to the homozygote nature of the Cr 7. Testing the assumption further by following some other of the offspring of the F 1 generation of this “Irish” 6 x Cr 2, to the F 3 generation, we may consider the behaviour of two of the F2 Cr2, which I mated with albinoes (Pedigree Chart I, experiments 73 and 74). If the assumption be correct that the Cr 2 parent (experiment 66a) carried B and P recessive, then the Cr 2 members of its offspring should be of several zygotic compositions, 7.¢., CecGgBSP, CGegBSsP, and CcGgBSsP. If the F 2 Cr 2 of CGgBSsP composition be mated with an albino of reputed cgBsP composition, two particular features are expected in the F 3 generation. There should be no albinoes, and black forms should appear. The expected offspring is :— Cr 2 4A Cri 3-4 Crepe emer ono: In experiment 73, an F 2 Cr 2 was mated with an albino derived from albino grandparents obtained from an ordinary dealer, and presumably, there- fore, of cgBsP composition. The actual result is :— 2 Cr 2+-4 Cr 3+5 Cr 5. 1907.] Transmission of certain Coat Characters in Rats. 111 The two special features predicted are therefore present, 2.¢., absence of albinoes and presence of black forms. We may therefore regard this experi- ment as being initial evidence that a Cr 2 of the predicted composition CGgBSsP was produced. Of course, the experiment must be repeated, and larger numbers reared before it can be accepted as proved. But it is also predicted that an F 2 Cr 2 of composition CeGgBSsP will be formed. The offspring of such a member when it is crossed with an albino of cgBsP composition will differ from the one just considered only in the presence of albinces. In experiment 74, the second F 2 Cr 2 was crossed with an albino which had been extracted from a Cr 2, and may therefore have been carrying the grey determiner, as well as the black one. If the albino carried the black determiner only, the expected offspring is— A Ora Oro el Cro Cr. 5-- il Cr ibid: If it carried the grey determiner additionally, it will be :— S.Cr4-eo (rl aor ot! Cro--1 Cr é & The actual result is -— 15 Cr4+1 Cr 2+1 Cr 3+4 pigmented eyes.* In the presence of albinoes, it satisfies one of the two special features of the prediction, though the absence of the black forms is disappointing, It is probable that the four individuals with pigmented eyes, which died quite early, included some black types. The presence of the albinoes is evidence that the Cr 2 contained albinism recessive. And such a type was predicted. Thus on the basis of the hypothesis of the nature of the Cr 2 of the F 1 generation (experiment 48), it is predicted that the Cr 7 type of the F 2 generation (experiment 66a) shall be homozygous. And prediction in this case has been completely proved to be fulfilled. It is also predicted that among the Cr 2 members of the F 2 generation (experiment 66a), three types shall be produced (supra, p. 110), and of these, two have been shown to very pro- bably exist, by the nature of the F 3 generation (experiments 73 and 74). (5) Zygotie “ pattern” Albinoes. There remains one feature of importance to mention. [It is one almost new to science, and, as far as I know, has been observed only by Haacke (19), and then only in the case of a single individual. In experiment 74, just considered, where a particular Cr 2 was x Cr 4, there occurs in the F 3 offspring 15 albinoes. Of these, six were born in the second litter. Now, it is predicted that albinoes of such parents may include six zygotic types, 7.c.,ceBsP, egBSsP, cGBsP, cGgBsP, cGBSsP, and cGgBSsP. * These died before the coat colour or pattern had developed. 112 Mr. G. P. Mudge. On the Hereditary [May 8, With regard to the positive pattern determinants which they carry, we may regard them as of two types, 2.e., those carrying only P, and those carrying both PandS. It should be noted that in pigmented individuals S is known to be dominant to P. Hitherto, we have had to rely upon breeding tests in order to ascertain what pattern determinants are carried by albinoes. But of the six albinoes in the second litter of this cross, one died early, while three of the remaining five had quite distinctly the pattern of the piebald rat. One had also the pattern of a Cr 2 or Cr 6 0 (these two patterns are the same, only the colour differs), and the remaining one had that of a self-coloured individual. I do not purpose going into details here as I shall publish a full account in ‘another paper. It will be sufficient to say that the pattern is rendered visible by some difference in the texture and closeness of arrangement of the hairs, so that where in pigmented individuals there is colour, in albinoes there is pinkness, due to the fact that the underlying skin shows through; and where ‘in pigmented individuals there is absence of pigment, in albinoes there is an opaque whiteness, the skin not showing at all. Thus, in the “self-pattern ” albino mentioned above, the whole surface of the body exhibited a pinkness. The visibility of the “pattern” early disappears, for I first noticed it in early August when the young were a month old, and it had disappeared in early October. In more recent cases I have noticed that it disappears when the individuals are about two to two and a-half months old. It is thus in the highest degree probable that some of the piebald marked albinoes were of the cgBsP, cGBsP, or of the cGgBsP type, that the Cr 2 or Cr 6 } pattern was of the cgBSsP or cGgBSsP type, while the self-pattern one may have been of the cGBSsP type.* If this conclusion is valid, then the prediction which forecasts that a homozygote black shall be formed, that certain Cr 2 types shall also be formed, and which also predicts that certain pattern bearing albinoes will be produced, has in all these cases been fulfilled. Haacke’s single case(18) was evidently a piebald-bearing albino. The other types are here described for the first time. Lock has described a some- what analogous case for the “maple” markings in peas (21). (6) Self-black x Albino = Cr 7 x Cr 4. (a) Black Variety of Wild Grey x Cr 4.—Two crosses of this kind were made (experiments 47 and 48). * While this paper was passing through the press, this conclusion has been tested by mating a “self-pattern” albino with a Cr 5. There were six young, all Cr6 6. Since self is dominant to piebald, the Cr 5 could not have carried the self-pattern determiner. This result is therefore conclusive proof that the albino did so. 1907.| Zransmission of certain Coat Characters in Rats. 113 In both matings, the albino parent was obtained from an ordinary dealer ; ithat in experiment 47 from one dealer, and that in experiment 48 from another. It is important to note this fact. In experiment 47 there were 13 young in one litter, all of the “Irish” 0d type. In experiment 48, there were seven young, all at first of the “Irish” 6 g; type, but one of these changed colour and became a Cr 2 in a few weeks after birth. This change of colour has several times occurred in my experiments and will form the subject of a separate paper. The further history of this Cr 2 member has been already considered, p. 108 and experiment 66a. From the “ Irish” 6 individuals, the extracted self-black types to be con- ‘sidered in the next cross were derived, as well as directly or indirectly, all the individuals of the “Irish” 6 crosses described in this paper. The details of the behaviour of these two types will, therefore, be found under their appro- priate headings. (b) HLxtracted Self-black x Albino —Four matings of this kind were made (experiments 43, 44, 45,46). Although there were four matings, only two black individuals were used, since the same individual ((1) 52/53) was crossed with three different albinoes. In experiments 43 and 44, this self-black individual was crossed with an albino which was derived from albino parents obtained from ordinary dealers. The zygotic composition of the albino may be, therefore, represented as coBsP. In each of these two matings there were eight young all “Irish” 8. Six of these from the second mating (experiment 44) were crossed inter se (experiments 51, 52, and 53). There is expected (supra, p. 103), on the assumption that their self-black parent is homozygote, albinoes and pigmented individuals in the proportion of 1:3 respectively. Actually it is as near as it can possibly be, ae, 4:11. The detailed expectation (Table I, p. 103), is :— 4 Cr 4-3 Cr5+2 Cr 6 a@+6 Cr 6 6+1 Cr 7. There are produced :— aera 3 Cr >) +0 CrG ao Cr 6 0-2 Cr 7 There is thus one Cr 7 too many and the expected “ Irish” @ is missing. But since some “Irish” a individuals have only a few white hairs on the chest, it is possible that they may be mistaken for Cr 7 until they are gametically tested. The result, therefore, may be regarded as confirming prediction in a very exact manner. 114 Mr. G. P. Mudge. On the Hereditary | May 8, These “Irish” members, combined with the similar offspring of other crosses, have been already considered (supra, p. 102). In experiment 45, the same self-black rat was crossed with an albino which had been extracted from a cross of Cr2xCr4 (éxperiment 71). The Cr 2 was derived from a cross of a wild grey x albino. In experiment 46, a very similar cross was made, but the self-black member was extracted from across between two “ Irish” 6 individuals (experiment 50). The albino used in this mating (experiment 46) was sister to that used in experiment 45. We may, therefore, consider experiments 45 and 46. together. The offspring will depend upon the nature of the Cr 4 parent. It will be assumed that the self-black parent in each case is homozygote, and in fact that used in experiment 45 has been proved to be so, by the previous results of 43 and 44. Albinoes extracted from a cross of Cr 2 x Cr 4, may be expected to include individuals of the following zygotic characters: cgBsP, cgBSsP, eGgBsP, and. cGgBSsP (supra, p. 111). If the albino is cGeBsP, and it is crossed with a homozygote Cr 7, the prediction (Table II, p. 109) is :— 1 Cr 2+1 Cr 6 8, If the albino is cGgBSsP, the prediction is :— SAN Ore VAT EOI ONG (2S Ore Gy ae In the two experiments (45 and 46) which we are considering, the total. offspring 1s :— 11 Cr 2+8 Cr 6641 Cr 6a. In experiment 46 no Cr 6 a type appeared, and it therefore suggests that. the albino parent is cGeBsP, and probably differing from that in experl- ment 45, in the absence of 8. If, therefore, we take experiment 45 alone the result is :-— 27Cr. 2-2 CrOuoe laCribea, This is as near as it is possible to get to expectation with the odd numbers obtained. The presence of grey offspring in both experiments is conclusive proof that the two albinoes carried the grey determiner. Thus in this case, prediction as to the nature of the Cr 2, of the two. extracted Cr 4 individuals, and of the two Cr 7* individuals, is tentatively fulfilled. The behaviour of the black individual in the manner described, in experiments 43, 44, and 45, shows in a marked manner the influence which the albino parent exerts upon the nature of the offspring. In experiments 43. * One of these two (ie, that of experiment 45) is the same individual as that. considered on p. 110. | 1907.| Transmission of certain Coat Characters in Rats. 115 and 44 it had none but “ Irish” ’ members in its offspring, 16 in number, because the albino with which it was mated had come probably from a piebald ancestry, and was carrying nothing but piebald pattern and black determiners. But in experiment 45 it was mated with an albino in which it was probable that the grey determiner was present, because of its extrac- tion from a grey parent. And grey individuals then appeared in its progeny. Grey individuals also appeared in the offspring of a sister albino when it was mated with a homozygote self-black. (7) Summary of Results. The figures within brackets indicate the page where the matter of the summary may be found. 1. That black is dominant to albinism (113). The dominance is not complete, some ventral white being always present. In relation to dominance this is perhaps not of particular significance, since ventral white patches are known to occur in both grey and black wild rats. This conclusion is confirmatory of that deduced from Crampe’s results, and arrived at in Doncaster’s experiments. It is theoretically conceivable that the incompleteness of the dominance is due to the influence of the factor s (= absence of self-pattern) brought in by the albino. 2. Expected types do not all always appear in any one litter of young, and frequently not until the third litter (106). Doneaster has also shown that there is considerable diversity in the proportions of the indivduals of each type in successive litters, but the examples published by him do not show whether a type present in one litter may be absent in another, as in several of my experiments. 3. Predictions based upon the doctrine of gametic purity and of dominance have not in these experiments (including those not yet published) been falsified by the appearance of unpredicted types. With but few exceptions, the predictions with respect to the kind of types expected are fulfilled, and approximately so with respect to proportions. The exceptions are due to the smallness of the number of offspring in certain cases. 4. The conception (hitherto a matter of deduction) that albinoes are the bearers of hidden factors, has been ocularly demonstrated for the piebald, “Trish” and self patterns (111). In peas a somewhat analogous phenomenon has been seen by Lock. 5. That completely self-black extracted torms are homozygous for black (110, 113). This conclusion appears not to harmonise with some of Doncaster’s 116 Mr. G. P. Mudge. On the Hereditary | | May 8,. results, 2.¢., experiments 34 and 37, where albinoes appeared in a cross when: one of the parents is described as black. Since the black parent was the same in both experiments, and its parents were unknown, it is not improbable that it was an “Irish” a, and not a. true black. If we make this assumption and accept the conclusion as to the nature of the Cr 6 @ given in this paper (Conclusion 7), then the results of Doncaster’s experiments 34 and 37 are in accordance with prediction. 6. The “Irish” “5d” type is a heterozygote. Two zygotic forms have: probably been experimentally recognised, ae, of the composition CegBSsP” and CgBSsP (104—107). With regard to this conclusion, the first symbolic representation confirms. that of Doncaster, while the second is new. 7. That the “Irish” “a” type is perhaps of the composition CegBSP. At present there is no direct evidence of this; but in the possible theoretical gametic combinations (supra, pp. 103 and 109) such a composition occurs.. The evidence shows that the “Irsh” 6 always carries piebald and s, and this being so, the composition CcgBSP can be ascribed to the “Trish” a form alone. Some of Doncaster’s experiments (15, p. 225, experiments 35 and 63) show that the “Irish” “a” does, in some cases, carry albinism recessive. Hence his own conclusion, that the Cr 7 is only an extreme type of Cr 6a@ and is not a separate variety, needs for the present to be accepted. with hesitation. My work indicates that the Cr 7 is homozygote for black (see 5). The above considerations suggest that the two varieties are: distinct. And, taken in connection with Conclusion 6, suggests that both “Trish” a and “Irish” 0 are heterozygotes. With regard to the composition CegBSP ascribed to the “ Irish” a, there is: to be remembered Doncaster’s conclusion, which I endorse, that the “Trish” @ and the Cr 7 have given no evidence that they carry P. But: there is nothing inconsistent between this conclusion and the symbolic representation of this form, because S and P are not allelomorphic to each other, and, therefore, though P may be present, it cannot be manifested so, long as S is also present in the same gamete or zygote. Evenif the “Irish” @ contains P, and it is crossed with an albino carrying P, no piebald forms. will be expected in the F 1 generation, because it (the “ Irish” a) also carries S.. Neither are they expected if “Irish ” a x “ Irish” a. Piebald forms cannot appear until s is introduced into the “ Irish” gamete: or zygote. The evidence indicates that when this is done the “Irish” 6} zygotic form is produced. It seems impossible, therefore, to obtain direct, experimental evidence that the “ Irish” a carries P. 1907.] Transmssion of certain Coat Characters in Rats. LL7 8. That of the various theoretically possible types of albinoes, the following four have in these experiments been shown to exist, ze, cgBsP (102, 106,. 110—111, and 112—113), cGgBsP (109, 112, 114), egBSsP (108, 112), and eGgBSsP (112, 114). To a lesser extent, the evidence indicates that two. other types, ze, cGBsP and cGBSsP, also exist (112). That is, of the four types experimentally recognised, so far, all carry both the black and piebald factors; one, and perhaps three, in addition, carry the grey factor, another the self-pattern factor, and one, or perhaps two, carry both the erey and self- pattern factors. The type cgBSP has not yet been demonstrated to exist. Of these four or six types, Doncaster has shown the existence of the two first: the others are new for rats. Hurst has shown the same thing for rabbits, 2.c., the existence of albinoes carrying B, and of those carrying BG. They also carry in some individuals the self character, in others the Dutch character, and in others both. The same thing has been shown for mice by Cuénot and Allen. There is thus a similarity in the carrying powers of albino rats, mice, and rabbits. In rats and rabbits it is to be noted no albino has yet been obtained which carries G alone, 2.¢., without B. 9. That self-grey types (Cr 2) may carry both biack and piebald recessive. The following two forms have been experimentally recognised, 7.¢., CGgBSsP’ (111) and CcGgBSsP (111). One thus carries albinism recessive in addition to the black and piebald determiners, and the other does not. Clearly they correspond to the two races of “ Irish” 6 revealed in these experiments. From some of Doncaster’s experiments it can be seen that the Cr 2 type carries B and P recessive, but he made no attempt to ascertain whether different zygotic varieties of this type existed. The discrimination of these two zygotic varieties is therefore new. 10. That piebald black-white types (Cr 5) may be homozygous for black and piebald, or may carry albinism recessive (105, 106). This conclusion can also be deduced from Crampe’s experiments; none of the Cr 5 types in Doncaster’s experiments appear to have been homozygous, and all carried albinism recessive. 11. Externally considered, similar zygotes may have a different gametic constitution (see above, 6, 8,9, and 10). In experiments not yet published, it can be shown that self-grey forms not distinguishable in external features. from those in 9, do not carry g, and therefore B, though present as a reces- sive character, cannot appear. Zygotic characters alone are not, therefore, a safe basis for prediction. , This confirms and extends the same conclusion which has been arrived at. in all experiments which give Mendelian results. 12. With regard to the number of allelomorphs for colour and pattern, the 118 Mr. G. P. Mudge. On the Hereditary [ May 8, evidence shows that there are two pairs for each, 7.¢., presence and absence of greyness, presence and absence of blackness, presence and absence of selfness, and presence and absence of piebaldness. , 13. Doncaster divided Crampe’s “ Irish” type into a and 6 sub-types (100). In this paper I propose a stricter definition of the “db” sub-type. REFERENCES. 1, Allen, G. M.—“ Heredity of Coat Colour in Mice,” ‘Proc. Amer. Acad. Arts and Science,’ vol. 40, No. 2. 2. Bateson, W., and Saunders, E. R.—‘ Experimental Studies in the Physiology of Heredity,” ‘Reports to Evolution Committee, Royal Society,’ Report I, 1901. 3. Bateson, W., Saunders, E. R., and Punnett, R. C._—‘ Report to Evolution Committee, Royal Society,’ Report IT, 1905. 4. Bateson, W., Saunders, E. R.,and Punnett, R. C.—‘ Report to Evolution Committee, Royal Society,’ Report III, 1906. 5. Bateson, W.—“ The Present State of Knowledge of Colour-heredity in Mice and Rats,” ‘ Proc. Zool. Soc. London,’ 1903, vol. 2. 54. Bateson, W.—“ The Progress of Genetics since the Rediscovery of Mendel’s Papers,” ‘Progressus Rei Botanice,’ Erster Band, Jena. 6. Castle, W. E.—“ Heredity of Coat Characters in Guinea-pigs and Rabbits,” Carnegie Institution of Washington, Publication No. 23, February, 1905. 7. Castle, W. E., and Allen, G. M.—“ The Heredity of Albinism,” ‘Proc. Amer. Acad. Arts and Science,’ vol. 38. | 8. Cotte, M. Jules.—“ Upon the Presence of Tyrosinase in Suberites domuncula, ‘Comp. Rend. Soc. de Biol.,’ Series IT, vol. 5, 19031904. 9. Cuénot, M. L.— La loi de Mendel et Vhérédité de la pigmentation chez les souris,” ‘Arch. Zool. Expér. et Gén.,’ Sér. 3, January 10; ‘ Notes et Revue,’ 1902. 10. Cuénot, M. L.—“ L’hérédité de la pigmentation chez les souris” (2me note), ‘ Arch. Zoo]. Expér. et Gén.,’ Sér. 4, January 1; ‘ Notes et Revue,’ 1903. 11. Cuénot, M. L.—“ L’hérédité de la pigmentation chez les souris” (83me note), ‘ Arch. Zool. Expér. et Gén.,’ Sér. 4, January 2 ; ‘ Notes et Revue,’ No. 3, 1904. 12. Cuénot, M. L.— Hypothése de ’hérédité de la pigmentation entre le croisement des noirs, des gris, et des blanc souris,” ‘Comp. Rend. Soe. Biol.,’ 1903—1904, p. 301. 13. Crampe.— Kreuzungen zwischen Wanderratten verschiedener Farbe,” ‘ Landwirth- schaftlichen Jahr.,’ vol. 6, 1877 ; zbid., vol. 12, 1883; zbed., vol. 138, 1884. 14, Crampe.— Die Gesetze der Vererbung der Farbe,” ¢bzd., vol. 14, 1885. 15. Doncaster, L.—‘‘ Inheritance of Coat Colour in Rats,’ ‘Proc. Camb. Phil. Soc.,’ vol. 13, Camb., 1906. 16. Ducceschi.—‘ Rendicontti della R. Accad. dei Lincei,’ vol. 2 ; ‘ Archivio di Fisiologia,’ vol. 1. 17. Durham, Florence M.-—“Tyrosinases in the Skins of Pigmented Vertebrates,” “Roy. soc. Proc? vol. 74. 18. Furth v. Schneider.—‘ Beitr. z. Chem. Phys. u. Path.,’ vol. 1. 19. Haacke, W.—“ Ueber Wesen, Urachen und Vererbung von Albinismus und Scheckung, etc.,” ‘ Biol. Centralbl.,’ vol. 15. 20. Hurst, C. C.—“ Experimental Studies on Heredity in Rabbits,” ‘Journ. Lin. Soc. Zoo.,’ vol. 29. 21. Lock, R. H.—‘‘‘ Ghost’ Maples in Peas,” ‘Roy. Soc. Proc.,’ vol. 79. Cr4gxCr4¢? +15 Cr 4 i Or 49 x Cr 5 try Table (pp. 119—1 PEDIGREE- CHarr I, THE DESCENDANTS oF No. 50x51. B. var. G.? xCr4¢ 48 Fil. 1Cr2+6Cr6“5” Cr29 xCr6“b”"¢ 66a F2. 1Cr142Cr2+10r441Cr5410r7" Cr7; xCr4é 43 Cr2 x Cr 4* Cr29 xCr4¢ 8Cr6“s” F8. F383, 2Cr2+4Cr3+5 Or5 aA ea la eyes Cr4gxCr49 Cr4gxCr7? <— Cr79xCr4g 44 19a Y 45 [ aa 715 Cr 4 FS. 2Cr2+2Cr6“6”"+1Cr6“a” 8Cr6“b” F3. fCr49 xCr5¢g Cr6“b" 3s x Or6b 2 Cr6“b"g xCr6“b”"9 Cr6“b"g xCr6“b"9 B7e 51 Se 52 538 i 1 ll 5 1 6Cr6 4” F4,. 1Cr4+2 Cr5+1Cr6 “ds” F4, 10r5+3Cr6“b”+1Cr7 F4. 30r4+2Cr6“5"”+1Cr7 Norr.—In those cases where the ancestry of only one of the parents can be traced on this diagram that of the other parent may be found on Pedigree Charts II or III, under the experiment bearing the same number, or it may be found in later papers. It can also be traced in the Ancestry Table (pp. 119—121) of this or later papers. * This albino was obtained from an ordinary dealer. LFS ; F + Seven of these exhibited the zygotic “ghost” of the self-pattern. The remaining eight died soon after birth. { This was one of the seven described in note fF. Wild G. g x Cr 6 F2. 4Cri+1Cr o+ PEDIGREE CHart II. THE DESCENDANTS oF No. 50x 51. B. var. G.? x Cr44 i 1 Cr 2+6 Cr6é “bd” re ae xCr4 fo Or 6“b"9 xCr4dg — Y 63 ( F2. 40r4+2 Cr5+10r6 “6” F2. 2Cr4+1 Or5+3 Pig. eyes F2. 4Cr1+1Cr2 ra Qo xCrig . F383. 10r2+20r3+4Cr4+3 Cri+2 Cr6“b” = een —— Cr2g xCrib? ee tide Crag x Cr6 5” Cri5g xCri? | 38a 66 28 F4. 20r3+2Cri+4Cr6“b” F4. 20r5+40Cr6“b” F4 3Cr4+5 Cr6 “6” F 4, 8 Or5 Novr.—In those cases, where the ancestry of only one of the parents can be traced on this chart, that of the other parent may be found on Charts I or III, under the experiment bearing the same number, or it may be found in later papers. It can also be traced in the Ancestry Vable (pp. 119—121) of this or later papers. et, Bs a i a r 7 ‘ j a al —- ——— iytaes ~ ‘ - “PEDIGREE CHaRT III. THE DESCENDANTS OF No. 750 x 152. B. var. G.? x Cr. 43 47 Or6“b"9 xOr5g Cr6“b"9 xCr5g Or6“b"9 xCrig 56 57 59 4 Cr 5+3 Or 6b” 12 Cr 4#8 Cr 5+15 Cr6 “5” 4 Cr 545 Or 6 “5” Orig xCr49? Cr 4x Cri Or6“b"g xCr6“b"9 Or 63" 9 x Cr*# 4g 24a 54 55 64 1Cr2+5Cr4+1Cr7 BiOe ae eICEe 8Cr6“b"+1Cr7 3Cr5+8 Or6 “b"+2Cr6a Cr2¢xCr4? 74a Cr 5x Cr 5t 1Cr2+2Cr3+1Cr5 28 Cr5¢ xCr2¢ 3Cr5 38a | 2 Cr3+2Cr5+4Cr6 “sb” Cr5¢? xCr6 “5” 59a 3 Cr5+2 Cr6 “5” B. var. G. 9 x Cr4 g 47 13 Cr 6 “b” Cr6“b”gxCr5? Cr6“b" go xCr6“b"9? Cr6“b"9 co nee ae 58 49 50 1 Cr 6+6Cr6 “5” 2 Or 441 Or 542 Cr6 “543 Or6 “a +1 Cr7 8 Cr¥ 44+2 Cr6 “6"+10r7 Sp eee Cr7g xCr4? Cr6“b"g xCr49 Cr6“b"? xCr4¢ Cr6“6"2 xCr4 46 60 ‘IC 61 We 66 9 Cr 2+6Cr6 “6” 4Cr4+4Cr5+1Cr6“6” 3 Cr4+5 Cr6 “3” 4 Cr 4+1 Or 6 “5” +3 Pig. eyes Cr 6 “3"2 xCr4 Cr2gxCr 49 65 72 2 Or 5+4 Or6 “5” 1 Cr 2+1 Cr3+5 Cr5+2 Cr6 “4” * The albino marked thus in Experiment 64 is from the offspring in Experiment 50. + The ancestry of this Cr 5 will be found in Pedigree 1000 x 263, Experiment 28, published in a later paper. ; cena ) La an ol an . ioe | , rs is Transmission of certain Coat Characters in Rats. 119 ANCESTRY AND OFFSPRING TABLES. Explanations. The “ Offspring Columns ” include Crampe’s seven types, 1.e.— Cr 1 = grey with no white. Cr 2 = grey with ventral white patches and white carpal and metatarsal bands. Cr 3 = grey-white piebald. Cr 4 = albino. Cr 5 = black-white piebald. : *Cr 6b = “Irish” 0; black with ventral white patterns or patches and ; | white carpal and metatarsal bands. , *Or6a= “Irish” a; black with little ventral white and no carpal or £ metatarsal white bands. Cr 7 = self black. * The division of the “Irish” type Cr 6 into“a” and “bd” is due to Doncaster, while I am responsible for the definitions of the two sub-classes as here given. The number of the parental pairing is given in the column headed Expt. The number of the ancestral pairings will be found in their respective columns, Experiments from 1 to 41 and from 67 onwards will be given in later papers. A few of these are to be found on the Descendants’ Tables (Pedigree Charts I to ITT). : | [May 8, Mr. G. P. Mudge. On the Hereditary 120 ee ae ieee S eee 8 een). t (Cea le ik pt eee ta! etl eresc| 0 0 eee iets Seelam "8 = |e ‘dutdsyzO zoz/zet/ocn(6)(F) ee ee Pe 7 q 5 9 Ip lige sto eae ae 992/ZST/OSL(4)() PB «@» 940 vee ecm ee cere ceseesseeece 992/ZST/0S2L(L)(1) 5 - Ga 9 Be) eee rec eve sesserceeeeeree 290/zST/0¢2Z(6)(1) 2 “# q 3 9 ID pee car tor/o0r(z)/e¢/z(9) & «%s» 940 oie eae Lor/00%(Z)/e9/29(9) 2 2s» 940 seseeersersssesss"""" 10 /006(2)/89/29(6) & «2» 940 Ts Sa ape So Tor/00F (z)/e¢/Ze(s) Pu @o 940 eg Se sie 10F/00F(Z)/E9/Z9(Z) & «%» 94D ace ey Tor /00F(Z)/e9/29(T) 2 ue» 940 Pee e eevee vee eee neers e vee vevere vee ves ZSt/oez(g) & ae) +5 9 IO SC ZST/0S2(g) P af ee 9 Ip SO ee ee a ZST/OSL(F) & 7 q - 9 Ip eer oe eee nes ees evevesvesesscesseseve ZSt/o¢4(1) P - q . 9 I9 ee 1S LP G2 Ip Peewee erence res ereserene os 5 (Z 19) Kars prea “TBA $I Cee ooe reese eres ees oes eos eoeoerees eve oereeeoeooee OED ZcL 2 P IM seer ee cee ees eer scesesees OSL ;S (Z I) faa3 prea SIDA Pitt Pee eee ee eee ner nee ser eee eeeerececees g0t/9/o¢t(z) (9) & F Ip oper zet/oe2(s)/egt/ogZ(e)(9) 2 440 Peo e rere eevee ree rereesveeeesveeees 80F/9/0St(Z)(Z) P p IQ Deemer cree recor eer ere ese nes eee veseeroeeresees ¢¢/¢9¢(T) $ Lag Pec eee r cere cence eee erseetversesersrerene Tor /00F(Z) P a) Poem rere mee eer eee sees er eee serves eer eresee ees e¢/29(T) g L140 Poe mee were renee eee reseeereeenerersereeeees tor /ooF (T) Pp ane) ee i i ii e9/2¢(T) 4 y In So REECE HE Lense Ma canna ono Cc a 0S8 P kaa PIEM. “SqUOLR 6S GZ pur LF g } g¢ LS GZ pus LF g LS. GZ pue LF g \ Fe 6S GZ pues Lip ¢ PP Gg pure v99 SF } gc PP g pus v99 SP PP Gg pus v99 SF } z9 PP G pue v9g SP PP G pus v99 SP } 1S PP g puv v99 8h Liv g == } LP @ = 08 LP g = i Lp e = 6V = = = i SF a a Wey 19h G pues EZ = \ og Lt = ay TZ G pur Ez = } ¥99 SP = aM : H een ¥99 Sh = Ww : = =, v99 St ae ey, ey = ia i CP —adxq o0g | —‘gdxq v0g | —"qdxq 00g ‘d-p-p-p | 3xq “squared—4 ‘d-)-) 121 N “ hd ~ 3 R : S ‘podojeasp pet Inojoo 4800 atojzoq perp Loy ynq ‘soko poquoursid ZIM spenprAtpur coy}0 vexyy otom ory], t * ‘porp soXo poqueursid YIM [enplAtpur eug + S ‘podopasap anojoo 4800 e1ofzoq porp Loy ynq ‘seXo poyuowsid YyIM s[eNprATpUr 10440 OM4 OOM OAT, » 8 | | = os ‘+ ne I ee ae ee eae i Zo Ps rane) OF am ee \ ec T T T G iE { May Gace aE Ne ee a IER QP = a: ‘oo S ae , Ge ee ee PST/est(F)/¢9(z)/e9z/0001(1)(¢) & F410 SE Z9 pue gF g g 99 = vere get/oge(8)/esT/og4(s)(s) 3 «@» 9 40 og LY g S Melee is |) |) Se { PST/eSt(%)/e9(z)/e9z/000T( 1) (Z) & ¥ 4D SE 39 g pus oF \ 69 a re Va a Mm Gn Ue 9 Ga eda CMe CG cee 9 4) 0s Lt g y ae ree ee ese pe @S1/0¢2/8/ZST/OSZ(e)(1) 2 PAD os Lb g \ me aes.) © { sa Z96/ZST/0SL(6)(Z) § «%@» 94D 6o Gg pus Ly — ke i) or e a: Hl : be a =| nature evelelev velo ela scelevetaveleYe stureyew leleie siete eeraielsieterett 9ST/9cT(T) i) ae) PL e —* \ an 5 + eee eee eee eee ee ee ee ee ee ee ey Gb u@n 9ID SP ae a 8 3 ae : ; : = > aa) SoC HOD UDO.OOC DCO DOO UCTATOCODROC HOC ours PST/ES1(F) ? paQ 1 e ie Be \ ES Ses He fee 8 SG aa SOU OR i G9 $ 9 940 QF mein See oy =? he : ‘ . oy = —{ Cece reece cece reer cece es see ees eeeereveeeeseerevence 20F P 19 c ==> he } 19 ie | | VY -| | ~~ | —~ | ~~} |ranesedcreseverccsascenseereessceres ZST/OGL(F) cis <9 040 LP pe eet eS oan E: tr = . = ke: —{ Se i i er i iii PST 5 ID e outa aes \ es | | | |. FL freterneseettereutneonnds vaceve ensues SST/OSL(T) P «%@» 940 Lb cn so 09 os PST/eS1(F)/ = — |—|l4e |e | — | — | — | —4 | 2¢(2)/e92/0001(1)(1)/e¢e¢/zet/o0gz(6)(¢)(1) 5 ¢ 419 8% ge pus gg | 9g pus yy | + VES | ae ee (OD FST/EST(F)/G9(3)92/000T(T)(4) 2 e@s» 940 ge 08 = J = 3 oh a3 ‘ ex rk a —{ elofare cic slejsiecclsrobelouaitaneistekeiersicterthateteharermietcetcvleleielehoteiercreisnetels ZGZ P Gig CZ = peas \ 6° rs, ee siojeveleieceveve ia\elcneveteTeletele clel nad eielstela eta (eis) cles ZST/OGZ(6) 4 pe ey 9 In LY ee wo 4 ey = ‘ d ‘: ae she —{ Wietaubieva olelwieis 01s lesa sie) siavelpin iG a Ghurere iw kine Gis votes wiete revels atelatoree 19% a G Ip CZ — oats ‘ ge rn crestor 0 te! me te TT 1 ies ecw tes coe cee ne aewe wine aces wcweslee ZST/0GZ(8) Pp _ oe 9 19 LY — a 1 Se a 6 Pa fe age Aetatiae oO EG poaes Reca 99% G49 eg =the we \ ON GI «8 GL { ecard eahecclan Sein RO wage TREES ors Zet/ogz(z) oe 9 10 LP oe sale Lg — a Dé . . his bac x | —{ Pome e ence nee eee ree sereeereecesseseseceneees e¢2/ZS2(T) P cca rg) 9% GZ es \ 9¢ SESE SIS ES | OSs ERI Se (bh th Aa Rm ST /09L(3) b u@s 94D le 6 ae ea 122 On the Result of Crossing Round with Wrinkled Peas, with Especial Reference to ther Starch-grains. By A. D. DARBISHIRE, Royal College of Science, London. (Communicated by J. Bretland Farmer, F.R.S. Received June 20,—Read November 14, 1907.) One of the characters with which Mendel dealt in his hybridisation experiments with peas was, as is well known, the shape of the ripe seed. Weldon, in his criticism* of Mendel’s interpretation of his results, showed that round were not discontinuously distinct from wrinkled peas, but that intermediate shapes connecting these two extremes were not infrequently exhibited. The answer which was made to Weldon’s criticism was that the intermediate shapes were due to spurious pitting or dimpling of the seed, and did not represent an intermediate condition of the germ which gave rise to them. And this answer was shown to be correct by the work of Gregory, who found that the starch-grains of round and wrinkled peas were quite distinct, and that they afforded an infallible test by which the real character of a pea with doubtful shape could be determined. Our knowledge of this subject has not advanced beyond the stage reached by Gregory in 1903; that is to say, we know no more about the inheritance of wrinkledness and roundness than Mendel did, except that each of these two characters is associated with a particular kind of starch-grain. What is the nature of the starch-grain in the hydrid; and how the characters of the starch-grains segregate, if they do so at all, in subsequent generations, are points on which we are at present ignorant. The observations, which have to record, form the first instalment of an attempt to fill up this gap in our knowledge. Part of the cost of these experiments was defrayed by a Government Grant. The Starch-grains of the Round Pea. The grains in a round pea such as “ Eclipse ” are, as shown in fig. 1,} single, potato-shaped grains. They will be referred to for the sake of brevity as p-grains. Their average length is 0°0322 mm., their average breadth * ‘ Biometrika,’ vol. 1, p. 231. + ‘The New Phytologist,’ vol. 2, No. 10, p. 226. { Fig. 1 is magnified slightly less than figs. 2 and 3. The actual sizes of the types of grains figured is given in the text. All the grains described have been derived (as far as possible) from the deeper layers of the cotyledon as distinguished from the superficial ones where the grains are smaller. Result of Crossing Round with Wrnkled Peas, etc. 128 00213 mm. These measurements are accurate to 2 pw. The length-breadth index (z.., 100 x breadth-length) is 66°14. The above figures were calculated from a series of measurements of 232 grains. Fia. 1.—Starch-grains of Round Pea (p-grains). Beside the large potato-shaped grains which have just been described there are always found associated with them extremely few very much smaller grains which are round. Two of these are shown in fig. 1. These small round grains occasionally exhibit traces of division, as in the case of the third small grain shown on fig. 1.. The division does not affect the contour of the grain and I have not found a small grain divided into more than two sub- divisions. The Starch-grains of the Wrinkled Pea. The starch-grains in a wrinkled pea like “ British Queen ” are, as shown in fig. 2, immediately distinguishable from those of the round pea, by the fact that they are compound. They will be referred to for the sake of brevity as c-grains. Each grain consists of a number of pieces which varies between 2 and 8. The separate pieces are loosely held together by a refringent yellow substance which, from the fact that it does not stain blue with iodine, appears to be of a different nature from the pieces themselves. These grains are very liable, as Gregory pointed out, to break up into their component pieces. I do not propose to go into the question of the relation between the number of component pieces and the size of a grain. Suffice it to say that the examination of a large number of samples reveals a rough 124 Mr. A. D. Darbishire. On Crossing Round wth [June 20, positive correlation between size of grain and number of component pieces. . The commonest types of grains are those with 4, 5 or 6 component pieces. Grains with 7 or 8 pieces are rarer; grains with 2 or 3 pieces are intermediate in frequency between those with 4, 5 and 6 on the one hand and 7 and 8 on the other. But whilst grains with 7 or 8 pieces are not much larger than those with 4, 5 or 6, grains with 2 or 3 are always conspicuously smaller than those with 4, 5 or 6. The average length of the grains is 0°0269 mm., the average breadth 0°0248 mm. The average length-breadth index is 92°19; that is to say, the grains are, as a glance at fig. 2 shows, almost round. The above figures were calculated from measurements of 105 grains. There are also in wrinkled peas a number of very small single grains which can be distinguished from the pieces, into which the compound grains break up, by the fact that they are circular. They are always smaller than grains consisting of two pieces. Two of them are shown in fig. 2. Fig. 2.—Starch-grains of Wrinkled Pea Fic. 3.—Normal c-grain of Wrinkled Pea, (c-grains). together with occasional p-grain. — Lastly, I have found very rarely indeed, in examples of “ British Queen,” isolated p-grains, as shown in fig. 3. At first I thought these grains had been introduced by using a razor with which I had been cutting round peas. But after taking every precaution to guard against this, by immersing the razor in strong potash before using it, I still found them: so that I cannot but believe that they really occur in wrinkled peas. I have also found them “Telephone,” and in extracted wrinkleds. 1907 .| Wrinkled Peas, and their resultant Starch-grains. 125 a The Starch-grains of the Hybrid. For the examination of the characters of the grains of F, cotyledons I had, unfortunately, only two seeds left from a number of crosses between “ British Queen” and “Eclipse” which Mr. J. T. Wadsworth made for me in the summer of 1905.* All the rest had been sown: the plants bearing the Fs cotyledons raised from them are now in flower. The starch-grains in the F, cotyledons produced by crossing a round with a wrinkled pea are not like those of the round pea, as might have been expected from the fact that round is dominant over wrinkled, but are inter- mediate between those of the round and those of the wrinkled pea. In the first place they are nearly round (see fig. 4), and will on this account and for brevity’s sake be referred to as 7-grains. Fig. 4.—Starch-grains of F, cotyledons (7-grains). In the second place, whilst the majority of the grains in the F; seeds are single the remainder are compound. In a count of 579 grains, 356 were single, 223 were compound. In the third place the degree of compound- ness exhibited by the compound grains in F, seeds is intermediate between * For the nature of the starch grains in other crosses between round and wrinkled peas, see pp. 132, 133. 126 Mr. A. D. Darbishire. On Crossing Round wth {June 20, singleness and the degree of compoundness in the grains of wrinkled peas. For whilst in these latter the number of pieces varies between 2 and 8 and the commonest is 6, in the F, grain it varies between 2 and 4 and the commonest is 3. Table I gives the average length, breadth, and length-breadth index of 166 7-grains, side by side with the measurements for p- and c-grains for comparison. Table I. Round. | 1p - Wrinkled. p-grain. r-grain. ce-grain. Average length ...........c00ce0s 0 :0322 0 :0276 00269 Average breadth ..............0008 0 02138 0 0236 0 0248 Length-breadth index ......... 66 *14 85 °5 92°19 | This table shows that, in the matter of shape, the F, grain is intermediate between the p- and the c-grain, but nearer the c-grain. | The Starch-grains in a Subsequent Generation. I have not at present any information as to the nature of the grains in Fs, but I have some data relating to F; which throw much light on the mode of segregation in this case. By a fortunate chance I happened, when I turned my attention to this subject, not to have sown the contents of 48 packets of F; peas;* each separate packet of which contained five seeds from a single plant. Thirty-six of the packets contained round seeds; 12 of them contained wrinkled ones. I possessed a record of the number of green and yellow, as well as, what concerns us now, the number of round and wrinkled seeds produced by each * The history of these seeds is as follows: In the spring of 1905, Mr. C. C. Hurst kindly gave me the seeds borne on an F; plant from a cross which he had made between “British Queen ” (yellow wrinkled) and “ Eclipse” (green round) (‘ Journ. R. Hort. Soe.,’ vol. 28, pp. 3 and 4). These seeds included yellow rounds (Y R), yellow wrinkled (Y W), green rounds (G R), and green wrinkleds(G W). I sowed these four sorts separately. The Y R’s gave either (i) plants bearing only Y R’s, or (ii) plants bearing Y R’s and Y W’s, or (iii) plants bearing Y R’s and G R’s, or lastly (iv) plants bearing Y R’s, Y W’s, G R's, and G W’s. The total number of yellow seeds, round and wrinkled, borne by plants falling in the last category (iv) was 232. These were sown, and 196 of them produced mature plants. 10 seeds from each of the 196 plants thus raised were preserved, in 2 packets of 5 from each plant. All of these seeds were sown this spring, except the contents of the 48 packets referred to in the text. Sa) ee 1907.| Wrinkled Peas, and their resultant Starch-grains. 127 of the plants, five of each of whose seeds were preserved in the packets. So that I could find out in the case of the seeds in any given packet whether they were borne on a plant which produced only rounds, or both rounds and wrinkleds, or only wrinkleds. I wish particularly to emphasise the fact that this information was not written on the packets, but was obtainable by looking up, in the records, the plant represented by the number on the packet containing its seeds. I started by writing down the catalogue-numbers of each of the packets. Then I recorded the nature of the grains in the seeds opposite the number of the packet containing them. Not until I had examined all the grains did I refer to my records for the characters (whether D D, DR, or RR in respect of seed shape) of the parent plants of the seeds in the packet. I postponed this reference to my records until after all the grains had been examined, in order to prevent any possibility of the determination of the nature of the grains being influenced by a knowledge of the gametic constitution of the plant which bore them. Our interest centres chiefly on the possible difference between the round peas borne on plants bearing both round and wrinkled, and those borne on plants bearing only round. I shall, therefore, postpone reference to the contents of the 12 packets containing wrinkled peas until after I have dealt with the rounds. There were 36 packets containing round seeds. In every case—16—in which all the five seeds in a packet contained nothing but potato-shaped grains (and occasionally the very small compound grains found in “ Eclipse ”’) the plants bearing them had produced only round seeds. Table IL gives the details of these families, and fig. 5 shows the appearance of these extracted dominant grains.* It will be seen that they do not differ sensibly from the -grains of “ Eclipse.” The small compound grains, which are often exceedingly minute, are likely to be overlooked: the absence of any record of them in the first four families in Table II may be due to the fact that I did not detect them because I was not on the look-out for them. The presence of these grains in a pea is indicated in the table by the letters 7c. There was a single case (54.129.11) of an apparently compound large p-grain divided longitudinally into two; but the rarity of such a grain points to the conclusion that this case was due to an accidental fracture; which would be quite likely to occur during the scraping of the soaked pea with a razor. * [ have not measured any grains in F;. But no difference could be seen, between the A p-, c-, and r-grains in this generation and the grains, of these three types, already described. 128 Mr. A. D. Darbishire. On Crossing Round with [June 20, Q) QSO Fie. 5.—-Starch-grains of DD Round Peas in F;. Table II. Number and characters of seeds borne by parent-plant. Ment Nature of starch-grains in each Catalogne- | (==> a | of these seeds. | seeds eh at Colour. Shape. | examined P ay : | for starch- | | grains. | | | Ne G. R. WwW. i | il. | ill | iv Vv | | 54. 18 31 13 44, 5 round p | p pp \ 2p p 54, 17 Pap ike 38 6 p P P P p 54, 25 S52 als AT A p Pp Pp Pp Pp 54. 50 207, 8 28 * Pp Pp Pp P Pp SMCeerme 0 Pp Pp dese 7D P 54. 83 TO Seat SO) i, D p \sp | p p of me a re ie 25 DIC | IPA NP Caer 2 o 0 P P P| Pp PAVE 54.129 30 15 45 at p pt | (Dem eeere Pp salva 128 al | 160 » | fel pse| pte pse| pfe : | » Pp Pp LE ie Pp eae a | 4 Bs ” a pie | RSC | Wyse . | “ 0 pse Pp Pp Pp Pp 54.190 132 alae 19 i; P Pp P |p fe P 54,191 2405 Waele eal eed a pfe| pfe| pfe | pfe|\ pfe p signifies potato-shaped grain. fe signifies the additional presence of the small compound grains found in “ Eclipse,” in very. small numbers. * When a discrepancy occurs in Tables II, III, and IV between the totals for cotyledon colour and totals for shape in a given plant, it signifies that in the case of some of the seeds of that plant the shape was determinable whilst the colour was dubious, or vice versd. + A large double grain in this pea. In every case in which at least one of the five seeds in a packet contained either a round or an irregular round grain, the plants bearing them had produced both round and wrinkled seeds. That the category irregular round 1907.] Wrinkled Peas, and their resultant Starch-grains. 129 is not due to a difficulty to distinguish between 7- and p-grain—even if it were possible for anyone familiar with these grains to have this difficulty—is shown by the fact that all peas containing grains falling into this category were borne by heterozygote plants, a fact which was not known when the grains were being examined. Table III gives the details of these families and fig. 6 shows some of the r-grains. ; Table ILI. Number and characters of seeds borne by parent-plant. | Character of Nature of starch-grains in each Catalogue- Bes of of these seeds. ae t Colour. Shape. Bice oT ee ate for starch- F grains. Ye G. R. We. re | i. ill, Iv. v. 54. 3 28 4, 22 9 5 round r sc p p p 7 SC 54. 18 20 9 , 6 i r sc p FSC || FSC.) .7-Se 54. 19 31 12 31 12 D 1p r sc p p 54. 23 26 6 22 6 5 rme|rme|rme|rme | rme 54, 33 26 10 26 9 ; a rmc;rme|rme| op fe 54, 52 33 13 30 13 . 4 SC p Pp p Tr SC 54. 57 23°) 8 21 10 i tse | tse | tse | tse |’ ise 54. 65 20 3 15 8 ; p Tr fe p tse p 54. 77 30- 9 35 2 ¥3 p rfe| rfe| rfe| p 54. 89 32 9 29 8 > ese | ife | rse p r se 54.105 32 8 27 13 = ime |ime | rme|rme | ime 54.112 27 6 16 5 % p i fe Pp p p 54.121 19 4 21 2 " p p rme| pfe| pfe 54,146 15 4, 14 5 Bs p Pp p p r me 54.150 18 6 17 7 ; rse | rse p p p 54.177 26 Zz 25 8 Ee rme| rse | rse | rse | rse 54.179 37 4 38 2 . ife |ime | pfe| pfe | rme 54.180 36 11 38 9 <3 p p fe be | Dye |p ve 54.181 38 9 36 i rs TASC UN Se eepipe | nese safe 54.182 37 9 30 13 4 aCe eerie: Nu fc in aton lea he ry = round grains (fig. 6). me = many compound. @ = irregular round. Other symbols as in Table IT. se = some compound, The difference between peas marked 7 mc and those marked 7 fc is some- times very great. A count of 508 grains from a pea marked r me revealed 203 compound and 305 single. Whilst in a pea marked 7 fc, there were only 28 c-grains in 304 counted. In the absence of any information as to the behaviour of the characters of the grains in F, it is not possible to make any definite statement as to the mode of segregation in this case; but the following points seem to be cleav.. We have seen that the grain in the F, seed is round. The evidence points to the fact that the heterozygote round peas in subsequent generations are characterised by the possession of 7- or r-grains and homozygote rounds by 130 Mr. A. D. Darbishire. On Crossing Round with |June 20, p-grains. For, in the first place, no round grains appeared in 80 seeds from. 16 plants which bore only round seeds. In the second place, of the 100 round seeds borne by plants bearing both round and wrinkled, 40 had p-grains as against the 33 or 34 expected by my theory—a discrepancy not so great that it cannot be accounted for by the smallness of the number of - ‘seeds. © | Fic. 6.—Starch grains of DR Round Peas in F;,. If the association of 7-grains with heterozygote round, and of p-grains with homozygote round holds good for the F, generation, we have a means of distinguishing between D D round and D Rf round in Fy»; instead of, as at present, having to wait until their progeny are mature in the following year. A further point is demonstrated by the nature of the grains in Fj, and borne out by those in F;. It is that the shape of the grain is inherited. separately from its composition—if we may use this term to cover the singleness or compoundness of the grain. In the round pea the grains are single and long; in the wrinkled they are compound and round; in the hybrid they may be either single or compound, but are more round than long. In a subsequent generation, F;, we have v-grains exhibiting much compound- ness (54.23) and others exhibiting little (54.182 1, 11, iv, and v). We have (possibly) p-grains either with no compounds, or with few ; and intermediate grains either with few compounds (54.179 1) or with many (54.179 11). The wrinkled peas contained, as was to be expected, c-grains, but some of . them contained in addition, very sparingly, p-grains, one of which is shown in fig. 7. 4 ee 1907.| Wrinkled Peas, and their resultant Starch-grains. 13k In the case of one plant, only one of the five seeds examined exhibited ‘them. In the case of two other plants, two out of the five seeds exhibited them, and in the case of both these plants the five seeds had been noted,. before soaking, as being only rather wrinkled. Fie. 7,—Example of occasional p-grain occurring, in extracted Wrinkled Pea in F;. The details of the grains and parentage of the wrinkled seeds are shown in: Table IV. Table IV. Number and characters of ' seeds borne by parent-plant. stein i Nature of starch-grains in each Catalogue- Sy caadks ¢ of these seeds. ee ee Colour. Shape. examined P aa for starch- grains. | x G. R. W. eae 2 il. iii. iv. v. | | ai 54, 38 24 7 82 | 5 wr. ¢ BoP ce ¢ c 54. 55 30 8 38 is c Gy ee c c 54. 59 18 3 21 | Me e en a c c 54, 61 19 2 21 | p C Chen ene ¢ C 54. 66 2g 3 30 | fa c ON Ge c c 54. 87 16 3 19 c CN ae c c 54. 97 32 12 41 | 5yratherwr.| ¢ csp csp c c 54.118 | 14 3 ile 5 wr. C c ce c c Bata7 | 1d 8 19 . c On eae c c 54.138 _ 80 15 45, c CA Xe c c 54.140 | 24 6 30 ie e c | c c c 54,187 | 13 a 20 | Srather wr.| ec esp | e ce sp c | e = compound grains. sp = some p-grains. The Amount of Water absorbed by the Seed on germination in the Various Generations in the Preceding Experiment. Gregory’s statement that, according to Denaiffe, “ wrinkled seeds take up. more water on germination than do round seeds,” made me curious to find a out how much more water wrinkled seeds take up than round; and, having: | determined this, to find out what relation the hybrid bore to its two parents q with regard to this character. I propose to refer to the capacity for taking up water as the absorptive capacity ; and the measure which I shall use throughout of this capacity is- 132 Mr. A. D. Darbishire. On Crossing Round with [June 20, the amount of water, taken up by a pea immersed in tap-water for 24 hours expressed as a percentage of the weight of the ‘dry pea. The average absorptive capacity of 15 “British Queen” (@¢., wrinkled) peas weighed separately is 122 per cent. The average absorptive capacity of 12 “ Kclipse” — (i.e, round) seeds is 86 per cent. The absorptive capacity of a single F, seed, produced by crossing “ British Queen” with “ Eclipse,” is 100 per cent. It will be seen that the hybrid is intermediate between the two parents. The only evidence I have concerning the absorptive capacity in succeeding generations is derived from the examination of the contents of two sample pods in the Fy» generation derived from the cross (already referred to) between “ British Queen” and “ Eclipse,’ made by Mr. Wadsworth. The facts are shown in Table V. And it will be seen that whilst the difference between the absorptive capacity of round and wrinkled is well marked, that the absorptive capacities of the two kinds of rounds, 7.¢. those with p-grains and those with 7- or 7-grains, is not in accordance with expectation. For in the pure round pea the p-grain 1s associated with an absorptive capacity 86, and in the F, round the 7-grain with one of 100. And it was not unreasonable to suppose that the absorptive capacity was in some way determined by or, at any rate, associated with the nature of the grain rather than with the shape of the seed, for it is in seed-shape only that we find dominance; whilst in both grain-shape and absorptive capacity the two parent forms blend. The expectation, however, suggested by this parallel between starch-grain and absorptive capacity in F, is not, as we see, fulfilled in Fe. Table V. Pod. | Individual seeds. ae rae Nature of grains. ei ES to oteo lea MSS RE Gece a GR p 96 B YR p me 100 Y GR p 97 | 6 YW c 141 £ GW c 141 Som Orc. ccs! a YR r me 100 B GR 7 mc 97 Y YR p 97 6 YR ame 97 E YW c 142 g GW c 139 The r-grains in other Hybrid Peas. The intermediate nature of F; grains was also observed in the following crosses (Table VI). The grains in these seeds were not measured ; but it was ~ 1907.] Wrinkled Peas, and their resultant Starch-grains. 138 evident, at a glance, that the grains were more round in some cases. than in others. Thus, M 303 and M 313 had the most conspicuously round grains that I have seen in F; seeds. Table VI. Catalogue- Number of number of cross. | seeds examined. Pistil-parent. Pollen-parent. M 299 74 “ British Queen ’”’?} Maple from Mr. R. H. Lock. M 303 1 55 a Grubb, corn dealer, Oxford M 312 u * Zucker Erbse. rotbl. kron.* M 313 3 ” ” ” M 316 1 hi Maple from Mr. R. H. Lock. * A maple sugar pea with pink flowers and “mummy” (i.e., fasciated) habit. Bought from Messrs. Haage und Schmidt, Erfurt, Germany (No. 1286, p. 24 in 1907 Catalogue). The Absorptive Capacity in other Hybrids. The absorptive capacity of the F, seed, as well as that of its two parents, was determined in eight cases.altogether, one of which is “ British Queen” by “ Kelipse,” to which reference has already been made. The results are shown on Table VII. The numbers at the left of the table refer to the absorptive Table VII. a [nw wae 70 Pa et (se COATT TAA TTT af SELL 14 “TTL rT TT af = aahe be ei a ge = 134 Mr. A. D. Darbishire. On Crossing Round with [June 20, capacity. The uppermost dot in a column gives the absorptive capacity of the round parent in a cross: the lowermost dot, that of the wrinkled parent. and the intermediate one, that of the hybrid. The absorptive capacity is, in every case, except in that of the round parent in M 299, calculated from: weighings made with the actual variety of round or wrinkled pea utilised in the cross ; and, indeed, in many cases from the seeds from the very plant which served as mother or father. Instead of the absorptive capacity of the actual round parent in M 299 is written the average of the values for the five different varieties of round peas we have dealt with. Table VIII gives the parentage of the crosses summarised in Table VII. Table VIII. | | v Catalogue- | Number Number pene number of Wrinkled parent. | of seeds Round parent. | of seeds f ee = cross. | weighed. | weighed. | °° tk | | weighed. a 2 British Queen ...... los oa Buichipsed se. ce ee ae 1 M 8 2 Laxton’s Aipha | 5 3 Sangster’s No. 1...... 4, 1 (Haage & Schmidt) | M 238 7) Fr rs | 5 6 Yellow round* sy) 1 (Genoa) M 29 2 Laxton’s Alpha | 8 3 45 a 9 if | (Sutton) | | M 67 2 x: 3 | 8 6 Bohnenerbset 3 1 | | (Haage & Schmidt) | M 96 3d Laxton’s Alpha | 5 2 Yellow round 9 6 | (Haage & Schmidt) | (Genoa) | M 137 2 Telephone (Haage & 3 3 Pisum arvenset 6 1 Schmidt) | hibernicum | M 299 2? British Queen ...... ee alia $ Maple from vide text 2 | | Mr. R. H. Lock | * Plants grown from (evidently unimported) yellow seeds which I bought in a small shop- in Genoa. + A large, long pea with a black hylum, which, however, is not situate in the middle of one side as in the bean, but at the end. { Pisum arvense hibernicum is a small pea seldom attaining 2 feet in height, though it is not. dwarf in habit, sold by Messrs. Haage und Schmidt (No. 1660, p. 30, 1907 Catalogue). Its seeds can be sorted into four categories. Maple and purple spots on grey : maple on grey; purple spots on grey, and grey. Vide “ Report II to Evolution Committee of Royal Society,” and R. H. Lock,. ‘Roy. Soc. Proc.,’ B, vol. 79, supra, p. 28. Summary. My investigations on this subject are being continued. The facts so far brought to light are :— 1. That, although roundness is dominant over wrinkledness in peas, the: starch-grain of the F, generation (the round or r-grain—fig. 4) is a blend between the type of grain of the round pea (the potato-shaped or p-grain—- ate ae -1907.] Wrinkled Peas, and thew resultant Starch-grains. 135 fig. 1) and the type of grain of the wrinkled pea (the compound or c-grain— fig. 2), in respect of three characters— (a) It is intermediate in shape as measured by its length-breadth index— that of the p-grain being 66, that of the c-grain 92, and that of the r-orain 85 (neglecting decimals). (6) It is intermediate in the distribution of compoundness, inasmuch as some of the 7-grains are compound and some single. (c) It is intermediate in the degree of compoundness, inasmuch as amongst those r-grains which are compound the most usual number of constituent pieces is three, whereas in ¢-grains it is Six. 2. In a subsequent generation—F;—the homozygote round peas contain p-grains; the heterozygote round peas contain r- or intermediate grains. But both r- and intermediate grains may be associated either with a high, or with a low degree of compoundness. 3. P-grains occasionally occur in wrinkled peas in F;, and the evidence (Table IV, 54.97 and 187) suggests that the existence of these grains in wrinkled peas tends to make them less wrinkled. 4, A wrinkled pea takes up more water when it germinates than a round one. The hybrid between a round and a wrinkled pea is intermediate in respect of this character between its two parents. . 5. But this intermediateness of the hybrid in absorptive capacity is not occasioned by the intermediateness of the starch-grain of the hybrid, because, in F,, peas containing r-grains and peas containing p-grains both have the same absorptive capacity as the F, pea (see Table V). 6. When, therefore, we cross a round with a wrinkled pea, we are dealing with four separately heritable characters :— .(i) The shape of the pea—whether round or wrinkled. (11) The absorptive capacity of the pea—whether low or high. (iil) The shape of the starch-grain—whether long or round. (iv) The constitution of the starch-grain—whether single or compound. REFERENCES. Mendel, G. J. “Versuche iiber Pflanzen-Hybriden,” ‘ Verhandl. d. Naturf. Vereines in Briinn,’ vol. 4, 1865. Weldon, W. F. R. “ Mendel’s Laws of Alternative Inheritance in Peas,” ‘ Biometrika,’ vol. 1, p. 231, 1901. Gregory, R. P. “The Seed Characters of Piswm sativum,” ‘New Phytologist,’ vol. 2, No. 10, 1903. VOL. LXXX.—B, L 136 Localisation of Function in the Lemur’s Brain. By F. W. Mott, M.D., F.R.S., and W. D. Hauuipurtron, M.D., F.R.S. (Received November 14,—Read December 5, 1907.) [PLates 2—4.] The brain of the Lemur, the lowest of the ape-like animals, does not appear to have been subjected previously to a thorough examination. Page May and Elhott Smith brought a brief communication on the subject before the Cambridge Meeting of the British Association in 1904.* Their experiments were apparently limited to stimulation of the cerebral cortex, and they have never published a full account of their work. Brodmannt has worked out some of the histological details of the structure of the cortex cerebri, and Max Volscht has performed a stimulation experiment upon one Lemur. The work of these investigators will be referred to again in the course of this paper. Our own investigation has in the main dealt with the motor centres, and the experimental methods adopted have been the usual ones of stimulation and extirpation. In animals so low in the scale, stimulation is to be regarded as the more decisive of the two methods for the purpose of localisa- tion. The extirpation experiments have, however, confirmed the results of stimulation, and in these experiments the course of the resulting degeneration was followed by histological examination of the brain and spinal cord. The results, moreover, agree remarkably closely with those obtained by a study of the histological structure of the various regions of the cortex cerebri. One of us (W. D. H) is responsible for the experimental part of the investi- gation which was carried out at King’s College, London; the other (F. W. M.) is responsible for the histological portion. We have to thank Miss Agnes Kelley for assistance in the histological work, and also for the drawings which accompany this paper.§ _ The species of Lemur employed was the Ring-tailed Lemur of Madagascar (ZL. catta) except in two experiments where the Black Lemur (LZ. macaco) was used. The results obtained in both species were practically identical. * British Association Reports, 1904, p. 760. + ‘Journ. f. Psychologie u. Neurologie, vol. 6, p. 272, 1906. { “Ein Rindenreizungsversuch an einem Halbaffen,” ‘Monatsschrift f. Psychiatrie u. Neurologie,’ 1906. § A full description of the histology of the cerebral cortex of the Lemur will be ‘published later by one of us (F. W. M) in conjunction with Miss Kelley. In the present paper only the motor area will be considered, / a Localisation of Function in the Lemur’s Brain. 137 Stimulation Experiments. These were five in number. In all cases the anesthetic employed was ether, and anesthesia was maintained until the animal was killed at the conclusion of the experiment by an overdose of chloroform. ‘The brain was exposed in the usual way, care, of course, being exercised not to injure its surface, nor to wound any large vessels. Its surface was explored by the usual two-point platinum electrodes connected to the secondary coil of a du Bois Reymond’s inductorium. The primary circuit was arranged for faradisation and included one cell. The approximation of the secondary to the primary coil was such that the current as tested by the tongue of the operator could be felt as a little more than a faint tickling. In some experiments Sherrington’s unipolar method* was used. Professor Sherrington was good enough to furnish us with one of his own electrodes for the purpose. The results obtained by this method were identical with those obtained by the two-point electrodes. It did not appear to us so easy to evoke movements by the single electrode, probably because the number of cortical cells excited is smaller than when two are used. The double-point electrodes, moreover, possess the advantage of being so readily tested in relation to strength of current by application to the operator’s tongue. Short protocols of the five experiments performed are as follows :— Lemur 1. Lemur catta.—Right hemisphere exposed in frontal and central regions, and explored with single-point and double-point electrodes, Resulting movements noted. Lemur 2. Lemur catta.—Experiment on Lemur 1 repeated, and some details of map made at the first experiment filled in and corrected. At the conclusion of this, the posterior part of the hemisphere was exposed, and its posterior pole excited ; no definite eye or other movements were thereby elicited. Lemur 3. Lemur macaco._,The same experiment repeated ; in this case the excitable area did not extend quite so far posteriorly as in previous experiments. Lemur 5. Lemur catta.—Stimulation of the occipital region, even with stronger currents than those usually employed, produced no effects, although stimulation of the anterior eye centres produced eye and head movements as in previous experiments. The occipital region on both sides was investigated. Lemur 6. Lemur catta.—Confirmatory experiment, the special object of which was to determine the posterior limit of the excitable area. No movements were evoked by stimulation of occipital regions, although these were better exposed than in previous experiments and explored on all surfaces. General fesults of Stimulation EHuperiments. The convolutional pattern of the brain, as will be seen by looking at the accompanying figure (Plate 2, fig. 1), is a simple one. , * As described in a paper by Fréhlich and Sherrington, ‘Journ. of Physiol.,’ vol. 28, p. 14, 1902. 138 Drs. F. W. Mott nail W. D. Halliburton. [Nov. 14, The diagram represents the cerebrum as seen from above. The fissure of Sylvius is easily recognisable ; beneath this is a well-marked parallel fissure, — and above it is the sulcus known as the lateral fissure. In the frontal region the best marked fissure is the sulcus rectus. On the mesial surface of the hemisphere (not shown in the diagram) the intercalary fissure and the © calcarine fissure are the most prominent sulci. An interesting morphological point is, which of the smaller fissures in the © fronto-parietal region corresponds with the central fissure (fissure of Rolando) of higher animals. These are four in number, and are labelled 1, 2, 3, 4 in the diagram. It will be noticed that these differ in length, and to some extent in position in the two hemispheres; the drawing of the right hemisphere, how- ever, more accurately represents their average size than the left. Fissure 4 is too far forward, and fissure 3 is too far back for the Rolandic fissure; and our experiments have given us no certain answer as to whether fissure 1 or 2 is the fissure of Rolando, for the excitable area extends behind both. Sherrington and Griinbaum have shown that, in the higher apes, the excitable cortex does not extend behind the Rolandic fissure. In one experi- ment we have performed on one of the lower apes (Aacacus rhesus), we have found by the unipolar method the same to hold. So far as physiological enquiry can give any reply to morphological homologies, we feel inclined to regard fissure 2 rather than fissure 1 as the representative of the central sulcus, since behind it movements are evoked with greater difficulty than in front of it. The precise posterior boundary of the excitable cortex is very difficult to determine with accuracy, and is variable in different animals. Our diagram (Plate 2, fig. 1) represents the extreme posterior boundary, but in one or two instances it was somewhat more anterior. The cayse of the greater difficulty of evoking movements here becomes clear on histological examination ; the large motor cells become more scattered as one passes backwards. We obtained the most energetic movements from this posterior region by placing the electrodes 3 mm. apart, and applying them in a longitudinal direction. Very marked results (extension of arm and fingers) were in this way obtained from the oval area so marked in the figure. This may be explained on the hypothesis that the series of psychomotor cells presiding over this particular movement are arranged in a longitudinal direction, so that when the electrodes _ are applied in the manner stated the current spread over the whole of the excitable area of cortex representing the extension of arm and fingers. The diagram represents better than any amount of verbal description the disposition of the motor centres. The trunk and, more posteriorly, the leg areas are uppermost, and extend on to the mesial surface as far as the inter- 1907.| Locahsation of Function in the Lemur’s Brain. 139 calary fissure. Below this and in front are the various head and face areas. The areas controlling the upper limb are posterior to, and somewhat higher than these. _ The excitation experiments show that, as in the primates, there exist in the Lemur three areas related to lower limb, upper limb, and head region respectively, from above downwards, in inverse order therefore to the spinal arrangement. Moreover, they substantiate and accord with the ablation experiments, which will be described later. The large size of the hand and finger area is not remarkable in view of the habits of the Lemur. It does not pick up its food with the hands in the same way in which a monkey does, but as a rule uses the hands to support the morsel which is picked up by the mouth. The hand movements are, however, undoubtedly more highly developed than in quadrupeds, and come chiefly into play in climbing. There is a well-formed and opposable thumb, which must be extremely useful in the arboreal life of the animal. We were unable to discover any special thumb area in the cortex. In the area marked “ upper limb mainly shoulder,” the movements which occur spread to the lower part of the limb and the digits on keeping up the usual weak stimulation, or by the use of a somewhat stronger current ; the resulting movement is a primary one of progression. The same is true for the lower limb, especially in those centres which, in the first instance, control movements of its upper segment. This spreading does not occur to any noticeable degree in the head, face, eye and ear movements. This, again, is explicable on histological examination, for the Betz cells in this region are much smaller than in the limb areas, and so do not possess so many con- nections with other cells. The difference is so marked that we have considered it worth while to contrast, in Plate 4, the two types of motor cortex. | It will be noticed in Plate 2 (fig. 1) that the head, face, and tongue area passes on to the outer side of the sulcus rectus. The extent to which this occurs probably varies in different animals, but in those subjected to micro- scopic study the Betz cells did not extend beyond the outer lip of the sulcus. It is probable that this is a more trustworthy guide than stimulation, for in the latter method it is often difficult to entirely eliminate spread of current. In the large area marked “ Pricking of ear” on the outer side of the anterior end of the lateral fissure, and which is also characterised by Betz cells of the small type, the shaded area near the Sylvian fissure was in some experiments the part, stimulation of which elicited the most marked move- ments ; this is quite intelligible because in this part one is approaching the 140 Drs. F. W. Mott and W. D. Halliburton. [Nov. 14, auditory centre, which analogy would teach us to place on the opposite side of the Sylvian fissure. This, however, did not obtain in all cases, and in one experiment the area passed upwards so as to slightly overlap the lateral fissure. As already noted in the protocols, repeated attempts to evoke eye move- ments from the occipital pole led to entirely negative results. It is quite certain that this region is, as in other animals, the visual area, for histo- logical examination of this part of the brain shows it to possess a well- marked line of Gennari, and the other structural features of the visual cortex. It is quite possible that if we had been able to stimulate in the depth of the calcarine fissure, where the solitary cells of Meynert are more abundant, we might have obtained eye movemeuts. The Histology of the Excitable Area. It is a comparatively easy matter to map out the area containing the large Betz cells; moreover, although, as in the primates, the region of cortex behind the motor area contains large pyramidal cells with Nissl granules, yet this can be differentiated from-the motor area proper by the existence of a granular layer, a sure sign of sensory function. Fig. 1, Plate 4, shows the type of cortex with the giant Betz cells; fig. 2, on the same plate, shows the type of cortex with the smaller Betz cells. The accompanying drawings in the text indicate the extent and boundaries cof these two types, the size of the Betz cells being roughly shown by the size of the dots. Diagrams A, B, and C are drawings of the outer and mesial surfaces of one hemisphere (A and C), and a drawing of the cerebrum as seen from above (B). The dotted area is the portion characterised by the possession of Betz cells, the size of which is roughly indicated by the size of the dots. The first type of cortex (with large Betz cells) covers that part of the outer and mesial surface which lies between the posterior end of the sulcus rectus, the anterior end of the sulcus lateralis, and the intercalary sulcus. It is continued further forward within the sulcus rectus than can be indicated 1907.| Localisation of Function in the Lemur’s Brain. 141 on surface diagrams. Anteriorly and posteriorly it gradually merges into the types of cortex which are respectively found in frontal and post-central regions. , The second type of cortex (with smaller Betz cells) covers the area which lies between the extremities of the sulcus rectus and sulcus lateralis, and extends downwards as far as the superior wall of the Sylvian fissure. Superiorly it merges into the first type of motor cortex ; anteriorly into an area intermediate to this type and the type of the frontal cortex proper ; and posteriorly into one intermediate to this type and the temporal and _ post- central types. Further histological details of these two types are as follow :— First type of motor cortex. (Plate 4, fig. :1).—The cortex is about 2 mm. deep; the depth of the molecular layer is 0°17 mm., that of the pyramidal layer and granules about 09 mm. ; that of the pallid zone in which the Betz cells lie about 0°2 mm. ; and that of the polymorph layer about 0°7 mm. The pyramidal cells are larger and have more pro- cesses than in other parts of the cortex (those of the post-central and temporal types most nearly approaching them in form). They are somewhat irregularly arranged, owing, perhaps, to the presence of the processes of the Betz cells. Granules are scattered in fair numbers at the bottom of the pyramidal layer, but they do not form a distinct layer. The infra-granular pyramids are the most typical feature of this area. They are, for the most part, well-formed giant pyramids (Betz cells), containing Nissl bodies and having several branched processes. They frequently measure as much as 60 p by 25 p, and are sometimes larger. This line of cells occupies a pallid zone in which only a few other cells are scattered, these being smaller Betz-like cells, faintly stained pyramidal cells, and a few granules. Some of the large Betz cells closely resemble the typical giant Betz cells of the cortex of the higher apes, but many are more pyramidal in shape. The tendency to arrangement in nests, which has been described in the human cortex, is not general, though it can sometimes be seen. The largest Betz cells are found immediately before and behind the small fissure (No. 1 in Plate 2, fig. 1), which lies in the middle of this area, and between it and the intercalary sulcus. Second type of motor cortex (Plate 4, fig. 2).—The pyramidal cells are smaller than in the first type. A line of darkly staining stellate cells is scattered above or among the granules, and the granules form a fairly well marked line. The cells which correspond in position to the Betz cells in the first type of motor cortex are not conspicuous either in size, shape, or number. But a somewhat scattered line of cells can be seen, measuring about 25 to 35p by 15, which are Betz-like in shape, having many branched pro- cesses staining more deeply than the other cells and possessing Nissl granules. The results of excitation and ablation of this area support the inference that this area is motor in funetion, although it differs considerably from the typical giganto-pyramidal motor cortex in the characteristics mentioned above. The definite layer of granules would suggest that this cortex is sensori-motor in function like the primary visual cortex of the calcarine region. This is best marked in the region from which ear movements were elicited. Brodmann* has also mapped out the distribution of the giant pyramids in the Lemur’s brain, and his results correspond fairly closely with ours. He points out that the little dimple (our sulcus 1 in Plate 2, fig. 1) is inconstant in position, and does not agree with * Loe. cit, 142 Drs. F. W. Mott and W. D. Halliburton. [Nov. 14, Ziehen, that it is homologous with the sulcus centralis of the primates, for the area giganto- pyramidalis extends behind it. In support of this argument he has also examined the brains of two monkeys, Periodictus potto and Propithecus coronatus. The former has a well-marked central sulcus, and this is the caudal boundary of the area giganto- pyramidalis, as well as of the excitable area. In Propithecus, the central sulcus is repre- sented by a small dimple only, very like what we have labelled sulcus 1 in the Lemur. In Propithecus, however, it forms the caudal boundary to the excitable area, and to the area giganto-pyramidalis. He therefore argues that in this monkey it is the true homo- logue of the central fissure, and that the apparently similar dimple in the Lemur’s brain cannot be the same, since it lies within the excitable area and the area of large Betz cells. Max Volsch’s* work was apparently undertaken to prove the correctness of Professor Ziehen’s view. He concludes that the general arrangement of the motor centres appears to agree with that of the primates, and that they are situated in front of the small sulcus we are discussing, and which he therefore regards as the representative of the central fissure. He, however, wisely remarks that the results of only one experiment cannot lead to far-reaching conclusions. Flatau and Jacobsohn} regard the anterior end of the lateral fissure as homologous with the central fissure, and look upon the little sulcus (1) as a rudimentary pre-central sulcus. Kxtirpation Hapervments. The extirpation experiments were four in number. The operations were performed under ether anesthesia, and with strict antiseptic precautions. Healing took place by the first intention in all cases ; the paralysis exhibited after the operation was not pronounced in any experiment, and soon passed off; this transitory nature of the paralysis has been frequently noticed before, especially in animals low in the scale. The animals were finally killed by chloroform about a fortnight after the operation, and the parts which were to be subjected to microscopic study were then placed in suitable preservative fluids. The following are short protocols of the four experiments performed :— Lemur 4. Lemur macaco.—The motor area was removed as completely as possible on the right side, except that the part below the sulcus rectus and lateral fissure, which governs mainly head, eye, and ear movement, was left intact. Immediately after the operation there was conjugate deviation of head and eyes to the right, and the left pupil was rather larger than the right. These effects were transitory. The paralysis of the left limbs had passed off to a great extent two days later ; the fingers and toes on this side were, however, not so well used in climbing as those on the right side. The animal was killed by chloroform 14 days after the operation, by which time it was difficult to recognise any paralysis. The grip of the left hand was not noticeably weaker than that of the right. Lemur 7. Lemur catta.—A large amount of the motor area was removed on the right * Loe: cut. + ‘Handbuch der Anatomie u. Vergl. Anat. des Zentral-Nervensystems der Saugethiere,’ 1899, 1907.] Localisation of Function in the Lemur’s Brain. 143 side. The extirpated portion was principally the area for the upper limb. Paralysis of the opposite side was marked immediately after the operation, but eventually passed off in the way described in the experiment on Lemur 4. The animal was killed 14 days after the operation. 3 Lemur 8. Lemur catta.—The leg area was removed on the right side. A good deal of hemorrhage occurred during the operation from the longitudinal sinus, a large branch of which was injured and had to be tied. The animal, nevertheless, made a good recovery, and paralysis of the leg had largely disappeared by the time the animal was killed, 14 days later. Lemur 9. Lemur catta.—Here the operation was limited to removal of the excitable (mainly ear, eye, and face region) area on the outer side of the lateral sulcus and sulcus rectus in the right hemisphere. No paralysis was noticeable ; indeed, after recovery from the anesthetic, the animal was climbing about the room, and eating fruit heartily within an hour of the operation. It was killed 12 days later. It will be noticed that the last three operations form a series in which the arm area, the leg area, and the head and face area (characterised by the small Betz cells) were respectively removed. We present, in the accompanying plates, diagrams of the degeneration tracts, selecting, of course, only a few important regions from the large number of sections which were made. It will be seen that, although the lesions were not absolutely limited to arm, leg, and face areas respectively, yet from our knowledge, derived from stimula- tion, we were able fairly accurately to perform the operations we have indicated. In each case the large drawings which come first are those of the brain, drawn on a somewhat smaller scale than the others. This shows the position of the lesion, and also the degenerated fibres (indicated by black dots and lines), passing in three directions: (1) to the internal capsule; (2) to the opposite hemisphere by the corpus callosum; and (3) to neighbouring convo- lutions by association fibres. The smaller drawings show the degenerated tracts in lower portions of the central nervous system.. Histological Kxamination. The brains and spinal cords were placed in Miiller’s fluid for three weeks ; appropriate slices 3 to 4 mm. in thickness were then placed in Marchi’s fluid for 14 days, the fluid being changed several times. After imbedding in celloidin, sections were then prepared. The Resulting Degeneration —In all cases there was: (1) A heavy degenera- tion of the fibres of the internal capsule on the side of the lesion, which could be traced to the crusta of the pedunculus cerebri. (2) A degeneration much less marked—with the degenerated fibres not so coarse—of the corpus callosum; most of these enter the radiating fibres of the opposite hemi- sphere, and-pass especially to that portion which corresponds to the region of VOL, LXXX.—B. M . 144 Drs. F. W. Mott and W. D. Halliburton. [Nov. 14, ablation ; some few may pass into the internal capsule (vide figs. 1 and 2, Plate 3). The degenerated myelin, in the form of black dots and rods, cannot be followed farther than the deeper layers of the opposite cortex. Appa- rently these fibres either end in the polymorph layer or the inner line of Baillarger, where the large psychomotor cells are situated (vide fig. 2, Plate 3). (3) A large number of coarse and fine degenerated association fibres pass from the lesion to the adjacent uninjured motor cortex; some run horizontally into the inter-radial association fibres, the majority, however, enter the radiating fibres. We were uncertain whether degenerated associa- tion fibres pass to remote regions of the cortex, but some certainly pass to the post-central and temporal regions ; there was no degeneration in the pre- frontal, hippocampal, or occipital regions. (4) The basal ganglia, especially the nucleus caudatus, exhibited great numbers of scattered fine black dots ; it is not improbable that this may be accounted for by degenerated collaterals given off by the degenerated efferent fibres contained in the internal capsule. The degeneration in the lower portions of the central nervous system may be most conveniently described by taking separately the individual cases. In Lemur 9 (Plate 3, figs. 1 to 8) the lesion involved the head and face region. ‘The degenerated peduncular fibres were traced. into the crusta of the mid-brain (fig. 5); they are most numerous in its outer and inner fourth, and least numerous in the middle two fourths. In Lemurs 7 and 8, where limb areas were removed, this relationship was reversed. In Lemur 9, _as might be expected, degenerated fibres can be seen leaving the pyramidal bundles in the pons, and crossing the median raphe, to arrive at the various motor nuclei of the pons, eg., the facial and fifth. This decussation of degenerated fibres can be followed all the way down the pons and medulla. Only a few degenerated fibres exist in the crossed pyramidal tract of the spinal cord in the upper cervical region. (See figs. 3 to 7, Plate 3.) These fibres have entirely disappeared in the cervical enlargement (fig. 8). In Lemur 7 (Plate 2, figs. 2 to 8), the lesion involved mainly the region of the forelimb, and we observe a heavy scattered degeneration in the pyramidal system of fibres in the pons and medulla; these fibres do not begin to decussate until the mid-portion of the medulla is reached, that is, they decussate at a much lower level than in Lemur 9, and at a higher level than in Lemur 8, There are no degenerated fibres in the lumbo-sacral region. (Fig. 8.) In Lemur 8 (Plate 3, figs. 9 to 16) the lesion has involved a part of the arm area and the whole of the area of the trunk and lower limb, except that lying on the mesial surface of the hemisphere; we observe the heavy pontine and medullary degeneration, but the fibres do not begin to decussate until i ae Ss) al ea 1907.| Localisation of Function in the Lemur’s Brain. 145 a lower level in the medulla is reached than in Lemur 7, and they can be followed all the way down the cord to the lumbo-sacral region, as shown in figs. 14,15, and 16. There is, indeed, almost as heavy a degeneration in the crossed pyramidal tracts of the cervical region (fig. 14) as in the pons and medulla. Moreover, the degenerated fibres have not left the crossed pyramidal tract in the mid-dorsal region, for there are nearly as many degenerated fibres seen in the sixth dorsal segment (fig. 15) as in the eighth cervical (fig. 14). Most of the degenerated fibres can be seen leaving the pyramidal tract in the lumbo-sacral region ; when fig. 16 (section at the fifth lumbar level) is com- pared with fig. 15 (section at the sixth dorsal level), a great diminution in the number of degenerated fibres 1s seen. We have not presented any diagrams of our examination of the central nervous system in the case of Lemur 4. It was thoroughly examined, and the results were confirmatory of those found in the other cases. Lemur 4 belonged to a different species from the other three, but this makes no differ- ence to the course of the degeneration. In this case, also, the lesion was more extensive, including both arm and leg cortical areas, so the degeneration was more extensive, and showed practically a combination of the results noted in Lemurs 7 and 8. In no case were we able to follow degenerated fibres to the anterior horn ; in some sections they could be seen lying as small compact bundles within the base of the posterior horn. As in the lower apes, there is no direct pyramidal tract in the spinal cord - of the Lemur, although it may be mentioned that one finds generally a few (homo-lateral) degenerated fibres in the crossed pyramidal tract of the same side as the lesion. Summary. 1. The brain of the Lemur has a simple convolutional pattern, and the fissures are few and for the most part shallow. 2. The motor areas are limited to the central region of the cortex, and details of the localisation by the method of stimulation are given in Plate 2 (fig. 1). 3. Extirpation of the excitable areas is followed by transitory paralysis of the corresponding regions on the opposite side of the body, and by degenera- tion of the tracts which pass to the bulbar or spinal grey matter which controls these movements. Degeneration also occurs in commisural (callosal) and association tracts in the cerebrum. . 4, The motor areas are characterised histologically by the presence of Betz cells. Localisation by histological study is therefore possible, and the close 146 Drs. F. W. Mott and W. D. Halliburton. [Nov. 14, correspondence of the results so obtained with those obtained experimentally — is well seen by comparing together the figures in the text with Plate 2 (fig. 7). 5. There are, however, two types of motor cortex in the Lemur’s brain, and these are shown side by side in Plate 4. The large type of Betz cell is found in the greater part of the motor cortex, particularly where limb and body movements are represented. The smaller type of Betz cell is found in the area governing face, tongue, ear, and eye movements, and in this excitable region there is a layer of granules; it is therefore probably sensori- motor. : 6. Although the investigation relates in the main to motor representation, histological examination of the occipital (and especially calcarine) region shows it to possess the structural characters of the visual cortex in other animals. That no eye movements could be elicited by faradic stimulation of this region is probably due to the difficulty of the experiment, as explained in the text. The expenses of this research have been in part defrayed from grants made to us by : . : £ : the Government Grant Committee of the Royal Society, and by the Science Committee of the British Medical Association. DESCRIPTION OF PLATES. PEATE 2, Fie. 1.—This is a map of the Lemur’s cerebrum from above. The names of the principal sulci, and the position of the subdivisions of the motor area, as ascertained by the stimula- tion method, are shown on the right hemisphere. The homologies of the small sulci, marked (1) and (2), are discussed in the text. Figs. 2--8.—These show a series of drawings made from the central nervous system of Lemur 7, in which the upper limb area was removed. The course of degeneration is indicated by black lines and dots. Fig. 2 is a section of the injured right hemisphere. The position of the lesion is indi- cated by a dotted line, and degenerated fibres are seen passing in three directions, namely, by the corpus callosum to the opposite side, by association tracts to adjacent convolutions, and by the internal capsule to the cerebral peduncle. Figs. 3, 4,5, and 6 are sections at various levels of the pons and bulb. Decussation of degenerated fibres begins at a lower level than in the case of Lemur 9 (in which the head area was removed, Plate 3, figs. 1—8), and at a higher level than in the case of Lemur 8 (Plate 3, figs. 9—16), in which the leg area was removed. Fig. 7 is a drawing through the thoracic region of the spinal cord ; a few degenerated fibres are still seen in the crossed pyramidal tract, but these have entirely disappeared in the lumbar region (fig. 8). Roy. Soc. Proc., B. vol. 80, Plate 2. <47gRAL_FiISSUp, LMERO GL ~ . NN 2 Gan Cerny a Wy ps cA LEMUR 7. eo &® OD) & LEMUR 8. : . -F a - . - - ! * = L 80, Plate 4, A . VO Roy. Soe. Proc., B Fic. ot Sign te een * - i . Z on r p . ‘ ' ss ? 4 . ‘ : 1 . ae - ; of ¥ ' 7 o d i o ! ' 1 : - . , 4 . } . . . : , , ae: t ( : hd] % 1907.] Localisation of Function in the Lemur’s Brain. 147 PLATE 3, Figs. 1—8.—These are drawings made from the central nervous system of Lemur 9, in which the face region was removed. The position of the lesion is indicated by a dotted line in fig. 1, which is a drawing of a section through the injured (right) hemisphere. The preparations were made by the Marchi method, and degeneration is indicated by black lines and dots. Fig. 1 shows degeneration passing by the corpus callosum to the opposite hemisphere, by association tracts to adjacent convolutions, and by the internal ‘capsule to the cerebral peduncle. Fig. 2 is a drawing of a section through the left (uninjured) hemisphere, and shows the degenerated fibres, reaching the grey matter, which have crossed by the corpus callosum. Some few degenerated fibres are seen in the internal capsule. Fig. 3 is the mid-brain. Degeneration is seen in the crusta of the peduncle, especially in its outer and inner fourths. Fig. 4 shows the degenerated fibres in the pyramidal tract of the pons. Figs. 5 and 6 show them in the pyramidal tract of the bulb. In figs. 4, 5, and 6 many of the degenerated fibres are seen decussating to reach the motor nuclei of cranial nerves. Fig. 7, through the upper cervical region of the spinal cord, shows a few degenerated fibres in the crossed pyramidal tract of the opposite side. Fig. 8, through the cervical eniargement, shows that at this level all degenerated fibres have disappeared. Fies. 9—16.—A similar series of drawings made from the central nervous system of Lemur 8, in which a large portion of the lower limb area was removed. Fig. 9 is a section through the injured (right) hemisphere. Figs. 10, 11, 12, and 13 show the degenerated fibres in the pyramidal tract at various levels in pons and bulb. Decussation of these does not begin until a lower level is reached than in the case of Lemur 7 (removal of arm area, see Plate 2). Figs. 14, 15, and 16 are drawings of the spinal cord—14 at the level of the 8th cervical and 15 at the level of the 6th dorsal segment. In fig. 15 the degenerated fibres are nearly as numerous as in fig. 14, and also nearly as numerous as in the pyramidal tract of the bulb. It is not until the lumbar region is reached that they begin to diminish (fig. 16, taken at 5th lumbar level). PLATE 4. This shows the two types of motor cortex described in the text. Fig. 1 is the first type of motor cortex, characterised by giant pyramids (Betz cells), and found in the limb and trunk areas. Fig. 2 is the second type of motor cortex with smaller Betz cells, found in the face, ear, and eye regions. Higher power views of typical cells from each layer are placed by the side of the low power drawings. Methylene blue preparations. VOL. LXXX.—B. N 148 On the Structure of Sigillaria scutellata, Brongn., and other Kusigillarian Stems, in Comparison with those of other Paleozoic Lycopods. By E. A. NEWELL ArpeEr, M.A., F.LS., F.G.S., Trinity College, Cambridge University Demonstrator in Paleobotany, and HucH H. Tuomas, B.A., formerly Scholar of Downing College, Cambridge. (Communicated by D. H. Scott, F.R.S. Received November 8,—Read December 5, 1907.) (Abstract.) Petrified stems belonging to the genus Sigilaria have hitherto proved to be extremely rare in the Upper Carboniferous rocks. The present paper contains the first full account of the structure of the Eusigillariz or ribbed Sigillarias of the Rhytidolepis section. Hitherto our knowledge of the anatomy of such stems has been limited to the account of the bark given by Williamson, and to brief descriptions of specimens, including the vascular cylinder, by Professor Bertrand and Dr. Scott. i The material, which forms the basis of the present study, consists of a petrifaction from the Lower Coal Measures of Shore-Littleborough in Lancashire, containing two well-preserved stems, lying side by side. The external surfaces of the ribs of both stems have been exposed by Mr. James Lomax, after much difficulty, and our thanks are due to him for his skill in preparing the sections. The characters of the ribs agree with those of the impressions known as Sigilaria seutellata, Brongn. In addition, other stems are described in which the ribs are not exposed, and which cannot, therefore, be determined specifically. Radial and tangential sections through the bark of all these specimens show, however, that they belong to species of the Rhytidolepis section of the Eusigillaric, which, like Sigillaria scutellata, possessed distant leaf scars. These additional petri- factions agree exactly with Sigillaria scutellata, and have been made use of to illustrate further the anatomy of that species. The stele has a well-marked pith, a tissue which is not, however, preserved in any of the stems which we have examined. The medullary cavity is bounded by a continuous ring of scalariform tracheides,—the primary wood,—the outer margin of which is crenulated. The protoxylem elements lie at the apices of the blunt, rounded teeth of the corona. The elements of the protoxylem and primary wood appear to consist entirely of scalariform tracheides. The elements of the secondary wood are also scalariform, and On the Structure of Sigillaria scutellata, ete. 149 rather smaller than those of the primary xylem, but, unlike them, they are arranged radially. The outer margin of this zone was crenulated, the ridges and grooves corresponding in position to those of the primary wood. The medullary rays usually consist of a single row of cells of varying height, of which the walls are sometimes thickened transversely. The phloem and inner cortex of thin-walled elements are not preserved. A well developed band of phelloderm is found near the surface of the ribs. This is regarded as having arisen on the inner side of a meristematic zone. No definite cambial layer is to be found, and it is suggested that the meristematic activity here took place periodically. Cells are to be seen in this region which appear to have undergone division shortly before preservation took place, and rings of growth are to be observed in the older portions of the phelloderm. The secondary tissue consists of prismatic fibres, often chambered. The ribs are really formed of cortical tissues, and not by fused leaf-bases. They consist largely of phelloderm, and externally what is probably a small zone of primary cortex, which lay without the region of secondary merister- matic activity, still persists. The stems were probably ribbed long before the formation of the periderm. The leaf-bases, consisting of thin-walled parenchymatous elements, merely form bracket-like projections from the ribs; those of the same vertical series being sufficiently distant from each other to leave a small gap of primary cortex between them. The ribbing of the stem in the Eusigillariz, being entirely independent of the form and arrange- ment of the leaf-bases, appears to be a natural feature of importance in classifying the Sigillariz. No sign of branching has been observed in any specimen. The presence of a heule and a lgular pit has been detected for the first time. The course of the leaf-traces in the leaf-bases and cortical tissues has been followed with important results. The bundle is collateral, and without secondary wood. In the leaf-bases the trace consists of a double xylem strand, the two xylem groups being widely separated. These two strands unite as - they pass through the phelloderm. The structure of the trace is almost identical with the foliar bundle of the leaf described by Scott as Sigillariopsis sulcata, which is obviously simply the leaf of a Eusigillarian stem. The parichnos—the two strands of thin-walled elements, which accompany the leaf-trace through the leaf-base and cortex—increases greatly in size, as we pass from the exterior of the stem to the inner margin of the periderm. The two strands further unite, first below and then above the trace, so that, at a deep level in the periderm, the trace is completely surrounded by a broad zone of this tissue. N 2 150 On the Structure of Sigillaria scutellata, etc. The leaf-traces pass through the secondary wood at first at an angle of about 60° to the vertical, but their course soon becomes almost horizontal, and this is maintained until near the inner margin of the wood, when they again bend sharply downwards, and eventually unite with the primary wood in one of the grooves of the corona. The Eusigillariz are compared anatomically with the Subsigillaric, and it is found that there are four points in which they differ. In the Eusigillarie, the stems are ribbed and the primary xylem always forms a continuous ring. The leaf-traces are monoxylic throughout their course. In the periderm, the xylem of the trace divides into two distinct strands, and these persist through the leaf-base, into the leaf, until near its apex, as the xylem of the foliar bundle. If, however, we regard Sigillariopsis Decaisnei, Ren., as a member of the Subsigillariz, a conclusion which seems inevitable, then this latter characteristic is common to both groups, though in the Subsigillarie it is combined with the diploxylic structure. The Eusigillarie are next compared with the various types of structure exhibited by Lepidodendron and Lepidophloios, with the conclusion that they correspond most closely to the Lepidodendroid trunks of Arran and Dalmeny. Anatomically they appear to be remote from Sothrodendron, so far as the structure of that genus is known. It is found that,in the absence of the cortical tissues, it is not possible to distinguish the stele of a Husigillarian stem by any definite characters from that of some Lepidodendree. The genus Mploxylon is discussed in this connection, and it is shown that it is by no means certain that all the decorticated stems, which have been referred to it, belong to the Sigillariz. It is more probable that the stems of several distinct genera are here grouped together, if only as a temporary expedient. 151 Dietetics in Tuberculosis: Principles and Economics.* By Nor, DEAN BaRDSWELL, M.D., M.R.C.P., F.R.S. (Hdin.), Medical Super- intendent, King Edward VII Sanatorium, and JoHN ELLIS CHAPMAN, M.R.C.S., L.R.C.P., Medical Superintendent, Coppin’s Green Sanatorium. (Communicated by Sir T. Clifford Allbutt, K.C.B., F.R.S. Received November 26, 1907,—Read January 23, 1908.) Object of Research.—The object of our research was to obtain reliable data upon which to draw conclusions as to the best lines upon which to base the dietetic treatment of pulmonary tuberculosis. In 1899, when this work was first commenced, the sanatorium treatment of consumption and other forms of tuberculosis was rapidly becoming adopted in this country. One of the most noteworthy features of this treatment was the systematic prescription of diets of a very high nutritive value. In the absence of any reliable authorities on the dietetics of tuberculosis, the practice of giving very large diets became very general, in spite of warnings from physiologists that such a method of treatment was probably unsound. Some preliminary observa- tions, which we made at Sheffield Royal Infirmary in 1899 upon the metabolism of several consumptive patients treated on very large diets, suggested to us that an extended series of such observations might enable us to place the dieting of tuberculosis upon a more scientific foundation. Our series of observations has extended over seven years, and this paper represents an abstract of our final report. Research 1. We hoped, as a result of our first series of observations, to establish :— (1) The best general principles upon which to construct diets for the treatment of tuberculosis. (2) A standard diet in terms of proteid, fat, carbohydrate, and total calorie value for the treatment of tuberculosis. Method of Observation—The progress made by 200 cases of pulmonary tuberculosis, representing well-marked stages of the diseases, were carefully observed whilst treated on definite diets, the general lines of treatment being the same in every case. * Towards the expenses of this research, the authors, on the recommendation of the Royal Society, were given a Government Grant. This paper is a Summary of the Final Report ; the full Report will be published shortly by the Oxford University Press. 152. Dr. N. D. Bardswell and Mr. J. E. Chapman. [Nov. 26, In the case of every patient that was observed, we first determined the diet which was physiological for the individual, when in ordinary health, up to his average body weight, and at physiological rest. This physiological diet was then increased in certain definite amounts of proteid, fat, or carbo- hydrate. The actual diet prescribed was carefully constructed so as to give the nutritive value decided upon. This diet was given in measured and weighed amounts, and anything left was also weighed or measured. In this way an accurate record of the food actually consumed by the patient was arrived at and its nutritive value calculated. Careful clinical observations were made on all the patients, especially as to the improvement in the morbid process in the lungs, gain of weight, and improvement in general health. In a large proportion of cases, metabolic observations were also made. Some- times these observations were made during a four-day period once a month; in other cases they were made daily for periods varying from a week to three months. The points especially studied were :— / (1) The absorption of fat and nitrogen. (2) The amount of the excretion of nitrogen. (3) The form in which the nitrogen was excreted, viz., whether simple or in the more highly elaborated forms, and their percentage relation. (4) The amount of intestinal putrefaction, as evidenced by the ratio between the aromatic and the alkaline sulphates excreted in the urine. Conclusions of Research 1.—The following are satisfactory principles upon which to construct dietaries for tuberculous patients :— (1) The physiological diet (viz., the diet which contains the exact amount of carbon and nitrogen necessary to balance the amounts of the substances excreted) for every tubercular individual when in normal health and at physiological rest, should first be ascertained, and this physiological diet should form the basis of the diet prescribed for the treatment. | (2) The amount of proteid in the physiological diet should be increased by 30 per cent., and this increase should be maintained until the disease is obsolete. (3) If the patient is much under weight, the calorie value of the physio- logical diet should also be increased 30 per cent. in the purely energy-giving foods, viz., in fats or carbohydrates, or in both. This increase should be maintained until the weight becomes stationary, at a point a few pounds in excess of the patient’s normal weight. A decrease of 15 per cent. can then be made, and the diet thus altered should be continued until the disease is. obsolete. (4) The meals must not be too bulky, but somewhat inclined to concen- tration, so as to give the comparatively large amount of nourishment in a but 1907. | | Dietetics in Tuberculosis. 153 slightly increased bulk of food stuffs. In the case of the consumptive working classes this last rule does not always apply, since they are used to taking diets of large bulk. (5) The meals should be given at considerable intervals; they should be well cooked and as varied as possible. Standard Diet for the Treatment of the average Tubercular Patient. The following table gives the nutritive value of the average diets taken by 49 tubercular patients throughout their course of sanatorium treatment. These 49 patients have been selected inasmuch as they all made very good recoveries. The average of these 49 satisfactory diets works out at Proteid. Fat. Carbohydrate. Calories. 150 150 250 3000 These figures, in our opinion, may be taken as representing the nutritive value of a diet which is satisfactory as a standard diet for the treatment of the average person suffering from tuberculosis. We have, as a matter of fact, adopted this standard in our sanatorium practice for the past few years, and found it to be most satisfactory. The standard diet which we have found to be best for tubercular women has a somewhat lower nutritive value, viz. :— Proteid. Fat. Carbohydrate. Calories. 126 150 220 2814 The following actual diets give the nutritive value of the above standard diets :— | Amount prescribed. ( Article of food as served. rane | t For men. | For women. ICI alata uc eias UU sacchari! 1500 c.c. 1500 c.c. MES eC eS es BA aicsehok Sica Haron « oelgeitedis eile 180 grammes. 135 grammes. | HEAIEGUG Ce Seer oh raf cs etnias ve viinsiacis Sept tiaists 120 us 2 3 PRUE TN Rees dont a kin.« scselswa ce uislossacnjeiSecanats 45 os 45 a Breakfast meat, such as bacon, fish, etc. 30, SOR ys (OA ONTOS 007 87 ce ne 180 % 150 ANGEL NR Praeteich a)? * 8.€9 x = 816 SPT SPL Lo = 0-19 mc [BULION Ble S) : €-6¢ a ~ poysorie Ajosae] Ara A G13 81 eS CoO 8. 6P “ G- 18—6.98 | @ | 8 ra , 8-69 “ : hee oie 0G GPL OPI 6a = 0.69 | podeduy | ¢.1e—6.98 | @ | 2 iar % 5.69 peaoiduy yonur A190 A 8G eI ST Ke 0.99 [BULION ele .9 pesoqsaa ATO Siw] peaordtay you OFS 98T OST 0-9 — 1.g¢ a G.L8—6.98 | @ | & [VULION 6.L1¢ poAoid ur yonut AIO A pesaoidut yonut Aue 4 88 PEL PEL 0-F — 9. GG e Pp. Le—-1. Le Calee : &- LE—6. 96 0.19 - i 098 Oot 81 et B.8S ss F. LE Gs q €. LE—6. 98 0.98 7U9][9OxET E ¥ 993 eG T9T Ol: 8m €-€L [BUdION [BUuIO N (as av) e. LE—6- 9g 0.92 petojsod ATQOoJ 10g qsodiv ojo[duloouy ILZ CCT COL 01-8 — 0.69 dyes Ata A 6-88 —6- 98 Scales = 0.09 “ “ qsoude aqojduro9 068 981 8ST C50 0.#¢ 100d Vole 0 | ee lee or [VULION 0-02 WOrPRIO}SOI ayo[duiog e PA LPL IST (ealy) 9.¢9 [BULLION v ey = Gc. Le—0. Le 9.Gg poos A193 A qsolie o3e]duLoouy 193 CCT L¥T 6-9 — L-6P Tey Ard A €- LE—0- LE € |g He| = 0.02 UOL}VIOJSOI 9Jo[AuLOD 4salie aja[dulog PTZ OST 09 G.F — G.19 poured uy 7. 18—1.18 G a S 0.9L = 5 ee es CS raat SFT 03 [enba 0-92 ie [BULION iB he [eULION G.9¢ poaordury Ap}vors Asa A es nn 3 C&G SST CCT [sts L.GP ‘ €.88—Z. LE i | ° Pp. LE—6. 9E 8. 8G quoe][Iox A peysotze ATQeiapIsuood Aro A. 682 SFL OST ¥.G — G.1¢ os P-1E—L.9e G —) 8. 18—G. LE 0-19 UOTFRIOJSaA VJaTdutoouT 4sodiv oJo[d WOoUuT CCZ IST eFL 9.61— 8.8¢ ]BULIO NT 0. 88—@. LE r) KS rs 8-18 - i a 088 0@I 8eL 8.8 — Z- 18 . a Zl Q 0. 9 6s 66 66 6 861 Eel SCL 04 [enbo 0-09 - I | 4 “ 9.5L i = - : raced oct at ovenbem|" =s-02 ’ a . 0-11 - = i i GLE @ST 091 Sola 9-89 . : 6 = e G-F9 i S is ‘ 93 8cI 8c1 VG = 0.89 - ‘ I ‘ 0-91 ee & : 9&3 9¢1 TLT kis €-89 ee G So ie 8- TL we ae qsotie oya[du0g 98Z SLI ZO 04 [enba 9.69 — yi s T | g..e9 os oC qsodae ojo]duoouy zGz 0st 9CL aS — 6.09 ss gt z co 66 9.29 ce 66 66 66 9G SFL Pan 04 jenbo G.9¢ 66 66 T Q ae 3 0.¢) 66 66 66 66 QZ OST ISL 04 jenbo QoGL 66 66 I b Oo i 9.69 - Ps . . 9FG Ost SPL €-9 — 0.09 . . Uke es L.€9 5 x : - 8G aa IST €.9 — ¢.6¢ = f Dele E y G- #9 - 9 - FS SB OST al 07 [enbe ¢. 6¢ i Lee me iE g. 19 Z - i . BES a eg oS L.¥9 ss [BULLION id bs = * g.89 ef - .6 ve 912 9ST O8T ¥-6 — 1.19 Re G-18—0.18 | 3 [8 = ss b. £9 ss a ee a B8S Gc 89T 0:8) = G.9G = v. 8E—6. LE | aa a ¥. 9 > ef e im and €ST ISI Leb 0.8¢ af 9. LE—B. LE Dales 6.9L - ce 3 . 9&8 SPL EST Lele 0.69 = 6-18—@.18 | 6 |e . = €.8 i a ‘ axa 8ST G91 8G = 1.9L is [BULION t | = 2 G-&9 UOT}VIOYSII 9}o1CuI0g qsolde aja[durop, OLE 961 O9T Nps 0-€¢ ‘ Gr lG——OoLe ¢ 16 i 0.1 po104sed Ajos1e7T qsodae ojo[/du00uy 096 SPT SCT 6: 6he= T-1¢ i 6. LE—9. 98 I rae tt ee ee eet | S ‘ 66 66 66 Z r) 79 °= B ~8e—6. L e 0-69 ys ve qyso1ie ojo] utoouy 89% 991 FST $.9) = 0.86 peaedwy [BULLION rd = 6.9L ¥ s ie i. 89 9ST BLT 0} yenba 0.92 9.18—-6.98 | T (za > 8.1L UCT}VIO}SaI aYo[AuUIOD qsorre 0jo[dui09 12 ZOL 891 Se 6.99 [BUuLIO NY 6.le—@.le | S$. 99 qua][oox ysorie oyo[duo00uy 98 ital GI 7g — 0. Lg IOOg [BULION zZ [RULLO NT 8.19 UOIPVIOJSOI VJo[dutog qsouie ojojdwog GGZ PSL I9L e.9 — 0-1G | yetudoN F. 88—6. Le e | | | | Qo “SOLD berry | “gqstam “SO[LY 00 ‘pasvasip os asura Ut “UPeay [erouoy -‘osvastp SUNT | A ae q “Rip ‘projorg | yeutou 04 UL | “Worysas1q aouvt Saqo] i> aINzVIIdU9aT, | IYBLOAA q7e): | UOTzEpoy | YYBIOM | ainqesedutay, | Jo “ON = ue inne | pene tl ze | ‘adieyostp uC | “401 ee “THOISSTTUpe UC ee eR PPE ETF PPV — ee - i — we erect ——— BESIIENY B507:) “. Dietetics in Tuberculosis. 155 Observations on Tubercular Patients treated with Very Large Nets. Careful observations have been made upon tubercular patients treated on the very large diets prescribed in many sanatoria, for instance, on diets with a daily proteid value of 200 grammes or more, and a total calorie value of 4000 or more. Clinical Results of these Observations.—Patients made much less satisfactory all round progress on the very large diets than on diets of considerably smaller nutritive value. Weight was gained in nearly every case, in some to a very large extent, and very rapidly, but this gain of body-weight was not associated with any more satisfactory progress in the tubercular lesions than was obtained with the smaller diets; on the other hand, general health suffered considerably, as evidenced by failure of appetite and marked digestive and intestinal derangements. Metabolism.—(1) The absorption of fat in the case of patients below their normal weight was higher than normal, even when very large quantities were ingested, eg., a patient taking 231 grammes of fat daily absorbed 96-4 per cent. The great majority of patients absorbed over 90 per cent., and in no case was the absorption below 87 per cent. (2) The absorption of nitrogen was also high in all cases, viz., 90 per cent., or over, but, when very large quantities were ingested, an increase in the amount excreted in the urine invariably occurred, and only a very small percentage of the increased amount of nitrogen ingested remained in the body. It was noticeable that the absorption, both of fat and nitrogen, was high, even in the cases suffering from acute dyspepsia. (3) The percentage of nitrogen excreted as urea decreased, and, conse- quently, the percentage excreted in less oxidised form increased, indicating less complete elaboration. Table I1—Diets and Metabolism of Three Patients treated by Forced Feeding. BSEORCI OU, 600), abe aencceness no 232 °5 Piffal al 251 °4 BRM t aeiciees ode PSE ew doe 183 ‘9 231-2 208 3 Nutritive value of Carbohydrate ..:............ | 321 °3 392 ‘1 297 °2 diet. Motal) calories ..:...6.66..05. 4126 °O 5026 -O 4187 °0 J PEIN os 288 eae Sb caiwa cae ney 2810 ‘0 3444 °O 3234 °O PSM PIEING och ecce cers s obs 30 “8 23 °8 18 *1 IGE (DE a ere cee 85 °5 40 °6 28 *4 INDRA EE COS. oo eces ces. 1. vou 7 4°3 2 °4 Metabolism figures (average of 4 days’ observation). LODUE ae Ne a 3°8 7°8 4, °4; | N absorbed (per cent.) ,.. 94 °2 89 9 93 9 | eh (seratet or 97 ‘9 96 4 She 156 Dr. N. D. Bardswell and Mr. J. E. Chapman. [Nov. 26, (4) The ratio of the aromatic to the alkaline sulphates excreted in the urine became smaller, indicating increased intestinal putrefaction. 7 The preceding table shows the nutritive values of the diets taken by three patients thus treated by forced feeding, and the results of metabolic observa- tions during a four-day period. Research 2. On the most Economical Lines upon which to Construct Diets having the same Nutritive Value as our Standard Diet. In view of the large expense of dieting in most sanatoria, and in order to bring the modern dietetic treatment of tuberculosis within the reach of the poorer classes, we made a series of observations, with a view to determining the most economical lines upon which an adequate diet can be constructed. An Economical Diet—An analysis we made of physiologically adequate diets, taken by 100 working-class families, showed us that an adequate diet for the working classes can be bought for about 10d. a day, and that, in such a diet, every penny spent buys some 12°7 grammes of proteid and 329 calories. The average of these 100 satisfactory diets worked out at: proteid, 119; fat, 114; carbohydrate, 417; calories, 3687; at a cost of 10d. (not including money spent upon beer or other alcoholic drinks). The aim of our work was to increase this average diet some 30 per cent. in proteid without appreciably increasing the cost. During the course of our preliminary observations on the subject, we found that the convalescent working-class consumptive, when taking a con- siderable amount of exercise, such as digging in the gardens, etc., requires a larger diet than our standard diet for men at physiological rest, and we aimed, in consequence, at constructing a diet with a nutritive value of Proteid. Calories. 150 and 3600 approximately. A trial of several dietaries of the above nutritive value, and constructed on the lines of ordinary dietaries, showed us that the expenditure on meat. accounted for some 40 per cent. of their total cost, and that, to construci a really cheap diet, the amount of animal proteid, especially meat, must be kept down as much as possible, and considerable use made of the cheaper forms of vegetable proteid, such as peas, beans, lentils, etc. We estimated that the replacing of meat by vegetable proteid would prove an economy of 30 per cent. In view of the widely accepted principle that animal proteid, especially meat, has some specific value in the treatment of tuberculosis, and may even be regarded as an essential in the diets for the tubercular, we decided first to Table III. Dietetics in Tuberculosis. 157 No. of Nutritive value of diet. | Percent- Weight days eee “| age of | Cost, | of food Weekly | Total Case. under | ee Te weee in taken at tag ue observa-| p,oteid | Fat Carbo- | Gajories. | | ik d table | pence. | daily, in yet i in Eel : | tion. aie ‘ | hydrate. aL e "| proteid. grms. sis mre ; | Cases of Early Disease with Normal Digestions. uy 71 175°2 | 146°8 | 550°5 4340 44°0 | 56°0 | 11°25 4331 1% 13 °5 2 55 156°1 149°6 | 501 *4 4085 46 1 | 53°9 | 10°81 | 4176 15 8°3 | 3 63 164°9 | 125°0| 456°5 | 3710 | 48:3 | 51-7 | 10°86] 3527 09 | 9-0 : Cases of Advanced Disease with Impaired Digestions. 4 47 eS )-7 99-4 | 289-0 2596 49 °2 50 °8 7°39 | 3047 | Stationary Nil 5 72 125 °8 | 110°8 366 °5 3049 43 °9 56 ‘1 Sl 3160 0°3 2°25 6 21 @120°9 96 -O 335 °O 2761 48 °O 52 °0 6°26 3111 1°75 2 ‘2 Average 55 143 °6 | 121°3 | 416°5 3473 46 °6 53 “4 9°11 3558 0°99 5 OF Table [V.—-Meat-free Diet taken by Case 2 for 55 days. Amount. Nutritive value. a AAgst} ; Cost, in eae pence. | Grammes. | Ounces. | Proteid. Fat. | ae ydrates. WWirole milk ..........0..: 2000 70 66 °O 80 -0 100 ‘0 5°28 SEN a eee Nil — — — — ou HUNGEOI a ss ile ce sieie vidoe vedens 71 23 — 56°8 — 1°86 GEREN rah. oken adie 44 obs — — —- — = ws Oise: dines nas ww bnsih ui 6:0 4,0 — 0 66 BACON! ch. seo deere dadases Nil — — — oe ee : Total animal food -— — 72 °0 140 ‘8 100 ‘0 7 ‘80 Tiare 268 9 23 8 2:6 | 182°5 0°72 ROtRTOES) 4.05. ..06000 068 hie — — —- — = ae Warmeal® ove... esos ees 50 2 80 3 °6 33.7 0°22 Peas, beans, etc. ......... 200 a 48 °6 2°6 120 ‘6 0 ‘87 POM Giels 0 sic osisisinc'sn <= 40 13 — — 40 °O 0°22 PRFEC OLE? |, H5j4.0 065.0% ones 28 if 2 °2 — 22 °4 0°12 POE CA ice crcrayss ervicis wanes 0 59 2 0°5 = 47 °2 0°46 Green vegetables......... q.s. ass 1:0 — 5°0 0°10 SUMGTICS 2. .2525..05+.2-- — — — —- — 0 °s0 (entirely un- nutritive) Total vegetable food — et 84 °1 8 °8 401 °4: 3 ‘01 Total food ............ — — 156 ‘1 149 6 501 °4 10°81 Total calorie value, 4085. Calories ain per penny, 378. Grammes of proteid bought per penny, 14°8. 158 Dr. N. D. Bardswell and Mr. J. HE. Chapman. [Nov. 26, satisfy ourselves as to the truth of the principle. For this purpose, we carefully observed six typical cases of pulmonary tuberculosis treated on an entirely meat-free diet. A summary of the results of these observations is shown in the following table, and a sample diet taken by one of these patients for a period of 55 days is also given. Results of Observations as to the Value of a Meat-free Diet for the Treatment of Tuberculosis. Our conclusions are as follows :— (1) Vegetable proteid, as the main source of the daily intake of proteid in a diet for the tubercular, is thoroughly satisfactory, so long as a sutticient amount of it is taken. (2) The clinical results obtained, when treating consumptives with good digestions upon meat-free diets of an adequate nutritive value, are quite as - good. as the results that are obtained when ordinary meat diets of a similar nutritive value are used. (5) Owing to the bulky nature of a meat-free diet, its use is restricted to patients with normal appetites and digestions; it is unsuitable for the treatment of those with marked impairment of the alimentary tract. (4) The use of vegetable proteid in the place of all the meat usually prescribed in an ordinary meat diet effects an economy of some 33 per cent. On the Construction of a Cheap Diet containing an Ordinary Amount of Animal Proterd, such as Meat, etc., to Ensure Palatability and Variety, and a Certain Amount of Vegetable Proterd for the Purpose of Economy. Our practical experience with meat-free diets showed us that, in spite of their great economy, they are not quite satisfactory, inasmuch as they require very careful cooking to make them appetising, and that even when well cooked they are not readily taken by the ordinary person accustomed to a meat dietary. To be really efficient, a diet must be to the liking of those to whom it is prescribed. _ We then constructed several dietaries in which the 150 grammes of proteid which we consider to be desirable in the treatment of tuberculosis is given, partly in the form of meat and partly in the form of vegetables. The following table shows one of these diets which was taken by three tubercular patients for an average period of 26 days :— 1907.) Dietetics in Tuberculosis. 132 Table V.—Diet taken by Three Men during an average period of 26 days. Meat* Margarine Cheese (American) Bread Potatoes Peas, beans, etc......... Oatmeal HENCE OL Cro ns. saiece's sone Flour Green vegetables Sundries Total vegetable food Total food eoecce Ce ee) eee eee oes ese reeves oes eresereeereeeesoe eceereereer eer ves eeeee eceee Amount. | Ounces. | Grammes. 42 1200 7 200 4 16 2 54 + 10 2 56 10 282 $ 210 a 97 2 56 14 40 2 56 4 16 2 56 6 170 Price retail. 1d. per pt. 7d. per |b. Gd. 3; Sd ts: Td. 39 6d. per lb. 23d. per 2 lbs. 8d. per stone 2d. per lb. Sr 22d. ,, Sad. 4 ‘Zed. 4; 1s. 6d. per stone | Nutritive value. Proteid. S| 153 Fat. 115 Cost, in Carbo- | pence. hydrate. 60 2°h1 — 3 16 -— 0:18 — 1-00 — 0°15 60 | 7°35 141 0:78 30 0°26 57 0 °40 37 0°25 40 0°22 33 O °44 13 0-08 39 0°16 8 0°25 10 0°50 408 3°34 | 468 10 69 Total calorie value, 3616. Ratio of cost of animal to vegetable food, 68 °7 : 31 °3. Grammes of proteid per penny, 14°3. Calories per penny, 337. * Uncooked and including bone, etc., and comprising beef, mutton, pork, tinned beef, mutton, etc. The above diet could be bought retail in London from 6s. to 6s. 6d. per head per week for an average sized family, according to the quality of food purchased. The results obtained in the three cases observed on this diet were most satisfactory. . After very considerable further experience in the treatment of tuber- culosis with cheap dietaries constructed on the lines which we have described, we have adopted the following dietary as being, in every way, the most efficient and satisfactory :— 160 Dietetics in Tuberculosis. Table VI.—Diet taken by 15 Patients at Coppin’s Green Sanatorium during the week of observation, per man per diem. — Ounces. |Grammes.| Proteid.| Fat. IMIG TS) NaS Se kore ree 24 *4, 690 23:0 28:0 Mieata Aeet- oceee eeees 8°65 245 4.4. °O 28:0 Liver, etc., fish .........| 20 56 100 7:0 Gheesehssccntee ee 0°5 14, ices 1 °4 Drip pins esses ers: 0°46 13 — 11 ‘2 IButtervewn cco ee eee 1°05 29 — 23:0 Egg (1 per week) ...... Te — 1:0 0°7 PB ACOUL. Vos eae 2°0 56 8:0 17:0 Total animal food — — 91 °3 116°3 ‘Bred’ xc ) shré 247 220 2°0 IROtatOCS eee eee 8:0 228 3 °0 — ‘Pulses, Sen See eee na) 85 20:0 — Oatmeal 2. jhe eee 2:°0 56 9:0 4 °O SUPA: Grane tichewenomeee 5°3 150 — — ACs eeenen eat aoee mac arGodt “ale <0) 28 — _ Cereals’ 2. bitr.con eee 0°3 10 — — OUI. oo eee 2:0 56 6:0 — Sundries: visor neeeees q.s — 2°0 30 Total vegetable food == == 62 °0 9°0 Total food — — 153 °3 125 °3 eee vce ere ces) C | Price Cost | arbo- Ai eer bea per lb. or i y e. gallon. pence. | | 34 1s. 187 — 63d. 3°00 — 5d. | 0°63 == 6d. | 0-19 — ls 0°68 | — — 0°14 | | =— Va O 87 | 34 — 7 38 | 123 lid. | 0 ‘69 | 30 | 6d. per 14 1bs.| 0°23 | 53 2d. 0°38 | 38 lid. 0°19 150 2d. 0 ‘66 21 3d. 0°19 mi Qhd. 0-05 39 | Is. 6d. per | 0°15 | stone 16 — 0:60 477 — 3°14 | 511 — 10 °52* Grammes of proteid bought per penny, 13°2. Calories per penny, 386. Total calorie value, 3889. * True cost, allowing 10 per cent. for waste in cooking as explained = 11 ‘57d. This dietary has a thoroughly adequate nutritive value for the treatment of tuberculosis. With a daily value of proteid, 154 grammes and 3889 calories, 1t 1s especially suitable for consumptives who are convalescent and doing a certain amount of muscular work. It is very palatable, easily digested, and allows of a considerable variety being made in the menu day by day. It is very cheap, considering the amount of nourishment which it contains, costing only 113d. per day. It is very economically constructed, every penny spent upon it buying’ 32 grammes of proteid and 336 calories. 161 On the Weight of Precipitum obtainable in Precyntin Interactions with Small Weights of Homologous Protein. By Professor D. A. WELSH and Dr. H. G. CHAPMAN. feted by Dr. C. J. Martin, F.R.S. Received December 17, 1907,—Read February 6, 1908.) (From the Physiological and Pathological Laboratories of the University of Sydney.) In a previous communication* on precipitin reactions, we brought forward observations which led to the conclusion that the precipitum is derived mainly from the antiserum and not from the homologous protein. In a recent reviewt of our paper the suggestion was made that gravimetric evidence might be more conclusive. Acting on this suggestion, we have carried out experiments in which a considerable amount of antiserum was allowed to interact with a known small amount of homologous protein. As soon as the interaction was completed, the deposits were collected, washed, and weighed. In evety case the amount of dried precipitum exceeded the amount of dried homologous protein. The smallest precipitum obtained weighed more than twice, the largest more than 25 times, the homologous protein employed in the interaction. Former observationst had revealed that antisera require very different amounts of protein to ensure the formation of maximal deposits. -It was, therefore, to be expected that considerable variations would be obtained in the weight of deposits produced by small amounts of protein in different antisera. Method of Expervment. The antiserum was obtained from rabbits which were bled on the day of the experiment. One milligramme or 2°5 milligrammes dried egg-white or blood serum was dissolved in 10 c.c. salt solution and added to the homologous anti- serum, together with about 90 c.c. salt solution. The whole of the available clear antiserum, with the exception of less than 1 c.c. reserved for control obser- vations, was employed. The dried protein was prepared under aseptic conditions, and every precaution was taken to exclude micro-organisms§ — * Welsh and Chapinan, ‘ Roy. Soc. Proc.,’ B, vol. 78, p. 297, 1906. + Mouton, ‘ Bulletin de l’Institut Pasteur,’ 1907. { Welsh and Chapman, ‘Journ. of Hygiene,’ vol. 6, p. 263, 1906. § We are unable to confirm Friedberger, ‘Centralb. f. Bakt., Orig.,’ vol. 43, pp. 490—494, March 5, 1907, in his statements regarding the prevention of bacterial growth by precipitin antisera, as our tubes tend to become infected in about 7 days. 162 Prof. D. A. Welsh and Dr. H. G. Chapman. [Dec. 17, during the experiment. After 48 hours the clear superfluids were removed, — and were usually subjected to further experiment, either by addition of more homologous protein, or by addition of fresh antiserum from another immunised animal. Since it had been shown* that different homologous antisera might interact to yield a precipitate, appropriate controls were carried out which showed that no trace of deposit occurred as the result of the interactions with one another of the antisera used in these experiments. The deposits from the interactions were shaken up three times with 50 c.c. saline solution, and separated by the centrifuge. They were then washed five times with 50 c.c. distilled water, whereupon the superfluid yielded no trace of soluble protein. After three washings with absolute ethyl alcohol, they were finally treated with ether free from water. On removal of the excess of ether with a pipette, the deposits were placed in an oven at 50° C. for 24 hours, and dried to constant weight in a desiccator over calcium chloride. The weighings were made in small glass test-tubes of about 7 grammes tare, in which the final washings with alcohol and with ether had been carried out. Amount of Precipitum obtainable from Large Quantities of Antiserum with Small Amounts of Homologous Protein. Five experiments were undertaken, and the results are summarised in Table I. Table I. | : lino. Weight : : of ee. Weight of homologous protein Amount of antiserum (fresh). precipi- | experi- | (dried). | Pepa | ment. | (dried) \ | milli- | | grammes. (eral | 2°5 milligrammes hen egg-white...... 19 c.c. hen egg antiserum (No. 40) ...,; 18:0 2 | 2°5 milligrammes hen egg-white...... 20 c.c. hen egg antiserum (No. 41) ...| 18:0 3 (a) | 1:0 milligramme horse serum......... 19 c.c. horse antiserum (No. 43) ...... 2°2 3 (6) | Supertiuidot 3 (a) si. .ci...- 2.4.2 .s508s 18 c.c. horse antiserum (No. 42) ...... 5°0 4 (a) 1:0 milligramme ostrich egg-white...| 20 ¢.c. ostrich egg antiserum (No. 44) 5 °0 4 (b) | 40 milligrammes ostrich egg-white | Superfluid of 4 (@) .....c.cseeeeesee eens 11 °0 5 (a) | 1:0 milligramme hen egg-white ...... 18 c.c. hen egg antiserum (No. 47) ...) 13:0 bu) a Supertiuid Of O00) Bes. .scte.scsscees 16 c.c. hen egg antiserum (No. 46) ... 8 °5 5 (c) | Superfluid of 5 (6) .t.......00..c0steses 18 c.c. hen egg antiserum (No. 48) ... 4 *4, In experiment (1) 2°5 milligrammes dried hen egg-white interacted once with 19 ee. fresh hen egg antiserum (No. 40) and caused the formation of a deposit * Nuttall, Hopkins, and Strangeways, quoted by Nuttall, ‘Blood Immunity and Blood — Relationship,’ p. 129, Cambridge, 1904. 1907.] Precipitum obtainable in Precipitin Interactions, etc. 163 weighing 18 milligrammes, or more than seven times the weight of the homologous protein concerned. In experiment (2) also 2°5 milligrammes dried hen egg-white interacted once with 20 c.c. fresh hen egg antiserum (No. 41), and yielded a precipitum of 18 milligrammes. | In experiment (3) 1 milligramme dried horse blood serum, interacting (a) with 19 c.c. fresh horse antiserum (No. 43) produced a deposit of 2°2 milli- gramines, or more than twice the weight of the homologous protein. The ‘ residual homologous protein in the clear superfluid, interacting (0) with 13 c.c. of a second fresh horse antiserum (No. 42), yielded a further deposit of 5 milligrammes, making 7-2 milligrammes in all, or more than seven times the amount of the original homologous protein. In experiment (4) (a) 1 milligramme dried ostrich egg-white, interacting with 20 c.c. fresh ostrich egg antiserum (No. 44), led to a deposit of 5 mulli- grammes, and (b) the subsequent addition of 4 milligrammes dried ostrich ege-white to the clear superfluid led to a further deposit of 11 milligrammes. In experiment (5) 1 milligramme dried hen egg-white, interacting (a) with 18 cc. fresh hen egg antiserum (No. 47), brought down a deposit of 13 mill- grammes. The residual protein in the clear superfluid, interacting (>) with 16 c.c. of a second fresh hen ego antiserum (No. 46), gave a further deposit — of 85 milligrammes. The resulting superfluid, again interacting (¢) with 18 cc. of a third hen egg antiserum (No. 48), gave a third deposit of 4°4 milli- grammes. Thus, by allowing 1 milligramme of the homologous protein to interact successively with three antisera (which did not mutually interact), a total deposit of 25°9 milligrammes was attained. The progressively diminishing weights of precipitum obtained from the successive antisera in experiment (5) (a), (0), and (c), do not necessarily indi- cate that the homologous protein is being appreciably used up in the inter- actions. A difference in weight of deposit may be an indication merely of a difference in precipitability of antiserum, a circumstance to which we have already referred. This interpretation is consistent with the control obser- vations made with each of these antisera and fresh homologous protein, and also consistent with the progressively increasing weights of deposit obtained in similar circumstances in experiment (3) (a) and (0) and its corresponding controls. Thus in the control observations in experiment,(5), when 0°1 c.c. of each of the three antisera concerned was tested separately with 0:00005 gramme homologous protein, it was found that the largest deposit was given by (a) antiserum No. 47, and the smallest by (c) antiserum No. 48. VOL. LXXX.—B. 164 Precipitum obtainable in Precipitin Interactions, ete. Conclusions. These observations reinforce our previous conclusions* regarding certain phenomena of precipitin reactions by showing :— (1) That the homologous protein is capable of throwing out of solution many times its weight of precipitum, and (2) That, in the interaction of minute quantities of homologous protein with large amounts of antiserum, the protein is not completely removed from the superfluid, but that sufficient remains to produce a second, and also to produce a third interaction with similar large amounts of fresh antiserum. It is apparent, therefore, that the serum (antiserum) of a rabbit, immunised with egg-white or with blood serum, acquires the capacity, not so much of precipitating, as of being precipitated by the homologous protein; in other words, that the antiserum, and not the homologous protein, is the main source of “precipitable ” substance. We desire to acknowledge the courtesy of Professor Anderson Stuart in placing his laboratory at our disposal. * Welsh and Chapman, ‘ Roy. Soc. Proc.,’ B, vol. 78, p. 312, 1906. : 165 Observations upon Phagocytosis carried out by Means of Melanin - to Ascertain more particularly whether the Opsonic Index vs Identical with the Hemophagocytic Index. By 8. G. SHATTOCK and LEONARD S. DUDGEON. (Communicated by Professor J. Rose Bradford, For.Sec.R.S. Received December 21, 1907,—Read February 6, 1908.) (From the Pathological Laboratories, St. Thomas’s Hospital.) The following work was taken up in the belief that interesting informa- tion might be obtained by the substitution of a finely divided inert substance in place of bacteria, wherewith to study the phagocytic index in particular infective diseases. The substance which we selected was melanin obtained from the eye of the ox. The questions which we set ourselves to test by this method we may put seriatum ; and we may, after each, recount the particular observations bearing upon it, and give the conclusions to be drawn from them. Question 1—Will an increased phagocytosis of melanin take place if melanin is added to the blood of a patient suffering from an infective disease, as compared with normal blood; orif melanin is presented to normal washed leucocytes in an immune serum, as compared with a normal serum ? To give first the results of the simpler of these two observations. The method consisted in drawing up 1 volume of blood into the capillary end of ,a pipette, and, immediately after this, 1 volume of citrated salt solution (1 per cent. sodium citrate, 9 per cent. salt), and thirdly 1 volume of melanin, suspended in salt solution. The three volumes were discharged from the tube by means of a rubber teat fixed to the wider end, mixed in a watch glass in the usual way, and finally drawn up the capillary portion of the pipette, the fine end of which was then sealed in the flame, and the pipette laid horizontally in the incu- bator at 37° C. for 20 minutes; the blood film was thereupon prepared as already described. In our later observations we simplified this technique by using only two volumes: viz., 1 volume of blood and 1 volume of melanin suspended in citrated salt solution, instead of in simple salt. Summary and Conclusions drawn from the Observations made to Test the First Part of Question (1). In a total of 32 cases, the phagocytic index is below par in 8; at par in 7; and above par in 17, ascending to 2, 3, 4, 5, 6, and even 8. 0 2 166 Messrs. 8. G. Shattock and L. 8. Dudgeon. [Dec. 21, It might be at first assumed that the heightened index showed that the “increased phagocytosis is due to the presence of a cytotropic substance exclu- sively, seeing that melanin, not being bacterial, cannot (so it might be supposed) be susceptible to the action of a bacteriotropic or opsonic substance. This, however, is not the case. The proof of this we will adduce later on, but briefly it consists in this, viz, that an immune serum can be largely deopsonised by means of melanin if the latter is used to saturation. And if melanin can deopsonise, it can only do so by becoming opsonised. We believe that in both cases the result so obtained is mechanical; the finely divided substance, though inert, entangling or becoming invested with the still more finely particulate material, of which the opsonin would thus appear to consist. In the six following observations, the ordinary method of estimating the opsonin index was carried out, 2.2, normal washed leucocytes were used : (1) in normal serum ; (2) in the patient’s serum. The cases comprised :—Acute pneumonia, lymphangitis, pneumonia, acute pneumonia with pleural effusion, urinary fever (colon bacillus infection), empyema. Renarks on the Six Foregoing Observations. The index in the third and fourth cases was taken by Wright’s method, and also by the simpler one of using normal blood against the blood of the patient. The index obtained by the second method is in both cases higher, being 1:2, as compared with 1; and 4°7 as compared with 1. Question 2.—Will saturation of an immune serum with melanin remove the r) ““opsonin” and reduce the phagocytosis of normal cells against either the bacillus or a suspension of melanin, or against both? That is to say, how far are “ opsonisation ” and “ deopsonisation ” strictly specific ? | In the following observation the action of the patient’s cells in the presence of melanin is compared in the patient’s serum, and in the patient’s serum after saturation with melanin. The serum was digested with a thick suspension of melanin for 24 hours, at 37° C. CasE.—Acute appendicitis ; peritonitis ; due to B. colv. 1 vol-cof the patientis: washed! cellesi11.& .gacssseee eee eee eee 50 cells 1 e e SOLU cine asin nestle ne omen eee eerie eeneares } contained 1 Pr MMOIAM ise Agee ae ences va\b.sin'n ecacacpvisin. ste POR RES erento 68 granules. I yol,ot the patient's avashed cells. (7) 1yayvanceeeeceeneenecee secre 50 cells il a Me serum after saturation with melanin } contained 1 AP PU ICAL PIN Eee rene ARMOR OR REMRE EEE fi 5cn sho BLALLoaNsg cnocHue eu 28 granules. The phagocytosis was reduced to nearly one-third. 1907.] Observations upon Phagocytosis. CasE.— Pulmonary tuberculosis ; untreated with tuberculin. A. 1 vol. of normal washed cells er aOR GS SOROS ona san on ndguameviosapinatecmnaanaceps 1 ,, tubercle bacilli (in 1°5 salt solution) Peotone sers senna seesseseeseseresesesenee Serum of patient saturated with thick suspension of tubercle bacilli ; digested for 44 hours, at 37° C., and centrifuged. (i) 1 vol. of the clear fluid thus obtained 1 Ss normal washed Cells... cscs dnorersw-neavesvocue 0 i ba Guperele Dacilli jerry. Get aanuwete ek weieeea ce voce cas (ii) 1 vol. of the clear fluid thus obtained 1 1 ar normal washed Celistheen. sess sate okt nese seset es » Melanin Dee SS HHSC HEHEHE HSH SEH HEHSHSH SHH HSE HSHHSTH SHH SEH STORES EES B. 1 vol. of normal washed cells 1, patient’s serum 1 Seem eeseseser esses reo aSFeSeeeeessee ga ye = We lami ays Beate Miers ge ce htisce sted cascade ch eae es ude Serum of patient saturated with thick suspension of melanin; digested for 41 hours, at 37° C., and centrifuged. (i) 1 vol. of the clear fluid thus obtained 1 * normal washed cells i »» melanin Peeeeereeeeessenseseseeeres eeoeeeee eee eee ee eeeeeeeeeeeeeeeres Poe eeereeesseeoeseeseseeneeeereseseet SEF sFeeeesesseneersenes (ii) 1 vol. of the clear fluid thus obtained 1 fs normal washed cells 1 4s tubercle bacilli eoerresesneeeseseeeereseses Poorer rt oeeseeteeeeesseeeeeeeseeese Peeneeseseesateseset FF eeverteseereneereaeene CasE.— Pulmonary tuberculosis ; untreated with tuberculin. A. 1 vol. of normal washed cells 1 ,, patient’s serum 1 ,, tubercle bacilli Poe eoeen eed ees eeeeeHeeeeeHeAaseoesseeeenes Poe ees sesr essere eeseesseereeseoeesesesesneeses CoC oecercesresereereoseeeeeeseeeseseerteseeeseseos Serum of patient saturated with an equa! portion of a very thick suspension of tubercle bacilli in 1°5-per-cent. salt solution ; digested for 24 hours, at. 37° C., and centrifugalised. © (i) 1 vol. of the clear fluid so obtained 1, normal washed cells 1 ,, tubercle bacilli eee eseeeresesceeeseereeeseneceseoe weaoeesesesseeeseeser ee esessenesecseseses (ii) 1 vol. of the clear fluid so obtained 1 ,, normal washed cells 1, melanin Pees eenesrersessceceesesesesese eoeeoeseereeeeeeeeneeseerseseceneeeseted Coe eeeeeet SFOS eseoertseeeeneeseseansesessseosene ee esenes B. 1 vol. of normal washed cells 1 , #patient’s serum 1,4, melanin eee vee tseeseseceeseneseeseeseeseseeeesese Pore eessseseseeeseteesesesseeseseeeesseneseee POPPER OOB ee HHT HEHEHE OSES EEE HH HSH H HEHE HOOH EHS EE HEO 50 cells contained 155 bacilli. 50 cells contained 46 bacilli. (50 cells contained 2 granules. 50 cells contained 22 granules. 50 cells contained 5 granules. 50 cells contained 113 bacilli. 50 cells contained 170 bacilli. 50 cells ‘contained 13 bacilli. 50 cells contained 5 granules. 50 cells contained 18 granules, 168 Messrs. 8. G. Shattock and L. 8. Dudgeon. [| Dec. 21, Serum of patient saturated with a very thick suspension of melanin ; digested for 24 hours, at 37° C., and centrifugalised. | (1) 1 vol: of the clear tluid solobtained ype. ere eee 50 cells I< ‘normal:washed cellsi.cit-0e- eee eee eee cere } contained _ Toy oy) MelanIM oA tee ce oe ae eee eee Eee ne coee 6 granules. , Gi) I vol. of the clear fiuid sojobtaimed-..pae neces 50 cells 1. %,,.. normal “washed cells? cea... ce eee eee } contained 1's, tubercle bacilli ..c2c5. ccc eee eee eee 56 bacilli. Summary and Conclusions from the foregoing Group of Observations. In Observation 1—The saturation with melanin of the immune serum from a case of acute peritonitis reduced the phagocytosis (using normal cells) from 68 granules of melanin to 28 granules. This result shows that a large amount of opsonin has been removed by the melanin, the phago- cytosis being reduced to 1/3. In Observation 2.—Pulmonary tuberculosis, untreated with tuberculin, when the serum was saturated with melanin the phagocytosis (using normal cells) was reduced from 22 granules of melanin to 5 granules, showing that a large amount of deopsonisation towards melanin had been brought about ; the opsonin was reduced to 1/4. Towards tubercle bacilli the phagocytosis (using normal cells) in the patient’s serum was 155 bacilli in 50 cells. After saturation with tubercle bacilli the phagocytosis was reduced to 46 bacilli. : How far did the saturation with tubercle bacilli reduce the phagocytosis of melanin? The phagocytosis of melanin was reduced from 22 granules to 2 after saturation of the serum with tubercle bacilli, 2.¢., the opsonin was reduced to 1/11. : How far did the saturation with melanin reduce the phagocytosis towards tubercle bacilli? The reduction here is from 155 to 113. This is consider- ably less than will appear from the following observation :— | In Observation 3.—The serum used was from a case of pulmonary. tuber- culosis, untreated with tuberculin. When the serum was saturated with melanin the phagocytosis (using normal cells) was reduced from 18 granules of melanin to 6 granules, showing that a large amount of deopsonisation towards melanin had been brought about. As in the preceding observation the opsonin was reduced to 1/3. Towards tubercle bacilli the phagocytosis (using normal cells) in the patient’s serum was 170 bacilli in 50 cells. After saturation with tubercle bacilli the phago- cytosis was reduced to 13 bacilli. How far did the saturation with tubercle bacilli reduce the phagocytosis of = oy 1907. ] | Observations upon Phagocytosis. 169 melanin? The phagocytosis of melanin was reduced from 18 granules to 5 granules after saturation of the serum with tubercle bacilli. As it was reduced after saturation with melanin from 18 to 6, the reduction is the same; 7.¢., the tuberculous serum is deopsonised towards melanin, equally by saturation with tubercle bacilli and with melanin. How far did the saturation with melanin reduce the phagocytosis towards tubercle bacilli? The reduction here is from 170 to 56. It will be noticed that the proportion here is very nearly the same as the reduction in phago- cytosis towards melanin after saturation with melanin; the phagocytosis is reduced to 1/3. Although the fall is only from 170 to 56 as compared with that from 170 to 13 (after saturation with tubercle bacilli), it is clear, nevertheless, that a large amount of the “tubercular opsonin” has been removed by the melanin; and to this extent the opsonin is not specific to the tubercle bacillus.* As already observed the fact than an immune serum can be deopsonised by means of melanin shows that melanin can be opsonised. The opsonisation and deopsonisation are probably in this case mechanical, the finely divided melanin, though inert, entangling or becoming invested with the still more finely particulate material of which the opsonin would thus appear to consist. Question 3.—Will heating an immune serum reduce the phagocytosis towards a suspension of melanin in the same way that it is known to do towards a suspension of a bacterium causing a disease ? ~The serum in the four following observations was heated at 60° C. for 10 minutes :— Cases: Erysipelas, acute pleurisy, streptococcus pyzemia, erysipelas. Summary and Conclusions from the Observations made to Test the Preceding Question. The observations show clearly that the phagocytosis of normal cells is greatly reduced towards melanin, in immune serum that has been heated, as it is towards bacteria. This. is a corollary of the observations recorded under Question 1, which * Muir and Martin (‘ Roy. Soc. Proc.,’ May, 1907) conclude that there is present in an immune serum a specific or immune (thermostable) opsonin, and in addition a normal (thermolabile) opsonin. The markedly increased phagocytosis of melanin in immune blood would, on such a view, imply that this was due solely to the increase of a “normal opsonin.” In the deopsonisation of an immune serum by melanin, the opsonin removed would be, on the same supposition, “ normal,” however much increased in amount. The authors cited show, indeed, that the thermolabile, “common,” or non-specific opsonin may be removed from the same immune serum by various different micro-organisms. 170 Messrs. 8. G. Shattock and L. 8. Dudgeon. [Dee. 21, show that in immune serum the phagocytosis of melanin may be largely | increased, 7.¢., that melanin is capable of being opsonised. So, when opsonin is removed by heating the immune serum, the phagocytosis of normal cells is notably reduced. The indices, commencing with that showing the least reduction, run :— 0°67, 0:23, 016, 0°16. Question 4.—Will an increased phagocytosis of melanin take place if melanin is presented to the washed leucocytes of an immune blood in normal serum, aS compared with normal washed leucocvtes in normal serum ? The cases selected comprised the following: acute erysipelas, chronic bone abscess, acute pneumonia, lymphangitis, streptococcal pyemia, acute cellulitis, acute peritonitis. Of some of these more than one example was used. Summary and Conclusions from the Observations made to test the Preceding Question. In the case where melanin is presented to normal washed cells and to the patient’s washed cells, both in normal serum, the phagocytosis is, in some, below par; in others, above ; in others, at par. The indices run :— 0:46.00", 21, AGolie Sale ali oe Gs ela me pono Question 5.—Will the phagocytosis be the same if melanin is presented to the washed cells of the patient, and to washed normal cells, in immune serum ? In the following experiments, the action of normal cells and patient's cells was tested in the patient’s serum. CasE.— Streptococcus pyogenes pyzmia. Index :7.,0-8-; USF CasE.— Acute pneumonia ; 5th day of disease ; male, zt. 23 ; temperature 103° F. Index tres.c. 3°3. In each of these observations, the work done by the patient's cells is greater than that done by normal cells, the indices being 1:7, 3°3. In the four following observations, the phagocytosis of leucocytes from different cases of Bacillus coli infection, in the same immune serum is set forth, as compared with that of normal cells in the same immune serum. The immune serum was from a ease of bone abscess in which the colon bacillus was present. The patient had been treated with anti-serum and also with vaccine. CasEe.—JB. coli infection of the urinary ‘tract; treated with anti-coli serum and with vaccine. | 1907. | Observations upon Phagocytosis. 171 CasE.—Bone abscess in which B. coli was present ; treated with anti-coli serum and with vaccine. ic > ae 12. Index... 41... OAR The indices in these observations run :— 0:9: eU Ls iult2 eae In the last of them the patient’s cells are doing considerably more work than normal cells in the same immune serum. In the three following observations, the cells from a standard case of acute pneumonia were tested against normal cells in immune sera from cases of different disease :— CasE.—Empyema. 1 vol. of normal washed cells, 1 ,, patient’s (empyema) serum, 1,4, melanin. 1 vol. of washed standard pneumonia cells 1 4, patient’s (empyema) serum, 1 ,,. melanin. Casr.— Urinary fever (B. cold infection). 1 vol. of normal washed cells, 1,4, patient’s (urinary fever) serum, 1 ,, melanin. 1 vol. of washed standard pneumonia cells, 1 ,, patient’s (urinary fever) serum, 1 eg emelamn. Index, 0.45 1°7 CasE.-—Acute pneumonia. 1 vol. of normal washed cells, 1 ,, patient’s (pneumonia) serum, 1, + melanin. j 1 vol. of washed standard pneumonia cells (from another patient), 1, patient’s (pneumonia) serum, ily a) cimelanin. In all these three observations, it will be seen that the washed pneumonia cells are doing more work than washed normal cells in the immune serum of three different patients, the most marked difference being that where pneumonia serum was selected. 172 Messrs. 8. G. Shattock and L. S. Dudgeon. [Dec. 21, The indices run :— Ley VaveunOsos In the following observations, the action of normal cells and patient’s cells, from a case of acute general peritonitis, was tested in the serum of another patient suffering from chronic pulmonary tuberculosis. 1 vol. of normal washed cells, 1 ,, tuberculous serum, 1 ,,. melanin. 1 vol. of patient’s washed cells (acute peritonitis), 1 ,, tuberculous serum, 1 ,, ~=+melanin. This observation, like the three that precede it, shows that the washed cells, from a case of acute infection, are more active that normal cells, in the serum of a patient suffering from another disease, the index being 1°4. In the two following observations, the action of normal washed cells is compared with the action of the patient’s washed cells, in the presence of melanin, in the patient’s serum, heated at 58° C. for 15 minutes. ANGE aceees 0°9. Casze.—Acute cellulitis of leg. index... 45 Qs In the two following observations, the action of normal washed cells is compared with the action of standard-pneumonia cells, in the heated serum of two other patients. CasE.—-Acute pneumonia. 1 vol. of normal washed cells, 1 ,, heated pneumonia serum, 1 ,, melanin. 1 vol. of standard pneumonia washed cells (z.e., from another case of pneumonia). 1 ,, heated pneumonia serum, 1, melanin. CasE.—Empyema. 1 vol. of normal washed cells, 1, heated empyema serum, 1,4, + melanin. 1 vol. of standard pneumonia washed cells, 1 ,, heated empyema serum, 15,088 rmaelanin: (Serum heated at 60° C. for 20 minutes.) index seers 1°6. 1907. | Observations upon Phagocytosis. 173 In the following observation, the action of normal washed cells is compared with the action of the same standard pneumonia cells as used in the pre- ceding observations, in normal heated serum. 1 vol. of normal washed cells, 1 ,, normal heated serum, 1,4, melanin. 1 vol. of standard pneumonia washed cells, ‘ 1,4, normal heated serum, I +, ‘melansa, Andie sss pans. te 2 In the five preceding observations, in the first four of which washed normal cells were tested against washed immune cells in heated immune serum, and in the fifth in normal heated serum, the indices run :— (o° 1 16 2 2. In two the immune cells are doing more work than normal cells in the heated immune serum, and the same is true in the case of the heated normal serum. ; In the following observations, colon bacilli were substituted for melanin, and the cells from a case of streptococcal puerperal fever, with multiple intramuscular and subcutaneous abscesses, were used in coli serum against normal cells in the same coli serum. The patient from whom the cells were taken died within 48 hours after the blood was taken. The blood was observed to be very watery when taken, and showed agglutination of the red cells. Case.— Urinary fever ; B. cold infection. - 1 vol. of normal washed cells, Ls - serum, 1 ,;,° -colon bacilli: 1 vol. of normal washed cells, ‘14, + patient’s serum (B. colz infection), 1 ,, colon bacilli. TndeX...0s sss OA This index shows that the patient’s serum is markedly active, i.c., it contains 2 good amount of an-opsonin which will prepare the colon bacillus for ingestion. | 174 Messrs. 8. G. Shattock and L. 8. Dudgeon. [Dee. 21, In the next place, the cells from a patient suffering from puerperal fever (streptococcal) were substituted for normal washed cells, thus :— 1 vol. of normal washed cells, 1,4, patient’s serum (colon infection), 1,4, colon bacilli. . 1 vol. of washed cells of patient suffering from puerperal fever, 1 ,, patient’s serum (colon infection), 1 ,, colon bacilli, This observation indicates that, although in colon serum, colon bacilli, when presented to normal washed cells, may give a good index (2°3), when presented in the same serum to the washed cells of another patient suffering from puerperal fever, the index fell to 0°5. This can only mean that the cells from the puerperal patient are incapable of carrying out the same amount of ingestion as normal cells in the same colon serum against the same colon bacilli, ze. the cells cannot make use of the phagocytic opportunity offered to them. Their decreased activity may be ascribed to damage sustained whilst in the circulating blood, or to the exhaustion brought about by the forced production of antibodies under the same circumstances. : | | It may be inferred from these data that a low index (as reached by the usual method) might be found still lower were the patzent’s cells used in the patient’s serum, in place of using normal cells in the patient’s serum against normal cells in normal serum. 1 vol. of normal washed cells, 1 ite Aes os serum, 1 4, ~~ colon bacilli. 1 vol. of patient’s washed cells (puerperal fever), 1,4, normal serum, 1 ,, ~colon bacilli. Indexte ns 0°56. This extension of the foregoing observation shows that the cells from the same case of puerperal fever do only half the work in normal serum against colon bacilli which normal cells do in the same normal serum against colon _bacilh. This is the same proportion which was obtained by presenting: (1) Normal cells, and (2) The same patient’s cells, in the patient’s serum, where the patient’s cells are doing only half the work of normal cells. In the following experiment, colon bacilli were substituted for melanin ; 1907. | Observations upon Phagocytosis. 175 and the washed cells, from the same case of puerperal fever as the preceding, were tested against normal cells in the patient’s serum. Casr.—Puerperal fever ; Streptococcus pyogenes ; death with pyzmia. 1 vol. of normal washed cells, Bea's, i serum, 1 ,, colon bacilli. 1 vol. of normal washed cells, 1 ,, patient’s serum (puerperal fever), 1 ,, colon bacilli (not from patient). fndex... 2.2... 0°8. 1 vol. of normal washed cells, 1 ,, patient’s serum (puerperal fever), 1 ,, colon bacilli (not from patient). 1 vol. of patient’s washed cells (puerperal fever), ere A serum, 1 ,, colon bacilli (not from patient). Index 22.2.0. 0°3. This observation shows that the patient’s cells (streptococcal infection) are less active in the patient’s serum, against colon bacilli, than are normal cells in the patient’s serum against colon bacili. The result corresponds closely with those which precede it, in which a colon serum was used in place of the above patient’s serum, but with the same puerperal patient’s cells. Summary and Conclusions from the foregoing Groups of Observations placed under Question 5. In the observations where melanin was presented to normal washed cells and to a patient’s washed cells, in the patient’s own serum, or in the serum of another patient suffering from another infective disease, the indices run :-— eo mele w se TAL 1p ay DAN 8:3 (Gb, In heated rmmune serum, the washed immune cells against normal washed cells give indices of :— Oo. en Oe a | In heated normal serum, the index, in the single observation made, was 2. In the majority of the cases, the patient’s cells take up more melanin, or more bacilli, than do normal cells in immune serum, whether the immune serum be that of the patient or that of another patient suffering from another infective disease ; the higher indices run :— | 2:4, 33, 6°5. When the patient’s cells are compared with normal cells in normal serum, the indices range from 0:46 to 2°9. 176 Messrs. 8. G. Shattock and L. 8. Dudgeon. [Dec. 21, The action of the patient’s cells would thus appear to be a factor which requires consideration if a full estimate of the patient’s hamophagocytic resistance is to be arrived at. Were the patient’s cells indifferent, normal cells would ingest to the same extent as the patient’s cells, in the patient’s serum. This is not the case. The patient’s cells may be less active, or they may be more active than the normal. The conclusion to which the observations lead is that the calculation of the opsonic value of the patient’s serum alone does not give a full estimate of the patient’s hemophagocytic resistance. It may be too high or too low, or, aS a coincidence, it may exactly represent it. In what may this greater activity of the patient’s cells consist? The greater activity might be ascribed to an elaboration and excretion of opsonin by the patient’s leucocytes in the patient’s serum, which brings about a still further increased preparation of melanin in the already opsonised serum. This would mean that the patient’s leucocytes, instead of playing only the secondary part of ingesting prepared bacteria, both prepare and then ingest. them. To gauge the curative capacity of the patient’s serum only, the method in commoner use is doubtless the correct one, for normal cells are used in the patient’s serum against normal cells in normal serum. But the question may be raised whether the action of the patient’s cells should be considered. For if their greater activity is due only to a further secretion of opsonin, in observa-- tions carried out in vitro, it may be urged that this exalted phagocytic index does not represent that of the circulating blood, but is too high. The patient's, cells are transferred to the patient’s serum in relatively abnormal numbers, and, during the 20 minutes’ incubation, they may be elaborating from the patient’s serum a further amount of opsonin which overcharges the serum. in the capillary tube and brings about a fictitious degree of phagocytosis. If any substance continues to be produced in the serum, 7 vitro, it must: be an opsonin. It cannot be a substance that would stimulate the cells to ereater activity: we cannot suppose that the cells produce and shed into. the serum a “stimulin” in order to stimulate themselves! The patient’s cells produce a further amount of opsonin, or they are simply “ more active” than are normal cells. One must remember that the process of thrice washing the cells in salt. solution does not wash out the leucocyte, it only washes the surface of it. One difficulty that arises in testing the alternatives just stated will be obvious :—During the process of incubation, in vitro, two phenomena may be proceeding simultaneously: the cells may be simply ingesting, or they 1907. | Observations upon Phagocytosis. — 177 may be producing opsonin and ingesting.’ If the’ phagocytosis increases upon allowing further time, it has to be determined whether this is due to. further opsonification, or simply to the fact that the cells are allowed a longer time in which to work. And even the termination of further phago- cytosis, after an extreme time-allowance, might mean equally either a termination of further opsonin production, or that the cells had taken up all for which they had capacity. Our observations bring out that the immune or active cells, as compared with normal cells, although they usually do more work in both immune and in normal serum, do more in the immune than in the normal. This points to some interaction between the patient’s cells and the patient’s serum; it indicates that there is something in the patient’s serum which is in excess, or which is not present, in normal serum. If we agree to call this “ something ” a stimulin, its presence will not, per se, account for the difference. For such a substance would equally stimulate the normal cells to do the same amount of work as the immune cells in the immune serum—which is not the case. In heated immune serum, again, the immune cell does more work than the normal: so that, whether the substance left in the serum after heating is specifically bacteriotropic and unable to affect the melanin or cytotropic (stimulin), it is clear that the immune cells behave differently from the ~ normal. We are led to hold, therefore, that the immune cell, as a cell, is in many cases more active, more irritable, or more sensitive, than the normal, as it may be less active than the normal cell, or active to the same degree. The immune cell has acquired a heightened activity in the body in response to the increased function demanded of it to cope with the infective process ; its full reserve power has been called out. The fact that more phagocytosis occurs in immune serum with immune cells than with normal cells indicates. that, in the living body, from a similar interaction, the patient’s cells are likewise doing more work than would normal cells, if we imagined, ey., the whole of the patient’s leucocytes suddenly replaced in his own plasma by normal cells. Question 6.—Is opsonin produced, in vitro, by the patient’s cells in the patient’s serum ? The only thing that would vitiate the foregoing conclusion would be evidence of an additional formation of opsonin, i vitro, during the 20 minutes in which the capillary tube containing the immune serum, the immune cells, and the melanin, or bacilli, was being incubated. It is conceivable that the immune serum contains a precursory substance —an opsinogen—which is converted into opsonin by a ferment produced by the cells, and that the increased phagocytosis of immune cells in immune 178 Messrs. 8. G. Shattock and L. 8. Dudgeon. [Dec. 21, serum is due to their greater secreting activity, ve, to their increased production of the converting substance; and that when melanin or bacteria are presénted in vitro, this interaction is set: going, or, rather, restarted in the drawn. blood. Observation. CasE.—Empyema. 1 vol. of blood, ; 1 ,, citrated salt suspension of melanin. Four tubes were prepared, and incubated for different periods, with the following results :— LS miMUbes |. Ate. cease oad 9 granules of melanin in 50 cells loti eee eke chee 21 3 a OD MOUTS dines. eee skes neat eee 36 i - AA Wee st icc caesecacea tener 76 * bs In such an observation it is impossible to determine whether the pro- gression is due to further formation of opsonin or to the increased time allowed for the continuance of phagocytosis. When bacteria are used in place of melanin, this progression may be more highly pronounced, as is shown in the three following observations :— CaszE.—Acute cellulitis ; temperature, 102° F. 1 vol. of patient’s blood, 1 ,, citrated salt suspension of colon bacilli. Incubated 20 minutes ............ 50 cells contained 126 bacilli. 1 vol. of patient’s blood, 1 ,, citrated salt suspension of colon bacilli. Incubated 30 minutes ............ 50 cells contained 195 bacilli. CasE.— Urinary infection due to colon bacillus ; untreated with anti-serum or vaccine. 1 vol. of patient's blood, 1 ,, citrated salt suspension of colon bacilli. Incubated 20 minutes ............ 50 cells contained 115 bacilli. 1 vol. of patient’s blood, 1 ,, citrated salt suspension of colon bacilli. Incubated 30 minutes ............ 50 cells contained 165 bacilli. Casre.— Urinary infection due to colon bacillus ; treated with anti-serum and vaccine. 1 vol. of patient’s blood, 1 ,, citrated salt suspension of colon bacilli. Incubated 20 minutes ............ 50 cells contained 116 bacilli. 1 vol. of patient’s serum, 1 ,, citrated salt suspension of colon bacilli. ‘Theabaved oO minutes 2 joraeseee 50 cells contained 196 bacilli. 1907.) Observations wpon Phagocytosis. 179 It is worth while to notice that such a progression does not necessarily occur under the same conditions of experiment. Observation. Case.—Suppuration of thumb. 1 vol. of blood, 1 ,, citrated salt suspension of melanin. Four tubes were prepared, and incubated for different periods, with the following results :— POPMUIMEES ge ccoessecececeses 31 granules of melanin in 50 cells. UMMM 6occceapesudicnse: 28 3 ane 11 010.5 Se eR 29 is 9 PAN OURS J coho eee noes 31 9 Here it may be inferred that the immune cells were of lowered activity. This we know to be so in some cases, where they do less work in their own immune serum than do normal cells. Observation. Normal Blood—In the case of normal blood, a slight progression in phagocytosis may be observed :— 1 vol. of normal blood, 1 ,, citrated salt suspension of melanin. Four tubes were prepared, and incubated for different periods, with the following results :— 1B WAIN WEES! 2 ists dei ee ecetes 18 granules of melanin in 50 cells. 30 99 @eesesseeseseeosece 16 99 99 J], Voor 1 n,n ee ee 26 43 os FEN OUTS RS es case we acenagen 31 Here, again, the progression might be due to a further formation of opsonin, or to the further time allowed for the process of ingestion. The progressive phagocytosis in normal blood is more forcibly shown in the following observation, where colon bacilli are used in place of melanin :— 1 vol. of normal blood, 1 ,, citrated salt suspension of colon bacilli. Incubated 20 minutes ............ 50 cells contained 151 bacilli. 1 vol. of normal blood, 1 ,, citrated salt suspension of colon bacilli. Incubated 30 minutes .........+.. 50 cells contained 399 bacilli. In considering whether the index obtained by using immune cells in immune serum, 7” vitro, correctly represents the phagocytic resistance of the patient’s VOL. LXXX.—B. P 180 Messrs. 8. G. Shattock and L. 8. Dudgeon. [Dec. 21, blood, z.e., of his serum and his cells, as distinguished from the resistance brought about by his serum alone, the question with which we are essentially concerned is whether a further formation of opsonin occurs in vitro, or whether the work done by the cells is done by means of the opsonin which was present in the blood when the latter was drawn. We endeavoured to determine this by means of the following experi- ments :— One volume of washed immune cells was thoroughly mixed with one volume of immune serum, using larger volumes than usual. The mixture was then drawn up into three capillary tubes of the usual calibre, and incubated for 20 minutes, 30 minutes, and 1 hour. At the end of these intervals, an equal volume of citrated salt suspension of lving colon bacilli was added to the contents of each capillary tube, mixed, and each tube then incubated for 20 minutes. By this means, a full supply of active immune cells was allowed to act in immune serum for different periods (without the presence of bacilli); the phagocytosis was then tested in each case after the same time-allowance, viz., 20 minutes. Case.—Colon infection of urinary tract ; treated with anti-serum and vaccine. 1 vol. of immune washed cells and immune serum, incubated 20 minutes, 1 ,, citrated salt suspension of colon bacilli. 50 cells contained 73 bacilli after 20 minutes’ incubation. 1 vol. of immune washed cells and immune serum, incubated 30 minutes, 1 ,, citrated suspension of colon bacilli. 50 cells contained 74 bacilli after 20 minutes’ incubation. 1 vol. of immune washed cells and immune serum, incubated 1 hour, 1 ,, citrated suspension of colon bacilli. 50 cells contained 81 bacilli, after 20 minutes’ incubation. Summary and Conclusions from the foregoing Group of Observations. It will be evident from the concluding observation that no difference in phagocytosis is brought about within 20 minutes, in different samples of the same immune serum, mixed with the immune cells from the same patient, when bacilli are presented, after periods of incubation of 20 minutes, 30 minutes, and 1 hour; the numbers of bacilli ingested by 50 cells, being :— 13, (WAS This crucial experiment proves that the marked difference in the phagocytosis observed when bacilli are added to immune blood, and incubated for different periods, must be attributed, not to further opsonifica- 1907. | Observations upon Phagocytosis. 181 tion of the serum, but to the increased time allowed for the cells in which to ingest. The practical conclusion we draw is, that in order to estimate the full phagocytic resistance of the patient’s blood, the cells should be taken into account as well as the serum. The method of making such an estimate is considerably simpler and much more rapid than the estimation of the opsonic value of the serum only, which entails washing normal cells and collecting blood in order to obtain both normal serum and the serum of: the patient. It is merely necessary to draw up 1 volume of the patient’s blood and 1 volume of citrated salt suspension of the micro-organism to be used, into the capillary tube, to mix, incubate for 20 minutes, spread the film, and compare the phagocytosis with that of a volume of normal blood similarly treated,—a slight modification of the technique practised by Leishman. The washed corpuscles, moreover, sometimes tend to cohere in clumps. The central cells of such clusters would fail to reach bacilli presented in the fluid. This is probably one explanation of the discrepancies obtained in the numeration of ingested bacilli, even in neighbouring fields of the same slide. It has been pointed out elsewhere in this communication that although the action of the immune phagocyte is usually higher than that of the normal cell, yet it may be lower, or it may be equal to it. The cells vary in value, like the serum, and the only method of arriving at a correct estimate of the patient's hemophagocytre resistance is to allow the immune cells to work in the ammune serwm. | By the method more commonly employed in this country, too low an index is obtained if the patient’s cells are acting above the normal level, and too high an index if they are acting below it. 182 A Contribution to the Study of the Mechanism of Respiration, with Especial Reference to the Action of the Vertebral Column and Diaphragm. By J. F. Hatts Datty, M.A., M.D. Cantab. (Communicated by Professor Sir T. Clifford Allbutt, K.C.B., F.R.S. Received January 24,—Read February 6, 1908.) In studying the alterations which occur in the shape, size, and position of the internal organs as the result of their functional activity, previous observers have worked at a disadvantage. During the past nine years X-rays methods, though indicating an advance in our knowledge of abdominal and thoracic visceral movements, have not been of absolute utility, since the rays, being divergent, produce magnification of the shadow of the object. Hence, exact measurements have been unattainable. In the present investigation the chief results have been obtained by means of Groedel’s orthodiagraph, which Dr. Hugh Walsham and myself have been the first, to our knowledge, to work with in this country, and of which we have already published a detailed description.* By means of this instrument it is possible, with almost mathematical accuracy, to measure motionless objects which he in a plane parallel with the vertical transverse plane of the body, and to measure moving objects with greater approach to exactitude than can be obtained in any other manner. SUMMARY OF THE RESULTS OF THE INVESTIGATION. I. Results obtained by Orthodiagraphic Measurement of Changes in the Trunk which occur during Respiration. One hundred healthy subjects, of ages varying between 15 and 35, were examined, the average measurements being recorded as follows :— (1) The neck is shortened 10 mm., and widened, on the right side 9 mm., on the left side 7 mm. (2) The shoulders are raised on the right side to a greater extent than on the left, the average on the right being 16 mm., that on the left 14 mm. (3) The presternum moves 30 mm. in an upward, and 14 mm. in a forward, diameter. ) (4) The clavicles execute a combined upward, forward, and outward * ‘Brit. Med. Journ.,’ September 14, 1907, p. 651. On the Study of the Mechamsm of Resprraton, etc. 188 movement, the vertical range of their inner ends on the right side being 28 mm., on the left side 27 mm.; of their outer ends, on the right side 21 mm., on the left side 16 mm. The divergence from the median line is, on the right side 7 mm., on the left side 6 mm. (5) The meso-metasternal articulation either may remain in the same horizontal plane, or may rise as much as 46 mm. The average ascent is 28 mm. (6) Widening of the infra-costal angle occurs, the interval between the costal margins, measured on each side at a level of 30 mm. below the meso- metasternal articulation, being increased by 26 mm. (7) The trunk is widened at the level of the meso-metasternal plane 9 mm. on the right side, and 8 mm. on the left, and midway hetween the meso-metasternal plane and lowest point of the costal margin 9 mm. on the right side, and 11 mm. on the left. (8) The umbilicus is retracted and drawn upwards in deep respiration for a distance of 13 mm., on account of the active recession of the abdominal wall produced by the contraction of the abdominal muscles, which, in this phase of respiration, act as antagonists of the spinal muscles. The upward displacement of the umbilicus is usually to the right, but may be median or to the left, the lateral deviation being 7 mm. (9) The heart and pericardium together undergo important changes in size and position as a result of the respiratory movements, being lengthened and narrowed in inspiration, shortened and widened during expiration. (10) The pericardium, at the level of its attachment to the central tendon in the adult, measures 80 mm. in antero-posterior diameter. II. The Movement of the Vertebral Column in Respiration. I can find no reference to this movement in eight of the latest and best known text-books of physiology. That this movement is actual and of mechanical advantage in breathing can be verified by visual and ortho- diagraphic examination. In the latter the subject is rotated through an angle of 30° to 45° into the “lateral oblique” position. The shadow of the vertebral column is seen clearly separated from that of the pericardium and great vessels by a transradiant triangle, the base of which is formed by the upper surface of the diaphragm. On inspiration the posterior wall of this triangle, formed by the vertebral column, recedes, to a greater extent below than above, and so opens out the interval from before backwards. With subsequent expiration the spine advances. Hiplanation of the Spinal Respiratory Movement.—A general rectification of the curve of the thoracic spine takes place. In eight orthodiagraphic 184 Dr. J. F. Halls Dally.. On-the. [Jan. 24 examinations of healthy adults, in deep inspiration, the average antero- posterior range of movement was as follows :— | At upper aperture of thorax opposite 1st thoracic vertebra, 6 mm. | Midway between upper aperture of thorax and level of diaphragm opposite 5th thoracic vertebra, 7°5 mm. At level of posterior part of diaphragm opposite 10th thoracic vertebra, 9 mm. These measurements show that on the average the spinal column is most displaced towards the lower part of the thoracic curvature, and that rectifica- tion lessens from below upwards. Individual differences, however, are not infrequent. ‘This straightening, which occurs especially in that segment of the spine which articulates with the 6th, 7th, 8th, and 9th ribs, happens as a consequence of the backward push of the sternal ribs when the sternum is raised upwards and forwards by contraction of the cervical and thoracic muscles, thus bringing a larger costal are into the place previously occupied by a smaller one, and so increasing the antero-posterior diameter of the thorax. Upon the ribs assuming a position of less obliquity, the spinal column, being far more limited in its possible range of movement than the sternum, on account of its multiple attachments can only execute a fraction of the sternal movement. Towards the end of inspiration, as the movement of the sternum reaches its dynamic limit, the remainder of the force of the respiratory cycle is spent upon the spine, which accordingly, during the latter half of inspiration, shows progressive mobility. In the lateral oblique position the apparent movement of tbe vertebral column is 6 to 9 mm.; the real movement may be readily calculated by means of a mathematical formula. Importance of the Spinal Movement.—The antero-posterior enlargement of the thoracic cavity produced by simultaneous extension of the thoracic vertebree in deep breathing has an important influence upon the aeration of the apices of the lungs, the forward and upward movement of the thorax, together with backward movement of the spine, being of far greater value than the lateral movement in promoting free access of air. IIL. Anatomical Dissimilarity of the Two Halves of the Diaphragm. ’ Anatomically the two halves of the diaphragm differ in size and shape, the right half being the larger and the more powerful, the reason being that this half has to overcome the resistance of the mass of the liver, whereas the stomach is much more easily compressed by the left half. Functionally, although the two halves of the diaphragm are of unequal size and strength, yet, owing to the difference in resistance on the two sides, their range of -1908.] Study of the Mechanism of Respiration, ete. 185 movement is but little dissimilar, the difference, if any, being usually in favour of the right half. IV. The Means by which the Diaphragm is supported. In another paper I have dealt with the superior, or thoracic, and the inferior, or abdominal, supports of the diaphragm. | V. Level of the Diaphragm. (1) After Death—tThe cadaveric position of the diaphragm indicates only the position of expiration. From the results of an examination of 80 dissecting-room and post-mortem-room cases, I find that the average highest point of the dome of the diaphragm is situate, on the right side at the level of the upper border of the 5th rib in the mid-clavicular line; on the left side, in the mid-clavicular line at the lower border of the 5th rib. This corresponds with the results of orthodiagraphic measurement in bodies of healthy persons who have met with sudden death. (2) During Life-—Owing to the great variability in position of the land- marks usually adopted in measuring the range of the diaphragm, I have taken the meso-metasternal articulation, 2.¢., junction between gladiolus and ensiform, as the basis from which to measure, and a line drawn horizontally through this point—the meso-metasternal plane—as the plane to which the level of the rise and fall of the diaphragm may be referred, and all ortho- diagraphic measurements of the position of the domes and central tendon have been taken in millimetres above and below this plane. VI. Absolute Range of Movement of the Diaphragm. The absolute range of movement of the diaphragm between deep inspira- tion and expiration in the adult male is, on the right side 34 mm., on the left side 32mm. The range in adult females amounts to 27 mm. on the right side, and 25 mm. on the left side, making the total average range 30 mm. on the right side, and 28 mm. on the left. The fact that these figures are only about half as great as those previously ‘given* is due to the greater accuracy of orthodiagraphic measurement. In quiet respiration the total average movement is 12°5 mm. on the right side, and 12 mm. on the left. Hence this movement is approximately equal on the two sides, whilst in deep breathing, the excursion is, for most people, slightly greater on the right side.than on the left. In diagnosis, the movement in deep respiration is the important one to observe. * The ‘ Lancet,’ June 27, 1903, p. 1802. 186 Dr. J. F. Halls Dally. On the [Jan. 24, VII. The Costo-phrenie Plewral Reflexion. According to the surfaces with which it is in contact the parietal layer of the pleura conveniently may be divided into costal, diaphragmatic, and mediastinal portions. The foldings of the pleura constitute three marginal grooves or recesses :— 1. Pericardio-phrenic groove. 2. Pericardio-sternal pleural reflexion. 3. Costo-phrenic pleural reflexion. 1. The pericardio-phrenie groove is a shallow recess formed at the junction of the pericardium with the diaphragm, and lodges the plice adipose. 2. The perrcardio-sternal reflecion forms the anterior marginal pleural recess. 3. The costo-phrenie reflexion must be considered in greater detail, since it facilitates to such a marked extent the action of the diaphragm. It is formed by the meeting of the costal and diaphragmatic portions of the pleura, and, beginning at the lower border of the 6th rib close to the termination of the gladiolus, it passes downwards and outwards, reaching the lateral aspect of the spinal column at the lower border of the twelfth thoracic vertebra. Practically the limit to which the lung descends in ordinary inspiration marks its upper boundary. Here, owing to friction between the pleural surfaces, a line of demarcation, best seen in the recent state, indistinct in the young child, but increasingly definite with advancing age, gradually forms. If evidences of pleurisy are present, not infrequently a ridge of organised lymph, concave on its upper surface, delimits the lower lung margin, forming a groove into which the lung fits. The lower border of the reflexion is wavy or festooned, the festoons being in relation with the intercostal spaces. In a series of 20 male and female subjects, of ages varying from 18 to 56 years, who had died from causes not involving the lungs, the average depth of the costo-phrenic reflexion, measured in the mid-axillary line from lower margin of lung to lowest limit of pleural cavity, on the right side was found to be 8°62 cm., and on the left side 834 cm. The greatest interval in the series was.11 cm. on the right side (corresponding to 9 cm. on the left), and the smallest, 5 em. on the left side (corresponding to 6 cm. on the right). In a female infant aged three months the depth of the reflexion on the right was 1°5 cm., and on the left 1:4 cm. Function of the Costo-phrenie Plewral Reflexion.—The two serous surfaces constituting the reflexion remain in apposition for a distance varying with inspiration and expiration, and are not separated until the wedge-shaped 7? 1908. ] Study of the Mechanism of Respiration, ete. L87 lower lung margin glides downwards in inspiration and insinuates itself between them. As the lung recedes the surfaces again come together. The diaphragmatic pleura, owing to its elasticity, accommodates itself to con- traction of the diaphragmatic circumferential muscle-fibres, and its smooth surface allows it to glide easily over the apposed costal layer, which is fixed in such wise that it cannot be displaced. Owing to negative intra-pleural suction and positive intra-abdominal pressure, together with molecular cohesion of the lubricated pleural surfaces, the pleural union thus formed is of sufficient strength to bind the diaphragm during its action closely to the chest-wall, and, moreover, is of mechanical advantage to the diaphragm, since in function it resembles the band or loop through which a muscle acts in order to change the direction of its line of force. VOL, IXxx.—n, Q ‘ " 1908. | Study of the Mechamsm of Respiration, ete. L87 lower lung margin glides downwards in inspiration and insinuates itself between them. As the lung recedes the surfaces again come together. The diaphragmatic pleura, owing to its elasticity, accommodates itself to con- traction of the diaphragmatic circumferential muscle-fibres, and its smooth surface allows it to glide easily over the apposed costal layer, which is fixed in such wise that it cannot be displaced. Owing to negative intra-pleural suction and positive intra-abdominal pressure, together with molecular cohesion of the lubricated pleural surfaces, the pleural union thus formed is of sufficient strength to bind the diaphragm during its action closely to the chest-wall, and, moreover, is of mechanical advantage to the diaphragm, since: in function it resembles the band or loop through which a muscle acts in: order to change the direction of its line of force, VOL. LXXX.—B. Q 188 The Influence of Temperature on Phagocytosis. By J. C. G. Lepincuam, M.B., B.Sc., M.A., Assistant Bacteriologist, Lister Institute, London. (Communicated by Dr. C. J. Martin, F.R.S. Received November 18, 1907,— Read February 27, 1908.) Recent work has shown that the phagocytosis of micro-organisms is a complex phenomenon, involving at least two factors, viz.: (1) the sensitisa- tion of the micro-organism by the opsonin of the blood serum, and (2) the phagocytic act of the polymorphonuclear leucocyte. The latter function has been known for many years to be markedly influenced by temperature. It appeared desirable, however, to ascertain whether, by the modern quantita- tive methods employed in opsonic work, some definite relationship could be found to subsist between degree of temperature and degree of phagocytosis. In the first set of experiments no attempt was made to separate the respec- tive functions of sensitisation, and amceboid activity of the leucocyte. Both actions were allowed to proceed simultaneously. Consequently, in Series I the leucocytes were put in contact with fresh serum and micro-organisms (staphylococci), and incubated together at different temperatures for the same period of time. Thereafter the number of cocci taken up per leucocyte was calculated on stained films in the usual way. The Staphylococcus aureus employed was an old laboratory strain. Series _—Experimment L. , Cocci per leucocyte with incubation periods. ‘Temperature. = ahs 15 mins. 30 mins 45 mins 43° C 15 °4 30 30 + 37 L3e2Z 23 25 18 Sali 3°3 1229 It will be seen that at a temperature of 18° C. the degree of phagocytosis for 30 minutes is not appreciably greater than that for 15 minutes. There is, in fact, a prolonged latent period. This was further brought out in the next experiment, where the temperature remained constant (18° C.) while the incubation periods varied. Experiment IT. Temperature. Incubation period. Cocci per leucocyte. 18° C. 15 mins. Ze9 18 30 3°8 18 45 2 18 1 hr, ONT, 18 1 hr. 15 mins. 12 °7 18 1 hr. 30 mins. 19 “2 The Influence of Temperature on Phagocytosis. 189 For 37° ©. this latent period is much shorter. To ascertain this, five- minute intervals were taken as in the following experiment :— Experiment III. Temperature. Incubation period. Cocci per leucocyte. 37° C. 5 mins. 2°0 37 ;, 20 W 37 15 4°3 37 20 4°0 37 25 5 °6 37 30 4 °9 - 37 35 5°0 37 40 6°5 37 45 8 °6 37 50 12°0 The five following experiments showing the influence of temperature on phagocytosis were carried out in a similar way to Experiment I. Coccal emulsions of varying strengths were purposely employed throughout the series :— Experiment IV. Experiment V. Experiment VI. Ped, = 15 mins. J.T. = 15 mins. ft. = 30 mins, Temperature. Cocci. Temperature. Cocci. Temperature. Cocci. 41° C, 17-1 48° ©. 15°7 42° CO. 81 37 15°3 37 12°3 37 9:0 17 4°2 31°5 10 °9 30 8°5 25 °7 4°0 25 6 °3 20 3 °2 Lire 3°7 13 0°8 6°5 ry Experiment VII. Experiment VIII. iL = 33 mins. poe de ie 30 mms: Temperature. Cocci. Temperature. Cocci. 43° C. 28 °0 41° C, 21 °4 37 20 °7 36 °6 11°8 31°5 22 °3 30 8 °2 25°7 18 *4 Ve 4°8 20 10 °9 13 3) 45 6°5 0°7 0 ? From the results quoted above, it will be apparent that the degree of phagocytosis rises, though in a somewhat irregular fashion, as the tempera- ture rises, but the results are not sufficiently constant to enable one to determine with any accuracy such a value for the temperature-coefficient as would allow one to calculate the amount of phagocytosis at a temperature A when the amount at a temperature B is known. The strength of the * I. T. = Incubation Time. . Ohi 190 Mr. J. C. G. Ledingham. [Nov. 18, coccal emulsion has probably.a great bearing on the rate of fall, especially during the upper temperature ranges (vide Experiment VI). A fairly constant ratio, however (4—5:1), is found to exist between the degree of phagocytosis at 43° C. and that at 18° C. . We have now to determine whether this increased phagocytosis following rise of temperature is due simply to increased activity on the part of the leucocyte. In short, what effect has temperature on the rate of combination of the serum with the coccus ? To settle this point, a second series of experiments was performed. The coccl were sensitised by contact with serum for a fixed time at a certain temperature. Thereafter leucocytes were added and incubation performed at different temperatures. By this method more or less completely sensitised coccl were exposed to the action of the leucocytes. Series I. Experiment [—A moderately thick emulsion of cocci in normal saline was added in equal volume to fresh serum, and kept in contact therewith at a temperature of 37° C. for 20 minutes in a small test-tube. One volume of leucocytes was then incorporated with one volume of the shaken up and partially centrifugalised serum-coccal mixture and incubated at different temperatures for 20 minutes. The result was as follows :— Temperature. Cocci per leucocyte. 37° C. 4, *4; 30 4: °4, ily 4 °3 (The low counts obtained here show that the centrifugalisation of the serum-coccal mixture had been carried a little too far.) Experiment IJ.—Technique similar Experiment I1I.—Combination at to above. 37. C. tor 2 0mmiie: | 1.2, = 30 aman: 1) = biamane: Temperature. Cocci per leucocyte. Temperature. Cocci. A1° C, 13 °8 37° C. 12 °5 37 8 atef 10°3 30 12-2 ef, 13 °5 (Again the fall of temperature had no effect on ’ the amount of phagocytosis.) Experiment IV.—Combination at 37° C. for 20 mins. Lo dibemmist Temperature. Cocci per leucocyte. 37° C, 5°7 28 5°2 18 5°9 3 °6 1907.| The Influence of Temperature on Phagocytosis. 191 Experiment V.—In this experiment the sensitisation was performed at 18° C. for 20 minutes, and the same coccal emulsion was employed as in Experiment IV, so that the figures obtained are quite comparable. Temperature. Cocci per leucocyte. 37° C. 6-2 28 4, *2 18 ; 2°6 7 0°8 Experiment VI.—A portion of the coccal emulsion was combined with serum at 37° C. for 20 minutes, and an equal portion combined with serum at O° C. for the same time. Leucocytes were then added, and the results of phagocytosis at different temperatures compared. Combination at 37° C. Combination at 0° C. Temperature. Incubation time. Cocci.' Temperature. Incubation time. Cocci. 37° C. 15 mins. 121 Si ©: 15 mins. 8°8 20 15 8°6 20 15 0°4 The very great difference between the amounts of phagocytosis at 37° C. and 20° C. after combination at 0° C. offers a marked contrast to what obtains under the same conditions after combination at 37° C. The employ- ment of very thin coccal emulsions, however, and the prolongation of the period of combination with the serum, have the effect of equalising the amounts of phagocytosis at high and low temperatures even after low- temperature combination. Throughout the experiments which illustrate this point, thin coccal emulsions were combined with serum for long periods, and the mixtures required no centrifugalising before use. Experiment VII.—Combination at 37° C. for 45 mins. LEE = loys: Temperature. Cocci per leucocyte. 3. 9°6 18 8 ‘0 Experiment VIII.—Combination at 37° C. for 2 hrs. 45 mins. loTy= 15 mime Temperature. Cocci per leucocyte. 37° C. 15 ‘0 18 14 °5 Two further experiments similar to Experiment VIII gave ratios of 12:°2:12°6 and 14:1:13°3 for the amount of phagocytosis at 37° C. to that at 18° C. | 192 Mr. J. C. G. Ledingham. [Nov. 18, Experiment IX.—Comparison of the Effects of Combination at 37° C. and at 18° C. Time of combination in each case, 1 hr. 40 mins. I. T. = 15 mins. Combination at 37° C. Combination at 18° C. Temperature. Cocci. Temperature. Cocci. 37° C. 8 °6 37°C. 4-9 18 8°5 18 6-1 It will be seen that after combination at 18° C. the amount of phagocy- tosis at 37° C. is even slightly exceeded by that at 18° C., though both fall very short of the corresponding figures obtained after combination at one C, Experiment X. Technique similar to that of Experiment IX. Temperatures of combination, 37° and 7° C. Time of combination, 3 hrs. 15 mins. Time of incubation, 20 mins. Combination at 37° C. Combination at 7° C. Temperature. Cocci. Temperature. Cocci. 3%, G3 : 23 °0 Sis 6°6 21 28°6 21 5°8 18 27 8 18 6°8 10 12:0 10 4:0 In each series, between the temperature range 37° C. to 18° C., the phagocytic values are practically identical, but the great differences between the absolute amounts in the two series afford a striking illustration of the superiority of combination at 37° C. Experiment XI.—This experiment was designed to show the effect of varying the combination times at the two temperatures 37° C. and 18°C. Samples for phagocytic purposes were removed from the serum-coccal mixtures every half hour and exposed to the action of the leucocytes. Combination at 37° C. Combination at 18° C. Temperature. Combination time. Cocci. Temperature. Combination time. Cocci. BY faa OF 30 mins. 50 37° C. 30 mins. 4, °7 18 30 mins. 4:1 18 30 mins. 5°4 37 1 hr. 8 °4 ay 1 hr, BS if 18 1 hr. 9°2 18 1 hr. 7:3 37 1 hr. 30 mins. 8°9 SH 1 hr. 30 mins. 7-4 18 1 hr. 30 mins. 8 °*4 18 1 hr. 30 mins. 7°0 37 2 hrs. 10°7 Si 2 hrs. 8°3 18 2 hrs. 10 °5 18 2 hrs. i2 The pairs of phagocytic values in the various time intervals approximate very closely in the two series, showing that, under the conditions indicated 1907.] The Influence of Temperature on Phagocytosis. 193 the degree of phagocytosis is independent of the temperature within the range 37° C—18° C. Further, when we compare the figures in the two series, those of the right hand series (combination at 18° C.) decidedly lag behind those of the left hand series (combination at 37° C.). The difference, however, is not nearly so marked as in a previous experiment, where the effects of combination’ at 37° C. and 7° C. were compared. One point is somewhat difficult to explain. In the case of combination at 18° C. it was. found in an earlier experiment’ where thick emulsions were’ employed that the amount of phagocytosis at 18° C. fell considerably below that at 37° C. In the present experiment, where thin emulsions were employed for combin- ing purposes, these values are equalised. The reason probably is that, although the rate of combination at 18° C. is slower than that at 37° C., prolonged contact of serum with cocci at the lower temperature (18° C.) effects a maximum absorption of opsonin by the cocci for that temperature, and, consequently, when phagocytosis takes place, the influence of temperature within the range 37° C.—18° C. is reduced toa minimum, just as in the case of combination at 37° C. Ina paper by Bulloch and Atkin (1905) it is stated that the combination of the opsonin with the micro-organism takes place as readily at 0° C. as at 37° C. If this were so, it would be difficult to explain many of the experimental results detailed above. Dean (1905), on the contrary, observed that combination at 0° C. was much slower than at 37° C. The problem was again attacked in the following way :— Experiment XIJ.— Emulsions of dead tubercle bacilli were incorporated with equal volumes of serum at the temperatures of 37° C. and 0° C. for a period of 30 minutes. The mixtures were then completely centrifugalised and the opsonic contents of the supernatant fluids towards fresh tubercle bacilli compared, fresh normal serum, in corresponding dilution, being employed a control. | Bacilli per leucocyte. Supernatant fluid after combination at 37° C. ......... 3°4 . Me 5) ig, Oe 6-4 kinesin serum (diluted). 0..fsis tase cis ag ae ke ds 6°2 Experiment XIII. Here the period of combination was prolonged for 11 hours. Bacilli per leucocyte. Supernatant fluid after combination at 37°.C. ......... 2°5 ” » ” 0° C. Deororteerelene 6°7 PRPS eS CMU CAMUGCH) .....c erence... ce 0-1 F. together with about 0°2 gramme of a mixture of sterilised magnesium and calcium carbonate. The solutions were in Erlenmeyer flasks, and had pre- viously been sterilised in the usual way; they were then incubated for at least a month at a temperature of 30°, the contents being gently shaken two or three times during that period. At the end an examination of the clear liquid was made for nitrates with diphenylamine, for nitrites with meta- phenylene-diamine, for ammonia with Nessler’s reagent. The soil samples were prepared as follows :— In four series holes were dug in the field and sterile brass cavenl 1 inch in diameter were forced into the sides, after breaking away some soil to expose a clean surface. The tubes were carried to the laboratory, the contents forced out into dishes and partly dried over strong sulphuric acid, suitable precautions being taken to avoid external contamination. The | partly dried soil was then roughly powdered and stones removed by passing through a 1 mm. sieve, sieve and mortar being sterilised each time. By a suitable spoon a quantity found by trial to be approximately 0:2 gramme * Since the above was written the authors have seen reason to suppose that some of the carbon dioxide, evolved on treating soils of this character with hydrochloric acid, comes from the organic matter. Several other processes agree to make the calcium carbonate in the surface layer amount only to 0°04 per cent.—Wote added March 19, 1908. 1907. | Nitrification in Acid Soils. 205 ‘was introduced into the culture solution. For the last two series the sample o ' . ’ > SS o> a of soil was extracted with an auger 2 inches in diameter having a slot in the side; at the selected depth in the slot a clean surface of soil was exposed and a small sample, approximately 0°5 gramme, was picked out with a sterile spatula and introduced into the culture flask, the operation being carried out in the field. Number of Times reacting after Four Weeks’ Incubation. Unlimed portion. Limed portion. Depth. — ~ oo ares re cm, : No nitrate , o nitrate Nitrate. ee Ce rary Nitrate. NE BFE. Plot 3.—Unmanured. 10—15 7 Uf ih 0 30 2 3 1 0 45 0) 1 75 0 1 105 0 1 135 0 1 Plot 4-2.—Ammonium Salts and Superphosphate only. 10—15 2 vi 1 2 20—30 5 1 1 1 45 I 0 0) 1). 75 0 1 0 E 105 0 1 0 1 135 0 if 0 Vi Plot 9.—400 Ib. Ammonium Salts and Complete Minerals. 10—15 0 11 3 A. 20—30 2 3 2 Z 50 it 0 iE 0. 70—76 ” 2 0 0 1 105 0 1 0) 1 135 0 ul 0 ai Plot 11-1.—600 Ib. Ammonium Salts and Complete Minerals. 15 0 2 0 2 30 0 2 0 2; 45 1 0 0 1 75 0 i 1 0 105 0 it 0) I 135 0 1 0 1 Plot 14.—550 lb. Sodium Nitrate and Complete Minerals. 15 30 45 75 105 135 SOM wee RKrEOSOOO C. The Nitrification of the Soils in Bulk. Large samples,of the soil down to six depths of 9 inches were taken in October, 1906, and one portion of each was at once dried at a temperature of R2 206 Mr. Hall, Dr. Miller, and Mr. Gimingham. _[Dee. 19, 70° to 80°, and the other placed in a thin layer under a bell jar over water. At the end of five weeks these second portions were similarly dried, and the two sets of samples, after removal of stones and reduction to a powder, were washed on a pressure filter with successive portions of water; the nitrates were determined in the extract by reduction and distillation of the ammonia. In the second trial the surface soil only, to a depth of 9 inches, was sampled in July, 1907; the exposure, made in the same way, was on this occasion continued for two months. The following table shows the amount of nitric nitrogen per million of dry soil both before and after exposure, also the differences between the two sets of figures to show the amount of nitrates formed during the exposure. Rothamsted Grass Soils. . October, 1906. Nitrogen as Nitric Acid per Million of Dry Soil. 1st 2nd ord Ath 5th 6th 9 inches. 9 inches. 9 inches. 9 inches. 9 inches. 9 inches. Plot. In Soil before Exposure. 3 Unlimed 1°06 = 0°54 — 2°19 1°33 4-2 Limed... 4°33 = 0°88 2°92 — 0°39 4-2 Unlimed 3°75 1°39 1-07 1°13 1 ‘09 0 67 9 Limed... 2°25 1°75 1°35 0°79 1:08 2 00 9 Unlimed 2°38 ee: 0°90 1°20 0°47 0°58 11-1 Limed... 2 °38 0-92 1 ‘09 0°79 0 ‘92 0°92 11-1 Unlimed 3 ‘00 1 ‘20 1°39 1 08 1°83 1°83 14 Unlimed 4°75 1 ‘33 2 ‘60 0°44 0°83 0°53 In Soil after Exposure. 3 Unlimed 1°41 0°97 0°67 1-08 0-96 0°63 4-2 Limed... 6°12 2 04 1:00 2-00 3°66 0°36 4-2 Unlimed 5°96 2°30 1°17 1°15 1 ‘04 0 “67 9 Limed... 2°70 2°54 1 ‘30 1°32 1°25 1°49 9 Unlmed 3 42 1°15 0°77 1°39 1°14 0°79 11-1 Limed... 2 67 2°10 1°53 1°12 0°83 0°58 11-1 Unlimed 2 ‘96 2 °08 1 ‘63 1 46 1 ‘92 0°92 - 14 Unlmed 8°42 1 ‘50 2°50 1 ‘09 0-79 0°53 Gain or Loss by Exposure. 3 Unlimed +0°35 — +0°13 — —1°23 —0°‘70 4-2 Limed... +1°79 == +0°'12 —0 ‘92 — —0°03 4-2 Unlimed +221 +0°91 +0°10 +0 °02 —0°05 0 9 Limed... +0°45 +0°79 —0°05 +053 +0°17 —O'al 9 Unlimed +104 +0 ‘04 —0°13 +0°19 +0 67 + 0:24 11-1 Limed... +0°29 +1°18 +0 °44 + 0°33 —0-09 —0°34 11-1 Unlimed —0°‘04 +0 °88 + 0°24 +0°38 +0 ‘09 —0°91 14 Unlimed +3 °67 +0°17 —0-°10 + 0°65 —0 °04 0) July, 1907. First 9 inches of depth only. Plot. At starting. After exposure. Gain by exposure. 3 1°78 : 2°51 0°73 4-2 3°39 9-01 5 62 9 6°17 6°79 0 62 11-1 12 °44, 13 °38 0°94 14 10°77 13 ‘03 2°26 1907.) Nitrification in Acid Sols. | 207 Two kilogrammes of an air-dried sample of the soil from Plot 11-1 were extracted with successive quantities of hot water and the extract concentrated _ to a volume of 500 cc.; 50 ce. portions of this were placed in the usual flasks for nitrification, 5 c.c. of a 1-per-cent. solution of ammonium sulphate was added, and each was seeded with 5 cc. of a cold water extract from a garden soil in a good state of fertility. The object was to ascertain if the acidity of such a medium, which would represent the solution in the soil when it contains 25 per cent. of water, would be sufficient to inhibit nitrification after a fresh stock of the organisms had been supplied. The extraction of the dried soil had, however, not been very effective, and the initial acidity was only equivalent to 0°6 cc. of n/10 alkali, or a little more than one-thousandth normal. In the check flasks the acidity was removed by the addition of a mixture of calcium or magnesium carbonates, and they were all incubated for three weeks at 30° C. Determinations were made of the nitrates before and after incubation, with the following results :-— 50 cc. Soil Extract+ 5 c.c. Soil Extract +5 c.c. 1-per-cent. (NH4)2SO4. Nitrogen as nitrates. . milligrammes, 1, 2,3 No calcium or magnesium carbonate, incubated ............ 0°95 4, 5,6 With calcium and magnesium carbonates, incubated ...... 2°38 7,8 INO DRM CUWATER) eRe psdedsccsatey alvdes Siviccededoheonennpned bodes aes 1°25 There was no formation of nitrates except when the acid had been neutralised by the calcium and magnesium carbonates. In 1, 2, and 3 weak growths of mycelium were visible, but none in the neutral extracts, 4, 5, 6. In a further experiment fresh soil was brought from Plot 11-1 and extracted rapidly with cold water; the extract had a slight acidity, equal to 0-6 c.c. n/10 acid per 100 cc. Portions of 50 c.c. were taken and 5 cc. of 1-per-cent. ammonium sulphate were added to each; in some cases the extract alone was incubated for three weeks, with or without earthy carbonates; in others, 5 c.c. of a garden soil extract prepared as before was added to introduce the nitrification organisms. Nitrates were determined after a month’s incubation, and in the checks before starting, with the following results :— } 50 c.c. Soil Extract +5 c.c. 1-per-cent. (NH,z)2SOx. Nitrogen as nitrates. milligrammes. 1 and 2 ING SIGUA LOR 5 2. . oshe pans ce ote + ss cduemenoMn agate sd ornae edb ane ets 0°25 3 ,, 4 No calcium and magnesium carbonate, incubated ......... 0°23 5 , 6 + Calcium and magnesium carbonate, incubated ......... 0°31 Der Ss +5 c.c. garden soil extract, no calcium and magnesium 0°39 carbonates, incubated Pes LO +5 c.c. garden soii extract, + calcium and magnesium 6°83 carbonates, incubated 208 Mr. Hall, Dr. Miller, and Mr. Gimingham. __[ Dec. 19, Again there was no formation of nitrates except when the acidity of the medium had been neutralised by the calcium and magnesium carbonates. Flask 4 showed a heavy crop of moulds in fructification, 7 and 8 showed mycelium in the liquid, 10 one or two colonies of a bacterium on the surface. D. The Nature and Extent of the Acidity. Fresh soil to the depth of 9 inches was taken in March, 1907,a month after the application of the ammonium salts, and about 7 kilogrammes of the soil as it came from the field was extracted on a pressure filter with successive portions of hot water. No attempt was made to remove all the acid, as previous trials had shown this to be impossible with water alone; the soil after extraction still reddened blue litmus paper. The extract showed an acidity equivalent to 1°71 grammes hydrogen per million of dry soil. Sulphates and chlorides were also determined in the extract as follows :— Chlorides equivalent to 1:91 grammes hydrogen per million soil. Sulphates ~ Br Os) n When the soil was extracted with a fifth normal solution of sodium chloride, the acidity of the extract amounted to 30 grammes hydrogen per million of dry soil; substituting potassium nitrate for the sodium chloride, the acidity was equivalent to 42°5 grammes hydrogen per million of dry soil. In these two latter cases humic acid, insoluble in the water extract, was also being estimated. Another sample was taken in November, 1907, and extracted in the same manner with the following results :— Acidity equivalent to 1:02 grammes hydrogen per million soil. Chlorides A 0°35 3 i Sulphates ¥ 1°60 i a | At this latter date a sample was taken from Plot 9 and gave the following results :— Acidity equivalent to 0°36 grammes hydrogen per million soil. Chlorides a 0°47 » ” Sulphates a 1:69 is 9 In this case some of the humic acids dissolved in the original extract were thrown out during the evaporation of the extract, hence the acidity measured is a little low. The acidity of the soil persists after drying in the air, but is measurably reduced when the soil is dried in the steam oven. If these measured acidities are calculated on the proportion of water usually found in the soil, which will vary between 10 and 25 per cent. of its dry weight, the following results are obtained :— 1907. | | Nitrification in Acid Sorls. 209 Approximate Acidity of Soil-water. Fractions of normal. When soil contains 10 per When soil contains 25 per cent. water. cent. water. Plot 11-1, March, 1907 ...... 1/60 1/150 Plot 11-1, November, 1907... 1/100 1/250 Plot 9, November, 1907 ...... 1/300 1/700 To ascertain if the acid soils contain any free humic acid, 10 grammes of the soil of Plot 11-1 were extracted with 500 c.c. of 4-per-cent. ammonia solution, and the amount of organic matter going into solution was determined. On a second 10 grammes the usual process of determining “humus” was followed; the soil was treated with hydrochloric acid, and the acid washed away before extracting with the ammonia solution. Parallel experiments were made with a prairie soil rich in humus but neutral, since it contained 35 per cent. of calcium carbonate. The following results were obtained :— Organic matter soluble in ammonia. ae a age ae Without acid After preliminary treatment. acid treatment. IPOD RISD, o. «us cessentnates oath 3°54 per cent. 4°51 per cent. PPAITIO OSE Lod cacescecvedeetaee Nil 4°65 The prairie soil yielded no humic acid to ammonia, unless the humates it contained had first been decomposed by hydrochloric acid, whereas the soil of Plot 11-1 contained 3°54 per cent. of humic acid immediately soluble Im ammonia. EK. Lnberation of Acid from Ammonium Salts. To ascertain if any of the organisms in the soil of these acid plots were capable of setting free ammonia from its salts, small Erlenmeyer flasks, containing a solution made up of 2 grammes ammonium chloride, 0°5 gramme monopotassium phosphate, 0°25 gramme magnesium sulphate, 1 gramme sodium chloride, and 10 grammes of glucose per litre, were sterilised, inoculated with the soil or soil extract, and incubated at 21° C. After seven days a vigorous growth of various moulds was observed, and the medium was found to be distinctly acid. In mixed cultures of this kind the acidity rose from about 1/166 to n/80 in a week, and then remained constant. Sub-cultures were made and the organisms isolated in the usual way: the dominant species were forms of Penicilliwm glauweum and a Mucor(?), which differs in several respects from those already known, and does not appear to have been described.* Samples taken from Plots 11-1, 9, and 4-2 at different times during the spring and summer showed the same dominance of Penicilliwm and the Mucor. Various other species of - * This mould has since been identified by Professor A. F. Blakeslee as Zygorhynchus Moeller, Vuill.—Note added March 19, 1908. 210 Mr. Hall, Dr. Miller, and Mr. Gimingham. . [ Dec. 19, Mucor, Trichoderma, and Acrostalagmus have also been observed, but the description of the fungus flora present in the soil of these plots will be given elsewhere. ‘The essential point is that both the dominant species, Penicillium and the Mucor, and several of the others examined, when grown into a nutrient medium containing a carbohydrate and an ammonium salt, draw the nitrogen they require from the ammonia and leave the acid ina free state. This production of acid by Penicillium growing in a medium containing an ammonium salt has already been observed.* The following experiments will serve to illustrate the extent to which the action takes place :— Experiments with Mucor sp.?—Flasks were made up with 60 c.c. of a nutrient solution containing varying amounts of ammonium chloride and cane-sugar as under; the flasks were incubated at 21° C., and the acidity determined after 34 days, with the results set out in the last columns :-— Gain of acidity as Percentage of ammonium. NEC Ranessugar. c.c. 2/10 acid. chloride converted. gramme. grammes. Axl 0° 1-2 11 °6 13 ‘9 A2 0°6 0°6 8-0 V2 Bl 0°3 1:2 8°1 14°5 B2 0°3 0°6 6°9 12 3 C1 0°06 1°2 ga 81-7 C2 0 ‘06 0°6 8°5 75 °0 The highest acidity observed at the end was equivalent to about a 1/50 solution of free acid. When a small quantity of asparagine was also added to the nutrient solutions the increase of acidity was negligible; the organism satisfied its requirements for nitrogen from the asparagine in preference to the ammonium chloride, which, in consequence, remained undecomposed. Experiments with Penicillium.—-One of the forms of Penicillium was seeded into a solution containing 1 per cent. glucose, 0:25 per cent. ammonium chloride, and the usual nutrient salts. Growth was fairly vigorous and was stopped after 14 days, when the yellow solution was filtered off and tested for acidity and ammonia, showing the following results for 100 c.c. of the original solution :— Acidity Nitrogen Ammonia removed. as c.c, 2/10. as ammonia. Check 0 0 063 A 5°5 0 059 0 004 = 2°8 c.e, n/10 acid B 52 0 058 0005 = 3°5 bP The acidity in this case has been increased, though not to the extent * Kohn and Czapek, ‘ Beitr. Chem. Physiol. Path.,’ 1906, vol. 8, p. 302. ae Nitrification in Acid Sorls. » 211 found with the Mucor; at the same time, ammoniacal nitrogen has been withdrawn from solution in amounts accounting for rather more than half of the observed acidity. - Another set of observations made by Dr. H. B. Hutchinson gave the following results :— Acidity produced and ammonia withdrawn from solution containing 1 per cent. dextrose and 0°2 per cent. ammonium sulphate, during 21 days’ growth of various moulds isolated from Plot 11-1. Cubic centimetres of Tenth Normal Acid per 100 c.c. Culture Medium. Acid equivalent of Organism. Acidity. ammonia withdrawn. ICON SPs 2 (25: gu0es coo aue 6-0 hb “E Mucor be Fake: eee 41 5 °6 MCI COP EL revs kaise eolaaenes 5 *44 9°6 (Penicillin sa. .css-s3.denne i 5 12 °4 Acrostalagmus ............ 10°1 11°9 Trichoderma I ............ 11°3 13 °9 Trichoderma II ......... 12 °54 13 °6 In these cases there was always a greater amount of ammonia withdrawn from solution than was equivalent to the acidity measured, which, as before, amounts to a concentration of from 7/200 to n/70 in the final solution. It is further significant that the degree of acidity developed in the culture solutions is of about the same order, between fiftieth and two- hundredth normal, as that which was found to exist in the soil water of the field plots. Summary. In the soil of certain of the permanent grass plots at Rothamsted, which is distinctly acid in consequence of the long-continued use of ammonium chloride and sulphate as manure, nitrification is greatly reduced, and the nitrifying bacteria are only found sparingly. In bulk, nitrification still goes on slowly, despite the acidity of the soil. Water extracts of the soil will not permit of nitrification unless they are previously neutralised. The amount of nitrate produced would not be sufficient for the nitrogen taken up by the crop, which must, in the main, utilise the ammonium salts without previous change. The acidity is chiefly due to sparingly soluble “humic” acids; free hydrochloric and sulphuric acids are also present, because the soil extract contains soluble acid in quantities comparable to the amount of chlorides and sulphates also present, and to the ammonium sulphate and chloride annually supplied as manure. The acidity is not brought about by purely chemical or physical actions of the soil upon the ammonium salts, but by various micro-fungi which are able to remove ammonia from a solution of its salts and set free the acids 212 Messrs. Dorée and Gardner. Origin and [Dec. 20, with which it was combined, the acidity attained in this way being equivalent to that of the soil water on the acid plots. | The authors attribute the continuance of the nitrification in these soils to the irregular distribution of the materials composing them; though acid as a whole, they still contain some calcium carbonate, each of the particles of which forms a centre for the nitrification process. The decline in fertility of the acid plots may be attributed to the repression of the normal bacterial activities of the soil and the encouragement of the growth of moulds. The Origin and Destiny of Cholesterol in the Animal Organism. Part IL—On the so-called Hippocoprosterol. By CuHaries Dorks, Lindley Student of the University of London, and J. A. GARDNER, Lecturer in Physiological Chemistry, University of London. | (Communicated by Dr. A. D. Waller, F.R.S. Received December 20, 1907,— Read January 23, 1908.) (From the Physiological Laboratory of the University of London.) Introductory. Since the discovery of cholesterol by Conradi in 1775, and its analysis by Chevreul in 1815, it has been found to be very widely distributed in the animal and in isomeric forms in the vegetable kingdom. It is found in small quantities in all protoplasmic structures, in blood, bile, sebum, and similar oily excretions of the skin, and is an especially abundant constituent of the white substance of brain and of the medullary sheath of nerve. But, although a considerable amount of work has been done, we have little or no definite knowledge of its physiological functions, and it is only in very recent times that a small glimmering of light has been thrown on its chemical constitution. In 1862, Austin Flint* published a series of experiments by which he attempted to show that cholesterol is always more abundant in the blood coming from the brain than in the blood of the general arterial system, or in the venous blood from other parts; that its quantity is hardly appreciable in venous blood from the paralysed side in hemiplegia, and that it is * “Kxperimental Researches into a new Excretory Function of the Liver,” ‘ American Journal of Medical Sciences,’ Philadelphia, 1862, new series, vol. 44, and “ Recherches Expérimentales sur une Nouvelle Fonction du Foie,” Paris, 1868. (1907.] Destiny of Cholesterol in the Anvmal Organism. 213 separated from the blood by the liver. He also stated that in cases of serious. structural disease of the liver, accompanied by symptoms pointing to blood poisoning, cholesterol accumulates in the blood, constituting a condition which he named cholesteremia. From his experiments he came to the conclusion that cholesterol is a product of the metabolism of nervous tissues, that it is carried from the brain by means of the blood and excreted by the liver through the bile, and, finally, that “we know of no function which it has to perform in the economy, any more than urea or any other of the excrementitious principles of the urine.” Flint’s methods of analysis were, however, open to grave objection, and he draws sweeping conclusions from differences so slight that had his method of estimating cholesterol been capable of considerable accuracy, we should have hesitated to attribute much signifi- eance to the figures. Flint also observead* that the cholesterol of the bile undergoes a modification in the intestine, and is found in the feces as “stercorine.’ Some support was lent to Flint’s views by the experiments of Picott in 1872 and Koloman Miillerf in 1873. Picot reported a fatal case of “orave jaundice,’ in which he found a great increase in the proportion of cholesterol in the blood; and Miller injected into the veins of dogs 2°16 fluid ounces.of a solution containing 69 grains of cholesterol, made by rubbing cholesterol with glycerine and mixing the mass with soap and water. In five experiments of this kind, he produced a complete representation of the phenomena of “ grave jaundice.” How far Flint’s views obtained general credence we do not know, for he is rarely quoted in physiological text-books, and we are unaware of any other extensive series of experiments on the subject; but B. Moore, in Schafer’s ‘Text-book of Physiology ’ (1898), states that “according to Hoppe-Seyler cholesterol is a cleavage product, constantly formed in the metabolic changes in the living cell; and for this reason it is that cholesterol is invariably found as a chemical constituent of both animal and vegetable cells. Cholesterol does not easily undergo decomposition in the animal organism when once formed, and is principally excreted in the higher animals in the bile. It is formed in increasing quantity in tissue which is undergoing pathological change . . . . It is probably formed most in the metabolism of nerve tissue, taken up by the liver cells from the blood, and passed as an excretion into the bile ducts. Cholesterol is purely an-excretion, and is not reabsorbed, but passes out of the body with the feces.” Some more recent observers * Ibid. | + ‘Journal de l’Anatomie,’ Paris, 1872, vol. 8. { “Ueber Cholesteriimie,” ‘Archiv fiir Experimentelle Pathologie und Pharmakologie,’ Leipzig, 1873, vol. 1, p. 213. | 214 Messrs. Dorée and Gardner. Ovigin and [ Dec. 20, appear to hold the view that the cholesterol of the bile is formed either in the liver cells or in the cells lining the bile passages or both, but the evidence: for this is not particularly convincing. That some cholesterol is formed somewhere within the liver, and not merely excreted by it, seems to be shown by an experiment of Jankau,* performed in Naunyn’s laboratory. He injected cholesterol into dogs, and also gave it in their food, and ascer- tained that it had been absorbed; but he failed to find any increase of cholesterol in the liver tissue or in the bile. The analyses of the liver and the bile published by Kausch+ from the same laboratory show no relationship between the amount of cholesterol in the gland and in its secretion. Thomas, also working under Naunyn’s direction, found that there is no relationship between the amount of cholesterol excreted and the kind of food taken. When, however, the dog under observation suffered from catarrh of the biliary passages, there was a marked increase in the cholesterol of the bile. [Goodman,§ on the other hand, in an experiment on a dog with a permanent fistula, observed that the quantity of cholesterol found in the bile varied considerably with the nature of the diet, but that the amount of cholesterol in the food taken was without influence. Thus white of egg and calves’ brain were equally efficacious in increasing the output of cholesterol, although the brain contains some 2 per cent. of its weight of cholesterol and the egg albumin practically none. He found, too, that intravenous injection of cholesterol did not increase the output in the bile, the result. being in agreement with that of Jankau. Pribram|| also has shown that. in the case of rabbits which have been fed with cholesterol for some days: and then killed, there is a decided rise in the cholesterol content of the blood, which, in consequence, exhibits an increased power of resistance to the hemolytic effect-of saponin. |‘ From these experiments, and from the fact that cholesterol is alee found where cells are disintegrating, Naunyn strongly supports the view that cholesterol is produced, not in the liver cells, but from the cells of the passages,** and that it is a product of disintegration of their protoplasm. More recently, V. Harley and W. Barratt,t} in a series of experiments on the * ‘Cholelithiasis,’ transl. by A. E. Garrod, New Syd. Soc., 1896. + Dissertation, Strassburg, 1891. t ‘Cholelithiasis, 2bcd. § ‘Hofmeister’s Beitrige,’ vol. 9, p. 91. || ‘ Biochem. Zeit.,’ 1906, vol. 1, p. 413. ul The passage widen square awe is added as the paper passes through the press. ** Cf. also Doyon and Dufort, ‘C. R. Soc. Biologie,’ 1896, vol. 10 (3), p. 487. tt “An Experimental Enquiry into the Formation of Gall Stones,” V. Harley and W. Barratt, ‘Journ. Physiol.’ vol. 29, p. 341, 1903. 1907. | Destiny of Cholesterol in the Animal Organism. 215 effect of introducing gall stones into the gall bladders of dogs, have shown that when the gall bladder is healthy the gall stones tend to disappear, while, on the other hand, when cholecystitis is present they remained unchanged. In his Text-book of Physiology, Schifer has also suggested that the constant presence of lecithin and cholesterin in the bile may well be associated with the destruction of the red corpuscles, which contain relatively considerable amounts of these substances. It will be seen from the foregoing sketch that we know little or nothing definite as to the functions of cholesterol, and we certainly know nothing whatever of the breaking up of cholesterol in the animal body. From the very general occurrence of cholesterol and its frequent association with lecithin, we cannot but think that it must play an important part in the cell economy, and must not be considered merely as a waste product. In this connection the experiments of Flexner, Noguchi,* Preston Kyes,t Abderhalden and Le Count, on hemolysis produced by cobra poison in the presence of lecithin and the inhibitory effect of cholesterin appear to us very significant. Whatever may be the value of Flint’s views on the cholesterol problem, he was correct in his statement that cholesterol is found in human feces in the modified form of “ stercorine.” | This body was rediscovered in 1896, by Bondzynski{—who gave it the name of coprosterol—and was thoroughly investigated by Bondzynski and v. Humnicki.|| It crystallises in long slender needles melting at 95° to 96° C. and behaves chemically as a saturated alcohol; it is dextrorotatory and gives colour reactions similar to those of cholesterol. Its formula, Co;H.s0, was confirmed by the analyses of a large number of derivatives, and its discoverers regarded it as a dihydrocholesterol formed by bacterial reduction in the intestine. They fed a man with cholesterol and found that it was excreted mainly as coprosterol, and, later, Miller proved that on a milk diet, in which * “Snake Venom in Relation to Hemolysis, Bacteriolysis, and Toxicity,” ‘Journ. of Experim. Med.,’ vol. 6, No. 3, 1902. + “Ueber die Wirkungsweise des Kobragiftes,” ‘ Beit. klin. Wochenschr.,’ Nos. 38 and 39, 1902 ; also “ Lecithin und Schlangengift,” ‘Zeit. f. physiol. Chemie,’ vol. 41, p. 273, 1904; also “Zur Kentniss der Kobragift aktivierenden Substancen,” ‘Berl. klin. Wochenschr.,’ Nos. 2—4, 1903. { “Die Beziehungen zwischen Cholesterin, Lecithin, Kobragift, Tetanuslaxin, Saponin und Solanin,” ‘ Zeitschr. f. experim. Path. u. Therap.,’ vol. 2, p. 199, 1905. § “Cholesterol of Human Feces,” ‘ Ber. der Deut. Chem. Ges.,’ 1896, vol. 29, p. 476. || “The Fate of Cholesterol in the Animal Organism,” ‘Zeit. Physiol. Chem.,’ 1896, vol. 22, p. 396. J “ Reduction of Cholesterol to Coprosterol in the Human Intestine,” ‘ Zeit. physiol. Chem.,’ 1960, vol. 29, pp. 129—135. 216 Messrs. Dorée and Gardner. Origin and [ Dee. 20, putrefactive changes in the intestine are reduced to a minimum, the cholesterol of the body is excreted unchanged. The reduction of cholesterol in the intestine thus seems established in the case of man, and Bondzynski and Humnicki, continuing, examined the feces of the dog and the horse.* In the case of the dog, they stated that the cholesterol of the bile was excreted unchanged, but in that of the horse intestinal reduction went much further than in man, and a cholesterol-like body, Co7Hs4 o, 50, was found, crystallising in microscopic needles melting at 74° to 75° C. This hippocoprosterol, as it was called, was further examined in 1905 by Wilenko.t The results of his work go to prove that the excrement of the horse contains two isomeric bodies, C27H52 or 540, one readily soluble, the other much less soluble, in 97 per cent. alcohol. These he designated as « and B- hippocoprostero] respectively. The « body crystallises in minute rhombic tables similar to cholesterol crystals. When dry it forms silky scales which are as soft as wax and melt at 66° to 67°C. The 6 isomer appears to be identical with Bondzynski and Humnicki’s hippocoprosterol, though Wilenko gives its. melting point as 56° instead of 74°. In order to throw some further light on the origin and functions of cholesterol in the animal economy, it appeared to us in the first instance essential, more especially considering the discrepancies and scantiness of the work of previous observers, to make a comparative study of the forms in which cholesterol is found in the feeces of different animals and to determine to what extent the substances thus excreted are dependent upon the food taken. In the present paper we give an account of our experiments on the feeces of herbivorous animals, those examined being the horse, cow, sheep, and rabbit. Method of Hxpervment. The material was obtained from grass-fed animals (Hampshire) and was. sometimes dried directly in the water oven, but generally spread out in thin layers and allowed to dry in the air. In order to deal effectively with such a light, bulky substance, we employed large metal extractors capable of holding 2 to 5 kilogrammes of material. They were made on the Soxhlet. pattern, fitted with long metal condensers, and the ether vessel was enclosed in an air chamber warmed with incandescent electric lamps. The dried material was extracted usually for five to six days with ether, the dark green solution obtained diluted, if necessary, with more ether and at once saponified with sodium ethylate in alcoholic solution according to the method of Kossel * Ibid. | + “Hippocoprosterol,” M. Gittelmacher Wilenko, ‘ Bull. International de l Académie: des Sciences de Cracovie,’ No. 1, Jan., 1906, p. 20. 1907.| Destiny of Cholesterol in the Animal Organism. 217 and Obermuller: care had, however, to be taken not to allow the alcohol added to amount to more than 1/10 to 1/12 of the volume of the ether, lest any of the hippocoprosterin should be precipitated along with the soaps. The mixture was well shaken, allowed to stand overnight,and the soaps filtered off and washed with ether. The filtrate was then shaken, first with an equal bulk of water to remove alkali and alcohol, and then with water containing potash to remove the last traces of soap. The ether solution was finally separated off, dried, and evaporated. The crude extract was dark red, liquid at 100° C., and had a pungent smell like that of wood spirit. It dissolved in boiling alcohol (with the exception of a small residue soluble in benzene), giving a deep red solution which could be partially decolorised by treatment with charcoal. The char- coal, however, subsequently required repeated extraction with alcohol, as it obstinately retained the substance. The pale yellow filtrate, on cooling, set to a bulky gelatinous mass which was difficult to filter and left a solid which, on drying, shrank up to light flakes of impure hippocoprosterol. This was dissolved in ether and precipitated by alcohol, the process repeated, if necessary, and the nearly white material dissolved in ether and allowed to crystallise by slow evaporation. The alcoholic mother liquors obtained in the above processes were subjected to further crystallisation till only oily matter remained. Examination of Horse Excrement. The hippocoprosterol obtained, as above, was purified by crystallisation from benzéne or ethyl acetate, and finally from ether. Sometimes it was necessary to use charcoal again before the last traces of colour could be removed, but often this could be dispensed with. The yield was 0:2’per cent. of the dry feces. Hippocoprosterol is a light, white substance, which may be powdered, but. cakes together under slight pressure. It dissolves at the boiling temperature in all the usual solvents (except water), but comes out again almost completely in the cold. Thus its solubility in benzene at 16° C. is only 0°32 part per 100 parts of solvent. From ethyl and methyl alcohol, ethyl acetate, glacial acetic acid and acetic anhydride, it comes out as a white jelly; from ether, petrol ether, benzene and chloroform in a more powdery form. From strong solutions it crystallises in microscopic needles grouped in stars, rosettes,and comet-shaped clusters, but on slow evaporation these clusters may be obtained 1 to 2 mm.in diameter. Ifa dilute ethereal solution be allowed to evaporate spontaneously, large transparent masses of crystals are obtained, consisting of fan-shaped groups of needles springing from a common base. It melts 218 Messrs. Dorée and Gardner. Origin and [Dee. 20, at. 78°5 to 79°5 C., and solidifies at 77°, a characteristic property. It is optically inactive, and does not give the cholesterol colour reactions. These observations are at variance with the statements of Bondzynski and Humunicki, who attribute to hippocoprosterol a slight dextrorotation and a green colour reaction in a modified Salkowski test, but seeing that their product melted at 74° to 75° C., it probably contained some impurity which produced these effects. Wilenko’s 8-hippocoprosterol, which is undoubtedly identical with ours, he describes as melting at 56°, a discrepancy we are unable to understand, since on elaborate purification of our product we failed to obtain any alteration in the melting point. Hippocoprosterol — does not absorb bromine in carbon bisulphide solution. Dry bromine in a sealed tube at 100° C. is without action, but at 170° C. various substitution products are obtained. For analysis the substance was dried 7m vacuo at the ordinary temperature, and was burnt in a copper boat filled with coarse copper oxide. I. 01936 gave 0°5823 CO, and 0:2473 H,0. et Urine us 0:4352' CO, _ ,;, 0:1738 HO. Found. Calculated for — HS FF ae — I. II. C,,H,,0. C,,H,,0. Cae. 82:03 82:03 S204: 81°73 Eee mee 14°13 14°11 13°80 14°24. A molecular weight determination, carried out by the cryoscopic method, showed that it possesses the simple formula :-— 08321 gramme in 13°36 grammes of naphthalene (constant — 70) gave a depression of 1°157. Molecular weight found, 377. C2;H;,0 requires 396. Hvppocoprosterol Acetate-——One part of hippocoprosterol was mixed with two parts of fused sodium acetate and five to six parts of acetic anhydride, and heated to boiling for half an hour. The product was poured into water, and the precipitated acetate dissolved in acetic ether. It came out from this in flocks, which consisted of microscopic needles resembling the original substance. The yield was almost quantitative. The acetate is moderately soluble in alcohol and acetic ether, easily in ether, benzene and petrol. From the latter it was obtained in shining flakes, which were sticky, caking together under the Jeast pressure. Melting point 61° to 62° C. On analysis, the following figures were obtained :— 1907.] Destiny of Cholesterol in the Anemal Organs. 219 I. 01719 gave 0°5035 CO, and 0°2061 H,0. Il, 01831 » 05333 CO, ,, 0°2181 H,0. Found. Calculated for ia ity G,,,H,,0.00.CH,. ,,H,,0.C0.CH,, ea Hee 79°88 79°43 OTS Tal i aati e352 13:24 12°93 1398 On saponification of the acetate with sodium ethylate, hippocoprosterol was obtained, melting at 79° C. Hippocoprosterol Benzoate—The hippocoprosterol was mixed with an equal weight of benzoic anhydride, and heated to 160° for two hours in an open vessel. The product was boiled with alcohol, and on cooling the benzoate separated in thick clots. These were recrystallised several times from petrol, and finally from ethyl acetate. Hippocoprosterol benzoate crystallises: in microscopic needles, which in mass appear as sticky lumps. It is difficultly soluble in alcohol and ethyl acetate (but more so than the mother substance), easily in ether, petrol, and benzene. The meiting point is 58°5 to 59°5 C. In concentrated ether solution it proved optically inactive, and on analysis gave the following figures :— 01799 gave 05395 CO, and 01975 H,0. Calculated for a aN, Found. C,,H,,0.CO.C,H,;. C,,H,,0.C0.C,H,. Shee eee 81°79 81°85 S152 Fe ee 12-20 lee 12°09 Two grammes of the benzoate were saponified with sodium ethylate and yielded hippoeoprosterol, melting at 79°. Hippocoprosterol Cinnamate.*—Five parts of hippocoprosterol were mixed with three parts of cinnamyl chloride and heated to 140° for one hour. The product was boiled out with alcohol, and the solution on cooling deposited the cinnamate in masses of minute needle-shaped crystals which, when dry, appeared sticky, caking together under pressure. It is very soluble in benzene, moderately in acetic ether and petrol, and difficultly in alcohol. The melting point is 62°C. On analysis the following figures were obtained :— 0°2040 gave 0°6143 CO, and 0:2132 H,0. Calculated for oie: Cy;Hy0.CO.C,H;. C,,H,,0.C0.C,H,, Ce 82:17 82:37 82:05 ete. 11°61 Th53 11°81 * This paragraph is added as the paper passes through the press. VOL, LXXX.—B. s 220 Messrs. Dorée and Gardner. Origin and [Dee. 20, Hippocoprosterol behaves, therefore, as a saturated alcohol. The hydroxyl - group, however, is not readily replaceable by chlorine. When ground up with phosphorus pentachloride in the cold, no action takes place, though on adding petrol and boiling some hydrochloric acid is evolved, but we have not succeeded in preparing the chloride, either by this method or by the use of thionyl chloride. Various substances appear to be formed, which we are at present investigating. Hxamination of the Alcoholic Mother Liquors. The residues remaining after separation of the hippocoprosterol were subjected to a careful examination, first with the object of isolating the a-hippocoprosterol (m.p. 66° to 67°) described by Wilenko, and secondly to discover whether any of the cholesterol of the bile was present. As an example, 2 kilogrammes of dry dung obtained in the summer yielded 3°65 grammes or 0°18 per cent. of hippocoprosterol, and 8:1 or 0-4 per cent. of a dark red buttery residue. The latter was dissolved in alcohol and allowed to evaporate. The first crops of material obtained gave melting points varying between 65° and 70°, but small quantities of pure hippocopros- terol could always be obtained from these. The second crop (0°2 gramme) gave a body which dissolved easily in all solvents, except dilute alcohol, from which it crystallised readily in microscopic hexagonal plates. It melted at 136° to 137°, absorbed bromine in carbon bisulphide solution, and gave the ‘Salkowski and Liebermann colour tests. It thus agreed closely with the ‘sitosterol of Burian.* The acetate of the body, however, did not confirm this. It was made in the usual way with acetic anhydride and sodium acetate, and came out of dilute alcohol in brilliant glistening leaves consisting of six-sided plates. In all its properties, however, it agreed with the original substance, except that it was less soluble in absolute alcohol. It gave a constant melting point of 136°, which could not be altered by repeated crystallisation. Sitosterol acetate melts at 127°5. The third crop (0'1 gramme) was white, and after several crystallisations from 80-per-cent. alcohol appeared under the microscope as long thin plates like sword blades. These melted sharply at 161° to 162° and gave Liebermann’s colour test. To identify it if possible with the caulosterine obtained by Schulze and Barbierit from the shoots of the yellow lupin (m.p. 158°), it was heated with benzoic anhydride, but no crystalline benzoate could be obtained. The concentrated alcoholic filtrate still showed traces of solid matter, but this could not be * From germinating wheat, ‘ Monatshefte f. Chemie,’ 1897, vol. 18, p. 551. + ‘Journ. Prak. Chem.’ (2), vol. 25, p. 159. ia 1907.| Destiny of Cholesterol in the Animal Organism. 221 obtained free from oil. The alcohol was accordingly evaporated off, and the buttery residue, which dissolved very easily in most solvents, was treated with _'75-per-cent. alcohol, in which it was more difficultly soluble. From this a white solid body was obtained in small quantity (less than 1 per cent.), which gave Liebermann’s test and crystallised from dilute alcohol in the _ sword blade plates described above. From ether and petrol it dried up to rosettes of needles which softened at 145° and melted clear at 154° to 155°. This melting point could not be raised by repeated crystallisation. To discover whether it was identical with Tanret’s ergosterol,* it was converted into the acetate by heating with acetic anhydride and sodium acetate. The product, which was very soluble in petrol, crystallised from dilute alcohol in microscopic rectangular plates. It, however, melted at 78° to 80° C., whereas Tanret’s acetate melted at 169° to 176°. After separation of the solid bodies as above, the alcoholic solution leaves, on evaporation, a very dark red thick oil, which, in the case of the horse, has not been further investigated. It is important, however, to emphasise that, although we have extracted some 15 kilogrammes of dried feces, we have not been able to obtain either the «-hippocoprosterol of Wilenko, or to detect any trace of cholesterol, microscopically or otherwise. Instead, we have obtained in minute quantity high melting bodies, which, so far as they could be examined, appeared to belong to the phytosterol, or vegetable cholesterol, group. Haiamination of the Haucrement of the Cow and Sheep. The material was obtained from grass-fed animals, and was extracted and worked up as before. The chief product was in each case a body identical in all its properties with the hippocoprosterol described above. The yield amounted to 0°15 per cent. of the dry dung in the case of the cow, and 0:3 per cent. in that of the sheep. The identity was proved by the crystalline form, solubility, melting points, mixed melting points, and analysis. i COW 2... nce 0:2383 gave 0°7168 CO, and 0°3025 H20. TI. Sheep Seat: 0:1578 ms 04780 CO. ,, 0°2028 H.O. Found — A-———, Calculated for | if II. Cy Elige: C&A... 82°02 82°61 82°14 DET gah 14°10 14:28 13°80 The cow also gave about 0°3 per cent. of a dark red oil similar to that of * The ergosterol described by Tanret (‘Annales de Chim. et de Physique,’ series 6, vol. 20, p. 289) crystallises from alcohol in plates, from ether in needles. M. p. 154°. s 2 22 2 Messrs. Dorée and Gardner. Origin and [Dee. 20, the horse, while the sheep gave 0:4 per cent. of a yellow vaseline-like substance smelling strongly of hay. Examination of the Eacrement of the Rabbit. The material was obtained in the summer from wild rabbits. Treated as before, from 3 kilogrammes of the dried feeces, 6°2 grammes, or 0°21 per cent., of pure hippocoprosterol were obtained. On analysis it gave the following results :— 01656 gave 04974 CO, and 0:2135 H,0. ; Calculated for Found. C,,H,,0. CR emi S191 82°14 Hs eee 14°33 13°80 The oils left after complete separation of the solid matter weighed 23°26 crammes, or 0°77 per cent. Mr. G. W. Ellis, at our suggestion, attempted to ascertain the composition of these residues by a fractional distillation 7m vacuo, but the process proved tedious and difficult and led to no very definite results. At first the liquid simply frothed over, but on returning the distillate and repeating several times the frothing became less marked, and under a pressure of less than 1 mm. it distilled over between 98° and the temperature at which the glass softened without the slightest charring. After elaborate and repeated fractionation, four main fractions were obtained, boiling around the following temperatures :—(1) 98°, (2) 164° to 168°, (3) 215° to 220°, (4) 260° to 265°. In the flask there remained a transparent, yellow, brittle, resinous substance which was not decomposed at the softening point of glass. | Fraction 1 consisted of about 2 c.c. of a pale yellow, fairly mobile oil, with a smell recalling that of pine oil.* On combustion it was found to contain 82°13 per cent. carbon and 11:96 per cent. hydrogen. Fraction 2 consisted of about 3 ¢.c. which, on long standing, deposited a trace of crystalline matter. It had a very faint turpentine odour and ‘reduced ammoniacal silver solution in the cold, markedly on heating. Fraction 3 consisted of about 5 c.c. of a very thick oil, smelling faintly of hay, and only just mobile at the ordinary temperature. Fraction 4 was the largest and consisted of a pale yellow, sticky, solid (at the ordinary temperature), which showed no signs of crystallisation after many months’ standing. On combustion it gave the following figures, which agree closely with those required for cholesterol :— * The rabbits from which these feces were obtained lived on the border of a pine wood. 1907.] Destiny of Cholesterol in the Animal Organism. 223 I. 0:2449 gave 0°7562 CO, and 0:2547 H,0. Ii, 02694 4 08343 CO. ,, 0°2908 H20. _ Found — A——_—, Calculated for I. II. CH; ,0. ene ae 84°21 84:05 84°37 1c pe ee 11°56 11°99 11°46 All attempts to prepare a crystalline acetate or benzoate of this substance failed. In the course of the tmvestigation we noticed that samples of faces collected during the winter season, when the animals were not fed entirely on grass, gave a lower yield of hippocoprosterol. Furthermore, an examination ‘of the feeces of domestic rabbits, fed on cabbage, made for us by Mr. G. D. Knox, showed that with this food a different product was obtained, an account of which we reserve for a future communication. We therefore were led to suspect that the hippocoprosterol might be a constituent of the grass on which the animals fed. Examination of Grass. 3°2 kilogrammes of the blades of grass obtained from the cuttings of a well- kept cricket pitch, fairly free from clover and other plants, were dried in thin layers in the air and extracted as before. The extract was dissolved in alcohol and decolorised with charcoal. The solution was now pale yellow and deposited a gelatinous solid mass which was readily worked up by methods previously described and obtained pure. The yield was 8 grammes, or 0°27 per cent., of the dry material. This body proved identical with hippocoprosterol, an important point which was confirmed by the following experiments :— (a) six grammes of the body were crystallised from benzene into three fractions, each of which gave a melting and solidifying point (79° and 77° respectively), the same as that of hippocoprosterol. (6) Equal weights of the body from grass were mixed separately aa equal weights of that from the horse, cow, sheep, and rabbit, The melting points remained unchanged. Finally, a sample from each of the five sources was mixed and the same result obtained. (c) The acetate and benzoate were made as before and proved identical with those from hippocoprosterol. They were saponified and yielded chippocoprosterol, melting at 79°. (ad) On analysis, the following figures were obtained :— 224 Messrs. Dorée and Gardner. Origin and [Dec. 20, I. 01924 gave 0°5758 CO2 and 0°2464 HO. i 0 Ae x 0()°5234 COz ,, 0°2230 HO. Found rr Calculated for I. 10G Cz HO: OR. eee 81°62 81°94 82°14 ds Deira 8 14°23 14°22 13°80 The mother liquors from this product yielded a considerable quantity of reddish oily matter, similar to that found in the feces. It would, therefore, appear that hippocoprosterol is not an animal product, but is a constituent of the grass food which is passed unchanged. In order to confirm this conclusion, and also to ascertain whether any cholesterol or derivative of it which we might have missed in our previous experi- ments was excreted by the animal, we made a series of experiments in which a domestic rabbit was fed on grass which had been thoroughly extracted with ether. Experiment 1—A rabbit, weighing 2-1 kilogrammes, was fed with 315 grammes of extracted grass, slightly moistened with water, during 14 days. A very small quantity of bran was given in addition. The animal took the grass readily, and at the end of the experiment had only lost 0:1 kilogramme in weight, and appeared to be in good health. The weight of dry feeces obtained was 128 grammes. This was extracted in the usual way, and the extract was found to have a somewhat fcetid odour not noticed with ordinary dung. One gramme of unsaponifiable matter was obtained, the greater portion of which was soluble in alcohol. On standing, a very small quantity of red crystalline matter was deposited, but the bulk of the substance eventually separated as a non-crystalline red oil. No trace of hippocoprosterol was discovered, and the small quantity of crystalline matter referred to after purification was obtained from dilute alcohol in the form of glancing white leaf-like crystals and from ethyl acetate as needles, which melted rather indefinitely at about 129°. Under the microscope the crystals from alcohol showed the form of hexagonal plates, recalling those of phytosterol, but the quantity was too small for further investigation.* Experiment 2—A rabbit, weighing 271 kilogrammes, was fed during 18 days on extracted grass with a little bran. The quantity consumed weighed before extraction 2°8 kilogrammes, and was of a somewhat coarser * This substance was derived from the bran given to the animals, as on extraction of a sample of bran we obtained a body, crystallising in the same forms, which melted at. 137°°5 C., and appeared to be identical with Burian’s sitosterol. . 1907.| Desteny of Cholesterol in the Anmal Organsm. 225 description than that previously used; 982 grammes of dried feces were obtained, and were treated as before. The residue obtained weighed 564 grammes. From this we isolated 0:25 gramme of hippocoprosterol, and the mother liquors on evaporation deposited an oil, which on standing showed under the microscope traces of crystalline matter in the form of six-sided plates and clusters of sword blades, but in too small quantity for further examination and identification.* General Conclusions. 1. Hippocoprosterol isolated from the feces of the horse by Bondzynski and others is not a product of animal metabolism, but is a constituent of the grass taken as food, and is passed unchanged by all herbivorous animals when fed on grass. The name is, therefore, misleading, and we propose to rename the substance chortosterol (yvopros, grass). 2. Chortosterol is an alcohol having the formula C2;H4O or Co7H5,0. It is not possible at present to decide definitely between these, though our analyses in every case agree better with the former. 3. If we consider the numerous vegetable cholesterols which have the properties of unsaturated monatomic alcohols and the formula C2;H4sO + H20 as isomeric substances constituting the phytosterol group, chortosterol cannot be regarded as a simple reduction product of any one of them in the same way that coprosterol is supposed to be related to cholesterol. It is possible that the substance may be derived from some member of this group, or vice versd, by some rearrangement of the ring structure during the development of the plant. We are at present engaged in some experiments on this point. This is, perhaps, supported by the fact that chortosterol, unlike other members of the cholesterol or phytosterol groups, gives none: of the usual colour reactions, for Windaust has shown that when the unsaturated open side chain of the cholesterol and phytosterol molecules is condensed to a ring, the products obtained show the colours feebly{ or not at all. . 4. In all the experiments we have made we have never found any cholesterol in the feces of the herbivora. If the view of Flint and other observers that cholesterol is an excrementitious product got rid of in the feeces through the agency of the bile, we should certainly have expected * This substance was derived from the bran given to the animals, as on extraction of a sample of bran we obtained a body, crystallising in the same forms, which melted at 137°°5 C., and appeared to be identical with Burian’s sitosterol. t ‘Ber.,’ vol. 40, pp. 2637 and 3681 (1907). { Cf. Diels and Abderhalden, ‘ Ber.,’ vol. 39, p. 884 (1906). 226 Origin and Destiny of Cholesterol in the Animal Organism. in the very large quantities of material examined to have obtained con- siderable quantities of cholesterol as such, or in a modified form as in the human subject. In the cow, for instance, every 100 c.c. of bile contains approximately 0°07 gramme of cholesterol, and supposing in this animal only 23 litres are poured into the intestine per day, this would mean a daily excretion of nearly 2 grammes, which we could not possibly have failed to discover. It follows, therefore, that cholesterol of the bile must either have been reabsorbed with the bile salts in the gut, or else destroyed. We are at present carrying out experiments on this subject with herbivora and other animals, an account of some of which we hope shortly to have the honour of laying before the Society. This work has been carried out with the help of a grant which was made to us by the Government Grant Committee of the Royal Society, for which we take this opportunity of expressing our thanks. 227 The Origin and Destiny of Cholesterol in the Anmal Organism. Part II].—The Excretion of Cholesterol by the Dog. By Cuartes Dorks, Lindley Student of the University of London, and J. A. GARDNER, Lecturer on Physiological Chemistry, University of London. (Communicated by Dr. A. D. Waller, F.R.S. Received February 10,— Read March 12, 1908.) . (From the Physiological Laboratory, University of London.) In a former paper* we showed that the so-called hippocoprosterol, which was considered by its discoverersf to be a reduced cholesterol, and the form in which the cholesterol of the bile of the horse is excreted in the feces, is contained in the solid excreta of all grass-fed herbivorous animals, and that it is not a product of the animal metabolism, but a constituent of the grass taken as food and passed unchanged. We also showed that cholesterol is not found in the feces of these animals. It therefore seemed to us desirable to extend the investigation to carnivora, and to ascertain, if possible, whether the cholesterol often found in their excreta is derived from the organism or from the food, and whether, under any circumstances, it is found in modified form as in the human subject. Of the various animals the dog appeared to be the most suitable for initial experiments, as it is easily trained and will thrive not only on meat but on vegetable diets. The animal selected for experiment was an Irish terrier bitch, between four and five years of age, and weighing 11°8 kilogrammes. It was quiet and used to a sedentary life. Preliminary experiments, using a mixed diet, showed that the quantity of cholesterol excreted per day was exceedingly small, much too small for even approximate estimation. We therefore decided to keep the animal under observation for the greater part of a year, and to feed it on particular diets for many consecutive days, varying from 14 to 30, according to circumstances. We did not think it necessary at this stage of the enquiry to make any determinations on the nitrogen metabolism during the experiments, but we took great care to keep the animal in good health during the whole time, by regular exercise, periods of rest, etc. During a part of the time the dog was kept at Messrs. Ridler and Hobday’s establishment, and we take this opportunity of expressing our * ‘Roy. Soc. Proc.,’ this volume, p. 212. t+ Bondzynski and Humnicki, ‘ Zeit. Physiol. Chemie,’ vol. 22, p. 396. 228 Messrs. Dorée and Gardner. Origin and [Feb. 10, thanks to Professor Hobday for the care and attention bestowed on the animal. while there. The feeces collected during each diet period were dried in the water oven, roughly powdered, and extracted thoroughly in a Soxhlet’s apparatus with ether. The extract in dilute ethereal solution was then saponified by sodium ethylate, and the precipitated soaps filtered off and well washed with ether. The ethereal filtrate so obtained was repeatedly shaken with water containing soda, to get rid of alcohol, some colouring matter, and excess of soap. It was then dried and the ether distilled off. The unsaponifiable matter differed widely with different diets in quantity and in character, being in some cases a viscid oil, and in others solid. . We hoped to be able to separate the cholesterol from this unsaponifiable matter by conversion into the dibromide, and taking advantage of the sparing solubility of the latter substance in petrol, but preliminary experiments showed that with the small quantities of cholesterol obtained the method was of no value from a quantitative point of view. We found it better to crystallise fractionally from absolute or from 85 to 90-per-cent. alcohol. The cholesterol was identified by microscopical examination, by the melting point after recrystallisation, and by conversion into the benzoate and the acetate. It was found better to convert the smaller crops of crystalline matter at once into benzoate, as this substance is very sparingly soluble in absolute alcohol. The acetate of cholesterol is readily prepared by boiling cholesterol with acetic anhydride and sodium acetate for a few minutes, pouring into water, and recrystallising from alcohol. The benzoate can be prepared by heating to 140° for half an hour or more with benzoyl chloride, treating the product with hot alcohol, and recrystallising from alcohol. We find, however, that it is a much better method in the case cf cholesterol to dissolve this substance in pyridine in the proportion of about 1 gramme to 10 to 20 ac. add a solution of a little more than the theoretical quantity of benzoyl chloride in pyridine solution, and allow to stand for several hours. The liquid is then poured into water, when the benzoate of cholesterol is precipitated, and may be recrystallised from absolute alcohol. The yield by this method is quanti- tative. The acetate of cholesterol melts at 114° C., and on cooling shows colour changes if pure. The benzoate melts at 145°5 C. to a turbid liquid, which suddenly becomes clear at 178° to 180°, and, in cooling, a brilliant display of opalescent colours is exhibited, among which a brilliant blue, appearing at about the temperature of the higher melting point, followed by a violet blue just before complete solidification, are most prominent. These colour changes are well marked and very characteristic of cholesterol. 1908.| Destiny of Cholesterol in the Anomal Organism. 229 The non-crystalline oily matters obtained from all the mother liquors in the above-mentioned fractional crystallisation processes were treated in pyridine solution with benzoyl chloride, in order to separate as benzoate any cholesterol that might have been retained in solution in the oils. In a few experiments, small amounts of cholesterol benzoate were obtained in this way from these oils, but in most cases none was found. The following is a brief account of the results of our experiments, using seven different diets :— (a) Oatmeai Porridge and Milk.—The oatmeal was well boiled with water containing a little salt, and milk added. During 21 days the dog consumed 3865 grammes of coarse oatmeal—or 184 grammes per day, and 2340 c.c. of cow's milk, z.¢., 128 grammes per day. During this period 1539 grammes of natural undried feces were obtained. On saponification in the manner described, a very large quantity of slimy soap was obtained, which was difficult to filter and wash with ether, and consequently had to be dried and re-extracted in the Soxhlet with ether. The unsaponifiable matter was obtained in the form of a red viscid oil, having a slight odour of peppermint. This oil was for the most part soluble in hot acetone, leaving a small amount of red slimy matter. On standing, the acetone deposited highly coloured and somewhat sticky crystalline matter. This was recrystalliged from alcohol, when 0:95 gramme of brown crystalline matter wes obtained. The alcoholic mother liquors, on standing, deposited some oily substance, and finally, on evaporating to small bulk, 0°35 gramme of sandy amorphous solid, which would not crystallise. The 0°95 gramme of coloured crystalline matter was recrystallised several times from 85-per-cent. alcohol, and was eventually obtained in three fractions, white in colour, and melting respectively at 134°, 137° to 139°, and 132° to 133°. The total weight was 0°5 gramme. A microscopic examination showed that these crystals consisted entirely of cholesterol. This was confirmed by conversion into the benzoate, which in each case melted correctly, and showed the colour changes in a well-marked manner. All the mother liquors were evaporated to dryness, and gave a stiff red oil. This dissolved for the most part in cold petrol, leaving 0-1 gramme of a red sandy solid melting at 120° to 130°. | On evaporating off the petrol, a viscid oil was obtained, which, on long standing, set to a crystalline waxy mass, weighing 1:25 grammes. This wax was dissolved in ethyl acetate, and, on standing, long white needle-shaped crystals separated, mixed with red colouring matter. The weight of crude crystals was 0°28 gramme. They were rather difficult to purify, but were eventually obtained in the form of glistening pearly leaflets easily soluble in 230 Messrs. Dorée and Gardner. Origin and [Feb. 10, ethyl acetate, but insoluble, or difficultly soluble, in petrol. This substance began to soften and sinter at 120° and melted sharply at 134°C. This was not cholesterol and was probably a phytosterol contained in the meal. The mother liquors yielded no further crops of crystalline matter, and dried to a viscid oil, which, on standing, showed no sign of erystallising. The yield of cholesterol was therefore about half a gramme or 0:023 gramme per day. (b) Cooked Beef and Mutton.—After the conclusion of the last experiment the dog was fed for a week or more on mixed diet, and given regular exercise, ' and appeared to be in good condition. It was then fed for 20 days on cooked beef and mutton with an occasional bone. The meat was fairly lean, but the fat was not specially taken out. The total weight consumed during the 20 days was 7470 grammes, or 373°5 grammes per day ; 413 grammes of undried feces were passed during this period, and, after treatment, yielded 2:1 grammes of unsaponifiable matter in the form of an oily solid. This was dissolved in alcohol, partially decolorised with charcoal, and fractionally crystallised ; 0°615 gramme of crude cholesterol was obtained. It was moderately pure, and, after recrystallisation from 85-per-cent. alcohol, melted at 135° to 138°. It gave a satisfactory yield of benzoate which melted rather high, 1475, to a turbid liquid which cleared at 160°, and, on cooling, showed the colour changes in a well-marked manner. The mother lquors deposited about 1 gramme of a brown sticky solid which would not crystallise. It was treated in pyridine solution with benzoyl chloride, but gave no trace of cholesterol benzoate. The yield of cholesterol was therefore about 0°615 gramme, or 0°037 gramme per day. (c) Sheep’s Bravn.—Atter the last experiment the dog was kept on mixed diet and allowed regular exercise for one month, when it was in excellent health. It was now given a diet rich in fatty matter and cholesterol—raw sheep’s brain—which was continued for 14 days. During this period the dog consumed 28 sets of brains, weighing in all 2129 grammes, and appeared to relish the food. It was also allowed 170 grammes of bone; 316 grammes of undried faeces were passed. The unsaponifiable matter was obtained as a buttery mass, weighing about 30 grammes. This, however, was not dry. On crystallisation from alcohol, three fractions were obtained, weighing respectively 9°8, 5°3, and 1:2 grammes. These were only slightly coloured and under the microscope appeared to consist entirely of long pointed needles : no typical cholesterol crystals could be seen. Each fraction, after recrystallising once from alcohol, appeared pure, and melted at about 96° C. On a careful fractional crystallisation no trace of higher melting bodies could be obtained. This coprosterol was saturated to bromine. The acetate melted at 88° to 89°, and the benzoate at 122° to 123° to clear liquids showing no 1908.|] Destiny of Cholesterol in the Animal Organism. 231 colour effects on cooling. Three crops of crystals, examined with the polariscope in chloroform solution, gave the following results :— (1) [a]p = +23°07, (2) [eJp = +23°7, (8) [a]p = 422°. It thus appears identical with the coprosterol of human feces described by Bondzynski and Humnicki.* To confirm this point, the whole of the material was converted to the acetate, which was repeatedly crystallised from alcohol and acetone. The highly pure ester was saponified with sodium ethylate in ethereal solution, and the product twice recrystallised from alcohol. A sample of human coprosterol was similarly worked up and purified, and in the following table we give a comparison of their properties. Coprosterol, dog. Coprosterol, human, Crystallises from alcohol in Long flexible needles Long flexible needles. IMeIGIMG; PONE... 65.00.5020 Falls together at 98°; melts, Falls together at 98°; melts, 99°—100° $9°—100°. Rotation [a ]pin chloroform + 23°.°7 + 23° °5. RCOEMUO se hes pasio es baceucees o¥eiepi Needles from alcohol; m.p., | Needles; m. p., 88°—89°. 88°—89° Benzoate (prepared by the Leaves, difficultly soluble in Leaves, difficultly soluble in pyridine method) alcohol; m.p., 122°—123° | alcohol; m. p., 121°—122°. The mother liquors, on standing, set to a brown oily mass. This was put on a porous tile and the oily part drained away. The dry solid matter was heated to 140° with benzoyl chloride, and then treated with hot alcohol. It dissolved readily with the exception of a small quantity which was only got into solution with difficulty. On cooling, about 0°2 gramme of white crystalline matter separated. This was recrystallised from alcohol and melted at 141° to a turbid lquid, which on cooling showed the brilliant colours of cholesterol benzoate. The mother liquors, on spontaneous evapora- tion, yielded a small quantity of brown crystalline matter. This was very easily soluble in absolute and dilute alcohol, but was not further investigated. ‘The yield of coprosterol was thus 16 to 17 grammes, or 1:14 to 1:2 per day. | (d) Cooked Horseflesh—After the last experiment the dog was allowed to rest for a month, and was then fed for a month on porridge made from coarse oatmeal, and water. The product was unfortunately lost. The animal was then given another month’s rest with aneordinary mixed diet. At the end of this period it was fed for 17 days on cooked horseflesh with scarcely any fat. In the middle of this experiment there was an interval of four days, *® ° Zeit. Physiol. Chemie,’ vol. 22, p. 396. 232 Messrs. Dorée and Gardner. Origin and [Feb. 10, during which the dog had a diet of dog biscuit, but the feces were not collected during this interval. During the period it consumed 6758 grammes | of meat, or 397 grammes per day. With a view to sweeping out the gut at the end, it was fed for two days on bread and bovril; 388 grammes of feeces were collected, which on drying at 80° weighed 155 grammes ; 347 grammes of unsaponifiable matter in the form of a red oil were obtained; this was very liquid at 100°, and had a soapy smell. With the exception of a little tarry matter this was soluble in alcohol, and, on evapora- tion, two crops of crude cholesterol were obtained, weighing respectively 0°67 and 0:074 gramme. This was identified by conversion into the benzoate, and the yields showed that it consisted practically entirely of cholesterol. The mother liquors, on evaporation, yielded oil, which, on treatment with benzoyl chloride in pyridine solution, yielded 0°54 gramme of brown erystalline matter which, after recrystallisation from ethyl acetate, melted to a turbid liquid at 142° to 145°. This cleared at about 180°, and on eooling showed the characteristic colour changes of cholesterol benzoate. The total yield of cholesterol was, therefore, approximately 1 gramme, or 0-06 gramme per day. (e) White of Egg, Bread, and Cream.—Immediately after the conclusion of the last experiment the animal was put on the above diet. The daily ration was prepared by mixing about 150 grammes of bread with the whites of three eggs and half a teaspoonful of cream. This was fried and moistened with a warm dilute solution of Liebig’s extract of beef. This diet was continued for 14 days, during which the animal consumed 2092 grammes of bread and the whites of 42 eggs. This proved a very nourishing diet, and at the end of the experiment the dog was in excellent condition ; 130 grammes of faces were passed, and weighed, after drying at 100°, 35 grammes. On saponification, the soap was white in colour and not large in amount. The ethereal solution of the unsaponifiable matter was pale yellow, and on ‘evaporation gave 0°59 gramme of a sticky yellow solid. This, with the exception of a small amount of tar, was soluble in 90-per-cent. alcohol, and on cooling the solution set to a mass of crystals. A small portion was examined under the microscope, and appeared to consist of small rosettes of needles, but no cholesterol crystals were at first observed. On dissolving again on the slide and recrystallising, a small patch of typical cholesterol crystals was observed, but the bulk consisted of the rosettes referred to. The total weight of crystalline matter was 0°3 gramme. ‘This was decolorised by animal charcoal, and crystallised from the least quantity of 85-per-cent. alcohol. 1908.] Destiny of Cholesterol in the Animal Organism. 233 The first and main crop of crystals, after drying at 100° C., melted at 137° to 140°. The acetate, after several recrystallisations, melted at 120° to 122°, and no colour changes were noticed. The benzoate began to soften at 138°, and melted to a clear liquid at 142° to 143°. On cooling, no colour changes could be observed. The substance was probably sitosterol, derived from the bread. The second and smaller crop from the mother liquors of the above was decolorised and recrystallised from alcohol. This was not pure, and the quantity was too small for further purification. It began to melt at 120°, but the process was not complete at 135°. It was converted into the acetate, which after recrystallisation melted sharply at 113° to 114° C. This was probably cholesterol acetate, though no colour changes were noticed with certainty. The soapy matter left on evaporating the mother liquors was treated with benzoyl chloride in pyridine solution, but no crystalline matter could be obtained. In this experiment, therefore, only traces of cholesterol were obtained. (f) Oatmeal Porridge.—As the dog at the conclusion of experiment (e) was’ in excellent health, it was at once put on a diet of porridge made from coarse oatmeal and water with a little salt. This diet was continued for 31 days, during which the dog consumed 2870 grammes of oatmeal ; ' 212 grammes of dried faeces were obtained. The ethereal extract, measuring about 1°5 to 2 litres, on saponification set to an almost solid mass of soap. This, however, filtered easily, and was readily washed. The unsaponifiable matter was a reddish brown oil, weighing 2°7 grammes. With the exception of 0°27 gramme of tarry matter this was soluble in alcohol. On standing, the solution deposited a slimy brown mud. This was taken up in ethyl acetate, and, on standing, the solution deposited several crops of white needle- shaped crystals, the total weight being 0°255 gramme. The ethyl acetate mother liquors, on spontaneous evaporation, yielded an oil. The needle-shaped crystals were readily soluble in petrol and in alcohol, but did not crystallise readily from these solvents. After several recrystal- lisations from ethyl acetate the substance was obtained in the form of long, flat, slender needles, which melted sharply at 133° to 134°C. The acetate began to soften at 70°, and melted to a clear liquid at 98° C., but we had not enough for a satisfactory purification. It was evidently the same as the phytosterol obtained in experiment (a). The alcoholic mother liquors _ from the above-mentioned muddy solid, on spontaneous evaporation, gave an oil through which crystalline matter was disseminated. This was recrystallised from 85-per-cent. alcohol when 0:172 gramme of whitish crystals was deposited. Under the microscope these were found to consist 234 Messrs. Dorée and Gardner. Origin and [Feb. 10, of long hexagonal plates, and no typical cholesterol crystals were observed. After recrystallismg from absolute alcohol until quite white, the substance melted at 124° to 125°. The acetate crystallised in microscopic plates, which melted at 118° to 122°. These properties agree with those of the para-sitosterol of Burian. All the mother liquors, from which the above-mentioned crystalline substances were obtained on spontaneous evaporation yielded viscid oils, which really constituted the bulk of the unsaponifiable matter. In order to ascertain whether any cholesterol or other crystalline substances were held in solution in these oils, they were dissolved in pyridine and treated with benzoyl chloride. After standing, the solutions were poured into water and the slimy deposits dissolved in hot absolute alcohol and left to crystallise. One oil yielded 0:094 gramme and another 0°04 gramme of crystalline matter, difficultly soluble in absolute alcohol. On purification, this was obtained in the form of glistening plates which melted at 145° to a turbid liquid. This: cleared at about 175° and on cooling showed the colour changes characteristic of cholesterol benzoate. The mother liquors, on spontaneous evaporation, dried to a viscid oil, which, on long standing, showed no sign of crystallising. Only about 0-1 gramme of cholesterol was therefore obtained in the month, or 6:003 gramme per day. (2) Rice, Gelatine, and Butter.—After experiment (f) the dog was fed for four or five days on dog biscuit and porridge and given regular exercise, and as it " appeared to be in good health, the final experiment was proceeded with. Each day’s ration consisted of 114 grammes of rice, which was well boiled and mixed with 114 grammes of a 10-per-cent solution of gelatine and 28 grammes of butter. The mass was flavoured with a dilute solution of Liebig’s extract of meat. During 18 days the dog consumed 2052 grammes of rice, about 205: grammes of gelatine, and 5V4 grammes of butter. The weight of dried faces: was 115 grammes. 1:316 grammes of unsaponifiable matter was obtained as a vaseline-like oil.. This, with the exception of 0°06 gramme of tarry substance, dissolved in 50 c.c. of hot 90-per-cent. alcohol. Three crops of impure crystalline matter were: obtained, (1) 0°37 gramme; (II) 0°16 gramme; and (III) 0:14 gramme. The mother liquors dried to an oil, which, on long standing, showed no sign of erystallising. Crop I, after decolorising and recrystallising from 90-per-cent. alcohol, was obtained in the form of white crystals not unlike cholesterol in appearance. On heating, these began to soften at 120° and melted at about. 129°. A microscopic examination showed that the substance was a mixture. We tried to separate the substances by means of the benzoates. The crude benzoate obtained by the pyridine method crystallised from alcohol, apparently 1908.] Destiny of Cholesterol in the Animal Organism. 235 in two forms, one light and floating in the liquid and the other granular and adhering to the sides of the beaker. These were separated as far as possible and recrystallised separately. The light form, after recrystallisation from alcohol, in which it was sparingly soluble in the cold, melted at 139° to a pale brownish liquid which was slightly turbid, but not so markedly as is usually the case with cholesterol benzoate and did not become any clearer at 170° to 180°. On cooling, no colour changes were observed at the higher temperature, but at the point of solidification a transient lilac blue appeared, but was not well marked. This substance might have been impure cholesterol benzoate, but we could not be certain. The granular portion, which was the greater quantity, after recrystallisation shrank together at 138° C. and melted sharply at 139° C. to a perfectly clear liquid. It showed no colour changes and was not cholesterol benzoate. Crop II was decolorised by charcoal and recrystallised from alcohol. It began to soften at 120° and melted at 135°. It was mostly cholesterol, for the benzoate made by the pyridine method melted at 140° to 143° to a turbid liquid which went clear at 178° and on cooling showed the colour changes in a well-marked manner. Crop III was very impure and greasy. It was therefore treated at once with pyridine and benzoyl chloride. A minute amount of crystalline matter was obtained which melted at 145° to a turbid liquid and appeared to clear at 178°. It was, however, yellow in colour, and we could not observe the colour changes. The oils from the mother liquors were benzoylated, but no crystalline matter was obtained. In this experiment, therefore, 0°667 gramme of impure crystalline matter was obtained. This contained some cholesterol, but we could not determine the amount. We do not think that more than one-third of it was cholesterol, certainly not one-half. The rest evidently consisted of phytosterols from the rice. Discussion of Lesults, Different observers have given very variable figures for the daily quantity _ of bile secreted by the dog, as the following table shows :— Quantity of bile in grammes secreted per kilogramme-weight Observer. in 24 hours. Hriedlander and Barisch® *......:....... 19°9 Biddercamd Schmidt ......csc.ccc-+ ccs -0< 24°5 Ietoe ninadmie No... c ss cod deeeeeecwec dees. Bh era Ce The... Sea caadt eas mavens 10:0 VOL. LXXX.—B. E 236 Messrs. Dorée and Gardner. Origin and [Feb. 10, If we take the mean of the above results as approximately correct, the daily secretion of bile by our dog would be 291 grammes. For the content of cholesterol in 100 parts by weight of dog’s bile, Hanne: Seyler gives the following figures :— (a) Bile from bladder. (6) Bile from fistula. (Ca A, Cae eS) i II. 1; i 0-449 0133 0-074 0-049 We do not know how far the bile poured into the gut would resemble either bladder bile or fistula bile, but, calculating on the basis of the lowest and highest values given, our dog should have received into its intestine, along with the bile, between 0°14 and 1°31 grammes of cholesterol per day. If it be the case that cholesterol is a true excretion product got rid of in the feeces through the agency of the bile, this quantity should have been found in the excreta along with that contained in the food consumed, whereas, as shown in the following table, in which we compare the amounts of cholesterol actually found in our experiments with those that should have been found on the assumption that the cholesterol of the bile is all excreted in the feces, the amounts found in most cases were less than one-fifth of the lowest of the above -values. It is clear from the above experiments that the whole of the cholesterol of the bile is not excreted in the feces. It must, therefore, have been either totally destroyed, which, considering the great stability of cholesterol, is highly unlikely, or reabsorbed in the gut along with the bile salts, which is the more reasonable explanation. It might be contended that the cholesterol had been changed in the intestine into the unsaponifiable oil found along with the cholesterol, but this is probably not the case, as the oils obtained were very variable, both in quantity and appearance, and further, the weights of such oils obtained were usually quite insufficient to account for the cholesterol. How far the quantities of cholesterol found can be accounted for as cholesterol contained in the food and passed unchanged is more difficult to answer, as we have little exact knowledge of the cholesterin content of the various food stuffs. In our opinion, however, it can be largely accounted for in this way. Considering the two een with cooked meat, in which the cholesterol recovered for 20 days was only 0°6 and 1:0 respectively, these amounts might well have been due to the food taken. As is well known, animal flesh contains cholesterol, though quantitative estimations have, as far as we are aware, not been made. Such estimations would be tedious and ve aw) «: 237 . “3 = = 18. I—FT- 0 410-0 KS T&- I—FI- 0 €00- O ~ 5 > Ss Te. I—FI- O So0BI] [[BUG a ‘T&- I—FT- 0 690- O re ~_ : S [&- T— VI. 0 «GT La) S = 18. I—*T- 0 180-0 ~_ A) ~ S Tg. I—FL- 0 &Z0- O = ‘£THo op1q UWLOL “SOULUL BIS > peonpoad useq | ut dep zed ‘S savy prnoys pessed % yey} Aep [OLe4soToYo Jo QR rod [ore4soToYo qunouLy jo yndgno 1908.] 9-€6— 9-6 9. OV—VE- D €-8I— 6-T LG. GE— BE: B &- 8I— 6-T 6-96— 8. G. LE—¥6. & ‘ToLaqsa[oyo 4ou ‘Toaoysoadog ri 6-0 UBY} ILOUL JON T-0 a0VL], 0-1 *J1—9T ST9- O c-0 ‘uoldumsse 940qe UO Sy Ot poyetoxe W90q oavy plnoys yey} [OLeqyseToyo jo qunoury "SOUULBAS UL punoy [oLaysaToyo Fo VysTom [Ry], &- 1 L-& 6¢- 0 LY. € (Aap oymb you) 0. O& 1-G "SOWULRIS UT 104yeu0 eTqeyruodesun jo qYSIoM [IO], JO FY S1OM | Loquin yy CIT = GIG =e cs O&§T SST 88E oa 9T€ = SIV os 6ESI *SOULUIBIS eae rita ur 0 OT | gy pelipun Tepe So0eT SORT JO JYSTOM 140], T?99.L 8I Té al Lt VI 06 1 ‘skep jo aa H 10994nq FOS oUlze[ES COZ 1aqynq pue e0IL ZGOS | ‘ouyRled ‘sony “1H OL86 vee" TBoulzeO “A UIRerO SozIyM S39 OF | puw ‘88a Jo pue 2606 | OFM ‘peorg “Wf 8GL9 “'"Ts9fesdoH “| ureaq 6212 sdooys Mey ‘Oo u0}jnu pue OLFL Jo9q = pe HOOK) “EL y[rut LVIP pus j[eomzyeTO “VY *SOTUUIRId Ul poof prjos Fo “PIT qystom [eqIOT, 238 Origin and Destiny of Cholesterol in the Anomal Organism. difficult to perform, as they would involve the artificial digestion of large quantities of material, for it has been shown that it is impossible to extract the whole of the fatty matter from meat without such digestion. Further- more, the quantities would probably have been variable. In the two experiments quoted, however, the cholesterol found would only have meant, in the case of beef and mutton, a content of 0-008 per cent., and in the case of horseflesh of 0°015 per cent. In experiment (f), with an oatmeal diet, we found only 0:1 gramme of cholesterol. Whether oatmeal contains any traces of cholesterol we cannot say, but on extracting three days’ diet we were unable to recognise any in the unsaponifiable residue. Taking this 0:1 gramme as a basis for calculation in experiment (a) (oatmeal and milk), the yield of cholesterol attributable to oatmeal would have been 0°135 gramme. Milk fat, according to Schmidt and Mulheim, A. Bomer, and A. Kersten, contains 0°5 per cent. of crude cholesterol, which Menozzi has shown to be identical with that of bile. If we take our milk as containing between 3 and 4 per cent. of fat, the amount of cholesterol from this source should have been for the quantities taken in experiment (a) between 0°35 and 0:468 gramme. The total cholesterol to be expected would therefore be between 0°485 and 06 gramme. We actually found about 0°5 gramme. In experiments (e) and (g) the traces of cholesterol found could be accounted for as due to the food. Experiment (c), on a diet of sheep’s brain, is of special interest, not only because of the quantities obtained, but because the cholesterol was entirely in the form of coprosterol. The brain substance is well known to be rich in cholesterol. The human brain contains about 2 per cent. Whether the content of cholesterol in the brain of other animals is as high as this we do not know. On the assumption that it is the same as in the human brain we should have expected in the feces of a period of 14 days between 25 and 30 grammes of cholesterol. We actually found 16 to 17 gr. of coprosterol. There can be no doubt that this was due to the diet. In man, according to Bondzynski and to Miiller(and in agreement also with our own experience), the change of cholesterol into coprosterol in the gut by bacteria, is the normal process, and if the gut is cleared of the particular bacteria by prolonged milk diet, cholesterol is excreted as such. In the dog, fed on either cooked vegetable or meat diet, cholesterol is normally excreted as such. In the case of raw brain we obtained coprosterol only. The cause of this we must reserve for future investigation. It may have had something to do with the fact that the food was uncooked, or that it was unusually rich in fat. Unfortunately, we were unable to ascertain - Bacteria as Agents in the Oxidation of Amorphous Carbon. 239 whether the state of the gut favourable to the formation of coprosterol persisted when the next diet (oatmeal) was tried, as, owing to an accident, the whole of the ethereal extract was lost. In the subsequent experiment with horseflesh, the gut had recovered its normal condition. Whether any of the cholesterol of the food is actually absorbed along with that of the bile in the intestine, these experiments do not show, but others are in progress, which we hope will throw light on this point, and this we hope to make the subject of a communication in the near future. The expenses in connection with this work were defrayed by means of a grant made by the Government Grant Committee of the Royal Society, for which we take this opportunity of expressing our thanks. Bacteria as Agents in the Oxidation of Amorphous Carbon. By M. C. Portrer, M.A., F.L.S., Professor of Botany, Armstrong College, in the University of Durham. (Communicated by J. B. Farmer, F.R.S. Received March 13, 1907,—Received in revised form, with additional matter, January 18,—Read March 12, 1908.) The problem which presented itself to my mind in commencing the following investigation was primarily one connected with agriculture. When considering the application of such insoluble substances as charcoal, cinders, soot, etc., to the land, an explanation was sought of their ultimate fate in the soil. What becomes of the carbon? Is it oxidised into COs, and if so by what agency ? Little is known at present as to the means whereby amorphous carbon is rendered available for plant life, except through its union with oxygen in the process of combustion, and further investigation upon this point offered an important field of enquiry. It is well established that carbon readily absorbs oxygen, and, in the case of coal, that carbonic acid is given off, but the cause of the latter phenomenon is still obscure, and in the theories advanced to account for it no consideration is ever given to the possible action of micro-organisms. My investigations have shown that under the action of certain bacteria a slow oxidation of amorphous carbon takes place, CQ2 is slowly evolved, and the carbon can thus be at once utilised for the nutrition of green plants. 240 Prof. M. C. Potter. Bacteria as [Jan. 18, This led to a wider consideration with regard to the action of bacteria upon certain carbon compounds such as coal and peat, and opened up the question as to whether it was possible that the vast supplies of carbon locked up in these formations could be utilised for plant life without the intervention of cirect combustion. That coal undergoes considerable wastage when exposed to the air is a fact very generally known, and in the case of large storages of coal the depreciation may reach a very high percentage. The conditions often preclude this loss being entirely attributed to weathering, and the question arises: are there then any other agents, such as bacteria, concerned in the process of disintegration ? In experiments dealing with substances of the nature of coal, peat, and charcoal, quite peculiar care had to be taken for the purpose of sterilisation, which proved to bea very difficult matter; while the chemical changes which take place during heating these substances to such a degree as was necessary, and the probable conservation of gases, were all points requiring due consideration for the elimination of possible sources of error. Further, the highly sensitive character of the electrical apparatus employed rendered special precautions necessary to guard against extraneous influences. It. will therefore be needful to give in detail, though as shortly as possible, the methods employed in conducting my research. The problem was attacked by three distinct methods, briefly stated as follows :— 1. By passing a stream of air, freed from all trace of COs, over the’material subject to investigation and determining the presence of COs by titration with standard oxalic and hydrochloric acids. 2. By determining the rise of temperature due to oxidation by means of a thermopile and galvanometer. 3. By detecting, in the case of charcoal, the presence of calcium carbonate in the flasks inoculated with the bacteria. Charcoal, Pieces of ordinary wood-charcoal were pounded and passed through a double sieve, the first having a mesh of 1/10 inch, the second of 1/20 inch, so that in this way small fragments of fairly uniform size were retained between the two. A quantity of this charcoal was next heated by means of a metallurgical furnace to a white heat (about 1200°), in a crucible protected from any access of atmospheric oxygen. Heating to this point was a necessary treatment, as commercial charcoal is seldom sufficiently charred to drive o all volatile compounds. The entangled oxygen would thus, in the presence of an excess of carbon, be mainly converted into CO, and the calcium salts '— 1908.] Agents in the Oxidation of Amorphous Carbon. 241 naturally contained in the woody tissue into the oxide. This process was also important as an efficient means of sterilisation, and for the destruction of any organic matter in the shape of dust with which the material might be contaminated. Determination of CO by Titration. The apparatus employed was in main outline that used by Sachs in his classical experiment on Respiration, with modifications to suit the special difficulties in this case. After some preliminary trials the form of apparatus finally adopted was as follows :— Air drawn by means of an aspirator was passed first through a Reiset absorption apparatus containing 100 c.c. of a strong solution of caustic soda, next through a similar apparatus containing an equal volume of baryta water, then through the flask containing the material under investigation (known henceforth as the research-flask), and finally through a Reiset containing 100 c.c. of baryta water. To avoid corks and the consequent difficulty of ensuring their perfect sterilisation, specially constructed research-flasks (Cloéz flask) were employed, in which the delivery tube, reaching nearly to the bottom, and the exit tube were fused into the neck of the flask. The flasks were thoroughly cleaned and subjected to the vapour of boiling nitric acid for some hours, a necessary precaution to remove any trace of organic matter, as such substances might give rise to CO, under bacterial action. All trace of nitric acid was then removed by water condensed in the flasks, this water being derived from the steam of distilled water. Such treatment rendered the flasks perfectly clean, and precluded the slightest fear of any contamination. Into such a flask about 5 grammes of charcoal, freshly heated as previously described, were introduced by means of a clean platinum spatula, the entrance and exit tubes were then plugged with asbestos heated to redness and short pieces of rubber tube fitted to them. The entrance tube was next connected with a flask containing distilled water, and steam from it blown through the research- flask. After enough water had condensed to cover the charcoal and all the air had been expelled from the flask, the apertures were securely closed by clamps while the steam was still passing through. The flask was then allowed to remain for 24 hours, during which time the charcoal would be exposed to a partial vacuum. After this interval air was allowed to enter the flask, the air being passed first through a Mohr’s bulb containing strong potash, and then through a short glass tube which was plugged with asbestos freshly heated. The entrance and exit tubes, after disconnection, were again heated to redness, steam passed through, and the flask closed as before. This 242 Prof. M. C. Potter. Bacteria as |. [Jane tat operation was repeated daily for at least three days. In this manner any gases entangled in the charcoal would be removed, and at the same time complete sterilisation would be effected. Finally the excess of water was removed by evaporation. Asbestos plugs, previously heated to redness, were always employed in place of cotton wool, as the latter might give rise to COs, and thus become a source of error. Several sets of apparatus, as described, were set up, and into some of the research-flasks bacteria were introduced, while the others were used as controls. The bactertwm was obtained from the soil. For the purpose of isolation a number of test-tubes were partially filled with the reheated charcoal moistened in distilled water and sterilised in a steamer. A small quantity of garden soil was shaken up with water, and, after allowing the coarser particles to settle, about 1 cc. of this water was introduced into one of the test-tubes, which was then placed in an incubator at 20°C. After two days a similar test-tube was inoculated from the first by means of a loop of platinum wire, the process was repeated in a third test-tube, and so on. Those bacteria which could not live on charcoal were thus gradually eliminated, and finally, by constant inspection, a Dzplococcus, diameter 1 w, was obtained in pure culture, which was employed for this research. (There is, however, no reason to suppose that this species alone is capable of oxidising carbon, probably it is a property possessed by many other species.) The bacteria were introduced into some of the research-flasks by removing the asbestos plug and pouring in a little distilled water containing the Diplococcus. The aperture was then closed as quickly as possible, the asbestos replaced, and the whole end of the tube heated to dull redness in the Bunsen flame. The inoculated flasks and the controls were then treated in a precisely similar manner. The apertures were closed with rubber tubes and glass stoppers, and all the flasks placed in an incubator at 20° C.; at intervals of a week a stream of air—about 5 litres—was drawn through the apparatus, and titrations were made of the baryta water, great care being taken to prevent the latter from absorbing any CO, from the air during this process. For the first week no CO, could be detected in the air from either the controls or the inoculated flasks. This result was not encouraging, and at this stage of the proceedings it appeared as though the investigation might. prove fruitless. However, after nearly another week the air contained in the inoculated flasks gave a distinct indication of the presence of COz, The amount detected in this way was never large, only amounting to 7 milligrammes: = 1908.| Agents in the Oxidation of Amorphous Carbon. 243 per week, but it was measurable and, continuing to be demonstrable while the controls exhibited no trace of this gas, it was sufficient to encourage the further prosecution of the research and the endeavour to confirm the results by other means. A parallel series of experiments with charcoal in which 5 litres of CQo- freed air was drawn each morning through the research- and control-flasks gave similar results. In this arrangement the baryta water was contained in Pettenkofer tubes and titrated every seventh day. The control-flasks showed no trace of COs, while from the research-flasks, after-the first week, an average of 8 milligrammes per week was obtained during a period of one month. An explanation of this delayed result may be found in the fact that calcium salts are contained in the plant cells from which the charcoal was derived ; the calcium oxide therefore present in the charcoal would combine with the COs as soon as formed, and until the process of neutralisation was completed no CO, would be present in the stream of air. Also a sufficient time was required for the growth and multiplication of the bacteria. Determination of Calcium Carbonate. If the explanation given above were true, calcium carbonate. should be present in the inoculated flasks but absent from the controls. Therefore the next step taken was an endeavour to detect the presence of calcium carbonate among the charcoal fragments. A small portion of charcoal was removed from the inoculated flask and mounted as a microscopic slide; a weak solution of acetic acid was then run under the cover-shp and an evolution of bubbles was immediately seen to take place, while no such evolution could be observed in the charcoal from the controls. A further confirmation was found in the fact that when freshly-heated charcoal was moistened with water and treated with acetic acid no bubbles. appeared, but after exposure for some hours to an atmosphere containing COs», a similar treatment with acetic acid resulted in a vigorous evolution of gas. An objection might be raised that these bubbles were due to the displacement of gases included in the charcoal, and proof is wanting that they were in reality CO This proof was supplied by treating the charcoal in one of the inoculated flasks with weak hydrochloric acid, and then passing a stream of air-free CO, through it and then through baryta water. When this was done, barium carbonate was precipitated, and titration showed that 33 milligrammes of CO2 had been evolved. 244 Prof. M. C. Potter. Bacteria as [Jan. 18, A further test in confirmation of the above was afforded by an artificial imitation of the conditions. About 5 grammes of freshly-heated charcoal was exposed to a partial vacuum in the manner before described, in two Cloéz flasks. One of these was filled with CO, and the other with air free from CO. After standing for 24 hours a stream of air freed from CO, was drawn through each of the flasks, and all traces of CO, would thus be effectively removed. About 5 cc. of weak hydrochloric acid was then introduced into each and connections made as speedily as possible with the Reiset absorption apparatus. After the lapse of 24 hours the first flask showed that 34 milligrammes of CO, had been given off from the charcoal, and in the second flask only 2 milligrammes, this small amount being possibly due to some residual carbonates which had escaped reduction in the charcoal. As the analysis of ordinary wood-charcoal gives about 3 per cent. of ash, and lime is one of the chief constituents, the above readings are in agreement with the amount of lime normally present in the charcoal and also with the amount of CO, given off from the research-flasks (inoculated with bacteria) when treated with weak hydrochloric acid. This further proves the truth of the supposition that CO2 is only evolved after the calcium oxide has been converted into the carbonate. Control experiments made with distilled water inoculated with bacteria, without any charcoal, etc., gave no evolution of COs, thus disposing of any criticism which might suppose the carbonic acid to be derived from the bacteria themselves, and not necessarily from their action upon the amorphous carbon. Moreover, the evolution of COs is not confined to the duration of the first experiment, and subsequent titrations made from the same material, which had been returned to the incubators for a further period, invariably demonstrated a further production of COs, thus indicating a continuous process of evolution. Hlectrical Method of Determining a Rise of Temperature. Since the phenomenon of oxidation is accompanied by an evolution of heat, it follows that if charcoal were undergoing oxidation it should be possible to detect any rise of temperature due to this process. The amount of CQz evolved, however, being small during the period of observation, the rise of temperature to be expected would at most be only a fraction of a degree. In order to discover whether there was any difference in temperature between the sterile and inoculated charcoal, the apparatus shown in the figure was designed. It consisted of two specially constructed flasks con- nected with a thermopile and placed in an incubator, with leads passing through a perforation in the side of the incubator to a galvanometer. “ot + fhe ‘ : ™ Cre 1908.] Agents in the Oxidation of Amorphous Carbon. 245 Measurement by means of the galvanometer of the E.M.F. produced by two thermo-elements at different temperatures, one placed in a sterile and the other in an inoculated flask, served to determine the difference of temperature between the two. | Diagram of Interior of Incubator. AA, Double-walled vacuum flasks. B, Thermopile, with terminals inserted in test-tube inside these flasks. , Jar packed with cotton-wool, containing junctions of thermopile wires D and galvanometer leads E. A thermopile with 20 junctions of the iron-nickel combination was employed and formed a highly sensitive instrument. The wires, silk-covered, 32 gauge, had the junctions carefully brazed together and coated with shellac, and the wires themselves were enclosed in thin rubber to give protection against moisture. The mirror galvanometer used was of the Broca type with a resistance of 50 ohms, and the electromotive force produced by a difference in temperature of 1/200° C. between the terminals registered a deflection of one scale- division. In an experiment of this nature, extreme precautions must be taken with all the junctions of different metals to avoid any thermal effects. To ensure that the junctions of the thermopile wires and leads to the alvanometer were maintained at the same temperature, these were connected with binding screws, insulated with thin rubber, and packed with cotton wool in a glass jar placed inside the incubator. The galvanometer leads passed through:a small perforation in the side of the incubator to a key of special construction and finally to the galvanometer. 246 Prof. M. C. Potter. Bacteria as [Jan. 18, A solid block of paraffin formed the basis of the key. Into this block four holes were bored to serve as mercury cups and these were connected in pairs by a metal bridge. One cup of each pair received the wires. from the galvanometer and into the others were fixed short glass tubes. Through these tubes the ends of the wires from the incubator could be raised or lowered to break or make a contact. The key was packed with dried cotton wool and enclosed in a box, the two glass tubes slightly projecting through perforations in the lid. Section of Special Key. A, Paraffin block with two mercury cups. B, Lead to galvanometer. C, Lead from incubator. D, Connecting bridge of copper wire. All the junctions of the different metals were thus carefully protected from light or changes of temperature which might set up an electric current, however small, and thus give rise to experimental error. | The accuracy of the measurements of temperature, as recorded by means. of the thermopile and galvanometer, is shown by the fact that the calculated. differences of temperature were found to correspond with those registered. by standard mercurial thermometers under the same conditions. The Hearson’s incubator was first regulated for 20° C., but as this. apparatus was heated from one side, it was necessary to ascertain whether the temperature inside was uniform. The terminals of the thermopile were packed with dry cotton wool in glass jars, and placed in different positions. inside the incubator, and by this delicate method a difference of temperature: of 0°09 C. was detected between the two sides. On discontinuing the heat. and allowing the incubator to assume the laboratory temperature, 14° C., this difference gradually disappeared, and I was able to determine, by | 1908.| Agents in the Oxidation of Amorphous Carbon. 247 renewing the tests, that the air contained within the incubator was then maintained at a strictly uniform temperature. The galvanometer leads passing through the incubator wall obviated the necessity of opening and closing the door, and permitted the research-flasks to remain quite undis- turbed. The glass flasks designed for this experiment were constructed with double walls. The inner flask, with a capacity of about 300 cc., was prolonged into two tubes diametrically opposite to each other, the upper one serving for the introduction of the charcoal and the lower for the escape of any CO, that might be formed. This inner flask was surrounded by a similar larger flask to provide a vacuum, the intermediate space being exhausted of all air in order to prevent any radiation of heat. Two similar vacuum-flasks were employed. After washing with fuming nitric acid, to remove any organic matter and for the purpose of sterilisation, they were filled with freshly-heated charcoal, which was moistened with sterile distilled water until it had absorbed all the water possible, the surplus being allowed to escape by the lower tube. It is important to note that the flasks were filled from the same supply of distilled water and with the same quantity. Into each of these flasks there was then inserted a thin test-tube previously washed externally in nitric acid and sterilised over a Bunsen flame, and the flasks were plugged with sterile asbestos wool. Finally, the iron-nickel thermopile was introduced into the test-tubes. The flasks were then placed in the incubator, the connections made with the galvanometer, and the apparatus was complete. Observations were taken at frequent intervals. When first arranged, the unavoidable handling of the metallic junctions necessarily produced thermo-electric currents, but these died away when the apparatus was left undisturbed for two or three days. When on depressing the key no movement of the spot of light could be observed, the whole arrangement was considered to be perfectly reliable, and the experiment could be proceeded with. One of the flasks was then inoculated with the bacterium, and thereafter readings with the galvanometer were repeatedly taken. At first no deflection was apparent on depressing the key, but after two days a move- ‘ment of the spot of lght could be observed, and by depressing the key synchronically with the swing of the mirror, a deflection of several divisions of the galvanometric scale could be registered. The thermo-electric current gradually increased until after an interval of six days the maximum deflec- tion of 38 divisions was attained, proving a definite rise of temperature in- the inoculated flask, while the sterile flask indicated no increase. To ascertain with certainty that the movement of the mirror was a correct 248 Prof. M. C. Potter. Bacteria as [Jan. 18, measure of the electromotive force, and in reality due to the difference of temperature between the two flasks, the terminals of the thermopile were interchanged, that is, the terminal in the inoculated flask was placed in the sterile flask and vice versé. The galvanometer then indicated an equal current in the reverse direction (after a sufficient time had elapsed to counteract the effects due to handling), thus proving that the deflection was due to the difference of temperature and not to any accidental error in the apparatus. By measurement, the maximum deflection indicated by 38 scale-divisions corresponded to a rise in temperature of 0°19 C., and this temperature was maintained for the further period of a week, when the apparatus was. taken down. This conclusively establishes the fact that a measurable rise of temperature takes place wm charcoai, owing to oxidation through the action of bacterva. Lamp-black. Experiments were also undertaken with commercial lamp-biack as another source of amorphous carbon, the results of which may be briefly stated. This substance in the first instance was heated to a white heat in the metallurgical furnace ; afterwards it was soaked in aqua-regia for nine days, then carefully washed in distilled water, and any excess of acid still remaining neutralised with metallic sodium cut from the centre of a block to ensure the absence of any naphtha. The lamp-black so prepared was: treated in the Cloéz flasks in the manner described for charcoal, and some were inoculated with bacteria, while the rest were kept as controls. For a period of 24 days the flasks inoculated with bacteria showed, upon titration, the evolution of a small quantity of CO, (17 milligrammes) as compared with the non-inoculated flasks. The result of the titrations agreeing so closely with those obtained in the case of charcoal, it was not deemed necessary to determine the rise of temperature by the thermo-electric method. Peat. A set of experiments upon peat, corresponding to those already described. for charcoal, was carried out in an exactly similar manner, except that the peat was not calcined; it will not, therefore, be necessary to do more than . briefly state the results. The peat was obtained from the Solway district, and was of the ordinary dried kind such as is used for fuel, Sphagnum being the chief constituent. 1908.] Agents in the Oxidation of Amorphous Carbon. 249 First, as regards the evolution of COs Several flasks were prepared containing small fragments of the dried peat, soaked with water, and sterilised by discontinuous boiling. A stream of air, carefully freed from all trace of COs, drawn through the research-flasks and then through baryta water, failed to exhibit any trace of this gas, even after many days. When this result was well established, some of the research-flasks were inoculated with bacteria, and within a day the cloudy precipitate appearing in the baryta showed that a considerable amount of CO, had been evolved as the result of bacterial action, and a copious precipitate continuing to be deposited indicated that a somewhat vigorous oxidation was taking place. Secondly, to measure any rise of temperature due to oxidation, the experiments with the double-walled vacuum-flasks, the thermopile and galvanometer, were again set in operation, substituting only peat for charcoal. The results were completely in accordance with those noted for the charcoal, except that, as might be expected from the nature of the substance and the consequent greater evolution of COs, the oxidation was more vigorous and a greater rise of temperature was recorded. It was | found that the inoculated flask maintained a temperature of 1°05 C. above the incubator for a considerable time. These expervments clearly show that when peat is exposed to damp air and to the action of switable organisms wt decays rapidly, with the evolution of COs, accompanied by a rise in temperature. Coal. The investigation of ordinary household coal presented many difficulties, chiefly on account of problems connected with sterilisation and the presence of occluded gases, and I have not been able to entirely overcome some special difficulties of the case. Obviously the inflammable gases contained within the coal and its combustible nature rendered sterilising by dry heat an impossibility. For the purpose of experiment the coal was taken from the centre of a, large piece to avoid contamination with foreign matter ; it was pounded and passed through sieves similar to those used for the charcoal, and then sterilised in the same manner by passing steam through the Cloéz research- flasks and subjecting the coal to a partial vacuum. The titrations showed that the flasks which were inoculated with bacteria gave off some 10 milli- grammes of CO: in excess of the non-inoculated flasks during the course of three weeks, but even with the strictest precautions traces of CO», were found in the non-inoculated flasks, owing to the escape of this gas from the occluded state. It was this difficulty which first suggested that the measure- 250 Prof. M. C. Potter. Bacteria as [Jan. 18, ment of any rise of temperature was a better means for testing oxidation than the collection and determination of the carbonic anhydride. The thermopile was therefore relied upon in order to determine whether coal sterilised by discontinuous boiling gives out any heat, and whether any thermal changes occur consequent upon the addition of bacteria. Small fragments of coal obtained as described were placed in a flask and immersed in distilled water, the aperture being covered with a small beaker, as cotton wool was inadmissible as a plug. The flask was steamed for five hours on two consecutive days, and on the third day the excess of water was partially driven off by boiling. The coal so treated was then introduced into a sterilised double-walled vacuum-flask, and placed in the incubator, a thermopile was inserted as before, and the connections made with the galvanometer according to the method previously recounted for charcoal. Immediately after setting up the apparatus, the thermopile indicated a temperature of the coal considerably above that of the incubator, this being due to its retaining some heat after boiling; but after 26 hours the research- flask had cooled down to the temperature of the incubator. Following this interval, the temperature of the coal gradually descended, which may be explained by the evaporation continuously taking place from the damp fragments of coal. Then for some three days it remained at 0°2 below that of the incubator, and it was quite clear that the coal thus sterilised generated no heat. This point being determined, the coal was inoculated with bacteria by pouring in distilled water containing these organisms, care being taken that the temperature of this added water was below that of the research-flask. The reduction of temperature was at once indicated by the thermopile, the spot of hght moving off the galvanometric scale in the direction opposite to the movement when the warm coal was first put in. Twenty-four hours after inoculation the temperature of the research-flask had not only risen to that of the incubator, but it had increased to 0°08 C. above it, and afterwards a marked rise of temperature amounting to 0°18 C. was steadily registered for 11 days. In the experiment just described, one terminal of the thermopile was inserted in the research-flask, and the other in a glass jar packed with cotton wool placed in the incubator, and in this manner any difference of tempera- ture between the research-flask and the incubator could be measured. But the experiment would not be complete without a control, and a second double- walled vacuum-flask, containing fragments of coal from the same source and treated in a precisely similar manner, was also placed in the incubator with a second thermopile, one terminal in each flask. The coal, however, in this 1908.] Agents in the Oxidation of Amorphous Carbon. 251 second flask was not inoculated with bacteria. Throughout the whole duration of the experiment, the control always maintained a lower temperature (0°38 C.), than that of the inoculated flask, and by means of a third thermopile it was proved to possess a temperature 0°19 C. lower than that of the incubator. For a period of 11 days the temperature of the control flask exhibited no upward tendency, while that of the inoculated flask rose from a point much below that of the incubator to nearly 0°4 C. above it, and steadily maintained that degree of heat. J¢ 2s thus clearly demonstrated that bacteria have a decided action upon coal, resulting in a distinct rise of tenvperature, and that this increase of temperature does not occur when the coal rs preserved from bacterial action. Some further experiments with the thermopile and galvanometer upon moist and dry coal emphasise these conclusions. At a temperature of 40° C, the coal moistened with distilled water and inoculated showed a difference of temperature of 1°25 C. above that of similar coal dried at 100° C. It was also found that at a temperature of 4° C., when the activity of the bacteria would be reduced almost to a minimum, the difference between sterile and ‘non-sterile charcoal was inappreciable, amounting to only 0°03 C.; while at 14° C, the amount registered was 0°19 C., which shows that the difference of temperature increases as the thermal conditions become more favourable to bacterial life.* Important additional evidence that the CO: production from the carbon is undoubtedly due to bacterial action would be afforded if it could be shown that the CO, is only evolved under conditions consistent with the life of these organisms. [ owe to Dr. F. F. Blackman the suggestion that it would be a critical test to show if the CO, production increased with a rise of temperature, and whether it goes up or down with causes that have the corresponding effects upon bacterial activity. With the object of elucidating this point, a further series of experiments was undertaken. Cultures of coal and charcoal were prepared under varying * In this investigation the actual measurements given must not be understood as repre- senting a quantitative analysis, which would be impossible under the conditions of treat- ment necessary to preserve the charcoal from contamination. The figures must be regarded only in a qualitative sense, and all that is claimed is that the titration method shows definitely that CO, is given off from the research material only when bacteria are present. From the equation C+ 0, = CO,+97650 calories we learn that 1 milligramme of CO, is produced by oxidation, with the evolution of 2:22 calories, and hence the rise of tempera- ture measured by the thermopyle is of the same order as the heat derived from oxidation. But as it is impracticable to suddenly destroy all bacterial action in a vacuum-flask and determine its rate of cooling, an exact equation cannot be obtained. VOL. .LUXXX.—B. U 252 Prof. M. C. Potter. Bacteria as [Jan. 18, conditions of treatment, and these were subjected to different temperatures of 20°, 30°, 40°, and 100° C. The first three were maintained by Hearson’s incubators and the last by a steamer continuously boiling. Cloéz flasks were employed as before, each containing 5 grammes of the research material. The conditions selected for the purpose of experiment were :— (a) The Provision of Moisture surtable for the Growth of Micro-organisms.— The charcoal or coal in each flask, after inoculation, was moistened with distilled water, and the flasks then sealed. One of each kind was placed in the steamer, and the others in the incubators at the different temperatures. (b) Absolute Dryness which would inhibit Bacterval Life-—The flasks, after washing, were placed in a drying oven at 100° C., and then heated with the Bunsen flame while a stream of air dried by H2SO,4 was drawn through them, this operation being repeated several times. The coal and charcoal were algo dried for some hours at 100° C., and, while still hot, inserted in the flasks. To remove any trace of COs, air was again drawn through them, first passing through a Reiset with a strong solution of caustic soda and then through H.SO4 The flasks were sealed as speedily as possible and placed in the incubators. (c) Treatment uith Antiseptics—A. 2-per-cent. solution of corrosive sublimate and a solution of iodine in potassic iodide, 95 ¢.c. of water with 5 c.c. of solution of iodine (12°59 crammes I+ 18 grammes KI +1000 c.c. H20) were the antiseptics preferred, and an excess of chloroform vapour was also employed in deference to the prevalent idea that this substance can be relied upon as an antiseptic. The use of any carbon-compounds, however, such as chloroform or prussie acid, etc., was unsatisfactory, as a special investigation might be required to determine whether they were themselves responsible for any COz2 production. The whole series of flasks remained in the steamer and the various incu- bators for a definite period, after which a stream of air-free COz was drawn through the flasks, then through baryta solution, and titrations made. The results obtained with uncalcined charcoal are given in the table for the sake of comparison. The results as set forth in the above table show that under the conditions suitable for the growth of bacteria increasingly higher temperatures indicated a, corresponding increase in the amount of CQO» given off, the production at 40° C. being greatly in excess of that at 20° C.; while at the temperature of 100° C., at which active bacterial life would be impossible, there was no evolu- tion of COz. Also, under dry conditions which prevented bacterial growth, no CO, was evolved at any of the temperatures tried. The treatment with antiseptics proved to be untrustworthy and it was evident that this method must be abandoned and the results discarded as ~~ 1908.] Agents in the Oxidation of Amorphous Carbon. 253 valueless for the purposes of the present investigation. There is a danger of chemical reactions taking place, and directly any antiseptic is employed the research becomes complicated by the introduction of an entirely fresh set of problems which need special investigation. Table of Results of Titrations. Duration of Experiment 20 Days, 5 grammes of Material used in each case, Miiligrammes of CO. Temperatures ...........000 20°C: | 30> &. | 40° C. | 100° C. Coal— Whoist:inoctilated .......c.sccccsceccs | 2°0 3°1 46 0:0 Moist sterilised by boiling ......... 0°0 0:0 0°0 0°0 OY soeede obee beer: cine ARBOR pre adotr | 0:0 0:0 0:0 0:0 Charcoal— MMOISG MOCUIAtE co. .c..0c..-ceecnenee OG 1 3 2°5 0:0 Moist sterilised by boiling ......... 0-0 0-0 0:0 0:0 ia? co, ee ie 0-0 0-0 0-0 0-0 Charcoal uncalcined— MMoistiimoculated ....c.iceseccccsescas 5 °4 8 ‘0 22 ‘8 — Neither corrosive sublimate nor chloroform were found to be effective as antiseptics. In the flasks treated with both these substances a microscopic examination at the conclusion of the experiment showed the presence of motile bacteria (not Brownian movement), which stained with gentian violet and grew feebly as stab-cultures on gelatine. In the flasks treated with iodine no movement of the bacteria could be observed and they appeared to have been entirely destroyed. It should be mentioned that a weaker solution of iodine proved to be less efficient, owing possibly to the smaller margin allowed for its reduction. I was not prepared to find the bacteria able to resist a 2-per-cent. solution of corrosive sublimate, though chloroform I well knew to be of little use. The value of antiseptics, however, in securing absolutely sterile conditions, is often doubtful, and as the author has previously pointed out, even very strong percentages of such poisons may be quite ineffective in destroying micro-organisms (8 and 9). The recent work of Adrian J. Brown (2) upon “The Existence of a Semi- permeable Membrane enclosing the Seeds of certain Graminee ” throws con- siderable light upon the action of antiseptics. Brown has shown that when these seeds are soaked for three days in a 5-per-cent. solution of cupric sulphate, silver nitrate, and potassium ferrocyanide, no trace of these substances penetrated to the interior of the grain, though water was freely 254 Prof. M. C. Potter. Bacteria ds [Jan. 18, absorbed. After this treatment the vitality of the seeds was not impaired nor their power of germination. Further, the semi-permeable covering enclosing these seeds permits the absorption of water from weak solutions of acids and alkalies, while excluding the latter compounds; but it does not prevent the passage of iodine. An important question raised by these investigations is : to what extent are plants in general protected by this means, and may it be that some bacteria may possess a cell-wall of a semi-permeable nature which acts as a protective envelope and so accounts for the immunity from injury which many species possess? Some observations I have made on the behaviour of certain bacteria in solutions of mercuric chloride seem to indicate that this might be so. Plainly, they have some means of protection against a strong solution of such a poison, but it proved too difficult a piece of microscopy to determine whether the sublimate was actually excluded by the enclosing membrane. The well-known oligodynamic theory of Nigeli(5) may also be cited as bearing upon the effect of antiseptics in certain cases. His observations show that the toxic action of poisons is reduced in the presence of certain insoluble substances such as graphite, etc. True and Oglevee (13) have more recently extended the work in this direction and entirely in favour of Nageli’s con- clusions. Their results establish the fact that the presence of certain insoluble substances tends to decrease the toxic activity of solutions of strongly toxic compounds; and their experiments with mercuric chloride prove that the presence of insoluble bodies modifies very markedly the toxic influence of this salt upon the roots of seedlings. The theory advanced is the attraction of the ions, or molecules, of the dissolved substances for the solids and their absorption by the latter. An explanation is thus afforded of the snecieney of a high percentage of corrosive sublimate and the behaviour of iodine as an antiseptic, in the presence of finely-divided coal and charcoal. The insoluble nature of the research material, and the possibility of a semi-permeable or selective property of the bacterial cell-wall, appear to offer an intelligible interpretation of the un- certain effect of the antiseptics employed in this research. The whole question of the dependence upon antiseptics requires considera- tion, and it is evident that the efficiency of any one cannot be taken for eranted, but must be tested for the special conditions under which it is employed. Lately, Stoklasa(11) has published a preliminary note upon the oxidation of coal and charcoal, and concludes from comparative researches upon “sterile and non-sterile ” coal that the evolution of CO is due to: (1) Auto- oxidation, (2) the action of an enzyme. The theory of auto-oxidation is not 1908.| Agents in the Oxidation of Amorphous Carbon. 255 in accordance with my own experiments, which show that while the amount of COz produced increases with a rise of temperature, CO is not given off at the supra-vital temperature of 100° C., nor under other conditions which prevent the growth of bacteria. In this preliminary note it is not clear what means Stoklasa employed for sterilisation. If he trusted to corrosive sublimate, the acid reaction commonly found in this salt would have to be taken into account as a source of. COz in the presence of any carbonates, as well as other causes affecting its reliability. In some previous experiments Stoklasa(12) himself notes that, notwithstanding all care, active bacteria appeared on the roots of sugar beet which had been steeped for 25 minutes in a ()'5-per-cent. solution of mercuric chloride, and this he attributes to the possible intro- duction of these organisms in a stream of vapour passed through the apparatus. It seems more probable, as I have proved, that the bacteria flourished in spite of the treatment with mercuric chloride. Further, Stoklasa does not state whether he re-calcined the charcoal. During re-calcination, various gases are given off, and microscopic examina- tion shows that the cell-wall in ordinary charcoal is often incompletely charred. Thus the combined carbon in ordinary charcoal would be readily attacked by micro-organisms, and on this account the evolution of CQ, is much greater from uncalcined charcoal than after reheating to about 1200° C. If the oxidation of carbon takes place through the action of an enzyme, this naturally assumes the presence of a living cell, or, in other words, the oxidation is due, primarily, to bacteria. General Conclusions. The methods of experiment which have been dealt with attack the problem from totally different standpoints, and the accumulated evidence affords convincing proof that amorphous carbon slowly undergoes oxidation through the agency of bacteria. The dependence on antiseptics is shown to be very treacherous, but it is clear that when complete sterilisation is secured by discontinuous boiling, there is no production of COs. The results obtained by the experiments with the varying degrees of temperature and under the dry conditions are also of critical importance and establish beyond question my contention that the amounts of COz given off are really due to bacterial activity, and not to any chemical action in the coal or charcoal. If the evolution of CO, had proceeded steadily beyond the supra-vital temperature, it would have pointed to a non- vital change, but the fall of CO. at the death point clearly indicated thecessation of a vital process. 2G Prof. M. C. Potter. Bacteria as [Jan. 18, The determination, by measurement with the thermopile, of the rise of temperature due to oxidation becomes a very valuable confirmatory experi- ment in conjunction with the method of testing the evolution of CO2 by titration. It has been conclusively established by means of the thermopile that a definite rise of temperature occurs when carbonaceous substances such as charcoal, coal, peat, etc., are subject to the action of certain bacteria. And in this connection it must be remembered that the rise of temperature is maintained for some considerable time, and that the double-walled vacuum- flask, with at least a portion of the wires composing the thermopile, are steadily preserved at this temperature above the surrounding medium, in spite of any loss from radiation. There must thus be a continuous dissipation of heat and the amount generated is therefore more than actually appears; it must also be taken into consideration that this rate only applies to conditions 7 vitro and in a laboratory. Probably, in the soul, the carbon would be attacked by micro-organisms under circumstances more favourable to their activity and the oxidation would proceed much more rapidly. It is of importance to recognise that every process of oxidation raises the temperature in an appreciable degree, and this is a factor which should be taken into account in all problems relating to the soil. Indeed, the action of bacteria in promoting exothermal changes is a subject too generally neglected. It must now be recognised as possessing a practical bearing upon investigations connected with oxidation of coal. The suggestion may also be made that in some cases of spontaneous combustion of coal, the heat generated by microbial activity is an influence to be taken into consideration, and may be a dangerous motive force acting upon explosive gases. That carbon should be proved to undergo oxidation by bacteria is not surprising when we consider the fact that nitrogen undergoes the same process, while the oxidation of sulphur by bacteria has been established by Beijerinck (1) and quite recently that of hydrogen by Kaserer(4). The author has previously shown that oxalic acid undergoes decomposition into COQ2 and H20 by the agency of a soil bacterium (6), a result which has since been confirmed independently by Hall (3). Incidentally the present investigation throws some lght upon the formation and «decomposition of coal. In the ordinary course of events vegetable matter gradually undergoes a process of decay, countless bacteria and fungi deriving their sustenance from it and gradually effecting the dissolution of the cellulose and other compounds, until, ultimately, the end- products are reached. This process demands, among other things, a constant supply of oxygen, and in the absence of this element only a partial reduction 1908.| Agents in the Oxidation of Amorphous Carbon. 257 can be attained. This is well exemplified in the case of peat, where the vegetable débris in a wet and sodden condition is excluded from a sufficient supply of oxygen, and therefore the oxidising organisms are unable to carry on their work, any further changes must be due to anaerobic forms, and the decay is necessarily incomplete. In a similar manner it may be supposed that the large deposits which form the basis of coal are due to plant remains which, in the first instance, were preciuded from complete oxidation owing to their submerged situation, where vegetable matter could only be acted upon by anaerobic bacteria. In this connection, Renault’s(10) researches upon Fossil Bacteria are of great interest. He brings forward evidence to show that bacteria have existed since Devonian times, and have played a considerable part in the destruction and decomposition of vegetable and animal tissues from this remote period. Microscopic sections have furnished remarkable proof of their presence inthe upper Jurassic beds, in the Permian Strata, the Upper, Middle, and Lower Coal Measures, in the Carboniferous Limestone, and in the Devonian, and these illustrate in a remarkable manner the destructive action of numerous micro-organisms upon organic remains imbedded in these formations. Renault states that in many cases the minutest details have been preserved in such perfection that it has been possible to detect the bacteria often more easily in the fossilised than in the living state. All stages in the disintegration of the cell-tissues are clearly exhibited, and it is proved that the rdle of these micro-organisms has been identical with that which they perform in the present day. According to Renault, “If in the formation of coal there are two distinct phases, one, purely chemical, which has brought the remains of plants to a certain composition answering roughly, in the case of ‘houille de bois pur,’ to the formula CyH30, the second, simply mechanical, due to a slow compression in a permeable medium, the first of these phases can be attributed to a bacterial fermentation developed in the marshes, ponds, deltas, and arrested by periodic floods, carrying away a portion of the macerated plants and transporting them into lakes and seas, where maceration became impossible.” In later times heat and pressure would convert this partially decayed vegetable déiris into coal. The decay bas, however, only been arrested, and, lke peat, when a sufficiency of oxygen is available and the necessary conditions for the life of aerobic organisms are presented, the decomposition proceeds, the elements are reduced to their simplest compounds, and the carbon is once more liberated in the form of CO, to play its 7d/e in the life eycle, Coal and peat are shown to be subject to the same laws as other organic 2.58 Prof. M. C. Potter. Bacteria as [Jan. matter, and such substances remain unsusceptible to change only so long as bacterial action is excluded. It is quite evident that where there is surface exposure bacteria must play a large part in the disintegration of coal, which is one of the most insoluble substances known, and that these organisms form an invaluable agency in assisting the circulation of carbon and again converting it to the uses of Nature. Summary. Under conditions of exposure to the air, a slow oxidation of amorphous carbon takes place through the agency of bacteria. This has been conclu- sively established by experiments upon such carbonaceous substances as charcoal, lamp-black, coal, and peat. When these substances are subjected to Petal action carbonic acid is given off, as estimated volumetrically by absorption in baryta solution and titration with standard oxalic and hydrochloric acids. : The amount of COz given off increases in proportion to the rise of tempera- ture and ceases to be evolved at a supra-vital temperature. There is no evolution of CO. under perfectly dry conditions such as preclude the possi- bility of bacterial life. A distinct rise of temperature occurs through the action of bacteria. The heat generated was determined by measurement, with a galvanometer, of the electromotive force produced by the difference of temperature between two thermo-elements, one placed in a sterile and the other in an inoculated flask. The evolution of CO, and the accompanying rise of temperature does not take place when carbonaceous substances are preserved from the intrusion of micro-organisms. The heat generated by microbial activity 1s an influence to be taken into account in connection with the oxidation and spontaneous combustion of coal ; it may be a dangerous motive force acting upon explosive gases. The oxidising action of bacteria must be largely responsible for the disintegration of coal and the high percentage of depreciation which it undergoes in store. Coal and peat, hke other organic matter, are lable to decomposition as soon as conditions are presented suitable for the life of aerobic organisms. The carbon is then once more liberated in the form of COs to play its rdle in the life cycle. It is thus conceivable that the vast supplies of carbon locked up in the world’s coal-fields may become available for plant nutrition without the intervention of direct combustion. 1908.| Agents in the Oxidation of Amorphous Carbon. 259 I have to express my indebtedness to my colleague, Dr. Morris-Airey, for much help which he has given me in the preparation and testing of the electrical apparatus employed in this research, and I take this opportunity of thanking him for the kind way in which he has always been ready to give me the benefit of his assistance in any technical difficulty. My obligations are due to the Government Grant Committee of the Royal Society for the use of a Zeiss 3 mm., 1-40 apochromatic oil-immersion lens. LITERATURE. 1. Beijerinck, ‘Ueber die Bakterien, welche sich im Dunkeln mit Kohlensaure als Kohlenstoffquelle ernadhren kinnen,” ‘Cent. f. Bakteriologie, abt. II, vol. 11, 1903 | 2. Brown, A. J., “On the Existence of a Semi-permeable Membrane enclosing the | Seeds of some of the Graminez,” ‘Annals of Botany,’ vol. 21, 1907. 3. Hall, “The Effect of Plant Growth and of Manures upon the Retention of Bases by | the Soil,” ‘ Roy. Soc. Proc.,’ B, vol. 77, 19085. 4. Kaserer, “Die Oxydation des Wasserstoffes durch Mikro-organismen,” ‘Cent. f. Bakteriologie,’ abt. II, vol. 16, 1906. 5. Nageli, “Ueber oligodynamische Erscheinungen in lebenden Zellen, mit einem Vorwort von 8. Schwendener und einem Nachtrag von C. Cramer,” ‘Denkschriften d. Schweizerischen Naturforschenden Gesellschaft,’ vol. 33, 1893. 6. Potter, “On the Decomposition of Oxalic Acid by Bacteria,” ‘ Univ. of Durham Phil. Soc. Proc.,’ vol. 2, 1903. ve “Bacteria as Agents in the Oxidation of Amorphous Carbon,” Brit. Assoc., Cape Town, 1905. 8. “On a Bacterial Disease of the Turnip (Brassica mae “Roy... Sec. Procs: vol. 67, 1900. PD: “On the Occurrence of Cellulose in the Xylem of needy Stems,” ‘ Annals of Botany,’ vol. 18, 1904. | 1 10. Renault, “Recherches sur les Bactériacées Fossiles,” ‘Annal. d. Sci. Nat.,’ Bot., series 8, vol. 2, 1895. 1l. Stoklasa, ‘‘ Ueber die anaérobe Atmung der Samenpflanzen und iiber die Isolierung der Atmungsenzyme,” ‘ Ber. d. Deut. Bot. Gesell.,’ vol. 25, 1907. 12. ~——— “ Der anaérobe Stoffwechsel der hoheren Pflanzen und seine Beriouine, zur . alkoholischen Garung,” ‘ Beit. z. Chem. Physiol. u. Path. v. Fr. Hofmeister,’ vol. 3, 1903. 13. True and Oglevee, “ The Effect of the Presence of Insoluble Substances on the Toxic Action of Poisons,” ‘ Botanical Gazette,’ vol. 39, 1905. VOL. LXXX,—B, xX 260 The Antagonistic Action of Calecum upon the Inhilitory Effect of Magnesium. By 8S. J. MELTZER and JOHN AUER. (Communicated by Professor E. H. Starling, F.R.S. Received March 10,— Read April 2, 1908.) (From the Department of Physiology and Pharmacology of the Rockefeller Institute for Medical Research.) Calcium and magnesium are chemically closely related elements. They are also close companions in the tissues of the animal body. It is the prevailing view that the physiological effects of both elements are similar in character. Many physiologists are at present of the opinion that calcium as well as magnesium exerts an inhibitory influence in the functions of the animal body. Loeb published, in 1899, his observations of the inhibitory action of calcium upon the twitchings of the frog muscles brought on by solutions of sodium chloride.* It was then assumed by Loeb that all the members of the group of alkali earths possess inhibitory properties, including, at first, even barium. In the numerous subsequent papers by Loeb and his pupils, the discussion turned, however, essentially around the inhibitory effect of calcium. As to magnesium, we have within the last few years published several studies in support of the hypothesis that magnesium salts favour inhibitory processes. The first fact which gave rise to that hypothesis was demonstrated in 1899 to the American Physiological Society, when an _ intracerebral injection of a.few drops of a solution of magnesium sulphate caused a state of paralysis in a rabbit, while the injection of other solutions brought on convulsions. In a series of recent studies which we have carried out upon the relations of the effects of calcium to magnesium, many facts came to light which demonstrate unmistakably that calcium is the most available agent to neutralise inhibitory effects of magnesium. We shall not enter here upon details; we wish only to report the following striking and instructive experiment. * (Note by E. H. Starling—The inhibitory action of calcium salts on the twitching brought on by sodium chloride solutions was observed by Dr. Ringer, F.R.S., many years before Loeb, and is fully described by him ina paper in the ‘Journal of Physiology,’ vol. 7, p. 291, 1886. In reference to the subject of the present communication, it is interesting to note that Ringer observed a similar antagonism between barium and calcium (vide ‘ Practitioner,’ vol. 31, p. 81, 1897).) Action of Calevum upon the Inhibitory Effect of Magneseum. 261 By subcutaneous injections of a magnesium salt (for instance, Epsom salt—about 7 cc. of a 25-per-cent. solution per kilogramme) rabbits are brought to a profound state of anesthesia and paralysis. The slow and shallow respirations indicate the approaching danger. Now 6 or 8 ce. of an M/6 or an M/8 solution of a calcium salt are given through the ear vein. Within a few seconds the respiration becomes quicker and deeper, and within one minute the animal turns over, sits up, and appears normai. Here calcium not only did not add an inhibitory effect, but completely neutralised the profound inhibitory effect of magnesium. The companionship of calcium and magnesium within the body means, at least in many instances, not a concerted action of similar effects, but rather a resultant effect of antagonistic actions. We may add that the experiment calls to mind similar relations existing in plant physiology ; the retardation of growth on account of the presence of too much magnesium in the soil is promptly corrected by the addition of a calcium salt; the process is termed “liming.” In animals, therefore, as well as in plants, calcium is antagonistic to magnesium. 262 Post-tetanic Tremor. (Supplementary Note. ) By Davin Fraser Harris, M.D., B.Sc. (London).* _ Since the publication of my paper on this subject,f my attention has been called to the fact that the phenomenon has been previously noticed by Dr. Sydney Ringer.} I regret that I was unacquainted with his paper until after mine had been published. Dr. Ringer states :—“This powerful prolonged faradisation, for one or two minutes, of the sciatic nerve of a cut-off leg of an undrugged frog, causes the lhmb to remain extended, and if it be held vertically, foot upwards, it falls more slowly than happens after only a momentary stimula- tion”; and again: “These fibrillary twitchings and this spastic condition can be produced in normal imprisoned muscle.” There can be no doubt that. what Dr. Ringer called “fibrillary twitchings” and I have termed “ post- tetanic tremor ” are identical phenomena. | In connection with the tremor which occurs in the muscles of the lobster during faradic stimulation, I ought also to have alluded to Professor C. Richet’s discovery§ of an identical tremor (“tetanos rythmique”) in the pincer muscle of the crayfish, but I was unable to obtain a copy of his paper until after my own had gone to press. [* This supplementary note was received March 11, 1908.—Sec. R.S. ] + © Roy. Soc: Proc., iB; vel) 80) p:737, 1908: + “Report on the Influence of Rhombic Sodium-phosphate and Sodium-bicarbonate on Muscular Contraction,” ‘ Brit. Med. Journ., July 19, 1884, vol. 2, p. 114. § “Contribution 4 la Physiologie des Centres Nerveux et des Muscles de l’Ecrevisse,” ‘Arch. de Physiologie Norm. et Path., fig. 22, p. 563, vol. 6, 1879. 263 The Glycogenic Changes in the Placenta and the Fetus of the Pregnant Rabbit : a Contribution to the Chemistry of Growth. By J. Locuueap, M.A., M.D., B.Sc. (Carnegie Scholar), and W. CrameEr,‘Ph.D., D.Sc. (Lecturer on Physiological Chemistry, University of Edinburgh). (Communicated by E. A. Schafer, F.R.S. Received January 1,— Read February 27, 1908.) , (From the Physiology Department, University of Edinburgh.) CONTENTS. PAGE IMAP OR UOMIOLION) Gage soe v ouies ova ceca cecunceot eck sencesnenemeemeaas Uaenedecivackestasbeessete ous 263 Experimental Part— DMM ES seis sot os creo ans ue guc inte Testa naA tate Sd en CeCe Tae Mei heb ulibodnceabeyesadidelnnt 264 Pre On COP CU: OF «IAC OMUA ate pact nsarcasepds conantasc seer soles vu SactadeesecvcneseUae 266 een come OF Mostra EaMeree tie. ccws vedas colideesdedsgentndsaceecceveresvinesseues 269 C.—Relationship of Placental to Feetal Liver Glycogen ..................684 271 iDi—-Grlycogen Of rest oF Postal Bodies | civcsceccscesncascncecssesecsssssoncocsouse 272 Relation of Foetal Growth to Glycogen Percentage ...............05. 272 Hihec tron MV aR IaulOnn Tl WEG 21 cauieigiac ices siececcs cay viewensincienerness saeoe 273 ie Pitrect of Enjections.of Phloridzin .... ccc. ccccceatscescessecscsecsadceaess 274 F.—The Glycogen-splitting Ferment of the Placenta ....................068 279 General Discussion— On the Fate and Function in the Fetal Organism of Glycogen SUSGrWed ThOmr CROMEITACENMUA, <0 adctecuesssssicwnscesteadroscvejiaseanedccseasegMeces 281 SS UMIMILIN AV AMER Mas ces aateteie te ilee tet ae cals desi a Rabbit F...... 1°81 0 -0109 0 ‘60 19 °25 0 0538 0-28 Control ...... 2°67 0 0392 1°47 30°17 0 1026 0 +34 1908.] in the Placenta and Fetus of the Pregnant Rabbit, 279 tissues. In the case of Rabbit F, the maternal part of the placenta is not diminished in weight, although its glycogen store has been greatly reduced, while the foetal part of the placenta, which normally is free from glycogen, is greatly reduced in weight. The results obtained by treatment with phloridzin confirm the conclusions which we have drawn from our observations on spontaneous cases of retarded growth. They show that the growth of the fcetus is dependent upon glycogen in such a way that the glycogen is not actually present in the growing cell, but that the foetus draws upon a deposit which is laid down at first in the * maternal part of the placenta and is transferred in the last week of pregnancy to the liver of the foetus. F. The Glycogen-splitting Ferment of the Placenta. Glycerine extracts, both of the maternal and of the foetal part of the rabbit’s placenta, were found to have a powerful hydrolytic action on glycogen. This action was destroyed by heating the extracts. Extracts of sheep’s placentze showed, even on qualitative examination, a very much less marked glycogen-splitting power. The activity of the ferment was measured quantitatively by determining the amount of sugar split off after incubating a mixture of measured quantities of the glycerine extract and of a solution of glycogen, purified by repeated precipitation with alcohol. Toluol was added. After 24 hours the glycogen was precipitated by alcohol and the amount of sugar estimated in the filtrate by Allihn’s method. In order to account for the sugar of the blood present in the glycerine extracts, control experiments were made with the heated extract. In these controls only very small and almost constant quantities of cuprous oxides were found, the figures ranging from 0:001—0:004 gramme. These figures were subtracted from the results obtained in the digests, and in this way a measure of the activity of the ferment was obtained. The results obtained for the placenta of the rabbit are given in the following table :— Glycogen-splitting Ferment in Placenta of Rabbit. Maternal placenta. Feetal placenta. Day of gestation. Weight of Cu,0. Weight of Cu,0. (Difference.) (Difference. ) gramme. gramme. 17 0 -0218 0 :0328 21 0 0040 0 1463 24, 0 0424, 0 -0476 30 0 °0196 0 0237 VOL. LXXX.—B. Z 280 Drs. Lochhead and Cramer. Gilycogenic Changes [Jan. 1, Extracts of the placenta of the sheep and the cow were examined in the same way, and gave the following result :— Glycogen-splitting Ferment in Placente of Sheep and Cow. Maternal placenta. Fetal placenta. Species. Weight of Cu,0. Weight of Cu,0. (Difference.) (Difference.) gramme. gramme. Sheep 0 0084 0 ‘0052 P 0 0068 0 0080 Cow negligible negligible a 0 -0060 0 -0040 The results reveal a marked difference between the placenta of the rabbit and that of Ruminants, the last-named animals yielding a much weaker ferment than the rabbit. It is a very interesting fact that this difference is reflected also in the glycogen content of the placentz of these animals. The maternal and foetal cotyledons of the placenta of the sheep were examined for glycogen in several cases. The material was obtained from the slaughter-house and extracted within half an hour after death. By extraction with hot acidulated water we failed to find any glycogen. Experiments with Pfliiger’s method of extraction with strong alkali, however, showed that the foetal cotyledons of the sheep’s placenta contain glycogen in traces to the amount of about 0°01 per cent. The glycogenic changes in the sheep’s placenta must therefore be very insignificant, and correspondingly we find a weak glycogen-splitting ferment in this case. In the rabbit, on the other hand, where large quantities of glycogen are dealt with by the placenta in a short time, we find a powerful ferment present in the placenta. The glycogen of the maternal placenta of the rabbit, which, as we have seen, is made use of by the embryo, must necessarily pass through the fcetal part of the placenta. This part contains such small traces of glycogen, if any at all, that they cannot be detected by microchemical methods. We must assume, then, that the plasmodium absorbs glycogen not as such, but in an altered form. At the same time we find that the foetal part of the placenta secretes a strong glycogen-splitting ferment, more powerful even than that of the maternal part. In view of these facts, it is difficult to avoid the conclusion that in the placenta the ferment is active during life. If we assume that it is found only after death, we would meet with the further difficulty, that a tissue—the foetal part of the rabbit's placenta—which contains no glycogen during life should suddenly develop a glycogen-splitting enzyme after death. 1908.] in the Placenta and Fetus of the Pregnant Rabhit. 281 The objection that the glycogen-splitting power of the placenta is due to the presence of blood, which is known to have a diastatic action, is negatived by our results. The extracts of the placenta contained as much bloodiin Ruminants as in rabbits, yet the diastatic power of the placenta of the former was almost negligible compared with that of the latter. We wish to point out specially that the glycogen-splitting enzyme is an enzyme secreted by the cells. It is the only enzyme which we have been able to demonstrate in glycerine extracts of the placenta, although a large. number of experiments were made to test for the presence of tryptic, peptic, ‘ ereptic, and lipolytic enzymes in these extracts. All these experiments gave: negative results. That such ferments are present within the cells of the: placenta as intracellular enzymes, and that their presence can be demonstrated, by using more severe methods than extraction with glycerine, we do not doubt. But such ferments, with the sphere of their activity limited to the cell itself, are present in the cells of many if not of all organs, so that their presence in the placenta would not throw much light on the function of this organ. On the Fate and Function in the Fetal Organism of Glycogen absorbed |. Jrom the Placenta. Whatever the source of the placental glycogen may be, its presence cannot be due to the accident of the anatomical site of the rabbit’s placenta, which, although identical in position with that of the cow and sheep, is distinguished: by its power of forming and storing glycogen. We cannot but recognise a definite function of the cells of the rabbit’s placenta of Hopeeiine) 2 a store. of carbohydrate for the foetus. There can be little doubt that the placental glycogen is absorbed by the plasmodium. It is situated, as we have seen, in decidual cells which are intimately related to the ectodermic tubules, and are, indeed, in Chipman’s words, “swallowed” by them, so that the glycogen contained in them must also be absorbed into the feetal structures. The quantitative relationship which we have proved to exist between the decrease in the placental glycogen and the increase in the fcetal glycogen affords strong direct evidence in support of the view that the placental glycogen forms a store for the foetus. A specific function appears to arise in the fcetal liver in the last week of gestation. If the hepatic store of glycogen were due to the favourable anatomical position of the foetal liver, we should expect to find the glycogen contents of the liver to be above that of the rest of the body during the whole period of gestation. We have seen, however, that up to the 24th day Z 2 282 Drs. Lochhead and Cramer. Gilycogenic Changes [Jan. 1, the function of storing glycogen for the foetus has not yet been acquired by the liver and is, in the meantime, fulfilled by the placenta.* The glycogen metabolism of the placenta and the foetus differs in some essential points from that of the adult organism. In the latter case the glycogen deposited in the tissues represents a store dependent upon external conditions and showing irregular fluctuations. The placenta and the fcetus, on the contrary, are characterised by the absence of individual variations in the amount of glycogen they contain at a given period of gestation. The glycogen metabolism of these organs shows a regular succession of changes which proceed almost regardless of external conditions, and which are independent to a great extent even of the glycogen metabolism of the mother. These facts point to the conclusion that in the development of the fcetus glycogen fulfils a definite function and is not merely an accessory source of nutritive energy as it is in the adult organism. Part of the glycogen which is absorbed from the placenta may be accounted for by the intense carbohydrate metabolism, which, according to Bohr’st experiments on the gaseous metabolism, proceeds in the foetal rabbit. Bohr’s results are not in agreement with those of Cohnheim and Zuntz.t But since the latter observers worked with sheep, which, in view of our observations, may have a different metabolism of pregnancy, and since Bohr’s figures show that his method entailed very little interference with the foetus, his observations may be taken as representing the normal condition in rabbits. The glycogen which is thus catabolised furnishes thereby the energy necessary for the formation of new tissues, the “ Entwicklungsarbeit ” of Tangl.§ The question arises whether glycogen performs also anabolic functions in the development of the foetus. : The absence of glycogen from some of the growing fcetal tissues, and the fact that the tissues where it is present do not contain even as much as the adult ones, leave little doubt that a definite formative power cannot be attributed to glycogen as such. On the other hand, the scarcity of glycogen * In order to account for the behaviour of the hepatic glycogen during development on ‘purely anatomical grounds it would be necessary to assume that at the beginning of the last week of pregnancy the vascular supply of the liver undergoes a complete change, so that after that date the foetal liver is freely supplied with blood from the placenta, while before that date very little blood from the placenta reaches the liver. We are not aware, however, that such a relation between vascular changes and the storing of glycogen can ‘be established. _ + Bohr, ‘Skandinavisches Archiv fiir Physiologie,’ vol. 10, 1900, p. 413 ; vol. 15, 1904, p. 23. { Cohnheim and Zuntz, ‘ Pfliger’s Archiv,’ vol. 34, 1884, p. 173. § Tangl, ‘ Pfliiger’s Archiv,’ vol. 93, 1903, p. 327. 1908.] a the Placenta and Fetus of the Pregnant Rabbit. 283 in embryonic tissues does not necessarily justify the conclusion that glycogen does not take part in the building up of the tissues. It is well known that embryonic tissues are rich in mucin, which contains a large amount of a carbohydrate group in its molecule. Although glycogen as such has no formative power, it may yield one of the “ Bausteine ” for the building up of the main protein body of foetal tissues. In this connection it is interesting to consider the conditions in the ben’s egg, which must necessarily contain in itself the material of which the embryo is built up. In the egg, carbohydrate as such, in the form of either glycogen or glucose, is almost absent. At the same time all the protein substances of the white of egg are distinguished by containing a large amount of glucosamine in their molecule. In the egg, therefore, the carbohydrate group has already entered into the protein molecule, and correspondingly there is a scarcity of free carbohydrate. Summary. In an age-series of pregnant rabbits from the 14th day of gestation to the end of pregnancy, quantitative estimations were made of the glycogen present in the placenta, in the liver of the foetus, and in the rest of the fcetal body. ‘The mechanical separation of the placenta into the maternal and the foetal part made it possible to determine the amount of glycogen in the peninsule of the maternal placenta, which are most intimately associated with the foetal placenta, apart from the glycogen in the rest of the maternal placenta. A marked feature of the results is the absence of individual variations in the glycogen contents of the various organs. Instead, there is an orderly sequence of changes, which allows of a graphical expression. Glycerine extracts of both the maternal and the fetal parts of the rabbit’s placenta contain an active glycogen-splitting enzyme. No evidence was obtained for the presence in these extracts of a proteolytic or lipolytic enzyme. Comparative observations on the placenta of the sheep show that the paucity of glycogen in the placenta of this animal is associated with the presence of a very weak glycogen-splitting enzyme in the glycerine extracts from this organ. The glycogen store of the rabbit’s placenta is not increased by feeding the mother animals on a diet rich in carbohydrates. Nor has such a diet any effect in increasing the hepatic glycogen reserve of the foetus either in the earlier or in the later stages of pregnancy. On the other hand, the placenta does not give up readily its store of glycogen to the mother organism when the hepatic reserve of the mother is exhausted by conditions such as injections of phloridzin. Neither during gestation nor at birth does the 284 Glycogenic Changes in the Placenta, etc., of the Rabbit. percentage of glycogen in the foetal organs reach the average amount found in adult tissues. - There is a distinct parallelism between the growth of the feetus and the percentage amount of glycogen which it contains. In two cases where growth had been spontaneously arrested, the percentage amount of glycogen in the foetal organs was diminished out of proportion to the diminution in weight. A similar condition could be reproduced experimentally by repeated injections of phloridzin. _ The interpretation of these results has led to conclusions which may be summarised as follows :— The placenta of the rabbit has the function of depositing glycogen as a store of carbohydrates for the needs of the foetus. The glycogen is absorbed from the maternal placenta in the form of a simpler carbohydrate. This transformation takes place in the placenta and is brought about by the action of an enzyme secreted by the placenta. --In the earlier stages of intra-uterine life the foetal liver is devoid of the power of storing glycogen, which it does not acquire until the last week of gestation. Before that date the placenta vicariously fulfils the hepatic function as far as glycogen is concerned. The glycogen metabolism of the placenta and the foetus is independent of that of the mother, and appears to be’ governed by conditions different in many respects from those which _ regulate the glycogen metabolism in the adult animal. There is a distinct relation between the glycogen metabolism and the growth of the foetus. Since the growing tissues of the foetus are not dis- tinguished by an abundance of glycogen, a definite formative power cannot be attributed to glycogen gud glycogen. The function of glycogen in the development of the foetal rabbit is probably to furnish material, on the one hand, for the intense carbohydrate metabolism that proceeds in the foetus ; and, on the other hand, for the building up of the protoplasm of fcetal tissues. The expenses of this investigation were defrayed by grants from the Moray Fund of the University of Edinburgh. 285 On the Maturation of the Ovum in the Guinea-prg. By J. E. Satvin Moors, A.R.C.S., F.L.S., Professor of Experimental and Pathological Cytology, Liverpool, and Miss F. Tozgr, B.Sc. London. (From the Cytological Department of the University of Liverpool.) (Communicated by J. Bretland Farmer, F.R.S. Received January 14,—Read February 27, 1908.) [PLates 5—7.] The following observations on the maturation of the ovum in the Guinea- pig are related to a more extended investigation. Certain results which we have already obtained while investigating the development of the eggs of the Guinea-pig are, however, of interest, and we think it desirable to give a brief account of them.* In the earlier stages of the development of the eggs within the ovary they appear as cells which present within their nuclei the characteristic synaptic contraction of the nuclear thread- work. Occasionally, during such stages, the centrosomes are clearly visible within a definite archoplasm. As the eggs increase in size the centrosomes appear to migrate from the interior of the archoplasm into the cytoplasm this migration corresponding to the migration of the centrosomes in similar stages in the development of spermatozoa. At a later period in the develop- ment, both archoplasm and centrosomes in the eggs disappear altogether. The development of the egg is nearly complete within the ovary. That is to say, the two maturation spindles corresponding to the maturation spindles in the production of the spermatozoa are found while the eggs are enclosed in the follicle and are still retained in the ovary. When the eggs have reached the large size represented in Plate 5, 1, the final stages in the evolution of a coarse spirem, corresponding to the coarse spirem of the spermatogenesis, are reached, and in the succeeding phase there occurs the usual development of gemini within the nucleus. Ina large number of the eggs the gemini appear to result from the pairing of the number of chromosomes usually found in somatic cells ; but it is very important to note that in many of the eggs with which we have had to deal the number of the aggregates of chromatin is largely in excess of what it ought to be, supposing these bodies * A detailed account of the early development of the egg in mammals is given by Winiwarter (“ Recherches sur ’Ovogenése et ’Organogenése de l’Ovaire des Mammiféres (Lapin et Homme),” par le Dr. Hans von Winiwarter, ‘Archives de Biologie,’ vol. 17, 1900). An account of the fertilisation and segmentation of the eggs of the Mouse is contained in a paper by Sobotta in the ‘ Arch. f. Mikr. Anat.,’ vol. 45, 1895. 286 Prof. J. E. Salvin Moore and Miss F. Tozer. [Jan. 14, normally occur in half the number of the somatic chromosomes. When the eggs have reached the above condition, the first polar body spindle is formed in the manner represented in fig. 2. There are no centrosomes, and several of the characteristic forms of the gemini correspond to the forms seen in the similar stages of spermatogenesis. The division of the gemini takes place in the same manner as that occurring during spermatogenesis and the diaster of such a division is represented in fig. 3. When the first polar body has been definitely extruded, as in fig. 4, the nucleus which remains in the egg immediately proceeds towards another division; the chromosomes appearing on this spindle (the second maturation spindle) being in the form of rods or diads, such as those represented in fig. 6. Here, again, as in the case of the first polar body spindle, we have found in some cases that the number of chromosomes appearing is greater than half the somatic number. In the second polar body spindle, as in the first, there are no centrosomes. At the time the second polar body is being produced, it is often possible to observe a division of the nucleus of the first polar body also taking place, and the final result is the production of four nuclei, one remaining in the egg, two derived from the first polar body, and one belonging to the second polar body. We are at present inclined to interpret the high number of gemini and chromosomes appearing during the first and second polar body divisions as possibly due to the fact that in the first polar body division the synaptic chromosomes have not all united in pairs to form gemini before the division ensues; whilst during the second polar body division the number perhaps appears to be high, owing to the fact that when the rods dnd diads are produced they often become prematurely divided, so that, in the act of counting, halves may be reckoned as whole individual chromosomes. In a certain number of eggs a very interesting process can be observed resulting in the parthenogenetic segmentation of the eggs, and the process may extend to the production of 12 or 15 irregular blastomeres; it then comes to an end, the eggs apparently degenerating. The spindles found in these dividing eggs are typically somatic in appearance. This process is probably the same as that described and figured by J. Janosik in his account of the “ Atrophy of the Follicles of the Guinea- pig,’* but it is necessary to draw attention to the fact that Janosik may possibly have missed the point upon which we lay stress. He has failed to separate the process resulting in the production of polar bodies from that ending in irregular segmentation and degeneration of the egg cell, and he apparently considers the stage showing a polar body and the spindle, for the * “Die Atrophie der Follikel und ein seltsames Verhalten der Hizelle,” ‘Arch. f. Mikr. Anat.’ vol. 48, 1897. Roy. Soc. Proc., B. vol. 80, Plate 6. Roy. Soc. Proc., B. vol. 80, Plate 7. 1908.] On the Maturation of the Ovum in the Guinea-pig. 287 production of the second polar body (which he figures), to be merely the first segmentation of an egg which has entered upon a process of degeneration and the follicle of which is about to atrophy. DESCRIPTION OF PLATES. PLATE 5. Fig. 1.—Ovum of Guinea-pig. The nucleus in a late stage of synapsis. Fie. 2—Ovum of Guinea-pig, showing the spindle (first polar body or heterotype spindle). PLATE 6. Fie. 3.—Ovum of Guinea-pig. The nucleus is in the diaster of the meiotic division (z.e., the formation of the first polar body). The intermediate bodies of Flemming are also seen on the fibres at the equator of the spindle. Fie. 4.—Ovum of Guinea-pig, showing the post-meiotic spindle (polar view) and the first polar body. ‘The latter is itself in division. PLATE 7. Fig. 5.—Ovum of the Guinea-pig, showing diaster of meiotic spindle. Fia. 6.—Ovum of Guinea-pig, showing the post-meiotic spindle, or second polar body spindle. The first polar body has divided and its two daughter elements are together on the upper margin of the cell. 288 The Infe-history of Trypanosoma equiperdum. By J. E. SALVIN Moorg, Professor of Experimental and Pathological Cytology, University of Liverpool, and ANTON BREINL, Director of the Runcorn Research Laboratories, Liverpool School of Tropical Medicine. (Communicated by Sir Rubert Boyce, F.R.S. Received March 9,—Read March 12, 1908.) [PLaTEs 8 AND 9.| In May, 1907, we* showed that the study of the parasite of sleeping sickness (Trypanosoma gambiense, Dutton), as it appears in the blood of rats artificially infected with the disease, revealed a cyclical metamorphosis, and that this cyclical metamorphosis corresponded closely to the alternating phases of presence and absence of trypanosomes in the blood. At the same time it was found that the cyclical metamorphosis in the parasites corresponded less closely, but still unmistakably corresponded, with the successive alternations of condition that characterise the clinical aspects of the malady in the host. The features of the life-cycle of the parasites of sleeping sickness as they appear in the blood during infection in rats are remarkable, and may be briefly repeated for reference. From the time of inoculation the parasites multiply in the blood through amitotic division of the nucleus,} and longi- * Note on “The Life-history of the Parasite of Sleeping Sickness,” ‘ Lancet,’ p. 1219, May 4, 1907; “The Cytology of the Trypanosomes,” Part I, ‘Ann. Trop. Med. and Parasitology,’ vol. 1, No. 3. What is called the nucleus of those trypanosomes with which we are acquainted, when fixed in Flemming’s fluid, or by any other appropriate method, does not appear to show any trace of chromosomes. It consists of a central sphere, intra-nuclear centrosome (Salvin Moore and Breinl, loc. cit.) enclosed by a mass of substance, which may be made to stain in a different manner from the sphere. When the nucleus divides, the interior sphere first elongates, then assumes a dumb-bell shape, and finally breaks into two spheres, the outer substance collecting these new centres (intra-nuclear centro- somes), so as to form two smaller nuclei with the same structure and appearance as the first. The reasons for regarding this structure as a nucleus, @.e, as equivalent in a morphological sense to the nuclei of other protozoa, and protophyte bodies, and to the nuclei of the metazoa and metaphites, are simply these: The structure in question bears a superficial resemblance to the nuclei with which biologists are familiar. It divides in the amitotic fashion, ze, as if it were a viscous drop, and owing to the existence of the intra-nuclear centrosome, and to the manner in which this body appears in. tiate the fission, the whole structure bears a close and striking resemblance to the undoubted nuclei of some unicellular organisms, such as Euglena. There is, how- ever, this difference: the nuclei to which that of the trypanosomes bears the closest resemblance, such as those of the Euglene, have been found to possess chromosomes. Chromosomes have been described as appearing during the divisions of the nuclei of The Infe-history of Trypanosoma equiperdum. 289 tudinal fission of the trypanosomes, until a vast number of trypanosomes are produced and the infection of the blood reaches a first maximum. (See chart, p. 290.) After such a period has been reached, the number of parasites in the blood falls until it may be impossible to detect their presence. But subsequently parasites reappear, and a second maximum is reached, and so on. The alternations of these maxima and minima in rats during an infection with T. gambiense are illustrated in the chart given on p. 290. Inthe case of man, trypanosomes by Schaudinn, Prowazek, Minchin, and others, but we have been uniformly unable to confirm these observations, and have reached a diametrically opposite con- clusion, namely, that in the case of Trypanosoma gambiense, T. equinum, T. lewist, T. brucei, and T. equiperdum, chromosomes are not present, and do not exist, at any rate during those forms of division which take place in the blood of a mammal infected with these trypanosomes. [Footnote added April 18, 1908.] Minchin, in the ‘Quart. Journ. Micro. Sci.,’ vol. 32, Part II, describes the structure we term extra-nuclear centrosome as the kineto-nucleus, and a different structure, a small swelling or bead, at the end of the stainable portion of the flagellum, as the blepharoplast or centrosome. The reasons for regarding the body we term the extra-nuclear centro- some as a centrosome are as follows :— The extra-nuclear centrosome appears to be derived from the intra-nuclear centrosome, _ and the intra-nuclear centrosome (karyosome nucleolus) appears to be a structure which is closely similar in its appearance and behaviour to the undoubted intra-nuclear centrosomic, or blepharoplastic, bodies within the nuclei of Euglena, and many protozoa. This conception is strengthened by the fact, originally observed by one of the present authors in 1894 (Moore, ‘ Internat. Monatschr. f. Anat. Physiol. vol. 11), that the true metazoan centrosome is regularly incorporated within the flagellated male gametes of these organisms. In such gametes the centrosomes become more or less definitely related to the flagella, just as the extra-nuclear centrosome is related to the flagella of the trypanosomes. In many metazoan gametes (reptiles) the flagellum abuts directly upon the centrosome. In others (some mammals) this is not so, the flagellum ending in a small bead corresponding to the blepharoplast of Minchin. In such cases the true centrosomes remain quite detached, as in some trypanosomes, for example in 7. lewosz. The bead, when it exists on the base of the metazoan flagellum, is not a centrosome, but simply an enlargement of the proximal end of the flagellum. For this reason we do not regard the blepharoplast of Minchin as equivalent to the blepharoplastic, or centrosomic, structures of other cells, but we regard the bead in question as possibly equivalent to the swelling at the end of the flagellum found among many metazoan cells and gametes. Similarly, we regard the name kineto-nucleus, when applied to what we call the extra-nuclear centrosome, as entirely inappropriate. In the first place, in so far as this structure can be homologised with any structure known in other cells, it appears, as we have said, to have the same relationships as the centrosome. In the second place, it does not appear to have any attributes, except the capacity to divide (a capacity which, of course, is shared by all centrosomes), in common with what is understood as a nucleus. Minchin draws attention to the large size of the extra-nuclear centrosome (kineto-nucleus, _ nucleolus blepharoplast) in some trypanosomes, as indicating that this structure is not of the nature of a centrosome ; but we are unable to see that the dimensions of this body affect the matter in any way, for the undoubted centrosomic or blepharoplastic structures of many male plant gametes are similarly large, if not larger. 290 Prof, J. E. Salvin Moore and Mr. A. Breinl. [Mar. 9, PP RECCE Co S2ec Cs cL suena BREET CEFEEPRERIE a WE ENE Er ee eae a ey The two different 29 30 31 32 33 34 35 36 37 88 39 40 41 42 | LA Hokie —RHK AR = aaa eR a CHEE | 2 a Ea ee Be eee se ia | of latent bodies aS ae | se Bele ae ET | iE inci a Chart of Two Male Rats inoculated with Trypanosoma gambiense. Parasites rising to Period of formation Ist maximum. ~ 2 Le = e ~ ae ate a Pe Ss Leeks a jee Ze iS 7 8 9 10 11 12 18 14 15 16 17 18 19 20 21 22 23 24 25 26 27 2 The horizontal figures represent the days after inoculation: the vertical figures the numbers of parasites in a micro- scopic field of blood, the curve representing the variation in this during the course of the infection. curves represent two different infections. Ou 25 a3: 2 4-25 where the infection runs a relatively prolonged course, the maxima and minima alternate again and again. The alternation is somewhat irregular, and does not possess the definite character of the analogous periods in a 1908.] The Infe-history of Trypanosoma equiperdum. 291 typical malarial infection. In the case of 7. gambiense it has been found* that amitotic division of the parasites proceeds up to the first maximum, but at this period other changes are also apparent in the parasites of the blood (see Diagram I, a, 6, c.) At this period numbers of parasites may be found in Diagram I.—Showing the cyclical metamorphosis occurring in Trypanosoma gambiense in the blood from an infected rat. a. The longitudinal fission of the parasites. 6. The interaction between the extra-nuclear centrosome and the‘nucleus. c. The formation of the latent bodies and the early stages of their redevelopment into trypanosomes. which a thick band is seen to be growing out from the extra-nuclear centro- somes (blepharoplast) which lies about the base of the flagellum (Diagram I, 0). This band extends down the interior of the body towards the nucleus. After a time the band enters into connection with the nucleus, and then breaks up and disappears.— At or near the maxima there is thus an interaction between the extra-nuclear centrosome and the nucleus. When the maximum has * Salvin Moore and Breinl, loc. cit. _ + Referring to our communication, ‘ Annals of Tropical Medicine and Parasitology,’ loc. cit., Swellengrebel (‘Compt. Rend. Soc. Biol., vol. 64, 1908, No. 2) appears to regard the formation of the stainable band as in some manner due to a form of degeneration. We are, however, entirely unable to agree to this conception for the following reasons. The formation of the band occurs only at a particular period of the infections, and at other periods it is not induced either by the administration of substances such as atoxyl or by allowing the trypanosomes to die. A clearer demonstration of the erroneous conclusion drawn by Swellengrebel is, however, found in the facts relating to 7. equiperdum ; here a similar process regularly occurs (see this paper) and after it has occurred the trypanosomes again pass through division, this fact demonstrating that the process to which we refer can have nothing to do with degeneration and is, on the contrary, part of a cycle occurring during the ordinary course of development. Swellengrebel appears to us to be again in error in supposing that the formation of the stainable band is connected with the production of trophic granules. That this is not the case can readily be seen by the use of stains such as that of Breinl or modifications of the iron hematoxylin method, whereby the remains of the band and the band itself stain quite differently to the trophic granules. Moreover, before any stainable band is produced the trophic granules are numerous and must have arisen from some other source. 292 Prof. J. E. Salvin Moore and Mr. A. Breinl. [Mar. 9, been reached, the parasites in the blood rapidly diminish in number, and during the period of diminution large numbers of trypanosomes may be encountered in the lungs, the spleen, and the bone-marrow (but at the same time also in the blood), wherein a profound and rapid change is taking place. The nucleus becomes more compact (see Diagram I, c). There arises a vesicle in connection with it, and eventually a complex structure (latent body) is produced,* consisting of the nucleus and vesicle, and enclosed by a delicate covering of cytoplasm. The latent body becomes detached from the rest of the protoplasm of the cell, and the whole remaining portions of the trypano- some now rapidly degenerate and disappear, so that in a short time we find nothing but numbers of the complex spherical latent bodies remaining. These eventually become chiefly lodged in the spleen, the bone-marrow, and other organs. The process we have just described, or, at least, parts of it, have undoubtedly been seen in other trypanosomes, but not in Gambiense, by various observers, and they have generally been interpreted as a form of degeneration. That this is not necessarily so seems now, however, to have become clearly demonstrated. In the case of 7’. gambiense we were able to find, during the period when no parasites were present in the blood, that the latent bodies still persisted in the organs, and that many of these gradually grew larger by developing a cytoplasmic investment, a new extra- nuclear centrosome, and afterwards a flagellum, such forms returning eventually to the form of ordinary trypanosomes. We have thus in the case of the sleeping sickness parasite a cyclical meta- morphosis going on in the blood of the infected animal. The parasites pass through divisions until the interaction between the extra-nuclear centrosomes and the nucleus. From this period they proceed to the formation of latent bodies, and subsequently to the development from the latent bodies of ordinary trypanosomes once more. | The interaction between the nuclei and the extra-nuclear centrosome (blepharoplast) may, as we have pointed out,} suggest a novel form of sexual process, and the whole series of changes may indicate that the life-cycle of T. gambiense is in reality completed in the body of the rat or man, and not necessarily related to the transference of the parasites to any other form of host. We know, however, that in the form 7. gambiense the parasites can be transmitted by the fly Glossina palpalts. The observations of Schaudinn apparently indicate that in the case of Trypanosoma noctue the sexual stage (which is described by him as quite unlike the process to which we have just drawn attention) occurs in the body * Salvin Moore and Breinl, loc. cz. + Salvin Moore and Breinl, loc. cit. 1908.| The Life-history of Trypanosoma equiperdum. 293 of a mosquito. This mosquito appears thus to stand in the same relation to the owl Athene noctua as Glossina palpalis does to man. It is perfectly natural therefore to suppose that whatever cycle we may have found in the blood during infections with 7. gambiense, the real sexual phase may occur within Glossina palpalis or some other fly. From the informaticn which at present exists this line of criticism cannot be answered through observations upon infections with 7. gambiense. On this account, and pending further investigation in relation to 7’. noctuw, we have turned our attention to the parasites of the horse disease known as “ Dourine,’ and caused by T. equiperdum. Dourine* is not necessarily transmitted through any fly or intermediate host, but by direct contact between animals that have become infected. In this case we have, then, a trypanosome the life-history of which is not necessarily complicated by transference through an intermediate host. Whatever sexual phase there may be in the life-history of this parasite must be passed through in the body of the horse. If 7. equiperdum be injected into rats, the parasites multiply and kill the animal in about four days after their first appearance in the blood, which occurs about three days after the inoculation.t The disease in rats thus reaches a first maximum, and the animal dies without being able to overcome the invasion even temporarily, as would appear to be the case in infections produced by 7. gambiense in rats. The method of investigation has been as follows:—From the time the trypanosomes appear in the blood a very large number of slides have been prepared at short intervals up to death, and for a short time afterwards. Owing to the manner in which the disease is trans- mitted, special attention was paid to the fluids which collect in the various superficial swellings that are produced, for it naturally seemed possible that a phase of the life-history might occur in such positions in relation to the transmission of the parasites. The results of a prolonged investigation of this matter have, however, revealed nothing but the presence of ordinary trypano- somes,} and it is thus indicated that the transference takes place by means of the ordinary trypanosome encountered in the blood, possibly through the existence of slight abrasions on the animals that become infected, or more probably through the capacity of the trypanosomes to invade a mucous membrane, even if it is intact. * The strain we have used was obtained through the courtesy of Geheimrath Professor Ulenhuth, in Berlin. + The best general account of dourine is contained in the works of Laveran and Mesnil’s ‘Trypanosomes et Trypanosomiases,’ Paris, 1904. t In rabbits, when few trypanosomes appeared in the blood it is interesting to note that in the fluids from the swollen vagina there existed many more parasites than in the blood. 294 Prof. J. E. Salvin Moore and Mr. A. Breinl. [Mar. 9, Preparations of the blood during early stages of infection show the trypanosomes to be increasing in numbers through rapid longitudinal fission, accompanied by amitotic division of the nucleus and the extra-nuclear centro- somes (Plate 8, figs. 1—4). As the disease advances, two series of structural changes in the parasite become apparent; one of these (figs. 6—10) consists in a gradual increase of the nuclear substance towards the side away from the extra-nuclear centrosome, until, after forming a distinct protuberance, the mass separates from the nucleus and passes away towards the free end of the flagellum in the manner represented in figs. 8, 9, and 10. It is only possible at present to describe the existence of this process. It may be related to the formation, or rather the renewal, of the so-called “trophic granules,’* but we cannot decide this matter at the present time. The other process to which we have referred is of a totally different order. Towards the end of an infection, that is to say, on the third day after the appearance of the parasites in the blood, numbers of trypanosomes are observed, wherein the extra-nuclear centrosomes become conspicuously large (figs. 11, 12), and at the same time there exist others, in which it is seen that the extra-nuclear centrosome is budding off a large mass towards the nucleus. This mass becomes detached, and can be found in many individuals passing away towards the nucleus (figs. 12, 13, 14). There is often a distinct, but faint, suggestion of a protoplasmic thread still connecting the detached body with the extra-nuclear centrosome at the base of the flagellum (figs. 12, 13, 14). Whiie this process is going on the intra-nuclear centrosome moves towards the extra-nuclear centrosomes, as in figs. 13, 14, and in a number of trypanosomes stages may be found in which the detached portion of the extra-nuclear centrosome is seen to pass completely through the body of the trypanosome until it becomes applied to the nucleus, as in figs. 15, 16. Some time later, the detached portion of the extra-nuclear centrosome merges with, and becomes indistinguishable from, the nuclear substance, and the trypanosomes again pass through division, as in figs. 16,17 (Plate 9). As division sometimes begins again before the detached centrosome is fused with the nucleus, the process can have nothing to do with any sort of degeneration. The process we have just described in the parasite of dourine is clearly analogous to the production of the stainable band in 7. gambiense. It differs only in there being a detached mass which passes from the extra- * It is curious to note that Laveran and Mesnil, Joc. cit., p. 273, are under the impres- sion that one of the specific characters of 7. eqguiperdum is constituted by the absence of protoplasmic granulations ; whereas, on the contrary, we have found them in abundance. It is impossible to say upon what this difference of condition depended. 1908.] The Life-history of Trypanosoma equiperdum. nuclear centrosome to the nucleus instead of a continuous band. As a matter of fact, the difference is really less than this, for in T. equiperdum the extra-nuclear centrosome not only seems to be connected with the detached portion, but also several portions may be detached, one after another. (Compare Diagrams I and IT, a, 0, ¢.) The phase wherein the detach- ment of a portion of the extra- nuclear centrosome which passes to the nucleus takes place is of very short duration, and at the same time appears to affect the great majority of the parasites present in the blood, just in the same way that an epidemic of conjugation among infusoria will affect at the same time a whole colony. In the specimens from which the figures aregiven the pro- cess was in full swing at 10 AM., but by 5 P.M. had come to an end. At the time this process is going forward, and immediately afterwards, still further changes take place, corresponding to the changes which in 7. gambvense precede the production of the latent bodies. But the latent bodies of dourine are very much larger than those in 7’. gambiense, and consequently their formation is proportionately less difficult to elucidate. When the process of translocation of a portion of the extra-nuclear centrosome has come to an end, and the parasites have again passed through 2A VOL. LXXX.—B. Diagram II.—Showing the cyclical metamorphosis occurring in Trypanosoma equiperdum in the blood of an infected rat. a. The b’. c. The formation The b. The interaction between the extra-nuclear centrosome and the nucleus. longitudinal fission which occurs after the interaction between the extra-nuclear centrosome and the nucleus. of the latent bodies and the growth of their flagelle. longitudinal fission of the parasites. 295 296 Prof. J. E. Salvin Moore and Mr. A. Breinl. [Mar. 9, several longitudinal fissions, a certain number of them are seen to become shorter, and when subject to the action of Breinl’s stain are relatively blue, in contrast to the purple coloration of the remaining parasites (figs. 19, 20, 21, 22). In such altered forms it is seen that the extra-nuclear centrosomes become related to a protoplasmic elongation, produced perhaps by the rounding up of the protoplasm of the animal’s body (figs. 20—22). This elongation becomes finally elub-shaped, and the extra-nuclear centrosome, together with the flagellum, becomes detached from the rest of the cell, which is now more or less round (figs. 21—23). In many cases quite a considerable portion of protoplasm is detached along with the extra-nuclear centrosome, and the detached structure may present, at first sight, very much the appearance of a spermatozoon (figs. 23, 24). In some cases it seems that when this process 1s going forward, the extra- nuclear centrosome having divided, one half of this structure passes down the stalk of the protoplasmic club and enters the rounded mass. In any case, however, at the time the extra-nuclear centrosome has become detached, a. new centrosome becomes visible in the remaining round cell (figs. 23, 24, 25). From this there grows out an exceedingly fine fibre, which is much more delicate than the ordinary flagellum (figs. 23, 24, 25). The new extra-nuclear centrosome divides, and after a time a second flagellum grows out from the second extra-nuclear centrosome contained in the round form (figs. 25, 27). One peculiarity in relation to these new flagella is their great length, this often being in the proportion of 7: 4 when contrasted with the length of the flagellum of an ordinary trypanosome. The changes we have just described appear ordinarily to take place before the death of rats infected with 7’. equiperdum, but may be encountered also at, and for some time after, death, from which facts it might be natural to suppose that they are related to the changed condition occurring during the approach of death. We have, however, found no evidence for this supposition. In none of the changes during the production of the round forms or latent bodies is there the least suggestion of degeneration. The growth of the new flagella and the division of the extra-nuclear centrosomes in the round forms are entirely against such a view. This latter conception is confirmed by many experi- ments we have made. Thus, if a rat be killed at the time the body derived from the extra-nuclear centrosome is passing towards the nucleus in large numbers of the trypanosomes, no changes analogous to those we have described take place in the blood of the dead animal. We have encountered in such cases, as time goes on, nothing but degeneration and disintegration of the trypanosomes. Again, in many cases the infection of dourine kills the 1908.] The Life-history of Trypanosoma equiperdum. 297 rats before the life-cycle in the parasites has reached the point at which the interaction between the extra-nuclear centrosome and the nucleus occurs, and here also it is found that after death no changes take place in the trypano- somes other than those related to their degeneration, or in any way corre- sponding to the formation of the latent forms. Still further, we have at various periods of the infections abstracted blood, and watched the condition of the parasites until degeneration is becoming general, and in these cases also have encountered nothing comparable to the changes we have described in relation to the formation of the latent bodies. The change is thus related to a particular stage of the development of the trypanosomes in the blood. It will be remembered that the formation of the latent bodies in 7’. gambiense takes place at, and immediately after, the periods of maximum number of the parasites in the blood, and the immergence from the latent bodies once more occurs a considerable time later. The infection with TZ. equiperdum in rats only progresses to a first maximum, during which the rat dies. We find also that it is only in those rats which have resisted the infection for a sufficient period that the formation of latent bodies in large numbers takes place. It should, however, be pointed out that, even on the second day after the appearance of the trypanosomes in a rat infected with dourine, a few trypanosomes with club-shaped projections, and a few latent bodies with their long, fine flagella, may occasionally be encountered. This corresponds to the occasional appearance of latent bodies during almost all the periods in an infection of 7. gambiense. It would be extremely interesting to ascertain what exactly happens during the successive periods of maxima and minima, which succeed one another when a horse is infected with dourine; but we have found that even at the maxima of such infections the parasites are so few in number as to render it practically impossible to utilise horses for this object. It would seem, then: that during the infection of rats with dourine, that is to say, with a form of trypanosome which under normal circumstances is not. related to two distinct hosts, there exists a life-cycle among the parasites closely analogous to that occurring during the successive positive and negative periods of infection of the same animals with 7. gambiense. The parasites, after introduction into a rat, multiply by longitudinal fission, accompanied by amitotic division of the nucleus. After this process, an interaction takes place between the extra-nuclear centrosome and the nucleus (sexual stage ?). Division again proceeds, and finally the trypano- somes are converted into round bodies, which correspond to the latent bodies of 7. gambiense, but possess two long and delicate flagella. 2 kB 298 The [nfe-history of Trypanosoma equiperdum. DESCRIPTION OF FIGURES. In both plates the coloured figures are stained with Breinl’s stain, and the extra- nuclear centrosome should be purple, but not so red as the intra-nuclear centrosome. PLATE 8. Fies. 1—5.—Stages in the longitudinal fission of 7. equiperdum. Figs. 1—4 showing the amitotic division of the nucleus, x. c, the intra-nuclear centrosome. In figs. 2 and 3 the intra-nuclear centrosome is dividing, below are the trophic granules. Figs. 1—4 are stained with Breinl’s stain, fig. 5, a late stage in the fission, shows the characteristic inequality in the size of the resulting cells. Fias. 6-—10.—Successive stages in the detachment of a portion of the nuclear substance. Figs. 7—10 stained with iron heamatoxylin. By this method the trophic granules are not shown. Fies. 11—15.—Successive stages of the passage of a portion of the extra-nuclear centrosome to the nucleus. PLATE 9. Fic. 16.—Late stage during the passage of the extra-nuclear centrosome to the nucleus. The detached mass is now practically fused with the nucleus. Compare figs. 14 and 15. Fic. 17.—T. equiperdum again dividing after the fusion of a portion of the extra-nuclear centrosome with the nucleus. Figs. 18—22.—Stages in the development of the latent body. Figs. 23 and 24.—Detachment of the old flagellum. and appearance of a new extra- nuclear centrosome, and a new flagellum. Fias. 25—28.—Complete latent bodies showing division of the extra-nuclear centrosome and the formation of two long delicate flagella. BVol.80PL. 8. Sae Eroe. S BO,PU. 9. 3 § a 299 The Alcoholic Ferment of Yeast-yuice. Part IL—The Function of Phosphates in the Fermentation of Glucose by Yeast-jwmee. By ARTHUR HARDEN and WILLIAM JoHN YouNG (Biochemical Laboratory of the Lister Institute of Preventive Medicine). (Communicated by C. J. Martin, F.R.S. Received March 6,—Read April 2, 1908.) In a previous communication the authors have shown* that when a soluble phosphate is added to a fermenting mixture of glucose and yeast-juice the following phenomena are to be observed: (1) The rate of fermentation is at once greatly increased. (2) This acceleration lasts for a short time and the rate then falls off, and returns approximately to its original value. (3) During this period the extra amount of carbon dioxide evolved and alcohol produced are equivalent to the phosphate added. (4) The phosphate is converted into a form which is not precipitable by magnesia-mixture, and is then probably present as a salt of a hexosephosphoric acid. (1) Lffect of the Addition of Phosphate on the Total Fermentation. The addition of phosphate, however, does not simply produce this initial decomposition of an equivalent of glucose, but also, as a rule, a greater total fermentation, after allowance has been made for the amount decomposed during the initial period. This is clearly shown by the results embodied in the following table. In each experiment two or more portions of 25 cc. of yeast-juice were taken, a solution of glucose alone, or one of glucose and phosphate, added, and the total volume made to 50 ¢.c. The solution of phosphate employed had a concentration of about 0°3 molar, and the concentration of glucose was 20 grammes per 100 c.c. in experiment 8, and 10 grammes per 100 c.c. in all the others. The fermentation was carried out at 25° in presence of toluene until the evolution of gas ceased. The numbers in the last column show the increase in the total fermentation produced during the period subsequent to the initial acceleration. It will be seen that the increase which occurs after the initial period varies from about 10 per cent. of the original fermentation to as much as 150 per cent. * “Roy. Soc. Proc.,’ B, vol. 77, 1906, p. 405. 300 Messrs. A. Harden and W. J. Young. [ Mar. 6, Cubic centi- E . |CO, evolved | metres of | CO; evolved Increase | CO, equiva-| Increase meee without phosphate | in presence due to lent to after initial eee phosphate. aap of phosphate.! phosphate. | phosphate. period. added. grammes. C.c. grammes. gramme. gramme. gramme. 1 0 °484 10 Orly 0 °233 0 °132 0°101 2 1 -280 5 1 °584 0 304: 0 ‘066 0-238 3a 0 °422 10 0 634 0 °212 0-132 0 -080 3b 0 °422 20 0 748 0 326 0 264 0 -062 4 0 °440 10 1 ‘258 0°818 0 °182 0 686 5 0-405 5 0-515 0-110 0-070 0 -040 6 0 603 5 0 ‘735 0-132 0 -066 O ‘066 a 0 °438 5 0 -593 0 °155 0 057 0 -098 8 1-016 15 1 632 0-616 0-198 0 418 C.c. c.c. Os OW, c.c. 9 369 10 629 260 63 197 10 337 10 569 232 56 176 (2) Recurrence of Phosphate. The reason for this increase in the amount of sugar decomposed in the long period following the short initial period of acceleration appears to be that the phosphorus compound first formed, which is a hexosephosphate of the formula CgHi04(POsRe)2,* is slowly hydrolysed, probably by an enzyme, with the production of a phosphate and a hexose. The phosphate is thus slowly regenerated and then again undergoes the reaction, causing an increased fermentation in the same manner as when it was originally added. This recurrence of phosphate is clearly shown by the following experiment. A known amount of phosphate was added to yeast-juice containing glucose, and the mixture incubated at 25° with toluene. At the close of the initial period a sample was removed, boiled and filtered, and the free and total phosphate present in it estimated, and this process was repeated at stated times. The results obtained are given below, the amounts of phosphate being expressed in grammes of MgeP20; per 10 c.c. Experiment 11.—215 c.c. of yeast-juice+ 20 grammes of glucose were made to 375 c.c. with a solution of potassium phosphate, the amount of the latter being equivalent to 0°133 gramme MgeP,0, per 10 c.c. of the resulting liquid. The slight increase in the total phosphate present is due to a corresponding ~ degree of evaporation during the experiment. It will be seen that the free phosphate per 10 c.c. gradually increases from 0°021 to 0°:226, so that 0:205 gramme is regenerated. Since the total phosphorus, expressed as phosphate in * Young, ‘Chem. Soc. Proc.,’ 1907, vol. 65. The Alcoholic Ferment of Yeast-juace. 301 | Time Free phosphate as Mg,P,07 | Total phosphate as Mg»P,O, | in hours. per 10 c.c. per 10 c.c. gramme. gramme. 5°5 0-021 0 266 18-0 0 093 0 ‘269 66 ‘0 | 0. 133 138°0 | 0°175 426 °O 0-226 0-273 the original juice, was 0°266—0°133=0°133 gramme, it follows that at least 0'205—0:133=0:072 gramme of this has been derived from the hexose- phosphate produced during the initial period from the added phosphate. Even in the absence of added phosphate a gradual production of free phosphate occurs when yeast-juice is incubated at 25°. In the absence of glucose, when the fermentation is very small, the increase in the amount of free phosphate is comparatively rapid, whereas, in the presence of glucose, the fermentation lasts for a considerable period, and the appearance of free phosphate is delayed, since it continually enters into fresh combination as rapidly as it is formed. Examples of this are the following. The various materials were digested at 25° with toluene for the time shown, and were then boiled and filtered and the free phosphate estimated. Experi- a oe Time Free phosphate as Gi. PS PSESS ING ee siat of digestion. Mie PLOMGEE! 25 ©€.c. days. gramme. 12 (a) Yeast-juice alone .................006 O 0 +128 b ‘ Ma ate Fete Na Leer 2 0-284 (c) : i Ok cee oe ane 10 0-283 (d) Yeast-juice + glucose ................4 2 0 +123 (e) , A ee eee ee 10 0 °255 13 (@) Neast-juice‘alone ......22....a0s0.6s0: 0 0 024 (d) ei a tie ee Pee satis | 1 0-053 (c) i Ay Ne eae § cere ge cn : 2 0 063 (d) 5 oP nudes ee ate se te tse) ale 4 0 069 (e) Yeast-juice + glucose ..............0065 1 0-011 i aM Dt et benders | 2 0 025 (9) 55 me etn oe aCe 4 0-051 hours. 14 (a) Yeast-juice + glucose + phosphate 1°25 0-063 (0°276 gramme per 25 c.c.) re 6 ob 46 ‘0 0°138 (c) A is a 96-0 0 °387 (d) ” ” ” 168 -0 0 °453 (e) ” ” ” 240 -O 0 -489 (f) ” ” » 336 -0 0 501 (9) ” 9 29 456 -O 0 °523 302 Messrs. A. Harden and W. J. Young. [ Mar. 6, In this last experiment (No. 14) sample (a) was taken at the close of the initial period and sample (0) immediately after the cessation of fermentation, these points being determined by observation of another sample of the yeast- juice. It is to be noted that during the fermentation only a small increase occurs in the amount of free phosphate (0:075 gramme), while after the cessation of fermentation the increase amounts to about three times as much (0°249 eramme) in approximately an equal time. The total phosphate of the original juice was 0°350 gramme per 25 cc. and the amount added was 0-276 gramme. Since only 0:104 gramme of phosphate remains combined at the close of the experiment, it follows that at least 0°276—0:104 = 0-172 gramme has been regenerated from the hexosephosphate. The recurrence of phosphate in these cases appears to be due to the action of an enzyme—whether a special enzyme or one of those already known to occur in yeast-juice has not yet been determined. After yeast-juice containing the sodium salt of hexosephosphoric acid has been boiled the amount of free phosphate remains practically unaltered when the liquid is incubated at 37°, as is shown by the following experiment. Experiment 15.—To yeast-juice containing glucose and an amount of free phosphate corresponding to 0°184 gramme of Mg.P20; per 25 c.c. was added sodium phosphate equivalent to 0°285 gramme of MgeP20; and the mixture incubated at 37° in presence of toluene. A sample was taken at the close of the initial period, and as soon as fermentation had almost ceased the whole was boiled, the free phosphate estimated and the remainder of the boiled liquid preserved at 37°, samples being taken at intervals. At the close of the initial period, 25 c.c. yielded only 0°04 gramme of Mg2P20;, so that practically the whole of the added phosphate must have been converted into a salt of hexosephosphoric acid. At the cessation of fermenta- tion the same volume yieided 0-131 gramme of MgeP20;, so that at least. 0:15 gramme was still present as a hexosephosphate. After incubation of the boiled liquid at 37° for an additional 144 hours, the amount of free phosphate had only increased to 0°138 gramme, so that the action practically ceased when the liquid was boiled. (3) Nature of the Chemical Change which occurs in the Fermentation of Glucose by Yeast-juice. The cycle of changes which is undergone by a phosphate in the presence of yeast-juice and glucose appears from the foregoing to be as follows :— (1) 2CgH120¢ + 2Re.HPO, = 2CO2 -- 2C2,H,O + CgH1004(PO1R2)2 a+ 2H.0. (2) CgHi04(POgRe)o+ 2H.2O a CgHi20¢6+ Diol ie Oa 1908. ] The Alcoholic Ferment of Yeust-juice. 303 The first of these equations does not include the fermenting complex, with- out which, however, the change does not occur, and it is probable that both the glucose and the phosphate form an intermediate association with this complex, which then breaks down, giving rise to the substances on the right- hand side of the equation, and at the same time regenerating the fermenting complex. Since free phosphate and a hexosephosphate are invariably present in the yeast-juice prepared by grinding yeast, it follows that at all events some portion of the fermentation is always clue to the foregoing reactions. During the initial period of rapid fermentation, as long as free phosphate is still present, the greater part of the change is certainly due to this reaction, whilst in the succeeding period of slower fermentation the constant production of free phosphate by the enzymatic hydrolysis of the hexosephosphate already formed, or by the action of proteoclastic enzymes on phosphoproteins, renders it equally certain that some portion of this greatly diminished fermentation must also be ascribed to the same reaction. The question at once arises whether it is not possible that the whole of the fermentation is due to this reaction. So far, no fact has been encountered which is inconsistent with this view. In previous communications™ the authors have shown that at least two substances are concerned in the production of carbon dioxide and alcohol from glucose: the ferment, which is thermolabile, and the coferment, which is thermostable; and, further, that phosphates are incapable of producing any fermentation when they are added to a mixture of the ferment and glucose in the absence of the coferment. If the theory suggested above be found to be correct, it will be necessary to complete this statement. Two possibilities present themselves. Hither a third substance, a soluble phosphate, is also necessary, and in the presence of the fermenting complex, made up of ferment and coferment, undergoes the reaction under discussion ; or the coferment may itself be a complex substance containing a group of unknown composition, united with the phosphoric acid group. The latter would then be passed on to the glucose during fermenta- tion, and a new group taken up from the phosphate, a continuous conversion of phosphate into hexosephosphate being thus effected. If this cycle of changes correctly represents the reaction which occurs, it follows that the rate of fermentation after the initial period of acceleration depends, in the first instance, on the rate at which phosphate is liberated according to equation (2). The most satisfactory method of testing the accuracy of this view would ‘be to free both ferment and coferment from phosphate and materials * *Roy. Soc. Proc.,’ B, vol. 77, 1906, p. 405 ; B, vol. 78, 1906, p. 369. “304 Messrs. A. Harden and W. J. Young. [ Mar. 6, ‘capable of yielding phosphates under the influence of the enzymes which -are present, and then to ascertain whether the mixture of these purified materials would ferment glucose; and, further, if they did not, whether the -addition of a phosphate would bring about fermentation. Many attempts have been made to realise these conditions, but hitherto without success. It is obvious that if the coferment be a hydrolysable derivative of phosphoric acid, as suggested above, success in this direction cannot be anticipated. Some idea of the extent to which alcoholic fermentation is due to this recurrence of phosphate could also be gained by ascertaining the amount of free phosphate produced from hexosephosphate in yeast-juice in a given time ‘in the absence of glucose, and comparing this with the amount of carbon dioxide evolved in the presence of glucose under otherwise identical conditions. In practice, however, two difficulties present themselves. In the first place, in the absence of fermentable sugar, or when the concentration of this is very low, free phosphate accumulates, and the rate of hydrolysis of the hexosephosphate is thus diminished; whereas in the presence of glucose the concentration of phosphate remains constant at a low value during fermen- tation, and the rate of hydrolysis of hexosephosphate accordingly remains at its maximum for a considerable period. This inhibitory effect of phosphate on the hydrolysis of hexosephosphate in the absence of all fermentation is shown in the following experiment, which also indicates the extent to which this hydrolysis occurs under these conditions. EHxpervment 16.—In order to avoid all fermentation, the inactive residue obtained by filtering yeast-juice through a Martin gelatin-filter was employed, and equal weights of this were incubated for 20 hours with: (1) water ; (2) a solution of sodium hexosephosphate free from glucose and free phosphate ; (3) a solution of sodium hexosephosphate + an equivalent amount of sodium phosphate. A solution of hexosephosphate was also incubated alone, the conditions of concentration and alkalinity being identical in all four solutions. At the expiration of 20 hours, the solutions were all boiled, and the free phosphate in the filtrates estimated. In the following table the numbers represent the weight of Mg2P,0, found. The numbers in the last column are obtained by adding together the -amount of phosphate produced separately by the incubation of the hexose- phosphate and the residue, and subtracting their sum from the free phosphate produced in the other two solutions. It appears from this that in this particular instance the enzymatic 1908. | The Alcoholic Ferment of Yeust-jwee. 305 Amount of free phosphate present. Amount of ; hexosephosphate Solution. hydrolysed by Before incubation. | After incubation. enzyme. 1. Hexosephosphate equivalent to 0:0 0 -0089 0 1626 gramme Mg,P,0, | 2. 0°75 gramme residue + water ...... 0-0 0 -0166 | 3. 0°75 gramme residue + hexosephos- | 0°0 0 1068 0 0813 phate equivalent to 0:°1626 gramme Mg,P.O, 4, 0°75 gramme residue + hexosephos- 0 1626 0 1884. 0 0003 phate as above, +sodium phos- phate equivalent to 0-°1626 gramme Mg,P.O0, decomposition of the hexosephosphate is almost completely arrested by the presence of an equivalent of free phosphate. The amount of phosphate actually produced in solution 3 from all sources is 01068 gramme Moeg2P20;, which corresponds to an evolution of about 22 cc. of carbon dioxide, and this in spite of the fact that the phosphate has been allowed to accumulate. The amount which would be evolved in presence of glucose and coferment would naturally exceed this, owing to the continued reconversion of the phosphate into hexosephosphate. This experiment also shows that the ferment which brings about the hydrolysis of the hexosephosphate is present in the residue obtained by filtering yeast-juice through a Martin-filter, and does not require a dialysable coferment. The second difficulty arises from the fact that when yeast-juice or a mixture of ferment and coferment is employed, a certain amount of fermentation always occurs, even in the absence of added sugar. This is due to sugar formed in the liquid, in part by the hydrolysis of glycogen and dextrins and in part by the hydrolysis of the hexosephosphate itself, which yields a fermentable sugar as one of its products. The practical result is that if an actual comparison be instituted between the production of phosphate in the absence of added glucose and the evolution of carbon dioxide in the presence of glucose, it will be necessary to take the sum of the carbon dioxide actually evolved and the carbon dioxide equivalent of the phosphate produced in the absence of added glucose, and this will always be found to be less than the volume of carbon dioxide observed in the presence of glucose. The result of such a comparison is shown in the two following experiments. A mixture was made of yeast-juice with a solution containing a suitable amount of sodium phosphate and just sufficient glucose to bring about the conversion of the greater portion of the phosphate into hexosephosphate, this 306 Messrs. A. Harden and W. J. Young. [ Mar. 6, being found by experiment to be rather more than an equivalent of glucose. The mixture was incubated until this conversion had been accomplished and the rate of fermentation had become steady. A sample was then taken and boiled and the free phosphate estimated in the filtrate. The remainder was incubated for a further period, the evolution of gas being noted, and a second sample was then taken. A parallel experiment was carried out with yeast-juice containing the same amount of sodium phosphate in presence of 10 grammes of glucose per 100 ¢.c., and the evolution of gas during the same period was observed. The solutions used and the results obtained were as follows :-— Experiment 17.—(1) 100 cc. yeast-juice+ 30 ¢.c. of a solution containing 1°3 grammes sodium phosphate and 2°62 grammes glucose. (2) 100 c.c. yeast- juice+30 c.c. of a solution containing 1°3 grammes sodium phosphate and 13 grammes glucose. Experiment 18.—(1) 100 ¢.c. yeast-juice + 40 c.c. of a solution containing 1°7 grammes sodium phosphate and 3:16 grammes glucose. (2) 100 cc. yeast- juice + 40 c.c. of a solution containing 1°7 grammes sodium phosphate and 14 grammes glucose. Solution 1 (low concentration of glucose). Solution 2 (excess of glucose). | Final Phospl CO CO i tal CO | Original | ina osphate | 2 2 ota 2 | phosphate. phosphate. | produced. | equivalent.| evolved. | equivalent. COsserolvad. c.c. c.c. 0 :0559 0 °1052 0 04938 10 °7 39 °8 50 °5 93 °9 0 *1036 0°1701 0 -0665 14°6 27 “7. 42 °3 69 °2 These experiments show that although the sum of the carbon dioxide evolved and that equivalent to the phosphate produced in absence of glucose is invariably less than the amount of carbon dioxide evolved in the presence of glucose, yet the difference is no greater than might be expected from a knowledge of the prevailing conditions, and is quite consistent with the view that the whole of the fermentation proceeds according to the equation proposed. (4) Influence of Concentration of Phosphate on the Course of the Fermentation. When a phosphate is added to a fermenting mixture of glucose and yeast- juice, the effect varies both with the concentration of the phosphate and with the particular specimen of yeast-juice employed. With low concentra- 1908. | The Alcoholic Ferment of Yeast~jwice. 307 tions of phosphate the acceleration produced is so transient that no accurate measurements of rate can be made. As soon as the amount of phosphate added is sufficiently large, it is found that the rate of evolution of carbon dioxide suddenly increases from 5 to 10 times, and then rapidly falls approximately to its original value. As the concentration of phosphate is still further increased, it is first observed that the maximum velocity, which is still attained immediately on the addition of the phosphate, is maintained for a certain period before the fall commences, and then, as the increase in concentration of phosphate proceeds, that the maximum is only gradually attained after the addition, the period required for this increasing with the concentration of the phosphate. Moreover, with these higher concentrations the maximum rate attained is less than that reached with lower concentrations, and, further, the rate falls off more slowly. The concentration of phosphate which produces the highest rate, which may be termed the optimum concentration, varies very considerably with different specimens of yeast-juice. All these points are illustrated by the accompanying tables and curves. Experiment 19.—The following solutions were employed :— (1) 25 cc. yeast-juice +15 c.c. sodium bicarbonate solution. (2) 25 c.c. yeast-juice +10 c.c. of potassium phosphate solution (0°3 molar) +5 ¢.c. sodium bicarbonate solution. (3) 25 ce. yeast-juice+15 cc. potassium phosphate solution. All the solutions contained 4 grammes of glucose, and the experiment was carried out at 25° in the presence of toluene. The solution of bicarbonate added contained an amount of this salt equal to that formed by the action of carbon dioxide on an equal volume of the solution of potassium phosphate employed. | Carbon dioxide evolved in preceding 5 minutes with n | Time after | cubic centimetres of 0°3 molar potassium phosphate added. addition in minutes. | m=O... n= 10 c.c¢, n = 15 c.c. 5 4 °O nT es Cae 10 3 °2 16 ‘0 9°7 15 A. 2 20 °2 12:1 | 20 3°6 22°4 16: 25 4 °3 17 °4 18 “4 30 3 °6 66 19 °4 35 4°3 4. °6 20-4 40 3 °2 47 16 °7 45 =a 4°5 12 °7 50 Ses 4°2 6°0 55 41 4, °0 308 Messrs. A. Harden and W. J. Young. [ Mar. 6, Hzpervment 20.— (1) 25 cc yeast-juice+ 5 c.c. phosphate+15 c.c. bicarbonate. (2) e2dne%c: “ +10 cc. Bs +10 ce. r. (3) 29: ¢.¢, . +15 ce. F + 5 cc. " (4) 25 ee, re +20 cc. re + 0c. 7 The concentration of glucose was 4°5 grammes per 45 c.c., and the experi- ment was carried out at 25° in presence of toluene. Carbon dioxide evolved in preceding 5 minutes with » cubic centimetres Time after of 0°3 molar phosphate added. addition in minutes. nm =.5 C.C. nm = 10 c.c. Ny —=wlanescs nm = 20 cc. _ -) ep me 7 0 bo 7 oo 30H a ern p we oom Ddy TWOOMOAKOHENW-T ONTIATSCOHODNDIHAMSTNbwWwnwe SDODHAAUIMHMWMOHROHAOKRG ANMIAHaHBWnAnKRRWOWWNYDNYDNNDOHE SEMODODNIDOLANHWAWDOOANSA Meealey all eae ected eet Pea ale biped et | Curves A, B, C, and D (fig. 1) show the rate of evolution per five minutes for the four solutions in experiment 20. The time of addition is taken as zero, the rate before addition being constant, as shown in the curves. It will be observed that in experiment 19 practically the same maximum is attained with 10 and 15 cc. of phosphate, whereas, in experiment 20, 5 and 10 cc. give the same maximum, whilst 15 ¢.c. produce a much lower maximum, and 20 cc. a still lower one, the rate at which the velocity diminishes after the attainment of the maximum being correspondingly slow in these last two cases. By calculating the amount of phosphate which has disappeared as such from the amount of carbon dioxide evolved, it is found that the maximum does not occur at the same concentration of free phosphate in each case. These results suggest that the phosphate is capable of forming two or 1908.] The Alcoholic Ferment of Yeast-jucce. 309: all “aa Te Se Ree sie A SCO SS acs SE a0408// S000 60} 2 ee eee em + 12 {2 C5 ay se a ae =o) ek) a n PAAR EERE EEE TH ree: IE Reo 2S Cea Ss, $ 7 COT 5 PCa a | Zane Doe es oe stony Pe on Ha ert ee eames, ys) Neg e- mie a e ‘ ae ee Pe ia 0505202530 40 50 60 7 &d 90 oo 0 Time in minutes Fig. 1. more different unstable associations with the fermenting complex. One of’ these, formed with low concentrations of the phosphate, has the composition most favourable for the decomposition of sugar, whilst the others, formed with high concentrations of phosphate, contain more of the latter, probably associated in such a way with the fermenting complex as to render the: latter partially or wholly incapable of effecting the decomposition of the sugar molecule. As the fermentation proceeds slowly in the presence of. excess of phosphate, the concentration of the latter 1s reduced by conversion: into hexosephosphate, and a redistribution of phosphate occurs, resulting in the gradual change of the less active into the more active association of phosphate with fermenting complex, and a consequent rise in the rate of | fermentation. In those cases in which the maximum rate corresponding to the optimum concentration of phosphate is never attained, some secondary cause may be - supposed to intervene, such as a permanent change in a portion of the fermenting complex, accumulation of the products of the reaction, etc. Experiments on this point are being carried out by varying independently — the concentration of the ferment, the coferment, the phosphate, and the. hexosephosphate. 3 * In agreement with these conclusions it is found that a high rate of evolution | of carbon dioxide can be maintained for a considerable period by the gradual addition of phosphate in such a way that the concentration of free phosphate - remains approximately at the optimum value. This may be effected by’ 310 Messrs. A. Harden and W. J. Young. | Mar. 6, adding in every interval of five minutes the amount of phosphate equivalent to the carbon dioxide evolved in excess of the normal rate of fermentation of the yeast-juice during the same interval of time. Experiment 21.—25 c.c. of yeast-juice + 2°5 grammes glucose were incubated at 25° in the presence of toluene until the rate became constant. A solution of potassium phosphate (2 mol. KzHPO. and 1 mol. of KH2PQ,) of 0°3 molar concentration was then gradually added from a graduated pipette provided with a tap and passing through the cork of the fermentation flask. Column 2 shows the c.c. of this added in each period of five minutes, and column 3 the evolution of carbon dioxide during each period of five minutes. The normal rate of the yeast-juice, in the absence of added phosphate, was 2 C.C. per five minutes. It will be seen that an average rate of about 15 was maintained for an hour and a quarter, 32 c.c. of the phosphate solution being added in all. | I. | 2 | 3. Cubic centimetres of Beolatonteraen Time phosphate solution ao d eee in minutes. added in , Lome rae eee a in each 5 minutes. c.c. 5 5°0 3 °1 10 5 a) 15 *4 15 2:0 16 ‘2 20 1:0 20 ‘2 25 2°5 1 30 5 127 35 2°0 15 <7 40 0:0 17 °4 45 0:0 9°6 50 3°0 8 °4 55 3 °0 15 °*4 60 3 °0 20 °-2 65 3 ‘0 72 70 3 ‘0 14. °8 75 153 14°9 Further experiments on this subject are in progress, particularly with respect to the relations between phosphate and coferment, and the bearing of these phenomena on the fermentation of sugars by living yeast. Summary. 1. The addition of a phosphate to a fermenting mixture of glucose and yeast-juice not only produces a temporary acceleration in the rate of fermentation, but, in addition to this, an increased total fermentation. 2. This last effect is due to the fact that the hexosephosphate formed during 1908. | The Alcoholic Ferment of Yeast-juice. 311 the period of temporary acceleration is continually hydrolysed by an enzyme, with production of free phosphate, which again enters into reaction and thus brings about an increased fermentation. 3. It appears probable that the presence of phosphate is essential for the ,alcoholic fermentation of glucose by yeast-juice, the reaction which occurs being the following :— (1) 2CsHiz0¢+ 2ReHPO, = 2CO.+ 2C2H,0 + CeH904(POsRe)2 + 2H20. This reaction is only realised in the presence of the ferment and coferment discussed in previous communications, phosphate alone being unable, in the absence of coferment, to bring about fermentation in a mixture of ferment and glucose. The hexosephosphate thus formed is then hydrolysed : (2) CgHip04 POsRe)o+ 2H20 = CgH1206+ 2R2HPO,. The rate at which this second reaction occurs determines the rate of fermentation observed when glucose is fermented by yeast-juice. 4, An optimum concentration of phosphate exists which produces a maximum initial rate of fermentation. Increase of concentration beyond this optimum diminishes the rate of fermentation. ho on) VOL. LXXX.—B 312 Studies on Enzyme Acton. XI.—Hydrolysis of Raffinose by Acids and Enzymes. By H. E. Armstrona, F.R.S., and W. H. Giover, Ph.D. (Received and read April 2, 1908.) [International Catalogue of Scientific Literature. Authors’ title slip :—D. Q. Subject slips :— D 1820 Cane-sugar. Hydrolysis by acids and enzymes. D 1830 Raffinose. Hydrolysis by acids and enzymes. D 8014 Invertase, action of, on raffinose and sucrose compared. D 7095 Cane-sugar and raffinose, hydrolysis of, by acids and enzymes compared. @ 1240 Invertase, action of, on raffinose and sucrose compared. | In a previous communication of this series (vol. 74, p. 191),* it is pointed out that the variouss glucosides are hydrolysed by acids at very different rates, the relative values being approximately of the order shown in the following table :— a-Methylelucoside .................. 100 B- ee We See ace a a ater 180 a-Methylgalactoside ............... 540 B- POLY Ps cnc re 880 Salicin (a @-glucoside) ............ 600 Milk-sugar (a 8-galactoside)...... 720 Maltose (an e-glucoside ) ......... 740 Cane-sugar, it is to be remembered, is hydrolysed at a rate vastly more rapid—at least 1000 times as rapidly as maltose, in fact. These differences, taking into account the peculiar specific behaviour of enzymes as hydrolytic agents, raise questions of interest from the chemical side and they are of no slight significance perhaps also from a biological point of view. The two stereo-isomeric methylglucosides are represented by the following formule :— ise Ouss ee OLE | I | | FON oe! a seer u on.o.da CZ Hepc_é_bon OEE Sh oe OP te OE gH.C—C—C.OH. \ OHH iN OH ~4 - Be We Nia a Qa a-Methylelucoside B-Methylglucoside * All references are to these ‘ Proceedings.’ Studies on Enzyme Action. 313 It will be seen that the two radicles H and OCHs shown as attached to the one carbon atom merely occupy different or reversed positions relatively to the two other radicles with which the carbon atom is connected. As to the manner in which the hydrolytic attack takes place, two views are possible: the one being that the compound behaves much as the simple ether CH3.0.CH; would and that the hydrolyst becomes associated with the oxygen atom to which the CHs group is attached; the other being that the attachment is to the oxygen atom in the ring. On the former view, it is to be supposed that the two isomeric compounds wouid be hydrolysed with equal readiness, as the CH;0 groups are equally weighted; but on the latter, it is conceivable that the methoxy-group is less easily accessible to hydro- lysis in the one case than it is in the other; or it may be that an inductive effect is exercised by the one oxygen atom upon the other which renders the oxygen atom in the ring either more or less attractive of the hydrolyst, as the case may be. In the galactosides, the CH30 group occupy the same relative positions as in the two glucosides ; but the contiguous oxygen atom in the pentaphane ring must be supposed to occupy a position slightly different from that which it occupies in glucose, its connection with the carbon atom on the right being slightly different, as shown in the two following formule :— Hom , oo SY Pe CH,0.C.H BH.C—C—C.OH. CH;0.C.H CC Or 4 VO 1 Xe / A OHH Aa WA a-Methylglucoside a-Methylgalactoside Apparently, seeing that the galactosides are the more easily hydrolysed, this difference is sufficient to render the hydrolyst—the attack of which in all probability is directed from the oxygen atom in the ring—more accessible to the neighbouring CH3;0 group than it is in the glucosides. The behaviour of the glucosidic acetates appears to be in accordance with such an interpre- tation.* In models of glucose and galactose constructed with tetrahedra to represent the carbon atoms, if these atoms are arranged in a vertical plane the oxygen atom in the ring occupies somewhat different positions more or less outside this plane ; the difference in the relative positions of the OCH; group and the oxygen atom in the ring, according as the position of these on either side of the ring plane is varied, then becomes apparent. * Of. Armstrong, E. F., and Arup, ‘Chem. Soc. Trans.,’ 1904, vol. 85, p. 1043. 2B 2 314 Prof. H. E. Armstrong and Dr. W. H. Glover. [Apr. 2, There can be little doubt that considerations of the order here pictured are more or less applicable to the explanation of differences such as those under discussion and that it may be possible eventually, proceeding on these lines, to discriminate between alternative formule applicable to compounds such as glucose and galactose. It is from this point of view that the study of hydrolytic changes is of supreme importance. Enzymes probably act much in the same way as acid hydrolysts; the attachment of the enzyme, however, appears to be more general and thorough, so to speak, than that of the acid hydrolyst and presumably extends over a large part of the molecule (compare III and X, B, vol. 79, p. 361). But in both cases the attack is directed, it may be supposed, from the oxygen atom in the pentaphane ring adjoining the group which undergoes hydrolysis. In the hope of obtaining further information bearing on this refined problem, the behaviour of raffinose towards acids and enzymes has been studied in comparison with that of cane-sugar, raffinose being a triose formed of cane-sugar weighted by the attachment of a molecule of galactose. Raffinose is a reserve material which accompanies cane-sugar in the sugar beet, in cotton seed, in barley and in wheat, for example. It can either be resolved into galactose and cane-sugar by a special enzyme or it can be resolved into fructose and a biose isomeric with cane-sugar—melibiose—by the action of invertase, the enzyme which resolves cane-sugar into glucose and fructose. Hydrolysis of Cane-sugar and Raffinose with the aid of Acids. Raffinose is easily hydrolysed with the aid of acids at ordinary tempera- tures, the products being fructose and melibiose, the latter, like maltose and milk-sugar, undergoing change only at higher temperatures. The experiments with acids were carried out at 25° C. in the manner described by Caldwell (A, vol. 78, p. 285), with an improved apparatus an account of which will be given at an early date by Messrs. Caldwell and Whymper. Except where stated otherwise, the polariscope readings were taken with the aid of a spectroscopic eyepiece, using a mercury lamp as the luminous source, the light being of the refrangibility of the dominant line in the green. As raffinose is less soluble than cane-sugar, solutions of quarter molecular strength were used. A complete record of two experiments is given on the left-hand side of Table I. The columns on the right contain the values of the velocity constant deduced by means of the formula a Ge Pere cae Ot oe K = — t 315 = S SS S a Sy 5 >> < RQ < S aw NY) © S = a) OFT a €6€ 198 &- 6IP OSs G. 8FS GOP G- GOP 89F 00s Gc. SIP an OOS, |S = EUs IAS = ae sage wa re a aX Medi ce ra Sac = ome G69. OT = 699.7 — 2 9VV 9b0 =" or SIP 67S en ae GOV TLV GOS ae as €0¢ oge. T— OIL OPV eH <5 ar 9TV SAE = = POP LOV POS 6p 086- 8T TOS OFT. [— SOT SVP 9FV = ye LIP StS a O9F 697 997 00g OCP 002. 61 c0G 6r6- O— OOT ShYV VPP a Sy OGY LVS = SSF VOV OLY TOS SIP SL¥- 6T cog SPL. O— $6 OPP 8tP a 98€ LI¥ Osg 6FS O9F cOV 997 867 Liv £9L- 61 €0S c6p-O0— | O06 Lvv LtP a: L8& CGV LES oss 6SP POV 997 OOS VIP 90T- 0G 00¢ €1é- O— g8 LUV SUP c6& 88 SIP CPs svg =| ~=19F 997 997 POS SIV LOF- 06 TOG OFO- O 08 STV a 6 988 6IP Ogg 9VG =| 09% COP 997 60¢ 16V LPL. 06 60S OTs. 0 iY Lvv SPP c6e 98& SIP 6gg SPS | 6S &9V 997 60G 61P PST- IZ 86P PE9- 0 OL 9bV GVV P68 L8& 6IP een CSG OOF SOV 89P 00g LIV TS9- 16 667 8E6- 0 g9 VPP SEP L6E 88E OSPF 9g¢ StS 6SP POV 897 967 SIP OFT. GZ 66P PLE. T 09 Livv 6hP €68 L8& OCP TSg 9FS €9P FOV 89P ZOS SIP OOL- 6 86F 869. T cs GrP Tv 16 8s SCV ogg SPS £9P 6S7 GLP 867 147 O86: &Z 867 000: @ Og LEY TSv F6E L8§ cv Gg OSS 997 OOv OLY 667 SIP O&9- €6 00S O88. 3 cv pal SPP 166 988 OcYV 6g¢G ig SOV SOV GLV POS SIV OL6-. &Z 867 VI8.% OV OST a 66E L8& OCP sage 8PS SOP 6SP PLY L6V SIP LEE. VS 667 PVS-& Gs =< SPT T6& 68 GCP ee 1gg 997 L9V TOV 867 GIP OOL- FZ 867 OGL. & O€ ovT - c6E 88E SIP . GGG SOP TOP 99P 667 SIV TOT. SZ 669 806. ¥ GG VPP tT 668 888 VOr seer ise GOV SOP ee 00g 6h SOG. 96 GOG OGd. ¥ 0G rz ar =< oo a Par ai as a a = Sze oot ey a O9T- 4 0 ; "SUTU "oso “ONH | 1OH | "OSH *ONH TOs ee 3 Pap a He | “unt, T- SPIT 6- 8CIT 9-860T c60T 9- P8OT 8. SLOT OO UT SMOTFNTOS YF JO souuNjoR “aSOUeYy “Iedns-ouRD ‘OH + ssougey ‘OH + tesns-ourp ‘proy jo uorsodoid repnoojour ourtme1s [ +o7e1pAyoqreg Jo uonsodoid avpnoejou otmuIeIs ¢7.9 + IOJB AA JO SOUIUIVIS YOOT SUTUTe{MOD SUOTINIOY UI ,Gzg 4e Sprloy Aq osouyyey jo pue rvSns-aueD jo stsAjorpAY—T oqey, 316 Prof. H. I. Armstrong and Dr. W. H. Glover. [Apr. 2, The values deduced for cane-sugar are in fair agreement with those obtained by other workers in our laboratory. Contrasting the mean values, they are as follows :— Nitric acid. Chlorhydric acid. Sulphuric acid. Cane=sucvar = sees 464 500 549 Rathimose. 2 .cceece: 390 419 446 The three acids differ in their activity towards both sugars: the cause of this difference will be discussed in a separate communication, dealing with the sucroclastic action of acids generally. It is clear that raftinose is less easily hydrolysed than cane-sugar, at a rate nearly one-fifth less than that at which the latter undergoes change. The ratios for each acid are nearly the same in the case of nitric acid and chlorhydric acid, but sulphuric acid is relatively less active towards raffinose —a result not without interest, the significance of which, however, need not be discussed here. Hydrolysis of Cane-sugar and Raffinose by Inwertase. In contrasting their behaviour towards invertase, comparative experiments were carried out simultaneously with the two sugars under similar conditions. A known volume of a very weak solution of invertase at 25° C. was added to a known volume of the sugar solution of definite concentration, also at 25°, contained in a Jena-glass flask; after adding a few drops of toluene, the flask was corked up and placed in an incubator kept at 25°. At stated intervals, 20 c.c. samples were withdrawn from the flask by means of a pipette and run into small Jena-glass flasks, each containing a single drop of a strong aqueous solution of sodium hydroxide. By this means the action of the enzyme was at once arrested and equilibrium established between the stereo-isomeric forms of the sugars in solution. The final values were obtained by keeping part of the solution at 25° C. during 24 hours, at the end of which time a drop of sodium hydroxide solution was added as before. In order to obtain the initial values, a drop of sodium hydroxide solution was added to a known volume of the enzyme solution, which was then mixed with the sugar solution in the same proportion as in the experiment. In the preliminary experiment the raffinose used was that supplied by Kahlbaum. The rotatory powers of the solutions were determined in sodium light. The results obtained are exhibited in Table II, the values given under K a being those deduced with the aid of the formula K = 2 logio =e apt Studies on Enzyine Action. 317 Table ILI. Cane-sugar. Raffinose (commercial). 342 grammes of cane-sugar (1/10 mol. 59°4 grammes of raffinose (1/10 mol. Fane, C,.H..0};) +4 ¢.c. of strong invertase ©gH3,0)5.5H,0) +4 ¢.c. of strong extract per 1000 c.c. invertase extract per 1000 c.c. Per cent. Per cent. “D- hydrolysed. ee = hydrolysed. x mins. 0 4°36 0°0 = 12°25 0°0 a 5 3°88 8°3 753 12°15 1°8 157 15 2°87 25 ‘9 868 11 °92 5°9 176 25 2°09 39 °5 865 11°51 13 “+ 249 40 0°78 62 *4 1062 11°14 20°1 243 60 —0°13 78 2 1102 10 °63 29°3 251 95 —0°87 oe 1106 9 95 41 °6 246 140 —1 02 93 °7 859 9°30 53 °*4 237 200 —1°10 955 656 8°57 66 °6 238 260 —1°15 96 ‘0 537 7°95 EES 252 x —1°38 100 ‘0 — 6°73 100 °0 — On plotting curves to represent the rates at which the two sugars are changed, it is seen that whilst one-half of the cane-sugar is hydrolysed in 33 minutes, the half of the raffinose is hydrolysed only after the lapse of Table III. | Cane-sugar. Raffinose (recrystallised). 34:2 grammes of cane-sugar (1/10 mol. 59°4 grammes of raffinose (1/10 mol. : C).H..0;,) + 4 ¢.c. of strong invertase C1gH30)¢.5H.O) + 4 ¢.c. of stron Time Lait oa 8 Odean ce 8 Ba «il extract per 1000 c.c. invertase extract per 1000 c.c. aro: Per cent. ayo: Per cent. 7 hydrolysed. Be ‘ hydrolysed. eS mins, 0 4 504 0°0 — 13 768 0-0 — 10 3 °332 17° 829 13 °462 4 °6 203 20 2 °100 35 °6 957 13-168 9-0 204: 30 1-074 50 ‘9 1043 12 ‘918 127 200 40 0 °306 62 °3 1058 12 668 16 °4 195 60 —0 848 79 °A 1143 — — — 80 —1 °490 88 °9 1193 11 814 29 °2 187 120 —1°810 93 ‘6 997 11 044 40 °6 189 160 —1 982 96 °2 888 10 °398 50 °3 190 200 —2:°114 98 *2 868 - 9 °850 58 °5 191 x — 2 *238 100 ‘0 — 7 ‘065 100 ‘0 = 318 Prof. H. E. Armstrong and Dr. W. H. Glover. [Apr. 2 y 126 minutes; in other words, it takes about 3°8 times as long to convert one-half of the raffinose into melibiose and fructose as it does to change one- half of the cane-sugar into dextrose and fructose. For the following experiments the raffinose was purified by dissolving it in hot water, filtering the hot solution, adding a large bulk of alcohol to the filtrate and allowing it to stand during several days. given in Tables III and IV. The results are Table IV. Cane-sugar. Raffinose (recrystallised). 68°4 grammes of cane-sugar (1/5 mol. 118°8 grammes of rafiinose (1/5 mol. ae C)oH90),) +4 c.c. of strong inver- C\gH3.015.5H,0) +4 c.c. of strong : tase extract in 1000 c.c. invertase extract in 1000 c.c. ayy. Per cent. p oe Per cent. ae hydrolysed. Is. ae hydrolysed. 1s mins 0 9 *886 0:0 — 28 °462 0:0 — 10 8 ‘818 lad 349 28 °156 2 °4 106 20 7 548 16 °9 402 27 °796 Del ae 117 30 6 °298 25 ‘9 434 27 °405 8°3 126 40 5 ‘198 33 °8 449 26 °972 7 136 55 — — — 26 °354 16 °6 144, 60 2 958 50 ‘1 503 oe — — 70 1 °895 57 °7 535 25 °743 21 °4 150 80 0 ‘985 64 °3 560 25 °293 25 ‘0 156 90 0 °262 69 °6 574 24-908 28 ‘0 159 100 —0 ‘366 741 587 24°517 31 ‘1 162 115 — = = 23 °962 35 ‘OD 165 120 —1 °528 82 °5 631 pea a 5 130 —1 932 85 °4 643 23 °352 40 °3 172 140 — 2 °266 87 ‘8 653 23 ‘001 43 1 175 150 — 2 °562 89 °9 666 22 °667 45 °7 ITZ 160 | —2°793 91 °6 674. 22 °346 48 °2 179 170; . pul — — — 21 -895 51°8 181 180 —3°172 94 °4, 694 == aan ar 190 — 3 °302 95 °3 699 21 458 55 *2 184 200 —3 °446 96 °3 719 21 °166 57 °5 186 220 —3 ‘524 96 ‘9 687 20 ‘662 61 °5 188 x — 3 °950 100 ‘O — 15 “787 100 ‘0 aes On plotting curves representing the rates of hydrolysis in the case of the solution of one-tenth molecular strength, it appears that whilst 50 per cent. of the cane-sugar is hydrolysed in 29 minutes, 50 per cent. of the raffinose is hydrolysed only after 158 minutes, the ratio being 1: 5'4 as compared with the ratio 1: 3°8 in the experiment with the commercial raffinose. Apparently, the recrystallised raffinose is less quickly hydrolysed than the crude material. In order to see if this were really the case, comparative experiments wer 1908. | Studies on Enzyme Action. 319 carried out, side by side, with the two kinds of raffinose and with cane-sugar ; moreover, with the object of ascertaining the effect of further purification on the stability of the sugar in presence of the enzyme, the recrystallised raffinose was dissolved in “conductivity ” water, the solution was mixed with alcohol and a stream of carbon dioxide passed through it during 50 minutes, this artifice having been found to be of service in purifying galactose; the solution was then filtered and a large bulk of alcohol added to the filtrate. The material obtained by this method is the “ Raffinose III” in the following table. It was again recrystallised from a mixture of alcohol and “conductivity ’ water; the material thus obtained is marked “ Raffinose IV.” Equimolecular proportions of these samples of raftinose having been dissolved, the liquids were boiled to drive off any alcohol present, and were then diluted to the proper volume. Equal volumes of these solutions were measured out and to each was added the same number of cubic centimetres of a dilute invertase solution. The results are recorded in Table V. Table V. Amount hydrolysed at 25°. | 1 hour. 2 hours. 3 hours. 4 hours. | Per cent. Per cent. | Per cent. Per cent. WRMICHB ISAT. Scras -pressure on injection by exciting dilator centres. In the normal animal the first dose causes a rise and subsequent ones a fall of pressure, since the first dose, if not too small, after exciting the vaso-constrictor centre, paralyses the synapses concerned, so that the simultaneous excitation of the dilator centres can now make itself felt. 9. The excitation of constrictors produced by reversal of inhibition is more resistant to the alkaloid than that produced in the normal way. 10. Asphyxial blood does not act directly on the efferent constrictor neurones, since it has no action at a stage of strychnine poisoning at which the depressor still excites constriction, by reversal of inhibition. 11. Chloroform converts pressor into depressor reflexes (in the rabbit), by reversal of excitation of constrictors into inhibition. 12. This effect of chloroform is not exerted on the efferent neurones directly, but at some point considerably earlier in the reflex arc. This is shown by the fact that asphyxial blood causes rise of pressure when excitation of sensory nerves causes fall. The expenses of this research were partially defrayed from the Government Grant administered by the Royal Society. [Note added March 23, 1908.—Since the preceding paper was written, I have received from Professor Mislavsky, of Kasan, a number of tracings showing a dilatation of the tongue of the dog and cat on excitation of the central end of the vagus, after section of the cervical sympathetics above the superior cervical ganglia. In one case there was no obvious change in the blood-pressure, a fact which perhaps makes the reflex origin of the dilatation somewhat doubtful; in the other cases there was the usual fall. In all cases, as Professor Mislavsky informs me, the effect was abolished by section of the lingual nerves, so that there seems no doubt that it was due to excitation of vaso-dilator fibres. These experiments were performed by Professor Mislavsky in conjunction with his pupil, Mr. Fofanoff. The results are of interest, in that they bring evidence of dilator-excitation in an organ on which my own experiments had been only partly successful. ] 376 The Action of Resin and Allied Bodies on a Photographic Plate an the Dark. By WiuuiAm J. RUSSELL, Ph.D., F.RS. (Received March 24,—Read May 7, 1908.) [PLates 10—12.] In former papers it has been shown that certain metals, woods, juices of plants, etc., have the property of acting on a photographic plate in the dark ; that a similar action is exerted by coal resins and allied bodies is proved by the following experiments. Ordinary resin or colophony is the solid, remaining on the distillation of crude turpentine, and the substance known in commerce as “ amber resin” is ordinary resin slightly purified, and is of a lighter colour. To prove the activity of these bodies it is only necessary to lay them on a photographic plate in the dark, and afterwards to develop the plate in the ordinary way. The plates used in the following experiments were in almost all cases “ Imperial Special Rapid.” At ordinary temperatures the action is but slow: the contact of resin and plate would have to be for two to three days in order to obtain a fairly good picture. The amber resin is, however, slightly more active than the ordinary resin. If the temperature be raised, and contact be at 30° to 40° C., the action is much more rapid, and three to four hours is long enough to give a good picture. In fact, in four hours, ordinary resin will give as much action at 40° as it would in three days at 15° to 20°. A still higher temperature cannot be used with safety, for then the resin softens and adheres to the photographic film. Absolute contact between resin and photographic plate is not necessary, for if the plate be held above the resin the action still takes place, and will, in fact, pass through a considerable distance. In one case when powdered resin was placed at the bottom of a glass cylinder and the photo plate on the top at a distance of 120 mm., and in another case when the distance was 210 mm., in both cases after 18 hours’ exposure at a tem- perature of 40°, a dark picture was produced. Another experiment which shows this action of resin was made by filling a glass tube, 1 inch in diameter and 10 inches long, and slightly contracted at one end with small pieces of resin, the tube being held in a horizontal position, and a photo plate placed vertically at 1 mm. from the open end of the tube. On passing a slow current of air through the tube, which was maintained at 40° C., a dark The Action of Resin, etc., on a Photographic Plate. 377 indication of where the air struck the plate was produced in two hours. On continuing to pass air through the tubes the activity of the resin gradually decreases, but if the resin be taken out and again broken up its activity is restored. If the tube containing the resin instead of being straight is bent at a right angle, and a photo plate be placed below the bend, on passing a slow current of air through the tube a large amount of action is produced upon the plate. | The presence of oxygen appears to be necessary for the action to take place. Two slabs of resin were placed separately in two desiccators: one was filed with dry air and the other with dry carbon dioxide; a photographic plate was fixed at 1 mm. below the resin plate, and both desiccators were kept at 40° C. for 18 hours. It was then found that, although considerable action had taken place in the desiccator filled with air, none had occurred in the one filled with carbon dioxide. A very marked and important character of this action of resin is that it is not able to pass through the thinnest sheet of glass or mica or aluminium. Glass 1/200 inch thick and mica 1/750 inch thick absolutely prevents the action passing through. This seems to separate this action from others of a somewhat similar character. Another important point with regard to the action of resin and other allied bodies is the form of the shadow which they produce. If, for instance, a glass screen is placed in front of a piece of resin on a photo plate the shadow is not bounded by straight lines, but the action, like that of a vapour, creeps in behind the screen, and in time meets from both sides. To prevent this action arising from any side action of the resin plate, a glass tube was filled with resin and directed against the centre of the screen. The experiment was repeated with the same apparatus, and a copper screen and ordinary light. Fig. 1 (Plate 10) shows the effect produced in the two cases. - With regard to other properties of the resin plate, a thin plate acts as energetically asa thick one; thus a plate only 0-017 inch thick gave a picture ‘of the same density as one 0°29 inch thick. : To obtain a suitable slab of resin for experiment it is best to melt the resin and cast it on a bright metal plate, and afterwards, to free the surface which has been in contact with the metal from air-bubbles, to pass a gas flame over it. Another way of using resin for experiments is to dissolve it in alcohol and saturate a card or paper with the solution and allow it to dry. Even very dilute solutions may be used: a card which has been soaked in an 378 Dr. W. J. Russell. The Action of Resin and [Mar. 24, alcoholic solution containing 0°25 per cent. of resin will give a dark picture, and with solutions of only 0°125 and even 0-086 per cent. of resin, faint pictures may be obtained. Again, another way of using resin is to pour the alcoholic solution on to a glass plate and allow it to dry there. There are, of course, other solvents which may. be used in place of alcohol. A card prepared with an alcoholic solution of resin was placed in the dark slide of a camera, and the lght of an arc lamp focussed upon it for five minutes. The card was then put up with a photo plate at 55° for one hour. A good and dark picture of the are was obtained. If resin be heated to a temperature of 40° to 50° for a short time it does not affect its activity, but if the heating be continued for 20 or 30 hours it slightly diminishes it. At higher temperatures the action is more marked ; for instance, at 140° the activity of the resin is much decreased after only four hours’ heating, and, although resin may be fused without appreciably diminishing its activity, still, if it be kept in a liquid state for three or four hours, its activity 1s much decreased. An interesting experiment is easily made with a slab of resin owing to its brittleness. A weight placed on the slab cracks it in all directions; this can be slightly warmed on the under side so as to prevent its falling to pieces, and then on putting it up with a photo plate for a short time a dark picture of the cracks is obtained (fig. 2, Plate 11). Another interesting experiment shows that the activity existing in resin can be transferred to a non-active body, making it as active as the original resin. A glass vessel was nearly filled with crushed resin, and a piece of inactive Bristol board placed on the top of it, at a distance of 5 mm. above the resin. This was left for a week at ordinary temperature, then on putting the Bristol board in contact with a photo plate at 55° C. for five hours a dark picture was obtained. | Theré are other ways in which this action of resin may be diminished or destroyed ; for instance, by the action of sulphur dioxide. A slab of resin was broken into two pieces; one was placed for five minutes in a saturated solution of sulphur dioxide, then very thoroughly washed and dried; the other piece was treated in the same way, but with water alone, and both pieces were put up at 40° for 18 hours. The one which had been washed with water alone gave a dark picture, and the one treated with the sulphur dioxide gave no picture. It has been shown in a former paper that wood, after exposure to sunlight, has its power of acting on a photographic plate in the dark much increased, and that this increase of activity is not permanent, but gradually passes away. Resin acts in the same way: expose it to sunlight or to the are light, and then bring it in contact or proximity to a photo plate, and it will be 1908.] Alled Bodies on a Photographic Plate in the Dark. 379 found that its activity has been greatly increased. Fig. 3 (Plate 11) shows the picture given by the resin in its ordinary state, and after exposure to the arc light for half an hour. In one experiment a slab of resin was exposed to a bright July sun for 5 seconds, and this caused no increase of activity ; but exposed for 15 seconds and a slight increase occurred, and after an exposure of 30 seconds the activity of the slab had greatly increased ; but on a still longer exposure no further increase took place, so that in about 30 seconds the resin was charged to its maximum amount. Another experiment, when the exposures were for 1 minute, 5 minutes, and 15 minutes; the 1 minute and the 15 minutes gave similar results. If the arc light be used in place of sunlight the same kind of action occurs, only more slowly. In one experiment the exposures were for 5 seconds, 15 seconds, and 30 seconds, and no marked increase of activity took place, but after 60 seconds’ exposure a great increase was evident. With another sample of resin exposed to an are light it was found that it required 5 minutes’ exposure to obtain its greatest amount of activity. If amber resin, in place of ordinary resin, be used, it requires a longer exposure to light to charge it to its greatest amount. Resin is, however, a body which varies so much in composition and constitution that exact measurements cannot be relied on for different specimens, and the above experiments are only intended to show the nature of the action which occurs. The effect of heat on resin in its ordinary state has already been described ; 1f, now, a resin slab, charged to its maximum by exposure to light, be heated to 55° for only one and a-half hours, all this extra activity is destroyed and it returns to its original state of activity. This increased activity induced by light acts generally in the same way as the original activity of the resin: it is destroyed by sulphur dioxide and does not pass through glass, mica, etc. In all cases of stimulating resin by the action of light a short interval occurs after the application of the light and before the increase of activity begins; when once begun, the increase takes place rapidly and it soon becomes charged to its maximum, so that longer exposure produces no further increase of its activity. In order to ascertain which rays of the spectrum were most active in producing this change, a spectrum obtained from an arc lamp, with a quartz ‘prism and lens, was allowed to fall upon a slab of resin for one and. a-half hours, and this gave, after contact with a photo plate for two hours at 40° C., evidence of action having taken place where the blue rays had fallen on the resin and not elsewhere. On placing slabs of resin in double bell jars with different coloured liquids, the blue, a solution of ammonia sulphate of copper, and the red, potassium bichromate, it was found that, even after an 380 Dr. W. J. Russell. The Action of Resin and [Mar. 24, exposure of one hour in strong diffused lght, the resin which had been exposed to the blue hght had been strongly acted on, and gave a good dark picture, while the one exposed to the red light gave no increase of activity. The amber resin acted in the same way. A beam of blue light or of red light thrown on a slab of resin or a card saturated with resin gave the same results. On exposing resin under different coloured glasses the same effects were produced: after an exposure of one hour to bright daylight, under a blue glass, the resin became very active, under a red glass no change took place, and under a green glass there was only a very slight increase of activity. It thus appears that the action of light in this respect, on resin, is similar to its action on wood, as described in a former paper. This increased activity of the resin slowly passes off, even at ordinary temperatures, on keeping in the dark or in red light. A slab was exposed to the are light for one hour and then cut up into eight pieces. One piece was put up with a photo plate at once at 40° for two hours: it gave a good dark picture; the other pieces were kept in the dark, at ordinary temperatures: after three days a piece was tested, the picture 1t gave was only slightly lighter than the former one. After nine days, again, a loss of activity had occurred and the same was the case after 18 days. The experiment was carried on for nine months, and at the end of this time although the picture it gave was much fainter than the first one still it was slightly darker than the picture it would have given before exposure. If the resin be only slightly stimulated by exposure to bright daylight, the same gradual decrease of activity was traced. In red light the decrease was apparently the same as in darkness. Although glass and some other bodies are opaque to the action of resin, porous bodies, of course, allow the action to pass through ; for instance, with ordinary paper, if a slab of resin be placed behind it, a very good sharp picture is obtained ; if, however, the paper be highly glazed and dressed, it is perfectly opaque. With ordinary papers interesting pictures, showing their structure and water-mark, and stencil pictures are easily obtained. If paper be treated with different substances in solution, it is made more or less transparent. As a general rule it would seem that acid salts, such as the sulphates, which do not act on the photographic film, make a paper opaque, but that neutral salts do not alter its transparency. If a paper be dried by warming it, 1t becomes rather less transparent. The principal constituent of resin‘is said to be an acid, known as abietic acid. It is not a body which has been very thoroughly examined, but it has the property of acting on a photographic plate in the dark to a remark- able extent. It can be obtained by dissolving resin in alcohol and passing hydrochloric acid gas into the solution; the acid then separates out in a ee ee 1908.| Allied Bodies on a Photographic Plate in the Dark. 381 erystalline form. By repeating this process it may be purified, and will then have a melting-point of 156°C. It is with an acid so prepared that the following experiments have been made. If a small glass vessel be nearly filled with the crystalline acid and a photo plate be laid on the top, not touching the acid, at ordinary temperatures, after two hours no action will have occurred, but after 18 hours the plate will give a strong dark picture. If, however, the acid be kept at a temperature of 40°, then a fairly good picture can be obtained in two hours, and with longer exposure a very dark one. Thus it acts in the same way as resin, and has about the same amount of activity. Exposed to sunlight or to the are light, its activity is much increased. Exposed to the are light for an hour, it gives a good and dark picture, and even on an exposure of half that time a picture only slightly lighter is obtained; in fact, in little more than half an hour it is charged to its maximum amount. Light acts upon it as it does on resin, The acid dissolves readily in alcohol, and if the solution be allowed to evaporate on a glass plate, it gives a film suitable for experimenting with. Paper saturated with the solution becomes very active. The acid also dissolves in ether, benzene, chloroform, etc., and behaves in the same way as with an alcoholic solution. If the acid be heated to 100° it slowly loses its activity ; after eight hours’ heating the picture it gives is only slightly fainter than before heating, but after 56 hours’ heating it has become much fainter, and after being heated for 152 hours it has lost entirely its power of acting on a photo plate. If the acid be fused it becomes quite inactive, but its activity is restored if it be powdered, or if its surface be rubbed with sand-paper—in fact, if the smooth surface be broken up. If exposed to sunlight or to the are light its activity is much increased, and different coloured rays affect it as they do resin. Exposed under blue or white glass to six hours’ sunshine it gives a dark picture, but under a red glass only a faint one. All the metallic salts of this acid are entirely without action on a photo plate: neutralise a solution of the acid with potash or soda and its activity has gone, and there is the same loss of activity with the copper and the lead salts, whether in solution or in the solid state. Decompose the metallic salts and the liberated acid is as active as before. To purify the acid the lead salt, which is very insoluble even in alcohol and other organic liquids, was boiled several times with pure alcohol, and afterwards treated with sulphuretted hydrogen, the acid well washed and dried, and recrystallised from alcohol, and it was found to be quite as active as before this treatment. Another specimen of the acid was treated with 382 Dr. W. J. Russell. The Action of Resin and [Mar. 24, an insufficient amount of alcohol to dissolve the whole of it. After boiling and digesting for a considerable length of time the undissolved acid was filtered off, washed, and dried, and was found to be quite as active as before this treatment, so that neither process of purification affected the activity of the acid. If the fused and inactive acid be simply exposed to light it will again become active. If the activity of turpentine depends to any appreciable extent on the presence of abietic acid, then if it be treated with an alkaline body its activity should be decreased. Turpentine is known to be a very active body, and a plate placed about one-eighth of an inch above it will, even at ordinary temperatures, in three hours give a black picture. Some turpentine was allowed to stand for 18 hours with a small amount of solid caustic potash; this was then filtered off, and the liquid distilled and put up with a photo plate for three hours; no trace of action was visible. Another photo plate was placed above the same turpentine solution and allowed to remain for 18 hours; even then only a very faint action took place. Another specimen of turpentine was shaken up with magnesium oxide and allowed to stand for 24 hours. The clear liquid gave a much fainter picture after this treatment. The same occurred when dry sodium carbonate was used, but lead acetate had no action on the turpentine. Amber, although classed as a resin, differs so much from the substance already described that it was of much interest to ascertain how it would act under similar conditions. It is a remarkable substance, known from the earliest times, and has been used for many purposes. Quarried at one time, like a stone, it was naturally looked upon as a mineral, but is now known to be of vegetable origin: the exudation of certain trees, probably mostly coniferous ones, which have been buried in the ground for ages. Even in the Green-sand formation some amber has been found. At the present time the principal supply of amber comes from the shores of the Baltic, but a small amount is still picked up on the east coast of this country. If a piece or pebble of amber, either in its rough state or cut so as to give it a flat surface, be laid on a photographic plate in the dark, no action takes place, even if the contact be continued for 18 hours and the temperature be at 40° to 50° C., thus differing from resin. This has been tried with a large number of specimens from different parts of the world, and with true amber has always been found to be the case. There are many bodies closely resembling amber in appearance, chiefly resins, which act strongly on the photo plate, and although readily distin- guished by an expert in the subject, can easily be mistaken for true amber. It often happens that a piece of amber, after long exposure to a plate, will 1908.] Alled Bodies on a Photographic Plate in the Dark. 383 develop on it small spots of action; these local actions are produced by fine cracks in the amber, which frequently occur, and it is above the opening of these cracks that the action takes place. If the amber be laid for a minute on a hot surface the opening of the cracks fills up and the action ceases. This resembles the action of resin, and apparently points to the collection of volatile matter within the cracks. Another way of showing that, although a flat surface of amber does not act on a photo plate, still there is a trace of active vapour connected with it, for if powdered amber is placed in a glass dish with a plate above it, but not necessarily touching the powder, after the usual exposure a dark picture is produced. Amber, as is well known, is practically insoluble in alcohol, but in all cases a very small amount of some substance dissolves out of amber ; now if this substance be collected by filtering the alcoholic solution and evaporating it to dryness, the residue is found always. to be a very active body and gives a dark picture, thus a lingering indication of the amber’s origin seems to be indicated. Following the same line of experiments as that applied to resin, amber was exposed to sunlight and to the arc light, and its activity was found to be much increased. Four pieces of amber were exposed to sunlight for different lengths of time, namely, for two, three, five, and seven hours. After two hours only a very faint picture was produced; after three hours the picture was much darker and strongly outlined; after five hours it was still darker, and after seven hours a very dark picture was produced. The are light acts in the same way. A specimen of good amber was cut into four pieces, and all of them were exposed at the same time, at a distance of 9 inches from the arc light: one piece for one hour, another for two hours, and the other two for respectively four and six hours. All of them were afterwards put up with photo plates at 55°C. for 18 hours. The amber exposed for one and for two hours did not act on the plate ; the one exposed for four hours gave a considerable amount of action, and the one exposed for six hours gave a dark picture. Another experiment of the same kind showed that the amber became slightly active in two hours, and was much increased after four hours, but after six hours and even after ten hours but very slight increase of activity occurred. As amber is a body which varies so much in constitution and composition, the action of light on it will vary slightly with every sample. for instance, five pieces of amber, all from different sources, were exposed at the same time for three hours to an arc light : two of them gave dark pictures, two only faint pictures, and one no picture at all. Amber, like resin, if stimulated to increased activity by the action of 384 Dr. W. J. Russell. The Action of Resin and [Mar. 24, light, gradually loses this increased activity on keeping it in the dark or in dull light, but for a long time retains a slight amount of its increased activity. If, however, the amber be heated, this loss of activity takes place rapidly, even when heated to only 50° C., and if a flat surtace of it be brought in contact with a piece of heated metal for one minute the amber loses entirely its activity. It has already been shown that resin is stimulated especially by the blue rays of the spectrum; the same thing occurs with amber. Specimens of different ambers were exposed both to sunlight and tu are light under different coloured glasses, and it was always found that under the blue glass it became strongly active and that under the red glass it remained quite inactive, and if black glass and colourless glass were used the black glass acted like the red glass and the white one like the blue, only rather stronger. When double bell jars with coloured liquids were used in place of coloured glasses, exactly similar results were obtained. One experiment of this kind was continued for four months and gave the same result. Lignite, jet, and peat have also been tested in the same way as resin and amber. Two specimens of lignite from the Museum of Practical Geology, Jermyn Street: one an ordinary brown coloured piece, the other a sample from Tasmania; both were quite inactive and light did not stimulate them to action ; even the alcoholic extract was inactive. Another specimen from Nigeria was also inactive, but one from Bovey Tracey was slightly active, and a specimen of “ Brown coal” from Victoria, after an exposure of 44 hours, was found to be also very slightly active. Several specimens of jet from different sources were tried. None of them, if simply laid on a photographic plate and warmed, gave any action, but if powdered and a plate placed at 1 mm. above it, at 55° for 18 hours, gave a faint picture. Again, if powdered jet was extracted with pure alcohol, the small amount of dissolved matter evaporated to dryness gave a dark picture. So that jet, although not in ordinary conditions an active body, still in the form of powder has the property of acting on - a photo plate. Light does not appear to have the power of making it active. Graphite from Ceylon did not act on a photo plate. A specimen of peat was found to have the property of acting on a photo plate, but its activity was not increased by exposure to light. One other substance belonging to this class of bodies, namely coal, remained to be examined, and it was interesting to find that all ordinary coals, if brought into contact with a photo plate at a temperature of about 50°, were capable of acting upon it and giving a clear and distinct picture; so sharp are these pictures that they may be enlarged five or six times and still show clearly all the 1908.] Allsed Bodies on a Photographic Plate in the Dark. 385 details. Fig. 4 (Plate 10) is the picture of a vertical section of a Nottingham coal enlarged three times. The vertical section of a coal gives usually a more interesting picture than the horizontal section. Figs. 5 and 6 (Plate 11) show vertical and horizontal sections of a Seaham coal. Through the kindness of Dr. Teall and Mr. Strahan, of the Museum of Practical Geology, I have had the opportunity of examining coals from different localities. Taking first the specimens of English coals, they all seem to be active, that is, have the power of acting on the photographic film in the dark. The best way of trying them is, first to saw off a piece from the rough block, and then rub it down first on coarse sand-paper and then on fine, till the surface is flat and true, then on laying this flat surface on a photo plate at about 50° C, for in most cases about 18 hours, but in some cases it may be well to continue the contact for as long as 48 hours, a good picture is obtained. If the coal contains much water, it must be dried, either by heating it for a short time at a temperature of about 40° C. or by drying it over sulphuric acid. In place of using a slab of coal, it is sometimes convenient to use it in the form of powder, and this is done, as in previous cases, either by simply placing the powder on the photo plate or by filling a small glass vessel with it and placing the photo plate on the top, either in contact with the powder or at a small distance above it. As long as the coal is used in form of a slab and is fairly dry, its action is very uniform, different pieces of the same coal giving pictures of the same density ; but when the coal is in powder, a small amount of moisture modifies the density of the picture to a very considerable extent. | The effect of slightly heating a coal is shown by the following experi- ment :—Four samples of a Seaham coal in powder were treated as follows : One sample was at once put up with a plate, and gave a fairly good picture ; another was heated for 24 hours at 100° C., and gave a much darker picture ; a third one was heated at 150° for the same length of time, and its picture was much lighter, only slightly darker than the first one; and the fourth sample was heated for 24 hours at 200°, and gave no picture. In another case the heating was continued for only three hours at 200° and it gave a faint picture. If the drying be effected by placing the powder over sulphuric acid, phosphorus pentoxide, or solid caustic potash, it seems in many cases to increase the activity of the coal to a very considerable extent, so much so that some coals which under ordinary conditions give only a faint picture can be made to give a dark one. But on the other hand there are coals which are not altered by this process of drying. One specimen of coal, a Seaham coal, powdered, was exposed 11 times in a glass vessel over sulphuric acid, each time for 24 hours, without any diminution of its activity ; 386 Dr. W. J. Russell. The Action of Resin and [Mar. 24, but if the coal was exposed to the air for 24 hours its activity considerably decreased, but was restored by again placing it over sulphuric acid. Coals exposed to sunlight or are light do not perceptibly increase in activity, as many other bodies do, nor does the small amount of substance dissolved out of them by boiling alcohol appear to be active. The following pictures are fair samples of coals from different English beds: figs. 7 and 8 (Plate 12) are both from South Wales. Fig. 9 is a Nottingham coal, fig. 10 a Derbyshire one, and fig. 11 is from Lancashire. In all cases the deposit of vegetable matter in long or short strips or patches is clearly shown and well defined, and the presence of vegetable matter appears diffused through the mass of the coal. Although there must necessarily be a strong resemblance between coal pictures, still it may prove that a certain specific and recognisable character belongs to coals from different beds. For instance, judging from the few specimens which have been examined, the South Wales coals appear to have their active strata fine and near together, whereas the coal from Derby and Nottingham has active strata which are much thicker and very sharply defined; but considering the small number of experiments made, this may be purely accidental. The pictures, however, clearly show differences in coals ; for instance, all the anthracites that have been examined have given pictures different from the foregoing: they are fainter in appearance, the structure they represent is more complicated and the active matter more evenly distributed through the mass of the coal, as shown in fig. 12. There always appear to be cracks in anthracites and these cracks are always white. There is also another curious point with anthracites: if they are dried over sulphuric acid the picture they give is much darker than the picture obtained in the ordinary way, fig. 13. Only a few Cannel coals have been examined : these gave pictures in character like the anthracites but with less detail and not so dark. Fig. 14 is a picture of the well-known Boghead Cannel coal. From coal plants of different kinds and from different localities no pictures have been obtained. If the soft powder so common in bituminous coals and known as “ Mother of Coal” is carefully removed and tested it is always found to be very active. The large amount of diffused action on the top of fig. 4 is owing to this substance; also the fibrous substance so often present and easily removed from coal is also very active, but the hard glistening surface of coal is only slightly active. Of the coals which have been examined, a Boora coal from the Lower Oolite, a Jurassic coal from Mexico, and some Argentine coals and a Tertiary coal from India are ones which have been found to have little or no action on the photo plate. No Russell. Roy. Soc. Proc., B. vol. 80, Plate 10. Russell. Roy. Soc. Proc., B. vol. 80, Plate 11. >, 4 pat Saar — Roy. Soc. Proc., B. vol. 80, Plate 12. | 1908.] Allied Bodies on a Photographic Plate in the Dark, 387 doubt the lomg exposure of small specimens in a museum may affect their activity. The foregoing experiments indicate the nature and to some extent the results which may be obtained by allowing coal to draw its own picture on a photographic plate, and in the hands of a geologist may help to explain the process of its formation. With regard to the nature of this action on photographic plates in the dark, it has been suggested in former papers that it is owing to the presence of hydrogen peroxide, and that the effects described can be imitated by means of this body. It now seems that actions of this same kind are obtainable from many other bodies, but still bodies of the same kind, and these additional experiments strongly indicate that the action is produced by a vapour rather than by any form of radio-activity. For instance, it is shown that the shadows thrown by resin are not bounded by straight lines, but curve round a screen; that the action is not capable of passing through glass, mica, or aluminium foil, even of extreme thinness, and does not affect an electrical field. The action can pass along a glass tube, even when it is bent at a right angle, and may be swept out of a tube by a slow current of gas; and, further, an experiment described above shows that the activity of resin can be transferred to a piece of perfectly inactive Bristol board, which will then give a black picture. Further, no action takes place in an atmosphere of carbon dioxide. On the other hand, resin dissolved in an inactive liquid, such as alcohol or petroleum spirit, causes it to become active. | The action which strong light has in increasing the activity of many bodies is important. For instance, it has been shown that pith may be in contact with a photographic plate at 55° for 48 hours and no trace of action is visible, but if the pith be exposed to sunlight for two or three hours it will then give a dark picture. The same action occurs with old printing, with pure india-rubber, etc., and many bodies which under ordinary condi- tions are but slightly active become very active after exposure to bright light or simply to blue rays. My thanks are due to my assistant, Mr. Bloch, who has made all the photographs and given me much aid in carrying out the experiments. The work has been carried on in the Davy-Faraday Laboratory of the Royal Institution. Von KXX.—B. Deo 388 On Some Features in the Hereditary Transmission of the Albino Character and the Black Piebald Coat in Rats.—Paper II. By GEORGE PERcIVAL MupceE, A.R.C.Sc. Lond., Lecturer on Biology at the London Hospital Medical College (University of London) and at the London School of Medicine for Women (University of London), (Communicated by Professor A. D. Waller, F.R.S. Received J anuary 14,— | Read May 21, 1908.) This paper contains a further record of experiments which were begun at the same time as those recorded in my first paper.* The method of interpreting results in the present paper is the same as that used in the first one, and is based upon the same working hypothesis, 7.¢., Bateson and Punnett’s “ Presence and Absence” hypothesis and Cuénot’s theory of colour. The cost of these experiments has been defrayed by a grant from the Government Grant Committee of the Royal Society. THE EXPERIMENTAL MATINGS. (1) (a) Cr 4x Cr 4= Albino x Albino. Twenty pairs of albinoes were mated. There was a total offspring of 174 individuals, all albinoces. Some of these albinoes had a near albino ancestry, while others had a pigmented one. The details of the ancestry may be seen upon reference to the Table of Ancestry, pp. 390—391. (b) “ Ghost” or Zygotic Patterns im Albinoes, I have already, in the first paper, given an account of the “ghost” pattern as shown in the offspring of one mating (experiment 74). I have now to describe a second case. In experiment 19A there was a total offspring of 15, in two litters of six and nine individuals. The first litter was at a much earlier date than the second, and I did not pay any particular attention to the appearance of the coat in the individuals of this litter. By the time the second litter was born, I had discovered the existence of the “ ghost ” pattern in albinoes. It is possible, therefore, that the first litter showed it, but since it is only a transitory matter of a few weeks, it will be unobserved unless looked for at the particular period at which it appears. Of the nine albinoes born in the second litter, two died quite young, and the remaining seven all * “On Some Features in the Hereditary Transmission of the Self-black and the “ Irish” Coat Character in Rats.—Part I,” ‘ Roy. Soc. Proc.,’ B, vol. 80, 1908. On the Hereditary Transmission of Coat Character in Rats. 389 showed the “ ghost ” of the self-pattern. One of these “ self-ghost ” individuals was mated with a Cr 5 (see experiment 37c)* and gave a coloured offspring, all of the self-pattern. The young consisted of nineteen “ Irish” 6 individuals, obtained in three litters. Since the self-pattern is dominant to the piebald, it is clear that the pigmented piebald parent (Cr 5) could not have been carrying the self-pattern determiner. The albino parent is therefore the only source through which it could have come. And it should be noticed that one of its G-Ps. (experiment 74), and three of its G-G-Ps. (experi- ments 71 and 664A) had the self-pattern coat. Now, on the basis of gametic purity and segregation, it is expected that one-half of the 15 offspring in experiment 194 of which the albino parent described above was one will carry the self-pattern determiner. By an ocular demonstration, corroborated by a subsequent breeding test, the expecta- tion is thus shown to be fulfilled. (2) Cr 4xCr 5 = Albino x Prebald Black-White. (a) Heterozygous Prebald x Albino. Experiments 29, 30, 31, 33, 34, 36, 87 and 37a. The expectation in this cross is 1 Cr4:1 Cr 5. The result is 44 Cr 4455 Cr5. The high proportion of the Cr 5 type is due to mating No. 30 (p. 391). If this is eliminated; the figures are 32 Cr 4+33 Cr 5. The disturbance of the proportions is due to the last two litters of this mating. They are:— Ist litter = 3 Cr 444 Cr 5 meh 6 fee EA, 210 Clear Ne-yo) a AECL De =O.) eke esthO! <5 In experiment 34, one of the four albino offspring showed the piebald “ chost ” pattern in its coat. (b) Homozygous Prebald x Cr 4. Hapervment 32. ° The expectation is that all the offspring shall be Cr 5. The result is 14 Cr 5. (c) Prebald x Cr 4 of more Complex Constitution. Experiments 35, 378, and 37c. In these crosses the result will depend upon the nature of the albino parent. If it be of cgBSsP constitution and the Cr 5 parent carries c recessive, * See also Pedigree Chart 1, in first paper. 2H 2 [Jan. 14, On the Hereditary Mr. G. P. 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a6 me a = \ 0g eee (| f eetteereeee niece teceineceieceeenteencen bere nn er Ve aie S Oba 8. | = { of hears Sates Ee ec SR ice as Ss nog § Gay on ae oad } 62 S ee erly af | 2), | {| re 80r/9/0s#(z) (¢)/80P/9/09r(z)(F)(T) 5 ¢ 20 pe TL G pur eZ b vee ee ae lc. | Le 80¥/9/0S+(z)(g)/80F/9/ogh(z)(F)(Z) PS 40 ve TL g pur ez | a ee el { ee: PST/eSt (F)/S9(z)/e9z/000T(T)(T) « d ¢ 40 Se 0z pur zg | ge pur ‘ez ‘BF \ oy " Ee Se | pee ee eoz/2gT/0e4(6)(g) 2 $40 69 eg pur LF = p > a a . és S 2s =} BGO O Ooo COU ROL OC OGD OUGD-0 OGD OCOD Ono SOLD OCOnEIDOn 6SZ 4 GIO CZ we as 3 de | oT (nee | nnn nnn VERS CISOCIOOUICICCIOR OIC IOOIOI OIC OC IOC OCI IO OIC 092 P c IQ CZ es ane on a ai at oa = sal ETE Ne iaTRibya: bieve) Wislovecgutee is ielevaieieis Fee eeeeeeneenseseneees eCZ 4 rca va) CZ “oer ae eieree Oran aya saterelsveleiaieiatavtereterols teretararatenevets pSO00U-000 009 Zos c IQ CZ elt as. i ee Ro cence ro fee oo a = a i ee ae te sex] — e i 392 Mr. G. P. Mudge. On the Hereditary [Jan. 14, the expectation is 2 Cr 4+1Cr5+1Cr60. The result (experiment 35) is 8 Cr 44+3 Cr 5+2 Cr 6 0. If the albino parent be cgBSP and the Cr 5 parent is homozygous, the expectation is that the offspring shall be all of the “Irish” 6 form. The result (experiment 37c) is nineteen “ Irish” 6 individuals. If the albino* is cGgBSP and the piebald carries ¢ recessive, the expectation is 2 Cr4+1 Cr2+1Cr60; the result is 3, +2 , +1 ,, (experiment 378.) A sister of this albino parent (experiment 378) was also mated with a black piebald (experiment 34). No grey individuals appeared in her offspring of eight. This, of course, is evidence that segregation of characters, ze. blackness and greyness, occurred. — (3) Cr 5x Cr 5 = Black Piebald x Black Piebald. (a) ILeterozygous Cr 5 x Heterozygous Cr 5. Expervments 25, 27, and 28a. The heterozygous Cr 5 carries c. The expectation is 1 Cr 4 :3Or5. The actual result is 16 Cr 4434 Cr 5. ; If we add the results of other observers, the figures stand thus :— f Cramper S4ce4. vee 12 Cr 4445 Cr 5 1 (Doncasbert: pec ce A yet Sto) elas, Mudge.) citee ss eeceee 16, +34 , Totals setae cade. s a2 vy +88 4, =1:35 nearly. With this total of 120 the predicted proportions are 30: 90. (b) Heterozygous Piebald x Homozygous Prebald. Experiments 26 and 28. The expectation in this case is that all the offspring will be piebald. The result is 11 piebalds. The further expectation is that some of the piebald offspring will be homozygous and some heterozygous. Experiments 31 and 56§ show respectively that the offspring of mating 26 was: one of them heterozygous and another homozygous. * The] previous behaviour of this albino is worth noticing (see experiment 45, first paper). + ‘Land. Jahr.,’ vol. 14, 1885, pp. 596 and 597. t ‘Camb. Phil. Soc. Proc.,’ vol. 13, 1906, p. 223. § See first paper. 1908.] Transmission of certain Coat Characters in Rats. 393 Summary. These experiments show that albinoes breed true to albinism, whether their ancestry is pigmented or not. They further show that, though externally albinoes may appear to be identical with regard to their coat characters, in reality they may be different. This fact has been previously known for both animals and plants, but it has hitherto been elucidated by means of breeding tests alone. These experiments add an ocular demon- stration of the actual presence of the coat-pattern in albinoes.. The interpretation placed upon the coat appearances in these albinoes is corroborated by the breeding results. It is further shown that when a piebald black rat (Cr 5) is mated with a similar one, two classes of offspring may be obtained. One of these contains all black piebalds and the other a mixture of black piebalds and albinoes in nearly equal numbers. When a piebald black rat is mated with an albino (=Cr. 5 x Cr 4), it may be said that, so far as these experiments have gone, five different results will be obtained. They may be stated as follows: (1) The offspring are all black piebalds. (2) They may be a mixture of black piebalds and albinoes. (3) They may be all “ Irish” forms (=a black self-coloured form). (4) They may be a mixture of albinoes, black piebalds, and “Irish.” (5) They may contain albinoes, “ Irish,” and a grey form (=Cr 2). It can be shown that the divergency of the results obtained when two individuals apparently similar are mated is due to the gametic nature of the albino employed. 394 Have Trypanosomes an Ultra-microscopical Stage in their Infe-history ? By Colonel Davip Brucz, C.B., F.R.S., Army Medical Service, and Captain H. R. BATEMAN, Royal Army Medical Corps. (Received June 6,—Read June 25, 1908.) By an ultra-microscopical stage in the development of a micro-organism is meant a stage in which the parasites are so small as to be invisible to the highest powers of the microscope, and to be capable of passing through the pores of a porcelain filter. For example, a drop of South African horse- sickness blood will give rise to the disease if injected under the skin of a healthy horse. If a similar drop is examined under the highest available powers of the microscope, nothing in the shape of a micro-organism can be seen. If this blood is filtered through a porcelain filter, the virus passes through, and the filtrate is found to be as infective as the original blood. Horse-sickness is therefore looked upon as a disease caused by an ultra- microscopical micro-organism. | For some time it has been reported by various workers that an ultra- microscopical stage exists among the trypanosomes. For example, Plimmer informs us that he found the filtered blood of nagana animals to be infective. Salvin Moore and Breinl write that the blood of animals suffering from Trypanosoma gambiense infection, although apparently containing no trypano- somes at all, and even if properly filtered, is still capable of infecting other animals into which it may be introduced. MacNeal also makes a similar statement in regard to Trypanosoma lewisi. He states that “in culture, on blood-agar, 7. lewisi may give rise to much smaller forms, and that such cultures, after passage through a Berkefeld filter, still infect rats.” Finally, it may be noted that the late Dr. Fritz Schaudinn, whose too early death we all lament, expressed the belief that trypanosomes may multiply by longitudinal division so rapidly as to become small enough to pass readily through a Chamberlain filter. This subject is an important one, as the discovery of an ultra-microscopical stage in these trypanosomes might throw light on the causation of some diseases in which no parasite can be found. In kala-azar, for example, the intra-corporeal form is small, and the extra-corporeal a fairly large flagellated organism. Let us imagine that the Leishman body lying inside the splenic cells had still further subdivided and become ultra-microscopical, then we would have an invisible parasite causing a serious disease in man and Have Trypanosomes an Ultra-microscopical Stage? 395 developing outside the body into a clearly visible flagellate. It is evidently important, then, in studying such diseases as South African horse-sickness, to try the effect of planting out the blood on various media, and looking for a visible stage of development in different insect hosts. The following experiments were made to test the truth of the statement that trypanosomes have this invisible stage. The filters used were Berkefeld’s ordinary filters for laboratory use. They were tested before use and found to readily keep back Micrococcus melitensis from the filtrate. The apparatus was attached to a Sprengel’s pump :— To ascertain if the Filtered Blood of Rabbits suffering from Nagana is unfective. Laperiment 23. March 20, 1908.—A rabbit which was inoculated on the 7th February, 1908, with Trypanosoma brucei, was killed to-day in an advanced stage of nagana. On microscopical examination of the peripheral blood and blood from the heart, no trypanosomes were seen. Portions of the heart, lungs, liver, spleen, kidneys, and bone-marrow were pounded up in a mortar with 1 per cent. sodium citrate in normal saline. The resulting emulsion was then filtered through a Berkefeld filter, and 1 c.c. of the filtrate injected sub- cutaneously into each of two white rats. April 30, 1908.—Both rats healthy. No trypanosomes have appeared in their blood. EHapervment 26. ot 24, 1908.—Rabbit inoculated with 7. bruce? on 26th March, 1908. Same proce- dure as in Experiment 23. May 21, 1908.—Both rats healthy. Experiment 35. March 31, 1908.—Rabbit inoculated 7th February, 1908. Same procedure as in Experiment 23. May 4, 1908.—Both rats healthy. Experiment 51. January 31, 1908.—Rabbit inoculated 10th January, 1908. Same procedure as in Experiment 23. _ March 25, 1908.—Neither rat showed trypanosomes at any time in its blood. Experiment 53. February 3, 1908.—Rabbit inoculated 10th January, 1908. March 30, 1908.—Result negative. Both rats healthy. Conclusion. From these five experiments it would: appear that the blood or organs of rabbits suffering from nagana does not contain ultra-microscopical forms: of 7. brucet. 396 Col. D. Bruce and Capt. H. R. Bateman. [June 6, To ascertain uf the Filtered Blood of White Rats suffering from Nagana is infective. Experiment 40. December 17, 1907.—A white rat, suffering from nagana, and whose blood was swarming with 7. brucet, was killed to-day. The organs and bone-marrow were made into an emulsion with 1 per cent. sodium citrate in salt solution and filtered in the usual way. Half a cubic centimetre of the filtrate was then injected into the peritoneal cavity of a white rat. March 16, 1908.—This rat has never shown trypanosomes in its blood. Haperiment 41. December 17, 1907.—This rat was also injected with the same quantity of filtrate as in Experiment 40. April 1, 1908.—Trypanosomes have never appeared in the blood. EHaperiments 42 and 43. December 24, 1907.—A nagana rat, whose blood was swarming with trypanosomes, was killed and the organs, etc., emulsified and filtered. One cubic centimetre of the filtrate was injected intra-peritoneally into two white rats. March 30, 1908.—Both rats healthy. Experiments 44, 45, 46, 47, 48, 49, and 50. This procedure was repeated seven times in exactly the same way and always with a negative result. Conclusion. From these 11 experiments it would appear that the blood of nagana rats filtered through a Berkefeld filter is not infective. To ascertain vf the Filtered Blood of White Rats suffering from Nagana or Surra, and treated for various Periods with Antimony, is infective. It was thought that the effect of treatment on animals suffering from nagana might lead to the development of small resting forms of the Trypanosoma brucei which might be capable of passing through a Berkefeld filter. The effect of certain drugs on animals suffering from nagana is marvellous. The blood may be swarming with trypanosomes, yet within an hour of the injection not a single one can be seen. They may remain out of the blood for weeks or months, in some out-of-the-way place, and, perhaps, in some resistant form. Experiment 28. March 26, 1908.—A white rat, whose blood was swarming with 7’. bruce, was treated with 3 cc. of a 4-per-cent. solution of sodium antimony] tartrate. The rat died half an hour after receiving this dose. The organs and bone-marrow were emulsified and filtered in the usual way, and 1 c.c. of the filtrate injected subcutaneously into a white rat. May 7, 1908.—Trypanosomes have not appeared in the blood. 1908.| Have Trypanosomes an Ultra-microscopical Stage? 397 Haperiment 29. March 27, 1908.—A white rat, whose blood was swarming with 7’. evansi, was injected with 4 c.c. of a }-per-cent. solution of sodium antimony] tartrate. This rat died half an hour after receiving the dose. Emulsion of the organs and bone-marrow made and filtered in the usual way, and 1 cc. of the filtrate injected into two white rats. April 29, 1908.—Both rats remain well. Experiment 33. March 30, 1908.—A white rat, whose blood was swarming with 7’. evansi, was injected subcutaneously with 4 ¢.c. of a }-per-cent. solution of sodium antimony] tartrate. This treatment was continued for a month, the animal receiving in all 11 doses. May 4, 1908.—Rat killed and its organs and blood emulsified and filtered. Half a cubic centimetre of the filtrate was injected intra-peritoneally into two rats. June 4, 1908.—Both rats healthy. Experiment 66. _ March 8, 1908.—A white rat, whose blood was swarming with 7’. brucez, was injected with two drops of a 1-per-cent. solution of sodium antimonyl tartrate. This treatment was continued for a month, the animal receiving in all eight doses. April 10, 1908.—Rat killed and its organs emulsified and filtered in the usual way. ‘One cubic centimetre of the filtrate injected into two rats. May 11, 1908.—Both rats healthy. Experiment 19. March 8, 1908.—A. nagana rat, whose blood was swarming with 7. brucez, was injected on the third day of disease with 2 minims of a 1-per-cent. solution of sodium antimonyl tartrate. March 9, 1908.—Repeated injection. A few trypanosomes in blood. March 11, 1908.—Repeated injection. A few trypanosomes in blood. March 13, 1908.—No trypanosomes in blood. March 18, 1908.—Blood swarming with trypanosomes. Injected 4 c.c. of a t-per-cent. solution of sodium antimony] tartrate. Rat died five minutes later. Organs emulsified and filtered and 4 c.c. of the filtrate injected into a white rat. March 26, 1908.—This rat’s blood is found to be swarming with trypanosomes. It is evident that something has passed through the filter capable of infecting a rat with nagana ; but it is possible that the filter has become defective on account of wear. It was tried again with a cultivation of Micrococcus melitensis in broth and failed to keep back the micrococci from the filtrate. It was, therefore, concluded that the filter was defective, and this experiment null and void. Conclusion. From these experiments it may be concluded that the blood of white rats suffering from nagana and treated for varying times with antimony salts does not contain ultra-microscopical forms of 7. brucez. 398 Have Trypanosomes an Ultra-microscopical Stage Z To ascertain of the Cultwation of Trypanosomes on Blood-agar will give rise to Ultra-microscopical Forms which are capable of passing through a Berkefeld ulter. Experiment 24.— White Rats. March 20, 1908.—The water of condensation from six flasks of blood-agar, upon which T. lewist had been planted out for 18 days, was to-day filtered through a Berkefeld filter, and 4 ¢.c. of the filtrate injected into two white rats. April 28, 1908.—These rats have remained in good health and no trypanosomes have appeared in their blood. Experiment 37.— White Rats. April 1, 1908.—A test-tube of blood-agar (2—1), which contained a luxuriant growth of T. lewist, was shaken up with 25 c.c. of normal salt solution, and the resulting emulsion - filtered through a Berkefeld filter. The filtrate was then injected into the peritoneal cavity of three white rats. April 30, 1908.—All three rats remained well, and trypanosomes never appeared at any time in their blood. Experiment 36.— White Rat (Control). March 31, 1908.—To ascertain if the culture used in Experiment 37 was virulent, three drops of the condensation fluid were injected into a small white rat. April 6, 1908.—T. lewist appeared in the blood of this rat. EHaperiment 64.— White Rats. April 9, 1908.—A blood-agar tube containing a growth of 7. lewisi, first generation, 46th day of growth, was shaken up with normal saline and filtered in the usual way. The filtrate was injected intra-peritoneally into two rats. May 11, 1908.—Both rats well. Trypanosomes have never appeared in their blood. Experiment 98.— White Rats. May 7, 1908.—Two blood-agar tubes, 27th day of growth. Same procedure as in Experiment 64. June 2, 1908.—Both rats healthy. Experiment 97.— White Rat (Control). May 7, 1908.—To ascertain if the culture used in Experiment 98 was infective, 3 c.c. of the condensation fluid was injected intra-peritoneally. May 18, 1908.—T7. lewisi in the blood. Conclusion. From these experiments it may be concluded that cultures of 7. lewase on blood-agar do not give rise to ultra-microscopical forms which are capable of passing through a Berkefeld filter. The final conclusion arrived at is that neither 7. bruce nor evanse develop in the body of animal forms so small as to be capable of passing through the pores of a Berkefeld filter, and that in cultures of 7. /ewist on blood-agar such small forms are also absent. 399 Diphtheria Antitoxim. By JoHn ME.uansy, M.D., George Henry Lewes Student. (Communicated by Professor J. N. Langley, F.R.S. Received June 3,—Read June 25, 1908.) (From the Wellcome Physiological Research Laboratory and the Physiological Laboratory, Cambridge.) CONTENTS. MEMO RCA ees a Heyes oo was vn cieswiatsaisen cats Scie a oat neem ad. «Sudo tence eaaeeich dans vasswadeecss 399 The Relation of Diphtheria Antitoxin to the Normal Proteins of Serum... 400 The Relation between the Antitoxic Potency of a Serum and the Amount MME LEH Ic COMUALME UD Mite. e occa teecamatte cameecidne cede cceatwodstenteecrdeenecCiecccsis 408 Penemunoory, Of Amtitoxin: PPOGUCHOM: .. 540.00. .s.cecvoceesscrsesentsenecorescsseeseenes 410 pe HEINE Uterine ote ce Seine ate opts Seen GRA te AAO, Son aac hale ois cieis'w Gis Scie tnle nbieme deine «dee biel 412 HISTORICAL. Many experimenters have endeavoured to isolate diphtheria antitoxin from fluids containing it. In 1893 Brieger and Ehrlich(1) investigated the properties of milk obtained from animals which had been immunised against diphtheria toxin. This milk contained an appreciable amount of antitoxin which could be precipitated between 27- and 38-per-cent. ammonium sulphate. By this method they were able to increase the antitoxic value of their fluid about five hundred times. But in milk only two proteins are present which differ widely in their physical properties—caseinogen and lact albumin—and their results could not be extended to horse serum, which is the main source of diphtheria antitoxin. Their experiments did not show that antitoxin is not a protein. Since horse serum is the main source of diphtheria antitoxin, experimental work has been done chiefly on this fluid. Aronson (2) stated that globulin precipitated from antitoxic serum by dialysis contained antitoxin. But Dieudonné showed that globulin precipitated by carbon dioxide from antitoxic serum did not possess this property. The protein precipitate obtained by both these workers corresponded to the globulin of serum as originally defined. Dieudonné further showed that if Hammarsten’s extended definition of globulin were adopted, then diphtheria antitoxin was a globulin, since it could be precipitated from serum by saturation with magnesium sulphate. 7 400 Dr. J. Mellanby. ) [June 3, Brodie (3) found that the same result could be obtained by half saturation with ammonium sulphate. Belfanti and Carboni confirmed the results of Brodie and Dieudonné. Freund and Sternberg (4) attempted the isolation of diphtheria antitoxin from serum by using 1°6-per-cent. potash alum. They stated that the filtrate obtained from serum after adding this salt, when treated with ammonium sulphate, gave a precipitate which contained the greater part of the antitoxin associated with only a small quantity of the original protein of serum. Pick (5) attempted to differentiate the antitoxic portion of serum by fractional precipitation with ammonium sulphate. Préscher first digested antitoxic serum with trypsin and then fractionated the resultant liquid by means of ammonium sulphate. He stated that in this. way he obtained a solution containing all the antitoxin free from protein. THE RELATION OF DIPHTHERIA ANTITOXIN TO THE NORMAL PROTEINS OF HorsE SERUM.* Three proteins possessing well-defined physical properties may be separated from horse serum. These proteins are present in serum in the following quantities :— (@)e Globulin eeeeee, oe About 3 per cent. (0)? Alum (a) BS Oa es Kc). “Albumin (3) ene Faint PaaS In the investigation of the properties of diphtheria antitoxin the first question to settle was whether it belonged to the protein groups (a), (0), Onc): The protein (a)—globulin—was obtained by precipitating antitoxic serum with 10 volumes of water and neutralising with acetic acid. The precipitate was adequately washed by decantation and filtration. This precipitate when dissolved in sodium chloride did not possess antitoxic properties. It may therefore be concluded that diphtheria antitoxin is not a globulin as rigidly defined. This result confirms the work of Dieudonné and Belfanti and Carboni. The protein (c)—albumin (8)—is probably that protein of serum which can be crystallised by means of ammonium sulphate and sulphuric acid. Crystalline albumin was prepared from antitoxic horse serum by this method. * The experiments detailed in the following pages are based upon results previously described in two papers: “The Physical Properties of Horse Serum,” ‘ Journ. Physiol.,’ vol. 35, p. 473, 1907, and “The Precipitation of the Proteins of Horse Serum,” ‘Journ. Physiol.,’ vol. 36, p. 288, 1907. 1908.| - Duphtheria Antitoxin. 401 The crystals obtained when dissolved in water did not possess antitoxic properties. It is evident, therefore, that diphtheria antitoxin belongs to or is associated with that great group of albumins which compose about 85 per cent. of the total protein of serum. For this class of albumin no method of physical differentiation has been obtained. It was interesting, therefore, to determine whether a delicate biochemical test such as is involved in the standardisation of diphtheria antitoxin would permit of a further differentiation. The Freezing of Serum. Serum containing 400 units per cubic centimetre of diphtheria antitoxin was frozen in a long cylinder and allowed to melt gradually. The resulting liquid was syphoned off in a series of layers. Antitoxin tests were made on some of the fractions and the relation of the antitoxic value to the percentage of protein determined. The following results were obtained :— Amount of solid in 100 ce. Antitoxic value. PPOA OUAMIMME SR: oe 6. eo de «e 50+ pm ee emma LS) 200+ 9-06 poe tent LS. 300+ 13°19 Pu PRAM NSS hon 450+ 17:08 Dae cent eas tlt 700+ Original serum... 9°4 AS Laie oar Hea ae 400 The + sign above indicates that the animals survived the tests without any serious symptoms. The results are conclusive,—the concentration of antitoxin follows the concentration of the protein on approximately parallel lines. The Heating of Serwm. The relation of diphtheria antitoxin to the proteins of serum coagulable by heat was determined. Serum containing 400 units of antitoxin per cubic centimetre?’ was coagulated at a series of temperatures—the coagulum at each temperature being filtered off before further heating. The following results were obtained :— Solids in each 100 c.c. Antitoxic value. PRIME SOLU, 560.0. 0in-02 nae sucnemannae oud 9-4 grammes 400 Filtrate after heating to 66° C. ......... Femi t ys 400 . sas ite Eiko Mea i te 3°04 50 ; ie: (PS ne ers Beahbes 3 50 402 Dr. J. Mellanby. [June 3, Thus heating to 66° C. coagulated a little fibrinogen, but destroyed no antitoxin ; but heating to 77° C. removed the greater part of the coagulable protein and also the greater part of the antitoxin. The fact that diphtheria antitoxin can be heated up to 66° C. without destruction is interesting. Ferments and complements are stated to be destroyed when heated to 56°C. The greater stability of diphtheria antitoxin to heat differentiates it from these classes of bodies. The Electrolysis of Antitoaic Serum. When a constant current is passed into serum through zine sulphate electrodes, a mass of insoluble protein accumulates at the anode and water comes out at the cathode, the relation between the water and insoluble protein being such that the concentration of solids in the electrolysed serum keeps constant. The influence of this procedure on antitoxic serum was determined. A constant current at a pressure of 100 volts was passed into antitoxic serum through zine sulphate electrodes for three hours. A considerable mass of insoluble protein accumulated at the anode and an equivalent quantity of water passed out at the cathode. At the end of this time the antitoxic value of the remaining serum was determined. This value was found to have remained constant; the antitoxin had neither accumulated nor diminished in the remaining serum. This fact is capable of only one explanation—that diphtheria antitoxin possesses the same properties as the normal proteins of serum when tested by means of an electric current passed into it through zinc sulphate electrodes. The protein mass which accumulated at the anode was also tested for antitoxin. This precipitate consists of a zinc sulphate compound of protein. The precipitate was dissolved in a minimal quantity of sodium hydroxide, and its antitoxic value determined. It was found to have an antitoxic value proportional to the amount of protein dissolved. ‘This fact is interesting— the combination of the protein with zinc sulphate, although causing an insoluble compound to be formed, had not produced any chemical change. The resulting precipitate possessed the antitoxin value of the original protein when adequately dissolved. The Precipitation of Diphtherra Antitoxin. (A) By Alcohol.—The alcohol precipitation limits of horse serum containing 400 units of antitoxin in each cubic centimetre were determined. The following results were obtained :— 1908.] Diphtheria Antitowin. 403 Percentage of Percentage of protein alcohol. precipitated. 12°5 3°6 25°0 76 37°5 48°7 50°0 97 75°0 99 The greater bulk of the protein is seen to be precipitated between 25 and 50 per cent.*alcohol. Antitoxin tests were made on the filtrate after precipitating with definite percentages of alcohol. It may be stated that alcohol in the dilutions used does not destroy toxin. From the antitoxin tests it was found that no antitoxin was precipitated by 25 per cent. alcohol; 25 per cent. was pre- cipitated by 37:5 per cent. alcohol; 30 per cent. by 40 per cent. alcohol; and 66 per cent. by 44 per cent. alcohol. From these figures it is legitimate to assume that antitoxin has the same precipitation limits as the general mass of protein of the particular serum used. From the antitoxin tests on the alcohol precipitation of other sera it appeared that the greater part of the antitoxin was precipitated between 35 and 45 per cent. alcohol. A point of some importance was to see whether the presence of antitoxin in serum had any general influence on the form of the alcohol precipitation curve. The quantitative precipitations of four sera of different antitoxin values were therefore determined. Antitoxin value Serum. per c.c. Total solids in 100 c.c. Be Melee ieee, 150 9°6 grammes | oie Meenas 250 11:0 ‘5 Or oe de 600 G29 jules h Aen SCARRED eric 800 9°7 3 Weight of protein precipitated from 109 c.c. of serum. Percentage of alcohol. | A B. C D grammes. grammes. grammes. grammes. 30 le 1‘1 — — 33 2°53 2°0 2:0 2°0 36 2 86 3 °4 3:1 3°3 39 3°8 4°6 41 4°7 42 4°83 6°0 5 °2 5°7 45 6°7 8°3 6:3 6:9 48 7-96 9 ‘92 es 8:0 50 8°52 — Vet d 8 °3 VOL. LXXX.—B. Zt 404 Dr. J. Mellanby. [June 3, There is some evidence from the form of the curves (which may be drawn ‘from the above figures) that the presence of diphtheria antitoxin does affect the precipitation values. Thus the Curve A is slightly concave to the left, and Curve D is slightly convex to the left. Curves B and C show features intermediate between A and D. The form of that part of the precipitation curve is affected in which the antitoxin is precipitated, and it may be seen that the degree of variation is roughly proportional to the amount of antitoxin present. : From this we may assume that although diphtheria antitoxin has the same alcohol precipitation properties as the great bulk of the proteins of normal serum, yet it is something added, and not merely a normal protein with an antitoxic group attached to it. Alcohol as a Protein Coagulant. An attempt was made to differentiate diphtheria antitoxin from the normal proteins of serum by means of alcohol as a protein coagulant. In the case of diphtheria antitoxin it was found that although alcohol added to serum did not affect its antitoxic value so long as no protein was precipitated, yet the rate at which precipitated antitoxin was destroyed by alcohol was the same as that at which the normal proteins of serum were coagulated. Therefore, so far as the coagulating action of alcohol was concerned, there was no evidence of any physical differentiation of diphtheria antitoxin from the proteins of normal serum. Precipitation by Alcohol below the Critical Potnt. The above results were obtained by the action of alcohol on diphtheria antitoxic serum at temperatures above the critical point. The alcohol precipitation limits of diphtheria antitoxin from serum were obtained at temperatures below the critical point. As a result of a series of alcohol precipitation experiments at 2° C., it was found that the greater part of the antitoxin was precipitated with other proteins of serum between 16 and 28 per cent. alcohol. These antitoxin limits, so far as the quantity of precipitated protein is concerned, are practically the same as those obtained at temperatures above the critical point. The only difference between precipitation above and below the critical point was that in the former case a portion of the antitoxin was lost owing to the coagulating power of the alcohol, whilst in the latter case the total protein precipitate was soluble, and the whole of the antitoxin could be recovered. (B) Ammoniwin Sulphate-—Brodie precipitated the antitoxin from horse i 1908.) Diphtheria Antitoxin. 405 serum in four parts by the gradual addition of ammonium sulphate to half saturation. He found no accumulation of antitoxin in any one fraction of protein precipitated, the result indicating that the antitoxin was not carried down in a purely mechanical way. The limits for the precipitation of antitoxin by ammonium sulphate were determined. It was found that the initial precipitation of the antitoxin was practically coincident with the first precipitation of the proteins of serum, and was complete only after about 22 per cent. of ammonium sulphate had been added. If these points be examined on the ammonium sulphate pre- cipitation curves, it will be found that they are practically coincident with the alcohol precipitation limits. (C) By Neutral Salts in the Presence of Acids.—The precipitation of anti- toxin by salts in the presence of acids was examined; 5 per cent. acetic acid and 0:25 per cent. hydrochloric acid were found to have no deleterious influence on diphtheria antitoxin at room temperatures. Controls showed that these acids in dilutions corresponding to the antitoxic value of the serum used did not destroy toxin. The precipitation of antitoxin by sodium chloride and 0°25 per cent. hydrochloric acid started when about 10 per cent. of the total protein of the serum was precipitated, ~.e., the first 10 per cent. of protein precipitate contained only a minimal amount of antitoxin. During the rest of the protein precipitation the antitoxin came down in a quantity proportional to the amount of protein precipitate produced. There was no indication of any differentiation of the antitoxin from the great bulk of the proteid precipitate. (D) The Mechanical Precipitation of Diphtheria Antitoxin from Serum.— It has been stated above that the temperature at which antitoxin is destroyed differentiates it from ferments. | A few experiments were made to determine whether it possessed the property common to ferments of mechanical precipitation. Various flocculent substances precipitated in serum did not diminish its antitoxic properties. This absence of mechanical precipitation might have been due to the simple nature of the precipitants, e.g.,calcilum phosphate and kieseleuhr. ) That antitoxin is produced in tissues remote from the seat of inoculation. 1908.] © Diphtheria Antitoxin. 411 _ (a) From a study of the lesions produced by diphtheria toxin, it is clear that the main tissues attacked by this poison are the heart and nervous system. Therefore, on Ehrlich’s theory, these tissues are mainly concerned in the production of antitoxin. From a general consideration of the metabolism of an animal it is improbable that two master tissues as the heart and nervous system are responsible for the protection of it. A horse had been immunised against diphtheria toxin for five years. At the end of this time it was killed, although it was suffering from no obvious disease. During its antitoxic life it had produced about 200 litres of antitoxic serum, containing, on an average, 500 units of antitoxin in each cubic centimetre, and had been injected with an appropriate amount of diphtheria toxin. A detailed post-mortem examina- tion of the various organs was made. There were no marked pathological changes to be noted—the heart showed a little brown atrophy, but no changes were found in the nervous system. It is inconceivable that the heart. and nervous system could have been attacked by such enormous quantities of toxin and have produced such a large amount of antitoxin without being more seriously affected. A consideration of the amount of antitoxin produced by a horse after the injection of a definite quantity of diphtheria toxin is of interest. During a period of four weeks 800 priifungs doses of toxin were subcutaneously injected into ahorse. At the end of that time the blood contained 400 units of anti- toxin per cubic centimetre. Suppose the horse contained 50 litres of blood. At the end of four weeks the blood of the animal contained 1000 x 400 x 50 = 2,000,000 units of antitoxin, or 800 priifungs doses of toxin produced 2,000,000 units of antitoxin; or each prtifungs dose of toxin produced 25,000 units of antitoxin. If this be interpreted in terms of Ehrlich’s hypothesis, it means that when the side chain of a cell is attacked by a molecule of toxin it manufactures more than 25,000 new side chains to protect itself. And if Ehrlich’s hypo- thesis be correct, the cells which are concerned in this extraordinary production are contained in those tissues which perform the main functions of the body—namely, in the heart and nervous system. (6) The production of antitoxin in a tissue remote from the site of injection of the toxin demands the assumption of other specialised mechanisms. Let us consider the case of a horse towards the end of its immunisation period. At the last injection, probably, an amount of toxin containing 200 priifungs doses is injected, and at this stage the blood of the animal contains 400 units of antitoxin in each cubic centimetre. This toxin, before it can produce any effect, must travel in the lymph stream to the thoracic duct, and so wd the jugular vein into the general circulation. When once in VOL. LXXX.—B. 2K ‘412 Dr. J. Mellanby. [June 3, the blood it will be ultimately carried into the neighbourhood of any tissue. But before it can attack a special tissue it must first pass through the capillary walls of the blood-vessels into the lymph bathing the cells of that tissue, — and such a passage demands a specialised mechanism of chemiotaxis. Further, from the union of toxin and antitoxin im vitro, it is probable that the toxin would unite with the antitoxin as soon as it got into the blood stream, and the hypothesis of the formation of antitoxin in a distant tissue would demand that this toxin-antitoxin compound should be broken up by the cell before it could stimulate the production of more side chains—in fact, that the formation of antitoxin by a cell should be of no use in protecting it from subsequent inoculations of toxin. From a general consideration of diphtheria toxin immunisation it is probable that the leucocytes are the immediate agents concerned in the pro- duction of antitoxin. After a subcutaneous injection of diphtheria toxin intoa horse there is usually produced at the site of inoculation a swelling of varying size. This swelling is of an cedematous character, and is crowded with finely granular oxyphil cells. The size of the swelling gives, to some extent, an indication of the degree of antitoxic reaction. The production of antitoxin by the leucocytes at the seat of inoculation would bring the mechanism involved into line with the facts observed by Metchnikow with pathological bacilli and the more recent work on opsonins. The antitoxin need not necessarily be produced at the seat of inoculation. After a leucocyte had ingested a toxin molecule the secretion of antitoxin by it would take place into any fluid in which it was present—the blood or lymph. This hypothesis affords a ready explanation of poisoning by excess of toxin. If the leucocytes are able to take up the toxin, none of it is carried to distant tissues, such as the heart and nervous system, and no pathological effects are observed. But if the toxin is injected in too great a quantity to be taken up by the leucocytes, then the excess is carried by the blood to the tissues, and general toxemia results. Again, Ehrlich’s hypothesis offers no explanation why subcutaneous injection of toxin yields much better antitoxic results than injection into the blood stream direct. The theory that antitoxin is secreted by leucocytes which have ingested toxin molecules affords a ready explanation of these results. SUMMARY. All the properties of diphtheria antitoxic serum indicate that diphtheria antitoxin is a protein which possesses characters identical with albumin (a) of serum, 2.¢., the protein forming 85 per cent. of the total quantity present. The alcohol precipitation curves of antitoxic sera of varying strength 1908. | Diphtheria Antitoxin. 413 indicate that this protein is added to serum during immunisation, and that the antitoxic property is not due to the addition of a group possessing this character to a normal protein molecule. This conclusion also follows from a study of the relation between total solid and antitoxic strength of the serum of a horse during its antitoxic life. It is suggested that one of the functions of the proteins composing the group albumin («) is to protect the tissues against disease. A theory is advanced that the production of diphtheria antitoxin is due to: an active secretion by the leucocytes, this secretion being stimulated by the- ingestion of toxin molecules. REFERENCES. (1) Brieger and Ehrlich, ‘ Zeitschr. f. Hygiene,’ vol. 12, p. 137. (2) Aronson, ‘Berl. klin. Wochenschr.,’ vol. 26, p. 425. (3) Brodie, ‘Journ. of Pathol. and Bacteriol.’ vol. 4, p. 460. (4) Freund and Sternberg, ‘ Zeitschr. f. Hyg.,’ vol. 31, p. 429. (5) Pick, ‘ Hofmeister’s Beitr.,’ vol. 1. (6) Szontagh and Wellmann, * Deutsche med. Wochenschr.,’ vol. 24, p. 421. (7) Hiss and Atkinson, ‘ Journ. of Exp. Med.,’ vol. 5, p. 47. (8) Ledingham, ‘Journ. of Hygiene,’ vol. 7, p. 65. ie a : eC ere ke pe) wow ane ae Pe 1908.) Diphtheria Antitoxin. 413 indicate that this protein is added to serum during immunisation, and that the antitoxic property is not due to the addition of a group possessing this character to a normal protein molecule. This conclusion also follows from a study of the relation between total solid and antitoxic strength of the serum of a horse during its antitoxic life. It is suggested that one of the functions of the proteins composing the group albumin («) is to protect the tissues against disease. A theory is advanced that the production of diphtheria antitoxin is due to an active secretion by the leucocytes, this secretion being stimulated by the ingestion of toxin molecules. REFERENCES. (1) Brieger and Ehrlich, ‘ Zeitschr. f. Hygiene,’ vol. 12, p. 137. (2) Aronson, ‘ Berl. klin. Wochenschr.,’ vol. 26, p. 425. (3) Brodie, ‘ Journ. of Pathol. and Bacteriol.,’ vol. 4, p. 460. (4) Freund and Sternberg, ‘ Zeitschr. f. Hyg.,’ vol. 31, p. 429. (5) Pick, ‘Hofmeister’s Beitr., vol. 1. (6) Szontagh and Wellmann, ‘Deutsche med. Wochenschr.,’ vol. 24, p. 421. (7) Hiss and Atkinson, ‘Journ. of Exp. Med.,’ vol. 5, p. 47. (8) Ledingham, ‘Journ. of Hygiene,’ vol. 7, p. 65. TOL. Lx kx. —B. Fei Al4 Croontan LecturE.—The Principles of the Minute Structure of the Nervous System as revealed by Recent Investigations. By Professor GustaF Rerzius, For. Mem. RS. (Lecture delivered May 14,—MS. received June 15, 1908.) When the flattering invitation to deliver this year’s Croonian Lecture before your far-famed Royal Society reached me, I first of all felt con- siderable hesitation as to whether I should be able to discharge so honourable a task. The very choice, out of the field of my investigations, of a subject which should be suitable for lecturing to you about, presented a very real difficulty. During the past 20 years I have been working principally in three depart- ments of scientific research—the Nervous System of Vertebrata and Invertebrata, Physical Anthropology in Sweden, and the Spernia of Animals of all Orders. By far the most interesting of these subjects is the first, dealing as it does with the chief organ in nature, that of the psychical functions. Upon this subject, of the minute structure of the nervous system, there has been a great deal of light thrown during the last two decades by the histological researches of a number of scientists, of whom may be specially mentioned the eminent Italian and Spanish neurologists Camillo Golgi and Ramon Cajal. The subject, however, has two real drawbacks: on the one hand it is exceedingly complicated, especially as several of its results are still under debate; and on the other it has already, in 1894, been treated of in a Croonian Lecture by Professor Cajal. Since his lecture, however, 14 years ago, the discoveries made in this department of science have been very numerous, many of them due to the researches of Cajal himself. At last I came to the conclusion that I might on this occasion continue and bring up to date the review of the subject which he then gave you. In pursuing this intention I have, however, been obliged here and there to glance back at work done prior to 1894, and also to speak of some aspects of the subjects upon which my predecessor only touched very lightly. I conclude that it is in accordance with the idea of these lectures that the lecturer may also mention some of his own researches. And I hope that you will pardon me for expressing my opinion more definitely in certain particulars, seeing that I have arrived at a conviction of my own respecting them, which is , based upon investigations which I have myself carried out. I regard this as. Minute Structure of the Nervous System. 415 unavoidable when the field of research in question consists of so much unbroken ground and such important problems are still not solved. It will all the same be necessary for me to limit my attention to a few of the most interesting chapters in this wonderful and fascinating department of biology. The scientific investigation of the histology and physiology of the central nervous system, above all of the brain, is surely one of the most difficult problems presented to human intelligence to solve. With good reason Emil Dubois Reymond’s famous ejaculation: “ignorabimus,” may be applicable here. “HH pur si muove.” How rapidly has our physiological knowledge of the: localisation of the motor and sensory centres in the brain—since Fritsch and: Hitzig first showed their existence by experimental proof—gone forwards: step by step, owing principally to the brilliant discoveries of the English investigators Sir Victor Horsley, Schafer, Beevor, Ferrier, Sherrington, and still others. It is indeed true that the proverb, “There is nothing quite new under the sun,” is not without an illustration here, too, for one cannot but be astounded to find that, as far back as the year 1744, the Swedish polyhistor and scientist, Emanuel Swedenborg, was able, in his famous work ‘ Gtconomia Regni Animalis,’ with his prophetic vision to set up as a goal for the scienee of physiology of the brain the following standard: “ Experientiz est et temporis, ut investigetur qui gyrus et qui serpens tumulus in cerebro hune aut illum musculum ut correspondentem suum in corpore respiciat,” and “Ergo inquirendum venit, qui tori corticei his aut illis musculis in corpore correspondent: quod fierl non potest nisl per experientiam in vivis. animalibus, per punctiones, sectiones et compressiones plurium, perque inde in corporis musculis redundantes effectus.” As we see, this is nothing short of a full programme in the experimental physiology of the brain which this. marvellous man here lays before us, and we are yet again amazed to read his. clearly worded statement, that the muscles of the lower extremities have their centre at the top of the cerebral cortex, the muscles of the abdomen and thorax in the central portions of the cerebrum, those of the head and face: at the bottom, “nam videntur ordine inverso sibi correspondere.” It has. been my purpose in quoting these theses of Swedenborg’s to point out that: grand scientific discoveries, of which our own age is rightly proud, may have been not only vaguely guessed at, but actually set forth in clear and definite terms by one or another brilliant enquirig mind of an earlier age. The theses cited are drawn up with such precision by Swedenborg that they cannot possibly be based on divination only, but must rest upon a real 2s Ean A16 Prof. G. Retzius. Principles of the [June 15, grasp of natural phenomena as well as on actual experiments and dissecting work. A more thorough knowledge of the minute structure of the brain and the whole nervous system was essential, if the physiology of those organs was to advance. To that end the perfecting of the microscope was a conditio sine gua non. Earlier anatomists, ¢.g., Leeuwenhoek and Malpighi, had paved the way, lt is true, to our present results, but did not proceed far themselves. In accord with the last-named great Italian scientist (Malpighi), Emanuel Swedenborg, though, put forward a remarkable theory regarding the composition of the cerebral cortex, which he—in opposition to so many anatomists of that day—definitely declared to be the seat of the psychical phenomena. Not until during the last century was it possible to get nearer the solution of this the most difficult problem of histology. It had by degrees been discovered that the nerve-tissues consist of nerve-cells in several different forms, and of nerve-fibres with and without myelin sheaths. It was also found out that the nerve-fibres are processes of the nerve-cells. The attempt was made to devise methods for tracing the nerve-fibres in their several courses, partly by hardening and staining them, partly by effecting their degeneration along certain paths, partly by studying the gradual development of their myelin sheaths in the embryo. By means of such researches scientists, before the close of the seventies, had arrived at the point of being able to establish theories concerning the minute construction of the nervous system in general. But, alas! how hypothetical, how uncertain these theories for the greater part were! I have still a clear remembrance of the hesitation and reluctance which I felt year after year, from 1877 onwards, in lecturing, as professor of histology, about this subject to my classes of students. I did not myself believe in a good deal of what I was constrained to teach them in accordance with the then accepted doctrines of science concerning the structure of the central nervous system. As a result of*the investigations which I conducted in conjunction with Axel Key in the years 1869 to 1876, into the lymphatic spaces, and the structure of the brain, and the rest of the nervous system, I had arrived at the conviction that the abstract theories then held by men of science as to the construction of that system must be incorrect and were inconclusive. It was at this period the Italian scientist Camillo Golgi made his invention for staining chrome-hardened nerve tissue with silver solution, by which the nerve-cells were selectively dyed brown. At first his communication, which was published in some smaller Italian journals with a very limited circulation in other countries, attracted very little notice, but on the appearance in 1885 1908. | Minute Structure of the Nervous System. 417 of his great work, “Sulla Fina Anatomia degli Organi Centrali del Sistema Nervoso,” men of science were surprised at this invention and its results. Now, von Kolliker, myself, and others repeated this method, but, though we carefully followed Golgi’s directions, the results were not really satis- factory. Ithen began working by the new method of P. Ehrlich, which he published in 1886, and which consisted in staining living nerve tissue methylene blue. Golgi’s chrome-silver method had, however, in the inventor’s own hands, yielded a series of fundamentally important results, which are already mentioned in the Croonian Lecture of Cajal. Of these the following may be specially mentioned here also. The occurrence in the central organ of two types of nerve cells, those whose axis-cylinder process does not branch and come to an end until after a long course, and those whose processes, after a short course, branch copiously, and come to an end in the central organ ; further, the discovery of fine collateral branches emerging out of the axis- cylinder processes in the central organs; a more exact knowledge of the forms of the cells in the cortex of the cerebrum and the cerebellum, and more especially also in the gyrus hippocampi; the investigation of the different types and methods of ramification of the dendrites, and an enquiry into the structure of bulbi olfactorii. Golgi had become convinced that in the grey substance of the spinal cord there exists an exceedingly extensive and delicate network, which he considered was due to anastomosing of the collateral branches, and not, as Gerlach before supposed, to the anastomosing of the dendrites. During the next following years, however, Golgi’s method did not yield any very remarkable results; the scientists who used it did not obtain any real success with it. Ehrlich’s methylene method, on the other hand, was tried and valued in some places, especially in Kazan by Arnstein, Dogiel, and Smirnow, and in Stockholm by myself. In the higher animals, the vertebrata, the method could be well applied to the peripheral nervous system, but to the central one only with the utmost difficulty. I then determined to try and find some lower animals which admitted of having their whole nervous system, or, at any rate, the principal parts of it stained, so that by experimenting with them one might obtain a comprehensive idea of their entire construction. At last, in 1890, I found, with a certain modification, that the method gave excellent pictures in the ganglions of the ordinary crawfish (Astacus). A general survey was obtianed of their construction. One could clearly perceive that their so-called “ Punktsubstanz” of Leydig was formed of the lateral twigs of the processes of the unipolar nerve-cells, which twigs could be traced in the 418 Prof. G. Retzius. Principles of the [June 15, substance in their most delicate ramifications, where they did not unite with each other, and on the whole did not form a network (reticulum), but a twist- work (plexus). One could also trace the stem-processes of the nerve-cells even in their various courses through the ganglions towards the periphery (fig. 1). ‘ Fic. 1.—A Part of the Ventral Nervous Chain of the common Crawfish (Astacus), showing a ganglion with coloured (black) nerve-cells and their processes. 1908. | Minute Structure of the Nervous System. A19 By similar researches on the spinal cords of Amphioxus and Myxine, I could also trace the condition of their central nerve-cells and the several paths of the cell processes. Still more beautiful and instructive pictures gave the nervous system of the Hirudines (fig. 2), where also Biedermann had studied them with success, and especially in that of a polychet annelid (Nereis diversicolor). Fig. 2.—A Ganglion of the Ventral Nervous Chain of Hirudo, showing the black stained unipolar nerve-cells with their processes. The figure below shows a part of such a ganglion with a nerve-cell and its processes. 420 Prof. G. Retzius. Principles of the [June 15, Now, in the years 1891 and 1892, the renowned Hungarian histologist Michael von Lenhossék, applying successfully the Golgi method in the common earth-worm, Lumbricus, discovered that the epithelium cells in the epidermis send fine fibres to the ganglions of the central nervous system, where they divide and terminate with free endings. These cells showed themselves very clearly to be a kind of sensory peripheral nerve-cells, which are in contact with the elements of the nervous central organ, and are capable of conveying to them impressions from the periphery of the body. This discovery of von Lenhossék was shortly after confirmed by myself and developed in conjunction with a closer investigation of the structure of the ganglions (figs. 3 and 4). I showed, by means of the methylene and chrome- silver methods, that similar cells exist in the epidermis of polychets and molluscs, and-also in the crustacea, though the cell body in these animals lies lower down under the epidermis (figs. 5 and 6). Besides these sensory cells there exist also in the epidermis and the other epithelial tissues ‘ramifications of free endings of tactile nerves, whose cells are to be found deeper. I have mentioned these investigations in connection, particularly as they have for the most part been carried out by the methylene method. Now, however, a new era had commenced for Golgi’s chrome-silver method, as applied to the central nervous system of the higher animals. In and after 1888 Ramon Cajal, the great Spanish neurologist, published a series of works on the minute structure of the brain and spinal cord of vertebrate animals. By means of Golgi’s method, which he improved and used in a masterly manner, Cajal succeeded in investigating the forms and the distribution of the nerve-cells and their processes in the different parts of the central nervous organ. We may date from this time almost a new epoch in our knowledge of the minute structure of the nervous system. Cajal’s first discoveries had an electrifying effect upon those who were working in the same field. For my part I shall never forget the overwhelming impression that the demonstration by Cajal, at the Berlin Anatomical Congress of 1889, of a large series of his preparations produced upon those of us who were specially interested in the subject. Albert von Kolliker and I were enchanted by the sight of the preparations which Cajal placed before us. Both he and I were converted, and we started home to begin working afresh with Golgi’s method, which was not in great repute among other anatomists of that day. Kdlliker, as well as von Lenhossék, working then in Kolliker’s laboratory as his prosector, succeeded in applying Golgi’s method, and published several excellent new researches. At the same time I, in Stockholm, and Van Gehuchten, in Louvain, were applying the same method, while 1908. | Minute Structure of the Nervous System. 421 : Pe a NL RY SS ae RS nel Gr) i, ASA FINE ae a\ as eee eZ ae | a — gz mm} es “ 4 SS a Fig. 3.—The Nervous System of Lumbricus. Parts of the ventral nervous chain of ganglions, with nerve-cells and their processes (nerve-fibres) stained black. 1. Two ganglia; at the upper the sensory nerve-bundles from the cells of Lenhossék are seen coming from the periphery and entering the ganglion, there dividing and terminating in free endings. Cajal himself went on with one investigation after another, and Golgi and a couple of his pupils continued pursuing their various researches. It may be said, nevertheless, that Cajal during this whole period was the most leading spirit in this research, so far as the central organ of the 422 Prof. G. Retzius. Principles of the [June 15, Fie. 4.—Nerves of Lumbricus, stained with the aid of the Golgi method. 1. A portion of a transverse section, with the skin and the cells of Lenhossék, with their central processes running to a ganglion. 2,3, and 4. Transverse sections of ganglia, with nerve-cells and their processes, and also with some central fibres of the cells of Lenhossék. 5. A little portion of the skin, with the cells of Lenhossék. 6, 7, and 8. Endings of nerve-fibres on muscles. vertebrate animals was concerned. Of Cajal’s works there may here be cited in the first place those dealing with the spinal cord, the cortex of cerebrum and cerebellum, and several of the interior cerebral ganglia, the retina, the olfactory organ, and the peripheral ganglia (the sympathetic and spinal ganglia). In all these organs Cajal not only made important discoveries of 1908. | Minute Structure of the Nervous System. 423 Fic. 5.—Portions of Antenne, Parapodium, and Skin of Nerezs diversicolor, with stained bipolar sensory cells and their processes, new cell types and ramifications of cell processes, he also established the relations of the cells to one another in many respects. It would take too long to give a detailed account here of these researches, and the investigator himself in his Croonian Lecture to this distinguished Society, in 1894, described several of his most important discoveries in this branch of science. Wilhelm His, of Leipzig, the eminent anatomist and histologist, had formed the conviction as long ago as in 1883, from his studies of the nervous system of the embryo, that the nerve-cells develop from the very first as organs 424 Prof, G. Retzius. Principles of the [June 15, Fic. 6.—Head of small Crustacean (Daphnia, male), from the right side, showing several nerve elements coloured (black). On the left is the brain ganglion, with nerve-cells, and on the right side of it the smaller optic ganglion, in which the optic nerve-fibres, coming from the eye, end. Below, there is seen in the figure the bipolar sensory cells in the first antenna, constituting the so-called organ of smell, with central fibres going to the basis of the brain-ganglion. In the second antenna two tactile cells, with leng central fibres are seen. independent of one another, and do not enter into connection one with another. In agreement with the statement of v. Kupffer, in 1857, His observed the nerve-fibres of the embryo growing as axis-cylinder-processes of the nerve- cells; v. Kupffer and His considered, moreover, every nerve-fibre in the body, both in the nervous central organ and in the peripheral parts of the body, to be processes of that kind more or less lengthened. a ‘. 1908. | Minute Structure of the Nervous System. | 425 This view was now also accepted by Cajal, on the basis of his own researches ; by the Golgian method he showed how the axis-cylinder process grows out of the cell body in the embryo, and how at its peripheral end it has as a rule a small thickening, the so-called bud of increscence (cono de crecimiento), which gropes its way, as it were, to make a path for the fibre. Shortly afterwards, v. Lenhossék, v. Kolliker, Van Gehuchten, and myself all arrived at the same opinion as a result of our own individual investigations (fig. 7). Weall of us also accepted, more or less definitely, the idea propounded Spinal cord of Lizard -embryo (transv. sect.) Spinal cord of Bat-embryo (transv. sect.) Spinat cord of Serpent - embryo (roan s ya sect) Spinal cord of Lizard-embryo (longitud. sect) Fic. 7.—Three transverse Sections and one longitudinal Section of the Spinal Cord with Nerve-cells and Nerve-fibres stained by means of the Golgi method. by His regarding the morphological independence of the nerve-cells in their first inception. Cajal led the way here, too, and the rest of us embraced the same theory on the basis of the results which we had obtained by the Golgian method. We did not discern, as Gerlach claimed, a network in the grey substance of the spinal cord and the brain by a union of the dendrites of the nerve-cells ; nor did we find, as Golgi assumed, and still seems to assume, any network which has arisen from a union of the collateral branches of the axons of the nerve-cells. As above stated, I also did not find by the methylene method any network 426 Prof. G. Retzius. Principles of the [June 15, in the grey substance (Punktsubstanz) of the invertebrata, but only a twist- work, a plexus, of delicate branches of nerve-cell processes, intricately intertwined. So we were all prepared, in accord with Cajal’s definite pronouncement, to regard the connection between the various nerve-cells and their processes as effected, not by continuity but by contact. In his excellent and concise review of the investigations in this depart- ment during the previous years, Waldeyer, in 1891, called this conception of a nerve-cell as an independent unit a neurone, a term which has since been generally adopted to imply the independence of the nerve-cells. This doctrine, the newrone theory, as enunciated by Waldeyer, appeared to gain more and more ground in neurology. His’, Cajal’s, v. Kolliker’s, v. Lenhossék’s, Van Gehuchten’s, and my own investigations had given the basis to and had confirmed this theory in many directions. As for the retina, Dogiel described the nerve-cells as anastomosing, but Cajal’s investigations by the Golgian method gave, as to this point, quite different results from Dogiel’s. And as regards the peripheral nerve-ends I may say that, though I have carried out extensive researches upon them, I have never been really able clearly to see any formation of network, either among the terminal ramifications of the different neurones or in those of one and the same neurone. Thus, I have never been able to observe a reticulum between the terminal ramifications of a motor nerve-fibre on a muscle, nor in those of a sensitive nerve-fibre, nor on a nerve-fibre of one of the sensory organs. Supposing such a connection were really to occur, as it is described by some histologists, I must regard a reticulum of that kind as rather abnormal, due to some sort of secondary coalescence. I have considered myself bound to specially notify these facts, as they are of importance for the neurone theory as a whole. The question of the relation and connection of the peripheral sensory cells to the nerve-fibres is also of fundamental importance. Scientists have long assumed, and in some cases asserted that they had observed, that in the higher animals each of those cells was directly connected with a nerve-fibre. That was held to be the case with the organs of smell and taste, and also with regard to the organ of hearing. Now, how was this fact to be explained by the principles of the neurone theory? This question came forward: Can a nerve-fibre be directly connected with both a central and a peripheral cell, or be actually a process of both of them ? This important question, thanks to the researches of the last two decades, found a perfectly natural explanation and one that fits in with the neurone 1908. | Minute Structure of the Nervous System. 427 theory. The different sensory organs are differently constituted, are constructed on different plans (fig. 8). First, as regards the organ of taste: A. Key, who was working ‘at the beginning of the sixties under Max Schultze, stated that he had seen in the LE EY can ys EEE i\! nerve -frelve Fig. 8.—Peripheric Nerve-endings in the different Sensory Organs, stained by means of : the Golgi method. frog a direct connection between the ends of the nerve-fibres and certain sensory cells situated on the surface of the so-called taste-papille; and in the higher animals, the mammals, the same mode of termination was taken almost for granted. I was enabled by the aid of the methylene method and, later, also by the Golgian method, to demonstrate conclusively, in [1888 428 Prof. G. Retzius. Principles of the [June 15, and 1892, that no mode of termination of that kind exists, that the nerve terminations find their way into the taste-buds, and copiously ramify there among the taste-cells without coming into direct connection with them. As regards the olfactory organ, it could be shown clearly and distinctly by the Golgian method that the olfactory cells of the mucous membrane send out a central process which makes its way into the glomeruli of the olfactory bulbs, and there copiously ramifies among the ramifications of one or more other axons proceeding from the central nerve-cells, forming with them the glomeruli. That has been shown already by Golgi, and was confirmed by Cajal, Van Gehuchten, myself, and Kolliker; the four last named did not discover any direct connection between the nerve-fibres that proceed from the centre and those from the periphery, though that there is none is often difficult to prove for certain by reason of the exceedingly complicated convolutions of the fibres. The sensory cells of the olfactory organ are to be regarded as a species of peripheral nerve-cells. They must also be regarded as corresponding fundamentally to those bipolar cells in the skin of the invertebrata already mentioned above. In 1881 to 1884 I had shown that in the macule and criste acustice of the auditory organs the nerve-fibres enclose the lower ends of the hair cells by means of, calyx-shaped extensions, and even send out fine fibrils that proceed in an upward direction on the surface of these cells. Here the matter proved more complicated, for by the Golgian method I succeeded. in 1892 in proving definitely that the terminations of the auditory nerve come to an end with free ramifications among the hair cells, and, to some extent, find their way up to the surface of the epithelium. More recently Cajal has shown by his new silver method that both modes of termination, calyx formation and free ramification, exist side by side in criste acustice. | It is in any case plain that sensory cells of the auditory organ (the hair cells) may in principle be compared with those of the gustatory organ. Their development makes manifest, too, that they do not send out any fibres which proceed towards the centre, but that the bipolar nerve-cells situated in the ganglion acusticum dispatch their peripheral processes to the sensory cells in the epithelium. By reason of these circumstances, constituting as they do an essential difference, I here distinguish sensory cells such as those of the olfactory organ, which I call primary, from sensory cells such as those of the gustatory and auditory organs, which I call secondary. The former are, of course, a species of peripheral nerve-cells, the latter are not. I do not propose here to enter upon a discussion of the complicated 4 1908. | Minute Structure of the Nervous System. 429 structure of the organ of sight, the retina, which is the less requisite as - Cajal described it in his Croonian lecture. As regards the termination of the sensitive and tactile nerves in the epidermis, the epithelia, and the specific sense organ, the Pacinian corpuscles, the Krause end-bulbs, etc., one may formulate a general rule as follows :— They terminate everywhere with free endings and. ramifications, though differing in arrangement in different cases, and sometimes among cells (Merkel’s terminal discs, Grandry’s corpuscles, etc.), which may, to a certain extent, be compared to secondary sensory cells. To give a survey of these different types of terminations I have drawn up a few diagrams of the various kinds of sensory cells (fig. 9). The olfactory cells and those sensory cells of the invertebrata that are here under discussion are consequently to be regarded as a kind of peripheral neurones, the gustatory and auditory cells on the other hand may not, strictly speaking, be so considered; the real neurones of the latter, as also of the terminal branches of the tactile nerves, are constituted by the nerve-cells situated in the cerebro-spinal ganglion system. | The neurone theory had consequently from a morphological point of view shown itself to be correct, both as regards the facts connected with the central organ and the peripheral one, and the Golgian method had yielded results agreeing in all essentials with those of the methylene staining one. The difference between our knowledge then and that we possessed, for instance, in 1880, was prodigious. Yet, in reality, it was a mere handful of investigators, dwelling far apart from one another, who had accomplished this work. In some quarters, even among eminent anatomists, physiologists, and especially neurologists, the work and results of those investigators were long regarded with a certain suspicion; now and then that suspicion found expression in a rather startling form. I might, for instance, read to you letters which I received from celebrated histologists abroad, who were other- wise favourably disposed towards me and my work, conjuring me in the most serious and moving terms not to go on experimenting with that wretched Golgian method which only resulted in art effects, impure chrome- silver precipitations within the tissues, and were misleading and dangerous for real scientific inquiry. Pretty much: the same verdict was pronounced, too, from a specially authoritative source upon the methylene method as we applied it. From the point of view of the history of science it may be of a certain interest to note that our labours by no means met with encouragement and recognition in all quarters. The new school of investigators found little difficulty in dealing with these antagonists. d, VOL. LXXX.—B. 2M 430 Prof. G. Retzius. Principles of the [June 15, 5 6 7 8 Fic. 9.—Schemes of the various Sensory Organs, with their different Sensory Cells and Nerve-endings. 1. The sensory nervous system of an oligochete worm (Lumbricus). 2. The sensory nervous system of a polychzte worm (Nereis). 3. The sensory nervous system of a mollusc (Limax). 4. The sensory nervous system of a mammal (the skin), 5. The organ of smell in the Vertebrata. 6. The organ of sight in the Vertebrata. 7. The auditory organ in the Vertebrata. 8. The organ of taste in the Mammals. 9. The tactile organ in the Vertebrata. But other opponents now came forward who had devised new methods and arrived at other conclusions regarding the minute structure of the nervous system. First came the Hungarian zoologist Stephan Apathy, of Kolosvar, who after some smaller communications published a larger book 1908. | Minute Structure of the Nervous System. 431 illustrated by elucidating figures, entitled: “Das leitende Element des Nervensystems und seine topographischen Beziehungen zu den Zellen” (1897). By the help of a new gold-staining method he had succeeded in showing in the ganglion cells of certain worms, and especially finely in the unipolar cells of the Hirudines (fig. 10), an intra- cellular reticulum of fibrils which form a continuous network round the nucleus and also near the cell surface ; from that network there proceeds through the single pro- cess an unbranched fibril which extends into the Punktsubstanz. In the vertebrata, too, Apathy had found fibril network in the ganglion cells; this he described, but did not illustrate by figures. In conjunction with this dis- covery of the neurofibrils, excel- lent in itself, Apathy built up a whole hypothetical theory regard- ing the minute structure of the nervous system. Of that theory it is not easy to give a brief summary, but these are the chief points in it:—The neurofibrils dis- covered by him form the specific constituent of the nervous system, the nervous portion proper, the conducting element; they are independent structural parts ; in the nerve-fibres they preserve their individuality; in three places, however, they form reti- cula, viz.,in the ganglion cells, in Fig. 10.—Two Unipolar Nerve-cells from a Gan- glion of the Ventral Nervous Chain of Hirudo. In the cells is seen the fibrillar net- work of Apathy, running down through the cell process into the Punktsubstanz, which consists of fine twisted fibres. the terminal organ (the sensory cells, etc.), and in Leydig’s Punktsubstanz, in which they subdivide into an exceedingly intricate reticulum, Apdathy’s “ Klementargitter,” in which the sensory nerve-fibres in particular dissolve and lose their individuality. Jowe 2 432 Prof. G. Retzius. Principles of the [June 15, In connection with this conception Apathy constructed his hypothetic theory regarding the minute structure of the nervous system. He maintains that there are two kinds of nervous cells, nerve-cells and ganglion cells. The nerve-cells produce the conducting substance, the neurofibrils, which grow both towards the centre into the ganglion cells and towards the periphery into the sensory cells, the muscle cells, ete. The neurofibrils constitute consequently an element foreign to the ganglion cells which has grown into them from without; they are further capable of independently emerging from the ganglion cells and their processes, the nerve-fibres ; they do not accordingly need to remain in them and their paths. They form, moreover, large continuous reticula in the organs of the body and above all in the central grey substance. It is not to be wondered at if the specialists were somewhat astonished at this new system of Apathy’s, which appeared to them to rest on very uncertain foundations. Apathy’s own preparations of Hirudo ganglions were exhibited at zoological and anatomical congresses. The existence of the intracellular fibril network in the ganglion cells of certain worms was clearly apparent in these preparations, but his “Hlementargitter” in the Punktsubstanz, on the other hand, was not confirmed ; there did not appear a reticulum, but a plexus of non-anastomosing delicate fibres. However, Apathy was now to obtain an ardent supporter in a German physiologist, A.* Bethe, who declared himself a believer in his theories. This scientist, who had been experimenting especially with the methylene staining method and had invented a good fixative for the preparations resulting from it, devoted himself to the question of the occurrence of fibrils in the ganglion cells of the higher animals, a question which Max Schultze had previously done a good deal to elucidate. Bethe invented a new method of staining these fibrils, which he represents as non-anastomosing among themselves as a rule. Bethe very vigorously championed the main article in Apdthy’s theory, both in lectures and essays, and also in a large book on the subject ; he was a particularly strong upholder of the idea that the nerve-fibrils are the true conducting element in the nerve-tissue and of their independence. Bethe became one of the chief opponents of the neurone theory and, generally speaking, of the school of ideas of which Cajal was the leading force. Bethe exercised for a time a not inconsiderable influence upon many of the Neurologists’ party. Some of the results he obtained by his experiments were especially effective in strengthening his position as a supporter of Apdthy’s theory. Bethe had removed certain groups of ganglion cells from some live crabs (Carcinus menas), but could nevertheless observe reflex activity in the processes of the nerve-cells 1908. | Minute Structure of the Nervous System. 433 helonging to them. Thus, he alleged he had hereby clearly shown the small importance the ganglion cells have as regards the activity of the nerves. I have myself tried to carry out these experimenis of Bethe’s on my own account, but have come to the conclusion that it is practically impossible to operate with such precision on the semi-transparent and soft ganglions of the living animal (Carcinus menas) that a removal of some particular groups of cells will be certain to result. Under such conditions I consequently regard it as exceedingly unsafe to draw the conclusions Bethe has done. The question is fundamentally of such moment that the utmost caution is necessary in arriving at any conclusions at all. I, at any rate, after the experience [ have derived from my experiments, must call in question the certainty and accuracy of what he has stated as his conclusions. Now, however, a new epoch of neurological investigation was to be inaugurated, for Cajal and Bielschowsky almost simultaneously invented methods very similar to one another for staining the fibrils in the nerve-cells and their processes by means of a silver solution. That not only confirmed the existence of the neurofibrils seen by Apathy, but added very materially to our knowledge, especially of the nervous system of the higher animals, the vertebrata. Thanks to these methods, it was shown by the inventors themselves and by other investigators, who had taken up the study of these problems, that everywhere in the nerve-cells and their processes there exist fibrils which belong to the cell substance and are very early developed in it. Cajal proved, too, that these fibrils form reticula in the cells, and that they increase or decrease in thickness according to the state in which the animal is, different in health and sickness (e.g., rabies), etc. ; he also pointed out that in the cell processes these fibrils always remain within their substance, and are not, as Apathy asserts, capable of emerging from it. Cajal asserted also that the fibrils do not anastomose outside the neurones, and specially not in the central substance and in the Punktsubstanz. This important point, which entirely agrees with the results I obtained before by the methylene method, was confirmed by the new researches I carried out with Cajal’s new fibril staining method which gives a very full and distinct colouring to even the most delicate ramifications of the nerve-fibres (fig. 11). Now these form everywhere, even in the most successfully stained parts, in the Punktsubstanz of the invertebrata as well as in the grey substance of the vertebrata, not anastomosing reticula, but only twistwork (plexus); divisions in the fibres occur, but no network is visible. I call attention to this fact once more, since the question has an important 434 Prof. G. Retzius. Principles of the [June 15, SLE GAING = BESS Sears == ZL ic ee F a= eo Be POT) AES 5 L LT” Sa Oi Sea ‘it 7, AY fF ay x Sa Z =. Cs Vy LZ LESS? : BAD 3 oe os e