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PROCEEDINGS

OF THE

ROYAL SOCIETY OF LONDON |

Serizes B

CONTAINING PAPERS OF A BIOLOGICAL CHARACTER

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PRINTED FoR THE ROYAL SOCIETY anp SOoLp By
HARRISON AND SONS, ST. MARTIN’S LANE,
PRINTERS IN ORDINARY TO HIS MAJESTY.

NovEMBER, 1909,

LONDON :
HARRISON AND SONS, PRINTERS IN ORDINARY TO HIS MAJUSTY,

ST. MARTIN’S LANE.

CONTENTS.

SERIES B. VOL, LXXXI

No. B 545.—March 13, 1909.
PAGH
Addvess of the President, Lord Rayleigh, O.M., D.C.L., at the Anniversary Meeting
Oi NOUS MINS SUIS) th padcesecbodcebSEno Goer On ROHEL DOE GEC CECB EEE Re Eee Tee EE OnE r er enTnE 1

A Trypanosome from Zanzibar. By Colonel Sir David Bruce, C.B., M.B., F.R.S.,
D.Sc., LL.D., Army Medical Service, and Captains A. E. Hamerton, D.S.O.,
and H. R. Bateman, Royal Army Medical Corps. (Plates 1 and 2) ............... 14

A Summary of further Researches on the Etiology of Endemic Goitre. By Robert
McCarrison, M.B., B.Ch., Captain, Indian Medical Service. Communicated by
Maj oUmonaldwhossy Cabse HaRiSis inset chcesesnaseacsecceceres scgednscédaceeccsersce Rey cep 31

The Proportion of the Sexes produced by Whites and Coloured Peoples in Cuba.
By Walter Heape, M.A., F.R.S., Trinity College, Cambridge. (Abstract) ...... 32

Electrolytes and Colloids.—The Physical State of Gluten. By Prof. T. B. Wood
emael Nive, IB B Tanne lre. 1.) RSH Searicate der aee Ha en dHe Sentence e sed oe eReee Etna nn CAScaneey BE ReaEeeronce 38

The Colours and Pigments of Flowers with Special Reference to Genetics. By
M. Wheldale. Communicated by W. Bateson, F.R.S. .............cccecsceseeeeeeeenes 44

An Experimental Estimation of the Theory of Ancestral Contributions in Heredity.
By A. D. Darbishire, Demonstrator of Zoology in the Royal College of Science,
London. Communicated by J. Bretland Farmer, F.R.S. ...........csccccseeeeeeeeeees 61

The Action of the Venom of Sepedon hemachates of South Africa. By Sir Thomas
R. Fraser, M.D., LL.D., Sc.D., F.R.S., Professor of Materia Medica, University
of Edinburgh; and James A. Gunn, M.A., BSc. M.D., Assistant in the
Materia Medica Department, University of Edinburgh. (Abstract)............... 80

No. B 546.—April 5, 1909.

The Selective Permeability of the Coverings of the Seeds of Hordewm vulgare. By
Adrian J. Brown, Professor of Brewing in the University of Birmingham.
Communicated by Prof. H. E. Armstrong, F.R.S. ....cc..0...ccceeccsecseccssesscoeceoes 82

The Origin of Osmotic Effects. II.—Differential Septa. By Henry EH. Armstrong,
TOTES, - cof cebu Cae BRROBHeticot LODCEDecda: SGetIe TERA OS SeEEE Ene Cc nne on JoCROEOEGDE ic Sateen acer rane se 94

iv

On the Determination of a Coefficient by which the Rate of Diffusion of Stain and
other Substances into Living Cells can be Measured, and by which Bacteria and
other Cells may be Differentiated. By Hugh C. Ross, late Surgeon R.N.,
Pathologist to the Royal Southern Hospital, Liverpool. Communicated by
MajoriRonalds Rossy Cab ehaha sean (labels) seesteeescear estes seieeteetec ee eeee eee eaeae

The Origin and Destiny of Cholesterol ia the Animal Organism. Part III.—The
Absorption of Cholesterol from the Food and its Appearance in the Blood. By
Charles Dorée, Lindley Student of the University of London, and J. A.
Gardner, Lecturer in Physiological Chemistry, University of London. Com-
muni cated aby Dy ravAweED SiWraillerENsenesaee eneeeeh eee cee reese eee eee eee EEE REe ee eee EERE

The Origin and Destiny of Cholesterol in the Animal Organism. Part [V.—The
Cholesterol Contents of Eggs and Chicks. By G. W. Ellis and J. A. Gardner,
Lecturer on Physiological Chemistry, University of London. Communicated
by Dr vASiD: Wialler, BRS. sansidassdesels oncolee setiechice sar ateten (ease eee ene ee eee

On the Cross-breeding of Two Races of the Moth Acidalia virgularia. By Louis B.
Prout, F.E.S., and A. Bacot, F.E.S. Communicated by Leonard Hill, F.R.S. ...

The Nerves of the Atrio-ventricular Bundle. By J. Gordon Wilson, M.A.,
M.B. (Edin.), Hull Laboratory of Anatomy, University of Chicago, Communi-
cabedsby: nih. IW). Mott, HAR Say (Blatesi4 6) smeeeenrtccen sian csteeer ee eisieeeeeticeae

No. B 547.—June 5, 1909.

The British Freshwater Phytoplankton, with Special Reference to the Desmid-
plankton and the Distribution of British Desmids. By W. West, F.L.8., and
G. 8. West, M.A., D.Sc. F.L.S. Communicated by D. H. Scott, P.R.S. .........

On the Presence of Hxem-agglutinins, Haem-opsonins, and Heemolysins in the Blood
obtained from Infections and Non-Infectious Diseases in Man. (Second
Report.) By Leonard 8. Dudgeon, F.R.C.P. Lond. Communicated by Dr.
1 an Vea Ko) 4 ea) de as Wage aaaanpmnina secaenasdopadéaacoedonescpddcadacbeabonoshGuonunoenadJoadoagecone

The Theory of Ancestral Contributions in Heredity. By Karl Pearson, I.R.S.......

On the Ancestral Gametic Correlations of a Mendelian Population mating at
IivewaKoloyonly Is\7 1ewdl Temes oI, WSIRASY ocnosconusuossaontonecas usaecaccunconcuceoecoonbadcoures

The Origin and Destiny of Cholestero] in the Animal Organism. Part V.—On the
Inhibitory Action of the Sera of Rabbits fed on Diets containing Varying
Amounts of Cholesterol on the Heemolysis of Blood by Saponin. By Mary T.
Fraser and J. A. Gardner. Communicated by Dr. A. D. Waller, F.R.S. .........

The Influence of Glucosides on the Growth of Acid-fast Bacilli, with a New Method
of Isolating Human Tubercle Bacilli directly from Tuberculous Material
contaminated with other Micro-organisms. (Preliminary Note.) By F. W.
Dore, Oloyovbanranraveznrerel ony Weorimenyel Tall IN IRISH Geacdebarossontenoessconuccncosrossacos

No. B 548.—July 8, 1909.

Reciprocal Innervation of Antagonistic Muscles. Fourteenth Note——On Double
Reciprocal Innervation. By C. 8. Sherrington, D.Se., PUAR.S. .....c.ccccceeeneeeeeees

PAGE

97

109

165

207

219

248

249

The Properties of Colloidal Systems. 1I.—The Osmotic Pressure of Congo-red and
of some other Dyes. By W. M. Bayliss, F.R.S., Physiological Laboratory,
lWithnersitysCollesew Mon G ony. 2-H. tatertet. iualecusdh asso eecerds sheeecsedes fecdes seamdenton lease

Some Effects of Nitrogen-fixing Bacteria on the Growth of Non-Leguminous Plants.
By W. B. Bottomley, M.A., Professor of Botany in King’s College, London.
Communicated by Prof. J. Reynolds Green, F.R.S. ............cccsceesececnenececeeees

The Intracranial Vascular System of Sphenodon. By Arthur Dendy, F-.R.S.
(GAs Geach) leer prema smreesnes orem. seeccyeeet aires ve sa biciaides Se ctiaculsk sans ols sean cieasienisinctanle sae

The Variations in the Pressure and Composition’ of the Blood in Cholera ; and their
Bearing on the Success of Hypertonic Saline Transfusion in its Treatment. By
Leonard Rogers, M.D., F.R.C.P., F.R.C.S., LM.S., Professor of Pathology,
Calcutta. Communicated by Sir T. Lauder Brunton, Bart., F.R.S. .............-.

The Effect of Heat upon the Electrical State of Living Tissues. By A. D. Waller,
NWLID., I IRASS Peep bbedcosasoognene Poradoaoic OBan GC NnCe HB gREC SeocrqeC HORT SEccU Rae Rbe dren eeee trac

The Incidence of Cancer in Mice of Known Age. By E. F. Bashford, M.D., and
J. A. Murray, M.D., B.Sc., Imperial Cancer Research Fund. Communicated
Ixy Tiron, dls Ise J Bima hostels SEG, UtaSia Bicdapododbodedodbe CHO SeUEONedsonenenerepeebone peesosage6

The Electrical Reactions of certain Bacteria, and an Application in the Detection of
Tubercle Bacilli in Urine by means of an Electric Current. By Charles Russ,
MEBs Communmicaved pyeAs = Waller WED: BM IRIS. ley.ccscacSessseccnsstersedearees

No. B 549.—October 9, 1909.

Trypanosoma ingens, vn. sp. By Colonel Sir David Bruce, C.B., F.R.S., Army
Medical Service ; Captains A. E. Hamerton, D.S.O., and H. R. Bateman, Royal
Army Medical Corps; and, Captain F. P. Mackie, Indian Medical Service.
(Gea) repeater race cece eer sameicetene rac cir ohiaalte haem ae oisin sacha omatee Heed aneslebih aisle ae

The Effect of the Injection of Intracellular Constituents of Bacteria (Bacterial
Hudotoxins) on the Opsonising Action of the Serum of Healthy Rabbits. By
R. Tanner Hewlett, M.D. Communicated by Prof. W. D. Halliburton, F.R.S.

The Alcoholic Ferment of Yeast-juice. Part [V.—The Fermentation of Glucose,
Mannose, and Fructose by Yeast-juice. By Arthur Harden, F.R.S., and
Wo dls: WORE ¢ caboebGuseocbodon boas Sood dpnocbo be ce enEccEGnEIe te meade conrcecaancerensra perrare

The Discovery of a Remedy for Malignant Jaundice in the Dog and for Redwater
in Cattle. By George H. F. Nuttall, M.D., Ph.D., Sc.D., F-RS., Quick
Professor of Biology in the University of Cambridge ; and Seymour Hadwen,
D.V.Sci. (McGill) of the Department of Agriculture, Canada ..............s.e1eeeeee

The Vacuolation of the Blood-platelets: an Experimental Proof of their Cellular
Nature. By H.C. Ross, late Surgeon Royal Navy. Communicated by Prof.
CE Sm Sbers i otom whee Somrmeeeener eco). cio usaas qeitco cis aseine etrtneteceamemiectecoeies ic nonsense sacs

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 W. b. Fry, Captain R.A.M.C. Communicated by J. Rose Bradford,
HYIP Co mE Cans tactarcrtct ac eaters TEC IGPORTe els al ax sie (aN rE alee ate Rte Maca char sible lcloreibatadiol eee

PAGE

269

310

314

336

348

351

vl

Observations on the Urine in Chronic Disease of the Pancreas. By P. J. Cammidge
(M.D. Lond.), Communicated by Sir Victor Horsley, FBS. .........ccscccneeneees

A Method of Estimating the Total Volume of Blood contained in the Living Body.
By J. O. Wakelin Barratt, M.D., D.Sc. Lond., and Warrington Yorke, M.D.
Liverpool. Communicated by Prof. Sherrington, F.R.S. ............cscceeseeeeeseeres

Preliminary Note on Trypanosoma eberthi (Kent) (= Spirocheta eberthi, Lithe) and
some other Parasitic Forms from the Intestine of the Fowl. By C. H. Martin,
B.A., Demonstrator of Zoology in Glasgow University, and Muriel Robertson,
M.A., Carnegie Research Fellow, Assistant to the Professor of Protozoology in
the University of London. Cormunicated by Prof. J. Graham Kerr, F.R.S.
(Plate '8) edagehetanes «dren atictesalonatieanas tacos ee heuer Senetoe Gets aleMt nis acetate ae atte ae een

The Possible Ancestors of the Horses living under Domestication. By J. C. Ewart,
MIDS, Jslesh, Winiensiny Oe Wieclimjomomla, (CAV OSAVCLE) soousngonhonsodooasacnonosononnenone

Hillhousia mirabilis, a Giant Sulphur Bacterium. By G. 8. West, M.A., D.Sc.,
F.L.S., and B. M. Griffiths, B.Sc. Communicated by J. Bretland Farmer, F.R.S.
(Elabeng) Rae suse onccegiee sere emncitty babs eso Jo edag-/ad Joudoseace coos aosgDERSCEas natmHoEOr oa.

No. B 550.—October 27, 1909.

The Development of Zrypanosoma gambiense in Glossina palpalis. By Colonel
Sir David Bruce, C.B., F.R.S., Army Medical Service; Captains A. H.
Hamerton, D.8.0., and H. R. Bateman, Royal Army Medical Corps; and
Captain F. P. Mackie, Indian Medical Service. (Sleeping Sickness Commission
OH ne eKonvenl Stoweicing, WoL) (eens; WO anal 111) «co nsccooananancnaonosoabonasananososoAa:

A Note on the Occurrence of a Trypanosome in the African Elephant. By Colonel
Sir David Bruce, C.B, F.R.S., Army Medical Service; Captains A. H.
Hamerton, D.S.0., and H. R. Bateman, Royal Army Medical Corps; and
Captain F. P. Mackie, Indian Medical Service. (Sleeping Sickness Commission

Of thesRoyalsSociety, 19082) (Plate l2) i reccrerecerepteteseceee reece neers ne keec eee eeseee
The Ferments and Latent Life of Resting Seeds. By Jean White, M.Sc., Victorian
Government Research Scholar. Communicated by D. H. Scott, F.R.S. .........

Croontan Lucrurn:—The Functions of the Pituitary Body. By E. A. Schafer, F.R.S.

On the Occurrence of Protandric Hermaphroditism in the Molluse Crepidula
fornicata. By J. H. Orton, A.R.C.S., Marshall Scholar in the Royal College of
Science, London (Imperial College of Science and Technology). Communicated
by. Brot. Au-thumsDen cya ARS Weemeee sect erioerccescte se cee riasemee eee eet caterer eee

No. B 551.—November 23, 1909.

The Hlasticity of Rubber Balloons and Hollow Viscera. By Prof. W. A. Osborne,
with a Note by W. Sutherland. Communicated by Prof. J. N. Langley,
HERISS a teac dati saiineckidenite asses chtec aeOweae eater oats case hier siete: abate sesh acta atten tiset mente

The Modes of Division of Spcrocheta recurrentis and S. duttoni as observed in the
Living Organisms. By H. B. Fantham, D.Sc. Lond., .Christ’s College,
Cambridge, Assistant to the Quick Professor of Biology in the University, and
Annie Porter, B.Sc. Lond., University College, London. Communicated by
Prot Gude We. Nuttally BARS 2.2, sea ssceneamannaelrh sects asee aan e ns enoebor teenie acticin eter

381

385

392

398

405

414

417
442

468

485

Vil

PAGE
The Origin and Destiny of Cholesterol in the Animal Organism. Part VI.—The
Excretion of Cholesterol by the Cat. By G. W. Ellis and J. A. Gardner,
Lecturer in Physiological Chemistry, University of London. Communicated
joy 1Dye, ZA, ID, Wwyeillllese, T8CIRUIS.“gosocousacecnesasGopbooon dadeporeanadansacnsevonadonasbooseadsoone 505

On the Supposed Presence of Carbon Monoxide in Normal Blood and in the Blood
of Animals aneesthetised with Chloroform. By G. A. Buckmaster and J. A.

Gardner. Communicated by A. D. Waller, M.D., FLR.S. .......ccccceceseesee senses 515
The Hexosephosphate formed by Yeast-juice from Hexose and Phosphate. By

W. J. Young (Biochemical Laboratory of the Lister Institute of Preventive

Medicine). Communicated by A. Harden, F.R-S. ...........cs.csscecesscsececcssoeoeees 528
The Comparative Power of Alcohol, Ether, and Chloroform as measured by their

Action upon Isolated Muscle. By Augustus D. Waller, M.D., F.R.S. ............ 545
Studies on the Structure and Affinities of Cretaceous Plants. By Marie C. Stopes,

Ph.D., D.Sc., F.L.8., Lecturer in Palezeobotany, Manchester University, and

K. Fujii, Ph.D., Assistant Professor of Botany, Imperial University, Tokio.

Commumicarediby Dr MEL Scot hak Ss CAdustract)seeueeseneceteecasesccssneanieeer 559
OBITUARY NOTICES OF FELLOWS DECHASED ............ceccccsececsscneceees i

ee oe
Je a ie
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PROCEEDINGS OF

THE ROYAL SOCIETY.

Szorron B.—BrotogicAL SCIENCES,

Address of the President, Lord Rayleigh, O.M., D.C.L., at the
Annwersary Meeting on November 30, 1908.

Since the last Anniversary the Society has sustained the loss of eighteen
Fellows and four Foreign Members.

The deceased Fellows are :—

The Right Hon. Lord Kelvin, died December 17, 1907.
Sir Alfred Baring Garrod, died December 28, 1907.
Robert Lewis John Ellery, died January 14, 1908.
Prof. James Bell Pettigrew, died January 31, 1908.
Wilham Ashwell Shenstone, died February 3, 1908.
Sir John Denis Macdonald, died February 7, 1908.
Lieutenant-General Sir Richard Strachey, died February 12, 1908.
Dr. William Edward Wilson, died March 6, 1908.

Dr. Henry Clifton Sorby, died March 9, 1908.

Sir John Eliot, died March 17, 1908.

The Duke of Devonshire, died March 24, 1908.

Dr. James Bell, died March 31, 1908.

Colonel Andrew Wilson Baird, died April 2, 1908.

Sir John Evans, died May 31, 1908.

Lord Blythswood, died July 8, 1908.

Arthur Lister, died July 20, 1908.

The Earl of Rosse, died August 29, 1908.

Prof. William Edward Ayrton, died November 8, 1908.
VOL. LXXXI.—B.

2 Annwersary Address by Lord Rayleigh. __[Nov. 30,

The deceased Foreign Members are :—

Pierre Jules César Janssen, died December 23, 1907.
Franz von Leydig, died April, 1908.

Henri Becquerel, died August 25, 1908.

Eleuthére Elie Nicolas Mascart, died August 26, 1908.

The list of deaths this year is exceptionally heavy, and includes the name
of one of the most eminent scientific men of our generation, who occupied
the Presidency of this Society from 1890 to 1895—I refer, of course, to
Lord Kelvin.

We are fortunate in having secured for our ‘ Proceedings’ a review of
Kelvin’s life and work, written by one who is especially well qualified for the
difficult task. I do not doubt that Professor Larmor is right in placing in the
forefront of that work those fundamental advances in Thermodynamics which
date from the middle of the last century. It was Kelvin who first grasped
the full scope of the principle known as the Second Law, a law which may
indeed well be considered to stand first in order of importance, regarded from
the point of view of man’s needs and opportunities. It would be futile to
attempt here a re-survey of the ground covered by Professor Larmor.

My acquaintance with Kelvin was limited, until about 1880, a time when
I was occupied with measurements relating to the electrical units, and
received much appreciated encouragement. From then onwards until his
death I enjoyed the privilege of intimacy and, needless to say, profited
continually from his conversation, as I had done before from his writings.
Our discussions did not always end in agreement, and I remember his
admitting that a certain amount of opposition was good for him. Such
discussions often invaded the officers’ meetings during the time that we were
colleagues, not always to the furtherance of the Society’s business. But
I must not linger over these reminiscences, interesting as they are to me.
We shall never see his like.

By the death of Sir Richard Strachey we have lost a man well known to
the senior Fellows, who served repeatedly upon the Council and whose
advice was always valued. He was a born administrator; and by his work
in India and afterwards at the Meteorological Office he rendered splendid
service,

Dr. Sorby’s researches extended over many fields, and in several of them
he was a pioneer. I suppose that his greatest achievement was the intro-
duction of the method in which thin slices of rock are examined under the
microscope. Among his many interesting observations are those upon the
retardation of freezing in capillary tubes. It appears that the walls exercise

1908. | Anniversary Address by Lord Rayleigh. 3

an influence at distances much greater than those usually regarded as
molecular—evidence apparently of structure upon an extended scale.
Dr. Sorby belonged to a class on whom England has special reason to
congratulate herself, men who pursue science unprofessionally. . The names
of Cavendish, Young, Joule, and Darwin at once suggest themselves.
It is to be feared that specialisation and the increasing cost and com-
plication of experimental appliances are having a prejudicial effect in this
regard. On the other hand, the amateur is not without advantages which
compensate to some extent. Certainly, no one who has the root of the matter
in him should be deterred by fears of such difficulties, and the example of
Sorby suffices to show how much is open to ingenuity unaided by elaborate
appliances.

The name of Sir John Evans must not pass without special notice. There
are few in recent years to whom the Society has been more indebted.
Many of our Fellows hardly realise how important and laborious are the
services rendered in the office of Treasurer. Evans’ scientific attainments, his
knowledge of the world and of business, and his personal characteristics
specially qualified him for office. An appreciation, signed by well-known
initials, has recently appeared in our ‘ Proceedings.’

On the Foreign List also the losses are heavy. We have especially to
condole with our colleagues in France upon the havoc caused by death within
the last year or two. Janssen, and Mascart, who was much missed at the
recent Electrical Conference, had reached a full age. But Becquerel was in the
full tide of life, and we had hoped to learn much more from him; as the
discoverer of radio-activity, he had opened up inquiries whose significance
seems ever on the increase. Science has lost a leader; his friends and the
world a charming personality.

During the time that I was Secretary, and so concerned with the passing
of mathematical papers through the Press, I was much struck with the
carelessness of authors in the arrangement of their manuscript. It is
frequently forgotten that a line of print in the ‘Transactions’ and in the:
new form of the ‘ Proceedings’ will hold much more than a line of ordinary
manuscript, unless, indeed, the handwriting is exceptionally small. Unless.
the authors’ indications were supplemented, it frequently occurred that.
several lines of print were occupied by what might equally well, and in my
judgment much better, be contained in one line. Even practised writers
would do well, when they regard their manuscript as complete so far as
regards matter and phrasing, to go over it again entirely from the point of
view of the printing. In this way much expense and space would be spared,
and the appearance of the printed page improved. Professor Larmor has.

B 2

4 Anniversary Address by Lord Rayleigh. [| Nov. 30,

drawn up a paper which has received the sanction of the Council and ‘is
appended to this Address, and will, it is hoped, be of service at once to
authors and to the Society. (The paper is printed in Series A only.)

Apart from questions of printing, the choice of symbols for representing
mathematical and physical quantities is of some importance, and is
embarrassed by varying usages, especially in different countries. <A
Committee now sitting is concerned with the selection of symbols for
electrical and magnetic quantities, but the question is really much wider.
One hesitates to suggest another international conference, and perhaps
something could be done by discussion in scientific newspapers. Obviously
some give and take would be necessary. When the arguments from
convenience are about balanced, appeal might be made to the authority of
distinguished men, especially of those who were pioneers in the definition
and use of the quantity to be represented. As an example of the difficulties
to be faced, I may instance the important case of a symbol for refractive
index. In English writings the symbol is usually uw, and on the Continent n.
By the early optical writers it would seem that no particular symbol was
appropriated. In 1815* Brewster has m. The earliest use of mw that I have
come across is by Sir John Herschel,t and the same symbol was used
by Coddington (1829) and by Hamilton (1830), both distinguished workers
in optics. On the other hand, x was employed by Fraunhofer (1815), and
his authority must be reckoned very high. As regards convenience, I should
suppose that the balance of advantage would incline to yp, since n is wanted
so frequently in other senses. Another case in which there may be
difficulties in obtaining a much to be desired uniformity is the symbol for
electrical resistance.

On a former occasion I indulged in comment upon the tendency of some
recent mathematics, which were doubtless understood as the mild grumbling
of an elderly man who does not like to see himself left too far behind. In
the same spirit I am inclined to complain of what seem unnecessary changes
in mathematical nomenclature. In my youth, by a natural extension of a
long established usage relative to equations, we spoke of the roots of a function,
meaning thereby those values of the argument which cause the function to
vanish. In many modern writings I read of the zeroes of a function in the
same sense. There may be reasons for this change; but the new expression
seems to need precaution in its use; otherwise we are led to such flowers of
speech as “zeroes with real part positive,’ which I recently came across.t
‘Phil. Trans.,’ 1815.

‘Phil. Trans.,’ 1821, p. 230.
‘Proc. Math. Soc.,’ vol. 31, p. 266.

++ +

1908. | Anmversary Address by Lord Rayleigh. 5

But though I may use a little my privilege of grumbling over details, I hope
I shall not be misunderstood as undervaluing the progress made in recent
years, which, indeed, seems to me to be very remarkable and satisfactory,
regarded from the scientific point of view. On the other hand I cannot help
feeling misgivings as to the suitability of the highly specialised mathematics
of the present day for a general intellectual training, and I hope that a careful
watch may be maintained to check, in good time, any evil tendencies that
may become apparent.

Among the notable advances of the present year is the liquefaction of
helium by Professor Onnes of Leiden. Itis but a few years since Sir J. Dewar
opened up a new field of temperature by his liquefaction of hydrogen, and
now a further extension is made which, if reckoned merely in difference of
temperature, may appear inconsiderable, but seen from the proper
thermodynamical standpoint is recognised to be far-reaching. The explora-
tion of this new field can hardly fail to afford valuable guidance for our
ideas concerning the general properties and constitution of matter. Professor
Onnes’ success is the reward of labours well directed and protracted over
many years.

The discovery and application by Rutherford and Geiger of an electrical
method of counting the number of «-particles from radio-active substances
constitutes an important step, and one that appears to afford better determina-
tions than hitherto of various fundamental quantities. It would be of interest
to learn what interpretation is put upon these results by those who still
desire to regard matter as homogeneous.

Another very interesting observation published during the year is that of
Hale upon the Zeeman effect in sun-spots, tending to show that the spots are
fields of intense magnetic force. Anything which promises a clue as to the
nature of these mysterious peculiarities of the solar surface is especially
welcome. Until we understand better than we do these solar processes, on
which our very existence depends, we may do well to cultivate a humbler
frame of mind than that indulged in by some of our colleagues.

A theoretical question of importance is raised by the observations of
Nordmann and Tikhoff showing a small chromatic displacement of the
phase of minimum brightness in the case of certain variable stars. The
absence of such an effect has been hitherto the principal argument on
the experimental side for assuming a velocity of propagation in vacuum
independent of frequency or wave-length. The tendency of the observations
would be to suggest a dispersion in the same direction as in ordinary
matter, but of almost infinitesimal amount, in view of the immense distances
over which the propagation takes place. Lebedew has pointed out that

6 Anmversary Address by Lord Rayleigh. |Nov. 30,

this conclusion may be evaded by assuming an asymmetry involving colour
in the process by which the variability is brought about, and he remarks
that although the dispersions indicated by Nordmann and Tikhoff are in the
‘same direction, the amounts calculated from the best available values of
ithe parallaxes differ in the ratio of 30 to 1. In view of this discrepancy and
of the extreme minuteness of the dispersion that would be indicated, the
probabilities seem at the moment to lie on the side of Lebedew’s explana-
tion ; doubtless further facts will be available in the near future.

I cannot abstain from including in the achievements of the year the
remarkable successes in mechanical flight attained by the brothers Wright,
although the interest is rather social and practical than purely scientific.
For many years, in fact ever since I became acquainted with the work
of Penaud and Wenham, I have leaned to the opinion that. flight was
possible as a feat. This question is now settled, and the tendency may
perhaps be to jump too quickly to the conclusion that what can be done
as a feat will soon be possible for the purposes of daily life. But there is
a very large gap to be bridged over; and the argument urged. by Professor
Newcomb and based on the principle of dynamical similarity, that the
difficulties must increase with the scale of the machines, goes far to
preclude the idea that regular ocean service will be conducted by flying
machines rather than by ships. But, as the history of science and invention
abundantly proves, it is rash to set limits. For special purposes, such as
exploration, we may expect to see flying machines in use before many years
have passed. ;

The Report of the National Physical Laboratory for the year again indicates
remarkable growth. The various new buildings, which have. been erected
and equipped during recent years at a-cost of about £33,000, are now
occupied; and the result is that both researches and test work can be carried
out with much greater ease and efficiency than previously. The Executive
Committee in charge of the Laboratory is indebted in the first instance to
H.M. Government, and then to the numerous friends whose assistance has
made this possible. At the same time, the needs for buildings are not nearly
satisfied. There has been during the year a very marked and important
growth in the demand by manufacturers and others for assistance in metal-
lurgical enquiries, which require investigations, frequently of a very complex
character; and with the present accommodation for much of the Metallur-
gical Department this demand is difficult to satisfy. Thanks in great
measure to the Goldsmiths’ Company, the chemical side of this department is
well provided for; but new buildings for the other branches of metallurgy
are au urgent want.

1908. | Anniversary Address by Lord Rayleigh. 7

The Report of the Treasury Committee of Inquiry referred to in the address
of last year was communicated by the Treasury to the Royal Society, with
the intimation that Their Lordships accept the recommendations of the Com-
mittee, and trust that the Royal Society may see their way to do the same.
In their reply the President and Council, with the concurrence and advice of
the Executive Committee of the Laboratory, expressed their readiness to use
their best endeavours to carry the Report into effect. The Report has since
been presented to Parliament.

The buildings of the Magnetic Observatory at Eskdalemuir are now
occupied ; but, unfortunately, difficulty has arisen in making the magneto-
graph rooms which are underground completely watertight, and the recording
apparatus is not yet properly installed.

The third and fourth volumes of ‘ Collected Researches’ of the Laboratory
have been published during the year, and testify to the vigorous scientific
activities of the staff. The third volume is occupied chiefly with the account
of the prolonged series of experiments on electric units carried out at the
Laboratory by Prof. Ayrton, Mr. Mather, Dr. Lowry, and Mr. Smith. These
researches proved of great value in the discussions at the International
Conference on Electric Units, for which recently the Society provided
accommodation and entertainment at the request of the Government.

The progress of the ‘ Royal Society Catalogue of Scientific Papers’ has
advanced a definite stage during the year, through the publication by the
Cambridge University Press of the Index Volume of Pure Mathematics for
the Nineteenth Century. Owing to the magnitude of the material to be
indexed in the several sciences, it has been necessary to adopt drastic
measures of compression, and the 40,000 entries involved in the present
section have thus been condensed into one royal octavo volume of some
700 pages. An essential element in this saving of bulk has been the
grouping of titles within each heading so as to avoid reprinting the leading
words. It was, perhaps, inevitable that this device would occasionally be
mistaken for an attempt at organic classification within the limits of the
main headings, which are substantially those of the yearly ‘International
Catalogue of Scientific Literature.’ This had, indeed, been foreseen in the
preface of the volume. As regards new actual sub-headings which have been
introduced occasionally, the Committee remark that “These minor classifica-
tions, being often made mechanically on the basis of the explicit mention of
the sub-heading, are not to be taken as exhaustive; cognate entries may be
found elsewhere under the same main heading. The unit of classification is
thus the complete numbered heading.”

The Committee of the Catalogue have indeed been fully conscious

8 Anniversary Address by Lord Rayleigh. __[ Nov. 30,

throughout of the difficulties of the task which they supervise; and it must be
gratifying to the Director of the Catalogue and his staff to have the support
of high authorities, not confined to this country, in their decision that in so
extensive an undertaking practical feasibility must be the aim rather than an
elusive theoretical perfection. One advantage, at any rate, will accrue from
bringing out a single volume well in advance, in that the Committee will be
able to profit in the future work from the experience they have acquired.

Through the kindness of Dr. Schuster I had the opportunity of submitting
to the Council, before the expiry of my term of office, a generous proposal
which he makes for instituting a fund of £1500, the interest of which is to
be applied to pay the travelling expenses of delegates of the Society to the
International Association of Academies. Dr. Schuster felt that the absence
of such a provision laid a burden upon delegates, and might operate to
limit the choice of the Society. I was empowered by the Council to convey
their cordial thanks to Dr. Schuster, and I have now the pleasure of making
his benefaction known to the Society at large.

In taking leave of the honourable office which I have occupied for three
years, I desire to thank the Society and especially my colleagues, the officers,
for the consideration which they have uniformly shown me. All the omens
indicate that the Society will be represented by one well versed in its
affairs, and whose scientific distinction and wide experience justify the
highest hopes for his tenure of the chair.

MEDALLISTS, 1908.

CopLEY MEDAL.

The Copley Medal is awarded to Dr. Alfred Russel Wallace, F.R.S.

It is now sixty years since this distinguished naturalist began his scientific
career. During this long period he has been unceasingly active in the
prosecution of natural history studies. As far back as 1848 he accompanied
the late Henry Walter Bates to the region of the Amazon, and remained
four years there, greatly enriching zoology and botany, and laying at the
same time the basis of that wide range of biological acquirement by which
all his writings have been characterised. From South America he passed
to the Malay Archipelago and spent there some eight fruitful years. It was
during his stay in that region that he matured those broad views regarding
the geographical distribution of plants and animals which on his return to
this country he was able to elaborate in his well-known classic volumes on

1908. | Annwersary Address by Lord Rayleigh. 9

that subject. It was there, too, amid the problems presented by the infinite
variety of tropical life, that he independently conceived the idea of the
theory of the origin of species by natural selection which Charles Darwin
had already been working out for years before. His claims to the admiration
of all men of science were recognised by the Royal Society forty years ago,
when, in 1868, a Royal Medal was awarded to him. Again, when in 1890,
the Darwin Medal was founded, he was chosen as its first recipient. He is
still full of mental activity and continues to enrich our literature with
contributions from his wide store of experience and reflection in the domain
of Natural History. As a crowning mark of the high estimation in which
the Royal Society holds his services to science, the Copley Medal is now
fittingly bestowed on him.

RUMFORD MEDAL.
The Rumford Medal is awarded to Prof. H. A. Lorentz, For. Mem. R.S.

Prof. Hendrik Antoon Lorentz, of Leiden, has been distinguished during
the last quarter of a century by his fundamental investigations in the
principles of the theory of radiation, especially in its electric aspect. His
earliest memoirs were concerned with the molecular equivalents which
obtain in the refractive (and dispersive) powers of different substances ;
in them he arrived at formule that still remain the accepted mode of
theoretical formulation of these phenomena. The main result, that
(v?—1)/(w?+2) is proportional jointly to the density of distribution of the
molecules, and to a function of the molecular free periods and the period of
the radiation in question, rests essentially only on the idea of propagation
in some type of elastic medium; and thus it was reached simultaneously,
along different special lines, by H. A. Lorentz originally from Helmholtz’s
form of Maxwell’s electric theory, and by L. Lorenz, of Copenhagen, from
a general idea of propagation after the manner of elastic solids.

The other advance in physical science with which Prof. Lorentz’s name
is most closely associated is one of greater precision, the molecular develop-
ment of Maxwell’s theory of electro-dynamics. This subject was never
entered upon by Maxwell himself, on the ground, probably, that the general
relations of the «ther, and in particular their dynamical bearings, offered
a definite field which must be fully probed and explored before the
uncertainties connected with molecular complexity became ripe for effective
detailed treatment. But the theoretical difficulties connected with the
simple law of the astronomical aberration of light, and particularly with
the entire absence of any effect of the Earth’s uniform motion in space on

10 Anmversary Address by Lord Rayleigh. _[Nov. 30,

terrestrial phenomena involving radiation, had more recently rendered this
problem urgent. Following on various purely optical papers on the
phenomena of moving bodies, Prof. Lorentz, in 1892, elaborated’ a general
molecular treatment in the memoir “La Théorie Electro-magnétique de
Maxwell, et son Application aux Corps Mouvants,” which appeared in the
‘ Archives Néerlandaises, and contains substantially the main root ideas of
the subject. In 1905 it was re-expounded with further development in a
tract entitled “Versuch einer Theorie der Electrischen und Optischen
Erscheinungen in Bewegten Korpern,” the main feature being the elimina-
tion of the. dynamical element in the previous discussion in favour of a
formulation by a system of abstract equations, after the way first set out by:
Maxwell himself as a summary of his final definite results as distinct from
the formative ideas underlying them, and afterwards brought into prominence
by the expositions of Heaviside and Hertz.

By these writings Prof. Lorentz has taken a predominant place in the
modern evolution of electric and optical theory. He has since been active
in special applications, of which the best known has been his theoretical
prediction of the physical features of the alteration of the lines of the
spectrum in a magnetic field, which had been discovered and has since been
developed by his colleague Zeeman.

Roya MEDALS.

The assent of His Majesty the King, our Patron, has been graciously signified
to the following awards of the Medals presented annually by him to the Society.

A Royal Medal to Prof. John Milne, F.R.S., for his work on Seismology.
In 1875, Dr. Milne accepted the position of Professor at Tokyo, which
was offered to him by the Imperial Government of Japan. His attention
was almost immediately attracted to the study of earthquakes, and he was
led to design new forms of construction for buildings and engineering
structures with a view to resisting the destructive effects of shocks. His
suggestions have been largely adopted, and his designs have been very
successful for the end in view. Incidentally he studied the vibrations of
locomotives, and showed how to obtain a more exact balancing of the moving
parts, and thus to secure smoother running and a saving of fuel. Here
again his suggestions were accepted, and his work was recognised by the
Institution of Civil Engineers.

He next devoted himself to the study of artificial shocks produced by the
explosion of dynamite in borings. He then studied actual shocks as
observed at nine stations connected by telegraph wires. A seismic study of
Tokyo, and subsequently of the whole of northern Japan, followed. In this

1908. | Anmnwversary Address by Lord Rayleigh. 3

latter work he relied on reports from 50 stations. The Government then
took up the matter, increased his 50 stations to nearly 1000, and founded a
Chair of Seismology for Mr. Milne. It is due to his energy, skill, and know-
ledge that the Japanese School of Seismology stands as the first in the world.

While still in Japan he attempted to obtain international co-operation
through the representatives of 13 nationalities. This first effort failed;
but subsequently, on his return to England in 1895, he succeeded, and
reports are now received by him from some 200 stations furnished with
trustworthy instruments, and scattered all over the world. On his return to
England he at once established his own observatory at Shide, in the Isle of
Wight, and the work has been carried on continuously from that time up to
now, mainly by his own industry and resources.

In Great Britain we owe everything in seismology to the British Associa-
tion. Their Committee was founded in 1880, and since that date Milne has
been the moving spirit in the long career of its activity. He has been the
author of 29 annual reports, and these form in effect a history of the
advance of seismology since it has been recognised as a definite branch of
science.

The knowledge which we have now acquired as to the internal constitution
of the earth is more due to Milne than to any other man.

The work of Dr. Henry Head, F.R.S., on which is founded the award of
the other Royal Medal, forms a connected series of researches on the Nervous
System (made partly in conjunction with Campbell, Rivers, Sherren, and
Thompson), published for the most part in ‘ Brain’ at various times since 1893
up to the present date, and constituting one of the most original and
important contributions to neurological science of recent times.

His first paper (1893), founded on minute and laborious clinical investiga-
tion, established in a more precise manner than had hitherto been done
the relations between the somatic and visceral systems of nerves. He
confirmed from the clinical side the experimental researches of Sherrington
on the distribution of the posterior roots of the spinal nerves.

An inquiry into the pathology of Herpes Zoster (1900), which he proved
abundantly to be due to inflammation of the posterior root ganglia, indicated
that the areas of referred pain in visceral disease corresponded specially with
the distribution of the fibres of the posterior roots subserving painful
cutaneous sensibility.

Continuing his investigations on the peripheral nerves, partly by experi-
ments on himself, in conjunction with Rivers, and partly by examination of
cases of accidental injuries to nerves, Head was led to formulate (1905) an

12 Anniversary Address by Lord Rayleigh. [Nov. 30,

entirely novel conception and differentiation of the functions of the peripheral
nerves, and of the paths for the respective forms of sensibility which they
convey—epicritic, protopathic, and deep sensibility. This is generally
regarded by neurologists as a research of quite exceptional originality
and ability.

Following the course of afferent impulses, Head next showed (1906) that
the sensory paths of the peripheral nerves at their first synaptic junction with
the spinal cord become re-arranged, and ascend in different relations in
certain definite tracts.

Davy MEDAL.

The Davy Medal is awarded to Prof. William Augustus Tilden, F.R.S.

_ The researches of Prof. Tilden extend into many domains. His work
on the specific heats of the elements in relation to their atomic weights,
described to the Society in the Bakerian Lecture for 1900 and in two later
papers published also in the ‘Philosophical Transactions, was of high
theoretical importance. The employment of liquid oxygen as an ordinary
laboratory reagent, rendered possible by the researches of Dewar and others,
enabled Prof. Tilden to test the validity of Dulong and Petit’s Law and
of Neumann’s Law over a much wider range of temperature than was
possible before, and gave a truer estimate of the nature of their validity.

In the region of organic chemistry, he has carried out important researches
on the terpenes, such as that on the hydrocarbons from Pinus sylvestris, on
terpin and terpinol, and on limettin.

In inorganic chemistry, his investigation on aqua regia and on nitrosyl
chloride are especially noteworthy. He has assisted much in clearing up
many points with regard to aqua regia about which obscurity remained.
His introduction of nitrogen peroxide and especially of nitrosyl chloride as
reagents has proved, in his own hands and in those of other workers, to be of
very high value.

Darwin MEDAL.

The Darwin Medal is awarded to Prof. August Weismann for his contribu-
tions to the study of evolution. He was one of the early supporters of the
doctrine of evolution by means of natural selection, and wrote in support of
the Darwinian theory in 1868. His great series of publications from that
date onward must always remain a monument of patient inquiry. In
forming an estimate of his work it does not seem essential that we should

- 1908. | Anniversary Address by Lord Rayleigh. 13

decide on the admissibility of his germ-plasm theory. It is in like manner
unimportant that he was, in certain respects, forestalled by Galton, and that
his own views have undergone changes. The fact remains that he has done
more than any other man to focus scientific attention on the mechanism of
inheritance. By denying the possibility of somatic inheritance, he has
compelled the world to look at this question with a closeness of criticism that
is wanting in all earlier inquiries. In the opinion of what is perhaps the
majority of naturalists, he has achieved much more than this—he has
convinced them that the solution of the problem of evolution must be sought
along the lines of his doctrine of germinal continuity. Thus the preformist’s
point of view, for which he has done so much, forms the basis on which
Mendelians and Mutationists are at work.

Weismann’s work was highly estimated by Mr. Darwin. Thus he writes,
in 1875 (‘More Letters, i, 356), of Weismann’s paper on Seasonal
Dimorphism: “No one has done so much as you on this important subject,
i.e., On the causes of variation.” Again (‘Life and Letters, iii, 198): “I
have been profoundly interested by your essay on ‘ Amblystoma,’ and think
you have removed a great stumbling block in the way of evolution.” And,
once more, in January, 1877 (‘Life and Letters, i, 231), Darwin wrote of
Weismann’s ‘Studien zur Descendenzlehre’: “They have excited my interest
and admiration in the highest degree, and whichever I think of last seems to
me the most valuable.”

HuGHEs MEDAL.
The Hughes Medal is awarded to Prof. Eugen Goldstein.

Prof. Goldstein was one of the early workers on the modern detailed
investigation of the electric discharge in rarefied gases, and by long continued
researches has contributed substantially to the systematic analysis of the
complex actions presenting themselves in that field. Of these researches may
be mentioned his observations of the effect of magnetic force on striations, of
the phosphorescence produced by the cathode rays, and of the reflection of
cathode rays.

By his discovery of the so-called Kanal-Strahlen, or positive rays, he has
detected an essential feature of the phenomenon, which, in his own hands
and in those of other workers, has already thrown much needed light on the
atomic transformations that are involved.

14

A Trypanosome from Zanzbar.

By Colonel Sir Davin Bruce, C.B., M.B., F.R.S., D.Sc., LL.D., Army Medical
Service, and Captains A. E. Hamerroy, D.S.0., and H. R. BaTEmAn,
Royal Army Medical Corps.

(Received September 18,—Read November 26, 1908.)
(From the Laboratory of the Royal Army Medical College, London.)
[Puatres 1 anp 2.]|

About the middle of April, 1908, Dr. J. Rose Bradford, F.R.S., had handed
over to him by Dr. Edington, F.R.S.E., a rabbit whose blood contained
a trypanosome. Dr. Edington stated that he had inoculated the rabbit with
blood from a horse he found at Zanzibar suffering from some obscure disease.
This rabbit was handed over to one of us (D. B.) by Dr. Bradford for the
purpose of keeping the strain alive and, if possible, identifying the species of
trypanosoma.

The following notes have since been received from Dr. Edington. The
trypanosome was found at Zanzibar, where no trypanosome has formerly
been known. It occurred in a horse in a stable among others, of which none
were infected. The animal was old, and had been many years in the place.
At death the symptoms were like those in surra and nagana, but the spleen
was not enlarged, nor was it coloured abnormally. The usual cedema was
apparent and most marked in the sheath, up the abdomen, in the chest, and
down the posterior limbs.

Dr. Edington inoculated a horse, an ox, and a goat successfully. The
disease ran a sub-acute form in the original horse, but in the inoculated one
it seemed rather more acute. Inoculated on February 18, trypanosomes
were seen in its blood on the 25th, and by March 1 the sheath was
swollen. There was no real fever (102°2 F.) until February 28, so that in
this case the appearance of parasites preceded the fever. On March 7 it had
greatly recovered, cedema had subsided, and the weakness of the preceding
few days was recovered from. Dr. Edington left on March 8, and fears the
animal was destroyed, as they had no further vote for funds for food, ete.

A young ox, inoculated on February 15, showed trypanosomes on the 27th.
It had fever fairly high, but had recovered before he left, and trypanosomes
were exceedingly few. A goat showed high fever, but its blood never showed
trypanosomes at any time, although Dr. Edington hunted with very great
thoroughness.

A Trypanosome from Zanubar. 15

Two rabbits were inoculated, one subcutaneously and one intraperitoneally.
The former was sent to Dr. Mesnil from Marseilles, but it has shown
nothing. It was twice inoculated with big doses, one from a horse and one
from an ox. The other Dr. Edington handed over to Dr. Bradford, and from
this rabbit the trypanosome under consideration was obtained and studied.

On examining the rabbit’s blood, the trypanosome was found to be a small
one, with poorly-developed undulating membrane, and no free flagellum.
The average length was only 13°5 microns.

Although it is impossible in some cases to name the trypanosomes from
their shape and size alone, still it is evident that a trypanosome of this size,
with no free flagellum, cannot be Trypanosoma brucei, evansi, gambiense, or
several other species which need not be enumerated. The names of such
small trypanosomes as Trypanosoma nanum (Laveran), Trypanosoma congolense
(Broden), or Trypanosoma dimorphon (Dutton and Todd), at once occur to.
the mind.

No doubt the tendency in naming these hematozoa is to multiply
unnecessarily the number of species. But, on the other hand, it is just as
great a mistake to lump too many species together, as has been done. If
there is some well-marked difference in two trypanosomes, even if alike in
shape, such as their power of setting up disease in certain animals, their
mode of spreading from the sick to the healthy—it may be in one by tsetse
flies, in another by stomoxys, or tabanus, or by other means—then, naturally,
it is of great practical use to distinguish them by different specific names.

Again, it might be argued, that if two trypanosomes were different
morphologically, but had the same effect on animals, the same distribution
and the same carrier, then the two varieties for practical purposes might be
included in the same species.

For example, when we have to do with Zrypanosoma gambiense we at once
know that man is susceptible, that the carrier is Glossina palpalis, and that
we must keep ourselves out of the area of distribution of this fly if we
would escape infection. Theories in regard to the spread of sleeping
sickness by mosquitoes, stomoxys, fleas, sexual intercourse, and such like,
may, for practical purposes, be ignored. If it is Trypanosoma brucei, then
we know man is not susceptible, but that we must keep our horses, cattle,
and dogs out of the area of distribution of Glossina morsitans.

The three most important questions to be borne in mind, in classifying
trypanosomes, are, what animals are they capable of infecting, the gravity of
the infection, and, thirdly, what is the carrier? To these may be added
the morphology of the trypanosome, its cultural characteristics, if any, and,
if possible, cross-inoculation experiments. If these several facts could be

16 Sir D. Bruce and Capts. Hamerton and Bateman. [Sept. 18,

set down for each trypanosome encountered in Africa, then some classifica-
tion of the African species might be attempted. But it is only for a few
species, such as Trypanosoma gambiense and Trypanosoma brucei, that we
have all these data. Take, for example, the case of Trypanosoma congolense
{Broden) and Trypanosoma dimorphon (Dutton and Todd)—most important
trypanosome diseases. Laveran thinks they are distinct on account of a
cross-inoculation experiment, but Broden himself, Rodhain, and Dutton and
Todd all seem to lean to their really being one and the same species.
With the data at our disposal at present it is impossible to come to a definite
decision.

At the present time the classification of the pathogenic trypanosomes is in
a state of chaos, and we have no desire to add to the confusion. Neverthe-
less, we think it will be well to give a description of Dr. Edington’s
trypanosome, as far as we have been able to study it, in view of the fact that
we are starting at once for Uganda to continue the investigation of sleeping
sickness.

MoreHoiocy oF Dr. Epineron’s TRYPANOSOME.

A. Ling, unstained.

Dr. Edington’s trypanosome in the fresh condition, as seen in a drop of
blood from an infected guinea-pig or rat, appears short and stumpy in
outline, about twice the diameter of the red blood corpuscles, among which
it slowly moves, with, as a rule, its rapidly-vibrating flagellum in front. The
posterior or non-flagellar extremity appears blunt and rounded off abruptly,
while the anterior tapers off to a fine point. In the fresh preparation the
undulating membrane is not much in evidence, though sometimes it can be
seen thrown into waves. The contents of the cell are homogeneous, except
for a small refractile body at the posterior extremity, which is evidently the
micro-nucleus.

B. Fixed and stained.

Method of staining—The method used for fixing and staining the
trypanosomes is usually as follows. The blood-film while still moist is
exposed to the vapour of a 4-per-cent. solution of osmic acid in distilled
water, to which a drop of glacial acetic acid has been added, for 45 seconds.
The cover-glass is then transferred to absolute alcohol for from five minutes
to half an hour. It is then passed through grades of alcohol from 80 per ceut.
to 10 per cent. in distilled water. Twenty-five drops of Giemsa’s stock
stain (Griibler’s) are now mixed with 29 c.c. of distilled water. The films
are placed in this, face downwards, for 8 to 12 hours, then washed in distilled
water, and rinsed quickly in solution of orange tannin (orange G. 1 per cent.,

1908. | A Trypanosome from Zanzibar 17

tannin 5 per cent., in distilled water). When sufficiently decolorised, the
films are washed in distilled water, dehydrated by passing through acetone,
cleared in xylol, and mounted in canada balsam.

Dr. Edington’s trypanosome when stained in this way appears of a pale
puce colour with reddish-purple nucleus and micro-nucleus. The following
detailed description must be understood to refer to this trypanosome as
found in the blood of the white rat.

Length—lt is no easy matter to measure these small irregularly-shaped
bodies, and doubtless the method of measurement used will govern to some
extent the result. The method used by us is simply to draw a sharp outline
of the trypanosome by means of a Zeiss camera lucida, at a magnification of
2000 diameters, and then to measure along the middle line of the body by
means of a pair of fine compasses, the points of which are separated
2mm. Kach step the compass takes is therefore equal to 1 micron. Twenty
trypanosomes, taken as they come, are measured in this way in each
specimen, and an average of the 20 measurements taken. The following
table gives some of the results :—

Dr. Edington’s Trypanosome.

In microns.
| No. of experiment. Day a saree of
oer apa Average Maximum | Minimum
length length length
| 184, mouse ......... 9 Giemsa ............ 15°3 20-0 13°0
le GOh ratios ses soos. 30 Leishman ......... 132) i 1G) 10-0
eb adece cae scibiese's vs 30 Giemsaie.--2-.-..-- 13 °9 17-0 10-0
np tie WEeBSeaECEoOd hele 30 Methyl green ... 13 “4 18-0 10-0
26 nokia CECA eCOG 30 Giemsa ........... 15°0 18-0 13 °0
Reese seest ds 30 Leishman ......... 13°53 170 11°0
S84. rabbit... .0.2....: | 22 Griemsayreeesecoees 13-0 16-0 9-0
| Guinea-pig ......... 18 Pe ere ore 12°5 | 16-0 8-0
[PET GG dog es.cce eeu! eta ees He-O ee | 1G0) 2 =: |e 10-0
|
Average ...... 136 171 10-4

VOL. LXXXI.—B. Cc

18 Sir D. Bruce and Capts. Hamerton and Bateman. ([Sept. 18,

For purposes of comparison measurements of Trypanosoma dimorphon and
Trypanosoma congolense are given in the following tables :—

In microns.

No. of experiment. Diy Gi Method of
CREEL planing: Average Maximum | Minimum
length. length. length.
Trypanosoma dimorphon.
Mouse (Laveran and ? Giemsa ......... 13 °8 17-0 12°0
Mesnil

116, rat (Breinl) ...... 15 gy. pbooad066 13 8 16:0 11°0
aN pA Wanodaac 9 Leishman ...... 11°3 140 9-0

é tae) 220) 9 OUP 4 eee 126 15 ‘0 11-0
Dog (Harvey, Sierra ? Sale tNeaer 12 °3 15-0 9:0

Leone) |
Cow (Smith, Sierra ? Ol ehctie 12 °4 15:0 10:0
Leone)
Average ...... 12°5 15 °3 10°3
Trypanosoma congolense.

142, mouse ...........000 Uf Giemsa ......... 12 °8 150 10:0
143, mouse .............-- 5 Leishman ...... 11°5 14-0 10°0
VG 2 rat ghccdesmsemecaeacee 11 Galemsayeeeecene 11 °6 15-0 9-0
ae er ee eth el 11 ecigite amet 11°5 14°0 10-0
GA frat, 2 eee 19 gues Ge 126 170 100
Average ...... 120 150 9°8

Breadth.—On an average the breadth at the widest part 1s 3 microns.

Shape.—Dr. Edington’s trypanosome when stained is seen to be of a short
and stumpy shape, somewhat reminding one of a miniature electric eel.
The posterior extremity is, as a rule, blunt, or rounded or obtuse-angled, but
sometimes, though rarely, it is prolonged into a sharp beak-like process.
The anterior end tapers more or less, and ends in a short stout flagellum.
The undulating membrane is narrow but distinct. The flagellum arises at or
near the micro-nucleus and passes along the edge of the undulating
membrane. There is no free flagellum, the protoplasm of the cell and the
undulating membrane extending as far as the tip of the flagellum.

Contents of Cell.—The protoplasm, which is stained a pale puce colour, is
homogeneous in structure. —

Nucleus.—The nucleus is oval in shape, about 2:5 microns in length, and is
situated at the centre of the trypanosome.

Micro-nucleus.—The micro-nucleus, centrosome, or kineto-nucleus, is small,

1908. | A Trypanosome from Zanzubar. _ x9

round, or rod-shaped, and is situated close to the posterior extremity. It
stains more deeply than the nucleus.

Undulating Membrane-—The undulating membrane is narrow. Asa rule
it is straight and simple, and does not show much tendency to be thrown
into folds.

Flagellum.—The flagellum stains intensely. It is well marked, and does
not project beyond the protoplasm of the cell and the undulating membrane.
Sometimes, in faintly-stained specimens, there is the appearance of a slight
projection of the flagellum beyond the body; but, speaking broadly, this
species of trypanosome may be said to have no free flagellum.

The conclusion to be drawn from a study of the morphology of
Dr. Edington’s trypanosome, Zrypanosoma dimorphon, and Trypanosoma
congolense, is, that the two first resemble each other very closely, whereas
Trypanosoma congolense seems to be of a somewhat shorter and stouter form.
It will also be seen that in the strain of Trypanosoma dimorphon used there
is only one form, and that, the short or tadpole form described by Dutton and
Todd. With regard to this, it may be of interest to quote some remarks of
Dr. Breinl, to whom I am obliged for his courtesy in sending me this strain.
He writes :—“ With regard to Zrypanosoma dvmorphon, you are aware that
some remarkable change has occurred in the strain between the time
Drs. Dutton and Todd brought it back from Africa and we started work
on it here. Whereas Drs. Dutton and Todd describe the long flagellated
forms with the free flagella, Thomas and myself, Laveran and Mesnil, could
not see these forms with a thin body and a long flagellum. The strain I send
you in a rat is the original strain.”

It is difficult to understand how this change in morphology has been
brought about. It may be that Dutton and Todd were dealing with a double
infection, of which one has died out. This point will require to be investi-
gated on the spot.

Another matter for consideration is whether this name Z7rypanosoma
dimorphon should be adhered to. It certainly seems a misnomer when
appled to the strain figured above. If it should be decided to drop it,
I think the compliment should be paid to Dr. Todd of naming it after him.

Inoculation Experiments on various Species of Animals.

The animals, in which the effect of the inoculation of Dr. Edington’s
trypanosome has been studied, have been horses, cattle, goats, monkeys, dogs,
rabbits, guinea-pigs, white rats and mice. The inoculations were made, as a
tule, intraperitoneally. Inoculation experiments with Trypanosoma dimorphon
are also given for purposes of comparison. ‘These are printed in italics -—

c 2

20 Sir D. Bruce and Capts. Hamerton and Bateman. [Sept.

18,

Source

No. of experiment. of Seas:
Edington ............| Unknown
Horse

Dutton and Todd| Natural
infection

chy ”

Laveran

Edington

Horse

Dutton and Todd

175
Thomas and Breinl
Dutton and Todd

165
166
167
179
180
181
Thomas and Breinl

Dutton and Todd |

sete cece tree eee see

Rabbit

ene ream etree nneeeecee

120
Thomas and Breinl

Dutton and Todd

| Period of

Duration
incubation,| of disease,
in days. in days.

Horses.
Unknown Unknown
i |

2”?

Unknown | 2°5 years, still

alive
os 1 year, still
alive
” aR
Cattle.
12 | Unknown
10°5 30
Goats.
Unknown Unknown
8°5 Well after a
year
Monkeys.
3 22
3 ee
4 and 6 160 and 75
4 ae
Dogs.
7 18
9 15
7 17
7 14
9 14
7 14
4to8 10 to 19
8 29
Rabbits.
25 _—
10 —
12 100
15 —
12 19
9 Acute, 26—35 ;
chronic, 78—157
13 53

Remarks.

Dr. E. thinks ran sub-acute course

Living after 18 days when Dr. H.
left

Blood still infective

No record

Horse recovered

Animal looked well on 21st day
Two cattle

Blood never showed trypanosomes
up to 18th day
Two goats

Spleen enlarged. General

glandular enlargement
Still alive (Sept. 11, 1908)
Two monkeys
Two never became infected

Spleen enormously enlarged

EP) th

Marked ulceration of stomach
Spleen greatly enlarged

” ”

Average of 4 days

Still alive after 146 days

bs, 136 ,,

Spleen 4 inches long and much
thickened

Still alive after 103 days

Spleen enlarged

One rabbit

1908. ] A Trypanosome from Zanzbar. 21

Sonrooroe Period of Duration
No. of experiment. F incubation,| of disease, Remarks,
virus. : :
in days. in days.
Guinea-pigs.
BP deopbcoobeqgndege0006 Rabbit. | 25 134 Liver and;spleen enlarged
MEN Ose Seeaeetesiniiaaitieis Rat | 12 43 f greatly enlarged
WRI B Ta re letatcrar sictatsiieciew co's ” | 19 43 ” ”
WM cosonqeandaqes0000 9») 21 = Still alive after 105 days
Zo yaciictcesciceisessciesi 3 21 69 Liver and spleen enlarged
IAS) aéopastesedandarno * 41 65 ” ”
Dutton and Todd — 6 380 Two guinea-pigs
Thomas and Breinl — | 4to15 9 to 60 |
White Rats
(8) coonsooodsandeooaL edd Rabbit | 13 30 Typical post-mortem appearances
13} cogosoanaqaagodagsono 3 10 = Killed for cultivation experiments
Sanne 4 {ltl a5 i
WAY oneocossdennponebo 3 7 Al Usual post-mortem appearances
WAS) soscnocacdtobocane 6) 7 36 » ey
is}: “soppageccoobbEsBEdaao Rat ii 5 20 2nd passage through rat
SOM eoicissasseccensses » U 5 — Killed for cultivation experiments
OB ccedaoacactonsuoce pa 5 — 3 5s
IO? Secanteeescnncobee aml 4 14 Typical post-mortem appearances
16S: eee a 7 35 ‘ is
MO De Tassie ctaverreisrorere ne Gas 5p ul 5 28 5 =
IIOP \pdedeadaehodadeode op ht 6 — Killed for cultivation experiments
TIOPA.- cnocaocoasosnbo00 py ul 7 37 Usual post-mortem appearances
TIZAIL eageogdseeeareacae » iil 4, 37 » 90
ZB ieee en one toiesisecats » li 5 44, » ”
iB" Teongadban eeosegeen » ul 4 — Rat lost
TUBIG. Se gdeonkaasecesdned 5 tl 7 20 Usual post-mortem appearances
SY ena anne ate 7 23 | 4 y,
Dutton and Todd _— 7 36 \
Thomas and Breinl | — | 4t07 7 to 42
Mice.
U8V4 — soeococdacsa000000 Rat 10 24
1B ly se Sa em Ret i 4 11
iISi0f) eee yetaeen ieee 4 10 11
Dutton and Todd — 5 16
Thomas and Breinl —_ 2t05 16 to 23
87 to 130

Conclusion.—The results of these inoculation experiments with Dr. Edington’s
trypanosome and Z’rypanosoma dimorphon show that they act on the various
animals employed in a strikingly similar manner.

CULTIVATION OF Dr. EpINGTON’s TRYPANOSOME, 7’rypanosoma dimorphon,
AND Trypanosoma congolense.

In June, 1903, Novy and MacNeal first announced the successful cultiva-
tion of Trypanosoma lewist. In the same year and in the following year they
also succeeded in cultivating Zrypanosoma brucet and Trypanosoma evans.

22 Sir D. Bruce and Capts. Hamerton and Bateman. [Sept. 18,

These gentlemen deserve the highest possible credit for this most difficult
achievement, an achievement which most workers in this subject thought
impossible. The amount of work they expended and the splendid intelli-
gence and pertinacity with which they pursued their object, refusing to
accept defeat, command the admiration of all their co-workers in this branch
of biological science. Since then the trypanosomes of birds, frogs, and fish
have been cultivated by the same and other workers; but these successes
have only been made possible, as a rule, by the pioneer work of Novy and
his assistants. Coming out of their work, mention may also be made of the
very interesting and important observation made by Rogers when he grew
Leishman’s bodies in ordinary citrated blood into trypanosome-like flagellates.

One of the chief interests attaching to this cultivation of trypanosomes is
that it may assist in separating the different species of these organisms. At
the present time trypanology is in a state of chaos on account of this difficulty
in differentiation. Many diseases of animals caused by trypanosomes have
been reported from all parts of Africa, Arabia, India, the Philippines,
Mauritius, etc., and it has often been found impossible to name the species
of trypanosoma causing them with any approach to certainty.

As mentioned above, the usual method of separating the different species
is by taking into consideration the morphology, the result of inoculation into
animals, the cross-immunisation methods and serum diagnosis of Laveran and
Mesnil, the mode by which the disease spreads from the sick to the healthy—
by a tsetse fly, a stomoxys, a tabanus, or by contact, as in dourine—by the
effect of various drugs, cultivation, etc. ; and, as already stated, the effect the
parasite has on animals and the mode of conveyance are probably, for
practical purposes, the most important. But to assist in separating the
various species, cultivation has been of use in the past, and, as the methods
become perfected, will be of still greater use in the future.

The following description of the cultural characters of Dr. Edington’s
trypansome exemplifies this, for, by comparing them with the cultural
characters of other pathogenic species, a fairly shrewd guess at its classifica-
tion may be made by this means alone. For the purpose of this comparison
a compilation of the cultural characters of Zrypanosoma lewisi, Trypanosoma
brucei, and Trypanosoma evansi has been made from the writings of Novy,
MacNeal, and Smedley.

It may be mentioned here that attempts have been made in this laboratory
to cultivate these three species. The cultivation of the first was found to be
a comparatively easy matter; but all attempts, and they were many, to
cultivate the last two have, up to the present, failed, although Novy’s
instructions were carefully followed.

1908. ] A Trypanosome from Zanxubar. 23

Cultivation Medium used.

The blood-agar medium used was made according to instructions kindly
sent by Prof. Novy. These need not be repeated here, as the details are fully
given by Novy and MacNeal in various papers.

CULTURAL CHARACTERS OF Zrypanosoma lewist.
A. Lwing, unstained.

Size.—Varies considerably in size. Some are not more than 1 or 2
microns long, not including the flagellum. Others are about the diameter of
a red blood corpuscle, while the usual length of the spindle-shaped cells is
15 to 20 microns. Some trypanosomes can be found at times which are
50 to 60 microns long. The greatest variation in size is found in young
cultures.

Shape.—Trypanosoma lewist varies greatly in shape, as well as in size.
Round, pear-shaped, fusiform and slender forms are present in the cultures,
The round forms are usually found in old cultures, and are probably
involution forms.

Contents of Cell_—tThe protoplasm in Trypanosoma lewisi, especially in young
cultures, is bright, glistening, and apparently homogeneous in structure in
the fusiform and slender forms.

Undulating Membrane—Not present as far as can be seen. The move-
ment of these cultural forms appears to be entirely due to the rapid motion
of the flagellum.

Flagellum.—These forms possess, as a rule, a long free flagellum. In the
slender forms this is sometimes twice the length of the body.

Motion.—The single, slender, cultural forms of Zrypanosoma lewist are
very active, and dart across the field of the microscope in a straight line. In
older cultures the round and other involution forms do not, as a rule, show
more than a slight swaying movement.

Colomes or Aggregations.—Growth commences in a first generation about
the fifth day by the appearance of small rosettes composed of a few
trypanosomes. The colonies rapidly grow, so that on the following day
masses of wriggling trypanosomes may be seen. These aggregations of
twenty or more are attached by their flagella. They grow larger and larger
until, about the twenty-fourth day, they are apparent to the naked eye, and
consist of many thousands of trypanosomes.

B. Fixed, stained,

Protoplasm.—Homogeneous, as a rule. Vacuolation is rare, but sometimes
a large highly-refractile vacuole is seen.

24 Sir D. Bruce and Capts. Hamerton and Bateman. [Sept. 18,

Nucleus—Round or oval in shape. Situated centrally or at the junction
of the anterior and middle thirds.

Micro-nucleus—Is placed either close to the nucleus or at a variable
distance anterior to it. In the free forms it is never seen lying posterior to
the nucleus. As a rule, it is a rod-shaped structure, lying transversely to the
long axis of the trypanosome.

Flagellum.—Arises from the vicinity of the micro-nucleus. The free
flagellum is often two, three or four times the length of the body of the
trypanosome.

Undulating Membrane.—In the cultural form of Trypanosoma lewisi this
structure is apparently absent.

Colonies or Aggregations.—There is little to add to the description of the
trypanosomes and of their arrangement in colonies. Stained preparations
show that the trypanosomes sometimes possess very long flagella. Novy
and MacNeal* have not apparently succeeded in staining the flagellum in
their preparations, though they noted the position of the centrosome. They
expressed the opinion that the end of the trypanosome pointing towards the
periphery of the colony was the anterior extremity, and that from it a
flagellum would arise if the cultural conditions were perfected (Smedley).

Measurements of the Cultural Forms of Trypanosoma lewisi.

Pear-shaped Forms.—(1) Body, 3:6 to 44 microns long, and nearly as broad.
(2) Flagellum, two to four times the length of the body.

Spindle-shaped Forms.—14 to 16 x 2-4 to 3°5 microns, flagellum not
included.

Smaller and larger forms are frequently found.

The adult parasitic form of Trypanosoma lewist measures 24 to 25 x
1:5 microns (Laveran and Mesnil) (Smedley).

CULTURAL CHARACTERS OF 7rypanosoma brucet.

A. Lnving, unstained.

Size.—Shows less variation in size than 7rypanosoma lewisi, and averages
15 microns in the living condition. Smaller than those found in the blood.

Shape.—Do not vary much in shape, and closely resemble the forms found
in the blood (Smedley).

Contents of Cell_—Show one or two very large, bright, and highly-refracting
globules, usually placed near the anterior or flagellar end, in the otherwise
homogeneous colourless cell. In size the globules may attain 1 micron

* ‘Cultivation of Trypanosoma brucei, p. 28.

1908. | A Trypanosome from Zanzibar. 25

At times the number of these globules is increased, as when the culture is
kept at 34°C. The presence of numerous large, highly-refractile globules
in the cultural forms of Trypanosoma brucei is attributed by Novy and
MacNeal to degeneration of the organisms, owing to imperfection of the
culture medium. These globules become more numerous as the age of the
culture advances. Do not seem to alter in position or shape if kept under
observation for several hours. Resist staining completely. Laveran and
Mesnil suggest that the globules are of the same nature as the refringent,
unstainable granules found in Trypanosoma rotatorium.

Undulating Membrane.—No detailed description available.

Flagellum.—the flagellum in the living cell is by no means as distinct and
as long as that of Trypanosoma lewisi.

Motion—The motion of Trypanosoma brucei is slow and wriggling, and
only exceptionally is a slowly-progressive form observed. The wave-motion
slowly passes along the thick, undulating membrane, and gives the appearance
of a spiral rotation to the entire cell. Scarcely departs from its place (Novy).
In a young culture the trypanosomes are found to possess very active
movements. Sometimes they advance across the field moderately quickly,
but their rate of movement is always much slower than that of the rat
trypanosomes, whose flagella are longer and more rapid in motion (Smedley).

Colonies or Aggregates.—Occurs in groups or rosettes. Rarely forms masses
of more than 10 to 20 cells. The individuals are long, narrow, and show the
peculiar writhing motion. The flagella are directed outwards, and the appear-
ance of the whole may be compared to the snakes on a Medusa head. The
stellate group with the bright, refracting globules within the cells, suggests
a jeweller’s “sun burst” (Novy). The active moverhents of the trypano-
somes, and the large glistening vacuoles with which they are studded, give
these colonies a singularly beautiful appearance (Smedley).

B. Fixed, stained.

Protoplasm.—The protoplasm invariably contains a few deeply-stained
granules of a red or violet colour. The vacuoles are seen as clear circular
spaces with sharply-defined outlines in stained preparations (Smedley).

Nucleus.—Round or oval in shape ; and in older forms it breaks into masses
of cromatin, which are found distributed through the protoplasm of the cell
(Smedley).

Micro-nucleus—This is much smaller than in Z'rypanosoma lewisi ; it is
usually circular, but sometimes elongated. It stains a deep red or purple
colour, and it is sometimes difficult to distinguish it from the other granules.

26 Sir D. Bruce and Capts. Hamerton and Bateman. [Sept. 18,

It is generally found close to the vacuole; sometimes it lies close to the
nucleus, but it is nearly always posterior to the latter structure (Smedley).

Flagellum.—Takes a tortuous course along the free border of the undulating
membrane, and projects for a short distance from the anterior extremity
(Smedley).

Undulating Membrane.—No detailed description given.

Colonies or Aggregates.—Most of the flagella are directed in an outward
direction. It is rare to find colonies of a large size (Smedley).

Measurements of the Cultural Forms of Trypanosoma brucei.

Length, including flagellum, 18 to 23 x 2°5 to 3°5 microns. Length of free
flagellum, 3 to 5 microns. Diameter of vacuoles, 1 to 2 microns. The adult
parasitic forms of Trypanosoma bruce measure, in the blood of rats, 26 to 27
x 15 to 2°5 microns (Laveran and Mesnil) (Smedley).

CULTURAL CHARACTERS OF Trypanosoma evanst.

A. Lnwing, unstained.

Size—The body of one large individual measured 21 microns, while the

flagellum was 28 microns in length.
' Shape—tThe slender fusiform body terminates at one end in a delicate
flagellum. The posterior end, especially when blunt, showed a rod-like tip
or stylet, which varied from 2 to 4 or even 6 microns in length. As the
cultures aged, pear-shaped or spherical, highly granular, involution forms
appeared. In the former type, measuring about 3 by 5 microns, the end was
often provided with a flagellum, 10 to 15 microns long, which still showed
a slow lashing movement, though the cell itself was motionless. The
spherical forms varied from 4 to 9 microns in diameter, were granular, and
often showed a remnant of the flagellum as a short, stiff, motionless whip.
These involution forms, as in the case of Trypanosoma lewist and Trypano-
soma brucei, eventually gathered into large groups or masses, which at times.
filled the field of an immersion lens. Later on, the round bodies broke up
into masses of very minute granules.

Contents of Cell.—Presence and peculiar arrangement of granules within
the cells, and a distinct yellowish or greenish colour of the granules and of
the contents. Large numbers of small granules or globules, which vary from
0:3 to 0°5 micron in diameter. These globules, as well as the contents of the
cell, possess a decided yellowish or greenish colour, and appearance quite
unlike that of either Zrypanosoma lewisi or Trypanosoma brucet. The
globules are usually massed in the anterior-third of the cells—that is, at the

1908. | A Trypanosome from Zanabar. 27

base of the flagellum, and only a few isolated granules are scattered through
the remainder of the organism (Novy and MacNeal).
Undulating Membrane.—Is not recognisable in the living organism.
Flagellum.—Usually as long and often even longer than the cell itself.
Motion—All single and actively motile, traversing the field of the
microscope at great speed. Travel with the flagellum in rear or in front.
Colonies or Aggregates——Entire absence of the groups or rosettes, which
are so characteristic of the cultures of Trypanosoma lewisi and Trypanosoma
brucer. The trypanosomes were all single and actively motile.

Measurement of the Cultural Forms.

- Length, including flagellum, 25 to 50 by 1°5 to 2°5 microns.

CULTURAL CHARACTERS OF Dr. EDINGTON’S TRYPANOSOME.
A. Living, unstained.

No difficulty is found in cultivating Dr. Edington’s trypanosome. As.
early as the second day,if kept at 25° C., it is found to have greatly
increased in numbers. The single individuals are in active motion, the
flagellum wildly waving, while the body slowly moves among the corpuscles.
Many dividing forms are seen with two or three flagella. Masses or
ageregations are also seen varying in size, from those composed of a dozen
individuals to those occupying a fifth of the field. These aggregation-forms
are all writhing and squirming, while the flagella at the periphery are
frantically waving. This incessantly moving mass, dotted over as it is with
many small bright vacuoles, makes a curious and beautiful microscopic object.
when brightly illuminated.

On the third day the trypanosomes have multiplied to an extraordinary
extent. Huge aggregations are now seen, each filling up several fields of
the microscope. The individual trypanosomes are still actively motile.
Single, double, and small aggregations are also seen.

By the seventh day they have reached the height of their growth and
begin to degenerate.

After the twelfth day living forms can no longer be recognised in the
culture tubes.

Size.—Dr. Edington’s trypanosome, examined in the fresh living condition,
varies considerably in size. Some of the large forms measure 32 microns in
leneth, whereas the smaller are only half that length, or even shorter.

Shape-—So also in regard to shape, these cultural forms vary extremely.
Round, oval, pear-shaped, and irregular forms are seen. Slender forms.

28 Suir D. Bruce and Capts. Hamerton and Bateman. [Sept. 18,

shaped like ordinary trypanosomes, with a beak or rostellum at one end,
a fairly thick flagellum at the other, and furnished with an undulating
membrane, are fairly common. Large irregular masses of any shape,
furnished with one or more flagella, are also frequent.

Contents of Cell—These cells have a remarkable appearance, as they are
filled with highly refractile granules, large in size, round in shape, and
numerous.

Undulating Membrane—The round, oval, and pear-shaped forms do not
appear to possess an undulating membrane, whereas the long, slender forms,
as also the huge fish-shaped or octopus-like forms, often show well-marked
undulating membranes.

Flagellwm.—the flagella in these living unstained cultural forms are thick
and coarse, and differ markedly from the slender structures usually associated
in the mind with trypanosomes. Just as in the parasitic forms found in the
blood, it is evident that the protoplasm of the body extends to the tip of the
flagellum giving rise to this thick stumpy appearance.

Motion—tThe slender forms are active and swim fairly quickly across the
field. The large, irregular forms are stationary, but exhibit actively
wriggling flagella and amceboid movements of the body substance.

Colonees or Aggregates.—Colonies or aggregations of 10 to 20 individual
cells are common. The cells are arranged irregularly. Some of their
flagella may be directed outwards, while others are seen entangled in the
mass and feebly wriggling. On the third day these aggregations may be
seen as large as three to five fields of the microscope, and must be composed
of many thousands of individual trypanosomes.

B. Fixed, stained.

Method of Fixing and Staining—The cultural forms of Dr. Edington’s
trypanosorhe were either prepared by mixing a drop of the cultivation fluid
with fresh serum, spreading on a slide, and staining by Leishman’s modifica-
tion of Romanowsky’s method, or the fluid was spread on a slide, fixed by
osmic acid and stained by Giemsa, and then treated with orange tannin to
differentiate the various structures.

In Leishman-stained preparations the protoplasm of the cells is stained
a pale blue, the nuclei and irregular masses of chromatin reddish or pink,
while the vacuoles stand out as unstained spaces with sharply-defined
margins. In Giemsa-stained preparations, on the other hand, the protoplasm
is stained a pale puce colour, while the chromatin material is stained reddish
purple.

Protoplasm.—The protoplasm of the cell is homogeneous, but contains

1908. | A Trypanosome from Zanubar. 29

irregular-shaped granules and masses of chromatin-staining material. There
are also present numerous well-marked vacuoles of various sizes, which are
unstained, and, as mentioned above, highly refractile.

Nucleus.—The nuclei are of every form and shape, and often broken up into
irregular masses.

Micro-nucleus.—The micro-nuclei are irregularly placed; in some cells are
not easily distinguishable from other granules contained in the protoplasm,
but in many are clearly seen as deeply-staining bodies, round or rod-shaped,
in close connection with the point of origin of the flagella.

Flagellum.—the flagella are, as a rule, thick and fleshy. In the irregular
forms they appear to spring from any part of the shapeless mass of proto-
plasm, and in any direction.

Undulating Membrane—The undulating membrane is also characterised
by its extreme irregularity. In many cells it appears to be absent, while in
others it is well marked, broad, and thrown into folds.

Colonies or Aggregations.—The individual trypanosomes which go to compose
the large aggregations are as a rule short and stumpy in form, with oval-
shaped nucleus and short stumpy flagellum. They are of irregular shape
and size, and are placed without any seeming order.

CULTURAL CHARACTERS OF 7'rypanosoma dimorphon (DUTTON AND Topp),

_ It is unnecessary to describe in detail the cultural characters of this
trypanosome, as they agree exactly with those of Dr. Edington’s. '

CULTURAL CHARACTERS OF Trypanosoma congolense (BRODEN).

Several attempts were made to cultivate Zrypanosoma congolense, but
none of them were very successful. There is certainly not the rapid growth
of this trypanosome which distinguishes Dr. Edington’s trypanosome and
Trypanosoma dimorphon. It is only after a long search that individual
trypanosomes can be found in the preparations. There is no formation of
masses or aggregations filling several fields of the microscope as in the others.
It is difficult to say whether there is any real multiplication or not. All that
can be said is that, for about eight days, living trypanosomes can be seen. At
first these are shaped like the ordinary trypanosomes found in the blood, only
larger and swollen in appearance; but by the fifth and following days these
change into most irregular and fantastic shapes. Nothing living could be
seen after the eighth day. This cultivation experiment would therefore seem
to strengthen Dr. Laveran’s opinion that Trypanosoma dimorphon and
Trypanosoma congolense are distinct species.

30 A Trypanosome from Zanzbar.

Conclusion.

The conclusion arrived at is that Dr. Edington’s trypanosome from Zanzibar
is probably Dutton and Todd’s Trypanosoma dimorphon. One link in the
chain of evidence, however, is wanting, and that an important one—the
identity or non-identity of the carrier.

DESCRIPTION OF PLATES.

PuLateE 1.

This plate represents the shape and size of the three Trypanosomes, viz. :—

1. Dr. Edington’s trypanosome. From blood of rat. 30th day of disease. Stained
Giemsa. x 2000. See p. 27.

2. Trypanosoma dimorphon. From blood of rat. 15th day of disease. Stained
Giemsa. x 2000. See p. 29.

3. Trypanosoma congolense. From blood of mouse. 7th day of disease. Stained
Giemsa. x 2000. See p. 29.

PLATE 2.

Fie. 1.—Part of an aggregation of Dr. Edington’s trypanosomes after 5 days’ growth.
Stained Giemsa. x 2000.

Fics. 2-4.—Dr. Edington’s trypanosome after 6 days’ growth. Stained Giemsa. x 2000.

Fras. 5-14.—Cultural forms of Dr. Edington’s trypanosome after 7 days’ growth. Stained
Leishman. x 2000.

Bruce and others. Io, (SOC: 1770C,, Je}, GOH, Si, IHCHwe I.

Wee. uthons Roa, Seo Beoe, 1S TGLGL EL

k
;
:

——_——

,

es en ee ee ae ee ee ee ee re eee

MP Perker hth Parker k& West imp.

31

A Summary of further Researches on the Etiology of Endemic
Govtre.
By Rovert McCarrison, M.B., B.Ch., Captain, Indian Medical Service.

(Communicated by Major Ronald Ross, C.B., F.R.S. Received October 24,—
Read November 26, 1908.)

The object of the research was to determine by experiment on man
whether goitre was caused by matter held in suspension in goitre-producing
waters; and to ascertain, as far as possible, the nature of the suspended
ingredient which had been surmised to be responsible for the production of
the disease.

Thirteen individuals, including myself, were given suspended matter, which
had been removed by filtration from goitre-producing water, every morning
before the first meal of the day. I and three others developed enlargements
of the thyroid gland. The experiment was repeated in the case of eight
individuals who were given the same suspended matter, which had previously
been boiled for 10 minutes; in no case did any enlargement of the thyroid
gland occur.

It is concluded from these results that goitre is due to a living organism
of disease present in the water. The incubation period of experimentally-
produced goitre was 13 to 15 days.

It is thought probable that the organism of goitre exists as an intestinal
parasite in goitrous individuals, since an intestinal antiseptic appeared to have
a marked curative effect.

Experiments were made on monkeys to test the possibility of the spread of
the disease by the feces of infected individuals, with negative results.

Plentiful amcebic infection of the intestine was found in the majority
of cases of goitre examined. It is not known, however, whether amcebe
have any relationship to the disease.

The research was carried out in Gilgit (Kashmir), and the results obtained
refer only to goitre as it occurs there.

32

The Proportion of the Sexes produced by Whites and Coloured
Peoples in Cuba.

By Water Heaps, M.A., F.RS., Trinity College, Cambridge.
(Received September 30,—Read November 26, 1908.)

(Abstract.)

Introduction.—Darwin, in his great work on the Descent of Man, deals
with the proportion of the sexes in various animals and the power of natural
selection to regulate the proportional number of the sexes. He recognises a
general tendency to equality of the sexes but remarks on the fact that this
equality is often greatly disturbed. In certain rare cases of marked
inequality he concludes they might have been acquired through natural
selection, but in all ordinary cases, such as, for instance, the difference in the
proportion of the sexes in legitimate and illegitimate children, it can hardly
be so accounted for and must be attributed to unknown conditions, although,
he adds, natural selection will always tend to equalise the relative number of
the two sexes.

About that time a host of writers were engaged in investigating various
possible causes for this inequality, and many theories were promulgated to
account for it, such as the relative age of the parents, the time of conception,
and so forth. Prominent amongst them was Diising, who set himself to show
that nutriment was the chief determining factor. He set forth his case with
great ability and brought an enormous mass of evidence in support of his view.

Students of heredity in those days claimed that the laws of heredity were
sufficient to account for all inequalities, but Diising emphatically denied this,
and in my opinion satisfactorily showed he had sound reason for doing so.

During the last few years much work has been done on sex, especially
regarding the factors which determine sex, and strong evidence has been
brought to show that both individual spermatozoa and ova are themselves of
definite sexuality. It is suggested that the sex of the individual resulting
from the conjugation of a spermatozoan and an ovum must be determined by
one or other of them, not by both, and it is claimed that, in order to fulfil the
conditions, a M. ovum must be fertilised by a F. spermatozoan and, vice versd,
a F. ovum by a M. spermatozoan. So far as the evidence available now goes,
it would seem possible that in some animals the sex of the offspring is
determined by the spermatozoan and in other animals by the ovum. The facts
are not clear, however, though itis to be hoped the efforts now being made by

Proportion of Sexes produced by Whites and Coloured Peoples. 33

the Mendelians will make it so; at present, perhaps, all that can be said
regarding mammals is that the evidence available is in favour of the view
that the ovum determines the sex of the offspring in these animals.

Now a female mammal produces only a limited number of her ovarian ova
during her life, others degenerate and are absorbed, in fact, some ovarian ova
survive at the expense of others and it would appear that this process goes
on with more or less activity at different times. Thus there is a struggle for
existence and a process of selection going on in the mammalian ovary, and
this 1s a very important fact, for the projection into the ovary itself of forces
which are undeniably produced by extraneous conditions shows that such
conditions must to some extent influence the output of the ovary.

A wealth of evidence has been adduced by many observers to show that
M. and F. larve are very differently affected by different foods and different
climatic conditions, and this evidence is overwhelmingly in favour of the view
that F. larve require more nourishment, more favourable conditions, than do
M. larve for their development. But if this is true for larve it is surely true
also for ovarian ova, and the conclusion may be confidently drawn, that the
selection of M. or F. ovarian ova, for production, is liable to be influenced by
the food supplied to the ovary by the mother and therefore by the conditions
of metabolic activity she experiences.

As all breeders know, the breeding power of an animal is in direct relation
to its metabolic activity, and the metabolic activity of a mother is
undoubtedly affected by the food supplied and the climatic conditions she
experiences ; thus it would appear that extraneous conditions must exert
influence on the proportion of the sexes produced by all animals in which a
struggle for existence takes place among the ova in her ovary.

This view, it appears to me, explains a variety of facts which have been
judged to be contradictory, and brings into line the results of many observa-
tions which have hitherto been supposed to favour now one, now another,.
quite different theory. For instance, all the contradictory evidence I have
examined regarding the effect on the sex ratio of the ages of the parents and
the times of conception, may be so accounted for, while upon the phenomena
concerning sex ratio observed in consequence of crossing or of in-breeding, a
new light is thrown which will, I think, go far to show adequate reason for
the results obtained for mammals. It must not be supposed that I attribute
the proportion of the sexes produced to these agencies alone; there can be no
doubt, in my opinion, that heredity is the main force at work, but it is.
incontrovertible that variations in that proportion constantly occur, and I
maintain that these variations cannot be accounted for by any law of
heredity and are referable to those extraneous forces which act as selective

VOL. LXXX1.—B. D

34 Mr. W. Heape. Proportion of the Sexes produced [Sept. 80,

agents on the ovarian ova. The evidence I have to offer in the following
paper is, I think, strongly confirmatory of this view.

1. Data dealt with—From a statistical point of view, human beings are
the only mammals for which sufficiently large numbers can be obtained with
any hope of ensuring sufficient accuracy, and for these there has always been
difficulty in assuring oneself of the completeness of the records at avy one
time for more than one race. When, therefore, my friend, Dr. F. H. H.
Guillemard, pointed out to me that the publications of the chief sanitary
officer of Cuba supplied separate details of the births and still-births of
whites and coloured people in the island, and that these records were further
subdivided into legitimate and illegitimate births and still-births, I commu-
nicated with that officer (Dr. Finlay), and he has very kindly supplied me
with a complete series of his monthly publications for the years 1904-5-6.
It is with these records I now deal. The numbers dealt with amount to—

Births—whites, 131,721 ; coloured, 39,576. Total, 171,297.

Still-births—whites, 4160 ; coloured, 2247. Total, 6407.

Total production—whites, 135,881 ; coloured, 41,823. Total, 177,704.

Deaths—whites, 52,087 ; coloured, 27,877. Total, 79,964.

Marriages—whites, M. 31,481, F. 31,240; coloured, M. 7598, F. 7839.
Total, M. and F. (each) 39,079.

These totals are arrived at from monthly records, for each of these three
years, for each of the six provinces into which the island is divided ; and in
each case, except for marriages, the proportion of M. per 100 F. has been
calculated. Altogether I have drawn up 64 tables of these and similar
details; they are dealt with more fully elsewhere, the results thus obtained I
now summarise.

2. The Racial Proportion of the Sexes—The first prominent fact
demonstrated is the difference in the proportion of the sexes produced by the
two races. The whites produce a larger proportion of M. than the coloured
people. For whites, the proportion varies during these three years from
106-8 to 110°52, in the total it is 108-44 M. per 100 F.; for coloured, the
proportion varies from 101 to 101-2, total 10112 M. per 100 F.; and this
racial difference is shown both for births and still-births.

Here, then, we have a marked racial difference which, after examination of
other statistics of coloured people and of the inhabitants of Spain (from
whence most of the white inhabitants of Cuba originally came), Iam of opinion
- may confidently be assumed to show that, in this particular, the influence of
heredity is clearly demonstrated.

3. The Sexual Ratio in Legitimate and Illegitimate Births—The second

1908. | by Whites and Coloured Peoples in Cuba. eB)

prominent fact is the consistent variation in the proportion of the sexes
produced in consequence of legitimate or illegitimate union. This is evident
both for births and still-births in both races, and emphatically shows that
legitimate union results in the production of a marked increased proportion
of F. For whites, the total legitimate births show 107°78, while the
illegitimate show only 1044 M. per 100 F. For coloured, legitimate births
show 106°76, and illegitimate 96°76 M. per 100 F. The records of still-
births show a difference of 10°06 more M. among legitimate still-births for
whites, and 16°63 more M. in that class for coloured people; but the
proportion of M. among still-born children is vastly higher than among births,
the totals for whites and coloured for the three years being in proportion of
144-45 M. per 100 F.; thus, when births and still-births are added together,
the result of legitimate union among whites gives 109, of illegitimate union
105°95 M. per 100 F.; among coloured, legitimate unions give 107°73, and
illegitimate 97°91 M. per 100 F. Thus this difference, while it is much
greater for coloured than for white people, is marked for both races in the
totals, and is shown to be a remarkably consistent variation throughout my
tables.

It is clear that illegitimate union amongst civilised peoples is due to
individual characteristics in the woman which have for their basis a specially
active sexuality. Thus the result of illegitimate union, the increased
production of F. in consequence thereof, is an individual matter, it cannot be
accounted for by any law of heredity and must be associated with physio-
logical conditions which induce this special activity, that is to say with forces
which affect the metabolic activity of the woman. I cannot here detail all
the arguments in favour of this view and will only add (the evidence admits,
I think, of no other interpretation) that, as I have already shown, an
exceptionally active metabolism in the mother should favourably affect the
development and ripening of F. ova, and that this is what is found to be the
case here.

4. Breeding Seasons—An examination of the monthly tables demonstrates
the existence of two sharply-defined breeding seasons each year, and shows
that they are experienced by both whites and coloured at the same time.
One breeding season is more marked than the other, this fact is also common
to both races, but both are quite unmistakably shown in the birth tables.
The records of marriages show that the marriage season, though it is also
quite definitely indicated, has no relation whatever to the breeding seasons.
On the other hand, reference to records of temperature, barometric pressure,
humidity, ete., shows that these bursts of reproductive activity always take
place at times when there is a marked change of climate; the one in the

D2

36 Mr. W. Heape. Proportion of the Sexes produced [Sept. 80,

autumn shortly after a sudden change from great heat to cooler weather, the
other in the early months of the year at a time when the cool winter weather
gives place to spring. The increased reproductive activity of the people is
suddenly acquired and almost as abruptly allayed, it is obviously not a
definite temperature but the experience of a change of temperature which
induces this boisterous generative activity.

The same conditions are found to influence, in a similar manner, other
animals which experience breeding seasons, and the effect on stock is
increased metabolic activity. There can be no doubt these breeding seasons
of the two races in Cuba are brought about by forces which tend to greatly
increase the metabolic activity of the individual.

5. The Effect of the Breeding Seasons on the Proportion of the Sexes produced.
—If, then, I am right in stating that the breeding seasons are induced in
consequence of increased metabolic activity, and if my reasoning is sound
regarding the increased output of F. among illegitimate births and the
influence of different degrees of metabolism on the ripening and production
of ovarian ova of different sexes, the result of the breeding seasons should
show this.

It does show it, emphatically; my tables demonstrate that the greatest
excess of F. is produced at times of greatest fertility, 2.e. during the breeding
seasons, when the metabolism of the mother is most active. This is true for
both races and it is clearly shown in all totals and in the totals for each
individual month, except for two months of one year for coloured people.

I feel convinced such a variation in the sex ratio cannot be ascribed to the
action of any law of heredity; it is clearly associated with the exercise of
extraneous forces on the ovary and is, I submit, due to those forces.

6. The Limitation of the Influence of Extraneous Forces.—In connection
with the above, another fact is shown which is of considerable interest,
namely, that while whites show a more marked sensibility to the influences
which induce the production of F., coloured people are more affected by the
forces which stimulate the production of M., and this condition is more
marked among illegitimate than among legitimate birth records.

This fact shows that the race which normally produces a considerable
excess of M. is most amenable to the forces which induce the ripening of
F. ova, while the race which produces the greatest proportion of F. reacts
more generously to the influences which favour the production of M. ova.
In other words, there is here demonstrated the exercise of a force which
limits the power to produce an excess of either sex, a force which makes
for some point near equality of the sexes, and which is, I take it, the force
of heredity.

ui

1908. | by Whites and Coloured Peoples in Cuba. 37

This exemplifies the nature of the claim I made; extraneous forces
undoubtedly exist which effect a variation in the sex ratio, but they are
to some extent subordinate to laws of heredity; nevertheless these former
forces cannot be ignored, they are certain to interfere to some extent with
the performance of the laws of heredity and with all calculations regarding
sex ratio which are based solely upon those laws.

7. The Effect of Town as compared with Country Life on the Sex Ratio—
Finally, on analysis, my figures show another fact, namely, that a quite con-
siderably higher proportion of F. are born in towns than in the country
districts. This is shown in my tables for both races and is evident as a rule
in the records for both legitimate and illegitimate births.

I have elsewhere discussed the reason for this; it is quite clear no law of
heredity can explain such a variation, and I have concluded that . the
extraneous forces which are accountable for it must again be associated
with the degree of metabolic activity experienced by the mother under
variable conditions.

8. Conclusion—Other facts of considerable interest in relation to this
' work are set forth elsewhere and I will not refer to them here. I have
given above three instances of conditions under which the production of
M. and F. children shows a marked variation from the normal. The results
are similar for both the whites and the coloured races in Cuba. These
people have hereditary qualifications which, in the main, govern the
proportion of the sexes they produce, but conditions undoubtedly occur
under the influence of which that proportion is varied. This variation is
similar in character but different in degree for the two races, and is directly
associated with definite extraneous forces, food and climate, which affect the
metabolic activity of the mother.

Taken singly any one of these instances might be thought to be in-
conclusive, but taken together they seem to me to present strong evidence
of the truth of my contention, that the variable metabolic activity of the
mother, acting upon the ovary, induces a struggle for existence between
the ovarian ova of different sexes, and affects the proportion of M. or F. ova
which ripen and which are produced for fertilisation.

It is worthy of notice that these same extraneous forces must affect the
proportionate produrtion of individuals possessing various kinds of different
characters (quite other than sex) which are associated with metabolism, and,
when better understood, may have valuable bearing on the means for selection
of healthy ova and for preventing the maturation of ova bearing the active
germs of disease. :

38

Electrolytes and Colloids.—The Physical State of Gluten.
. By Prof. T. B. Woop and W. B. Harpy, F.R.S.

(Received October 24,—Read December 10, 1908.)

Gluten, as ordinarily prepared by washing wheat flour in tap water, forms
a coherent stringy mass insoluble in water. It consists essentially of
a mixture of two proteins, gliadin and glutenin, but even when very
thoroughly washed it always includes some starch. Gliadin, which forms
rather more than half of the total protein, is soluble in dilute alcohol, and
gives to the gluten its peculiar physical properties.

The power which dough possesses of retaining the gas formed during
fermentation is due to the tenacity and ductility of gluten.* Therefore,
the property of forming a light and well-shaped loaf, which is so variable
a feature of different flours, is determined by the amount and the physical
state of the contained gluten.

The physical state of gluten, like that of other colloids, is conditioned
by the electrolytes which are present. Gluten washed out of flour with
distilled water obviously is more friable and less tenacious than gluten
washed out with tap water which contains salts. It is this influence of
electrolytes upon the physical state of gluten which we propose to discuss. —

Gluten is peculiarly sensitive to low concentrations of acid or alkali.
A tenacious ductile mass suspended in a large volume of, for instance,
00001 normal acid, begins almost at once to show signs of disintegration,
and is at once dispersed by slight movement to form a stable opaque
colloidal solution or hydrosol.

Action of Acids—This action was investigated quantitatively by
suspending a small mass of gluten on a bent glass rod in a_ beaker
containing 120 c.c. of a solution of acid of known strength, and noting the
concentration at which cohesion was so far reduced as to allow the protein
to fall off the rod and disperse in a cloudy “solution.” It was found that
while very dilute acid causes dispersion, a solution of a strong acid above.
a certain concentration maintains the cohesion. Gluten, therefore, is
coherent in distilled water, and in strong acids above a certain critical
concentration. A weak acid, such as acetic acid, brings about dispersion up
to as high as twice normal, the highest concentration tried. Inspection of
a series of beakers with concentrations of any strong acid from zero to the
eritical point makes it clear that, starting from the lowest concentration,

* Wood, ‘ Journ. Agric. Sci.,’ vol. 2, part 2, p. 139, and part 3, p. 267.

Electrolytes and Colloids. 39

dispersion increases to a maximum and then falls to zero at the critical
point. In other words, the power of destroying the cohesion and dispersing
the gluten as a cloud varies with the concentration of the acid, so that the
relation can be shown by a curve. The form of the curve will be seen later.

The dispersion of the gluten is not due to a change in the protein molecule
of the nature, for instance, of hydrolysis, since it can be recovered as a
tenacious stringy precipitate by neutralising the acid or by the addition
of salt.

The following table gives the mean of several determinations of the
concentration at which the gluten retains its coherence. The exact point is
the concentration at which gluten just breaks under its own weight when
suspended in the solution of acid; and the results obtained in different
experiments are fairly consistent. It is remarkable that there should be no
simple relation between the observed concentrations and the strengths of
acid used as measured by electric conductivity. The conductivity of the
solutions after the gluten had been immersed in them was measured, and the
results are given in the second column of figures, the value of the sulphuric
acid solution being taken as unity :—

Table I.
Normality of critical Relative
Acid. concentration. conductivity.
HELE S © gestern ciate isa: Stu oe tke 0-017 1:0
Camphorsulphonic ............ 0:62 1:59
ETEINO)5 ones fos semsiaiian Green clealeiateg 0:03 eg)
1a (Gl aa adeescue sapocceneeenes 0:05 3°8
Oar erce tec tas scicimelstae thie 0-15 3°8
lols Ore cee see sre nanrpesbne saan enee 2:00 —

Action of Distilled Water—Gluten breaks up when washed very
thoroughly in many changes of ordinary distilled water. The distilled
water used was acid to litmus owing to the presence of carbonic acid; and
the dispersion of the protein is due to this acidity, since (1) it is precipitated
by the addition of a trace of alkali, and (2) the protein when dispersed is
electro-positively charged—that is to say, it displays the characteristic
relation of protein to acid.

The Influence of Salts—Salt in small concentration precipitates a hydrosol
of gluten whether it be formed by acid or by alkali. Therefore, salts lessen
the power which acids or alkalis possess of destroying the cohesion of
gluten, and, in sufficient concentration, completely neutralise it. The
concentration of salt necessary completely to nullify the dispersive power

AO Prof. T. B. Wood and Mr. W. B. Hardy. [Oct. 24,

of particular acids was investigated in the manner already described, namely,
by suspending approximately equal pieces of gluten in varying concentra-
tions of acid and salt, and noting the point at which cohesion was so far
reduced as to allow the protein to flow off the rod. The relations appear in
ithe following curves (fig. 1), which show that for all strong acids and for all
Salts the concentration of the latter needed to balance the former increases to
a maximum as the concentration of acid increases, and then declines to
zero at the point where the acid alone is sufficient to maintain cohesion.
‘The curves all agree, therefore, in showing that, measured by the concentra-
tion of salt needed to prevent dispersion, the dispersive power of an acid
increases with increasing concentration, and then falls until the critical
concentration is reached, where dispersive action is nil.

LACTIO ACID

OXALIC ACID
Na Cu

PHOSPHORIC ACID

1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 B00 200 100 0)

Fie. 1.

These curves are so characteristic that they afford a means of testing
a point of general theoretical interest. One great class of colloidal solutions,
the aqueous solutions of characteristically insoluble bodies such as metals,
some proteins, sulphides, and gums, are characterised by the fact that round
each particle of the solute there is an electric double layer, and on the
potential difference between which the stability of the solution depends.
Coagulation or precipitation of such a solution is approximately coincident
with the reduction of the potential difference to zero, the most complete
coagulation, 1.e., mechanically the densest and most coherent coagulum, being
formed at the isoelectric point.*

* Hardy, ‘Roy. Soc. Proc.,’ vol. 66, p. 110, 1900; Picton and Linder, ‘Chem. Soc.
Trans.,’ 1905—1906, vol. 87; Perrin, ‘Journ. de Chim. Physique,’ vol. 2, p. 601, 1904 ;
vol. 3, p. 50, 1905.

1908. | Electrolytes and Colloids. 41

On this view the formation of the hydrosol of gluten is due to the
development of electric charges round the particles of the protein owing to
chemical interaction between the protein, the acid or alkali and the water;
and the tenacity, ductility, and water-content of a solid mass of moist gluten
depends upon the total or partial disappearance of these electric double
layers, and the reappearance of what is otherwise obscured by them, namely,
the adhesion or “idio attraction,” as Graham called it, of the colloid particles
for each other, which makes them cohere when they come together.

It is possible to put this hypothesis to the proof. We can measure the
potential difference between the water face and the protein face of each
particle in the hydrosol of gluten by determining the rate of transport of
the particles in a uniform electric field. The method adopted has been
described by one of us.* Briefly it consists in the use of a graduated
U-tube, the opalescent hydrosol is introduced as the lower layer, the upper
layer in each limb being a clear solution of the same electrical resistance.
Electrodes are immersed, in the upper layer, a field established, and the rate
of movement of the boundaries between upper and lower layers observed.

The hydrosol was prepared either by washing gluten in distilled water
containing carbonic acid, a process which occupied at least two days, or in a
few hours by washing in a few changes of 0:0001 normal sulphuric acid. It
was freed from all starch by centrifuging. To successive lots of the hydrosol,
acid was added in varying amounts, and water when necessary, so that the
concentration of protein was constant, while the concentration of acid varied.
Finally the resistance was measured, and a fluid to form the upper layer
was prepared either by adding the same acid to water or by adding sodium
chloride. Hydrochloric, sulphuric, and acetic acids were used, and the results
were in all cases the same. The figures for hydrochloric acid are plotted in
the following curve, the ordinates being specific conductivity of the solution
x 107°, the abscisse the specific velocity in centimetres per second for unit
potential gradient x 107 (fig. 2).

The curve agrees in form with those already given for the effect of salt
upon cohesion, and we. may therefore conclude that acids, and by inference
alkalis also, destroy the cohesion of gluten by forming double electric layers
round the particles, and that the potential difference between these layers
rises with increasing concentration of acid to a maximum, and then falls.

Action of Alkalis—The action of alkali in destroying the cohesion of
gluten is essentially similar to that of acid, except that the electric sign is
reversed. Inahydrosol of gluten formed by carbonic acid or any other acid

* Hardy, ‘ Journ. Physiol.,’ vol. 33, p. 251, 1905.

42 Prof. T. B. Wood and Mr. W. B. Hardy. —[ Oct. 24,

the protein is charged positively ; when formed by any alkali it is charged
negatively.

It is interesting to note that, when alkali is added, it not only neutralises
any acid present, but also reacts directly with the protein as though the latter
were itself an acid. The alkali, therefore, disappears as such; it is, in point

400

500

200

100

1000 500
Fig. 2.

of fact, neutralised by the protein with the formation of new ions. For
instance, in a particular hydrosol formed by carbonic acid, the particles of
gluten were charged positively, and had a specific velocity 46 x 10~® em. per
second, Sodium hydroxide was added in quantity sufficient to make the
entire solution contain N/1600 of NaOH. The fluid was not alkaline to
phenolphthalein, but in spite of this the protein was now charged negatively
and had a specific velocity of 23 x 10~® cm. per second.

Approximately pure gliadin, dissolved in 70-per-cent. alcohol, shows
relations to acid and alkali the same as those described for gluten. Dropped
into 98-per-cent. alcohol or distilled water, it forms an opalescence, and is
then electro-positive ; in presence of N/4160 of NaOH it is electro-negative.

Conclusions.—The experimental results seem to prove beyond question that
the physical state of gluten—that is to say, the degree of coherence or
dispersion as a hydrosol, is determined by the potential difference between
the particles of the protein and the fluid.

1908. ] Electrolytes and Colloids. 43

The development of such a potential difference between colloid particles
and fiuid has been accounted for in two ways. The first, which may be
described as a purely physical hypothesis, ascribes it to differences in the
speed of the ions of electrolytes present. The colloid particles at any moment
contain within themselves an excess of the most penetrating and rapidly
moving ions present, and they therefore have the charge of that ion. In
presence of acid they will have the charge of the hydrogen ion, in the
presence of alkali that of the hydroxyl ion. This hypothesis was advanced
by one of us to explain the properties of certain proteins of the globulin
class when in solution.* It was also advanced independently by Perrin to
explain the electrical properties of colloidal solutions in general.

The second hypothesis is frankly chemical in nature, and, as applied to
proteins, it may be put as follows:—The protein molecule contains H and
OH groups. Proteins, therefore, as a class are, like their chemical allies the
amino-acids, amphoteric electrolytes. They react with acids and alkalis to
form salts, but the reactions are not precise, an indefinite number of salts of
the form (B),BHA being formed where the value of 2 is determined by con-
ditions of temperature, and concentration, and of inertia due to electrification
of internal surfaces within the solution.

The salt so formed is ionised by the water. Positive or negative ions, as
the case may be, leave the protein face to enter the water face, and form an
electric layer there, while the protein face is left charged respectively
negative or positive.{ On this view, in the particular case under consideration,
the decrease and final disappearance of the potential difference which occurs
when the concentration of acid rises above a certain value would be due to a
suppression of the feeble ionisation by the excess of acid.

The first view seems to be incompatible with certain experimental facts—
such, for instance, as the fact that salts such as LiCl or LiBr, the velocities
of whose ions are in the ratio of about 1 to 2, do not confer any change on
proteins, nor, as Perrin noticed, do they produce any contact difference of
potential between a water and a solid wetted byit. It also ignores the purely
chemical nature of the conditions which govern the formation of colloidal
solutions of metals.§

* Hardy, ‘ Journ. Physiol.,’ vol. 29, p. xxvii, 1903.

+ Perrin, ‘Journ. de Chim. Physique,’ vol. 2, p. 601, 1904 ; vol. 3, p. 50, 1905.

{ Hardy, ‘Journ. Physiol., vol. 33, p. 251, 1905 ; ‘ Roy. Soc. Proc.,’ vol. 79, p. 413, 1907.
§ Burton, ‘ Phil. Mag.,’ vol. 11, p. 425, 1906.

44

The Colours and Pigments of Flowers with Special Reference to
Genetics.

By M. WHELDALE.

(Communicated by W. Bateson, F.R.S. Received October 31, 1908,—Read
January 28, 1909.)

Investigations, of which the following is some account, have been
undertaken with a view to being of assistance in the interpretation of the
phenomena observed in the inheritance of flower-colour. An attempt has
been made to classify, of necessity roughly, the pigments, more especially
those soluble in water, found in flowering plants, and at the same time to
ascertain whether there is any connection between the genetic behaviour of
pigments and their chemical reactions and constitution. On the basis of
this classification it was thought that, at some future time, further investiga-
tions might be carried out in greater detail among the various classes of
pigments.

This account deals more with yellow pigments than with red; attention
was first directed to the yellow group, because of a certain correlation
between reds and yellows observed in the inheritance of flower-colour in
Antirrhinum majus, the relationship suggesting that a greater knowledge of
yellows might be useful in classifying the reds. No detailed examination
has been made of any one pigment, but merely a general survey of the
colouring matters of genera from various natural orders.

A classification in outline of the pigments, other than chlorophyll, found
in flowering plants has been given by many authorities,* but perhaps
a repetition will not be out of place here, as follows :—

A. Pigments in solution in the cell-sap.t

(1) Soluble red-purple-blue pigments known as “ anthocyanin”; that this
term includes several different classes of pigments seems probable
when observations are made as regards their behaviour towards
various reagents. The sub-classes will be described later. :

(2) Soluble yellow pigments known as “aanthein.” Again, various sub-
classes, to be described later, may be made according to their
reactions towards reagents.

* General classifications and properties of pigments, as far as they are known, are

given by Czapek (4) and Zimmermann (13).

t+ Said to be occasionally precipitated or crystallised out on natural concentration of the

cell-sap.

Colours and Pigments of Flowers, etc. 45

B. Pigments associated with specialised protoplasmic bodies—chromo-
plastids—the colour in this case being usually yellow, orange-yellow, orange,
or orange-red. Insolubility in water appears to be a constant characteristic
of this group. Two well-known pigments are included here :—

(1) Carotin—a hydrocarbon of definite and characteristic properties.
According to Zimmermann (13), it is insoluble in water, almost so in
alcohol, slightly soluble in ether, more readily in benzine, and most so in
chloroform and carbon bisulphide. It occurs naturally and can be obtained
artificially in crystalline form, and is microchemically recognised by certain
reagents ; with concentrated sulphuric acid it gives an indigo-blue colour,
at first momentarily violet, and with iodine a green or greenish-blue colour.

(2) Xanthin—again, according to Zimmermann (13), occurs in the plastid
in amorphous form. It is insoluble in water, somewhat soluble in ether,
chloroform, and benzine, but more so in alcohol. Microchemically xanthin
can be recognised by giving with concentrated sulphuric acid a blue colour,
at first momentarily green, and with iodine a green colour.

In addition to the above, there appear to be other plastic pigments, which
do not give a blue colour with sulphuric acid, but a yellow or brown.

Anthocyanin.

Classification of the soluble red-purple-blue pigments has always been
a difficult problem, but there seems to be evidence that anthocyanin is
a general term, including several different pigments. The differentiation
made here is based on the inheritance of colour in certain genera, on the
sequence of flower-colour in cultivated varieties, and finally on the behaviour
of red pigments towards chemical reagents.

There are present in most plants colourless or pale yellow substances,
soluble in water, but insoluble in ether; with strong acids and alkalis they
give a canary-yellow colour, and a similar coloration or most frequently
a precipitate of the same colour with basic lead acetate. In some cases,
Eschscholtzia californica (Courchet(3)), Argemone grandiflora, and yellow
species of Viola, these substances crystallise from extract solutions in
needle-shaped crystals aggregated in clusters or spherules; solutions of the
erystals from the above genera reduced Fehling’s solution slightly, but after
prolonged boiling with dilute acid, a deep yellow substance, together with
a reduction of Fehling’s solution, was obtained, suggesting the glucoside
nature of the crystalline bodies. A similar glucoside is probably present in
Narcissus Tazetta (Bidgood (2) ).

The colour reaction with alkalis and acids is most obvious in parts free

A6 Miss M. Wheldale. Colours and Pigments of [Oct. 31,

from chlorophyll; in unpigmented genera, Galanthus nivalis, for instance,
or in pigmented (anthocyanic) genera such as many Umbelliferw, with white
flowers, the petals turn bright canary yellow with ammonia.

The inheritance of colour in Antirrhinwm (Wheldale (12) ) has led to the
suggestion that anthocyanin is possibly a compound of such a glucoside-like
body with a reddening substance. The original type of Antirrhinwm has
magenta (anthocyanin) flowers.* Loss of the reddening substance, which
may be represented by a Mendelian factor (M), gives a variety bearing
ivory-white flowers containing no pigment (except in the palate and hairs),
but a glucoside-like body giving the reactions with acids, alkalis, and lead
acetate described above. Further loss of a substance, again represented by
a factor (1), from the glucoside-like body in the superficial cells of the lips
gives a yellow xantheic pigment, and the variety thus bears yellow flowers.
Loss of yellow pigment, represented by yet another factor (Y), gives an
albino, containing no pigment and no glucoside-like body. Local decom-
position produces the same xantheic pigment on the palate and in hairs on
the inner surface of the tube in all varieties except the albino. The albino
may carry I or M, or both, since these factors are invisible unless the
fundamental colour Y is present. Moreover, the reddening factor can exist
with Y, the decomposition product, giving a mixed colour, 7. crimson.
Each variety may breed true or may throw itself and one or more varieties
below it in the scale of colour, according as it is homo- or heterozygous in
the various factors. Magenta can throw all varieties; crimson can throw
yellow and white ; ivory, yellow and white; and yellow, white only.

At this point it is interesting, perhaps, to give views regarding the consti-
tution of anthocyanin, based on results obtained from a totally different
kind of investigation. Overton (9) found that in plants supplied artificially
with excess of sugar and other carbohydrates there is a correlated increase in
the production of anthocyanin, and he concludes that the latter, im many
eases, is a glucoside compound of a tannic acid. Mbolisch(8) and Heise (6)
are also of the opinion that some red sap-pigments are glucosidal in nature.
Tannin is by no means always present in plants containing anthocyanin; the
magenta pigment of Antirrhinum gives a tannin reaction, but no tannin has
been found in the albinos.

A similar range of colour to that found in Antirrhinum, 2.c., various shades
of purple or magenta and crimson, together with ivory, yellow, and sometimes
white, also occurs in Althwa rosea, Azalea, Dahlia variabilis, Dianthus
Caryophyllus, Helichrysum bracteatum, Innaria, Nemesia, Phlox Drummondit,

* With yellow pigment locally on the palate and in hairs on the inner surface of the
tube.

1908. | Flowers with Special Reference to Genetics. A7

and Rosa. It is probable that the inheritance of, flower-colour will be found
to be similar in these genera.

The inheritance of colour in Lathyrus and Matthiola (Bateson, Punnett and
Saunders (1)) differs from Antirrhinum, in that two factors (C and R) are
required to produce colour, and the loss of either gives a white containing
only a glucoside-like substance. An additional factor, B, gives the purple
varieties.

A similar range of colour to that in Lathyrus, 1.e., shades of blue or purple
and red, together with white, is shown by Campanula, Digitalis purpurea,
Lberis, Lobelia, Nemophila insignis, Pisum, and many others; no xantheic
variety occurs in this series.

It is evident, then, that there are two classes of anthocyanin as regards the
series of varieties to which each can give rise; in one case the decomposition
possibly of the glucoside-like constituent gives a yellow xantheic form, and
in the other case no such decomposition is possible, and no yellow variety
exists.

The close relationship between xantheic pigments, the glucoside-like bodies,
from which they may be derived, and the anthocyanin, of which these bodies
are themselves possibly constituents, is suggested also by the fact that yellow
xantheic varieties almost always have an anthocyanic type. This connection
is well exemplified among the genera of the Composite. Usually plastid
pigments in addition are present in this order, but these may be disregarded
for the moment. Yellow varieties of Coreopsis, Chrysanthemum carinatum,
Dahlia variabilis, Helichrysum bracteatum, contain xanthein, while the type
has anthocyanin ; other genera, Zinnia elegans, Gaillardia, Hieracium rubrum,
have anthocyanin of the kind which gives no xantheic varieties, while,
finally, Calendula officinalis, Helianthus annuus, Erigeron spp., and Senecio spp.
have no xanthein and no anthocyanin.

When we consider the behaviour of anthocyanin towards such substances
as acids, alkalis, certain salts, etc., we find this term includes at least several
groups of pigments. Two classes have been described by Weigert (11), and
termed “weinrot” and “riibenrot” respectively. The former is soluble in both
alcohol and water, giving with basic lead acetate blue-grey or blue-green
precipitates, and with concentrated sulphuric acid a bright red colour. It is
found in the leaves of Vitis, Ampelopsis quinquefolia, Rhus typhina, Cornus
sanguinea, and others. The latter, riibenrot, Weigert found in leaves
of the Amarantacee and Chenopodiaceee (Beta vulgaris, Iresine, Amaranthus,
Atriplex), and in fruits of Phytolacca decandra. Though readily soluble in
water, the pigment is insoluble in alcohol; with basic lead acetate it gives a
red precipitate, with sulphuric acid and with ammonia a deep violet, but with

48 Miss M. Wheldale. Colours and Pigments of [Oct. 31,

other bases a yellow colour. He further distinguishes a pigment, “Malven-
violett,” occurring in the leaves of Coleus Hero, Perilla nankinensis, Corylus
avellana atropurp., red Brassica, and Malva sp. It is present as a compound,
which, on the action of acids, produces the weinrot pigment.

In the present paper, conclusions are based upon the examination of
relatively few red pigments, and even these present great complexity. Yet, .
on the whole, a rough classification may be given as follows :—

1. A purple anthocyanin, which is characterised by giving a deep blue or
violet colour with ferrous sulphate and ferric chloride, a green colour with
alkalis and a blue or blue-green precipitate with basic lead acetate. Such a
pigment is found in the deep purple and crimson varieties of Dianthus
Caryophyllus, Lathyrus odoratus, Phlox Drummondiu, the purple and plum
varieties of Matthiola (Saunders (1)), purple Fuchsia, and in the berries of
Atropa Belladonna and Rosa pimpinellifolia.

2. A purplish-red anthocyanin, corresponding probably to Weigert’s wein-
rot. This pigment does not give the above reaction with iron salts; but
with alkalis a green colour and a green precipitate with basic lead acetate.
This form occurs in the magenta varieties of Antirrhinum majus, Dahlia
variabilis, pale magenta and violet varieties of Phlox Drummondu, red
varieties of Lathyrus odoratus, Salvia Horminum, and Verbena; also in the
crimson Cheiranthus Cheiri, in Tradescantia virginiana and in Rhus aromatica.

3. A red anthocyanin characterised by giving a red precipitate with basic
lead acetate. Two sub-groups may be further identified as—(qa) those giving
green colour with alkalis—red, copper, and rose varieties of Matthiola, and
a red variety of Delphinium,; (6) those giving a reddish yellow colour with
alkalis—pink varieties of Dianthus Caryophyllus, flesh and terra-cotta
varieties of Matthiola, certain scarlets of ZLathyrus, salmon-rose varieties of
Dianthus barbatus, Phlox Drummondii, Verbena, and the “rose dorée” variety
of Antirrhinum majus.

The above classes seem to show a gradual diminution in the amount of
blueness present as we pass from the very blue purples of Lathyrus, through
the magentas and blue-reds of Antirrhinum and Lathyrus, to end finally in
such varieties as the “rose dorée” Antirrhinum, from which blueness is prac-
tically absent. The bluer form is usually the original type and the reds, as
derivatives, are probably components of the original anthocyanin.

There is some indication of a connection between the chemical behaviour of
the classes and their inheritance. The purple anthocyanin appears to be that
form, which, in Zathyrus and Matthiola, is given when the B factor is present
in addition to Cand R. The extreme purple is not found in Antirrhinum ;
hence in the latter case, when a blueing factor is present, as in the magenta

1908. | Flowers with Special Reference to Genetics. 49

type, purplish-red anthocyanin is the ultimate form. The red of Antirrhinum
is represented by the “rose dorée” type, which finds its parallel in the flesh
and copper of Matthiola, certain scarlets of Lathyrus, and the salmon-rose of
Phlox Drummondit.

It is of interest to note also that in the reds of Antirrhinum and the
salmon-rose of Phlox,* of which several shades exist, the deeper are dominant
to the paler, while in the purples, purple-reds, and magentas of Lathyrus
Matthiola, Phlox, and Antirrhinum, the paler shades are dominant to the
deeper. As regards shades of one colour, a fuller investigation has been
made in Antirrhinum, in which genus every shade is a definite zygotic form,
and the chemical reactions of these shades are fundamentally similar though
differing in degree. The precipitates with basic lead acetate, for instance,
are of varying shades of green, yet these remain unaltered on artificial
concentration or dilution of the extract. This would appear to indicate that
the shade of colour was but an outward indication of some definite organic
compound in the sap.

All the red pigments so far described give, with strong sulphuric acid,t
bright red and yellow colours, becoming orange when mixed; it seems
possible that the red is due to the reddening factor, and the yellow to the
glucoside-like constituent of the anthocyanin. With alkalis, the reddening
factor of the bluish-red turns blue and the other body yellow, the result
being green, sometimes rapidly fading to yellow. Basic lead acetate gives
blue-green or green precipitates, due again to the same mixture.

The scarlet pigment of some genera—Lobelia cardinalis, Phaseolus multi-
jlorus—is again different, in that it gives a bluish colour with alkalis and
a red precipitate with basic lead acetate.

With regard to natural orders and relationships, as far as these investiga-
tions have gone, it appears that the red pigments of the Papaveracee differ
from others. Also those of the allied orders Aimarantacew, Nyctaginacee,
Phytolaccacee, and Portulacacee (included in the Centrosperme by Engler),
form an isolated group giving reactions essentially different from any hitherto
described.

Of the Papaveraceew, the red pigment of Glauctum pheniceum and Papaver
Rheas gives a purple colour with basic lead acetate.

The red pigment of Amaranthus (and other genera of the same order
according to Weigert(11)) is characterised by its insolubility in alcohol.
With concentrated sulphuric acid it gives a purple colour, with ammonia

* T am indebted to Miss Killby for this information.

+ Reactions are best seen by dropping acid on to the pigment on a white porcelain
plate. Mixtures of colours are thus more readily detected than in bulk in solution.

VOL. LXXXI.—B, : E

50 Miss M. Wheldale. Colours and Pigments of (Oct. 31,

a reddish colour, but with other bases a clear yellow, and with basic lead
acetate an orange-red precipitate. In the stems and berries of Phytolacca
decandra, the magenta and crimson flowers of Mirabilis Jalapa and Portulaca
grandiflora, two pigments apparently exist. One, insoluble in alcohol but
soluble in water to a magenta solution, gives reactions on the whole similar
to those given by the red pigment of Amaranthus. The other pigment is
soluble in alcohol to a crimson solution, which gives a yellow colour with
acids and alkalis and a reddish precipitate with basic lead acetate.

Overton (9) is also of the opinion that anthocyanin contains several classes
of pigments, of which he gives—(1) the Amarantaccw and Beta vulgaris,
(2) Papaver Rheas and other species of Papaveracew, (3) Tradescantia
discolor* and other Commelinacee.

The alcoholic solution of the red pigment in some genera is colourless, the
colour returning on evaporation or on addition of acid. In other cases, again,
the alcoholic solution is as deeply coloured as the flowers. It is possible that
these phenomena may indicate a difference in the nature of the pigments.

Lastly, it might be well to mention that anthocyanin is said to occur in
solid and crystalline states in the cell. Many instances are cited by
Molisch (8) in his work on crystalline anthocyanin.

Xanthein.

Xanthein, like anthocyanin, includes at least several pigments varying in
their reactions towards acids and alkalis. They may be classified as follows :—

1. Those giving a deeper yellow, orange, or orange-red colour with acids
and alkalis and similarly coloured precipitates with basic lead acetate.
Such pigments are found in yellow varieties of Althwa rosea, Antirrhinum
majus, Calceolaria, Coreopsis, Dahlia variabilis, Dianthus Caryophyllus,
Helichrysum bracteatum, Phlox Drummondu and Tagetes signata.

2. Those in which the yellow colour becomes paler with acids and alkalis
and basic lead acetate gives, as a rule, no precipitate or a precipitate of the
same colour as the pigment. Such is the case in Mirabilis Jalapa,
Montbretia sp., Nemesia strumosa, Papaver nudicaule, and Portulaca grandiflora.

3. Those in which the yellow colour remains unaltered in the presence of
acids and alkalis, and basic lead acetate gives a yellow precipitate, as in
Mesembryanthemum pomeridianum, Verbascum.

If xantheic pigments are derivatives of anthocyanin, the dissimilarity
of the former among themselves strengthens the view that the reds, from
which they are derived, are also dissimilar.

* T have not been able to detect so far any divergence in this case.

1908. | Flowers with Special Reference to Geneties. 51

Albinism.

‘It has been suggested that there are two forms of anthocyanin giving
respectively two colour series, one containing a yellow xantheic variety, the
other not. Whites occur in both, and it seems probable that the term
albinism should be used in a different sense when applied to each of the
two series.

The extract from most white flowers (also from flowers coloured only with
plastid pigments, when these have been removed), gives a canary colour with
strong acids and alkalis, as stated previously. Without exception, as far as
observations have gone, whites of genera having no yellow sap type, have
given this yellow colour-reaction. These whites may, without hesitation,
be declared to be recessive to the red-purple-blue types, and they are albinos
as regards anthocyanin.

On the other hand, in the case of Antirrhinum, Azalea and Phlox
Drummondii,* belonging to the series giving yellow sap-colour, whites exist
which do not give the same yellow colour-reaction. Moreover, these whites
are recessive to yellow in Antirrhinum and Phlox, and are albinos as regards
both anthocyanin and xanthein. It is the ivory in this series which contains
the glucoside-like body, and gives the yellow colour-reaction.

Whites giving no colour-reaction have not yet been observed in the other
genera mentioned in the anthocyanic-xantheic series, though relatively few
types have been examined. It is possible that the true albino, as con-
trasted with ivory, is rare in commercial samples, since the albino type in
Antirrhinum and Phlox has been found to set poor seed unless fertilised
artificially.

What appears at first an exception to this view is the case of Mirabilis
Jalapa. Here we find a range of colour similar to that in Antirrhinu, te.,
shades of magenta and crimson, together with deep and pale yellow and
white. The white (when it does not carry a reddening factor) is recessive
to yellow,f and yet gives a colour-reaction with ammonia, etc. The
explanation lies in the fact that both the yellow and red pigments in
Mirabilis (see p. 50) are of an entirely different nature from those
in Antirrhinum and Phlor. For the same reason, the inheritance in
Portulaca grandiflora will, if worked out, doubtless prove to be similar to
Mirabilis. |

Shull(10) also gives the case of Verbascum Blattaria, in which a very

* T am indebted to Miss Killby for the information that in Phlox Drummondi the
ivory type may throw both the yellow and the albino.

+ I am indebted to Miss Marryat for this information from results obtained in cross-
breeding of Mirabilis.

E 2

52 Miss M. Wheldale. Colours and Pigments of [Oct. 31,

pale form, perhaps an albino, is recessive to the full yellow. He also states
that Correns found white Polemoniwm cerulewm to be dominant to yellow
Polemonium flavum. If the former contained a glucoside-like body, and the
latter a xantheic pigment, the result might be analogous to Phlox. As
material of these genera was not available, the pigments have not been
investigated, and consequently this conjecture must remain unverified for
the present.

Plastid Pigments.

The plastid pigments, carotin and xanthin, are well-known substances,
of which the properties and characteristics have been investigated. Both
may be present in the same plastid, when the coiour is orange-yellow,
orange or orange-red, and this condition is very widely distributed ; or
xanthin only may be present, when the colour is yellow. In the orange-
yellow or red type the loss of the power to produce carotin in the plant
may give rise to a lemon-yellow variety. This is the case in flowers of
Argemone grandiflora, Calendula officinalis, Tagetes signata, Tropeeolwm
majus, and probably Cheiranthus Cheri and Salpiglossis grandiflora.

In other cultivated genera, where the type contains xanthin, this pigment
appears to give rise to paler yellow varieties containing derivative plastid
pigments, probably decomposition products of xanthin, and giving a yellow
or brown colour with strong sulphuric acid. At present these derivatives
have not been thoroughly examined. They are found in the pale yellow
varieties of Helianthemum spp., Chrysanthemum carinatum, in the autumn
cultivated Chrysanthemum, and in Zinnia elegans.

There is evidence from cross-breeding in Chewranthus and Tropeolum* that
presence of carotin is dominant to its absence, that is the orange-yellow
variety is dominant to the lemon-yellow.

The plastid pigment in cream varieties of Lathyrus odoratus, Matthiola,
Rosa, and Eschscholtzia caniculata rosea, is again different from carotin
and xanthin as regards its chemical reactions. There is evidence from
cross-breeding in Zathyrus and Matthiola (Bateson and Saunders(1)) that —
cream plastid pigment is recessive to its absence, 7.e., colourless plastid.

Combinations of Soluble and Plastid Pigments.

Anthocyanin and plastid pigments are frequently found together in plants.
When the red sap occurs with plastids containing both carotin and xanthin,
the resulting colour is some shade of brown, crimson, or orange-red ; with
plastids containing xanthin only, or some derivative product of xanthin the

* J am indebted to Miss Saunders for information regarding Tropeolum.

1908. | Flowers with Special Reference to Genetics. 53

resulting colour is maroon, purple, or salmon-pink. Hence we find in
cultivated genera containing plastid pigments and anthocyanin a colour
series brown, crimson, or orange, purple, magenta, or salmon-pink, deep
yellow and pale yellow.

Such is the case in Cheiranthus Cheri, Chrysanthemum spp., Heli-
anthemum spp., Salpiglossis grandiflora, Tagetes signata, Tropeolum majus, and
Zinma spp.

This series differs from the anthocyanic-xantheic series in one respect ;- in
the former the type is crimson and the purple or magenta is the derivative,
whereas in the latter the purple or magenta is the type, while crimson is
a derivative. These two contrasting series cannot be better exemplified than
by the two indigenous genera, Antirrhinum and Cheiranthus, and their
cultivated varieties. The wild Chetranthus is deep yellow tinged with
brown; cultivation has produced from the original, a pale yellow type, to
which the addition of anthocyanin gives purple. The wild Antirrhinum is
magenta, which, on loss of some constituent, has given a yellow xantheic
type, and this gives, further, in presence of the reddening substance, a crimson.

Stress should be laid, in connection with colour, on the conception of the
pigmentation of a plant as a whole. The power to produce colour is the
property ot every cell of a pigmented plant; frequently the flowers are
white or show but little colour in piants which are really pigmented, as, for
example, Solanum nigrum, Geranium Robertianum, var. album. In a plant
having red, purple, or blue flowers, anthocyanin may invariably be detected
in the vegetative parts, such as cotyledons, under surfaces of leaves, wounded
or exposed areas, etc. The diffusion of colour throughout the plant is
manifested in the correlation so frequently found between fruit- and seed-
colour on the one hand and flower-colour on the other. De Vries (5) gives
as examples the green-flowered variety of Atropa Belladonna and the white-
flowered variety of Daphne Mezerewm with yellow fruits; also the white-
flowered Zinwm with yellow seeds as contrasted with the brown seeds of the
blue variety. The colour and pattern of seed-coat in Matthiola (Bateson and
Saunders (1)) and Pisum (Lock (7) ) is also correlated with flower-colour in
the same way.

Method for Examination of Pigments.

The material to be examined is ground very finely with powdered glass in
a mortar, extracted with methylated spirit and filtered. If from the colour
of the residue, or from a microscopic examination, the presence of carotin be
suspected, a further extraction is made with benzine or chloroform. The
alcohol extract contains the pigments soluble in water and such plastid

54 Miss M. Wheldale. Colours and Pigments of [| Oct. 31,

pigments as are soluble in alcohol (chiefly xanthin). After evaporation to
dryness on a water-bath, the xanthin is separated by ether from the xantheic
and anthocyanic pigments. The chloroform extract contains both xanthin
and carotin ; the latter can be washed free from xanthin by means of alcohol.

DETAILS FOR GENERA OF VARIOUS NATURAL ORDERS.

The following particulars chiefly concern yellows, the reds having been
dealt with in detail previously.
Aizoucee.

Mesembryanthemum pomeridianum has a xantheic pigment, unaffected by
acids and alkalis and precipitated by basic lead acetate.

Amaryllidacec.

Alstrameria aurantiace has plastids containing carotin and xanthin ;
anthocyanin is present in addition.

Caryophyllacce.

Dianthus Caryophyllus, the parent form of the Carnation, shows the
anthocyanic-xantheic series. The yellow pigment is intensified to orange
yellow by acids and alkalis. Magentas and crimsons are probably produced
by the addition of red sap toivory and yellow respectively. Ivory gives the
yellow colour-reaction with acids and alkalis, but whether it is dominant to
yellow, and whether an albino, recessive to yellow, exists has not yet been
ascertained.

Cistacee.

Varieties of Helianthemum vulgare show the anthocyanin-xanthin series
and pale yellows, which are derivative plastid pigments, probably from
xanthin. The type is crimson, 7.¢. anthocyanin on xanthin, and the magenta
and pink types are due to the same sap-colour on the pale yellow derivative
forms.

Composite,

Calendula officinalis exists in two varieties, orange and lemon yellow; in the
former the colour is due to plastids containing carotin and xanthin, in the
latter xanthin only. There is apparently no anthocyanic form.

Chrysanthemum carinatum shows the anthocyanic-xantheic-xanthin series
with pale yellows containing derivative products of xanthin, and whites
giving the yellow colour-reaction with acids and alkalis, and containing
colourless plastids. Anthocyanin on whites, pale yellows, and deep yellows
gives the usual magentas and crimsons. In the yellows, from which antho-

1908. | Flowers with Special Reference to Genetics. 55

cyanin is absent,a xantheic pigment exists, which gives an orange colour
with acids and alkalis and an orange precipitate with basic lead acetate.

The autumnal cultivated forms of Chrysanthemum resemble the above
species, except that the anthocyanin appears to be of the kind which does
not give a xantheic derivative.

Coreopsis Drummondvi has a brown patch of anthocyanin-containing cells
at the base of the ray-florets. The orange-yellow of the florets is due to
plastids containing ecarotin and xanthin. A xantheic pigment, probably left
as the anthocyanin retreats to the base of the florets, is also present in their
upper portions; it turns orange-red with acids and alkalis and is pre-
cipitated by basic lead acetate as an orange precipitate.

Dahlia variabilis shows the anthocyanic-xantheic series. The yellow turns
a brilliant orange colour with acids and alkalis, and is precipitated as a
deep orange-red precipitate by basic lead acetate. The ivory gives the
yellow colour-reaction. Magentas and purples are anthocyanin on ivory,
and crimsons are anthocyanin with xanthein.

Gaillardia spp. have usually orange-yellow ray-florets, with anthocyanin at
the base. The orange-yellow is due to plastids containing carotin and
xanthin. There appears to be no xantheic derivative.

Gazania splendens has orange-yellow ray-florets with a dark basal patch of
anthocyanin cells. Orange-yellow is again due to carotin and xanthin.

Helianthus annuus has plastids containing xanthin; a pale yellow variety
exists in which the plastids probably contain some derivative product of xanthin.

Helichrysum bracteatum shows the anthocyanic-xantheic series. The white
variety gives the yellow colour-reaction with acids and alkalis. The yellow
contains a xantheic pigment, which gives an intense orange colour with
acids and alkalis, and a similarly coloured precipitate with basic lead
acetate. Magentas and crimsons are due to anthocyanin on white and yellow
respectively.

ieracium rubrum has anthocyanin in addition to plastids containing
carotin and xanthin.

Picris pauciflorus, Senecio Jacobwea, and Taraxacum officinale have only
plastids containing carotin and xanthin.

Tagetes signata shows the anthocyanin-xanthein-plastid series. There is
an orange-yellow variety with plastids containing carotin and xanthin, and
a lemon-yellow variety with plastids containing xanthin only; both yellows
have in addition a xantheic pigment derived from anthocyanin; it is
intensified in colour by acids and alkalis, and gives an orange precipitate
with basic lead acetate. Anthocyanin on the deep yellow gives brown; on
the lemon-yellow maroon,

56 Miss M. Wheldale. Colours and Pigments of [Oct. 31,

Zinnia elegans shows the anthocyanin-xanthin series, with pale yellow
derivative plastids from xanthin. Anthocyanin gives the usual magentas,
pinks, crimsons, and orange on the pale and deep yellows.

Crucifere.

Brassica sinapis has plastids containing xanthin.

Cheiranthus Cheiri has two yellow varieties, a deep and a pale ; the former
has plastids containing both carotin and xanthin. It is probable that the
pale yellow contains xanthin only, though it has not been tested. Antho-
cyanin gives the crimson or brown on the deep yellow, and the purple
varieties on pale yellow respectively.

Matthiola shows the anthocyanin series with purples, reds, and whites,
the latter having colourless plastids; anthocyanin may also exist with plastids
containing a “cream” pigment differing from both carotin and xanthin.

Cucurbitacee.

Cucurbita Pepo has plastids containing carotin and xanthin.

PFumariacece.

Corydalis lutea appears to have plastids containing xanthin and a yellow
xantheic pigment precipitated by basic lead acetate as an orange-yellow
precipitate.

Hypericacee.

Hypericum Hookerianum has plastids containing xanthin.

Legunvinose.

Coronilla viminalis has plastids containing carotin and xanthin ; some
anthocyanin is also present.

Lathyrus odoratus shows the anthocyanin range, purple, red, and white.
Also a “cream” plastid pigment similar to that in Matthiola.

Spartium junceum has plastids containing carotin (?) and xanthin.

LInliacee.

A variety of Liliwm tigrinwm was found to contain plastids and antho-
cyanin. From the plastids a brick-red pigment was extracted giving a blue
colour with sulphuric acid, though no purple was detected; it is probably a
form of carotin.

Linacee.

Linum flavum has plastids containing carotin and xanthin.

1908. | Flowers with Special Reference to Genetics. 57

Loasacec.

Bartonia aurea has plastids containing carotin and xanthin.

\

Malvacece.

Althea rosea shows the anthocyanic-xantheic series. The yellow pigment
is intensified in colour by acids and alkalis and is precipitated by basic
lead acetate as a brownish-orange precipitate. The ivory gives the yellow
colour-reaction. It has not yet been ascertained whether the yellow is
recessive to ivory nor whether an albino exists. Anthocyanin gives various
purples, mauves, magentas, pinks, crimsons, and orange, according as it is
present on an ivory or on a yellow ground.

Nyctaginacee.

Mirabilis Jalapa shows the anthocyanic-xantheic series. The yellow
pigment becomes paler with acids and alkalis, and is not precipitated by
lead acetate. The white variety gives the yellow colour-reaction, but is
recessive to yellow. The red pigment, as already stated, differs from most
other forms of anthocyanin.

Onagracee.

Enothera Lamarckiana has plastids containing xanthin and, in addition,
a pale yellow xantheic pigment intensified to orange-yellow by acids and
alkalis.

Papaveracec.

Argemone grandiflora exists in three varieties, deep and pale yellow and
white. The deep yellow is due to plastids containing carotin and xanthin ;
in addition, a crystalline glucoside, similar to that in Eschscholtzia, is present
in the sap. The pale yellow appears to contain xanthin and the glucoside,
and the white gives the yellow colour-reaction.

Eschscholtzia californica has plastids containing both carotin and xanthin ;
sometimes the margin or outer half of the petals is yellow or the orange
petal is striped with yellow. Examined microscopically the yellow colour
of the streaks and margin is seen to be due to yellow plastids, containing,
undoubtedly, xanthin only. The orange portions contain orange plastids,
earotin being present in addition in these. The sap contains a glucoside
(the soluble yellow pigment of Courchet(3)), crystallising in spherules of
needles. #. Caniculata rosea is cream tinged with pink. Plastids are
present containing only a little xanthin; most of the plastid pigment
appears to be similar to that in cream Matthiola. The glucoside is also

58 Miss M. Wheldale. Colours and Pigments of [Oct. 31,

present, though in smaller quantity than in the orange species. The pink
tinge is due to anthocyanin.

Glaucium luteum has plastids containing carotin and xanthin.

G. pheniceum, an orange-red species, has anthocyanin in addition.

Papaver nudicaule exists in three varieties, orange, yellow and ivory-
white. The pigment appears to be of the xantheic type, which becomes
paler on addition of acids and alkalis, and is not precipitated by lead
acetate.

Polemoniacee.

Phlox Drummondii has already been considered. The xantheic pigment
is deepened by acids and alkalis and precipitated by basic lead acetate as
a deep yellow precipitate. The ivory gives the yellow colour-reaction, and
the albino, which does not give this reaction, is recessive to yellow.

Ranunculacee.

Ranunculus spp. have plastids containing carotin and xanthin.

Rosacece.

Potentilla fruticosa has plastids containing xanthin.
Cream tosa spp. contain a plastid pigment similar to that in cream
Matthiola.
Scrophulariacec.

Antirrhinum has already been fully described.

Calceolaria spp. contain a xantheic pigment intensified in colour by acids
and alkalis, and precipitated by basic lead acetate as an orange-brown
precipitate. In some cases plastids containing xanthin are also present.

Varieties of yellow Nemesia strwmosa have a xantheic pigment, which
becomes paler on treatment by acids and alkalis.

Verbascum Lychnites has a xantheic pigment giving a blue colour with
sulphuric acid, fading to yellow, a yellow colour with alkalis, and a yellow
precipitate with basic lead acetate.

Solanacee.

Hyoscyamus Chloranthus, a yellow species devoid of purple veining, has
plastids containing xanthin.

Physalis Alkekengi has plastids containing carotin and xanthin in the
orange calyx.

Salpiglossis grandiflora shows the anthocyanin-plastid series. The deep
yellow is due to plastids containing carotin and xanthin; the pale yellow

1908. | Flowers with Special Reference to Genetics. 59

probably contains xanthin only. Various purples and crimsons are given on
the addition of anthocyanin.
Tropeolacece.

Tropeolum majus shows the anthocyanin-plastid series. In the pale
yellow variety the plastids contain only xanthin, in the deep yellows carotin
and xanthin. Anthocyanin may be present in addition at the base of the
petals or diffused throughout the flower. Various concentrations of antho-
cyanin on pale and deep yellow give orange-red, salmon-red, crimson, etc.

T. canariense has plastids containing xanthin only.

T. speciosum contains anthocyanin.

Violacce.

Viola tricolor shows the anthocyanin-plastid series. The deep yellow has
plastids containing carotin and xanthin, and in addition a crystalline
glucoside, similar to that found in Hschscholtzia. A paler yellow variety
appeared to contain the glucoside only.

Summary of Results.

1. “ Anthocyanin,” the term used in connection with the red sap-colour in
plants, includes several pigments differing as regards their inheritance, the
colours to which they give rise in variation, and their behaviour towards
chemical reagents.

2. The colours of the varieties arising from an “anthocyanic” type may
be regarded as components of the original “anthocyanin”; the type,
conversely, may be supposed to lose its components (which are expressible
as Mendelian factors) in succession, thus giving rise to a series of colour
variations.

3. Broadly speaking, there are two series of colour variations, one con-
taining a “xantheic” derivative, as, for example, in Antirrhinum majus, the
other no such derivative, as in Lathyrus odoratus.

4. Albinism, in the first series, is a lack of both “anthocyanin” and
“xanthein” ; in the second series of “anthocyanin” only.

a. “ Xanthein,” a term used in connection with yellow sap-colour, includes
several different pigments. This is to be expected if the view that “ xanthein ”
is a derivative of “anthocyanin ” be accepted.

6. There is evidence, as far as investigations have gone, of a correlation
between the behaviour of pigments in genetics and their reactions towards
chemical reagents.

7. In the case of plastid pigments, the type may contain carotin,
xanthin, or both. Varieties arise in some cases from loss of power to

60 Colours and Pigments of Flowers, etc.

produce carotin, or in others from loss probably of some of the constituents
of xanthin.

8. “ Anthocyanin” may exist together with plastid pigments in the type,
in which case derivative products of both forms of pigmentation are found
among the varieties.

I wish to take this opportunity to express my sincere thanks to
Prof. Bateson for his kindness and help throughout this research. I am also
indebted to Mr. John Parkin, who has kindly made various suggestions.

BIBLIOGRAPHY.

1. Bateson, Saunders, and Punnett, ‘Reports to the Evolution Committee of the Royal
Society,’ I, II, and ITI.

2. Bidgood, J., ‘Floral Colours and Pigments,” ‘Roy. Hort. Soc. Journ.,’ December,
1905.

3. Courchet, ‘“‘ Recherches sur les chromoleucites,” ‘ Ann. des Sci. Nat.,’ série 7, 1888.

4. Czapek, ‘ Biochemie der Pflanzen.’

5. De Vries, ‘Species and Varieties, their Origin by Mutation.’

6. Heise, R., “Zur Kenntniss des Heidelbeerfarbstoffes,” ‘ Arbeiten Kaiser]. Gesund-
heitsamt,’ 1894.

7. Lock, R. H., “The Present State of Knowledge of Heredity in Pisum,” ‘ Ann. of the
Roy. Bot. Gardens, Peradeniya.’

8. Molisch, H., “ Uber amorphes und Kristallisiertes Anthokyan,” ‘ Bot. Zeit.,’ 1905.

9 Overton, “ Beobachtungen u. Versuche iiber das Auftreten von rothem Zellsaft bei
Pflanzen,” ‘ Prings. Jahrb.,’ 1899.

10. Shull. ‘New Cases of Mendelian Inheritance,” ‘ Bot. Gaz.,’ vol. 45.

1l. Weigert, “ Beitriige zur Chemie der rothen Pflanzenfarbstoffe,” ‘ Jahresbericht der
K. K. Oenologischen u. Pomologischen Lehranstalt Klosterneuberg bei Wien,’
1894—95.

12. Wheldale, M., “Inheritance of Flower-colour in Antirrhinum majus,” ‘Roy. Soc.
Proc.,’ B, vol. 79, 1907.

13. Zimmermann, ‘ Botanical Microtechnique.’

61

An Experimental Estemation of the Theory of Ancestral
Contributions in Heredity.

By A. D. Darsisuire, Demonstrator of Zoology in the Royal College of
Science, London.

(Communicated by J. Bretland Farmer, F.R.S. Received December 7, 1908,—
Read February 11, 1909.)

The experiments described in the following pages were undertaken with
the object of finding out if the proportions in which characters segregate in
the F2 generation are affected by the distribution of those characters over
the parentage and ancestry of the forms crossed. To this end I crossed a
yellow pea with an extracted green in F;, and obtained a purely negative
result. The proportion of yellows and greens in the F2 generation from this
cross did not differ sensibly from that obtaining in the Fy, generation from
a cross between a pure yellow and a pure green. The nature and result of
the experiment may be summarised in the following pedigree, in which the
two forms which I crossed are enclosed in brackets :—

75°12 per 24°88 per
cent. cent.*
DETAILS OF THE EXPERIMENT.
The Parent Forms.—(i) The Greens.

The original cross which provided me with the extracted F; greens used in
my experiment was made by Mr. C. C. Hurst, and has been fully described

* See Summary on p. 78.

62 Mr. Darbishire. Experimental Estimation of the [Dee. 7,

by him.* In the spring of 1905, Mr. Hurst kindly sent me some seeds, the
cotyledons of which belonged to the F3 generation resulting from the cross
which he had made. All the seeds were borne on a single plant (No. 31), of
which the following is the pedigree, which is copied exactly from the
MS. pedigree which Mr. Hurst sent to me with the seeds :—

“No.1, 2 x Eclipse ¢
B. Queen,+
wy RG.
= > |
(No. 5) RY (F,)
ae pe tS a EWE eed
(No. 31) RY RG wy WG (F,)
aE ae Slee ra ml tant ee
RY RG wy WG (F3)

(Sample here).’’t

I sowed the yellow round seeds of the sample which Mr. Hurst sent me
(i.e., the YR cotyledoned plants of the F, generation) in the spring of 1905,
and harvested F, in the autumn of that year. Of this F, generation only the
YR were, as before, sown in the spring of 1906, and F;§ was harvested in the
autumn of that year. I kept some green round seeds from three plants of
this generation, and it was this seed which produced the plants on which I
made the crosses the results of which are recorded in this paper. The
characters of these three plants are summarised in the following table, which
gives the catalogue numbers of the parent plants and of the “ green” plants||
raised from their seeds, the numbers of the various kinds of seeds borne on
the three parent plants, etc.:—

v S
Catalogue numbers of— ake an aries by <
umber Naeao
of ma er
3 0
Sowing of eee H88H19 plants raised.
Parent plant. | green seeds borne} YR. | YW.| GR. | GW. sete
on it.
|
B55.138......... 483 58 = 21 — 7 i
B54.180 ...... 484, 29 9 a 2 6 5)
B54.1838 ...... 485 41 == 14 — 12 11

* ‘Journ. R. Horticultural Soc.,’ vol. 28, pp. 483—494.

+ British Queen.

t Y signifies yellow, G green, R round, and W wrinkled. Mr. Hurst writes the shape
of the seed first. Throughout the present paper, however, the colour is written first.

§ The proportions of “ yellows,” “ greens,” “rounds,” and “‘ wrinkleds ” in these genera-
tions will be published at a later date.

|| By a “green” plant I mean one raised from a seed with green cotyledons.

4

1908.| Theory of Ancestral Contributions in Heredity. 63

(ii) The Yellows.

I shall now give a brief account of the various strains of yellow peas used
in the experiment. I had been endeavouring for some time previous to 1907
to bring together a collection of peas from widely separated sources, with a
view to repeating the observations of Mendel and others on a broader basis
of material. The yellow strains thus obtained were used in this experiment.
I shall briefly describe them in the order in which they occur in my records,
writing the catalogue number before the description of them.

(477) This is a native Chinese cultivated pea, and was procured for me
in a village not far from Pekin by Dr. Korsakoy, of the Russian Legation in
Pekin. Wen-dow is the name written on the packet in which they were sent.
The peas are yellow and round. The pod and the peas themselves are very
small, and have evidently not been imported from Europe during the last
half century, if, indeed, there is any reason to suppose that they have been
imported at all.

(478) This is one of a set of peas which my colleague, Mr. F. J. Bridgman,
procured from a shipping merchant who was kind enough to put up a few
packets of seeds from cargoes which had been shipped from various parts of
the world. 478 was a particularly fine seed, resembling Victoria marrow,
from a cargo which had been shipped from Germany.

(479) Nineteen seeds of this variety came into my possession at a meeting
of the Natural History Society at the Royal College of Science during
December, 1906. It is a small yellow round pea. |

Through the same channel as 478. All yellow

(481) From Canada.
round,

(480) From Calcutta.
(482) From Russia. }

(492) A yellow round pea which I bought in Genoa in April, 1906. The
parents of the plants which were used in this experiment were grown in my
garden in 1907. A yellow round pea.

(493) The same as 492, from a later sowing in 1907.

Explanation of Tables I—VILI.

Cross-pollination was effected in the following way:—A flower of suitable age was
selected to function as pistil-parent ; one side of its carina was torn transversely, the
whole carina then temporarily slipped off the bunch of stamens, which were removed with
forceps, after it had been determined that they had not yet dehisced. A flower of suitable
age to function as pollen-parent was selected and plucked from the plant which bore it ;
the tip of its carina was cut off and the stigma of the pistil-parent was inserted into the
aperture in the carina made by the cut, and consequently into the mass of pollen which it
contained. The carina of the pistil-parent was then slipped back, and the standard and

64 Mr. Darbishire. Experimental Estimation of the [Dec. 7,

wings folded into their original position. The flower was then labelled. Asa rule, I
pollinated four flowers with the pollen of a single flower, in order to save time. When
more than one flower has been pollinated from a single flower, this fact is indicated in
Tables I and II by writing all the crosses made with the pollen of a single flower on the
same line ; except in cases in which different kinds of pistil-parents were fertilised from
the same flower, when they are connected by a vertical bracket, as in crosses 121—124
(Table I).

The flowers of the pistil-parent were not enclosed in bags after they had been pollinated.
I did not do this, because I had observed that bees did not visit the flowers until they
were open, 7.¢., a day or two after they had been pollinated. The neglect of this precau-
tion was justified by the event. With a single exception, all the crosses which “took”
proved successful, as could be determined in the autumn of the year in which the cross
was made in the case of G? x Y g, and in the following autumn in the case of Y2 xGé.
The exception referred to occurred inacross between Wen-dow and a pure green—Zupress,
viz., cross No. 71 (Table II). When the pod containing the F, cotyledons was opened, it
was found to contain four yellow and one green seed. This was presumably due to the
fact that the flower functioning as pistil-parent was operated on a little too late.

All the crosses between the pure yellow and extracted green are recorded in Table I.
Those which gave no seed are indicated by being printed in ztalics. The date at
which the crosses were made is written against the first of the crosses made on a given
date. Table II is a list of the (successful) control crosses, to be described later on (p. 69).

Tables III to VI give the numbers of yellows and greens in individual families com-
posing F, from the cross between the yellow and the extracted green, and a summary of
all the families composing this F, is given after Table VI. Tables VII and VIII give
details of F, from the control crosses. When there was any doubt as to the colour of a
seed, it was recorded as doubtful. The number of such seeds, if they occur, in each family*
is given in the column headed witha ?. The families of F, cotyledons from the cross
between the pure yellow and the extracted green are given in two lots, in one of which
(Tables III, IV, and V) it has been possible to classify the families according to the green
and. also according to the yellow parent forms from which they have sprung, and in the
other of which (Table VI) this classification has not been possible, owing to an accident.
This occurred during harvesting. The plant or plants bearing the F, families from
cross 166 were accidentally incorporated in a bundle with the plants from some other
cross. I discovered this by finding the peg with 166 on it, after the day’s harvesting,
rather deep in the ground (the top was just covered), as I was hoeing. So that, knowing
the mistake had occurred that day, I was able to know in which bundles 166 had been
incorporated. If I had kept a record of the number of seeds I sowed from each cross, I
should have been able to discover the plants bearing the F, from cross 166 during the
process of recording ; but I had not done this, in these particular sowings, partly because
the ground had not been used for peas before (not in itself a sufficient justification), and
partly because the seeds containing the F, cotyledons had been sown exactly 9 inches
apart, so that if stray seeds did come up, they could not escape detection. (The rows were
carefully examined during the process of weeding, which was done with great minute-
ness with the hand, the smallest seedlings being removed).t The families of F, cotyledons
which cannot, as a result of this accident, be classified according to their yellow and
green parent forms, have been given under a separate heading ; but it must be under-
stood that one of them (which, there is no means of discovering) contained an additional

* By “family ” I mean, in the present case, the number of peas on a plant.
+ I have reorganised the method of carrying out my experiments in such a way that no
further mistakes of this kind can occur.

1908.| Theory of Ancestral Contributions in Heredity. 65

family borne on one or more plants. I tried to identify it by the general look of its seeds,
but although the F, cotyledons produced from the different types of yellows are often
strikingly like their yellow grandparents, I did not succeed. The only drawback,
however, which this accident entails is the impossibility of employing these families for
the purpose of determining the percentage of greens from crosses from the several green
and yellow parent forms. The total result from the F, families not classifiable by parent
forms has been incorporated in the grand total ; as also has a plant which is recorded
as having been “accidentally left behind between 216 and 28 and not belonging to
either.” There were 161 yellow and 50 green seeds on it (see Summary after Table V1).
Fortunately, all the plants from crosses between the pure yellow and extracted green were
grown on a plot by themselves, so that there can be no question as to the parentage of a
plant like this.

The Percentages of Greens in F2 produced from the several Yellow and Green
Parent-forms.

I have classified all the F2 families, from the cross between the pure yellow
and extracted green, into groups according to their yellow grandparents, and
also according to their green grandparents, where this was possible (see
previous section), and have determined the proportions of greens in each of
these groups with a view to finding out whether the close approximation to
25 per cent., viz., 2488 per cent., which was the proportion of greens in
the whole population (including those families which could not be classified
into groups), was also exhibited in each of these groups. The percentage of
greens, calculated from the total numbers of yellows and green in the
classifiable families is 2482. Now it is quite conceivable that this total
proportion may be compounded of widely divergent proportions, ranging
perhaps from 10 to 40 per cent., and each, possibly, characteristic of families
from particular yellow or green grandparents. In order to find out whether
the deviations which might be expected to, and do, occur, are significant, or
attributable to chance, it is necessary to calculate the Probable Error of the
percentage. Half the deviations should fall inside and half outside the
Probable Error in the plus or minus direction; and deviations greater than
four times the Probable Error should not occur often. The Probable
Error of the ratio, expressed as a percentage, of any part to a whole (in this
case the ratio of the greens in a generation to the total number of seeds in
that generation) is calculated by the following formula, which I have used

here :—
100 | o-67449 x Af, ao ( 1 =|
pp eee Sih» Vina S diel Ae

b)

in which « is the whole (or the total number of seeds) and a is the part (or
the number of greens). The table on the next page shows that of the
VOL. LXXXI.—B. F

66 Mr. Darbishire. Eapermmental Estimation of the [Dec. 7,

11 groups, 5 exhibit a deviation in a — direction, and 6 in a + direction.
Also that 7 of the deviations (478, 479, 481, 482, 493, 483, and 484) fall
within the Probable Error and 4 outside, in only one of which is the deviation
greater than thrice the Probable Error—viz.,in 480 in which the actual
deviation is a little over three and a-half times the Probable Error. The
application of this test, therefore, justifies us in concluding that the deviations
from 25 per cent. which occur in each of these 11 groups are attributable to
chance.

Catalogue Per- Actual Probable
number* aa ieee centage | deviation | Error of
of grand- of of per- per-
parent. ROGER at i greens. centage. centage.
Classification by yellow :
grandparents ......... 477 14,081 4,532 24°35 —0°65 +0 '212
478 7,172 2,405 25°11 +011 +0 °299
479 20,761 6,951 25 :08 +0 ‘08 +0°176
480 3,978 1,224 23 53 —1°47 +0397
481 2,777 942 25 33 + 0°33 +0481
482 11,140 3,715 25-01 +001 +0 °240
492 14,157 4,658 24°76 | —0-24 | 40-212
493 1,408 | 486 25 66 +0 “66 +0 °677
Totals ......... == 15,474 24,913 24°82 —0:18 +0092
Classification by green
grandparents ......... 483 38,565 12,788 24°9 —O°'1 +0129
484, 17,150 5,719 25°01 +0°01 +0193
485 19,759 6,406 24, -48 +0°52 +0179
Totals ......... = 75,474 24,913

* For signification of these, see pp. 62 and 63.

The totals have been added up and written under the grouping by yellow
grandparents and under the grouping by green grandparents. But the per-
centage of greens, the actual deviation and Probable Error of the percentage
calculated from these totals, have only been written once, 7.e., under the
grouping by yellow grandparents.

A Special Case.

In the F,2 families raised from five of the crosses there occurred
wrinkled as well as round seeds. All these occurred in crosses made with
the extracted green No. 484, which is the only one of the three races
of extracted greens used, from which, on the Mendelian theory, wrinkled

1908.|: Theory of Ancestral Contributions in Heredity. 67

seeds could arise ; for, as the table on p. 62 shows, neither of the other green
rounds used occurred in a family in which wrinkleds also occurred.
The nature of the mating enclosed in brackets in the pedigree on p. 61 and
of its subsequent progeny is of the type
RR ODD

DR

Pala a
RR DR DD

as far as the colour of the cotyledons is concerned. But with regard to the
shape of the cotyledons, the nature of the mating, in those cases which gave
wrinkleds in F2, was of the type
DR x- DD,

giving equal numbers of DR and DD individuals, the former of which, when
allowed to self-fertilise, should produce rounds and wrinkleds in the pro-
portion of 3 to 1. 14 families from 5 different crosses contained wrinkled
seeds; in the case of 5 of these families I did not record the numbers of
wrinkled, because I was not immediately alive to the interest of the case,
and contented myself with recording the fact that both round and wrinkled
did occur. In the remaining 9 families I recorded the numbers, and the
totals were 2605 round and 914 wrinkled, which gives a percentage of
25°97 wrinkleds. Let us look for a moment at the ancestry, that is to say,
the characters of the ancestors of this cross. The round hybrid, which gave
rise to the F2 in which this proportion of wrinkled occurred, was the result
of the union of two rounds, one of them, the yellow, presumably a pure
round, and the other, the extracted green, a hybrid round. This hybrid
round was descended from an unbroken line of 4 “round” ancestors (each
of them hybrids in the Mendelian sense); whilst behind that point half of
the ancestors were round and half wrinkled. It will be convenient to
display this ancestry in the form of a pedigree (Pedigree A), in which the
crossing which gave rise to the F2 in question is enclosed in a square
bracket. Each ancestor is represented by a circle, and its character is written
immediately to the right of the circle; the nature of the gametes borne by
each ancestor being written within the circle.

Now let us write the pedigree on p. 61 out again (Pedigree B) in the same
way as this, and compare the two (see p. 68).

The result obtained in F2 from the experiment with cotyledon shape
(Pedigree A) affords a more convincing @ posteriori demonstration than that
afforded by the experiment with cotyledon colour (Pedigree B), of the
correctness of the course of leaving the soma out of account in the attempt
to predict the result of a given mating. For, as is clearly seen by a

F 2

68 Mr. Darbishire. Experimental Estimation of the [Dec. 7,

~ 7 SS VA
Neal NI 8 YA
> -
R and R ye} ¥
x
Ts OS Ags
Agee
» © Ox Oa
Ney
74:03 per 25:97 per 75°12 per 24°88 per
cent. cent. cent. cent.
PEDIGREE A. PEDIGREE B.

comparison of the two pedigrees, whereas in the case of cotyledon colour one
of the parents of the hybrids, from the self-fertilisation of which the F»2
generation in question arose, bore the recessive character which reappeared
in the proportion of 25 per cent. in that generation; in the case of cotyledon
shape, neither of the parents exhibited the recessive character. Indeed, in
the latter case the wrinkleds which appeared in F»2 had no ancestors bearing
the wrinkled character for six generations behind them.

Subjoined are details of the families in which segregation into wrinkled
and round occurred. Those in which I did not count the number of
wrinkleds are simply marked DR. The families 158.1 to 6, and 175.1 to 4,
fall into the unclassifiable group (see p. 64). They can, of course, be included
in reckoning the percentage of greens in total of families in which segre-
gation occurred, but not for calculating the relative SULTS of nae wk
DD and DR parents.

1908.| Theory of Ancestral Contributions in Heredity. 69

The actual deviation of the percentage of wrinkleds from expectation is
+097; the Probable Error of this percentage is +0°498: so that the actual
deviation is just less than twice as great as the Probable Error, and is
certainly not significant. Moreover, it is in the opposite direction to that
which would afford any support to the theory of Ancestral Contributions.

Number Nature of parent | Number Number
of with regard to of of
| family. cotyledon colour. round. wrinkled.
| |
Classifiable families ...... 124. 1 DR 249 96
124, 2 DR 219 64
124.3 DR 360 125
165. 1 DR
165. 2 DD
165. 3 DD
165. 4 | DR 393 142
218. 1 DR 162 61
218. 2 DR 408 147
218.3 DR 219 75
218. 4 DD
218.5 DD
Uncelassifiable families ... 158. 1 DD
158. 2 DR
158. 3 DR
158. 4 DR 297 103
158.5 DD
158. 6 DR 298 101
175.1 DR
175. 2 DD
175. 3 DR
175. 4 DD
2605 914
Percentage of greens ............:..... 25°97
Probable Error of percentage ...... +0°498

The number of families (falling into the categories classifiable by yellow
and green grandparents) in which segregation occurred was 8; the number
of families from the same parents in which segregation did not occur was 4.
There should, on the Mendelian hypothesis, be equal numbers of the two
(DE and DD give 50 per cent. DR, and 50 per cent. DD). But I do not
think it desirable to base any conclusion on numbers so small as these.

Control Crosses.

The evidence on which the statement that the greens occur in the pro-
portion of 25 per cent. in the F. generation from a cross between a pure
yellow and a pure green pea is based, has been collected by Mr. Lock in his
most useful paper on “The Present State of Knowledge of Heredity in

70 Mr. Darbishire. Experimental Estumation of the [Dee. 7,

Pisum.”* Subjoined is the table in which the details of this evidence are
summarised by Mr. Lock. I give it in full because it embodies the result
in F, from the cross made by Mr. Hurst, in F; from which the extracted
greens which I used in my experiment occurred. The percentage of greens
calculated from the total is 24:9. The actual deviation is less than the’
Probable Error.

Observer. Yellow. Green. GuEeE,
per cent.

Mendel ene 6,022 2,001 249
Correns) ieeeepecehecesees 1,394 453 24°5
Mschermalkdeseesescs-esces 3,580 1,190 24-9
Bateson .....00.ss0eeleees 11,903 3,903 24-7
U5 hivb5:| cunqHopncenaneatcenseans 1,310 445 25 *4
1 OGY0 ee apenprocesanpacatop 1,438 514 26 °2

25,647 8,506 24-9

Besides the above, I have some evidence based on observations made by
me which more strictly deserve to rank as control observations. This evidence
is based on the result of crosses made between the identical yellow races used
in the experiment, and, with the exception of the few crosses made in 1906,
from tbe pollen from the identical crop of plants which were the yellow
parents in the cross between the pure yellow and extracted (F;) green (see
Table II). The details of the F2 families raised from these control crosses are
given in Tables VII and VIII. The total number of yellows raised was
4015, and of greens 1394, the percentage of greens being 25°77. The Probable
Error of the percentage is +0°401, so that the actual deviation, being less
than twice the Probable Error, is certainly not significant.

The expense of carrying out this experiment was defrayed by a grant from
the Government Grant Committee of the Royal Society, and I take this
opportunity of expressing my indebtedness to them. I also wish to express
my thanks to Mr. Udny Yule and to Dr. E. Schuster for some preliminary
statistical assistance, and to Mr. Charles Biddolph for clerical help.

Summary.

An experiment has been devised to test the truth of the theory of Ancestral
Contributions: the results of the experiments prove :—

(a) That the phenomena of dominance, and, what is more important, of
the segregation of characters in definite proportions, are independent of the
ancestry (and of the geographical source) of the parent-forms mated.

_ * © Annals of the Royal Botanic Gardens, Peradeniya,’ vol. 4, Part III,

1908.] Theory of Ancestral Contributions in Heredity. 71

(0) That the recessive character which reappears in F; is as pure as that borne
by a pure race, as tested by the results obtained from its union with a pure
dominant character.

(c) That there is nothing like Ancestral Contributions within the limits
of a single unit-character.

(d) That in the attempt to predict the result of a given mating the somatic
characters not only of the parents and of the ancestors* of the individuals
mated, but of the individuals themselves,t may be entirely left out of account ;
and that the expectation based on a theory of the contents of the germ cells
of the two individuals mated is fulfilled.

Synopsis oF ConTENTS oF TABLES AND SUMMARIES.

Those tables (viz., II, VII, and VIII) which embody the result of the control
observations (see p. 70) are printed in italics.

Page

Table I.—Matings between Pure Yellows and Extracted (F;) Greens ... 72

*l Table I[.—Matings between Pure Yellows and Pure Greens..............-..: 73
( Table III.—Particulars of F, Families from Crosses made between Pure

| Yellows and Extracted (F,) Green No, 483 ......0.........00e0es 73

Mabey EVE Ditton (NOs 484 yi wi. Me anc sctins caccescuscseaceseeeenet cess ccetuseeece scones 75

Mablewe WED ittow WNOVASON ceascncaicssscosesees sseecestlenocecedasatcesesecsecetasee 7

Table VI.—Ditto. Greens, not Classifiable by Yellow and Green Grand-

parents. (See p. 64) ........csceeseeneees Mey dacdae es acuncaniacan oaks aU

FR, Summary of F, Families from Crosses between Pure Yellows and Extracted
| CB) Cee pd alse Ghee I al ae Un EN eed 78
Table VII.—Particulars of F, Families from Control Crosses made in 1906 78
| Table VIII.—Particulars of F, Families from Control Crosses made in 1907
between the Pure Yellow Types used in the Experiment
and “ Express,” ‘a Pure Green (Round) Type...............s-006 79
L Summary of F, Families from Control Crosses (Tables VII and VIII) ......... 79

* See Pedigree B on p. 68.
+ See Pedigree A on p. 68.

72

Table I—Matings between Pure Yellows and Extracted Greens in F;.

Mr. Darbishire.

Experimental Estimation of the

(See pp. 61—65).

[Dec. 7,

Number'ot ‘Grsecriocstase Number of | Number of | Date of making
2 parent. | ¢ parent. | cross (all 1907).

20, 21 485 493 VI.2
22, 23 492 485 VI.9
24, 25, 26, 27, 28 485 492
75, 76, 77, 78 483 ATT VI.16
79, 80, 81, 82 483 477
83, 84, 85, 86, 87 483 492
88, 89, 90, 91 485 492
108, 109, 710, 111 ATT 483
112 483 4T7
118, 114, 115, 116 483 ATT V1.22
117, 718, 119, 120 483 A477
121 483 479
122, 123, 124 484 479
125, 126, 127, 128 485 479
129, 130, 131, 132 485 492
143, 144, 145, 146 483 479 V1.26
147, 148, 149, 150 483 479
151, 152, 153, 154 483 479
155, 156, 157, 158 484, 479
159, 160, 161, 162 483 492
163, 164 483 480
165, 1667 484, 480
167, 168, 169, 170 484 481
171, 172, 178, 174 484 477
175, 176, 177, 178 484 478 VI.29
179, 180, 181, 182 483 479
183, 184, 185, 186 483 480

| ime
187, 188, 189, 190 485 492

a>

191, 192, 193, 194, 195, 196 484 479
205, 206 483 ATT VIL1
217, 218 484 493

Eo=N
223, 224 485 482 VII.2
225, 226 484, 482
227, 228 478 484
229, 230 485 478 VIL6
231 A8 4 A478
232, 233, 234, 235 478 483
236, 237, 238, 239 482 483
240 483 479 VII.7
241, 242, 243 485 479
263, 264, 265, 266 482 483
267, 268, 269, 270 482 485
271, 272, 273 485 494

(Gait
274, 275, 276, 277 481 484. VII.14

* See p. 64.

Norr.—The association of crosses (such as 129 and 130) by a horizontal bracket indicates that
the two stigmas pollinated were in flowers on the same peduncle.

1908.]

Table I1—Matings between Pure Yellows and Pure Greens.

Theory of Ancestral Contributions in Heredity.

| Number of cross Number of Number of | Date of
| or crosses. | 2 parent. & parent. making cross.
| | | 2
H 29 | 329 | 404. 1.10
| 19064 31, 32 323 | 404
| (40 477 491 VL.9
| 4d. 477 491
69, 70, 71* 491 | 477 | V1I.10
97, 98, 99 480 491 | V1.16
104, 105 491 | 4T7
200 491 479 V1I.29
204 491 Ai
1907 4 208 491 479 VIL.1
210 491 AIT
244 491 479 | VIL7
| 250 | 491 477
252, 254, 255 | 491 478
256 | 491 480
258, 259 491 481 |
(260, 261 491 482 |

* See note on p. 64.

73

(See p. 70.)

Note.—tThe significations of the numbers indicating the parents in the 1907 crosses will be
found on p. 63. The following are the parents of the 1906 crosses (the first three on the list) :—
323 = “ Express” (green round); 329 = Laxton’s “ Alpha” (green wrinkled) ; 404 is the 1906
sowing of the yellow round type which appears as 492 and 493 in the 1907 crosses (see p. 638).

Details of F, Families, produced from Crosses in Table I, classifiable by
Yellow and Green Grandparents.

Table I1].—Particulars of F, Families from Crosses made with

Extracted Green No. 483.

Number Number | Number
of Ye G. 2 of ye G. 2 | oF We Ge ?
family. family. family.
75.1 146 52 77. 4 13 11 | 85.1 74 26
75. 2 82 20 77.5 103 37 Sul Son 73 28
75.3 172 55 2 79.1 107 31 85. 3 138 43 2
75. 4 142 42 1 79. 2 96 30 86. 1 203 54
75.5 149 50 4 79. 3 32 10 86. 2 157 58
75. 6 160 63 1 79. 4 129 4) | 86.3 103 48
73.7 24 14 79.5 172 47 | 86.4 85 31
76.1 196 68 1 81.1 38 22 87.1 38 9
76. 2 133 41 81.2 153 54 87.2 73 27
76.3 64 27 81.3 190 52 87.3 al 27 1
76. 4 118 36 81. 4 152 52 87. 4 122 44. 1
76. 5 283 95 2 81.5 141 44 2 87.5 173 57
76.6 100 31 81.6 63 26 | 87.6 83 32
76.7 114 34 1 83. 1 95 36 | 108. 1 360 114 2
76.8 158 48 84. 1 31 18 108. 2 343 115
Ufleak 8 2 84. 2 67 23 109. 1 41 15
77.2 52 12 84. 3 50 17 | 109. 2 174 58 3
77.3 69 21 84. 4 122 38 109. 3 36 16

74 Mr. Darbishire. Experimental Estimation of the [Dec. 7,

Table [11—continued.

Number Number Number

of Y. G. ? of XG G. ? of ¥- G. ?
family. family. family.
109. 4 33 13 144. 1 40 12 186. 2 300 97
109. 5 66 15 144, 2 20 9 186. 3 216 5 1
109. 6 4l 10 144. 3 249 V7 il 195.1 184 58
112.1 18 15 148.1 361 103 1 195. 2 231 85
112. 2 343 93 148. 2 266 98 3 195. 3 181 65
112.3 241 77 148. 3 19 5) 195. 4 169 63
112.4 163 43 148. 4 175 78 206. 1 346 115 3
a4, 655 129 39 152.1 349 120 5) 206. 2 173 68
113.1 132 47 152. 2 286 95 6 206. 3 608 201 6
113. 2 149 56 152.3 194 58 3 206. 4. 103 39
113.3 75 26 4 153. 1 118 48 4 232. 1 528 180 il
1138. 4 115 45 158. 2 56 20 232. 2 229 84. 2
113.5 124 40 1538. 3 53 8 232.3 241 84. 2
113. br.* 38 15 1538. 4 97 27 3 233. 1 224 86
114, 1 86 22 1 154. 1 270 93 3 233. 2 224 76
114, 2 172 68 154. 2 158 46 233. 3 164 64
114.3 159 45 2 154.3 190 66 3 233. 4 221 79 1
114. 4 279 84 4 154. 4 265 80 233. 5 79 24
114.5 119 52 154. 5 307 96 233. 6 201 75
114. 6 130 45 1 159. 1 91 28 233. 7 59 18
114.7 169 44 159. 2 138 55 233. 8 76 39 1
115.1 1238 36 159. 3 169. 42 235. 1 173 57
115. 2 135 54 159. 4 265 83 235. 2 203 57
115. 3 176 58 3 161.1 360 86103 2 235. 3 99 26
116.1 154 50 161. 2 176 69 3 235. 4 127 47
ileal 376 §=110 161.3 290 78 8 235. 5 206 61 6
117.2 257 97 2 162. 1 156 56 235. 6 139 36
117.3 305 98 2 162. 2 167 65 5 PBY (6 I 483 200 I
119.1 246 72 2 162.3 295 11 2 237.2 364 125 2
119, 2 150 43 162. 4 155 48 238. 1 378 127 1
119.3 72 22 1 162.5 212 71 1 238. 2 430 1384 3
119. 4 30 6 162. 6 159 66 1 240. 1 321 102 4
119.5 317 93 6 164. 1 150 56 2 240. 2 299 98
119.6 375 127 3 164. 2 286 68 4 240. 3 127 37
120.1 161 53 164. 3 151 45 240. 4 263 110
120. 2 87 32 1 164. 4 194 59 2 240. 5 267 76
120. 3 127 46 164. 5 BY) 15 265. 1 165 59
120. 4 121 25 164. 6 247 81 2 265. 2 291 74
120.5 209 64 5 164. 7 222 62 6 265. 3 408 123 5
120.6 250 68 1 164. 8 263 91 10 265. 4 449 138 3
121.1 198 65 164. 9 119 35 8 265. 5 296 98
121.2 288 97 179.1 3384 112 3 265. 6 204 67
121.3 253 97 1 179. 2 481 157 8 265. 7 429 184. 2
121. 4 244 79 179.3 285 108 266. 1 355 128 3
121.5 324 94, aft 179. 4 442 163 266. 2 403 116 6
121.6 379 131 179. 5 391 152 I 266. 3 25 6
121.7 45 10 179.6 504 181 266. 4 465 152 1
121.8 362 109 2 179.7 387 142 af 266. 5 202 64 a i
143. 1 191 56 186. 1 234 72 266. 6 379 129 3

* A branch.

1908.] Theory of Ancestral Contributions in Heredity. 75

Table [V.—Particulars of F, Families from Crosses made with Extracted
Green No. 484.

Number Number Number

of VG G. P of YG G. ? of ae G. ?
family. family. family.
124. 1 260 85 4 191. 2 253 102 lL’ |, 225. 2 1384 44,
124, 2 205 78 2 191.3 310 1038 2 225. 3 339 ©6108 1
124. 3 378 107 1 192.1 269 89 222, 1 1382 51 2
156. 1 279 96 192. 2 311 109 1 | 228.1 207 58 5
156. 2 A419 125 192.3 497 175 228, 2 100 37 1
156. 3 311 90 192. 4 408 144 1 228.3 165 52 1
165. 1 546 168 3 193.1 72 22 228. 4 207 54
165. 2 226 19 6 193. 2 270 87 1 228. 5 206 63 1
165. 3 353 120 193.3 318 126 2 228.6 271 85 5
165. 4 416 119 2 194. 1 215 79 1 228.7 245 89 1
172.1 436 112 194. 2 108 37 23a 133 40
W725 2) 273 84 194. 3 166 48 See Zola? 108 36
172. 3 81 17 1 194. 4 34 vi 231.3 878 151 1
172. 4 265 92 1 194. 5 168 65 6 274, 1 439 141 3
172.5 316 99 1 194. 6 125 34, 1 274. 2 334 88
174. 1 278 97 218.1 165 58 10 275.1 326 §6108
174. 2 237 74 218. 2 422 133 2 275. 2 342 127
178.1 239 98 2 218. 3 213 81 3 275. 3 179 13
178. 2 150 48 2 218. 4 514 178 4 | 275. 4 399 153 1
178.3 339 §=6.114 4 218. 5 94, 36 275.5 875 «127
178. 4 176 58 1 225. 1 3880 154 1 275.6 383 125
191.1 253 82 2 |

76 Mr. Darbishire. Experimental Estimation of the (Dee. 7,

Table V.—Particulars of F. Families from Crosses made with Extracted
Green No. 485.

Number Number Number
of AY és: G. ? of VG G. 2 of re G. ?
family. family. family.

22.1 86 35 91.3 388 128 1 188. 1 165 45
22. 2 28 aati 91. 4 271 86 1 188. 2 172 59
22.3 118 43 125. 1 83 28 1 188. 3 192 72
22. 4 138 30 126.1 72 31 188. 4 195 64
22.5 205 65 il 127.1 371 112 3 190. 1 14 1 2
22.6 220 59 1 127. 2 354 114 1 190. 2 25 4
22.7 206 71 127.3 25 98 190. 3 82 19 3
23. 1 33 12 127. 4 273 94. 1 223. 1 255 83 2
23. 2 174 50 127.5 370 123 223. 2 434 142 6
23.3 78 21 127.6 257 88 223.3 264 82 3
23. 4 230 64. 128.1 262 717 223. 4 411 128 A
23. 5 112 44 128. 2 394 137 229. 1 270 88
24.1 213 78 128. 3 297 100 229. 2 202 56
24, 2 206 66 1] 128. 4 270 75 2 229.3 137 37
25.1 129 35 128.5 328 §6113 229. 4 314 97
25. 2 97 42 128. 6 337 107 242. 1 129 43
26. 1 282 109 128. br 51 16 242, 2 26 4,
26. 2 155 54 1 129.1 216 70 242. 3 1138 3l 1
26.3 237 87 iL 129. 2 275 89 5 242, 4 265 84
26. 4 123 52 130. 1 295 78 267.1 85 28
28.1 153 45 2 130. 2 89 35 2 267. 2 412 122 1
28. 2 96 38 130. 3 269 88 2 267.3 212 73 a
88. 1 123 40 130. 4 65 10 267. 4 234 84 1
88. 2 34 10 130. 5 227 80 3 267.5 179 58
88.3 255 56 131.1 268 87 3 267.6 267 101
88. 4 209 55 131. 2 341 122 6 268. 1 29 5 1
88. 5 1238 36 il 132. 1 153 45 268. 2 79 24 5
88. 6 91 20 132. 2 71 27 1 270. 1 407 118 3
89.1 186 ol 2 132.3 157 52 il 270. 2 133 46
89. 2 149 53 1 1382. 4 225 6) IL 270.3 414 155 5
89.3 33 17 132. 5 260 88 7 270. 4 3820 107 4
89. 4 156 55 1 Spel! 88 33 270.5 22 10
91.1 197 64 187. 2 187 71 4 270.6 404 119 2
91.2 201 72

1908.| Theory of Ancestral Contributions in Heredity. 77

Table V1I.—Particulars of F, Families, from Crosses between Pure Yellow
and Extracted (F;) Greens, not Classifiable by Yellow and Green

Grandparents.
Number Number | Number
of XZ G ? of Xe, G ? | of ave G. ?
family. family family.
idee 194. 68 1 170. 3 189. 3 253 67 1
27.2 141 49 and } 382 123 1 189. 4 200 74.
80.1 169 56 170. 4* 189.5 146 57 A
80. Z 133 35 1 170.5 291 91 1 189. 6 21 9
80. 3 111 29 175.1 5 26 189. 7 247 74 2
171. 237 70 3 UWF 153 49 196. 1 459 170 5
123.1 518 175 7 175. 3 138 34 196. 2 271 91 6
123. 2 216 74 1 175. 4 263 99 196. 3 194. 50 1
128. 3 333 126 3 Ue 205 76 196. 4 270 91 3
123. 4 295 106 0 177.2 248 70 196. 5 122 38
123.5 386 124 2 ides 251 92 196. 6 150 49 1
123. br. 36 16 180. 1 178 53 1 196. br. 12 1
145.1 228 ail 3 180. 2 325 109 3 205. 1 117 34
145. 2 132 35 2 180. 3 283 106 3 205. 2 146 37 1
145. 3 147 59 | 180. 4 264 89 205. 3 261 97
145, 4 174 57 180.5 226 64 2 205. 4 335 118 if
145. 5 214 69 5 181.1 134 43 3 205. 5 265 71
145. 6 311 121 181. 2 45 163 5 224. 1 463 144 3
145. 7 82 27 182. 1 212 61 3 224. 2 71 27
145. 8 260 106 1 182. 2 292 114 4 224.3 455 143 1
149.1 251 87 7 182. 3 290 92 224. 4 447-186
149, 2 490 165 12 182. 4 126 51 239. 1 232 73 1
149.3 192 50 5 182.5 278 79 4 239. 2 105 24
149, 4, 326 =109 5 182.6 351 127 239.3 490 167
149.5 176 42 6 182. 7 406 128 3 239. 4 323 96 1
149. 6 309 106 11 183.1 233 70 2 239.5 136 42
150.1 225 5 ip 183. 2 164 62 239. br. 73 26
155. 1 318 110 183. 3 158 46 1 241.1 388 126 2
155. 2 516 =. 201 183. 4 16 7 241. 2 312 106 1
158. 1 282 109 183. 5 117 39 241.3 119 39
158. 2 132 AT 183. 6 234 74 3 241. br. 25 9
158. 3 146 40 184. 1 254 89 2 243. 1 186 70 1
158. 4 296 104 184, 2 346 108 1 243. 2 153 53
158. 5 268 88 185. 1 283 94, 2 243. 3 189 66
158. 6 304 95 185. 2 350 106 243.4 204 83
167, 1 359 #128 185. 3 309 87 iba 192 68
167. 2 270 93 185. 4 271.2 243 69 2
167.3 519 203 1 and } 560 185 2 271.3 186 73
167. 4 314 86 185. 5* 272. 1 269 96 2
170.1 416 133 2 185. 6 270 112 272. 2 259 66 1
| 170. 2 138 48 1 189. 1 164 52 272.3 133 57
189. 2 19 7 273. 1 419 123

* By an accident the two families bracketed were recorded together.

Nore.—A glance at Tables III to VI (Experimental F,) on the one hand, and at VII and VIII
(Control F;) on the other is, alone, sufficient to reveal an enormous difference in the average size
of the family, z.e., in the average number of seeds ona plant, in these two groups. The superiority
of the former group is due to the fact that the seeds which gave rise to the plants recorded in it
were sown 9 inches apart, that the plot on which the plants grew was dug two spits deep, a heavy
dressing of stable manure being thrown on to the top of the lower spit; whereas the plot on which
the Control F, families (those recorded in Tables VII and VIII) was only dug one spit deep and
received no manure, except a dressing of superphosphate of lime and steamed bone-flour which
was also given to the other plot. I have evidence, as yet unpublished, that the proportion of
yellow and green seeds is not affected by the nutrition of the plant.

78 Mr. Darbishire. Haperimental Estumation of the [Dee. 7,

Summary of F. Families from Crosses between Pure Yellows and Extracted
(F;) Greens.

| Yellow. | Green
Table res see ee seo cdeesos 38,565 12,788
at STV Neat santa ned 6 17,150 5,719
SE rss CR a aN 19,759 6,406
ie ames BO es UN 29,410 9,829
Family referred to on p. 65... 161 50
Motals).cncnss seers 105,045 34,792
Percentage of green) <....s.scenc-coesatecere 24°88
Actual deviation ..........-..-0s-seeeerores —0°12
Probable Error of percentage ........... +0 078

Table VII.—Particulars of F; Families from Control Crosses made in 1906.

Number Number Number
of Y. G ? of Ni G. ? of Ye G.
family family. family.
29.1 108 29 31.4 103 33 32. 4 46 18
29. 2 95 22 32.1 22 9 32.5 72 25
al. 1 73 26 32. 2 60 19 32.6 70 23
31. 2 227 78 32.3 76 27 32. 7 110 ol
31.3 84 19

Norze.—For details of parentage, see Table IT.

1908.] Theory of Ancestral Contributions in Heredity. ve

Table VIII.—Particulars of F2 Families from Control Crosses made in 1907
between the Pure Yellow Types used in the Experiment and “ Express”
a Pure Green (Round) Type.

Number Number | Number
of aye G. ? of We G. ? of XG G. ?
family. family. family.
40.1 24 12 105. 1 12 2 255. 2 32 19
40. 2 52 13 105. 2 12 3 255. 3 64 19
40. 3 49 17 105. 3 38 14 255. 4 27 tf
40.4 ine 4 105. 4 40 12 255. 5 31 10
40.5 49 20 105. 5 18 8 255. 6 20 14
44,1 12 4 105. 6 24 6 25Det 45 18
69.1 15 6 200. 1 22 7 256. 1 34 9
69. 2 9 6 204. 1 44, 14 256. 2 61 16 1
69.3 23 8 204. 2 28 16 2 256. 3 33 22
69. 4 2 2 208. 1 23 7 256. 4 37 11 2
69.5 30 11 208. 2 18 6 256. 5 52 20 1
70.1 23 Wf 208. 3 48 16 258. 1 34 22
70. 2 vi p 210. 1 15 8 258. 2 15 8
70.3 4l 16 210. 2 32 6 258. 3 7 4
71.1 2 1 244, 1 39 13 2 258. 4 43 15
71.2 45 14 244. 2 7 5 259.1 3 2
97.1 i —— 1 244.3 64 24 | 259. 2 8 1
97.2 24 10 250. 1 8 4 259. 3 14 2
97.3 52 19 250. 2 26 11 259. 4 35 11
98. 1 40 9 250. 3 34 18 | 260.1 87 raf
98. 2 51 14 1 250. 4 iii 8 260. 2 21 6
99.1 86 39 250. 5 5 2 260.3 26 7
104.1 15 10 252.1 43 LY 260. 4 69 20
104, 2 29 12 252. 2 29 19 260.5 24 8
104. 3 58 20 254. 1 25 8 261.1 7 2
104. 4 9 2 254. 2 31 16 261. 2 104 32
104. 5 29 11 254. 3 25 12 261.3 74, 22
104. 6 18 Uf 254, 4 88 31 261. 4 51 14
104. 7 28 13 255. 1 53 15 261.5 109 37

Norr.—For details of parentage, see Table IT.

Summary of F2 Families from Control Crosses.

| Yellow. Green.
|
Table aVil lances secnc.n. | 1146 359
sige WLLiyy seers 2869 1035
Totals ...... 4015 1394
iBercentageloisgreenycscccsccssscnsacnsecsse- 25°77
PR CLUSE QEVISDION, fare c cc eee tsaaateeaeeosnees +0°77

80

The Action of the Venom of Sepedon heemachates of South Africa.

By Sir Tuomas R. Fraser, M.D., LL.D., Sc.D., F.R.S., Professor of Materia
Medica, University of Edinburgh; and James A. Gunn, M.A., BSc.
M.D., Assistant in the Materia Medica Department, University of
Edinburgh.

(Received June 30, 1908,—Read January 28, 1909.)
(From the Pharmacology Laboratory of the University of Edinburgh.)

(Abstract.)

The venom used was an extract from the dried venom glands of the
Sepedon hemachates. Its minimum lethal dose by subcutaneous injection per
kilogramme was found to be: for the frog, 0:0012 gramme; for the rabbit,
0:001 gramme; for the rat, 00016 gramme ; for the cat, 0015 gramme; for
the pigeon, 00033 gramme; and, by intravenous injection, for the rabbit,
0:00055 gramme.

In the case of all these animals, the venom primarily and with greatest
intensity affects the respiration. Respiratory paralysis is the cause of death
in mammals and in birds; in frogs, the respiratory movements are early
paralysed, but death occurs after several days from gradual failure of the
circulation. Other conspicuous effects produced by lethal doses in mammals
are drowsiness, ataxia, Impairment of reflexes, and fall of temperature. In
frogs, the venom produces diminution of reflex excitability, motor paralysis,
and progressive increase in weight due to cedema.

In warm-blooded animals, the venom has a marked enfeebling action on
the brain and spinal cord, which is only slightly, if at all, produced on the
motor nerve ends. In frogs, however, motor paralysis is due to a paralysing
action both on the central nervous system and on the motor nerve ends, the
former action being characteristic especially of large doses, the latter being
more pronounced in the late stages of poisoning with smaller doses.

The venom has, comparatively with its action on nerve structures, a very
slight action on skeletal muscle.

From the point of view of lethality, the effects of the venom on the circula-
tion are of minor importance compared with those on the respiration. Perfused
through the frog’s heart, strong solutions of Sepedon venom bring about an
increase of the rate, followed by arrest of the heart in systole; and weaker
solutions slow the heart and arrest itin diastole. The latter effect is the only
one manifested after injection of even 10 times the minimum lethal dose.

Action of the Venom of Sepedon heemachates of South Africa. 81

Strong solutions slightly constrict the frog’s blood-vessels when perfused
through them.

In rabbits, the venom injected intravenously causes a slight fall of blood-
pressure. This is soon recovered from, and thereafter the blood-pressure
rises and remains high till the end of life. The transient fall of blood-pressure
is probably mainly due to a weakening of the heart’s contraction. When
pronounced embarrassment of the respiration comes on, the blood-pressure
rises above the normal level. ‘This is mainly due to stimulation of the vaso-
motor centre by the venous condition of the blood, the heart being at the
same time slowed through stimulation of the vagus. The venom also slightly
slows the heart by a direct action on it, and the direct but slight constriction
of the vessels may be a contributing factor in maintaining the level of the
blood-pressure.

In the course of poisoning in frogs, the lymph hearts are paralysed tardily,
but long before the blood heart.

Sepedon venom has little action on the blood. It does not definitely affect
the coagulability, and neither hemorrhages nor intravascular clotting are found
post mortem. UHemolysis is not found im vivo.

Respiratory failure in mammals is due to erase of the respiratory
centre, the excitability of the iii. -ends being practically unim-
paired. Stina

Non-lethal doses of Sepedon venom cause a rise of temperature ; lethal
doses cause a fall of temperature, with sometimes an initial rise.

VOL. LXXXI,—B, G

82

The Selective Permeability of the Coverings of the Seeds of
Hordeum vulgare.

By AvrIAN J. Brown, Professor of Brewing in the University of Birmingham

(Communicated by Prof. H. E. Armstrong, F.R.S. Received January 23,—
Read January 28, 1909.)

The seeds of the variety of -barley known as Hordewm vulgare var.
cwrulescens owe their colour to the presence of a blue pigment in the aleurone
cells; this pigment, like litmus, is turned red by acids. Such seeds, when
immersed in a dilute solution of sulphuric acid, if their coverings are damaged,
soon turn pink in colour, which is a proof that acid diffuses into the endo-
sperm; sound seeds, on the other hand, although they imbibe water freely
from the solution, becoming soft and swollen, retain their colour, showing that
the covering has the property of resisting the passage of the acid, whilst it
allows water to diffuse freely into the interior of the grain. So much is this
the case that a dilute solution of sulphuric acid may be concentrated by
steeping barley in it. Thus in an experiment with a solution containing
4-9 grammes of acid per 100 c.c. it was found that the concentration of the
acid was increased to 7°6 grammes per 100 c.c. In another case, in which
the weight of water absorbed was ascertained, it was observed that the con-
centration effected was in direct proportion to the amount of water absorbed
by the seeds.

Having made the discovery of so remarkable a “semi-permeable ”
membrane, I have endeavoured to ascertain its behaviour towards substances
generally. In my earlier experiments, of which an account has been given
elsewhere,* it was found that sulphuric acid could not penetrate into the
grain, not only from volume normal solutions, but also from solutions con-
taining 9, 18, or even 36 grammes of acid per 100 c.c. In the case of the
seeds immersed in the strongest acid, however, the interior remained dry,
presumably because the power of the seed contents of imbibing water was
insufficient to overcome the osmotic pressure of the liquid.

The vitality of the embryos was not destroyed by steeping the seeds in the
acid solutions ; when placed under suitable conditions they all germinated.t

When the blue seeds were immersed in a volume normal solution of

* © Annals of Botany,’ vol. 21, p. 79, 1907.

+ Recent observations show that the barley corn displays a most remarkable power of
withstanding the action of sulphuric acid. A number of blue corns, z.e., those containing

the neutral indicator for acid, were steeped in a volume normal solution of sulphuric acid
during 48 hours, those corns which showed traces of red after this treatment being rejected

Permeability of Coverings of Seeds of Hordeum vulgare. 83

hydrogen chloride, the colour remained unaltered, showing that there is no
diffusion of acid into the grain.

Solutions of caustic soda containing 1 per cent. or more of alkali disintegrate
the seed covering; this resists the action of the alkali, however, if the liquid
contain only 4 per cent., and after steeping seeds during several days in the
solution, although water diffuses into the grain, no alkali enters.

Salts such as cupric sulphate, ferrous sulphate, potassium chromate, and
silver nitrate were all found to be impenetrant substances.

Up to this point, it appeared that the covering of the seeds was a perfect
“semi-permeable” membrane. Using iodine dissolved in a solution of
potassium iodide, however, observations were made which indicated that the
membrane possessed the power of selection—iodine was found to pass slowly
into the seed until after several days it permeated the whole of the starchy
endosperm, staining it a deep blue colour. That this result was not a conse-
quence of the destruction of the membrane was proved by steeping seeds thus
impregnated with iodine in a solution of sodium thiosulphate. So long as
the seed coverings remained intact the iodine was unaffected, but when the
coverings were ruptured the thiosulphate diffused rapidly into the seeds
decolorising the iodine.

At this point my earlier studies were directed to an investigation of the
nature and position of the particular covering of the seed of A. vulgare, which
acted as the “semi-permeable” membrane. The experiments already
described demonstrate that the embryo and endosperm of the seeds are
enclosed within an envelope through which water and iodine diffuse readily,
but through which salts and strong acids do not diffuse. As there appeared
to be no recognised instance in the vegetabie kingdom of a membrane other
than one composed of living protoplasm possessing marked “semi-permeable ”
properties, 1t was obviously desirable to ascertain if the selective permea-
bility of the seed-coverings were a function of the living tissue. From the

as faulty. Subsequently, the corns which remained blue were steeped continuously in the
acid, and observed from time to time, with the following results :—

Time Percentage of corns
of steeping. remaining blue.

3 days 100

Dy 99

T a5 95

10 4, 89

TA 74

19) 25

24, 1

84 Prof. A. J. Brown. Selective Permeability of the | [Jan. 23,

first this appeared to be very improbable, as the property was exhibited by
the seeds in the presence of highly toxic acids and salts, which could hardly
fail to arrest its vital activity if brought into contact with protoplasm.
Experiments made with seeds which have been immersed in boiling water
during varying periods extending to one hour have afforded conclusive
evidence that the semi-permeability of the seed-coverings is not a function of
living protoplasm.

Histological study of the seed-coverings indicates that their selective power
is confined to the testa, and probably to that portion which is derived from
the epidermis of the nucellus during the development of the seed.

It appeared to be very desirable that the coverings should be removed and
their behaviour studied apart from that of the seed contents. Although this
has been attempted, the many experimental difficulties met with have not
been satisfactorily overcome, and hitherto no other means of investigating
their activity has been found than the somewhat unsatisfactory one of
experimenting with whole seeds.

Behaviour of the Seed of H. vulgare as a Semi-permeable System possessing a
strong affinity for Water.

When sound seeds of H. vulgare are immersed in an aqueous solution con-
taining a substance which cannot pass through the outer semi-permeable
membrane, and water is absorbed, presumably the seed contents enter into
competition with the solute for the water. From this point of view, it
appeared to be important to ascertain to what extent water would be absorbed
from various solutions, and to compare the amounts with that absorbed when
the seeds were placed in water alone.

The method adopted in all the experiments to be referred to was as follows:
—A known weight of selected air-dried seeds, usually about 5 grammes,
having been steeped in the solution under examination during some desired
period, the seeds were separated from the liquid by means of a small wire-
gauze strainer. After draining for a few minutes they were placed between
the folds of a soft, dry cloth and were rubbed gently to remove as much of
the adherent liquid as possible; they were then weighed. It is obvious that
such a method cannot afford absolute values; but experience shows that
concordant results may be arrived at without difficulty if all operations in
connection with draining and drying the seed are carried out in as constant
a manner as possible. The water absorbed is expressed as a percentage weight
calculated on the original dry weight of the seeds.

The results of a series of observations with solutions of common salt con-

1909. | Coverings of the Seeds of Hordeum vulgare. 85
taining from 2 to 32 grammes of salt per 100 grammes of solution are
recorded graphically in the accompanying diagram.

Hours of steeping.
10 20 30 40 50 60 10) 60 90

$0 | ——_+____+____1__ (SEAS ea 2A

Va

Percentage of water absorbed by the seeds.
3
|
Per cent. solution sodium chloride

SATURATED] SOLUTION GF SAL

It will be noticed that the amount of water absorbed by the seeds when
equilibrium is established is less the more concentrated the solution, as it
varies from about 14 per cent. in the case of a saturated solution to about
41 per cent. in the case of a solution containing only 2 per cent. of salt;
this latter amount is much below that absorbed when the seed is steeped in
water alone (over 70 per cent.).

It should be pointed out that the determinations on which the curves are
based are affected by an unavoidable error due to the manner in which the
values are arrived at. Some allowance should be made for the amount of
water absorbed by the outer covering of the seeds. It is not possible to
evaluate this amount very closely; apparently, however, it may be taken as
equal to about 8 per cent. of the original weight of the dry seed. Making
the extreme assumption that the amount absorbed by the outer covering is
independent of the concentration of the solution, the quantity absorbed by
the starchy contents of the seed from a saturated solution of salt is only
about (14 — 8 =) 6 per cent. calculated on the weight of the dry seed. It
appears, therefore, that the power of the seed contents of attracting water
from a saturated solution of salt exceeds the osmotic attraction of the latter
to only a slight extent; as the “osmotic pressure” of a saturated solution

86 Prof. A. J. Brown. Selective Permeability of the [Jan. 23,

of sodium chloride is about 125 atmospheres, the power with which water is
absorbed by the seed contents probably corresponds to a pressure somewhat
in excess of this value.

Table I contains the results of a further series of determinations, made
with a variety of solutes, in which seeds of H. vulgare were steeped in the
liquid until equilibrium was established; in all cases the solutions were of
volume normal strength, 7.¢., they contained a molecular proportion in grammes
of the dissolved substance per litre of solution.

Table I.
Percentage of water
stalkuss. absorbed.
NaCl caren seesets tations 37 “4
IN@INO3) Wags sceeess cee 37°9
KRG) vi asencce nneececmsiv eens 37-1
KINO a iscrecnsesneenseitees 40 °5
CuSO pee sacneccnisence 41-7
HgSO 4 dsnaveeeeeeaoscess 37 °8
Tartaric acid ............ 42-2
Cane-sugar ............++« 39 °3
Water (control)......... 743

Although the various solutes appear to regulate the diffusion of water
into the seeds in a very similar manner, minor differences are observable ;
thus, contrasting sodium chloride with potassium nitrate, there is an excess
of 3 per cent.in the amount of water absorbed from the solution of the
latter. This small excess was at first regarded as an experimental error,
the method being open to suspicion when small differences are concerned ;
but as all experiments made subsequently with the same salts have consistently
afforded similar results, there can be little doubt that the small departure
is real. Small differences are also noticeable between the values for cupric
sulphate and tartaric acid in comparison with that afforded by sodium
chloride.

As the amount of water present in a volume normal solution varies from
substance to substance, experiments were now made with weight normal
solutions equivalent in strength, prepared by dissolving the solute always in
the proportion of 1 gramme molecular proportion to 1000 grammes
(55°5 molecular proportions) of water. The results obtained are recorded
in Table II, the values in the last column being those observed when
equilibrium was established. Although they are in close general agreement
with those obtained on using volume normal solutions, small specific
differences between the salts become apparent, the potassium salt being less

1909. | Coverings of the Seeds of Hordeum vulgare. 87

Table II.

Percentage of water absorbed after steeping the seeds during—

Solute.
2 days. 4, days. 6 days. 8 days. 11 days.
RW a asthe stam tete coke 31 °2 36 °5 36 °9 37 °3 38 4
Nn Clee es neene ne ee. 30°5 34 °2 Spit 37 °3 37 °2
ANGEL, Gline Ase bs teehee: 31°7 34 6 36 °4 36 6 37 °*4
IKON Oe as rcshesenciseteceace 341 38 °7 40 °5 Al ‘1 41°6
NAN OS sek eae k sf eek 32°5 36 °3 387 °5 38 °7 38 ‘5
INNO ooe ccs cso dicvsoss 32°3 36 ‘1 38 °3 38 “4. 38-9
Water (control)......... 43-1 55 6 64:1 68 °3 70°0

active in the case both of the chloride and nitrate than either the sodium or
ammonium salt, which behave alike.

In Table III are recorded the results of a direct comparison of the
behaviour of the seeds towards dextrose and cane sugar in comparison with
sodium chloride, in weight normal solutions. It will be seen that, although
distinctly less active than salt, these two substances both inhibit the
absorption of water to a very marked extent, being about equally effective.

Table ILI.

Percentage of water absorbed in—

Solute.
2 days. 4, days. 6 days. 7 days. 11 days.
Cane-sugar ............ 29°5 34°3 36 °2 36 ‘9 38 °*4
Dextrose) | 7. dsssssne-% 30 °2 35 °8 38 “1 38 °3 39 ‘8
INGO) e decetueeceesentaav 28 1 31°9 34 °2 84: °4 35 °5

Selective Permeability of the Seed-coverings of H. vulgare.

Mercuric Salts—When barley seeds are steeped in a 3-per-cent. solution
of mercuric chloride in water, the salt may be detected within the seed
covering after a few hours; after two or three days it is usually found
diffused throughout the whole of the interior portions of the seeds.

At first it seemed possible that the passage of the salt into the seed
ought not to be regarded as proof of a selective property of the seed-
covering, but perhaps merely as an indication that the action of the salt
destroyed their semi-permeable character. Experiment, however, has proved
conclusively that this is not the case. When seeds which have been steeped

88 Prof. A.J. Brown. Selective Permeability of the [Jan. 23,

in a solution of mercuric chloride during several days and then dried were
steeped in a normal solution of sulphuric acid, it was found that the
coverings still retained their original power of resisting the diffusion of
sulphuric acid while permitting the diffusion of water into the seed.

Still more conclusive evidence of the possession by the seed-coverings of
a differentiating power was furnished by an experiment in which seeds were
steeped in a mixture of half a volume of a saturated solution of mercuric
chloride with half a volume of normal sulphuric acid. After three days’
steeping in this solution the mercuric salt was found to be diffused
throughout the contents of the seed; even after five days’ steeping, however,
no trace of sulphuric acid could be found within the seed-coverings. The
seeds of H. vulgare therefore possess the very remarkable property of
absorbing mercuric chloride and rejecting sulphuric acid when steeped in
a solution in which both are present. The exhibition of this property by
the seeds appears to be of very special interest from a physiological point
of view.

To ascertain their behaviour towards mercury salts generally, seeds were
steeped in solutions of mercuric chloride, cyanide, nitrate and sulphate of
equimolecular strength. The cyanide diffused as readily as the chloride
into the seeds, but after several days no trace of mercuric salt could be
recognised in those placed in the solutions of nitrate and sulphate. Moreover,
the amount of water absorbed by seeds from solutions of mercuric chloride
and cyanide did not differ from that taken up from water alone.

It should be noticed in passing that chloride and cyanide of mercury—
which diffuse through the seed-coverings—are commonly regarded as but
very slightly dissociated in aqueous solution, whilst mercuric sulphate and
nitrate—which cannot penetrate the membrane—are salts which are supposed
to be freely dissociated.

Cadmium Salts—On steeping seeds of H. vulgare in volume normal
‘solutions of cadmium iodide, chloride, and sulphate until equilibrium was
established, the following results were obtained :—

Table IV.
Percentage of water
oe absorbed.
(ill Pyssenagasaeabedcseso00e 54:2
(Gist O) Fi ecReanaswoadcusaGece 46 °3
CASO eteseccaseeeerescene 46 -O
NaCl (control) ......... 39 °8

1909. | Coverings of the Seeds of Hordeum vulgare. 89

It will be noticed that the gain in weight in presence of the iodide is
markedly greater than in presence of the chloride or sulphate, but that none
of the salts has so considerable an effect as sodium chloride.

In the case of the seeds steeped in the solution of cadmium iodide—a salt
which is supposed to be very slightly dissociated when dissolved in water—
small quantities of the salt diffused through the coverings; cadmium was
not detected, however, in the seeds which had been steeped in solutions of
either the chloride or the sulphate, which are supposed to undergo dissocia-
tion to a somewhat limited extent, although to a greater extent than the
iodide.

Acetic and other Weak Acids—On searching for compounds capable of
passing through the seed-coverings, when it was found that acetic acid
possesses the property, it at first seemed probable that the acid might be
capable of destroying the semi-permeable layer of the seed. Seeds were
therefore steeped in solutions containing both acetic and sulphuric acids or
acetic acid and cupric sulphate; in both cases only acetic acid and water
diffused through the coverings.

Water also passes freely together with the acid into the seeds. The
results of experiments with a volume normal solution of the acetic acid
are recorded in Table V; it will be noticed that the acid has but a slight
influence in diminishing the amount of water which is absorbed when
equilibrium is established.

Table V.
Percentage of water
sole, absorbed.
JNGHIO BOG! scoracgooepseoonencoo 400 73 °8
Water (control).................. 78 °2
Sodium acetate (control) ...... 39 8

On examining the behaviour of the seeds towards organic acids other
than acetic, it was found that formic, propionic and butyric acids also
enter the seed system, and that they affect the introduction of water much
as acetic acid does.

Glycollic acid, although excluded during about 48 hours, subsequently
diffuses slowly into the seed. Lactic acid did not enter most of the grains
until after the lapse of 72 to 96 hours. The amount of water absorbed by the
seed is diminished more by these acids than by acetic acid, thus :—

90 Prof. A. J. Brown. Selective Permeability of the [Jan. 23,

Table VI.

Percentage of water absorbed during—

Solute.
2 days. 4, days. 7 days. 9 days. 10 days. | 11 days.
Glycollic acid ......... 335 43 “9 51°5 57 6 595 63 °4
NEKO) en ee hanes eeacteedanes 28 2 31°9 341 35 °2 35 °7 36 °0
Water! cscceusnelerses: 40-1 53 °6 63 ‘8 68 6 69-0 70°3
11 days 13 days.
Lactic acid ............ 387-4 45 ‘1 52°8 55 ‘1 58 “1 61 °4
BNEW O1 Ia scapcodororee ccabre 309 34 °4 35 8 36 °8 36 °5

Trichloracetic Acid.—This acid was chosen on account of its similarity in
configuration to acetic acid, from which it is distinguished, however, by being
a strong electrolyte, acetic and the other acids and the salts which diffuse
through the seed-coverings being all weak electrolytes.

On immersing seeds in a solution containing 5 per cent. of the acid, it was
found to enter them very rapidly, so much so that after 48 hours they were
saturated with it. This result was clearly not due to any destructive action
of the acid on the seed-coverings, as when seeds saturated with a solution of
trichloracetic acid were immersed in a solution of sodium bicarbonate, the acid
within the seeds remained unaffected even after the lapse of 10 days. In
a control experiment, seeds impregnated with acid, of which the coverings
were intentionally damaged, were placed in a solution of the bicarbonate ;
this soon entered the seed and in a few hours neutralised the acid. Trichlor-
acetic acid is the only strong electrolyte which has been found to possess the
property of diffusing into the seed system.

Ammonia.—The membrane is more or less injured by exposure of the
seeds in solutions of ammonia of weight normal strength, as acid penetrates
into the corns after they have been steeped in such a solution. On the other
hand, when corns which had been steeped in one-half or one-quarter normal
solutions of ammonia were dried and then exposed in a normal solution of
sulphuric acid during 48 hours, no acid was found to enter. The velocity
with which water is absorbed from solutions of ammonia is remarkable, as
shown by the results recorded in Table VII.

The ammonia passes into the corns with the water; on the other hand,
when the corns impregnated with ammonia are placed in a normal solution of
sulphuric acid, after 24 hours they are no longer alkaline internally, the
ammonia having passed out in the reverse direction.

1909. | Coverings of the Seeds of Hordeum vulgare. 91

Table VII.
Percentage of water absorbed.
2 days. 4 days. 6 days. 8 days.
Ammonia—

Normal solution............0c000e00 53 °5 70:1 74°8 17 °2
oy 7 As lt cednonadenaenporentda 53°9 68 °5 73°5 74.°2
= & Bot i BRS DRESHBREAEoeaacucn 51°5 65 °9 4()B} 72 °2

Sodium chloride (control)—

iNormalesolutioneeceeesssstcieeeeter 29-4 32 2 333 34 °6
dea, 1h COINS Ln OP aan 32-2 37:1 39-4 40 °4
Bg es ieee ne eae 35:1 41-7 45-7 48 *4

{WW GUS? Ghaee dacanones boRB EE cee Rae Hae CERO Haar 43 °1 55 ‘6 641 68 °3

Non-electrolytes—As previously pointed out, cane-sugar and dextrose
resemble the electrolyte sodium chloride in their power of diminishing the
extent to which water is absorbed by the seed system, and in being unable to
penetrate the seed-covering.

Experiments made with a number of non-electrolytes of much lower
molecular weight than the sugars show the behaviour of these to be com-
parable with that of weak electrolytes.

The following table contains the results of experiments with volume normal
solutions of ethyl alcohol, aldehyde, acetone, and ethylic acetate. For purposes
of comparison, the results obtained at the same time with water and with
volume normal solutions of acetic acid (representing a freely diffusible solute)
and of sodium acetate (representing a non-diffusible solute) are given :—

Table VIII.

Percentage of water
StohnUGs absorbed.

IDR ALENKEING onocacsoonensbreshenosn
JNA EE “pon acoobosaboccubonaApenasopee
PAICELON Clan eeenespieteeaciancdacsescsce
Ethylic acetate ...........0....01cs000
Acetic acid (control) ...............
Water (control) .......,........-----
Sodium acetate (control)............

owt
OODWWwNoOn
SyYoEIWAS

The results indicate that water is absorbed by the seed-system from solutions
of alcohol, aldehyde, acetone, and ethylic acetate approximately as it is absorbed
from that of acetic acid, or when in contact with water alone.

Very similar results were obtained on using weight normal solutions.

Experiments in which seeds were placed in contact with alcohol, aldehyde,
acetone, and ethylic acetate in the anhydrous condition have shown that these

92 Prof. A. J. Brown. Selective Permeability of the [Jan. 23,

Table IX.
Percentage of water absorbed during—
Solute.
2 days. 4 days. 7 days. 9 days. 11 days.

Ethyl alcohol............ 43 *4 54 °9 66 °9 68 “7 69 6
Ethylic acetate ......... 63-9 70 °7 728 (4 aL 71°8
ALGAE OC oossnasna9079 53 °3 67 °6 68 °3 68 °5 68 ‘0
Water (control)......... 45-0 55 6 65 °5 68 ‘9 705
NaCl (control) ......... 309 34 °4 35 8 36 °8 36

substances do not diffuse through the seed-coverings in the absence of water,
although they all diffuse readily into the interior of the grain from their
aqueous solutions.

It is also interesting to note the manner in which the velocity with which
the different solutions are absorbed varies. The solution of ethyl alcohol
enters comparatively slowly, at about the same rate as pure water; that of
acetic acid enters more rapidly ; whilst the rate of entry of the solution of
ethylic acetate is markedly the most rapid of the three. Nevertheless,
despite the differences of velocity, equilibrium is established between the
seeds and the three solutions at approximately the same point.

A further series of observations with solutions of non-electrolytes are
recorded in the following table :—

Table X.
Percentage of water
sole. absorbed.
Glycerol... ccsesccsetausssedeneseancse 41 °5
GAR E® pnaooaosc0apsnn90880008000009 41 ‘8
WW tearm To ernsecin sores eaciesisieeatecseneiaaress 45 °S
Ethylene glycol.................00+ 52-7
Sodium chloride (control) ...... 36 °5
Water (control)................060e+ 20 °5

The results obtained with glycerol and glycine resemble those afforded
by cane-sugar and dextrose (see Table III), but differ very markedly from |
those obtained with such compounds as alcohol and acetic acid. The
behaviour of glycine or amino-acetic acid is particularly interesting, as this
compound differs to so slight an extent in constitution from acetic acid.
Urea and ethylene glycol have less influence than either glycerol or glycine ;
glycol, however, although it differs to so slight an extent from alcohol in
constitution, is far more effective in preventing the entry of water.

1909. | Coverings of the Seeds of Hordeum vulgare. 93

Summary of Conclusions—The investigation of the selective properties of
the semi-permeable seed-coverings of H. vulgare described in this paper should
be regarded as pioneer work only ; much further study is required in order
to explain the varying actions of the seeds in the presence of different solutes
in aqueous solution.

At present, the general trend of the evidence tends to show that solutions
of the solutes which diffuse readily through the seed-coverings differ in some
essential manner from solutions of non-diffusible solutes, although the nature
of the difference remains unexplained. The results of some of the earlier
experiments described above appear to support the view that the property of
diffusion is intimately associated with a low degree of “ionisation” of the
solute; yet the conspicuous instance which has been noticed of the ready
diffusibility of trichloracetic acid, a highly “ionised” acid, tends to show
that such correlation, if it exist at all, is not an intimate one. Further, the
view does not appear to be favoured by those experiments which have
demonstrated that certain non-electrolytes, such as ethyl alcohol, are readily
diffusible, whilst others, such as glycerol, are non-diffusible.

In connection with the same question, it seemed possible that differences
in the surface tension of solutions of diffusible and non-diffusible solutes
might perhaps be associated in some way with the different behaviour of the
two classes of solutions towards the seed-coverings; but it appears from
a study of the surface tensions of the two classes of solutions that there is
no such intimate connection between them. Neither can any indication be
found that viscosity is associated with the manner in which diffusible and
non-diffusible solutes behave differently towards the seed-coverings.

The only explanation of the observed difference in activity of the two
classes of solutions which at present suggests itself as a working hypothesis
is, that some unrecognised peculiarity in the manner in which the molecules
of the two classes of solutes are combined with the molecules of the solvent
water may constitute the factor which orders their different behaviour with
respect to the seed-coverings. This hypothesis appears to be supported by
the experiments which demonstrate that, whereas readily diffusible solutes
enter the seed together with a large amount of water, seeds placed in
solutions of non-diffusible solutes absorb water with some difficulty.
Moreover, the observation that an aqueous solution of alcohol diffuses
readily through the seed-coverings which are impervious to this solute
in the anhydrous state, appears to show that some form of combination of
solute and water is necessary to condition diffusion of the solute through the
seed-coverings.

94

The Origin of Osmotic Effects. Il.—Differential Septa.
By Henry E. Armstrone, F.R.S.

(Received January 23,—Read January 28, 1909.)

I have had the privilege of following the progress of the inquiry of which
an account is given by Prof. A. J. Brown in the previous communication and
of watching the development of the exquisite and invaluable method of study-
ing the osmotic process which he has devised; I trust that I shall not be
presuming if I discuss the results which he has arrived at and attempt to
interpret them in the light of views already placed before the Society in my
communication on “The Origin of Osmotic Effects”* and in the series of
“ Studies of the Processes Operative in Solutions.”

Prof. Brown’s observations appear to be extraordinarily significant, as
affording the means of dividing substances broadly into two classes accord-
ing as they will or will not diffuse through a membrane such as that which
forms the outer covering of the seed of barley (Hordewm vulgare) and with the
aid of the classification thus secured of arriving at an explanation of the
selective process.

Inasmuch as the barley grain contains but a small amount of soluble
erystalloids, the absorption of water by the grain may be regarded as mainly
conditioned by the extremely minute granules of starch enclosed within it ;
presumably these have great attraction for certain molecules in the liquid
and become coated superficially therewith. From this point of view the
method developed by Prof. Brown involves the study of a struggle for hydrone
between a mass of fine particles of solid and the solution of a substance
present in the liquid state in solution in water; the observations are the
first of their kind, I believe.

It is clear, although the method affords only approximate results, that the
conclusions to be deduced as to the relative “ concentrating” efficiencies of
the several solutes are in general accordance with those arrived at in other
ways. The observations made in my laboratory show that chlorides are more
active than nitrates in solution and that sodium salts are more active than
either potassium or ammonium salts—more active, that is to say, in the sense
that they exercise a greater concentrating effect; this is precisely the result
arrived at by Prof. Brown.

No division of the substances into electrolytes and non-electrolytes can be
made in any way corresponding to the extent to which water is absorbed

* ‘Roy. Soc. Proc.,’ 1906, A, vol. 78, p. 264.
+ Ibid., p. 272 (I); II—V, vol. 79, 1907, pp. 564—597; VI—X, vol. 81, 1908,
pp. 80—140.

The Origin of Osmotic Effects. 95

from the solutions by the grains—cane-sugar is nearly as active as common
salt. In like manner, not only are strong acids and most salts indiffusible
through the membrane covering the grain but also not a few non-electro-
lytes; the membrane is slowly permeated by certain weakly oxygenated
organic acids, by salts such as mercuric chloride and cadmium iodide, by
iodine, by ammonia and by a number of non-electrolytes of low molecular
weight.

The compounds which penetrate the membrane, whether electrolytes or
non-electrolytes, are all substances which attract water presumably only to
a minor extent and which exist to some extent in solution in an unhydrated
condition ; those which cannot penetrate it, on the other hand, probably all
form hydrates of considerable stability in solution.

I picture surfaces generally, colloid surfaces in particular, as not merely
wetted by water but as more or less hydronated and hydrolated—using these
terms in the specific sense explained in No. VIII of my “Studies on
Solutions”; that is to say, they are not merely wetted by water complexes*
but associated with hydrone, the simple fundamental molecule of which water
is composed. The intramolecular passages in a colloid membrane, if thus
hydrolated, would be guarded by the attached hydrone molecules; molecules
in a solution bathing the membrane which attempted to effect an entry
through such passages, if hydrolated, would be seized upon and held back in
virtue of the attraction which the two hydrolated surfaces—that of the
membrane and that of the solute—would exercise upon one another. The
hydrolated passages, however, would be indifferent to molecules which were
not hydrolated—consequently, a substance such as acetic acid, of which
probably only a small proportion is present in solution in the hydrolated
State, would gradually pass through them.

The apparently exceptional behaviour of trichloracetic acid, which must be
more fully if not more firmly hydrolated than acetic acid, is very striking and
may be taken as proof that the hydrolation must extend over a certain area
to secure protection against penetration ; it should be noted, however, that
the result is in accordance with the behaviour of the acid as a substitution
derivative. Hydroxy- and amino-acetic acids (glycollic acid and glycine),
which are far weaker acids, are nevertheless far less easily diffusible—pre-
sumably because not only the carboxyl group of the acid but also the adjacent
hydroxyl- or amino-group is hydrolated. The behaviour of glycol, C.H,(OH)»,
in comparison with that of alcohol, C2H;(OH), may be interpreted in a similar
manner. The concentrating effect exercised by the sugars has already been

* Compare ‘Chemical News, Jan, 15 and 22, 1909, pp. 28 and 37; ‘Science Progress,’
Jan., 1909, No. XI, p. 484.

96 The Origin of Osmotic Effects.

considered from this point of view in our previous “Studies” (VIII,
pp. 111112; X, p. 130).

The exceptional rapidity, to which Prof. Brown directs attention, with
which ethylic acetate acts in promoting the entry of water into the grain is
also easily explicable from the same point of view. Entering together with
water, it should render water within the grain more active and more attractive
of external water (by promoting its dissociation, (H,O), — «H.O) than the
water would be which entered alone from a solution of an indiffusible solute,
as in such water (on account of its homogeneity) the osmotic stress would be
at a minimum.*

It is obvious that the argument now put forward may be applied to the
discussion of a great number of more or less obscure physiological phenomena.
It may be desirable to consider the rise of the sap in trees from such a point
of view. The argument affords an explanation of the well-known efficacy,
for example, of mercury salts, of iodine and of alkaloids as drugs. It should
point the way to the production of medicaments adjusted to their purpose—
according as it is desired that they should penetrate this or that membrane.
It may lead to the discovery of a method of using stains as the means of
determining whether this or that membrane or layer in a cellular tissue is
to be regarded as a mere sieve or as differentially penetrable, masmuch as
stains—whieh hitherto have been used all but empirically—must vary greatly
in penetrative power and it should be possible to grade them, according to
their diffusibility, by observations similar to those made by Prof. Brown.

* [February 15, 1909.—Attention has been specially drawn in No. VIII of our Studies
(p. 108) to the behaviour of methylic acetate as a weak hydrolyte in comparison with the
strong hydrolyte cane-sugar; the observations now under discussion appear to afford
complete confirmation of the argument there put forward that in discussing the phenomena
of hydrolysis it is necessary to take into account not only the condition of the medium
but also the nature both of hydrolyte and of hydrolyst, which are reciprocally concerned
in the change. The argument should be extended to colloid and other surfaces. Sir
James Dewar has shown that solids differ greatly in their power of attracting and holding
gases at low temperatures; hydrolytes and dissolved substances generally, we must
suppose, also differ in the extent to which they undergo “hydration” ; wetted surfaces
generally must also differ in the extent to which they become hydrolated ; consequently,
it is to be supposed that more or less considerable variations will be met with when
differential septa are studied comparatively. Appaxently the barley septum is not
penetrated even by ammonium chloride, so that it is more exclusive than that of red
blood cells, which are rapidly penetrated by this salt but scarcely if at all by ammonium
sulphate. The difference between ammonium chloride and ammonia is very striking, the
latter resembling ethylic acetate in passing rapidly into the seed and in promoting the
ingress of water; this behaviour is easily understood, as it exists in solution partly in
the free state and partly, it may be supposed, as the hydrone H,N : OH,, the hydroxide

being present in only very small proportion. If ammonia were contained in solution as
the hydroxide, its behaviour would undoubtedly be that of caustic soda. ]

97

On the Determination of a Coefficient by which the Rate of
Diffusion of Stain and other Substances into Living Cells can
be Measured, and by which Bacteria and other Cells may be
Differentiated.

By Hueu C. Ross, late Surgeon R.N., Pathologist to the Royal Southern
Hospital, Liverpool.

(Communicated by Major Ronald Ross, C.B., F.R.S. Received December 9,
1908,—Read February 11, 1909.)

[PLaTE 3.]

In former papers (3, 4, 5) it has been shown that when blood is spread
upon a film of agar jelly which contains Unna’s stain and certain salts, the
cells will absorb the stain, and that the absorption increases with the tempera-
ture and the time during which the cells have been resting on the film. The
following facts have also been published :—(1) That alkalies, like heat and
tvme, increase the diffusion of stain into the cells; (2) that acids and neutral
salts delay the diffusion; and (3) that the staining of the nuclei of leucocytes
is a sign of death. Soon after death the staining ceases, and the cells rupture
or lose their stain.

Evidence has also been given that these phenomena are due to the diffusion
of stain into the jelly-like cytoplasm being hastened or delayed, as the case
may be, by the agency of these factors, and that death, coincident with the
staining of the nucleus, is followed by liquefaction of the cytoplasm and
other changes which cause the cells to lose their stain and enter a phase which
has been called the condition of achromasia (6).

I have made further investigations in this subject, and have ascertained
that if the constituents of the agar film are arranged in a constant manner
and the other factors are constant, the staining of the cells will be constant
provided that the latter are in the same healthy condition when placed on
the agar. It has also been found that when one class of blood cell stains on
a given agar film, others do not. By altering one or more of those factors
which hasten or delay diffusion of stain into the cytoplasm, that class of cells
which previously refused the stain will now absorb it. Therefore the rate of
diffusion of stain into the cells differs with the class of cell. Cells other than
blood cells, especially bacteria, have also been tried and have been found to
be subject to the same conditions ; and it has been possible, by altering the
arrangement of the factors, to differentiate cells by their rate or coefficient of

diffusion. The object of this paper is to give methods by which the coefficient
VOL. LXXXI.—B. H

98 Mr. H.C. Ross. On a Coefficient of (Ween

of diffusion of living cells can be obtained, and the differentiation accom-
plished.

Scheme of the Method employed—tThe cells to be tested are placed on a film
of jelly on a slide. The film is prepared from a test-tube which contains
10 cc. of jelly. The 10c.c. of jelly contains, besides stain, some of the factors
such as alkalves, acids, and salts, which hasten or delay the diffusion of stain
into the cells. In order to simplify matters, I measure these factors in units
and I endeavour to arrange them in sucha way that 1 unit of any one factor
is equal in value, as regards hastening or delaying diffusion, to 1 unit of any
of the other factors. The factor heat, which hastens diffusion, is applied after
the cells have been spread upon the film by keeping the slide at various
temperatures for various periods of time. This factor, as well as that of time,
is also arranged in units, so that 1 unit of either of them is equal in value
to 1 unit of the other factors contained in the agar. Hence if 1 unit of a
given substance delays diffusion of stain into a cell, the delay it causes can
be exactly neutralised by the addition, either to the agar or to the slide, of
1 unit of a factor which hastens diffusion.

Consequently an equation is formulated by means of which the total
number of units which, with a certain amount of stain, will cause given, or
stipulated, staining of the cells, 1s equivalent to, or said to be equivalent to,
their coefficient of diffusion. Therefore to find the coefficient of a cell, it is
necessary to prepare films from a succession of tubes of 10 c.c. of jelly, each
tube having a certain, known, number of units added to it, and to examine
each film until the stipulated staining is obtained. Since the units are equal
in value, it matters little, within reasonable limits, which factor is added,
provided that the total number of units is known. Then, after subtraction
of those units which delay diffusion, the remainder added to the quantity of
stain is the coefficient of diffusion of the cell experimented with.

Conversely, if the coefficient of diffusion of a cell is known, one is enabled,
by means of the equation, to know how many units of one or several of the
factors it is necessary to add to the jelly, or apply to the slide, to obtain
stipulated staining of that variety of cell in a given time. J have mentioned
that the staining of healthy cells, only, appears to be constant. When the
rate of staining of cells of persons suffermg from disease has been found
experimentally, the equation indicates in a moment the difference between
the coefficients of healthy and diseased cells, and this difference can be
expressed in grammes, degrees, or minutes, etc., according to the nature
of the factor into which the coefficient of diffusion may be ultimately
resolved.

Definitions—When a film of agar jelly contains stain and other substances,

1908. | Diffusion into Inving Cells. 99

its Index of Diffusion (7x) may be defined as the sum of its constituents
which delay diffusion subtracted from the sum of constituents which
accelerate diffusion added to the quantity of stain contained in the jelly.

The Coefficient of Diffusion (cf) of a cell is that index of diffusion plus the
time and temperature required to cause staining of the nucleus, or staining of
the cytoplasm in unnucleated cells (¢.g., red corpuscles), when the specimen is
prepared by a standard method.

Standard Method of Preparation—tThis consists in: (1) Mixing the cells
with a neutral solution containing 3-per-cent. sodium citrate and 1-per-cent.
sodium chloride.* If blood is experimented with, it is mixed with an equal
volume of the solution. In the case of bacteria and other cells, the mixture
is made as convenient.t (2) The mixture is then placed on a cover-glass,
which is inverted and allowed to drop flat on a film of agar jelly containing
Unna’s stain and salts, and which, after boiling, has been allowed to set on a
slide. Since the surface of the film is convex, the solution spreads to the
periphery of the cover-glass, leaving the cells gently pressed out between the
glass and jelly, and this affords an excellent means of examination by the
microscope.t Only the cells in the centre of the preparation should be
examined.

It is stipulated that the jelly contains stain, but the amount of stain added
to the agar may be variable. The chemical nature of methylene blue may or
may not affect diffusion. The point is difficult to determine accurately, but
in this procedure it is of little importance, because the stain employed is
always the same, namely, Unna’s polychrome methylene blue (Grubler). On
the other hand, it is obvious that the more concentrated it is—that is, the
more stain there is in the 10 cc. of jelly, the more rapidly, ceteris paribus,
will the cells stain. It is also obvious that the effect of an increase of the
concentration of the stain can be neutralised by the addition of one or several
factors which delay diffusion. Consequently I also measure the stain in
units, so that an increase of its concentration by 1 unit can be neutralised
by the addition of 1 unit of a factor which decreases diffusion.§

* Merck’s reagents have been used throughout these researches.

+ The mixture of the cells with this solution is merely used as a vehicle to keep them
alive. As the solution spreads to the periphery of the cover-glass, it does not materially
influence the diffusion of stain from the agar, a point which has been tested experi-
mentally by rendering it alkaline. The cells, however, should not be kept in it longer
than necessary.

t The suggestion of mixing stain with the jelly was made to me, as already noted
elsewhere, by my brother, Prof. Ronald Ross.

§ Unna’s polychrome methylene blue is only supplied in solution, which is standardised.
It cannot be made in a powder.

H 2

100 Mr. H. C. Ross. On a Coefficient of [Dec. 9,

Convenient Method of Preparing the 10 c.c. of Jelly—tI have found it more
accurate and simple if the factors which hasten or delay diffusion are added
to the jelly from standard solutions, and I make the tubes of 10 c.c. of jelly
as follows:—50 c.c. of a 2-per-cent. solution of powdered agar in water,
filtered and sterilised, is prepared. This solution has such a consistency that
another 50 ¢.c. of water could be added to it without preventing it from
setting on a slide when cold.

Since I have shown that blood cells will not live on agar jelly unless it
contains a combination of sodium citrate and sodium chloride (3, 7), I add to
the 50 c.c. of jelly 1 gramme of sodium citrate and 0°8 gramme of sodium
chloride, and accurately neutralise to litmus with citric acid. The whole is
then rendered acid by the addition of 0:083 gramme of citric acid. The
reason for this will be given in the next paragraph but one.

The molten jelly is then decanted into test-tubes, each of which contains
5 e.c., so that it is possible to add the stain and certain quantities of the
standard solutions which contain the factors to these tubes, and provided the
total quantities of the several solutions added do not cause a tube to contain
more than 10 cc. of jelly, its ultimate setting on a slide is assured. The
standard solutions are so arranged that their total quantity required in an
experiment never does exceed 5 c.c. On the other hand, if the amount is
less than 5 c.c., the balance up to the maximum in the tube of 10 ce. is
made up with water. In other words, a test-tube originally contains 5 c.c. of
jelly which is acid and holds a certain quantity of salts in solution. The
stain and quantities of the standard solutions, which correspond to the
number of units of factors which it is intended to try, are added. The total
content of the tube is then made up with water to 10 cc. and boiled. So
that a test-tube never contains more nor less than 10 cc. of jelly when a film
is prepared from it, though it may contain a variety of units of stain and
standard solutions.

I have stated that the agar is rendered acid at the outset ; this is done to
reduce the number of factors. Acids and alkalies delay and accelerate
diffusion respectively. Since they neutralise each other, the neutral point
would also have to be taken into consideration. As this would complicate
the equation, I render the agar acid at the outset, so acid that I cannot get
any cell to stain on it with 1 unit of stain, and deal only with alkali The
neutral point I ignore, although by knowing the initial acidity of the agar,
and that the units of all the factors are equal, the point can be readily
determined by referring to the equation. I therefore deal with one factor,
alkali, instead of two and a neutral point.

To recapitulate shortly: 50 c.c. of a 2-per-cent. solution of agar is prepared

1908. | Diffusion into Inving Cells. 101

which contains 1 gramme of sodium citrate and 0°8 gramme of sodium
chloride. It is neutralised and rendered acid with 0°083 gramme of citric
acid. It is then collected in quantities of 5c.c. In order to determine the
cf of a cell,a tube of 5 cc.is melted and certain quantities of stain and
standard solution of alkali added. The content of the tube is then completed
with water up to 10 c.c. Consequently the tube contains 1 per cent. sodium
citrate and 0°8 per cent. sodium chloride in addition to the acid, stain, and
alkali, and this content of salts allows leucocytes to live on the jelly. The
whole is then boiled until it froths up the tube and a film prepared from it
by pouring a drop on a slide and allowing it to set. The cells are placed on
to the film and the slide is kept at a convenient temperature for a period of
time. If the nuclei or cytoplasm are not yet stained, a higher temperature
may be tried combined with a longer period of time, or a fresh tube prepared
with more units of alkali added, and so on until staining is obtained. Should
the contents of a tube cause the cells to stain very deeply, or if they soon
become achromatic, a fresh tube is made containing less stain, or more salts,
or less alkali, or acid may even be employed, and so on. But provided the
arrangement of the contents of the tube which just causes staining of the
nuclei is known, and if the time and temperature are also known, the
equation will give the ¢f required.

Units—In preparing these units I have mainly considered their practical
application in the endeavour to curtail the procedure as much as possible.
In the instance of alkali and salts, I give the actual amount in grammes
which 10 c.c. of jeliy should contain as1 unit. I also give a convenient
standard solution and the amount of it in cubic centimetres to be contained
in the 10 cc. of jelly to constitute 1 unit.

Alkali, Sodium Bicarbonate, hastens diffusion—Unit, 0°005 gramme.
Standard solution 5 per cent., unity being 0°1 cc. It is convenient to
remember that this solution is neutralised by a 4:175-per-cent. solution of
citric acid, and that 1 unit of alkali is neutralised by 071 c.c. of such
a solution. Since the agar at the outset is acid to the extent of
0:083 gramme to 50 cc. a tube of 10 c.c., made up as described, must
contain 0:0083 gramme of acid. This is exactly neutralised by 0:2 ce. of
the standard alkali solution ; that is, the agar at the outset, before any stain
or other factor is added, delays diffusion to the extent of 2 units. Or, the
addition of 2 units of sodium bicarbonate will render the agar neutral.

Sodium Citrate, delays diffusion—Unit 0°03 gramme. Standard solution
10 per cent., 0°3 c.c. being unity. Since 50 cc. of agar contains 1 gramme at
the outset, the 10 c.c. of jelly may be said to contain about 3 units.

Sodium Chloride, delays diffusion. Unit 0°08 gramme. Standard solution

102 Mr. H. C. Ross. On a Coefficient of [Dee. 9,

10 per cent., unity being 0°8 c.c. The 10 cc. of jelly contains this from the
outset.

Heat, hastens diffusion.—Hach unit 5° OC. 10° C. is unity, 15° C. is
2 units, 20° C. 3 units, ete. For practical purposes I call 37° C. 7 units.

Time, increases diffusion. 10 minutes being 1 unit, 20 minutes 2 units,
and so on.

Stain, Unna’s polychrome methylene blue (Grubler), behaves as if it
increased diffusion.*—Unit 0:1 e.c.

Hquations for ascertaining the Coefficient of Diffusion.—The nuclei of poly-
morphonuclear leucocytes recently shed from a healthy person, just stained
in 10 minutes when resting on a film of agar, 10 cc. of which contained
0:2 c.c. of stain, 1 per cent. sodium citrate, 0°8 per cent. sodium chloride, and
6 units of sodium bicarbonate. The slide was kept at a temperature of
37° C. What was their coefficient of diffusion ?

Then cf = that fe+h+t which just causes staining of the nuclei,
but ju = (sta)—(e+n);
of = (28+ 6a)4+(7h4+t)—(8e+n) = 16-4 = 12.

Where s is the unit of stain, a the unit of alkali, / the unit of heat, ¢ the
unit of time, ¢ the unit of sodium citrate, and 7 the unit of sodium chloride.

The 10 c.c. of agar in this case was made up as follows :—5 c.c. from the
original 50 ¢.c. of agar which contained sodium citrate 1 gramme, sodium
chloride 0°8 gramme, and citric acid 0°083 gramme. The jelly was melted
and the following quantities of standard solutions added: 0:2 cc. stain,
0°6 cc. 5-per-cent. solution of sodium bicarbonate, and 4:2 c.c. water.
Total 10 c.c.

The eosinophiles, however, did not stain under quite the same conditions
for it was found in the foregoing experiment that they were either achromatic
or ruptured. A fresh tube was made with 1 unit less alkali, when it was
found that the eosinophiles would just stain in 10 minutes. What was
their cf?

of = (28+ 5a+7h+t)—(8¢e+n) = 11.

The lymphocytes, large and small, required 0:2 c.c. of alkali more than the

polymorphonuclear cells, the other factors being as before, what was their cf?

of = (28+ 8a+7h+t)—(8e+n) = 14.
The foregoing tubes contained a very low content of stain, the chromatin

* As already pointed out, stain should not be a unit of diffusion, for it is doubtful
whether it affects diffusion. It contains salts, and is alkaline when made, and alkalies
and salts are antagonistic. However, for reasons already given, it may be included in
the category.

1908. | Diffusion into Living Cells. 103

network stained better if its concentration was increased. In order to obtain
staining of the nuclei of lymphocytes in 10 minutes on agar from a tube
which contained 4 units, instead of 2 units, of stain, the amount of alkali
added was of course less in proportion to the increased concentration. With
4 units of stain, the other factors, except allali, being as before, the equation -
now stood as :—

of = (48+ 6a+ 7h+t)—(8e+n) = 14.

The red corpuscles appear to have a very high ¢f. I caused them to stain
on agar which contained 1 c.c. of stain (10 units) and 11 units of alkali, in
the presence of 1-per-cent. sodium citrate and 0:8-per-cent. sodium chloride
at 37° C. in 10 minutes. This was the equation :—

of = (10s+1la+7h+t)—(8e+n) = 25.*

Hxamples—A growth of staphylococci had a cf of 16.

How much alkali must 10 cc. of jelly contain to cause the germs to
stain in 10 minutes if the jelly already contains 5 per cent. of stain, 1°5 per
cent. sodium citrate, and 0°8 per cent. sodium chloride, when the slide is
incubated at 37° C. ?

a = (16ce/+45e+n)—(5s+ 7h+12),
a = 85 units, or 00425 gramme of sodium bicarbonate.

A strain of typhoid bacilli had a ¢f of 21. A tube of agar contained 6 units
of alkali solution and the usual quantities of sodium citrate and chloride.
How much stain should be added to the tube to produce staining of the
bacilli in 20 minutes at 37° C. ?

8 = (21cf+ 3¢e+n)—(6a+4+ 7Th+4 20),
s = 10 units of stain, 2.2. 1 ce.

* I do not think this is strictly accurate, for it depends on the coloration of the
stroma, Nucleated red cells have a comparatively low ef, resembling that of the poly-
morphonuclear cells, though I am also doubtful of this point, because I have only been
able to obtain these nucleated cells from persons suifering from disease, and, as I have
already shown (8), all the blood cells in chronic illnesses, especially phthisis, malaria, and
Hodgkin’s disease, have so far shown a general fall in their coefficient of diffusion.
Again, attention may be drawn to the comparatively low cf of the granular erythrocytes
which constitute about 1 per cent. of all the red cells in healthy blood as demonstrated
by this method of examination (1). The rate of staining of the granules is about the
same as those of the polymorphonuclear leucocytes, but the rate of staining of the stroma
of these granular cells is much higher than that of their granules, yet lower than the rate
of staining of the stroma of ordinary erythocytes. This fact is the more interesting,
because it is almost in these granular red cells alone, which have a low ef, that “red
spots” are to be seen, although I have on three occasions seen them in ordinary red cells,
and the spots appear to be dependent on diffusion (4). These cells also become achromatic
more readily than ordinary erythrocytes. The granules have been described (1) as the
remains of a nucleus. The question, therefore, of the cf of the red cells I will leave for
the present as indicated.

104 Mr. H. C. Ross. On a Coefficient of [Dee. 9,

Precautions As regards Life and Death: In a previous paper (3) it has
been shown that the staining of the nuclei of leucocytes, when examined by
this method, is a sign of death, and that the nuclei of dead cells will stain,
ceteris paribus, before those of living cells. Consequently all the experiments
given in the present paper have been made with fresh normal cells, and in
the case of micro-organisms with cultures not more than 48 hours old. It
may also be mentioned that the liquefaction of the cytoplasm which occurs
after death materially alters the conditions of staining of leucocytes, and that
the ¢f of living blood cells falls gradually after the blood has been shed. The
fact has already been mentioned that in chronic wasting diseases the coefficient
of blood cells may be very low.*

As regards Excess of Alkali causing rapid death and liquefaction of the
cytoplasm with consequent prevention of staining (achromasia): The
addition of excess of alkali may cause death, staining of the nuclei
liquefaction, and the loss of stain on the part of the cells (3, 6). This
may occur before a preparation can be focussed, in which case the cells
appear unstained and will refuse to stain, no matter how much more stain
or alkali are tried. Therefore it is better to begin with a low index of
diffusion and to try tube after tube, each containing a little more alkali, until
staining is obtained. Further, the amount of sodium bicarbonate should
not exceed 20 units, because, as has already been pointed out in a former
paper (3), if added to excess, it may act as a neutral salt and delay diffusion.

As regards Deficiency of the Salts sodium citrate and sodium chloride: If
the jelly contains no salts, the blood lakes and the leucocytes are killed
outright. If it contains sodium chloride only, the cells are killed rapidly, and
the same may be said if sodium citrate only is employed (7). In examining
blood, therefore, the combination is essential.

As regards Excess or Deficiency of Heat: A temperature above 40° C. may
allow the cells to diffuse through the agar (2). A temperature below 13° has
not been experimented with, because, even at a temperature of 20° C., it
requires a minimum of 3 units of stain to cause staining of the nuclei of
leucocytes in spite of the addition of a large amount of alkali, for the alkali
is not sufficient, per se, to cause the cells to absorb sufficient stain to colour
the nuclei unless the stain is concentrated.

As regards excess of Time: A period of more than half an hour has not
been employed for fear of death and liquefaction of the cytoplasm, for the

* I have found that the life of leucocytes of persons suffering from some chronic
diseases, when bathed in their own plasma, is considerably shorter than the life of
healthy persons’ leucocytes bathed in their own healthy plasma(8). I have reasons for
believing that there is an association between this and a low ¢f.

1908. | Diffusion into Inving Cells. 105

cells may die and become achromatic before there has been time for sufficient
stain to diffuse into them to cause staining of the nuclei, in which case, of
course, the cells will never stain.

As regards Excess of Stain: More than 10 units of stain may cause pre-
cipitation of the agar as the film cools on the slide, and the precipitate carries
some of the stain down with it, vitiating the results, for it has been shown
that agar is not very soluble in cold stain (3).

As regards Examination: The observation of cells floating near a bubble
under the cover-glass should be avoided. The fact that blood cells in such
a situation will stain before others has already been noted(3). I consider
this to be due to these cells floating in a small quantity of alkaline citrated
plasma collected round the bubble.

Consequently the experiments have all been made within the compass of
the above restrictions. So far no cells, whether blood, bacteria, or other cells,
have been met with which would not give a coefficient of diffusion by this
method. It may also be advised that when unnucleated cells contain granules
in their cytoplasm, the staining of the granules gives a more constant rate
than the staining of the cytoplasm. By this fheans it is seen that the ¢f of
the blood-plates is identical with that of the polymorphonuclear cells.

The Construction of other Units—It may be necessary to add cther
substances to the jelly to test their effect on cells. For instance, it may
be useful to try other salts, in which case their rate of antagonism to diffusion
must be found and a unit made. This may be done by comparing their
action with that of a unit of one of the other factors, after which the new
unit may be added to the equation. In the case of sulphate of atropine, it
was found that a tube of 10 c.c. of agar, which had a correct fx to cause
staining of lymphocytes in 10 minutes, but which also contained
0013 gramme of sulphate of atropine, required the addition of 1 more
unit of alkali to cause the nuclei to stain in 10 minutes. Consequently
the unit of atropine sulphate may be said to be 0:013 gramme.

The Determination of the Coefficient of Diffusion of Leucocytes involves
Death.—Since the staining of the nucleus is the moment by which the ¢ of
leucocytes is obtained, and since the staining of the nucleus is a sign of
death (3), the cells are necessarily dead at the expiry of the time involved in
finding their coefficient.

The Reconciliation of their Coefficient of Diffusion to Cells which may be
Alive at the Termination of the Time required—It has been shown that
leucocytes will live for a considerable period and show amceboid movement
with their granules stained (3, 5). If 1 digit is subtracted from their cf and
the jelly arranged according to such an equation, the granules but not the

106 Mr. H. C. Ross. On a Coefficient of [ Dee: 9;

nuclei will stain in the given time. By this means death is not necessarily
involved.

Example to show that the Diffusion of Substances other than Stain may be
Dependent on the Coefficient of Diffusion of Cells—Given eosinophiles have
a of of 11. They are resting on agar which contains the usual quantities of
sodium citrate and chloride, viz., 3 and 1 units respectively, but it also
contains 0:02 gramme of sodium bicarbonate (4 units), 0°6 c.c. of stain, and
0:007 gramme of atropine sulphate. The fact that a mixture of atropine
and methylene blue will excite a remarkable exaggeration of amceboid move-
ment in leucocytes has already been published (5). How long will it take
to produce marked exaggerated movements in the given eosinophiles at
a temperature of 20° C. ?

Then, since it is necessary for the given cells to be alive at the expiry of
the time required, 1. digit must be subtracted from their e/—

t = (10c/+ 38e4+n+0°52z)—(6s+ 4a4 3h),
¢ = 1°d units of time or 15 minutes.

Where 2 is the unit of atyapine sulphate, 0-013 gramme. A 1-per-cent.
solution was found convenient as a standard, and 0°7 c.c. was used.

The Coefficient of Diffusion may be resolved into the Value of any one of the
Units—Since by the foregoing equations any one of the units can be
resolved into the value of any one of the other units which go to make
the fz, and since fx+h+t = cf, therefore any ¢f can be expressed in the
value of any of the units; into alkali for instance. But the unit of alkali is
5 milligrammes of sodium bicarbonate. Consequently the coefficient of
diffusion of the bacteria contained in the growth of Bacillus typhosus, used in
one of the examples, may be said to be equivalent to the alkalinity of
105 milligrammes of sodium bicarbonate.

Summary and Suggestions.

The difficulty has been in the construction of the units. So far I have
found them to be sufficiently accurate for practical purposes within the
compass of these experiments, of which a very large number have been
made, extending over a period of several years, involving the use of many
varieties of cells.

The determination of the coefficient of diffusion is brought about by
allowing living cells to rest on a jelly which contains stain. Several factors,
some of which may be contained in the jelly, hasten or delay the diffusion of
the stain into the cells, and the coefficient of diffusion is the sum of the
factors which causes the stipulated staining of the cells added to the amount

1908] Diffusion into Living Cells. | 107

of stain employed. The factors which hasten diffusion are heat, alkalies, and
tvme; and those which delay it are acids and neutral salts. But the rate of
diffusion depends also on the concentration of the stain, for if this is weak
a large sum of factors to produce staining is required; but if it is very
concentrated the cells may stain even in the presence of acid. Since all
these items are variables, I have constructed a formula by which, if some of
them are known quantities, the others can be readily determined.

By this means the coefficient of diffusion of a cell can be obtained, and it
varies with the class of cell.

I have also stated that the diffusion of substances other than stain may
appear similarly to depend not only on their concentration but on the
coefficient of diffusion of a cell. But other substances may be alkalies, acids,
or salts, and may affect the diffusion of neighbouring substances and be so
affected themselves. I have given a means by which this effect can be
determined and aunit made. Then, provided the unit of a given substance is
known, and provided the coefficient of diffusion of a given cell is known, the
comparative rate of effect of the given substance on the given cell can be
ascertained by referring to the equation.*

In medicine, for instance, drugs and sera are frequently given with
a view to affecting certain cells, yet, as far as I know, no steps are taken
either to ascertain the rate of effect of the drug on the cells, or suitably to
modify the alkalinity of the blood by treatment, in order to produce
maximum results according to the temperature of the patient. The point
appears worthy of consideration.

In the case of bacteria, it is frequently heard that certain bacteria are
more resistant to antiseptics and drugs than others. It is possible that this
varied resistance may be summed up in the expression “coefficient of
diffusion.” If antiseptics could be rendered alkaline according to the
coefficient of the bacteria which they are intended to kill, and according to
the temperature, it might lead to a reduction of the concentration of the
antiseptic, with consequent saving of cost and increase of efficiency.

Since the blood fluids affect bacteria, it seems desirable to know the
coefficient of diffusion of the bacteria when estimating the effects of the
fluids on the cells. Again, since erythrocytes will diffuse bodily into agar
jelly and remain suspended in it (2), and since droplets of liquid will diffuse
into the colloid cytoplasm of leucocytes and remain suspended in it (red

* The knowledge that heat accelerates the diffusion of substances into cells has already
been applied in some researches by Dr. C. J. Macalister and myself in order to
demonstrate an excitant for leucocytes in the plasma of cancer patients (‘ Proc. Roy. Soc
Med.,’ December, 1908).

108 Coefficient of Diffusion into Liwing Cells.

spots, 4), it seems possible that germs which proffer the same jelly-like
properties as red cells may enter the phagocytes by the process of diffusion
and be subject to the same factors which influence it, for the temperature
and alkalinity of the blood vary in health and disease. Therefore, it may be
important to know the rate of diffusion of leucocytes when estimating
phagocytosis.

I have noted that there appears to be a relationship, in leucocytes at.
least, between the coefficient of a cell and the length of its life as measured
by a procedure which I have already published (5, 8). It has also been
shown in a former paper (4) that if stain is passed over a jelly such as agar,.
the rate of the coloration of the jelly depends on its consistency, that is, as
to whether it is solid or diffluent. In the same paper it was stated that the
effect of stain on cytoplasm also depended in a like manner on its consistency.
Since there may be a relationship between the coefficient and vitality, the
consistency of a cell may depend to some extent upon its vitality. There-
fore, the determination of the coefficient of diffusion may prove important in
the prognosis of tumours if the cells can be suitably kept alive, since it may
give an indication of the consistency of the cytoplasm, and a lowered
coefficient, as occurs in the blood cells in anemia, may foretell a lowered
vitality.

Further experimentation is also required to determine the property on
which depends the varying influence of alkalies, salts, etc., in hastening or
delaying diffusion.

I hope this method may ultimately prove of value, not only in bacteriology
as a means of differentiating bacteria, but also in the investigation of the
diffusion of substances into living cells.

REFERENCES.

(1) R. Ross, C. E. Walker, Salvin Moore, “Some New Diagnostic Methods,” ‘ Lancet,”
July 27, 1907.
(2) H. C. Ross, “‘ Diffusion of Red Blood Corpuscles through Agar,” ‘ British Medical
Journal,’ May 5, 1906.

(3) “On the Death of Leucocytes,” ‘Journal of Physiology,’ vol. 37, p. 327,
1908.

(4) 55 “On the Vacuolation of Leucocytes, etc.,” ‘Journal of Physiology,
vol. 37, p. 333, 1908.

(5) 9 “On a Combination of Substances which Excites Leucocytes, etc.,”
‘Lancet, January 16, 1909.

(6) 9 “On the Cause of Achromasia in Leucocytes,” ‘Lancet, January 23,
1909:

(7) %p “On the Modification of an Excitant for Leucocytes, etc.,” ‘Lancet,’
January 30, 1909.

(8) as “Some Comparative Measurements of the Lives of Leucocytes, etc.,”

‘Lancet,’ February 6, 1909.

c

Soc. Proc BL. vol. Of, Fi /. <

Y-

J

Feo

POSS.

Origin and Destiny of Cholesterol in the Animal Organism. 109

DESCRIPTION OF PLATE 3.

Coefficient of diffusion. Drawn by P. Nairn from a preparation of fresh blood cells which
have been resting for 10 minutes at 37° C. on an agar film with an index of diffusion
of 4, The nucleus of one polymorphonuclear leucocyte bas just stained and the cell
is showing three small red spots. The nuclei of two large lymphocytes have not yet
stained, one cell is showing 1 centrosome and the other 3 centrosomes. The film also
demonstrates an eosinophile leucocyte which is becoming achromatic, 7.e., its nucleus
has lost its stain; and one granular red cell which contains two red spots. 2 mm.
apochromatic objective, No. 4 eye-piece, 250 mm. draw-tube, 1 amp. Nernst lamp.

The Origin and Destiny of Cholesterol in the Animal Organism.
Part Ill.—The Absorption of Cholesterol from the Food and
its Appearance in the Blood.

By CHarLes 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 18, 1908,—Read
February 11, 1909.)

In his ‘ Text-Book of Physiology’ Schafer has suggested that the constant
presence of lecithin and cholesterol in the bile may well be associated with
the destruction of the red blood corpuscles which contain relatively large
amounts of these substances, the latter, according to Hepner,* being present
in the free state and not in the form of esters. This idea has recently
received strong support from the investigations of Chasoburo Kosumotot
on the influence of toluylene diamine on the output of cholesterol in the
bile. This reagent was found by Schmiedeberg to produce icterus, and
Stadelmann, working in Schmiedebereg’s laboratory,t observed that at the
beginning of the action of the drug an increased production of bile took
place. This, however, was only temporary, and soon the normal physical
properties of the bile underwent an alteration ; it became sticky, darker, and
more concentrated.

Afanassiew§ showed that the effect of the drug is to destroy the red blood

* ©Pfliig. Archiv f. d. Ges. Physiol.,’ 1898, vol. 73, p. 595.
+ ‘Bioch. Zeit.,’ 1908, vol. 13, p. 354.

t ‘ Arch. f. experim. Pathol. u. Pharmak.,’ 1881, vol. 14, pp. 231, 422.
§ ‘Zeit. f. klin. Med.,’ vol. 6.

110 Messrs. Dorée and Gardner. Origin and [Dee. 18,

corpuscles. The increased viscosity, which Afanassiew correlated with
certain changes observed by him in the liver tissue, causes a hindrance
to the flow of bile, and since the bile formation is, perhaps, increased beyond
the normal, leads to icterus. When the action of the toluylene diamine
ceases—according to Afanassiew owing to the removal of the hindrance
which opposes the bile flow—the amount of bile increases again, possibly
even above the normal. These statements made it appear probable that the
cholesterol of the red corpuscles destroyed would appear in the bile, along
with the bile colouring matter. By careful measurements of the quantities
of bile produced in dogs in which permanent fistule had been established in
a satisfactory manner, and by estimations of cholesterol in the bile, by
methods which do not seem to us open to objection, before and after the
administration of toluylene diamine, Kosumoto showed that this was the
case. The conclusion seems to be justifiable that a part at any rate of the
cholesterol of the bile arises from the débris of the normal destruction of red
blood corpuscles in the liver.

On the other hand, the percentage of cholesterol in the fistula bile of dogs
does not appear, according to the investigations of Goodman,* to depend
upon the cholesterol content of the food taken by the animals, a result in
accordance with the work of previous observers. Goodman made the
interesting observation that with a diet of 725 grammes of coagulated white
of egg, which contains little or no cholesterol, he was able, during five days,
to collect 477 grammes of bile containing 0°208 gramme of cholesterol, whilst
with a diet of 488 grammes of calves’ brain, which is very rich in cholesterol,
he obtained in four days 367 grammes of bile containing 0:145 gramme of
cholesterol, so that the percentage content in cholesterol of the bile excreted
in these two diets was 0°0436 in the case of the white of egg, and 0:0395 in
that of brain. It seems probable, therefore, that the cholesterol of the bile
is not derived directly from the food, and some of it at any rate is the result
of the elimination of the cholesterol of dead blood corpuscles, and possibly
also the débris of other tissues, by the liver. On the other hand, this
cholesterol can in no sense be regarded as a waste product, as we have
shown in previous papers? that herbivorous animals do not excrete
cholesterol or any recognisable derivative of that body in their feces,
although their bile contains considerable quantities, in the case of the cow
for example 0-07 per cent.t Further, in the case of the dog any cholesterol
found in the feeces can be entirely accounted for by the cholesterol contained

* ‘Hofmeister Beit.,’ 1907, vol. 9, p. 91.

+ “Roy. Soc. Proc.,’ B, vol. 80, p. 12.
t ‘Journal of Physiol., Proc.,’ 1907, vol. 36, p. ix.

1908.] Destiny of Cholesterol in the Animal Organism. iki

in the food taken. In experiments in which cholesterol free food was given
no cholesterol was found; thus a dog fed for 31 days on oatmeal and water
passed no more than 0:1 gramme of impure cholesterol in the feces.

From these observations we were led to the conclusion that the cholesterol
of the bile must either be destroyed, or absorbed, along with the bile salts in
the intestine, and taken into the blood stream.

The latter hypothesis is more in accordance with the great stability of the
cholesterol molecule, and is supported by the observation of Pribram* that
- emulsions of cholesterol with olive oil, when injected into the stomach of the
rabbit, cause an increase in the cholesterol content of the blood.

From a consideration of these facts we have been led to put forward with
regard to the origin and destiny of cholesterol the following working
hypothesis :—

1. Cholesterol is a constant constituent of all cells, and when these cells
are broken down in the life process the cholesterol is not excreted as a waste
product, but is utilised in the formation of new cells.

2. A function of the liver is to break down dead cells, ¢.g., blood corpuscles,
and to eliminate their cholesterol in the bile.

3. After the bile has been poured into the intestine in the process of
digestion the cholesterol is reabsorbed, probably in the form of esters, along
with the bile salts, and carried by the blood to the various centres and
tissues for re-incorporation into the constitution of new cells.

The question arises whether the excretion and subsequent absorption of
the cholesterol of the body form a regular and exact cycle, or whether there
is any wastage of cholesterol which would require to be made up by the
animal either by actual synthesis in the body from simpler substances or by
the utilisation of that taken in the food. With reference to the wastage of
cholesterol it must be pointed out that not inconsiderable quantities are
excreted through the skin; the sweat and sebum of men have been shown
to contain cholesterol, and in the case of the sheep the sweat, being absorbed
by the wool, enables us to demonstrate the presence of large quantities of
cholesterol and isocholesterol. How is the wastage made up? Considering
the remarkable chemical nature of cholesterol it would appear less probable
that it is synthesised by the animal than that the loss is made up by the
absorption of cholesterol obtained from the food. In the case of carnivora
the cholesterol is contained in their food as such, and might easily be
utilised. On the other hand, the food of the herbivora contains no
cholesterol, but instead the closely related phytosterols, and the question
arises whether this closely allied substance can be utilised by the animal.

* ©Bioch. Zeit.,’ 1906, vol. 1, p. 413.

sa Messrs. Dorée and Gardner. Origin and [Dee. 18,

If the wastage is made up in this way we should expect to find variations
in the cholesterol content of the blood according as the food was free from,
or rich in, cholesterol. If, further, it were able to take in from its richer
diets more cholesterol than was at the moment required, storing it up in the
intervals of feeding to replace loss we should expect considerable variations
in the cholesterol content of the plasma with the variety of the food. If,
however, the animal only takes up what is required to supply immediate
waste we should not expect more than a slight variation on different diets,
and this variation might easily be entirely masked owing to the different
quantities of bile caused to flow into the intestine under the influence of
foodstuffs of different kinds. With regard to the mechanism of the
absorption of cholesterol in the intestine it would seem probable that it is
first esterified, being converted into the oleic and palmitic esters. These
compounds, which are stated to possess the property of formimg with aqueous
fluids lanoline-like emulsions,* were found by Hirthle to be constantly
present in the blood plasma of various animals.

With a view to testing the validity of these considerations we planned
a series of experiments, the first instalment of which is described in the

present paper.

Experiments to Ascertain whether Cholesterol is Absorbed by Herbivorous
Animals when given urth their Food.

The animal selected for this investigation was the rabbit. Preliminary
experiments showed that the bulk of the phytosterols of bran can be
extracted along with the fat by means of ether, without altering the
appearance of the bran and without impairing its feeding value, except for
the elimination of the fat. It was found that rabbits could be kept for long
periods on this diet without apparent injury to health, and often without
much loss in weight, though individuals varied considerably in this respect.

The general method adopted in these experiments was as follows :—A rabbit
was fed for several days previous to the commencement of an experiment on
extracted bran. It was then given each morning 0:25 gramme of cholesterol
mixed with a few grammes of extracted bran, care being taken to see that
the animal ate the whole. After eating the cholesterol-bran mixture the
animal was allowed during the rest of the day as much extracted bran as it
would eat. This procedure was followed until the animal had eaten
2 grammes of cholesterol, after which it was fed on extracted bran only for
three days in order to sweep all cholesterol from its gut. The feces during

* Ivor Bang, ‘ Ergebnisse d. Physiologie,’ vol. 2, p. 180.
+ ‘Zeit. Physiol. Chemie,’ vol. 21, p. 331.

1908.] Destiny of Cholesterol im the Animal Organism. 113

the experiment were carefully collected, dried in the oven at 80° to 90° and
weighed. The feces were then extracted with ether in a Soxhlet’s apparatus
for a week or ten days. The ethereal solution was saponified according to the
method described in our former paper* by means of sodium ethylate. The
precipitated soap was filtered off and washed thoroughly with ether. The
ethereal filtrate and washings were freed from excess of alkali and alcohol by
repeatedly washing with water, dried, and the ether distilled off. The dry
residue was weighed and then fractionally crystallised from alcohol until no
further crystalline matter could be obtained. The oily residues were then
dried, dissolved in pyridine, and treated with excess of benzoyl chloride and
after standing over night poured into water. The precipitated matter was
filtered, taken up with ether, dried, and the ether again evaporated. The
residue was boiled with a little alcohol and any cholesterol that had remained
in the oily residue was thus obtained in the form of the highly insoluble
benzoate.

Experiment I—In order to ascertain whether it was possible to extract the
whole of the cholesterol from feces by the method used, 2 grammes of
cholesterol were ground up with moistened feces that had already been
extracted, in the proportions usually found. The mixture was dried and
subjected to the whole process detailed above, when 27098 grammes of
slightly brown-coloured cholesterol were obtained—a quantitative recovery.
As the cholesterol recovered from natural feeces is often highly coloured and
can only be readily purified by treatment with animal charcoal, it was also
desirable to ascertain what loss occurred under the conditions usually
followed. The two grammes of recovered cholesterol were therefore
dissolved in about 50 c.c. of alcohol, boiled with about half the weight of
animal charcoal, and filtered by means of a hot funnel. The charcoal was
then washed with hot alcohol. On evaporating the alcohol and crystallising
the cholesterol 1:8 grammes were recovered. The loss due to boiling with
charcoal was therefore about 10 per cent.

Expervment II—In order to ascertain (1) how far it was possible to
extract the phytosterol from bran by simply extracting with ether for
several days; (2) whether any cholesterol could be detected in the feces,
after phytosterol had been eliminated as far as possible from the diet; and
(3) how far the quantity of the oily unsaponifiable matter was affected by
the use of ether extracted food; and (4) whether an animal could be kept in
a healthy state on a prolonged, ether extracted diet, a rabbit weighing
24 kilogrammes was fed from December 30,1907, to January 15, 1908,
inclusive on extracted bran, moistened with a little water. The feeces were

* ‘Roy. Soc. Proc.,’ B, vol. 80, p. 212.
VOL. LXXXI.—B. I

114 Messrs. Dorée and Gardner. Origin and [Dee. 18,

collected from January 2nd to the 15th inclusive and weighed, after drying,
462 grammes. During this period the animal was given 1:12 kilogrammes of
extracted bran, most of which it ate with apparent satisfaction. On
January 15th its weight was 2:4 kilogrammes and during the period of the
experiment varied from 2°4 to 2°5 kilogrammes.

The feces were extracted with ether and the ethereal solution saponified in
the manner described; 1:215 grammes of dry unsaponifiable matter was
obtained as a viscid stiff oil. This dissolved ina small quantity (20 c.c.) of
absolute alcohol, with the exception of 0:05 gramme of insoluble tar. On
adding enough water to make the alcohol about 85 to 90 per cent. strength
and allowing to stand, some crystalline matter separated and, after filtration
and drying, was obtained in the form of dark brown greasy crystals weighing
0-4 gramme.

This brown crystalline matter was decolorised by animal. charcoal and
carefully recrystallised fractionally from alcohol. The first and main crop
melted at 135° to 136°, and under the microscope had the form of trans-
parent long hexagonal plates. It was identical with the “ phytosterol”
which we had isolated from the bran. The mother liquors yielded a small
crop of crystals which under the microscope were more indeterminate, being
grouped in masses and stars. On recrystallisation, however, long hexagonal
crystals were again obtained melting at 133° to 135°. Wo trace of cholesterol
could be discovered.

Experiment I/7—Immediately on the close of the above experiment the
sane rabbit was fed from January 16 to January 25 inclusive, on
80 grammes of extracted bran with 0:25 gramme of cholesterol per day,
except on two days on which no cholesterol was given. It received therefore
in this period 2 grammes of cholesterol. It was then fed for three days on
extracted bran alone, and the feces were collected during the whole period.
After drying, the feeces weighed 497 grammes. The weight of the animal
remained constant all through the experiment.

The ethereal extract was reddish in colour, with a green fluorescence,
which was just as marked after saponification; 2°56 grammes of dry
unsaponifiable matter were obtained. This dissolved in absolute alcohol
leaving 0:06 gramme of insoluble tar. Water was then added to reduce the
alcohol to 90 per cent., and on standing, brown crystalline matter was
deposited weighing 1:14 grammes. On further standing another crop weighing
0:28 gramme was obtained. The mother liquors were then evaporated to
dryness, dissolved in pyridine and treated with benzoyl chloride;
0:1035 gramme of crude benzoate of cholesterol was obtained. The total
weight of crude cholesterol was thus 15 grammes. The two crops of

1908.] Destiny of Cholesterol mm the Animal Organism. 115

cholesterol were decolorised by animal charcoal and recrystallised from
aleohol. The larger crop melted at 139° and was practically pure cholesterol,
but the smaller crop softened at about 125° and melted at 135°. Under the
microscope the latter appeared to consist mainly of cholesterol, but was
contaminated with phytosterol. The benzoate after recrystallisation melted
at 144° to 145° to a turbid liquid which cleared at 180° and on cooling
showed the characteristic play of colour in a well-marked manner. About
1 gramme of purified cholesterol was in this way obtained. Evidently
therefore the rabbit had absorbed between 0°5 and 1 gramme of cholesterol
during the time of the experiment. The animal remained in good health for
some weeks afterwards, its weight remaining constant, when it was killed for
another purpose.

Experiment IV.—The rabbit used in this experiment weighed 1:7 kilo-
grammes, and was very thin. It was fed for the three days prior to the
commencement of the experiment on extracted bran; it was then given
0:25 gramme of cholesterol and 60 to 70 grammes of extracted bran daily
for eight days, and extracted bran alone for three more days. Care was
taken that the animal took the whole of the cholesterol, but it wasted a
good deal of the bran. The animal lost weight during the whole experiment,
and died the day after, when its weight was 1°3 kilogrammes. A post-mortem
examination showed that the animal was very thin, and in poor condition.
The intestine was filled with a watery fluid, the liver very dark in colour,
and the stomach dilated with gas. It may be noted that during this
experiment the weather was very cold, and for a few days the heating
apparatus was out of order, so that possibly this may have had something
to do with the death of the animal, which was not in the best of condition
at the start.

Three hundred and twelve grammes of dry feces were obtained. The
ethereal extract was pale yellow in colour, and on evaporation gave
146 grammes of unsaponifiable matter as a greasy brown solid. This
dissolved in 85 to 90 per cent. alcohol, with the exception of a small
amount of tar which was insoluble in absolute alcohol, though soluble in
ether. On standing, the solution deposited 0°79 gramme of brown crystalline
matter. A further crop, weighing 0°32 gramme, was obtained on long
standing. This was more granular in appearance, and rather sticky. The
mother liquors were evaporated to dryness and benzoylated in pyridine
solution, but no matter difficultly soluble in alcohol could be isolated.

The two crops were decolorised by animal charcoal and recrystallised.
The first deposit melted at 142° to 143°, and under the microscope was seen
to consist of practically pure cholesterol. The second crop of crystals

I 2

116 Messrs. Dorée and Gardner. Origin and [Dee. 18,

melted at 141° to 142°, but a microscopic examination showed that it
contained some phytosterol, as the shape of the crystals deviated from those
of cholestero] in the well-known manner occasioned by traces of phytosterol.
A final crop melted at 138° to 139°, and contained a larger amount of
phytosterol. In this experiment it is evident that at least 1 gramme of
cholesterol was absorbed.

Experiment V.—A rabbit, weighing 1°7 kilogrammes, was treated exactly
as in Experiment IV. At the end it weighed 1°6 kilogrammes, and appeared
to be in good health; 277 grammes of dry feces were obtained, which were
extracted for 14 days in a Soxhlet’s apparatus with ether. The ethereal
solution was deep red, with a strong green fluorescence. The dry unsaponi-
fiable matter weighed 2:275 grammes, and was dissolved in 70 cc. of hot
alcohol, leaving 0:05 gramme of insoluble tar. After adding 6 c.c. of water to
the hot alcohol solution, 1°39 grammes of red crystalline matter were
deposited, from which 1125 grammes of fairly pure cholesterol were obtained.
The mother liquors, on standing, deposited a small quantity of crystalline
matter mixed with oil, from which, after boiling in alcoholic solution with
animal charcoal, 0:11 gramme of white crystals was isolated. All the mother
liquors were evaporated and benzoylated, but only a small trace of difticultly
soluble matter resulted. The weight of cholesterol recovered was therefore
1-23 to 1-4 grammes, which, however, would contain any phytosterol present.

Lxpertment VI—A rabbit, weighing 1°7 kilogrammes, was fed on extracted
bran and cholesterol as in the previous experiments. The weight remained
practically constant, but after the experiment, on being put on ordinary diet,
it began to lose weight, and died on the eighth day. The weight of the
dead animal was 14 kilogrammes, but a post-mortem examination revealed
nothing abnormal.

The weight of dry feces collected was 283 grammes, and they were
extracted for 14 days. The ethereal extract was pale yellow in colour, but
without any fluorescence. The unsaponifiable matter weighed 3:13 grammes,
but was not free from calcium chloride. It was boiled with absolute alcohol,
when 07145 gramme of insoluble matter was left. The alcoholic solution
after dilution with water until the strength was 85 to 90 per cent., gave
1:47 grammes of not very coloured crystalline matter. The mother liquors
yielded between 0-1 and 0°2 gramme of impure crystalline matter. After
recrystallisation of the whole from the least amount of 90 per cent. alcohol,
the melting point was 135°.

The results of these experiments are summarised in Table I.

It is clear from these experiments that (1) cholesterol is not excreted
by rabbits unless they are fed on it, which is in agreement with our previously

1908.] Destiny of Cholesterol in the Armmmal Organism. kA
Table I.
Nl Se Algae | |
| Duration | Weight of Weicht of | Weight of | woioht of Tee | Le ;
OE | xabbitvat Weight Sg © bran, in ene e fiable | cholesterol
| Exp. experi- beginning, Eeouals cholesterol Lies dry motes |) mecoreedl,
ee in ui kilo- | given. | erammes. | feeces. dry, in | including
aye: grammes: | | | grammes. phytosterol.
ie 14, 2°4 2 °4 — 1°12 462 1:215 | O4Aoft |
| phytosterol
III. 11 2 A 2°4 2 1°01 497 2°56 1°5
IV. 11 7 1°3 2 0°67 312 146 | 1-11
Vv. 11 17 16 2 0°73 277 2°27 | 1-23—1-4 |
VI. 11 il % Ie 2 0°73 283 38:13 | 1°4-1°6 |

published results; (2) that when cholesterol is administered with the food a
It is
also clear that vegetable food such as bran or grass can be freed from fat

portion of it is absorbed, in our experiments about 50 per cent.

and phytosterols by extraction with ether without impairing its feeding
value.

Is Cholesterol Absorbed from the Food by Carnivorous Animals ?

In our former paper on the excretion of cholesterol by the dog* we
described experiments which, although they were carried out primarily for
the purpose of showing that the cholesterol content of the feces was a
function of the cholesterol in the food taken, may yet be considered as
evidence for the absorption of some cholesterol from the food in this animal.
But such evidence cannot be regarded as of the same conclusive nature as
that afforded by the experiments on the rabbit, because the cholesterol
content of the various foodstuffs given is not known with any degree of
certainty. The estimations of Dormeyery, for the cholesterol content of dry
muscle (0:23 per cent.), are perhaps as satisfactory as any, though they
probably err, if anything, in being too high. However, if we take such a
value and apply it to our own data we arrive at such results as the following.
In one experiment a dog in 20 days ate 7470 grammes of cooked beef and
mutton, the percentage of solids in which we found to be about forty.
Allowing for the fact that the meat did not consist entirely of muscle and
for variations of other kinds, we may perhaps halve Dormeyer’s value in
this case. On this assumption, then, the animal consumed 3°4 grammes of
cholesterol, whereas 0°8 gramme only was found in the feces, so that a
disappearance of about 2°5 grammes of cholesterol is indicated.

* “Roy. Soc. Proc.,’ B, vol. 80, p. 227.
+ ‘ Pfliig. Archiv,’ vol. 61, p. 341.

118 Messrs. Dorée and Gardner. Origin and [Deec. 18,

In another experiment the animal ate 6758 grammes of horseflesh in
17 days. Making the calculation as before, we find that the dog may have
eaten 4 grammes of cholesterol, whereas only 1 gramme was discovered,
pointing again to an absorption of possibly 3 grammes.

We did not institute any further experiments with the dog on these lines
on account of the numerous uncertainties involved. We have, however,
lately been successful in discovering a cholesterol-free diet comparable with
extracted bran on which cats can be fed, and have begun an elaborate series
of experiments on the question which we hope to communicate in the near
future. Preliminary experiments on cats showed us that, as in the case of
dogs, the cholesterol of animal food is passed in the feces as such, but that on
a brain diet these animals also convert the cholesterol into coprosterol. The
two following experiments may be quoted here as bearing on this interesting
point.

Experiment VII._—A cat weighing 2°8 kilogrammes was fed on raw sheep’s
brain for 14 days. The feces were somewhat liquid, and dried at 100° to a
soft, sticky, glue-like mass, which was ground up with excess of sand before
extracting with ether. Weight of dry feces, 245 grammes. During the period
of diet the animal lost 0°5 kilogramme in weight, which loss, however, it
subsequently regained on ordinary diet; 28 grammes of unsaponifiable matter,
in the form of a dark red viscid oil were obtained. By fractional crystallisation
from acetone between 18 and 19 grammes of brown crystalline matter were
separated. This consisted of coprosterol, and after further purification from
dilute aleohol 12 grammes of perfectly pure coprosterol were obtained. This
melted at 99° to 100°C., and had a specific rotatory power (in chloroform)
[a ]}§ = + 20°4.

On a diet of raw sheep’s brain, therefore, the cat changes cholesterol into
coprosterol in the same way as we showed was the case with the dog.* If
we assume that sheep’s brain contains 2 per cent. of cholesterol, our cat
should have consumed in the 14 days some 34 grammes of cholesterol, which
would correspond with an absorption of about 15 grammes, or 1 gramme
per day. Owing to the difficulty of crystallising coprosterol completely from
the oily matter with which it is mixed in the unsaponifiable residue, one
cannot claim for this estimation any high degree of certainty. It is, how-
ever, significant that the total weight of unsaponifiable matter obtained was
less than the total cholesterol that should have been consumed, and there
can be no doubt that the extraction of the feces by ether was a very
thorough one, as it was allowed to go on for 14 days, the ether distilling over
during the day and being allowed to soak on the material during the night.

* “Roy. Soc. Proc.,’ B, vol. 80, p. 227.

1908.| Destiny of Cholesterol in the Animal Orgamsm. 119

Experiment VIII—In’' this experiment the sheep’s brain was lightly fried
in its own oil before being given, in order to ascertain whether the cooking
process had any influence on the conversion of the cholesterol to coprosterol.
The cat selected weighed 2°9 kilogrammes, and during the feeding period of
14 days lost 0-4 kilogramme in weight; 1913 grammes of brain (weighed
uncooked) were consumed, and 365 grammes of dried feces obtained. These
were of very much the same constituency as before, and were treated in a
similar manner ; 23 grammes of unsaponifiable matter in the form of a red
oil were obtained. This proved very difficult to purify, owing to the tarry,
sticky oils present, and we were only able to isolate 7-4 grammes of white
erystalline matter, which proved to be a mixture of cholesterol and coprosterol.
The cholesterol taken (on the above assumption) should therefore be
38 grammes, the total unsaponifiable matter being only 23 grammes. The
cooking and consequent partial sterilisation of the brain seem, therefore, to
have interfered with the conversion of the cholesterol into coprosterol.

EXPERIMENTS TO ASCERTAIN WHETHER ANY OF THE CHOLESTEROL WHICH
DISAPPEARS FROM THE FOOD CAN BE FOUND IN THE BLOOD.

Herbworous Animals.

Pribram* has stated that on administering cholesterol to rabbits per os, an
increased percentage of this substance could be found in the blood. His
method consisted in injecting into the stomach of the animal an emulsion
of cholesterol, cholesterol oleate, or cholesterol palmitate, made up with
olive oil. After some hours the animal was killed, and the cholesterol in the
blood determined in the usual way.’ But from the point of view of proving
that cholesterol taken by the mouth can be absorbed into the blood stream
these experiments seem to us for several reasons by no means conclusive.
In the first place, the rabbits studied were far from being under normal con-
ditions of diet. A comparatively large dose of oil was put into the stomachs
of animals who are not accustomed to take or assimilate fats in this form.
Pribram mentions that the oil passed into the blood as the serum became
opalescent, and it seems to us not improbable that, with a quantity of oil in
the stomach which cannot be assimilated in the ordinary way, some of the
oil might percolate, if one may use the term, into the blood, carrying the
cholesterol with it. The supposed increase, therefore, found in the blood
might not unreasonably be due to a mechanical rather than to a metabolic
process. But a further consideration of the data given by Pribram led us to
doubt whether they could be considered as showing that there was an increased
percentage of cholesterol.

* ‘Bioch. Zeit.,’ vol. 1, p. 414.

120 Messrs. Dorée and Gardner. Origin and [Dec. 18,

The standard of comparison employed by him was the cholesterol content
of the blood of a starving rabbit, which again is an abnormal case, since con-
ceivably some cholesterol might disappear from the blood during starvation,
and if this is so it is probably a variable standard, as different animals would
vary in the rate at which the cholesterol was removed from their blood.
Again, the quantities of cholesterol isolated and weighed were extremely
small, and were admittedly not pure. No melting points or other constants
were given, and we are consequently left uncertain as to whether the matter
weighed was cholesterol in a more or less pure state, or whether it was largely
composed of crude unsaponifiable matter, or whether it contained any
cholesterol at all. As the percentages found were so small, a very slight
variation in the amount of impurity present would invalidate and even
entirely reverse the conclusions deduced by the author. In a second series
of experiments, however, which bring out the increased inhibitory power of
the serum of the cholesterol-fed rabbit towards the hemolytic effect of
saponin, the results are more satisfactory, and certainly speak for an assump-
tion of cholesterol by the blood. But our objection to the method of dosage
adopted still holds, and the number of hemolytic experiments carried out
was too few. Our own experiments in comparing the action of sera in this
respect have shown us that there is a very considerable yariation in the
action of the sera of individual animals of the same species, when treated
under precisely similar conditions. The discussion of this point, however,
we leave for the present, as we hope to make it the subject of a communi-
cation in the near future.

In the experiments we have carried out to ascertain the fate of the
cholesterol which disappears from the food we have endeavoured to avoid
the difficulties pointed out in the preceding paragraph in the following
way —

1. We adopted as a standard of comparison a rabbit which was fed for
a long period on a cholesterol-free diet, viz., bran thoroughly extracted with
ether. The blood of such an animal was compared with that from others
which had been fed in an exactly similar way as to times and quantities of
extracted bran, but whose food contained in addition a measured daily
quantity of cholesterol. In this way we had two rabbits feeding on
practically the same diet under the same conditions, and accordingly the
chance of variations, especially in the bile flow, due to differences in the
food taken was reduced to a minimum, and the only variation likely to
interfere was that due to the individual peculiarities of different rabbits
which are inevitable in such experiments.

2. With regard to the estimation of cholesterol in the tissues and the

1908.| Destiny of Cholesterol in.the Animal Organism. 121

blood, we must again emphasise the fact that the weight of crude unsaponifi-
able matter obtained from them gives little or no idea of their cholesterol
content. In our experience, the ether extract of animal tissues always
contains relatively large quantities of low melting oily or resinous bodies,
which may prove to be of very considerable importance in biochemistry, but
they are non-crystalline and cannot be considered as cholesterol. Further-
more, their amount is very variable, so that even for purposes of comparison
the weights of crude unsaponifiable matter are useless. Our own procedure
was as follows: The blood, if dried in the ordinary way, becomes a very
hard horny mass which even if powdered is difficult to extract. We there-
fore mixed the blood after whipping to prevent coagulation with sand and
plaster of Paris in sufficient quantity to form a friable mass. This was
ground up and extracted for 14 to 30 days, the heating being stopped
during the night so that the ether might thoroughly soak into the material.
The extract was saponified in the manner we have previously described, and
the non-saponifiable residue converted directly to benzoate in pyridine
solution, the cholesterol being thus weighed in the form of cholesterol
benzoate.

Experiment [LX.—Rabbit A. A rabbit, weighing 2°8 kilogrammes, was fed
for 21 days with 70 to 80 grammes of extracted bran per day, and then
killed 24 hours after the last supply of food had been placed in the cage.
A post-mortem examination showed that the stomach still contained some
food. The animal during this period lost 0:3 kilogramme in weight. The
weight of blood obtained was 73 grammes, from which 0:14 gramme of
unsaponifiable matter in the form of a stiff oil was obtained. The quantity
of cholesterol contained in this was so small that it was not found possible
to isolate any in a pure state.

Experiment X.—Rabbit B. This rabbit, weighing 2-2 kilogrammes, was
fed for three days on extracted bran, then during 10 days on 540 grammes
of extracted bran, mixed with 24 grammes of cholesterol, care being taken
that the whole of the cholesterol was eaten. The weight of the animal
remained unaltered during this period, and it was killed 24 hours after the
last meal had been placed in the cage. The blood obtained weighed
71 grammes and yielded 0:29 gramme of crude unsaponitiable matter, from
which 0:0375 gramme of pure cholesterol benzoate was obtained. The
specimen, which was actually weighed without further crystallisation, melted
at 142° to 148° to a turbid liquid which became clear at 170°, and on
cooling showed the characteristic play of colours. This quantity corresponds
to a yield of 0:0295 gramme of cholesterol or 0:0415 per cent.

Experiment XI—Rabbit C. In order to compare the cholesterol content

122 Messrs. Dorée and Gardner. Origin and [Deec. 18,

of an animal fed on a normal diet which contained phytosterol but not
cholesterol, a rabbit weighing 2°8 kilogrammes was fed on a liberal mixed
diet of cabbage, oats, and bran for a month, and killed after 24 hours as in
previous experiments. The blood obtained weighed 75 grammes, which
yielded 0-117 gramme of unsaponifiable matter as a brown oil mixed with
crystalline material. After treating with benzoyl chloride in the usual
way 0°028 gramme of greasy crystals was obtained, which were obviously
not pure. Under the microscope these appeared as star-shaped aggregates
of needles mixed with indeterminate matter, but no typical crystals of
cholesterol benzoate were observed, and the substance could not be further
purified. It is obvious that there is not sufficient cholesterol in the blood
of a single rabbit, when fed on a non-cholesterol, or on a normal diet, for an
accurate quantitative estimation. We therefore fed six rabbits, weighing
15, 1:7, 1:4, 1:9, 1°5, 1:9 kilogrammes respectively, on a liberal diet of oats,
bran, and greens for a week. They were then killed and the total blood
taken. This weighed 500 grammes. On treatment in the usual way
0464 gramme of unsaponifiable matter as a brown, slightly greasy solid
was obtained. This was crystallised from alcohol; the first crops, weighing
respectively 0:091 and 0:049 gramme, consisted, as a microscopic examination
showed, mainly of cholesterol, plate-like crystals of which were mixed with
minute spherules of some other substance. These crystals were dried and.
treated in pyridine solution with benzoyl chloride. All the mother liquors
remaining were evaporated to dryness and treated with pyridine and benzoyl
chloride. The benzoate found was recrystallised from a measured quantity
of absoluté alcohol; 0°1523 gramme of pure cholesterol benzoate in all was
thus obtained, which, without further purification, melted correctly, and gave
the colour play of cholesterol benzoate; 0°1199 gramme of cholesterol
was thus obtained from the blood of six rabbits or 0°024 per cent.

It is clear from these experiments that Pribram was correct in his
conclusions, and that cholesterol can be absorbed from the intestines into
the blood of the animal, since in the case of the rabbits which had been fed
on cholesterol we were easily able to prepare and weigh pure cholesterol
benzoate, whereas in the case of a rabbit fed on extracted diet, or on normal
diet, the quantity was so small that we were unable to obtain any
cholesterol from it in a pure state. In order to get a precise figure for the
cholesterol content of the blood of the rabbit under normal conditions we
were obliged to deal with the blood of six rabbits.

In the case of rabbits A, B, and C we made estimations of the cholesterol
contained in the brain and spinal cord, and in the rest of the animal
respectively. We can, however, draw no conclusions from the results of the

1908.] Destiny of Cholesterol in the Anomal Orgamsm. 123

experiments, but we think the actual determinations are of sufficient interest
and accuracy to be placed on record. The method we adopted for the brain
and cord was to mix with plaster of Paris in a mortar, and, after the mass
has hardened, to powder it thoroughly. This was then thoroughly extracted
with ether (21 days) and the extract treated in the usual way. The rest of
the rabbit carcase (including the fur) was finely minced in a machine, mixed
with plaster of Paris, and again passed through the machine. After it had
set to a dry mass it was ground up in a mortar with coarse sand and plaster
of Paris, and extracted for three weeks with ether. The results are collected
together in the following table :—

Table II.
| |
| - lt “p | Pure
| _ Weight _Unsaponitiable | Bie eee Cholesterol
in grammes. | matter. | a per cent.

Rabbit A, weight 2°5 kilos., fed on extracted bran for 20 days.

181 G)a)6| «Sah rooe espe ener eReee rere | 73 0-14 | (trace) | —
Brain and spinal cord ...... | 14°3 0°68 0-427 | 3°0
Rest of rabbit ..........-.......| 2413 | 3°75 | 2-168 | 0-09

| Rabbit B, weight 2-2 kilos., fed on extracted bran +21 grammes of cholesterol
for 10 days.

Blood nen GOL BOOCORSADS HEC ROACEC | (al | 0°29 0 :0295 | 0 0415
Brain and spinal cord ...... | 13°31 | 1°31 (lost) | =
Rest of rabbit .....4..-.:....--s 2116 3 50 | 1-908 0-09

| Rabbit C, weight 2°8 kilos., fed on a mixed diet of cabbage, oats, and bran

| for 1 month.
HUIGUG! pebeecentapanenedeccusedes 75 0°117 | (trace) —
Brain and spinal cord ...... | 175 0-776 0 5225 | 3°0
Rest of rabbit ...............+- | 2708 | 5-11 | 2-716 | 0-10
Six rabbits, fed on above mixed diet.
GOCE ea sectesa ewe lectter a te 500 0-464 | 0 °1199 | 0-024.

I | | |

EXPERIMENTS TO ASCERTAIN WHETHER THE CHOLESTEROL CONTENT OF THE
BLOOD CAN BE CORRELATED WITH VARIATION IN THE CHOLESTEROL
CONTENT OF THE FooD IN CARNIVOROUS ANIMALS.

A. Experiments in which the Animals were killed two to four hours after
a Meal.
Experiment XII—A dog, weighing 7:36 kilogrammes, was fed for 10 days
on a daily ration of 200 to 300 grammes of bread, the whites of two eggs,

124 Messrs. Dorée and Gardner. Origin and [Dee. 18,

and a teaspoonful of cream, the whole being moistened with a solution of
Liebig’s extract of beef, and lightly fried. The animal was killed three and
a half hours after the last meal. The blood weighed 480 grammes, and from
this 0°687 gramme of unsaponifiable matter was obtained. This was at
once benzoylated in pyridine solution, and a total crop of cholesterol benzoate
weighing 0°4865 gramme was separated. This corresponds to 0°3892 gramme,
or 0°0811 per cent. of cholesterol.

A post-mortem examination showed that the stomach was practically empty.
The gall-bladder contained 5:2525 grammes of bile which on evaporation
yielded 0:392 gramme of solid matter. From this 0-002 gramme of
cholesterol benzoate was obtained, or 0°18 per cent. of cholesterol (calculated
on the dry solids).

Experiment XIII—A dog, weighing 8:13 kilogrammes, was fed for nine
days ona daily ration of 250 grammes of raw brain. At first it did not
take kindly to this food, but in the last few days it consumed the whole of
the brain given. The animal was killed two hours after a meal. The
weight of blood obtained was 430 grammes, which yielded 0°74 gramme of
unsaponifiable residue. ‘This was benzoylated directly in pyridine solution,
and 0-484 gramme of pure cholesterol benzoate was obtained, melting
correctly. This corresponds to 0°3872 gramme, or 0:09 per cent. of
cholesterol.

A post-mortem examination showed that the stomach contained some
undigested food. The gall-bladder contained 2°06 grammes of bile of a pale
yellow colour, and left 0:2245 gramme of solid matter. From this
0:004 gramme of unsaponifiable matter was obtained, but only a trace of
benzoate could be separated from it.

Experiment XIV.—A dog, weighing 9°77 kilogrammes, was fed for nine
days on a daily ration of 200 grammes of dry oatmeal made into porridge
with water. The animal ate about half the last meal only, and was killed
four hours afterwards. The blood weighed 680 grammes and yielded
0-91 gramme of unsaponifiable matter. This, on benzoylation in pyridine
solution, gave 0°6775 gramme of pure cholesterol benzoate, corresponding to
0542 gramme, or 0:0797 per cent. of cholesterol in the blood.

A post-mortem examination showed that the stomach contained a quantity
of undigested food. The gall-bladder contained 6°36 grammes of bile which,
on evaporation, yielded 1:1065 grammes of solid residue. From this only
0:001 gramme of cholesterol benzoate could be obtained, corresponding to
0:07 per cent. of cholesterol.

Experiment XV.—A dog weighing 8°5 kilogrammes was fed for six days on
a daily ration of 200 to 300 grammes of brain, mixed with some bread. The

1908.| Desteny of Cholesterol im the Animal Organism. 125

animal was killed four hours after the last meal. The blood was unfortunately
lost. The gall-bladder contained 702 grammes of bile which, on evaporation,
yielded 1:3947 grammes of solid residue. From this, 0:°0028 gramme of
cholesterol benzoate was obtained, or 0°16 per cent. of cholesterol.

B. Experiments in which the Animals were killed 24 hours after a Meal.

Lxperiment XVI—A dog weighing 7°8 kilogrammes was fed for 7 days on
porridge made by boiling about 100 grammes of oatmeal with water, daily. It
was killed 24 hours after the last meal. The quantity of blood obtained was
524 grammes, from which 0°9845 gramme of greasy unsaponifiable matter
was obtained. The first crop of crystals from alcohol weighed 0°2915 gramme,
and a microscopic examination showed that these consisted of practically
pure cholesterol. The residues were benzoylated in pyridine solution and
yielded 0°1795 gramme of crystals, which melted correctly and gave the
characteristic colour play of cholesterol benzoate. The total cholesterol
obtained was therefore 0°435 gramme or 0-083 per cent.

A post-mortem examination showed that the stomach was quite empty.
The gall-bladder was distended and contained 4°55 grammes of bile, which,
after drying at 100° C., left 0°8257 gramme of solid matter. We attempted
to estimate the cholesterol in this quantitatively, but the amount was too
small for an accurate determination. However, 0:0025 gramme of choles-
terol benzoate was isolated, or 0:24 per cent. of the total solids.

Hiperiment X VIT.—A dog weighing 9:1 kilogrammes was fed for 14 days on
raw brain. It was given 300 to 500 grammes of fresh brain per day, but the
animal did not take the food very well and often left a portion uneaten. On
the last day of the period the dog developed feverish symptoms and was
killed 24 hours after the last meal. The quantity of blood obtained was
007 grammes and yielded 1:45 grammes of unsaponifiable matter. This
was not completely soluble in alcohol. The first crop of crystals weighed
05075 gramme and melted at 145° C. A second crop weighed 0:0785 gramme
and melted at 144° C. From the residues on benzoylation 0:2115 gramme of
cholesterol benzoate, melting at 145°, was isolated. The total cholesterol
obtained was therefore 0°7526 gramme, or 0:1486 per cent.

A post-mortem examination showed that the stomach was empty and
distended with gas, and the lungs appeared to be congested. The gall-bladder
contained 0°844 gramme of bile, which was very thick and stringy, and
contained 01617 gramme of solid matter. From this 0:0025 gramme of
cholesterol benzoate was obtained, or 1:2 per cent. of the total solids.

Liperiment XVIII.—A large dog weighing 19:2 kilogrammes was fed for
24 days on raw brain together with a little bread. This animal took the

126 Messrs. Dorée and Gardner. Origin and [Dee. 18,

food readily and consumed daily from 500 to 800 grammes of brain, and at
the end of the period was in good health. It was killed 24 hours after
a full meal. The weight of blood obtained was 1140 grammes, which yielded
1:5 grammes of unsaponifiable matter. On crystallisation from alcohol a first
crop, weighing 0°304 gramme, melting at 144° to 145°, was obtained, and
a second weighing 0184 gramme and melting at the same temperature. The
mother liquors and residues, on benzoylation, gave 0°41 gramme of choles-
terol benzoate. The total cholesterol obtained was therefore 0°816 gramme,
or 0°072 per cent.

A post-mortem examination showed that the stomach was empty. The
gall-bladder contained 15°82 grammes of bile, which gave 3-947 grammes of
solid matter, and from this 0:017 gramme of cholesterol benzoate was
obtained, or 0°34 per cent. of cholesterol.

Experiment XIX—A dog weighing 13:4 kilogrammes was fed for 10 days
on a diet practically free from cholesterol. The daily ration consisted of
250 to 300 grammes of bread, the whites of two eggs, two teaspoonfuls of
cream lightly fried together after moistening with a solution of Liebig’s
extract of beef. The animal continued in good health and was killed
24 hours after the last meal. The blood weighed 760 grammes and yielded
1:326 grammes of unsaponifiable residue. On crystallising from alcohol the
first crop weighed 04615 gramme and melted at 145°, the second weighed
02135 gramme and the same melting point. After benzoylation, the
residues yielded 071728 gramme of cholesterol benzoate, melting at 145°,
and showing the-colour changes. The total cholesterol obtained was there-
fore 0°8132 gramme, or 0°107 per cent. A post-mortem examination showed
that the stomach was empty. The gall-bladder contained 10°29 grammes of
bile which, on evaporation, gave 2°632 grammes of solid matter. From this,
0:003 gramme of cholesterol benzoate was obtained, or 0:09 per cent. of
cholesterol.

These results are collected together in Table III.

The experiments differ fundamentally from those carried out on the
rabbit described in the earlier part of this paper. In the case of these
animals a standard diet free from cholesterol and similar bodies, to which
measured portions of cholesterol could be added, was available ; and as they
are practically continuous feeders the bile flow and consequently the
cholesterol content of the blood, due to this source, would remain practically
constant. In the case of the dogs we had no such cholesterol-free standard
diet and were obliged to make use of a number of entirely different foods, ,
differmg as far as possible in cholesterol content, though even the non-
cholesterol ones contained phytosterol. Little is known concerning the

1908.|] Destiny of Cholesterol in the Anmal Organism. 127

Table III. |
| : F Per- |
E : weigh of Diet Weight of | Weight centage of |
xperle nie Diet. period, in | blood, in | “7? cholesterol |
ment. kilo- | in the 3 |
| days. grammes. | 13044 in the
| grammes. | ood. Biecdl
| |
| |

ob oo |

|

A.—Animals killed two to four hours after a meal. (Terriers.)

XII. 7°36 | Bread, egg-white, cream 10 | 480 0 -3892 | 0-0811 |
XIII. 8-13 Raw brain.................. ©) | 430 0 -3872 0:0900 |
XIV. OL Tiaw til Micals esimeerstidssesuco ce: ee Ri enanGSO 0-5420 | 0-0797

B.—Animals killed twenty-four hours after a meal.
XVI.* 7°8 Ie Mears. syed ccnsettateiieaisesss | 7 | 524 | 0 °4350 | 0 083
XVII.+ 9-1 | Raw brain...............++: 14 | 507 | 0 “7526 0°148
XVIILt | 19-2 | Ranw brain bread an. 2d. 1140 | 0-8160 | 0-072 |
XIX.§ | 12°67 Bread, egg-white, cream 10 760 | 0 8132 | 0-107
| |
* Terrier. + Terrier ; dog jaundiced.
{ Retriever dog. § Collie dog.

influence of food on the excretion of bile, but from experiments that have
been made by various observers there is good reason to suppose that the
nature of the diet would not be without influence. Furthermore, the dog is
a discontinuous feeder and the flow of bile into its intestine would be
intermittent. Under these circumstances the portion of the floating (as
distinguished from the constitutional) cholesterol in the blood due to the
reabsorption of the cholesterol of the bile, would not necessarily be strictly
comparable in these different cases. As to what would be the limits of such
variation, if any, we have no data at present from which to form an opinion,
but a variation of the kind suggested might wholly or partially mask any
variation due to the cholesterol absorbed from the food, which, at best, would
not be great in absolute magnitude. In the first series of experiments (A)
in which the animals are fairly comparable in weight and variety, the blood
of the dog fed on brain shows a small increase in the percentage of
cholesterol in its blood, though it would not be unreasonable to ascribe this
to an extra bile flow due to the fatty nature of the diet. In the second
series of experiments (B) in which the blood was taken after the digestive
process was completed, the percentages found were of much the same order
of magnitude as in the first series, with the exception of Experiment XVII.
It will be noticed, however, that the percentage in the blood of the retriever
dog fed on brain was slightly less than that in the blood of the dogs fed on
meal, and bread and egg-white respectively. These animals were, however,
very different in weight and were of different varieties.

128 Origin and Destiny of Cholesterol in the Animal Organism.

Little value can be given to the high figure obtained in Experiment XVII,
as the animal was ill at the time its blood was taken.

It seems to us very doubtful whether the chemical methods of estimating
cholesterol, which we have endeavoured to make as perfect as possible, are
sufficiently accurate to enable us to draw definite conclusions without making
an enormous number of experiments of this type. We have, however,
recently found a material suitable for the food of cats, which can be rendered
cholesterol-free, and a series of experiments are in progress to compare the
effect on the blood of the addition of cholesterol to such a diet both by
chemical analysis and by comparisons of the anti-hemolytic effect of the
sera. The results of these experiments, which we expect to give more
definite information on this subject, we hope to make the subject of a com-
munication 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.

129

The Origin and Destiny of Cholesterol in the Anuomal Organism.
Part IV.—The Cholesterol Contents of Eggs and Chicks.

By G. W. Exuis and J. A. GARDNER, Lecturer on Physiological Chemistry,
University of London.

(Communicated by Dr. A. D. Waller, F.R.S. Received January 15,—
Read February 11, 1909.)

(From the Physiological Laboratory, South Kensington, University of London.)

In a paper recently communicated to the Royal Society* the hypothesis
was advanced that cholesterol is a substance which is strictly conserved
in the animal organism. As it is difficult to conceive how a body of
the constitution of cholesterol can be synthesised in the organism from
proteids, carbohydrate or fat, it was suggested that the waste of cholesterol
might be made up from the food taken by the animal. In order to test the
correctness of this view we thought that evidence of fundamental importance
might be obtained by comparing the cholesterol content of eggs and newly-
hatched chicks, and also by ascertaining whether chicks could be reared and
would thrive on food deprived of its cholesterol or phytosterol. In this
paper we give an account of our estimations of cholesterol in hens’ eggs and
newly-hatched chicks.

Method of Estumation—The weighed egg or chick (including broken shell)
was pounded up in a mortar with sand and sufficient plaster of Paris to
cause the whole to set after a time toa dry mass. This was powdered and
extracted in a Soxhlet’s apparatus with ether for about twelve days. The
ethereal solution of the extract was saponified in the cold by means of an
alcoholic solution of sodium ethylate. After standing overnight the
precipitated soap was filtered off and thoroughly washed with ether. The
filtrate and washings were repeatedly shaken with water to get rid of
alcohol, excess of alkali, traces of soap, etc., dried with calcium chloride and
the ether distilled off. The residue was dried at 100° C. and weighed. In
the case in which single eggs or chicks were analysed, the dry residue,
dissolved in 10 cc. of pyridine, was mixed with about three times its weight
of benzoyl chloride also in solution in 10 e.c. of pyridine. After standing
overnight the liquid was poured into water, and the precipitated cholesterol
benzoate after drying was boiled with 10 c.c. of absolute alcohol and allowed
to stand some hours. The crystals were filtered off, washed with a little

** Roy. Soc. Proc.,’ this vol.
VOL, LXXXI.—B. K

130 Messrs. Ellis and Gardner. Origin and [Jan. 15,

absolute alcohol and weighed. In all cases these crystals were colourless, or
nearly so, and melted approximately correctly. The filtrate and washings
were separately measured, and corrections, which had been ascertained by
previous experiments, made for the solubility of cholesterol benzoate. When
cholesterol benzoate was crystallised from absolute alcohol the mother liquor
at 21° C. contained 0°12 per cent. When, however, ready formed crystals
were shaken for a short time with alcohol at 20° C. and filtered, the filtrate
was found to contain only 0:04 per cent. When a number of eggs or chicks
were analysed together, the unsaponified residue was crystallised from
alcohol, and as much pure cholesterol as possible was isolated, melting at
145°—147° C. The mother liquors were then evaporated to dryness,
benzoylated in a pyridine solution as described above, and the benzoate
weighed. The soaps precipitated on saponification were collected together in
two lots, comprising respectively the total amount obtained from all the eggs
examined, and the total amount obtained from all the chicks. These were
separately mixed with about twice their weight of salt, water added, and
after evaporating to dryness were thoroughly extracted with ether. In
neither case could any appreciable quantity of cholesterol be isolated.

In Table I the analysis of eight eggs is given, and in Table II the analysis
of eight chicks.

It is obvious from these figures that no increase in the quantity of
cholesterol takes place during the change from ovum to newly-hatched chick,
the average percentage of cholesterol in eggs being 0°3827, and in chicks
03693, or in terms of the weight of the original eggs 0°3172. The same
result follows,no matter whether we take the figures for crude unsaponifiable
matter, or those for pure cholesterol.

At first sight 1t would appear that a loss of cholesterol occurs, but taking
into account the facts that the difference between the average percentage of
cholesterol in eggs and chicks (column e)—0:066—is of much the same order
of magnitude as the average deviation from the mean in the two cases,
viz.,0°057 for eggs and 0°075 for chicks. That individual eggs differ
considerably in the loss in weight which takes place during incubation, that
there is no reason to suppose that the proportion of yolk to white in
different eggs is very constant, and that the method of estimation of
cholesterol does not possess a very high degree of accuracy, it would seem
probable that no change in the quantity of cholesterol takes place, and that
all the cholesterol of the egg is contained in the newly-hatched chick. In
order to obtain a more accurate value for the cholesterol content of eggs and
chicks, six eggs and six chicks were analysed together, as the greater the
quantity of cholesterol weighed in an analysis the more accurate is the

1909.| Destiny of Cholesterol in the Animal Organism. 131

Table I.—Analysis of Separate Eggs.

| : Weight of F
N WYGIIED ©! musaponitable Micugets Ox Percentage
O. eggs in cholesterol in
gs _ matter ses of cholesterol.
ore "| im grammes. 8 =

1 67 °33 0 °4024. 0 2243 0 3331
2 57°57 0 3490 01978 0 3436
3 53 °32 0 °3655 0 °2104 0 3946
4 58 °48 0 :3768 0 -2162 0 -3697
5 52 “70 0 3265 0 1356 0 -2563
6 55 *40 0 -3628 0 -2582 0 -4661
7 57 °30 0 3923 0 -2570 0 -4485
8 55 *45 0 °3735 0 °2514 0 -4534

Total ...| 457 °55 2 9488 1 ‘7509 0 °3832

Table I1—Analysis of Separate Chicks.

P : Weight of : Percentage Cholesterol
No pioepee Bere unsaponifiable Micigts et reterred referred
i 88 2 matter 2 _ | to weight of | to weight of
grammes. | grammes. ees taniestes (ne eaomes. eee Aen
a. b. C. d. e. Ife
9 59 -80 55 20 0 -3490 0 -2014 0 3368 0 -3649
10 58-14 53 °20 0 °31380 01819 0 °3129 0 3419
11 67 -55 61-10 0-44.15 01433 0 -2121 0 °2345
12 58°10 46 °20 0 °3753 0 -2592 0 4461 0 °5610
13 58 °30 52 °30 0 -2965 0 -2951 0 5062 0 °5642
14 53°23 49 *55 0 2690 01074 02018 0 -2168
15 52°56 48 :05 0 -4815 0 1298 0-24.70 02701
16 55 16 45 *50 0 °3850 0 °1502 0 -2723 0 °3301
Total 462 °84 411-10 2 -9108 1 °4683 0 °3172 0 3693

result. In Tables III and IV the figures thus obtained are compared with
the total values for the eight eggs and eight chicks dealt with in Tables I
and II. As in the latter case the eggs used were taken indiscriminately
from various farmers, whereas the eggs analysed together were specially
selected hatchable eggs obtained from the dealer, we give in another column
the total values for the eggs analysed separately after eliminating the
abnormally heavy and abnormally light eggs, Nos. 1, 5, 11, and 15.

The percentages of cholesterol in eggs and chicks calculated from the data
obtained by analysing a number of eggs or chicks together are nearer the
truth than the averages obtained from the analyses of single eggs or chicks,
as in the latter case the errors of the various estimations would be

K 2

132 Origin and Destiny of Cholesterol in the Anmal Organism.

Table III.
. Weight of é
Weight of : Weight of | Percentage
eggs pos poneple cholesterol of
in grammes.| . in grammes. | cholesterol.
in grammes. ore
6 eggs analysed together............... 359 -00 2 4025 1°7578 0 4896
8 eggs analysed separately ............ 457 °55 2 9488 1°7509 0 °3827
6 eggs analysed separately ; same 337 52 2 °2199 1 3910 0 °4121
as above, eliminating 1 and 5
Table IV.
Ks Weight of |
eight o Were! F
: : - ght of | Percentage| Cholesterol
WSN Gs eet OF) maipon cholesterol| referred referred
eggsin | chicks in fiable | a to weight | to weight
grammes. | grammes. | matter in grammes. | of egg. ef chick:
grammes.
6 chicks analysed together ...| 340-2 302 1 -7805 1°5914 0 °4677 0 5270
8 chicks analysed separately | 462 -84 A411 “1 2 ‘9108 1 -4683 0°3172 0 3693
6 chicks analysed separately ;| 342 °73 301 “91 1 9878 1°1952 0° 3487 0 3958
same as above, eliminating
11 and 15
accumulated. A comparison of the figures again shows that no gain in

cholesterol takes place during the incubation of the chick. Whether the:
cholesterol of the egg remains unchanged, or whether some loss occurs,
cannot be definitely decided.

Conclusions.
In the differentiation of the ovum into the complex aggregates of cells.
constituting the chick, no formation of cholesterol] takes place. This is in
accordance with our view that cholesterol is not synthesised in the organism.

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.

133

On the Cross-breeding of Two Races of the Moth Acidalia
virgularia.
By Louis B. Prout, F.E.S., and A. Bacort, F.E.S.

(Communicated by Leonard Hill, F.R.S. Received January 8,—Read
February 25, 1909.)

A. INTRODUCTION.

The general interest which has been aroused of recent years by the various
researches which have been undertaken in investigation of the working of
Mendel’s Law of Heredity, and the adaptability of the Order Lepidoptera to
such investigation, have led us not only to reconsider the results of some
earlier and undirected experiments in moth-breeding, but also to seek out
some peculiarly suitable species in order to take in hand a more exhaustive
course of study along the lines most likely to yield further results in eluci-
dation of Mendelism.

Résumé of some Previous Rearing Hxpervments.

Perhaps a brief reference should be made to our previously recorded
attempts at pedigree-breeding.

Lasiocampa quercis.—Crossings of the various local races were carried out
extensively by A. Bacot and J. C. Warburg in 1896-1900, and the results
of their work detailed in ‘The Entomologist’s Record,’ vol. 13, pp. 114-116,
237-240, 256-259, 313-317, 338-342. The outstanding feature, as regards
a possible bearing on Mendelism, is _ that two races from the same
geographical region, when hybridised, produced progeny that segregated
into the two parent forms, whereas when the southern French var.
meridionalis, Tutt, was crossed with the Scottish var. callune, Palmer, no such
segregation occurred, the larvee being of an intermediate type.

Forres — Triphena comes (Agrotis comes, Stgr. Cat.).—Some rather
unsystematic breeding experiments with the interesting Forres forms of this
species were made in 1902-1903 by ourselves and others, and are recorded
in ‘The Entomologist’s Record,’ vol. 15, pp. 217-221; vol. 16, pp. 1-5.
The progeny from two wild melanic females segregated, that from ? A
being divisible into 74 typical and 93 melanic, that from 9 B into 39 typical
and 22 melanic. From brood A offspring was obtained, namely, a batch
from a single melanic pairing and a batch from melanic “stock” ; the former
gave 25 typical and 52 melanic, the latter 20 typical* and 48 melanic;

* “Nine” in ‘ Ent. Rec.,’ vol. 16, p. 3, line 19, is a laps. cal. or misprint for “seven.”—
1b, 18), 12

134 Messrs. Prout and Bacot. On Cross-breeding of (Jan. 8,

the total, therefore, 45 typical and 100 melanic. Thus the segregation was
Mendelian in its completeness, but less so in its proportions.

Cluny.—A little later, A. Bacot followed up this experiment with another
on the same species, this time with material from Cluny, Aberdeenshire.
The results are recorded and discussed in the ‘ Proceedings of the Entomo-
logical Society of London’ for 1905, pp. 1xvii—lxxi,* and more briefly in ‘ The
Entomologist’s Record,’ vol. 17, pp. 340-341. In the generation F* 60 per
cent. of non-melanic against 40 per cent. of melanic were reared from
melanie g x non-melanic ?. In F? 100 per cent. non-melanic appeared
from non-melanic parents, the melanic g grandparents showing no influence ;
on the other hand, from extracted melanie pairings of like parentage, F?
consisted of: in two broods, 30 per cent. non-melanic to 70 per cent. melanic ;
in another brood, 21 per cent. non-melanic to 79 per cent. melanic. In F%,
so far as tested, both forms bred true, 7.¢., two pairings of non-melanic x non-
melanic produced non-melanic offspring only (6 and 22 specimens
respectively), and two pairings of melanic gave melanic only (24 and
12 specimens respectively). The strain was becoming weakly through in-
breeding, and here died out. There is some suggestion here that non-melanic
is recessive to melanic, though some of the proportions are rather inexact.

Xanthorhoé ferrugata (Coremia unidentaria)—A long series of experi-
ments, extending from 1894 to 1898, was undertaken by L. B. Prout with a
view to obtaining light on the curious colour-dimorphism of this species,
and the results have been published in considerable detail in the
‘Transactions of the City of London Entomological and Natural History
Society’ for 1897, pp. 26-34, and Tutt’s ‘ British Lepidoptera, vol. 5,
pp. 61-64, and summarised in a later memoir, entitled “ Yanthorhoé ferrugata
and the Mendelian Hypothesis” (‘ Trans. Ent. Soc. Lond.,’ 1906, pp. 525-531).
These showed, as Mr. L. Doncaster pointed out in an interesting supple-
mentary note (‘ Proc. Ent. Soc. Lond.,’ 1907, pp. xx—xxii), roughly Mendelian
proportions on the assumption that the black form was recessive to the
purple. It seems to us curious, however, in spite of the large percentage
of deaths which introduced a factor of indefiniteness, that one pairing of
heterozygotes (No. 3 on p. 528 of the paper) should have yielded in Fy
11 specimens showing the recessive coloration as against only 6 showing
the dominant—the “expectation” being 4 recessive against 13 dominant, or
at best 5 against 12. At any rate, the species shows nearly complete
segregation, and will be a valuable one for future Mendelian research.

C. dominula—We may further mention that Mr. L. W. Newman, of Bexley,
a careful and successful breeder of Lepidoptera, has recently observed

* On p. 13, line 8, “45 7%” is a misprint for “40 7/.”—L. B, P.

1909. | Two Races of the Moth Acidalia virgularia. 135

apparent Mendelian dominance in the typical form of Callimorpha dominula
over its yellow-hindwinged aberration rossica, and of Abraxas grossulariata
over the aberration varleyata. He has very obligingly furnished us with
statistics, so far as the experiments have yet gone; and as they are hitherto
unpublished, we take this opportunity of putting them on record. Of
C. dominula, a type g X rossica ¢ paired in 1906 produced in 1907 a brood
consisting entirely of typical specimens; a pair of these gave in 1908 the
following result: 34 typical, 10 ab. rossica—there was a great mortality
among the larve before and during hibernation, fully 60 per cent. dying.
Of Abraxas grossulariata, a type § x varleyata 9 paired in 1907, produced
in June-July, 1908, a brood consisting entirely of typical specimens;
pairings from these gave, as a partial second brood, October-November,
1908, 24 of the type (including one aberrant but not varleyata) and 7 ab.
varleyata—the rest of the larve now hibernating.

After some consultation we decided upon the small geometrid moth known
as Acidalia (or Ptychopoda) virgularia, Hiib., as meeting the essential condi-
tions. ‘There are, be it observed, practical difficulties to be encountered with
many species, which have been overlooked by theorisers on the nature of the
work that ought to be done by Lepidopterists. Thus many moths are
exceedingly difficult to pair in confinement; many are extremely averse to
inbreeding, so that an inbred strain cannot be continued beyond two or
three generations ; many are difficult to bring through the winter, or require
food-plants which are not always obtainable.

Convenience in Rearing.—Acidalia virgularia, on the other hand, will feed,
apparently, on almost anything belonging to the vegetable kingdom, leaves
of all sorts—whether fresh or withered—sliced carrot, etc., proving equally
acceptable to it; it pairs very readily, is continuously-brooded throughout
the summer, feeds up rapidly and generally without need of hibernation,
and does not deteriorate through continuous inbreeding; moreover, its
small size is a practical convenience both for the accommodation of large
numbers of larve in a small space and for bringing large numbers of the set
moths under the eye at the same time. Few, if any, other British species
would offer all these advantages to the same degree; and as Acidaha
virgularia produces in the south of France a race so different-looking from
the British that more than one British field naturalist on seeing it has failed
to recognise even the species, it is not difficult to trace the influence of the
respective parent strains in crossings.

Origin of Stock.—Ova and pupze of the southern French form were kindly
supplied by Mr. H. Powell, F.E.S., from Hyeres; wild moths of the London

136 Messrs. Prout and Bacot. On Cross-breeding of [Jan. 8,

form by Mr. J. E. Gardner, of Clapton, N.E. The former race is distin-
guished by its white or cream-coloured ground, almost devoid of grey
dusting, and scarcely variable except in the intensity of the transverse black
lines, which may be strong (the tendency in the particular strain with which
we worked), or broken up into dots, or obsolescent; the latter is variable
within limits, but always with the pale (not white) ground-colour profusely
dusted with dark grey atoms, whether these be uniform throughout or more
concentrated in certain areas. The Hyéres form is therefore described in our
experiments as “light” (L), the London form as “dark” (D). It is necessary
to add that more or less intermediate phases of variation occur in some
parts of Germany, Italy, etc., so that we have been dealing with local races
rather than fixed recurrent “aberrations ” or with incipient species.

The first cross-pairing was obtained on June 21,1906, and filial generations
I-X (F; to Fj) on the well-known Bateson method) appeared from August,
1906, to November, 1908. Altogether, 5531 specimens have been analysed
in preparing these notes, so that they may be regarded as fairly compre-
hensive as an indication of the behaviour of this particular cross-pairing.

B. STATISTICAL RESULTS.

General Remarks.—Altogether our breeding of Acidalia virgularia has been
carried out to the tenth filial generation, and 5531 specimens have been
subjected to careful analysis, exclusive of a few which have been more
indefinitely summarised but which are confirmatory of the general results.
We feel that we may, therefore, speak authoritatively on the general course
of inheritance in the cross-breeding of these races, and that the imperfection
of our statistical analysis is not due to ignorance of the forms with which we
are dealing, but to the fact that their hybridisation really gives no segrega-
tion capable of analysis by the human eye. It is necessary to dwell somewhat
on this point. At first sight it might appear a confession of incompetence to
have to state—as we do quite frankly—that our figures are only approxi-
mations, and that in many cases a re-count (either by another entomologist or
even by ourselves) might easily result in a slight modification of them ; but
when it is understood that there is, in the cross-breeds, every conceivable
intergrade, it must be manifest that the distinction between “dark” and
“intermediate” on the one hand, and “light” and “intermediate” on the
other, becomes purely one of degree, and it is absolutely impossible to draw
a perfectly consistent line throughout. Having made a special study of the
family Geometride for nearly twenty years, one of us (L. B. P.) can at least
claim to have acquired that eye for slight differences in them that will have

1909. | Two Races of the Moth Acidalia virgularia. 137

safeguarded him against any material error of judgment in the present
investigations.

As regards the pure stock, or presumable homozygotes, inbreeding for
ten generations, under more or less artificial conditions, has not had the very
slightest influence on the pretty Hyeéres form nor, in the aggregate, on the
London form. Excepting a single dark specimen, which was obviously an
accidental importation (perhaps on food-plant, as the moth is so common in
Mr. Bacot’s neighbourhood), upwards of 400 specimens show not the slightest
deviation from the clean whitish ground-colour which characterises the Hyéres
race. The dark form, which varies more in a wild state, naturally showed
a greater range, and one or two broods, apparently by some accidental
selective agency, became lighter than the normal; but here, again, we can
contidently aftirm that, among some 400 specimens, none have occurred
which could possibly be mistaken for the “light.”* It follows, therefore, that
the bulk of those which we have classed as “intermediate” can be with
certainty explained as blends originating from the hybridisation.

Crosses obtaimed.—Cross-pairings were obtained in each generation, usually
in reciprocal crosses, and in not a few instances in duplicate. In many cases
the progeny of the crosses was also carried on down to the generation
collateral with that of Fy) from the original cross. The complete scheme
upon which we intended to work may be indicated as follows :—

Pure dark (D).—F, to Fy.

Pure light (L).—F; to Fro.

Dark by light (called cross-pairing A).—F to Fyo.
Light by dark (cross-pairing a).—F\ to Fi.
Dark by light, ex Fy (B).—F2 to Fi.

Lnghi by dark, ex ¥, (b).—F»2 to Fyp.

Dark by light, ex Fz (C).—F3 to Fio.

Laght by dark, ex F2 (¢).—F3 to Fi.

Dark by light, ex Fs (#).—F, to Fy.

Light by dark, ex Fs (e).—F sz to Fyp.

Dark by light, ex Fy (£).—Fs to Fy.

Lnght by dark, ex Fy (f).—Fs to Fi.

Dark by light, ex F; (G).— Fg to Fy.

Light by dark, ex Fs (g).— Fe to Fr.

Dark by light, ex Fg (H).—F; to Fy.

* One curious strain is dealt with separately below, as its actual origin is altogether
problematical.

138 Messrs. Prout and Bacot. On Cross-breeding of [Jan. 8,

Light by dark, ex Fg (h).—F; to Fy.
Dark by light, ex F; (L).—Fs to Fro.
Light by dark, ex F; (2).—Fg to Fr.
Dark by light, ex Fs (J).—Fy to Fyo.
Light by dark, ex Fs (7 ).—F» to Fyo.
Dark by light, ex Fy (K).—F yo.
Light by dark, ex Fo (k).—Fyo.

Or in tabular form (see p. 139).

The actual hiatus in the carrying out of this scheme will be seen from
the details which follow. The number of the dark~x light crosses that
faded out would suggest some inherent tendency to weakness in this rather
than in the reciprocal cross ; yet the oldest hybrid of all was a dark x light (A)
and continued vigorous to the last. None of the hybrid strains was labelled
“ D,” this letter being reserved for the pure dark strain.

In addition to these systematic crossings, a few quadroon broods and
complex crossings of hybrids were obtained.

Most of the continuations of the broods were from single pairings, but
occasionally—as when a number of specimens had emerged simultaneously
and we could not be sure that they had not mated unobserved—we bred from
stock. The question of the infiuence of individual parental characteristics as
opposed to broader racial ones was not the least interesting in connection with
our work.

It is to be remarked that the percentage of deaths in the early stages was
generally quite insignificant, and that many of the broods reared to the imago
state were so rich in individuals relatively to the fecundity of the species,
that our statistics are incontrovertibly much more representative than those
obtained from Xanthorhoé ferrugata, where the pupal deaths were often
enormously numerous.

Since it is impossible to forecast what statistics may ultimately assume
unexpected importance, the following record tends to err, perhaps, in the
direction of over-completeness. Such deductions as we have been able to
draw from the mass of figures will be reserved for the next section of this
paper.

Broop A.—This was started in duplicate, one of the strains being lost at
F;. The reciprocal cross (a) was not obtained, as we had no dark 2 of
assured virginity.

(1) In the generation F, there were 62 specimens, all true intermediates
with variation inconsiderable. In F»2, 66 specimens, variation considerable,
5 quite dark (perhaps less brownish than the pure Clapton race), others

®

139

Two Races of the Moth Acidalia virgularia.

1909. ]

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140 Messrs. Prout and Bacot. On Cross-breeding of [Jan. 8,

approaching this or mottled or banded with dark; none approaching the
pure light form. In F3, 21 specimens, variation slight, follow their actual
parents very closely—z.e. all were intermediates. In Fy, 54 specimens, variation
rather considerable in ?’s, less in ¢’s; 2 2 dark, much as in Fy; several
quite as light as F,, none pure light. In F;, 47 specimens, variation rather
considerable, none very light, 7 (4¢, 39) dark.

(2) In generation F, all were true intermediates, though somewhat
variable ; a subordinate race-characteristic was perhaps adumbrated, which
became more pronounced in a few specimens of each brood from A» to Ag,
namely, a tendency to darkening in the outer area of the wings (a common
characteristic of some species of Acidalia, such as A. politata, etc.). In
F,-F the variability increased, but according to no fixed rule; in Fg (20
specimens) the range was from almost pure light (very weakly marked) to
almost pure dark, with intergrades. Two pairings were obtained in F, one
of light x light, the other dark x light; the former yielded, as F;, 47
specimens of remarkable constancy, most of which might be called pure
light, three perhaps light-intermediate ; the latter yielded, as F; (from fine
dark ¢ x light ¢), some half-dozen specimens only. Fs, from the former of
the two F, broods, consisted of 13 pure light and one (¢) intermediate ;
another Fs, from stock (of the latter of the two F, broods), also of 14
specimens, differed strikingly from its cousin brood in tone, all being inter-
mediate, and somewhat variable. Fy, was again duplicated, one batch
(labelled A ix ©), from light parents, consisted of 81, the majority light, but
only about 30 per cent. pure light; the other batch (A ix +), also from light
parents, gave 6 only, all pure light. Fy» (ex. A ix ©), 21 specimens, ranged
from light (1 or 2) to true intermediate (2 or 3), the majority light but very
slightly dusted.

Broop £&—Carried only to the fifth generation (strictly speaking, the
fourth generation ; but, as shown in our “scheme,” it has been thought better
throughout to give uniform numbering to collateral lines, 7.¢., to regard as Fe
the grandchildren of the original stock even though with strain B the
crossing of the two races only commenced a generation later, with strain C
two generations later, and so on). In generation F2 there were 14 specimens,
variation inconsiderable, all intermediate, lines rather weak. In Fs, 34
specimens, variation not great, similar to parent brood, but 3 or 4 distinctly
light—beginning to “throw back” towards ? grandparent. For Fy a
duplicate pairing was obtained ; one brood, 31 specimens, averaged distinctly
paler than Fs, several closely approaching the pure light; the other brood,
52 specimens, also varied little, but more closely resembled the parent brood,
only 3 or 4(?’s) being whitish the rest intermediate. In Fs, from the

1909. | Two Races of the Moth Acidalia virgularia. 141

former of the two Fy broods, 32 specimens varied little, though not absolutely
inappreciably, all light, yet not quite so pure as the original Hyeres strain.

Broop 6.—Obtained in duplicate, one strain carried on to generation F;,
the other to Fy.

(1) An interesting strain on account of an apparently hereditary pre-
dominance of the female sex, figures therefore given in full. In F)
(28 3, 36 2) all were intermediates, though somewhat more variable than
most first crosses. F; was duplicated; one brood (13 3, 17 ?) showed
considerable variation, ranging, in both sexes, from pure dark, through
intermediates, to nearly, but not quite, pure light; the other brood
(13 g, 24 2) also varied rather considerably: 3 almost pure dark, a few
others approaching these, others intermediate or lightish, 7 with a
characteristic facies, almost of the pale, well-lined Hyeres form, yet less
extreme and less white. 4, from the former of the F3 broods, consisted of
14 g, 25 @ : both sexes quite variable, several ¢’s dark, 1 or 2 9’s light (not
strongly lined), the 9’s thus averaging somewhat the lighter. In F,
(17 g, 27 2) the range of variation was much asin Fy. Adding the above
numbers together, we find that this strain yielded only 85 ¢ against 129 9,
a proportion of 2: 3.

(2) This proved on the whole a very stable strain, though generations F,
to Fs, varied more. F2 was very uniform, intermediate. Fs; (7 only) similar,
may have been a shade darker and a few showed a dark border, which
became a feature of the strain. F, distinctly variable, though not quite
reaching either extreme; a dark central shade, quite a feature of some,
lacking in others. F; (19 only) very similar to Fy, but smaller, and perhaps
hardly so variable. Fg (66) strongly variable, particularly in the expression
or suppression of the two rows of transverse dots, there being a sudden
outcrop of specimens in which they are very pronounced—none such being
observable in F2 to F;. Where these dots are on a white ground
(14 specimens), “ pure light ” is produced ; the rest are intermediate to dark,
none very dark. F; consisted of 3 only, intermediate, weakly marked, but
2 with the borders darkened. Fs, 23 specimens, a singularly uniform brood,
a phase of “intermediate” without strong dusting or lines of dots, the distal
margin often darker. Fy was duplicated, but both the broods (46 and
11 specimens respectively) closely followed Fg, though a few in the larger
brood were a little more heavily dusted. Fi) was almost a failure, only one
(intermediate) specimen coming through.

Broop C.—Continued to generation Fs. In F3 there were only 3 poor
specimens, apparently intermediate, but no exact analysis possible. Fy,
a large batch, moderately variable, range from almost pure light to darkish

142 Messrs. Prout and Bacot. On Cross-breeding of [Jan. 8,

but hardly dark ; about half tend toward the light side. F; are similar to
F'4, but perhaps less variable, very few pure light; might in the aggregate be
termed light-intermediates. ¢, 36 specimens, are pretty variable, about
11 light (only with a stronger central shade than in the pure Hyéres strain);
1 or 2 others nearly as light, weaker-marked ; the rest intermediate to darkish,
nothing extremely dark. The darkest pair available was used for parentage
of Fs. Fs (4,16 ?) are rather large, variable, the average dark, could
perhaps be classified as 11 (3 ¢,8 ?) dark, 5 (2) intermediate, 4(1 g, 3 2)
light, though not quite pure, but there are very gentle gradations ; duplicate
pairings out of F; (labelled C viii (2) and C vii (3)) vary somewhat less,
C viii (2) (33 specimens) being intermediates, slightly variable in detail, and
C viii (3) all being possible London forms (z.¢., dark), variable only in detail.
F, was obtained only from the second of the above (C viii (2)); the specimens
numbered 35, still intermediates, not unlike the parent brood.

Broop ¢.—Continued to generation Fy. F3 consists of 7 only, inter-
mediates, apparently not variable. Fy, varies from almost extreme light to
almost extreme dark, but with intermediates which preclude any possibility
of splitting up into darks and lights. FF; (16 only) is similar, but the
preponderating tendency is on the dark side, only one being really light with
strong lines of dots. ¢, 29 specimens, is perhaps even more variable, 4 or
5 at least being pure dark, 6 or 7 at least pure light, others nearing both
extremes (especially the light side) and a few intermediate. F, (from light
3d x dark ?) showed quite moderate variation, most being rather uniform,
lightish intermediate, 2 or 3 (¢) darker, without being strikingly dark.
Fs, 52 specimens, are very variable: about 12 pure dark, about 12 pure light
(only with well-expressed central shade), the rest grading through. Fp, was
bred in triplicate; brood ¢c ix ©, 30 specimens, from intermediate parents,
are very uniform, all being possible Clapton forms (dark), only 2 or 3 a little
paler than would be normal for Clapton ; ¢ ix *, 7 specimens, from lghtish
parents with distinct dot-lines, follow the parents closely; the remaining
brood (stock ?), 56 specimens, shows moderate variability, but mainly inter-
mediate, the few darks and the few lights hardly quite pure. Fy) was nearly
a failure, but 5 specimens ex brood ¢ ix © are all dark, 6 ex ¢ ix ¥ all
lightish, reproducing their parents’ facies.

Broop #.—Only continued for two generations. The original pairing, ex
generation F3, was obtained in duplicate.

(1) In generation F, 55 specimens were reared, all intermediate, the
variation not considerable. In Fs, 83 specimens, the variation is con-
siderably greater, ranging from darkish (not extreme) to specimens closely
approaching the pure light strain, though slightly less pure, with central
shade better indicated.

1909. | Two Races of the Moth Acidalia virgularia. 143

(2) In generation F, 58 were reared, all intermediate and remarkably
constant. Excepting the slight sexual dimorphism, the variation might be
said to be practically nil. Progeny not obtained.

Broop e.—Continued to generation F;. F, consisted of 77 specimens, all
intermediate except, perhaps, one brownish 2, which resembles some of the
lightest London forms; the other 76 exceedingly constant. In F;,
49 specimens, the variation is much greater, ranging from a few of each
sex quite resembling the London forms to a few whitish, though certainly
not pure. Fs, 78 specimens, extremely variable, though not definitely
segregating ; a few very dark, several darkish, one darkened in outer area,
numerous intermediate, numerous light or lightish, the black dot-lines then
generally (not always) well expressed, some with, some without, the dark
central shade, 2 or 3 agreeing fully with the pure light strain. In F; only
6 moths were bred, from stock, variable from dark to light.

Broop #.—Continued to generation Fs. The original pairing was
duplicated.

(1) F;, 29 specimens, singularly enough, acted differently from all the
other first crosses, being virtually a pure light brood, and we hoped
that, for once, the light ¢ parent had acted asa dominant. Fortunately a
large ofispring was obtained (from stock) consisting of 150 specimens. These
(#’¢) are much more variable than F;, but cannot be split up into light and
dark definitely; roughly classified, we made 45 light (perhaps a dozen pure
light), 95 intermediate, 10 dark (none extremely), but the gradations are so
extremely slight that a re-count would be almost sure to modify the
figures somewhat. Two pairings were obtained: one brood of F, (ex
light g x dark 2) yielded 7 specimens, all more or less intermediate,
3 more dusted than the other 4; the other brood (ex light ¢, with strong
dot-lines), 6 specimens, all rather light, but only one with the lines sharp.
Fs, from a pair of the lightest specimens in the former of the last-mentioned
broods, again proved numerically inadequate, only 7 coming through; these
are rather variable, 6 being on the lighter side (2 or 3 pure, the others grading
towards intermediate), the seventh strongly dusted (dark intermediate).
Attempts to continue the strain proved unsuccessful.

(2) F; here consisted of 47 specimens, the variation not considerable, the
general facies being very uniform, but the colour ranging from lightish (not
pure) to a lightish intermediate. Offspring was not obtained.

Broop 7—Continued to generation Fi, though then on the verge of
extinction. F;, not variable, would certainly be classed as true intermediate,
though rather on the light side. Fs, 64 specimens, is much more variable,
two or three being pure light, several others closely approaching it, many

144 Messrs. Prout and Bacot. On Cross-breeding of [Jan. 8,

intermediate, and a few rather dark. F, (ex light ¢ x dark 2) consists of
32 specimens, extremely uniform, all intermediates, with fairly distinct dot-
lines. Fs, 44 specimens, again vary, yet with no absolutely pure white, and
only 3 or 4 very strongly dark-dusted, nothing extraordinarily dark. Duplicate
pairings were here obtained ; from a rather dark ¢ x rather light 9, sprang,
as Fy (labelled fix ©), 28 specimens having a similar range to Fs; from an
apparently intermediate pair, the ¢ darkened in outer margin, 33 specimens,
rather constant, intermediate, nearly all well dusted on a whitish ground.
Fy, 2 specimens only, agree with the Fy brood last mentioned, from which
they sprang.

Broop G.—This cross was obtained, but not propagated beyond the single
generation (Fs). The brood consisted of 69 specimens, slightly more variable
than most first crosses, yet in no way startling. Most are quite normal
intermediates, 2 or 3 might better be classed as dark, yet not extreme,

Broop g.—Continued to generation Fy. In Fs, 35 specimens, the variation
is inconsiderable, all being intermediate, though such variation as there is is
towards the “light” side. FF, was obtained from a lightish pair, and yielded
4 lightish specimens. Fs, 47 specimens, was again rather constant, a light-
intermediate. Fy was obtained in duplicate; one batch (labelled g ix ©,
parents rather weakly marked) consisting of 18 specimens, intermediate,
nearly all weakly marked, the colour ranging from darkish to lightish without
extremes ; the other batch (g ix :%, from a better-marked pair) considerably
variable, 49 specimens, mainly well-lined, about 12 almost the pure Hyéres
form, 3 or 4 approaching the London form, many intermediate. Fo,
47 specimens from the last-named brood, follows it well on the whole, nearly
all being well-lined, though there is much variation in tone and many
(especially of the darker ones) are rather strongly darkened towards the outer
margin. :

Broop H.—Continued to the second generation of the cross, that is, to Fs.
F,, 56 specimens, is very constant, and very typical of the normal “ first
cross ’—all intermediate. F's, 49 specimens, is very variable; hardly any are
quite pure light, only 1 or 2 pure dark (and not very intense); but there is
almost every other variation, in size, strength of markings, general facies,
and ground-colour.

Broop /.—Continued to generation Fy. In F;, 12 specimens, the variation
is very slight, all being lightish intermediate. Fs, 36 specimens, is decidedly
variable, the range being from pure light to darkish intermediate, with the
usual intergrading. Fy was duplicated; one batch (from light, well-lined
parents) yielded 17 specimens, variable from pure light (though not intensely
white) to intermediate, 11 or 12 having the lines rather strong; the other

1909. | Two Races of the Moth Acidalia virgularia. 145

batch yielded 16 specimens, hardly variable, intermediate to light-inter-
mediate, weakly lined for the most part. From the former of these batches
sprang, as Fo, a brood of 17 specimens, rather constant, with a uniform facies
which struck one as recognisable even when they were emerging; all are
intermediate in colour, the dusting weak, the lines rather strong.

Broop 7-—Obtained but not carried on. The single family (Fs) consists of
46 specimens, intermediate, decidedly constant.

Broop 7.—Continued to generation Fy. Fs consists of 22 specimens,
rather constant, normal intermediates. Fg, was obtained in duplicate; from
one pair (labelled 7 ©) resulted a very variable brood of 32 specimens:
5 or 6 pure light, others near, 7 or 8 pure dark (some quite extreme), others
near, and various intergrades; from the other pair (labelled 7 <)) another
variable brood, of 12 specimens only, mostly lightish-intermediate, 1 almost
pure light, though slightly brown tinged, 1 pure dark, 2 darkish. Fi) was
reared from both these broods; that from the former consisted of 44
specimens, intermediate to dark, presumably from some of the darker
examples among the parent stock; the latter of 25 specimens, varying in
colour from white to intermediate, yet with a most conspicuously definite
facies, all being well lined, with the central shade strong and clear cut in
addition.

Broop J.—F 5, 29 specimens, intermediate to lightish-intermediate, fairly
constant. Fy, 5 only, certainly variable, though without extremes—
altogether too few for generalisations. The reciprocal cross (j) was not
secured.

Broop &.—Obtamed in duplicate. Both batches (56 and 38 specimens
respectively) normal intermediates, the variation slight. Some undersized
specimens look a little pale, but this is because of their weak scaling.

Broop k.—40 specimens, variation moderate, from light-intermediate
to dark-intermediate. i

The quadroons and other irregular crosses have next to be briefly dealt
with. In the first filial generation pure light ¢ was crossed with hybrid ¢
(out of the brood described as A, number (2) in this paper), and the strain
earried on for four generations (F2 to Fs). It continued “intermediate,”
with the variation appreciable but not considerable, only in F¢ there were
more of the whiter specimens. In generation F, this quadroon race was
erossed with the hybrid race called f in this paper; the variation in the
offspring (F;) was only very moderate, ranging from light to lightish-
intermediate.

Also in generation Fs, crosses of bxc, bx/f, and Gx C were obtained, but
only bx was followed up to subsequent generations, All these three were

VOL. LXXXI.—B. L

146 Messrs. Prout and Bacot. On Cross-breeding of [Jan. 8,

_interesting, as in each case both the parents were more or less extreme, the
6’s light and the 2’s dark; the offspring of xc (16 specimens) varied
little, all being intermediate or lightish; that of Gx C (4 specimens only)
much more, the single ¢ bred being darkish, the 3 ¢’s light, weakly marked.
In this generation (F;) the specimens of )x/(34 in number) varied little,
the range being from lightish-intermediate to lightish, almost reaching the
pure Hyeres form; the characteristic dark border of the parent strain } (2) -
entirely disappeared. In Fs, 47 specimens, the variation was considerably
greater, ranging from pure light (about 8) to pure dark (2 or 3), the majority
intermediate, and the extremes not very intense. Fy) was obtained in
triplicate; from a light pair (especially the ¢@) sprang a brood of 34
(labelled 6f Ky ix), hardly variable, all light or lightish; from a somewhat
intermediate pair a brood of 44 (labelled 0/© ix), variable, from lightish
(not extreme) to dark—about 8 that might be likened to average London
specimens ; from stock a batch of 23 (labelled 0/ (3% 1x), slightly variable, all
light or lightish except 1, which is intermediate, brown. In generation Fy
one brood was raised, simply labelled 0/ x, the note of its exact parentage
having unfortunately been mislaid ; it consists of 32 specimens, nearly pure
light and not varying much, a few virtually of the Hyéres form, but the
larger number with a fairly distinct central shade.

In generation Fs a pairing was obtained between a ¢ out. of brood H
(intermediate or darkish, weakly-marked) and a @? out of brood ¢ (inter-
mediate or rather light, the central shade distinct). In generation Fo
a brood of 33 appeared, rather variable, from light-intermediate to dark (not
intense), mostly weakly lined, a few strongly freckled. Their progeny
(Fy, 19 specimens) are also variable, from light (3) to dark (5); 4 are inter-
mediate, fairly well scaled, the rest more or less poorly scaled, weakly-marked.

It remains to notice a strain which must be treated as of uncertain
ancestry, and which originated in Fg and has been carried on to Fy. It was
believed to have sprung from pure dark ancestry, a number of hibernating
larvee of F3 in that strain having fed so slowly as to be still in the larval
state when their nephew-brood of larve (7.e. pure dark F,) arrived, and
having been mingled therewith; but in Fg the behaviour of the strain was
so unprecedented that we feel forced to imagine there must have been some
accidental importation of hybrid or light material, inexplicable though it is,
considering the care that was taken. Of course, it is open to those who so
desire to assume that there was here a true mutation, but as the white form
has never been known in Britain, and inbreeding has not changed the rest
of our pure dark stock, we ourselves cannot regard such a view as even
worthy to be provisionally entertained, unless confirmation be forthcoming.

1909. | Two Races of the Moth Acidalia virgularia. 147

Broop D %.—This aberrant stock in Fs, which we called D ¥ vi, con-
sisted of 41 specimens, 18 of them pure light, 2 nearly pure but browner in
ground-colour, a few normal intermediates, and about 12 typical dark. By
analogy with the rest of our material this would suggest being a second
generation from a hybrid. On account of the riddle of its origin, 5 pairings
from this brood were obtained, 2 others attempted proving infertile.

(1) From a light pair sprang, in F;, a brood of 16 (labelled D % vii (2)) all
light, about half being quite extreme, the rest slightly more dusted.

(2) From another light pair, F, consisted of 47 (labelled Ds vii (3)),
rather variable from pure Hyeres form (4 or 5) to intermediates. Their
progeny in Fs (64 in all, from different pairings) varied conspicuously, the
majority light to intermediate, perhaps only one really dark, and that not
very extreme. In Fy, (two broods, 67 specimens) the variation was less, only
ranging from light to intermediate; all the four actual parents were more or
less light. In Fy (three broods, 96 specimens) the variation again increased
somewhat, but with the lighter forms still in the ascendant and thoroughly
dark ones only occurring, and sparingly, in one of the three broods—labelled
Dx(1) ©, and noteworthy for its darker average tone than its parent brood.

(3) From yet another light pair, F; (26 specimens, labelled D vii (4))
bred absolutely true to the extreme parent form (= wild Hyeres type).
One pairing produced, in Fs, a further brood of 24, all equally pure,
unfortunately lost here. Another pairing produced, in Fs, a very variable
brood of 45—15 pure light, about 15 others lightish to light-intermediate,
the rest darker, 1 or 2 practically “dark.” From a pairing of rather light,
well-marked specimens in the latter of these (the variable) was obtained, in
F,, a brood of 38 varying much less than the parental one, indeed pretty
constant light-intermediate, rather well-lined. Their offspring (Fy,
42 specimens) would nearly all be classed as “light-intermediate” in some
sense, yet wonderfully variable within this limit; whitish weak-marked,
similar examples but greyer behind the outer line, moderately light strong-
marked, intermediate (2 or 3 strongly-marked, 2 or 3 weaker-marked) are all
represented.

(4) From a dark pair, only three specimens were reared in F, (labelled
D * vii (6)). These were darkish intermediate. Fortunately a ¢ and a ?
emerged together and copulated. The resultant Fs (49 specimens) varied
a good deal, one only being pure light, the rest about half intermediate
(a few light-intermediate) and half darkish to dark, but with intergradations.
Fy (78 specimens, 3 broods) was moderately variable, but all should be
classed as broadly “intermediates.” Fi) (49 specimens) was variable, ranging
from intermediate to dark, the average darker than in Fy; perhaps about

L 2

148 Messrs. Prout and Bacot. On Cross-breeding of [Jan. 8,

20 would be called dark, but there is no clear line of demarcation. The
return to a darker type might be called atavistic, but more probably the
actual parents—which are not known—happened to be among the darkest
ones of Fo.

(5) From a dark ¢ xlight 2, F, consisted of 21 specimens (labelled
D * vii (7) ), all normal intermediates, with no appreciable variation. The
strain was unfortunately lost.

C. GENERAL CONCLUSIONS.

From the foregoing mass of detail a few facts emerge with conspicuous
clearness, and certain other points are sufficiently suggested to be worth
putting forward, at least tentatively.

In the first place, there is most certainly no Mendelian dominance in
coloration in the cross of the dark (London) race of Acidalia virgularia with
the light (Hyeres) race. With remarkable persistence, a first cross of the
pure races produced a form intermediate in coloration. The sole exception,
out of two dozen such crossings, is the brood noticed above as F, No. (1).

But, in the second place, it is perfectly well known that colour-dominance
is not the essential feature of Mendelism. As Mr. Bateson says,* “The
essential fact which Mendel discovered is the segregation of characters in
gametogenesis.” Now, as the intermediate form, which was so nearly universal
in the first crosses, did not appear in either of the “ pure ” strains, 1t may well
be taken as the normal manifestation of hybridity in this blend, correspond-
ing to the “blue” Andalusian fowl and other well-known cases; and it is
certainly noteworthy that a rough resolution into a wider range of forms
proved quite general in the F, generation. That proportions did not agree
with expectation might be due to defective analysis. For example, the said
“hybrid ” or “intermediate” might have a wider range of variation than had
been discovered by the investigators, who might thus have referred some
hybrids to one of the “pure” forms. But a glance at our actual results
convinces us that it is not generally too few intermediates that we obtained
in F, but too many; and fortunately we know very accurately the limits of
the variation of at least one of the pure races (L), so that there seems no
chance, on the assumption of gametic purity, of our having classified pure
“lights” as “intermediate.” It is, however, further noteworthy that some,
at least, of the extracted strains (light x light, ex hybrid, viz, A (2) in
generation F,,t ?B in generation F; (1), 0x/ [O] in generations Fy and ? Fio,

* ‘Progressus Rei Botanic,’ 1906, p. 368.

+ But if this was really “pure” whence came the single “intermediate” ¢ in its.
offspring ?

1909. | Two Races of the Moth Acidalia virgularia. 149

? D * (1) in generation F;, ? D * (3) in generations F; and one section of
Fg ;* dark x dark, ex hybrid, viz., C (3) in generation Fs, c © in generations
Fy, and Fi) attained a considerable standard of purity ; and also that a few
of the extremest (light x dark) pairings among hybrids (such as ) xc, bx/,
D * (5), and ? A (2) in generation F7) were the most reliable in producing
again genuine intermediates. (Gx C was possibly an exception, but the
parents here were not so extreme in colour as to render hybridity unthinkable.)

Another fact that can be stated with certainty is that our experiments have
revealed no other decisive “ reversion to type ” than the kind which Mendelism
would demand; the intermediates have been quite as stable as Mendelism
would expect in hybrid pairings. Whatever be the explanation, it would
appear that the hybrid form cannot be “bred out”; except in cases where
a selective mating has been employed and the rest of the brood allowed to
die out, intermediates have continued to appear through all the generations.

Without desiring to dogmatise, we feel it is necessary to remark that
neither of the points last considered—the obtaining of a comparatively
uniform type by selective mating and the persistence of intermediates under
other circumstances—belongs exclusively to any one theory of heredity, while
such occurrences as those noticed in the footnotes on broods A» and D x (3)
are harder to reconcile with Mendelism than with, for example, the Galtonian
view. On the whole, the apparently large responsibility of direct parenthood
suggests to us the idea of some such principle as is involved in the well-
known formula of one-half the characters from the parents, one-fourth from
the grandparents, etc.

Over and over again some trifling race-characteristic has interested us in
a particular strain, including—besides the tendency for some broods to favour
the slightly darker variations and others the lighter—obvious differences in the
expression or suppression of the transverse “dot-lines,” tendency to develop
a dark central shade or a dark marginal area (for instance, brood 6 (2)), and
so forth. Any of these would have been well worthy of minute study, either
from a Mendelian or a non-Mendelian point of view, had time and oppor-
tunity allowed. We suspect, however, that in large measure they also would
be found traceable to direct parentage, for it is certain that in some cases
cousin-broods differ quite materially in some of these characteristics, and that
a reference to their parents shows how closely these are followed; see, for
instance, some of the references under the statistics of broods ¢, g, h, etc.
The sex-predominance in brood 6 (1) was another peculiarity which deserved
more attention than it received.

* But how would Mendelism account for the (very variable) other section of F,?

150 Cross-breeding of Two Races of the Moth Acidalia virgularia.

We noticed also that the larvee were very variable, and it is not impossible
that an analysis of their variation might yield some results of value.

As a final impression, we would suggest that our failure to find Mendelian
inheritance at work was due mainly to our bringing together two compara-
tively remote geographical races (as with Messrs. Warburg and Bacot’s
Lasiocampa meridionalis x callune) and that we, personally, now only expect
to find segregation in the case of crosses of two forms occurring together (like
the two forms of Zviphena comes or those of Xanthorhoé ferrugata), where
a long course of natural selection has presumably eliminated the inter-
mediates. We pointed out in the introduction that just such intermediates of
Acidalia virgularia as were produced artificially by crossing our specimens
from London and Hyeres (localities where they are apparently quite
unknown in a wild state) do occur in a state of nature in other parts of its
geographical range.

A few pairings which occurred in generation Fy) produced ova which have
been handed to Mr. W. Bateson in the hope that he may be able to follow up
our researches in the species. Unfortunately both the pure strains have been
lost, but possibly Mr. Bateson will be able to extract them, by selective
pairing, from the new hybrids, which we labelled J/ xi and m xi.

ow

The Nerves of the Atrio-ventricular Bundle.

By J. Gorpon Witson, M.A., M.B.(Edin.), Hull Laboratory of Anatomy,
University of Chicago.

(Communicated by Dr. F. W. Mott, F.R.S. Received January 21,—Read
February 11, 1909.)

[PLates 4—6.]

Physiologists, in explaining the transmission of the wave of contraction in
the heart from atrium to ventricle, have alternately leaned towards either
the myogenic or the neurogenic hypothesis. Many of the discussions on this
subject have been useless and many of the deductions false, because of the
misconception of the anatomical facts. For long it was held that though in
cold-blooded vertebrates the experiments of Gaskell had settled the question
in favour of the myogenic theory, yet in mammals there existed an interrup-
tion between the atrial muscle and the ventricular muscle at the atrio-
ventricular groove, and that this was opposed to a general acceptance of the
myogenic theory. Later anatomical investigation, however, has definitely
shown that there exists in all mammals, between the atria and the ventricles,
a pathway of modified muscle fibres along which the contraction wave
appears to go. This is the atrio-ventricular (auriculo-ventricular) bundle, or
bundle of His. The discovery of this muscular connection gave very decided
support to the myogenic hypothesis, and at present it would appear that
prevailing opinion favours this theory. It receives additional support in so
far that in this bundle only a few nerve fibres have as yet been recognised
by one or two observers; some assert that nerves are not present, or, if so,
are too few in number to be of any moment. More definite statements have
been made by Tawara and Retzer. Tawara(1) found that “in the heart of
the calf the atrio-ventricular bundle is accompanied by a very considerable
nerve bundle, which runs with the muscle bundle, and in the left ventricular
septum nerve.cells are present 1-2 cm. below the aortic valve.” In the
atrio-ventricular bundle of the sheep only a few nerve bundles were seen,
but in the dog, cat, and in man he could find none, though he expressly states
that these cannot be excluded, since fine nerve fibres accompany the bundle.
Retzer(2) has pointed out that the first ganglion cells that appear in the
embryonic heart lie in the atrial septum, immediately above the beginning of
the conductive system; further, that the Purkinje fibres are surrounded by
a plexus of non-medullated nerves.

Such is the present position of our knowledge of the nerve constituents of

152 Mr. J. G. Wilson. [Jan. 21,

the atrio-ventricular bundle. Although a vast amount of work has been
done on the nerves of the heart generally, yet, so far, but little attention has
been directed to this particular and definite strand. In this preliminary
paper I have limited the report to the muscle band as it passes from the
atrium to the ventricle, and shall not discuss the nerve constituents in the
connections of the atrio-ventricular bundle with the ordinary muscle of the
atrium or of the ventricle; that is, 1 limited the investigation to that part
which extends from a point in the bundle towards the coronary sinus over
the bifurcation into right and left branches and down these into the right
and left ventricles. These parts are represented in figs. 1 and 2 (Plate 4).

Historical.—The first specific demonstration of the presence of a muscular
connection between the atrium and ventricle was given by Gaskell in 1883.
Previous to that time many writers had declared that such a connection
existed, but their statements were indefinite. For instance, Paladino(3) is
referred to by Bardeleben as having found that “die Vorhofsmuskulatur
endet nicht an den Annuli fibro-cartilaginosi, sondern geht grossentheils in
die Ventrikelwand und die Papillarmuskeln weiter.” It was Gaskell (4) who
first definitely showed that in the tortoise the contraction wave spreads from
the sinus over the auricle to the ventricle by means of the muscular connec-
tion which exists between the three parts of the heart at the sino-auricular
and auriculo-ventricular grooves. He found that at the sino-auricular
junction the fibres of the sinus form a circular muscle-ring from which the
fibres of the auricle take origin. From this origin the fibres of the auricle,
after ramifying in all directions, approach and get attached to the upper and
middle part of the auriculo-ventricular groove, forming a ring of muscle
fibres from which in turn the fibres of the ventricle take origin. By experi-
mentally sectioning between the two auricles, and by removal of the visceral
pericardium, he was able to show that the “ventricle contracts in due
sequence with the auricle, because a wave of contraction passes along the
auricular muscle and induces a ventricular contraction when it reaches the
auriculo-ventricular groove.’ The integrity of the whole muscle at the
auriculo-ventricular groove is unnecessary for this sequence, for there exists
a definite track along which the wave of contraction passes. Histologically
he found that the muscle fibres present in the auriculo-ventricular muscle-
ring differed from the muscle cells of the auricle and ventricle both in the
size of the nucieus and the character of their striation.

In spite of Gaskell’s work, the hypothesis generally accepted was that the
contraction wave cannot be myogenic because in mammals there occurred a
distinct break between the muscle of the atria and the muscle of the
ventricles. It was held that the atrial fibres and the ventricular fibres

1909. | The Nerves of the Atrio-ventricular Bundle. 153

belonged to independent systems, and were separated by a considerable
amount of connective tissue at the atrio-ventricular junction. In 1893,
ten years after Gaskell’s work appeared, Stanley Kent (5) showed that
muscular connection did exist in the mammalian heart. He found in young
rats that there was at birth a well-defined continuity of atrial and ventricular
muscles. As age advanced there took place a considerable development of
connective tissue in the atrio-ventricular groove; nevertheless, in adults
there still persisted well-marked bands of muscle tissue between the two
chambers over a considerable ohn of the atrio-ventricular groove, and in
particular at the junction of the atrio-ventricular septum. This muscular
connection varied in amount in different animals, yet could be demonstrated
in all; but the exact location of these bands he did not definitely determine.

In the same year there appeared the work of Wilhelm His, junior (6),
“ Die Tatigkeit des embryonalen Herzens und deren Bedeutung fiir die Lehre
von der Herzbewegung beim Erwachsenen.” Here for the first time was
given the definite location of the atrio-ventricular bundle. His demonstrated
that in the earliest stages of development of the mammalian heart there
exists a continuous muscle union between the heart segments, and that the
primitive contraction goes on without the presence of ganglion cells.
Further, it was erroneous to suppose that this primitive continuity of
muscles was completely interrupted in the adult by connective tissue at the
atrio-ventricular groove ; that while a break did occur it was by no means
complete, for at a particular place in the atrio-ventricular septum muscular
union persists. This union is brought about by a strand of muscle fibres
which springs from the posterior wall of the right atrium, passes forward to
the atrio-ventricular groove lying in the upper part of the ventricular
septum, soon forking into right and left branches. The presence of this
bundle of His has been confirmed by many subsequent writers.

The most important work on the atrio-ventricular bundle is that of
Tawara(1), “ Das Reizleitungssystem des Siugetierherzens.” This Japanese
investigator, working in Aschoff’s laboratory in Marburg, published, in 1906,
a careful and exhaustive monograph on the macroscopic and microscopic
appearances of the bundle in a series of mammals. In it he demonstrated
the connections of the fibres of the bundle with the ordinary cardiac muscle
and the relation of the Purkinje fibres throughout the heart to the atrio-
ventricular fasciculus. His findings (7) may be summarised as follows :—

(1) In man and all the animals examined, the Purkinje fibres or their
equivalents form the outspreading of a muscular system which unites the
atrial muscle to the ventricular muscle. This muscular system constitutes
the atrio-ventricular bundle.

154 Mr. J. G. Wilson. [Jan. 21,

(2) This muscular system has a uniform arrangement in all mammals,
though slight individual differences appear. It runs from the atrial wall
through the atrio-ventricular fibrous septum to the point where it spreads
out in the ventricular wall, at first as a bundle enclosed in connective tissue,
then spreading out into tree-like end branches. The closed strand never
enters into relation with the ventricular muscle, but the end branches fuse
with the usual ventricular muscle.

(3) This uniting system is early developed in the embryo. From this
time on, leaving growth out of consideration, it remains unchanged during
life. It is not affected by hypertrophic and atrophic processes in the heart in
the same way as the ordinary cardiac muscle.

(4) The topographical, histological, and biological peculiarities of this
system are opposed to the suggestion that its function is that of a heart
pump, like the ordinary cardiac muscle. Moreover, the physiological
experiments of Gaskell, Engelmann, Herring, and others suggest that in this
system can be found a conducting path for the co-ordination of the heart
muscle.

Macroscopic Description of the Bundle-—To dissect out rapidly the atrio-
ventricular bundle, I have found it best first to identify the pale pink muscle
fibres of the right and left branches. These are more or less subendocardial.
The left is readily observed at a varying distance beneath the junction of the
posterior semilunar (non-coronary) with the right semilunar valve. The
right branch, less readily found in some mammals because covered by a thin
layer of ventricular muscle, can be found lying near the line which joins the
moderator band to a point under the medial (septal) cusp, adjacent to its
junction with the anterior (infundibular) cusp of the tricuspid valve. From
either of these points the entire atrio-ventricular band can be dissected out.

As seen in the calf, where it is very large, the bundle originates in the
posterior wall of the right auricle in the region of the sinus coronarius (Plate 4,
figs. 1 and 2). At this point there is a mass of pink fibres—not red like the
ordinary cardiac fibres, and less pale than the main continuation of the band.
It is cone-shaped in appearance, with an ill-defined base merging into the
auricular muscle and a well-marked apex passing into the narrow band of
white tissue which goes forward into the atrio-ventricular septum in a groove
on the under surface of the cardiac cartilage. Reaching the upper part of
the interventricular septum, it divides into a right and a left branch. The
right branch passes downward on the septal wall of the right ventricle more
or less superficial towards the septal attachment of the moderator band.
Here it begins to separate out and send branches into the septum. If a
transverse section be made of the moderator band, Purkinje fibres are seen in

1909. | The Nerves of the Atrio-ventricular Bundle. 155

small bundles isolated by more or less definite connective tissue sheaths.
Through the moderator band it reaches the lateral wall of the ventricle and
the papillary muscles. The left branch appears under the endocardium of
the septal wall of the left ventricle about 1:5 cm. below the junction of the
posterior with the right semilunar valve. Immediately under the aortic
opening it is covered with cardiac muscle. On reaching the surface of the
heart it spreads out and sends branches downwards and outwards to the
septal and lateral walls of the ventricle. —

In mammals generally, the course of the atrio-ventricular bundle agrees in
the main with this, but some slight differences are observed. Thus in the
sheep the band is smaller and less distinct. The right septal branch is
covered by cardiac muscle from the atrio-ventricular septum to the moderate
band; the left branch appears under the endocardium 1:2 cm. beneath the
junction of the right and posterior aortic valves. In the pig there is a close
resemblance to this. It is interesting here to note how in all mammals the
bundle is in close apposition to the insertion of the aorta, behind its posterior
valve.

Microscopically, as Tawara pointed out, one must also distinguish an atrial
and ventricular part. The atrial part begins as a complicated network of
branching fibres smaller than the atrial muscle cells with a nucleus lying in
undifferentiated protoplasm and with fibrillee less well developed and more
irregular than in the ordinary cardiac cell. Within this network lie
connective tissue with fat, blood-vessels, and nerves. From it emerges a
series of more or less parallel muscle fibres similar to those of the network
surrounded by a well-marked connective tissue sheath. The appearance of
these cells and the amount of connective tissue between them readily
distinguish them from the ordinary cardiac muscle.

The ventricular section begins immediately where the atrio-ventricular
bundle breaks through the fibrous septum of the atrio-ventricular groove. It
consists of an irregular network of muscle cells surrounded by connective
tissue. Histologically it has no similarity with the atrial portion of the band
nor with the ordinary cardiac muscle cell. One, two or more of the cells of
the atrial strand pass into a much larger cell of irregular size and shape which
possesses many features in common with a Purkinje cell. As the ventricular
strand passes down the septum, these initial cells gradually pass into typical
Purkinje fibres which constitute the muscle cell component of the two arms
and their outspreading branches.

Technique.—As a first step, it is necessary to be able rapidly to cut out the
bundle either after staining or in the fresh condition. This can be done after
a series of preliminary dissections. For the purpose of this research I limited

156 Mr. J. G. Wilson. [Jan. 21,

my observations to the main bundle surrounded by its connective tissue
sheath, including the part directed to the coronary sinus and the two arms
running into the septal walls of the right and left ventricles (figs. 1 and 2).
My results have been obtained by the methylene blue “vital” method.
Occasionally the Cajal method or gold impregnation was used, but with
results less satisfactory, chiefly, I believe, because the methylene blue gave
such definite results that it was not felt necessary in this preliminary report
to ascertain experimentally the modifications which appeared necessary to
apply either of these methods, especially the Cajal, to the heart muscle.
The strength of the solution injected was :—

Methylene blue, }-per-cent. solution ......... 10) cre:
Salt solution, 0°9-per-cent. solution ............ 90m

The coronary arteries were injected with the solution either directly or
indirectly from the aorta. When the heart was well injected the bundle was
rapidly dissected out, placed on a slide and examined in the usual way ; or
the bundle was cut out from the fresh heart, partly immersed in a methylene
blue solution slightly weaker than the above, and kept in a hot chamber at a
temperature of 37° or 38° C. The full description of the technique in use has
been so often presented in previous papers, Wilson(8), that it seems
unnecessary to repeat it here. Fixation was always done in 8-per-cent.
ammonium molybdate and sections cut in paraffin.

The limitations of the methylene blue technique are well known. This dye,
though neurotropic, isnot monotropic. A difficulty encountered in the atrio-
ventricular bundle is the affinity of the methylene blue for the elastic fibres—
a tissue sufficiently abundant in the bundle to give trouble at first.
Recognising this possible source of error, one readily gets accustomed to
distinguish between the relatively coarse wavy fibres of the elastic tissue and
the fine varicose fibres of the nervous strands branching and anastomosing
irregularly.

The nerve fibres are in the main non-myelinated. A few medullated
fibres are to be seen, especially in the calf: these appear usually as isolated
fibres and do not enter as a rule into the strands of fibres which pass directly
through the bundle. ;

The staining of all the nerve elements in the bundle in one and the same
preparation is unusual. Thus it is not common to get in the same prepara-
tion the finer network together with the ganglion cells and their processes.
If one gets good ganglion cells with processes, the finer varicose fibres
distributed through the muscle are usually poorly stained. This agrees with
the observations which I have satisfied myself with time and again, that the

1909. | The Nerves of the Atrio-ventricular Bundle. ay

dye will at one time select motor endings in preference to sensory or vaso-
motor; at another time, under the same external conditions, the sensory or the
vasomotor are the better, and may be the only ones stained well. This
appears to me to be due to the particular chemical state of the nerve ending
at the moment the dye reaches it.

The animals used in the investigation have been the calf, sheep, and pig,
and to a less extent the dog. Tissue was also obtained from the human
heart. I 4m not prepared at this time to report on the results of the
investigation on human material or in the dog, but so far as these have gone
I have no reason to believe that they will not bear out the results reported in
this paper on the three first-mentioned animals.

The examination was confined to the part of the bundle extending from
near the coronary sinus through the fibrous septum and down both arms.
Though this constitutes but a part of the entire bundle, it forms an important
section ; it is a well-defined structure and includes the path across the atrio-
ventricular septum. To avoid confusion, it is referred to in this paper as the
atrio-ventricular bundle.

Even a very superficial examination convinces one of the important part
taken in its composition by nerve elements. Nerve cells are scattered in
profusion along its course, and nerve fibres pass in strands along with its
muscular elements or intricately interlace around its cells. There is no part
of it, from the coronary sinus to the end of its right or left arm, bereft of
groups of ganglion cells or devoid of nerve fibres, The neurologist might
well refuse to recognise in it a muscle bundle; to him it might become
conspicuously a nerve pathway of very intricate structure.

In the bundle the nerve elements divide themselves for descriptive
purposes into three groups:

I. Ganglion cells.
II. Nerve fibres and plexuses.
III. Nerves directly associated with the blood-vessels.

I. The ganglion cells are naturally first described, for they are the most
conspicuous nerve structures present, both from their abundance and their
size. They are found usually in groups of varying number; some of these
in the calf have as many as 16 nerve cells, but more may easily be present.
Individual cells, scattered either in the course of the nerve strands or
isolated in the fibrous tissue around the bundle, are frequently seen.

Nerve cells are abundant in the atrio-ventricular bundle of all the animals
studied, but I have examined them chiefly in the calf, where, from their large
size and from the facility with which they stain, they are especially suitable

158 Mr. J. G. Wilson. [Jan. 21,

for investigation. All three varieties of cells are found (see Plate 5) unipolar
(fig. 3), bipolar (fig. 4), and multipolar (fig. 5). Ganglionic groups are
scattered in the connective tissue not only around the muscle band but also
in the interstices between the muscle fibres (fig. 4). They are not especially
located in the subendocardial area; when the bundle reaches the surface of
the heart, they are seen not only between its tissue and the endocardium, but
are also equally conspicuous within the bundle and in the fibrous tissue on
the side remote from the endocardium. :

They can be seen all the way from the coronary sinus to the distribution in
the right and left walls of the ventricular septum. In the particular part
examined they appear to be most abundant near the point of bifurcation and
in the course of the right and left ventricular divisions. Their abundance
may be roughly estimated by saying that in a series of sections of one of
these arms, especially the left, cut 50 microns thick, it is no unusual thing
to find in a section three or four ganglionic masses containing from five to
nine nerve cells. Were one roughly to indicate any one area more than
another where they are conspicuous, one might select the upper border of the
bundle just before its division into right and left limbs. Here there is a
large group, easily seen in the calf and sheep, lying in the fibrous tissue out-
side the bundle whose processes pass into the muscular pathway.

The processes of these nerve cells can often be traced for a long distance
gradually dividing in their course (fig. 3). Many of them ultimately become
varicose fibres, and some at least go into the nerve plexus around the muscle
cells. But the mode of termination of the majority, from the distance they
traverse and from the failure to stain referred to above, I have so far been
unable to determine. Fig. 6 shows a large nerve cell in some respects akin
to Dogiel’s type I. It has one long process and several smaller ones, which
latter project only a short distance from the cell-like sharp prickles. The
long process gan be traced for a very considerable distance ; it frequently
divides and ultimately ends as very fine varicose fibrils which enter into the
plexus around the muscle bundle. Fig. 5 shows the interlacing of the
processes of several nerve cells in one pericellular plexus. Here a multipolar
cell, G1, gives off a branch, one of whose rami enters into the pericellular
nest of cell G.2, into which also enter twigs from two other more distant
nerve cells by fibres C and D.

II. In the atrio-ventricular bundle the nerves present themselves

(a) as strands of fibres ;
(6) as plexuses of fibrils.

(a) The nerve strands have a general course along the length of the bundle.

In preparations of the entire bundle they are seen to break into the fibrous

1909. ] The Nerves of the Atrio-ventricular Bundle. 159

tissue around it at various parts of its course, but chiefly prior to or near the
point of division into the right and left arms. Thus several strands appear
towards the coronary side of the division, corresponding to the position of
the ganglionic group referred to above. The question of the source of the
fibres lies outside the field of the present investigation, but their course
indicates an origin near the insertion of the aorta and the lower part of the
atrial septum. Sections of the bundle bring out more clearly the relation of
these strands to the ganglion cells. Throughout their course nerve cells are
scattered individually or in groups and the processes of these cells enter into
the nerve strand (fig. 4). The fibres of these strands are not in close
apposition. This is especially observed in the left septal part (fig. 4). They
are found in the connective tissue both around the bundle and between the
muscle cells. They have irregular connection with each other. Often one
can see a strand send off a single fibre or a group of three or four fibres
which pass across the intervening muscle cells to an adjacent strand and then
continue their course along this. Occasionally a fibre may be observed to
turn backwards ; however, the general tendency is for the strands to pass
downwards in the direction of the atrio-ventricular bundle. The fibres are
as a rule non-medullated, most of them with varicosities. These varicosities
occur at less frequent intervals than in group (0). The medullated nerves
seen have been chiefly in the cow. It is with fibres of this group that at
first the elastic tissue is apt to be confounded, but enough has been said to
show how they may be easily differentiated.

It is difficult to tell what becomes of these strands. Many of them pass
through that part of the bundle I have examined. Some, however, may be
seen to break up into very fine varicose fibrils which enter into plexus (0d)
(fig. 4, A.).

(b) The nerve plexuses are composed of very fine fibrils with varicosities
at frequent intervals. They can be seen with the 8-mm. objective and
4 ocular, but require a higher power for distinct observation. The fine
branches lie in close apposition to the muscle cells and have absolutely no
resemblance to elastic tissue. In well-stained preparations they are so dense
and intricate from frequent branchings and anastomoses that it is impossible
to trace individual fibres for any distance. I have chiefly studied these
plexuses in the pig and sheep. Here they form a continuous network which
can be traced for long distances over a series of sections and appear to
extend the whole length of the atrio-ventricular bundle. Their general
characters can be well seen in figs. 7 and 8. As will be noted, it is not a case
of a nerve breaking up and surrounding individual muscle cells, but of
a complicated network lying around both single cells and groups of cells.

160 Mr. J. G. Wilson. | [Jan. 21

The source of the plexuses is not easy to determine. The difficulty is due
partly to the length of the varicose fibrils before they enter intimately into
the plexus, and partly to the difficulty of staining at the same time
sufficiently well ganglion cells and muscle plexuses. Occasionally, however,
a non-medullated fibre can be seen to pass out from the strand and break up
into fine twigs, which ultimately become very fine varicose fibrils and enter
into direct relation with the muscle plexus. (See Plate 6.)

III. Nerves related directly to the blood-vessels of the atrio-ventricular
bundle. These have no special significance in this locality and differ in no
way from nerves found in arteries elsewhere, so well described by Dogiel and
others. Two distinct varieties present themselves :—

(1) A vasomotor plexus of fine non-medullated varicose fibrils (fig. 9).
These are most abundant in the large vessels and gradually get fewer as the
smaller arterioles are reached. At the point where a vessel divides or
a branch is given off, the plexus becomes more dense; it is as if the fibres
became concentrated at the point of division. Then the plexus divides or
sends offshoots along the arterial ramus, the amount sent off varying with
the size of the branch. The main plexus runs in the adventitia, but a part of
it passes into the tunica media to form anastomosing branches directly
related to the muscle fibres. These do not appear to me to form definite
endings; the knob-like endings on the muscle cell sometimes described
appear to be artefacts due to a stoppage of the dye. This appears to be
confirmed by relatively few appearing in well-stained preparations and their
abundance in badly-stained tissue.

(2) Distinct from these are the so-called sensory endings. These are
definite end organs situated in the fibrous coat around the vessel, and difter
from the above described anastomosing plexus formations. They are so
called because of their resemblance to sensory endings elsewhere. A nerve
fibre thicker and less varicose than the ordinary vasomotor nerve and at
times faintly medullated is seen to break up in the tunica adventitia into
a more or less complex arborisation which is non-capsulated. In the smaller
arterioles they are very simple (fig. 9, S.), in the iarger arteries of the bundle
they become extremely complex (fig. 10).

Conclusions.

I. Anatomically the atrio-ventricular bundle contains not only a special
form of muscle fibre distinct from the ordinary muscle of the atrium or the
ventricle, but, as I have shown, is an important and intricate nerve pathway
in which we find :—

el

1909.] The Nerves of the Atrio-ventricular Bundle. 161

(1) Numerous ganglion cells—monopolar, bipolar, and multipolar—whose
processes may pass—

(a) To adjacent ganglion cells in the bundle ;
(6) To the muscle fibres in the bundle ;
(¢) Through the muscle bundle so far as it was examined.

(2) Abundant nerve fibres running through it in strands, the processes of
which may end (a) in ganglion cells in the bundle; (@) in the muscle plexus,
or may pass through the part examined.

(3) An intricate plexus of varicose fibrils around and in close relation to
the muscle fibres of the bundle.

(4) An abundant vascular supply with well-marked vasomotor nerves and
sensory endings.

II. Physiologically it has been shown that the atrio-ventricular band
constitutes the pathway which assures the communication of the atrio-
ventricular rhythm. When the bundle is sectioned or crushed the ventricles
cease momentarily to beat, though they soon regain pulsation, but with
a rhythm much more slow than that of the atrium. Pathological anatomy
supports this view; the allorythmia or Stokes-Adams disease can be
explained satisfactorily by lesions involving this pathway. As a result of
these physiological experiments and from these pathological conditions, it has
been asserted that the contraction wave must be myogenic. To such
a deduction my anatomical findings are opposed. They demonstrate that in
these experiments and pathological conditions an important nerve pathway
is equally involved with the muscle bundle. Considering the neurogenic as
opposed to the myogenic hypothesis from the anatomical standpoint, one
must acknowledge that the very complex nerve constituents of the bundle
indicate an important nerve pathway and are very suggestive of an intricate
nerve mechanism.

Can the atrio-ventricular bundle be regarded as a neuro-muscular spindle ?

It has recently been stated that “the conclusion derived from the study of
the development and cytological structure of the conductive system is that it
is a neuro-muscular apparatus akin to the neuro-muscular spindle of voluntary
muscle” (Retzer (Y)). Similar suggestions have been made by others, and
readily present themselves as a possible explanation of the position and
structure of the atrio-ventricular bundle. But have we anatomical data on
which to base such a conclusion? To answer this it is necessary to consider
the structure of the neuro-muscular spindle.

Were one to accept Golgi’s definition, made in 1880, that it is “a bundle of
incompletely developed muscle fibres surrounded by a special sheath,” one

VOL. LXXXI.—B. M

162 Mr. J. G. Wilson. - [Jan. 21,

might be tempted to acquiesce in the above view, at any rate provisionally.
But the work since then, especially of Ruffini(11) and Sherrington (12),
has so widened our knowledge of the neuro-muscular spindle that Golgi’s
definition is now inadequate, and it is with the complex nerve ending which
they describe that the atrio-ventricular bundle must be compared.

The essential anatomical points in the structure of the neuro-muscular
spindle may be summed up as follows :—

(1) The fibres which go to form the muscle bundle (Weissmann’s bundle)
in the neuro-muscular spindle have a diameter less than that of the ordinary
muscle fibre.’ The fibres are directly in apposition, and no connective
tissue lies between them, though connective tissue lies around Weissmann’s
bundle:and constitutes the axial sheath of Sherrington. The striation of the
fibre is usually only marked in the marginal area, and so on transverse
section it looks like a Purkinje fibre. Regarding the two ends of Weiss-
mann’s bundle, one, the wider end, is muscular; the other tendinous where
the axial fibres of the bundle are attached to the fibrous tissue of the
capsule or to the tendon of the muscle.

(2) Around it lies a lymphatic space.

(3) There is a distinct capsule of concentric superposed lamelle of con-
nective tissue. At the tendinous end of the muscle bundle it is thin and
adheres to the tendinous portion of the spindle: at the muscular end it is
thin and may be absent.

(4) In the vast majority of cases it is fusiform in shape.

Contrast this with the anatomical description given of the atrio-ventricular
bundle in this paper, and it will be seen that, apart from the muscle cells being
similar to Purkinje fibres, they have nothing in common. This lack of agree-
ment is further emphasised when we compare the nerve constituents of each :—

(5) To the distribution and termination of the nerves in the neuro-
muscular spindle, with its three distinct kinds of endings described by
Ruffini, there ‘is nothing comparable in the atrio-ventricular bundle.

(6) Ganglion cells are not present in the neuro-muscular spindle, whereas
they are a marked feature in the atrio-ventricular bundle.

‘From the above, one must conclude that, whatever the physiological
significance of this bundle may be, it has anatomically nothing in common
with the neuro-muscular spindle.

In conclusion, I wish to express my great indebtedness to Dr. Mott for
allowing me to carry out a considerable part of this investigation at the
Pathological Laboratory at Claybury, and for his kindness in assisting me
to vrocure a large part of the material which has been used. :

1909. | The Nerves of the Atrio-ventricular Bundle. 163

BIBLIOGRAPHY.

1. Tawara, S., ‘ Das Reizleitungssystem des Saugetierherzens, Jena, 1906, S. 189
2. Retzer, R., “Some Results of Recent Investigations on the Mammalian Heart,”
“Anat. Record,’ 1908, vol. 2, p. 149.
3. Paladino: Bardeleben, ‘Jahresbericht iiber die Fortschritte der Anat. und Physiol.,
1876, vol. 5, p. 251.
4. Gaskell, W. H., “On the Innervation of the Heart, with especial reference to the
Heart of the Tortoise,” ‘Journ. of Physiol.,’ 1883, vol. 4, pp. 43, 64, 70.
5. Kent, A. F. Stanley, “Researches on the Structure and Function of the Mammalian
Heart,” ‘Journ. of Physiol.,’ 1893, vol. 14, p. 233.
6. His, Wilhelm, Junior, “ Die Tatigkeit des embryonalen Herzens und deren Bedeu-
tung fiir die Lehre von der Herzbewegung beim Erwachsenen,” ‘ Arbeiten aus
der Med. Klinik zu Leipzig,’ 1893, SS. 14—49.
7. Tawara, loc. cit., S. 190.
8. Wilson, J. G., ‘Journ. of Comp. Neur. and Psychology,’ 1904, vol. 14, p. 1; ‘ Brain,’
1905, p. 339.
9. Retzer, R., “Some Results of Recent Investigations on the Mammalian Heart,”
‘Anat. Record,’ 1908, vol. 2, p. 154.
10. Sherrington, C. §., “On the Anatomical Construction of Nerves of Skeletal Muscles,”
‘ Journ. of Physiol.,’ 1894—95, vol. 17, p. 211.
11. Ruffini, A, “On the Minute Anatomy of the Neuro-muscular Spindles of the Cat, and
on their Physiological Significance,” ‘Journ. of Physiol.” 1898—99, vol. 23,

p. 190.
EXPLANATION OF PLATES.
A.Y.B. = atrio-ventricular bundle. C.S. = coronary sinus.
R.B. = right branch of A.V.B. M: = muscle.
L.B. = left branch of A.V.B. G. = ganglion cell.

B. = bifurcation.

PLATE 4.

Fic. 1.—Dissection of right atrium and ventricle of calf to show A.V.B. The band is
seen passing from under atrial muscle near C.S. to B., then continued under
medial cusp over right ventricular septum at R.B. towards the insertion of
moderator band (M.B.).

V.C.[. = inferior vena cava. M.C. = medial cusp.
A. = auricle. A.C. = anterior cusp.

Fic. 2,—A.V.B. of calf looking down from above. The great portion of atrium has been
removed toshow bifurcation in the upper part of ventricular septum.

R.V.S. = right ventricular septum.
L.V.S. = left ventricular septum.

164 The Nerves of the Atrio-ventricular Bundle.

Puate 5.

Fic. 3.—Monopolar ganglion cell; one of six lying among muscle fibres of A.V.B. on
septal wall of calf. Zeiss comp. oc. 4, obj. 8 mm.

Fia. 4.—Group of ganglion cells lying among nerve fibres in A.V.B. on left septal wall of
calf.

A. = nerve fibre breaking up near muscle fibre and entering into muscle
plexus (not distinguishable with this low power). Zeiss oc. 6, obj. AA.

Fig. 5.—Ganglion group in A.V.B. of calf, showing pericellular plexus around ganglion
cell (G.,) containing fibres from three nerve cells.

G., = nerve cell which sends off process (dendrite) B, which branches
repeatedly within ganglionic group. One of these branches breaks up into a
network which embraces the adjacent ganglion cell G...

C. = nerve fibre from an adjacent ganglion—not shown—which breaks up
into two branches : from these, twigs pass into plexus around G.,, others into
plexus around G..,.

D. = nerve fibre from nerve cell in same group as C, which breaks into
plexus around G...

E. = varicose fibril clinging to A—neuraxis of G.,.

Fic. 6.—Nerve cell in A.V.B. calf, containing a well-marked nucleus with halo. The cell
has several small processes which project, like sharp prickles. It has one long
process, A, which divides frequently ; its ultimate branches become varicose
and lie in close proximity to muscle fibres of A.V.B. Zeiss comp. oc. 4,
obj. 2 mm.

PLATE 6.

Fig. 7.—Plexus around and adjacent to muscle fibres of A.V.B. of pig, immediately to
coronary side of its division into right and left septal branches. Zeiss oc. 4,
obj. 8 mm.

Fic. 8.—Plexus around muscle fibres of left septal branch of A.V.B. of pig. Zeiss comp.
oc. 4, obj. 2 mm.

Fig. 9.—Vasomotor fibres in small artery in A.V.B. of pig.

S.N. = sensory nerve, S. = sensory ending, V.M.P. = plexus of fibres at
bifurcation of artery.

Fie. 10.—Sensory ending in wall of artery in A.V.B. of cow.

. = nerve which breaks up into complex ending.

Roy. Soc. Proc., B. vol. 81, Plate 4.

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165

The British Freshwater Phytoplankton, with Special Reference to
the Desmid-plankton and the Distribution of British Desmids.

By W. West, F.L.S., and G. S. West, M.A., D.Se., F.L.S.

(Communicated by D. H. Scott, F.R.S. Received October 30, 1908,—Read
January 28, 1909.)

PAGE

Marla tro duc hi OMe rary sac eijaiseiss Neem arerctaesietele bas veleGialsaasts sane deiainlsvoie bh snueiciee 165
JOT: Wag Stoo uarisla) IDEN seecsensocsgsocostobosuasoousebasbudaasdunpadseHuceeeonenipsaboonas 168
III. Lakes of the Orkneys and Shetlands ...............cseccceceeeereeneeeeeeetens 170
IV. The Irish Lakes (West and South-west) .............eeeeeeeesee eee eee eens 171
WemlLo ual iNiea chin reset eatesattind ic ayastecamauticnce seas mascoeniaiciad duaetencaenwaweeeas 172
NV Alepribo Wiel shitltalke=aneayencacssjst peti sae cere ae sa aen cee acc aires sis tetecits osje flelselans 173
Walt Dhesbinelishy Malkesanea ncn cua dawaie se sere ciiecinccuen aeteseesesarem ea ae iaers vacate eel 174
Vel Malham arn iWestmMorksbine ms ssscc.cuccsccenseccrecnssosccscscerencase sats 175
Xe Rhee Britis npkiver-plamlcconmpeeeterent cece secntaccaaene keener tee ncsccie 175
X. General Comparison of British Lake-areas..............:cceceeeeeneneeee eens 176
XI. General Summary, and Discussion on the Dominance of Desmids ... 190

I. INTRODUCTION.

Not until much work had been done at the phytoplankton of the fresh-
waters of Western Hurope were investigations of a similar nature begun in
the British lakes and rivers, and it is during the last ten years that almost
all our knowledge of this branch of freshwater biology has been acquired.

We have otirselves conducted nearly all the British investigations, and
we now think the work has progressed sufficiently to enable us to
summarise the results, and to institute comparisons between the British
phytoplankton and that of continental Europe and other regions.

Since 1900 we have collected plankton from a large.number of lakes in
the west of Scotland, from some of the lowland Scottish lochs, from
practically all the lakes of the English Lake District and most of those in
North Wales, from nearly all the lakes of the west and south-west of
Treland, from Lough Neagh and Lough: Beg, from Malham Tarn in West
Yorkshire, and from the Rivers Ouse, Lochay, and Bann. In the collection
of material for these investigations we have been greatly assisted by four
grants from the Government Grant Committee of the Royal Society and two
from the Fauna and Flora Committee of the Royal Irish Academy.

From a biological standpoint the British lakes are of great interest, and
since the publication of the first reports on their plankton, the diversity of
the Algal constituents has been a revelation to the freshwater biologist.
In the number and diversity of the species constituting this phytoplankton

VOL, LXXXI.—B. N

166 Mr. W. West and Dr. G. 8. West. [Oct. 30,

the lakes of the western British areas stand ahead of any other lakes which
have so far been investigated.

The numerous Scottish lakes are almost all of a montane character.
They are situated in the vicinity of high mountains and in a northern
latitude, and many of the higher plants occurring in these localities in
abundance, at almost sea-level, are typical montane species.*

The lakes of the four principal areas examined, the North-west Scottish,
the West and South-west Irish, the Welsh, and the English lakes, are
situated in the most mountainous parts of the British Islands. As these
areas contain the great majority of the British lakes, and are all
geographically in the west or north-west, they can be spoken of as the
western and north-western lake-areas. The mountains amongst which they
are situated are composed almost entirely of old formations, and the lake-
basins are for the most part drainage areas on outcrops of Older Palzeozoic or
of Precambrian rocks, often with associated intrusive Igneous masses.

Malham Tarn, West Yorkshire, and Lough Neagh are somewhat isolated
lakes, and their phytoplankton is considered separately.

Wherever possible, boats were used for the collection of the plankton, and
for one part of the investigation of Lough Neagh, the Lough Foyle and
River Bann Fisheries Company kindly lent a steam launch. The smallest
tow-net had a mouth-diameter of 6 inches and a length of 15 inches, the
largest was 12 inches diameter and about 36 inches in length. The nets
were usually towed after the boat at a speed of about 1$ to 2 miles per hour,
but on Lough Neagh the large nets were used up to a speed of 4 miles per
hour. They were generally kept in the water from 20 to 30 minutes.

We have come to the conclusion that the best fixing agent is 2 or 3 per cent.
formalin, but tt should always be replaced before the material 1s examined.

Neither picric acid nor chromic acid should be used in plankton-work, as it is
almost impossible to wash the material free from these reagents by ordinary
methods, and all the gelatinous colonies are broken wp. Alcohol is also to be
avoided, as it causes too great a shrinkage and distortion of the more delicate
plankton species.

During the past 18 years we have been acquiring a very extensive
knowledge of the distribution of British freshwater Algz, having made many
thousands of collections in all parts of the British Islands. As the lake-
areas are the richest parts of the country for freshwater Algze, these districts

* Occurring in abundance around these lakes are :—/estuca ovina, var. vivipara, Andreca
Rothii, A. petrophila, Bryum alpinum, various species of Rhacomitrium, Anthelia julacea,
Platysma triste, Oladonia cervicornis, Stereocavlon coralloides, Spherophoron coralloides,
Lecidea geographica, etc. The whole district is almost treeless.

1908. | The British Freshwater Phytoplankton. 167

have been worked very extensively, and as we did not begin plankton
investigations until 1900, we have a much more complete knowledge of the
general Aloze-flora of these areas—such as is found in the bogs, lake-margins,
streams, on the wet rocks of the glens, etc.—than of the phytoplankton, In
consequence of this previous detailed acquaintance with the Algz of the
lake-areas, we are enabled with a considerable degree of accuracy to state
which species should be considered as true constituents of the plankton and
which should not. The other constituents are casual or adventitious, and
their sojourn in the plankton is both temporary and accidental.

The work has been entirely of a qualitative character. We have had
neither the time nor the necessary funds to conduct quantitative investiga-
tions such as those carried out by Apstein,* Zacharias,+ Lemmermann,t
Volk,§ etc.,in Germany, by Wesenberg-Lund|| in Denmark, by Huitfeldt-
Kaas{i in Norway, and by others in Switzerland and the United States.**

We have also met with many difficulties in attempting to acquire a
knowledge of the periodicity of the plankton of the British lake-areas.
Neither academic duties nor funds have permitted the necessary number of
visits to these areas which would be required in the course of a year in
order to obtain an adequate idea of the periodicity of the plankton. At the
very least, monthly visits would be imperative for reliable results to be
obtained; and to collect material from a number of lakes, even in one
district, would require several days, not to mention the difficulty, and in
some cases the impossibility, of obtaining boats in the winter months. It is
likewise no easy matter to find suitable men on the spot of sufficient
intelligence to carry out detailed instructions at regular monthly intervals
throughout the year.fT

* Apstein, ‘ Das Siisswasserplankton, Methode und Resultate der quantitativ. Uuter-
suchung.,’ Kiel und Leipzig, 1896.
+ Zacharias in ‘ Forschungsber. Biol. Stat. Plon,’ vol. 3, 1895, etc.
{ Lemmermann, “Das Plankton der Weser bei Bremen,” ‘ Archiv fiir Hydrobiologie
und Planktonkunde,’ Bd. 2, 1907.
§ Volk, “Hamburgische Elbe-Untersuchung., I u. VIII,” ‘Mitteilungen aus dem
Naturhist. Mus. Hamburg,’ Bd. 19, 1903 ; Bd. 23, 1906.
|| Wesenberg-Lund, ‘ Studier over de Danske Séers Plankton, Kjébenhavn, 1904.
4] Huitfeldt-Kaas, ‘Planktonundersigelser i Norske Vande,’ Christiania, 1906.
** C. Dwight Marsh, in ‘ Wisconsin Geol. and Nat. Hist. Survey Bull., No. XII, 1903 ;
Kofoid, in ‘ Bull. Illinois State Laboratory of Nat. Hist.,’ vol. 8, 1908.
tt We are at present receiving regular periodic collections, with some necessary daa,
from several of the Scottish lochs and from some of the lakes of the English Lake
District. We have been able to arrange for these collections by means of a further grant
from the Royal Society. Periodic collections have also been made for a period of two
years from one of the large pools of the Midlands. The details of all these collections
will be published shortly.
N 2

168 Mr. W. West and Dr. G. 8. West. [Oct. 30,

Il. Scortisu Laks.

Extensive collections of phytoplankton were made in various parts of
Scotland, but especially in the west and north-west, in August, 1901, May
and August, 1902, April, July, August, and September, 1903, and August,
1907. The areas comprised Perth, Inverness, Ross, Sutherland, and the
Outer Hebrides. We have also examined a number of collections made by
Mr. James Murray of the Scottish Lake Survey (Pullar Trust). In all, we
have examined phytoplankton from 38 of the most important of the Scottish
lochs. The results of these investigations have already been published,*
the previous work being represented by a short report by Borge on some
phytoplankton from the Island of Mullt Mr. James Murray has also
examined the plankton of avery large number of the Scottish lochs, and has
at different times commented upon the occurrence and distribution of the
phytoplankton. Further remarks upon the Scottish phytoplankton have
been made by Wesenberg-Lund in comparing it with the plankton of the
Danish lakes,§ and still more recently a paper has appeared by Bachmann
comparing the results of Scottish material with plankton from ther Swiss
lakes.

The Scottish phytoplankton is largely Chlorophyceous, and is conspicuous
for the large number and great variety of its Desmids, among which the
following are perhaps the most noteworthy: Yanthidiwm subhastiferum,
Staurastrum anatinum, St. Ophiura, and St. jaculiferum. In some of the
lochs, Mesoteniwm macrococcum occurred as a normal constituent of the
plankton, This is a most interesting adaptation of a colonial wet-rock
species to a limnetic life, with an accompanying reduction in size of the
colonies and their assumption of a spherical form. A similar adaptation of
the mucous colonies of Cosmocladium saxonicum to a limnetic existence is
also found in both the Scottish and the Irish lakes, but much less frequently.

In the smaller and more elevated lochs, M/icrospora amena is a characteristic

* W. and G. S. West, in ‘Linn. Soc. Journ. Bot.,’ vol. 35, Nov., 1903; in ‘ Roy. Soc.
Edin. Trans.,’ vol. 41, part 3, 1905.

+ Borge, ‘ Algol. Notis. 4, Stissw.-Plankton aus Insel Mull,’ Botaniska Notiser, 1897.

{t Vide James Murray, in ‘ Roy. Physical Soc. Edin. Proc., vol. 16, June, 1905 ; and in
various reports, under the direction of Sir John Murray, on the Bathymetrical Survey of
the Freshwater Lochs of Scotland in the ‘Geograph. Journ.,’ 1900—1908.

§ Wesenberg-Lund, in ‘Roy. Soc. Edin. Proc.,’ vol. 25, part 6, 1905; also in appendix
to a subsequent paper, zbid., part 12, 1906.

|| Bachmann, in ‘ Archiv fiir Hydrobiol. u. Planktonkunde,’ vol. 3, 1907. It should be
mentioned, however, that some of Bachmann’s records are open to serious doubt. His.
identifications do not appear to be strictly accurate. We might ask, among many other

questions, “ What is Cosmariwm lunaria?” He also copies Tanner-Fulleman’s records,
some of which appear to be equally doubtful.

1908. | The British Freshwater Phytoplankton. 169

constituent of the plankton, and sterile filaments of slender species of
Spirogyra, Zygnema, and Mougeotia occur in abundance. The more slender
species of Mougeotia are generally the most abundant of these filamentous
Algz, and in some instances they exhibit a coiling of the filaments such as is
known to occur in certain of the plankton-forms of Melosira. (Consult fig. 1.)

Fic. 1.—A Coiled Filament of one of the Sterile Species of Mougeotia from the Scottish
Plankton. x 300.

The Protococcoidez are not very abundant in the deeper Scottish lochs
most of them having a decided preference for shallower and warmer water.
The most frequent are Botryococeus Braunit, Spherocystis Schroeteri, Gleocystis
gigas, and Ankistrodesmus falcatus.

Diatoms are conspicuous, largely due to the occurrence in quantity of
a few species. They occur throughout the spring and summer in large
numbers in the deeper lochs, as the temperature of the water never becomes
very high. Many of them are adventitious constituents washed into the
plankton from the bogs and shores, but some have become established as true
plankton-species. There are about 18 well-established species in the
plankton, but only the three species of Rhizosolenia are of exclusive limnetic
habit, all the remainder occurring in other situations.

The Myxophycee (Blue-green Algz) are very poorly represented in the
deeper lochs, and are by no means abundant in the smaller, shallower lakes.
The phenomenon of “ water-bloom” is very rarely met with, and discoloration
of the water, even of the smaller lakes, does not occur either very often or
with any great regularity as the result of the accumulation of large
quantities of Blue-green Alge.

The only periodic collections which have so far been reported upon were
those made in Loch Ness and examined by Bachmann,* and our own

* Bachmann, Joc. cit., 1907, pp. 85—88.

170 Mr. W. West and Dr. G. S. West. [ Oct. 30,

observations relative to the periodicity of the Scottish phytoplankton are
amply confirmed by Bachmann’s brief report.

Desmids are the dominating constituents in the late summer and early
autumn, and there is a preponderance of Diatoms in the colder months, but
compared with the seasonal variations of the phytoplankton in some lakes,
these differences are not very conspicuous.

Specimens of Dinobryon occur in quantity in the spring and summer, and
the Peridiniez are represented by 11 species, although in the larger lakes
they are by no means numerous.

Of a total number of 354 species observed in the phytoplankton, 49-4 per
cent. are Desmidiace, 17°7 per cent. are Bacillariez, and 8°7 per cent. are
Myxophycee.

IIL. LAkES OF THE ORKNEYS AND SHETLANDS.

We have examined material from one freshwater loch in the Orkneys and
from six in the Shetlands. The collections were made in August, 1903, and
the results published in 1905.* Mr. James Murray has collected material
from 34 lochs of the Orkneys and Shetlands, and has briefly noted a few
species of the phytoplankton.t+

Although containing a considerable number of species, the phytoplankton
was not very rich. Most of the lochs were shallow, and contained quantities
of Asterionella formosa. Pediastrum Boryanum and Scenedesmus quadricauda
were both fairly common. Colonies of a Crucigenia, described by Wille from
the Norwegian plankton as C. wrregularis,; but most probably only irregularly
developed forms of C. rectangularis, occurred in several lochs of the
Shetlands.

Desmids were frequent, and in some cases numerous, but the species were
mostly those of shallow lowland lakes. The characteristic western British
types were absent, although further investigations in the Shetlands would no
doubt bring some of them to light.

Peridinieze were abundant, as is so frequently the case in shallow lakes,
and the ubiquitous Ceratium hirundinella was much more generally abundant
than in the larger Scottish lakes.

Out of 52 species of Alge described by Borgesen and Ostenfeld§ as

* W. and G. S. West, in ‘ Bot. Soc. Edin. Trans.,’ Nov., 1904 [1905], pp. 5—10.

+ James Murray, in ‘ Roy. Soc. Edin. Proc.,’ June, 1905, pp. 55, 56.

{ Wille, in ‘Nyt Magazin for Naturvidenskb.,’ Bd. 38, Heft 1, 1900, p. 10, t. 1, f. 15.
Consult also the remarks of G. S. West, ‘Treatise Brit. Freshw. Alg.,’ 1904, p. 217; and
Ostenfeld in ‘ Hedwigia,’ vol. 46, 1907, p. 383.

§ F. Borgesen and C. H. Ostenfeld, “ Phytoplankton of Lakes in the Faeroes,” ‘ Botany
of the Faeroes,’ Copenhagen, 1902.

a

1908. | The British Freshwater Phytoplankton. 171

occurring in the plankton of the Faeroese lakes, 28 were observed in the
plankton of the Orkneys and Shetlands.

Of a total of 178 species observed in the phytoplankton, 47-4 per cent.
were Desmidiaceze, 20°9 per cent. were Bacillariez, and 9°6 per cent. were
Myxophycee.

IV. THe Irish Lakes (WEST AND SOUTH-WEST).

_In 1906 we published an account of the plankton of some of the more
important lakes of the West and South-west of Ireland,* from collections
which we made in May, August, and September, 1904.

Jn the summer the phytoplankton is greatly in excess of the zooplankton,
and the Entomostraca are only dominant in the early spring months. Asin
the Scottish lakes, the phytoplankton of the lakes of the west and south-
west of Ireland is to a large extent Chlorophyceous, but the Bacillariez, the
Peridiniee and to some extent the Myxophycez, are also conspicuous. The
latter are much more noticeable than in the Scottish plankton, especially
species of Anabena, Oscillatoria, Gomphospheria, Celospherium, and Chroo-
COCCUS.

The Desmids are numerous, and include many of the characteristic western
types. There is a great abundance of Spondylosium pulchrum, var. planum,
Staurastrum anatinum, St. jaculiferum, St. Arctiscon, St. pseudopelagseum, and
St. paradoxum, var. longypes, but a curious absence of St. Ophiura. The latter
is a feature of a large proportion of the Scottish phytoplankton, but we have
not yet observed it in the Irish lakes. In the lakes examined, the Desmid-
flora of the plankton was not quite so rich as that of some of the Scottish
lakes, but we think that an investigation of many of the smaller lakes of
Galway and Mayo would bring ‘to light a phytoplankton not at all inferior
to that of the western Scottish lochs.

Diatoms are very abundant, and form a relatively large part of the Irish
phytoplankton. Centric Diatoms are more numerous than in the Scottish
plankton, and are represented chiefly by species of Melosira and Cyclotella.
Tabellaria, Asterionella, and the narrow forms of Synedra are very conspicuous
in the Irish lakes.

The Peridiniez are generally abundant, and are represented by 10 species.
Much the most interesting of these is Peridiniwm limbatum (fig. 2), a charac-
teristic horned species which occurred in some of the small lakes of Galway.
It has only been found elsewhere in the United States.

* W.and G. 8. West, “A Comparative Study of the Plankton of some Irish Lakes,’
‘Roy. Irish Acad. Trans.,’ vol. 33, sect. B, part 2, 1906.

2 Mr. W. West and Dr. G. S. West. [Oct 305

Of a total of 246 species observed in the phytoplankton, 41:7 per cent.
were Desmidiacex, 19 per cent. Bacillariez, and 13-3 per cent. Myxophycee.

Fie. 2.—Peridinivi “imbatum (Stokes), Lemm., from the Plankton of a small Lake between
Clifden and Roundstone, Galway. 500. The reticulated surface markings of the
cell-wall are only indicated on one of the plates.

V. LoucH NEAGH.

The first contribution to the plankton of the Irish lakes was an account of
the plankton-Aloz of Lough Neagh and Lough Beg,* the material having
been collected in May, 1900, and July, 1901. This material has since been
subjected to a further examination. The lake is so isolated, and, moreover,
so differently situated from the other Irish lakes examined, that we have
kept the records in a separate column in the tabulated list of British phyto-
plankton. It is also of interest as being the largest lake in the British
Islands; but although covering a large area it is very shallow, its average
depth being only 45 feet, and the deepest sounding (made in the north-west
corner), is only 96 feet.

The phytoplankton consists largely of Diatoms, Peridiniee, and Chloro-
pbhycez. Of the latter, a few of the Desmids characteristic of shallow lakes
are abundant, and Stauwrastrum paradoxum, var. longipes, occurs in prodigious
quantity. St. pelagicwm was first described from this lake, and has since been
found in the lakes of Germany and Iceland. The Protococcoidee are very
well represented, species of Celastrum, Pediastrum, and Oocystis being
especially noticeable. Of the Diatoms, Tabellaria fenestrata, var. asterionel-
lowdes, is the most conspicuous, but Coscinodiscus lacustris, Cymatopleura
elluptica, and species of Surirella are also abundant. The Myxophycee are

* W. and G. 8. West, “ A Contrib. to the Freshw. Alg. of the North of Ireland,” ‘Roy.
Trish Acad. Trans.,’ vol. 32, sect. B, part 1, 1902.

1908. ] The British Freshwater Phytoplankton. 173

well represented by species of Anabena and Oscillatoria, the narrow limnetic
species of Zyngbya, species of Celospherium, Gomphospherium, and Aphano-
thece; and Chroococcus limneticus is abundant. Three species of Dinobryon are
fairly common, but we have no evidence to show whether they ever become
dominant or not.

On the whole, the phytoplankton is a combination of that which occurs in
shallow lakes with that of large pools and ponds. Out of a total of
128 species observed in it, 171 per cent. were Desmidiacezx, 25°7 per cent.
other Chlorophycez, 32 per cent. Bacillariez, and 16-4 per cent. Myxophycez.

VI. THe WetsH LAKE-AREA.

Plankton collections were made from 19 of the Welsh lakes, mostly those
of Carnarvonshire, during June, 1905, August and September, 1906, and
April, 1908.*

The phytoplankton of the spring and summer is essentially Chloro-
phyceous, and as in the case of the Scottish lakes, is especially noteworthy
for the abundance of its Desmids. There is little bulk even in the summer
plankton, and in general it has practically no effect on the colour of the
water. The Desmid-flora in certain of these lakes is equal to that found in the
richest lakes of the north-west of Scotland, and in one case—the Capel Curig
Lakes—is superior to that known from any other lake in the world which has
been biologically investigated.

Of a total of 162+species which we have observed in the phytoplankton
of the Welsh lakes no fewer than 101 are species of Desmids.

The Protococcoideze are relatively few, both in number of species and
individuals, Ankistrodesmus falcatus and Botryococcus Braunii being the most
frequent.

Diatoms are not conspicuous, and there are fewer species in the Welsh
plankton than in either the Scottish or Irish lakes.

Apart from the spasmodic occurrence in fair quantity of one or two species
of Anabena, and the moderate abundance of Oscillatoria Agardhii, the blue-
green element is distinctly scarce in the Welsh plankton.

Species of Dinobryon and various Peridiniew occur abundantly in the
Welsh lakes. Cerativm cornutum is more abundant than in any other part
of the British Islands.

There is no great development of peat in this Welsh lake-area, and the
water of the lakes is for the most part clear and limpid. The drainage is
mostly down steep mountain sides, with occasional bogs and boggy pools.

* The details of these collections are being published separately.

174 Mr. W. West and Dr. G. S. West. (Oct. 80;

The Capel Curig lakes merit special mention. The summer plankton is
almost a pure Desmid-plankton, and consists largely of those rare and
handsome species which are almost exclusively confined to the west-coast
districts of the British Islands. The following species occur in great
abundance :—MWicrasterias radiata, Stawrastrum anatinum, St. aversum,
St. Aretiscon, St. Cerastes, St. longispinum, and a stout variety of St. Ophiwra.
It is interesting to note that S¢. anatinwm is present in prodigious abundance,
as it was from the littoral region of this lake, near the outlet, that it was
originally described. Micrasterias radiata exists in myriads in the plankton
of this lake, occurring in a profusion unknown in any other of its British
localities.

Mixed with the Desmids are numbers of Ceratiwm cornutwm, and a very
few individuals of Tabellaria fenestrata and T. flocculosa.

Of a total of 162 species observed in the Welsh phytoplankton, 62:4 per
cent. were Desmidiaces, 11:1 per cent. Bacillariee, and 74 per cent.
Myxophycee.

VII. Tot ENGLISH LAKE-AREFA.

Plankton collections were made from 18 of the English lakes in June,
1903, and September, 1906; and since then periodic collections have been
commenced in Windermere, Ennerdale Water, and Wastwater.*

As in the Welsh lakes, the phytoplankton of the spring and summer is
essentially Chlorophyceous, and contains numerous Desmids, but although
most of the typical British plankton-Desmids occur, they are not represented
by so many species as in the Scottish or Welsh lakes. The most frequent
are the spiny species of Stawrastrum, St. lunatum, var. planctonicum,
St. Arctiscon, Xanthidium antilopeum, Cosmarium subtumidum, var. Klebsi,
and Spondylosiwm pulchrwm, var. planum. The presence of Staurastrum
Ophiura in the plankton of Easdale Tarn is particularly interesting, as this
Desmid is not known to occur in any of the bogs of the English lake-area.

The Protococcoides are somewhat scarce, Glaocystis gigas and Spherocystis
Schrocteri being the only generally distributed species, and these only in
small quantity.

The Bacillariese and Myxophycee are represented by relatively few species,
but in some of the lakes Diatoms are dominant constituents of the plankton.
Particularly is this the case in Ullswater, in which Asterionella formosa
was the dominant constituent of both the May and September plankton.

* As no work has previously been done at the plankton of the English lakes, only the
list of species is given for comparison with those of the other British lake-areas. The
details of the investigations are reserved for special publication.

1908. | The British Freshwater Phytoplankton. 175

Species of Dinobryon are common, especially in the early summer
plankton. In the May and June plankton of Crummock Water and
Derwent Water Dinobryon cylindricum, var. divergens, completely dominated
all other constituents.

Comparison of precisely similar plankton-samples from the various lakes
of the English Lake District shows clearly that the proximity of habitations
has a distinct effect on the relative bulk of the plankton. Those lakes which
are contaminated by the presence of numerous dwellings and villages along or
near the shores possess a relatively greater bulk of plankton than those free from
contamination. The explanation of this fact is most probably the increased
amount of nitrates in the water of the contaminated lakes.

Out of 188 species observed in the phytoplankton, 51 per cent. were
Desmidiacez, 21 per cent. were Bacillariez, and 9°5 per cent. Myxophycez.

VIII. Maryam Tarn, WEST YORKSHIRE.

This lake is the largest natural sheet of water in Yorkshire and covers an
area of 153 acres. It is situated on a limestone plateau at an altitude of
1250 feet, and there is an extensive peat-bog at its northern extremity.

The material was collected by boat on July 23, 1904.

Out of a total of 20 species observed in the phytoplankton nine were
Desmids. Spherocystis Schroeteri was very abundant and Volvoz aureus
rather common. Only one Diatom was observed, and four Blue-green Algz.
Ceratiuim hirundinella was very common.

The following is a complete list of the species observed :—JJougeotia sp.
(sterile), Gonatozygon monotenium, Cosmarium Botrytis and var. depressum,
C. depressum, Staurastrum Avicula, var. subarcuatum, St. brevispinum, St.
Sureigerum, St. Manfeldivi, St. paradoxum, St. teliferum, Volvox aureus,
Pediastrum Boryanum, Spherocystis Schroeteri, Surirella biseriata, Oscillatoria
Agardhii, Microcystis eruginosa, Merismopedia elegans, Chroococeus limneticus,
Ceratium hirundinella, and Peridinium sp.

IX. THe British RIVER-PLANKTON.

The first account of British river-plankton (potamoplankton) was the
comparison of that found in the Upper River Bann with that of Lough
Neagh,* and since then Fritsch} has published an account of the phyto-
plankton of the Rivers Thames, Trent, and Cam. We have also examined

* W. and G. S. West, in ‘ Rey. Irish Acad. Trans.,’ vol. 32, sect. B, part 1, 1902.

+ Fritsch, in ‘ Ann. Bot.,’ vol. 16, Sept., 1902 ; zbid., vol. 17, Sept., 1903 ; zbid., vol. 19,
Jan., 1905.

176 Mr. W. West and Dr. G. S. West. [Oct. 30,

the plankton of the Rivers Ouse, Avon, and Cam in England, and the River
Lochay in Scotland.

It would appear that some phytoplankton occurs all the year round in the
British rivers, due mostly to the absence of severe winters, and that in the —
winter months the living constituents are mostly Diatoms. In fact, Diatoms
dominate throughout the entire year, the most important genera being
Asterionella, Synedra, Melosira, Surirella, and Fragilaria. Fritsch* summarises
the phytoplankton of the Thames in the course of a year as follows :—

Mixed Plankton (with Asterionella-phase) — Melosira — Synedra — Mixed
Plankton.

We find Melosira varians conspicuous in the spring-plankton of British
rivers and persisting in quantity through the summer until the late autumn.
Synedra Acus is one of the dominant summer species in the British rivers,
although it is a spring or an autumn form in the Oder, the Danube, and the
Illinois River. Swurirella biseriata and S. robusta, var. splendida, are often
conspicuous in the summer-plankton, the former being the more abundant,
which is never the case in the lake-plankton.

Certain Protococcoideze occur in the late summer, but never in great
quantity. Species of Pediastrum, Scenedesmus, and a few other genera are
the most frequent, and occasionally odd specimens of a Cosmarzwm or
a Clostervwm may occur.

Flagellates and some of the Volvocacez occur mostly in the spring months
and generally attain their greatest abundance before the maximum summer
temperatures. Hndorina elegans is most irregular in its occurrencé, and is
often found in quantity in midsummer or even in early autumn. We have
never found the Volvocacee so numerous in river-plankton as they often
become in the plankton of lakes and pools. Pandorina morum is the most
frequent both in the rivers and small pools.

The backwaters of the rivers are largely the breeding-places of the
plankton-organisms, and as pointed out by Kofoid, their contributory
function to the plankton of the river is at its maximum during the decline of
the floods. It is during such times that vast accumulations of plankton-units
are carried into the main stream.

Melosira varians, Fragilaria capucina, and Cyclotella Kiutzngiana are
perennial plankton-organisins in the rivers we have examined.

X. GENERAL COMPARISON OF BRITISH LAKE-AREAS.
In summarising our present knowledge of the phytoplankton of the
British lakes, it has been one of our first duties to obtain a record of the
* Fritsch, oc. cit., 1903, p. 637.

1908.]

species which occur in the different areas.

The British Freshwater Phytoplankton.

U7

As these records have never

previously been correlated, we have drawn up the following tabulated list of

all the species observed in the phytoplankton of the British Islands.

In this list those Algz, which, so far as is known, are exclusively confined
to the plankton are marked “P”; those which are exclusively plankton-
varieties of species which frequently occur in other situations are marked

“Py”; and those species which are more abundant in the plankton

elsewhere are marked “ p.”

than

| | o = ! = | a
| BIC ca a ene
| Species. SB) UF lee |e ie
| an Omere Peis
|
CHLOROPHYCER. |
@dogonium spp: (stertle)) 2...<.-2 2 -cu-eerrs-oeeeeeseececese sees geile ce pil Xa x
| punctato-striatum, De Bary .............00..005 x |
Ulothrix zonata (Web. and Mohr), Gita bon kddesaaporBhcaees x | — — »— | x
| ib SLOG UES RGU Zest rout ctor coiracrar ers stsaeie neers ese aes eaTE oe —| — — —_—
tH » var. variabilis (Kiitz.), Kirchn. ......... x | x Xai ox
33 TOMI GOTPATOUS, VR. ccocdseccncdebeoconcoceneneecseacec x x
| Geminella interrupta, Turp......-....0000-00- 0000-2 dessenneeees x == J|> x
| Myxonema subsecundum (Kiitz.), Hazen. ...............02.05- (=|) = | x
3 aioe, (Na, Weloferalli, opbccencoceacaerme cee cccioca cer |
| Microspora amena (Kiitz.), Lagerh. ............00....0cc0ee ee ite — | x Sa |) 3
| a -,, var. irregularis, W. and G. S. West Saari tet (fo
5 AYHALOC TI, WEAN SIT. o- cocassceonncbececedconeeucoDace ;=| = | = x x
| WWongconie TiS (Cietstle)) | eeccénacaesocoseecsconcsecduceoarcosececee| i roa aller x x | x
| A ClegomiUla NNLGLEY | Seccxensels- ioe oasiesaseae cece rise: xe , — ae
IpZa gemma spp (sterile); ccs aeadenedatecceenecticeoecitsies ces souecs exe ell ae x | x
"6 ericetorume (Kurtz: )jeblanns peer cee eee ee bye | |
SVOGKOULIFE Bia. (SETHI) -ceconsodendeoeceon’ eaneconcoeeeerocaoUPAAce Wexies| e—e Ns ox Se |i
| Debarya glyptospermu (De Bary), Wittr. .................+-2- | — x
| Gonatozygon monotenium, De Bary eax x x Si)
| 95 var. wloccllone, Nordst. | = x x | x
i Brevissonii, Der Banya cisoaeccsnk senan ceca —} — — x
| 3 Kinahani (Arch. Veokalbenth® qc qaccceescete sce hx x x x) |
| deuleatumm, Wastin gs se...c.-cceeecsssecseneecre-r lex
| Genicularia elegans, W.and G. S. West (G29) ieee bontieneeoae | x
| Spirotenia condensata, BréD. ...........1.0000e eee ee ceenectons ;}—| — — x
| Mesoteniwm macrocoecwm (Kiitz.), Roy and Biss............. x =| ss
| Cylindrocystis diplospora, Vrond.......-...2.000.00-0.000000-0+-2:| X
| var. major, WeSt .........2-.-.----| | x
Netrium Digitus (Ehrenb. ), Etzigs. and Rothe ............... | x i p< Ne
Peniwm Libellula (Rocke), Nordst. ..............----eereneeeeees x Say eres |
5 5 var. interruptum, W.and G. S. West...; — | — — x |
Se meunutim’ (alts); (Clever. ncsereis ne -oc-se-eseresssee-6 Kee ea lle =
| » margaritaceum (Khrenb.), Bréb., var. LEnOMELATG | —| x |
W. and G. S. West | |
| Bal SEU COLUM BECO Srna ctea- ane daca eee secain onieae «eee see eee [Se ee)
N Clostenvum @oruptunt, WIESU vor dsccecscasscccecssccuceoorsressscsss satlhy eg |
S QCULUMRSNOD: feet Neate mer cr eneebios nesecaeeaeeeneteeen| x} — | — | — Ny
5 aciculare, T. West, var. subpronum, W.and| x | — x —|—
| G. 8. West (p) |
7" acerosum (Schrank), Hhrenb. ..................02. — x |
5 53 var. minus, Wamtzsch .....2-++-.---9-5- x _ x |

Lough Neagh (and

178 Mr. W. West and Dr. G. S. West. [Oct. 30,

' | ize}
| | | las
a | e | a =
a) 23% 5) 2
Species. S| og |.60 | So Seo
a)/ ee) Bel|e)a| a3
“a oo Ss oa 2 ao ~Q
= | Ss eee, | cay | SPS
8 | Re Sieh NS | er | Ba
m | O = a
Closterium Ceratium, Perty =| = — |—|— x
5 Cornu, Khrenb. — x
5 Cynthia, De Not — x
- ma var. curvatissimum, W. and G. 8.} x
West |
6 Cecorwm SBLEDA \.cuasivsiastoes cectienaeeee areeee ene =| = x |
* Diane wWhrenb son: sac seesawncncee cea veneer eaee ee x — — |—|x
a JURADO WISDEN saoodocsoasascoconocsoobnDsoad x
- TNCUNDUTIU NESTED spr ssleiseretracphicslacteeseetees ons ee eeee ee = x
7 HENNENIN CRANLS se jscreyesaiecternarsene seis ates Mower ea eReeeG x
a GOTO GUUO Ty INENES “Ss oopscedon500q050008ede6" 598000005008 =| = — x |
Bs GANG BLED nastenneeeeeceerseceec ere roere eee x = x =| *
Fa x var. onychosporum, W. and G. 8.| x = x
West (Pv)
* Wyevo Lerni,, ROUbZ. 5.cnclalsaec cetera cele seeoeseen nares —| x x
5 USPPHOTO, IBITERAO0), oasosonsonca HosososoqonnoqcKe.nOne: x |
Topypetley, (WIGHIL,)), INWAEOID, cosaaccooe8000 osanann00000 = x
a MACIENTUM EBLE DaeReeReE eee cere rere ener x x — x
i moniliferum (Bory), Ehrenb. .............2....05. x
a GOCIFOMUOTTOS INN, ShocspesssoonpsenansdeonbesesoodoEgane x == x =| =
* PHONUM BLED. ccicasacccgaucese ves uescantahaneesecaeece x = x
8 TESCO OGIIEHIED, INOS} os0ac06000%G000000090000900000000006 x = x
Ps POSARGLLOy ABIONYEOIO), 590 05000000 900000 G0Eheno0D000008 x
- SOMA TO» IDNR) soasacooneanaqsdacpeacod snoodea0ne> x = — | x
ps - var. elongatum, W. and G.S. West | x = = |=—|/| = x
(Py)
5 POM, WANA, gacoosasogoossacceaacoonoceans0see x
COLOR: Westie sean saccceconaane saeusectenes eset essence x
. HUET, ATOMS, y50nongoc00asnd0040040nd 04000000009 =|) = — |—|]|x
¥ turgidum, Khrenb., forma glabra, Gutw. ...... x
ay UWilna WMOcke vcs-musucee sete tranetaccce ceca x
VAT OUTS RIVA, os origond bop bdcdeG Lodo odBogonqoonToadedaudeaG x
Docidium Baculum, BBE Ds artedeeiity seri cise eel ee erebeucoialach —| — — x
Plewrotenium coronatum, Bréb. .........0.c.eececceneee ee venes x = — x
* var. fluctuatum, West ............ x
a Ehr enbergii (GReghes)), IDYe TRE Fococcocconsonconse x — x x x
nodosum (Bail.), Lund. ..........0..00.0eseeee —| — — x
Tetmemorus Brébissonii (iene chs) MiRallishe.eeeereeeearre x
3 granulatus (Bréb.); Rallis) 7. ..0.c00.s-+.eesece ee x x x || 32
UezebS (EGA) 5 TR AMWESo55690063000000008500 obnaa9000008 = x
Euastrum CPUDG) WEIGES 00 copao05c0nD6 nope HES s09 909200 009E6n009000 =| — — x
- GmpullaceumpRealisience- ner eeesecheseeeteeeercecece ee x
Bs CHISCATADy SRE IS 90006 200 cog obo 9obbon CDOREO NOD SOB AHODOUNEE x x x x x
A SOONG TD, INEVE5 ooaccn soadbobocons ban GnosooconSsoDDNDeS x x x =< x
3 binale (Turp.), Hhrenb. ...........,....::0eeeee eens x
0 crassum (Bréb)), Kaitz. . 0. oecencsccscseeoeensesce sees x — — x
s denticulatum (Kirchn.), Gay.............00.2002000. x x x
ap LDyeratger (AUbEF0),))y IAIBES poscosootesasco0005000804@0N00 —| — — x
elegans) ((Bréby)) :Keubz cen eereeeeemeaeee sees sneer cts x x x on es
A gemmatum,sRalisn nen areccce cece easceecres x x
3 montanum, W.and G. S. West.........-2.-1.2ceeee —}; — = |—] *
5 oblongum (Grev.), Ralis.......5.......0..0000-.0000- x x
es POEEAO IER Dos 189) )y509050909000000000800008 000085 200000008 —}| — = |=] *
43 ‘i var. tnevolutum, W.andG.S. West| x x
53 pinnatum, Rallis ice aseeeencpen ne ceetocceeree crete x
si SUMOSTH7Dy WENN, “SoanasecodescodsovccoosonuaDoaz0N G8 = x
f DARRCOTHL Oy ADVE), Sxanononaoooonoosas00c00qG9000000 x

1908.]

The British Freshwater Phytoplankton.

179

| oo
mies (3
| a aS) | = | j a | a
2/28)" | 2)4) 2.
Species. a ae | oe a | | 7 eo
a| 63 | 28/2 /2| 28 |
= | 2p Sis | eo] sa |
S ra ) Sle me =| ° =|
n|O = a
Euastrum verrucosum, var. reductum, Nordst. (p) ......... x x x x xia
i Pp var. planctonicum, W. and G. 8.| x |
West (Pv) |
Micrasterias Americana, Ehrenb. ...........::1cceseece eens neces x | |
me apiculata, Menegh., var. fimbriata, Ralfs ...| x | | |
pe. rs var. brachyptera (Lund.), Nordst.| x
Fs COIR WILEAG|, \oascdonposoqe000000006-17000000000 000 x
#3 ETUC DUGTG, VBIREN95 sco oadvadaodas0990990000000000000 x x i Ih Sele
3 AUPE Py UEAIEES o00600900605660000000309009200 900000000 x |
4 Mahabuleshwarensis, Hobson, var. Wallichii| x x — |—| x
(Grun.), W. and G. 8. West | |
if Murrayi, W. and G. 8. West (P).............. x
4 <5 var. ena W.andG.8. West} x
(P
- FROGDUMOGPARE, VIN, econo o3¢90000000000008 020560 x sel | ose || x
hs 4 var. glabra, Nordst. ............... —}; — | = x
a pinnatifida (Kiitz.), Ralfs............c:ecsseeeeee x — | — 3.
ie radiata, Hass. [= WM. furcata, Ralfs] (p) ...| x — x st se |
. POSH, (EHNIo)) IRAVHES, Gog on500c05ab0ac009%0000 005006 x — — x |
5) Sol (Ehrenb.), Kiitz. | = W. radiosa, Ralfs|(p)| x x x 2 be
‘, tmumcata (Corda) Bred. eeresseeteeeee- sense: x — x 3 ll x
Cosmariwm abbreviatum, Racib. .........sccceeceneee een eeeeeenes x x
3 3 var. planctonicum, W. and G.8.| x = x il x ||
West (Pv) |
i angulosum, Bréb., var. concinnum (Rabenh.), | — x | |
W. and G. S. West |
i (OCH, BIS), coo oc sco bnaccoconsnbocKS00HbecoODds x x eee ee |
ie eB lyctit, NV alle bee eaten saersecannbresciseicnacehiceielesce x — = |—| x |
is EBOccHeieMNVAlll ome ecco umrenisa-eecmes i ataceananeseeai — x
a DEO GRS; MISC BN, — souncngoodooddaconcoaodsoe0b00000 x x x x Sciex
r 5 var. tumidwm, Wolle .............0.+5- x
= » var. depressum, W.and G. 8. West| x | — | x |
(Pv) |
4 DAWSON MIGBEE!M, — cooenade0es9000 ce0v00D00q0000C x _— x
; capitulum, Roy and Biss., var. grenlandicum, | x — x = || x
Borges. (p)
- Cxlatumpaliisir. actin essnaise st nar aieonesaieeiecn x |
=) COMMA Gs, IITA,” \copodeooesonooanceobsoonnoodéHbedode x — | x = || = |
Ms COMPAL Dey UAKOGO, omonanveccocuceuoveccosocecot0C00 x — | x x
ie 5 var. ellipsoideum (Hlfv.), W.and} x ane aes x | x |
G.S. West |
43 COMM OVENSUIMAINVESE Matieeenee eects x —- |} — |—]| x
BS Corribense, W. and G.S. West (P) ............ —| — x x
* COST, ISIOKGIS Gs So0n0000080000080000000000000009000 =| = | = x x
= depressum (Nig.), Lund. [= ©. Scenedessmus, | x x x x | x
Delp.] (p)
fe depressum, var. achondrum (Boldt), W.and| x _ — |—|} x
G. S. West (p) |
* ChypCGale, TUMOKET Soocoovousvovncedeododedoodedoo00ed x
3 op var. subleve, Liutkem)............-.....++- x eae Ml oe —|x
+5 OU EM Dy INO 6,» igoboagoondnsa0954c6d ads gboGde peu sbGcoD —-;| — | — x
4 EPUORM iis, WEVOVEE, coovns avodnoqvosqaoa D0 a8e000300000 x eo ee eH
F GHOST, IBM, 565 00c059000 900809904500 26000000N900900 — | = — x
OPED, WEI soceeqooccopss0nen004ab00b4000090000 x — x
3 By var. subgranatum, Nordst............. — x — fi] — x
3 Fenvibe Cray. caatectetersec ace cee eer EEE ee sone x x x 54. i] x
Fr UR ESSOAMLTO:, VBNINZ, cecd0n0000 39000 4000uKa90093CR0G00 x

180 Mr. W. West and Dr. G. S. West. [Oct. 30,

| | ic
Rae a
€|e2\%. | sian
Species. = Pata) eee | ic as
4/82/88 |) | ae
2| 83 | °¢ )2 eae
8 | 22 | > | Soe
ao | 2 \ eae
° |
Cosmarium Kjellmanni, Wille, var. grande, Wille ......... es
= Lundellii, Delp., var. xthiopicum, W. and| x | ‘
G. S. West
Fs lzve, Rabenh., var. septentrionale, Wille ...... x
; 5, torma octangularis (Wille) nob. ......... x x |
- LO GLENSE: BRISSEUD) yr) oake nel cose sone seetee seer eee f= |= — |—!| x
is margaritatum, Roy and Bissett ..............0++ x | Se -= x
a margaritiferum (Turp.), Menegh. ............... x x x x | x
s MeneqgHimit Bred. cacsessteaeseceety seerieeseadtce se sale OX
3 montliforme (Turp.), Ralfs ..............0..022000- a x x
e Or NAW RALEB .22;.eiedsicsenseisaseesisicis anissibeaeoeees <a x Hex
is or bhostichiune, AiunG ee) aac deesseseceeneoeeeeereeree {== = — x
fs OUALE> MALES: tsSucdsmeaabececenen ttoneeaee Geeta || xi) == ee
A Phaseolus. reba! letua sia seaaeeiesielace slsevaceseesore == || x
i pseudopyramidatum, Lind. .......0006.00eeeeeee ees =|) =) x
5 punctulatum, BED. .s.c.s.ssnesccrssoorcasneecotee: Smee AI —| x
a 3 var. subpunctulatum (Nordst.),|— | x | x
Borges. | | |
a pyramidatumm, BYED.....c s+... e2sronneserecsrscoess Ve—ahy Beas a x
* quadratum, Ralfs, forma Willeit, W. and}; — |} — | — |—| x
G.S. West |
a JAP Siiy IBXGAD, 354 cop o2g0s00p0nGEMEopODIBADNDINSI0NN0 x | — — x
ss reni forme (Balis); Arches, Mi cesrcces se saseneepe ate x <i ee
‘ SI TEMOS TE, WTEC, cecnsnaconpsposoq090000900000959008 ee
f subawersuim, BOLQC ..-seeeeesescecdeneeenteeeecesees <a |
a subarctowm (Lagerh.), TRENCH 95 agHadeo>poonbo0edase al tll one = x
i = forma punctata, W. and G. S.|—| x |
West | | | |
‘ SUDCOSEGLUMU NOTOSU.A- sen paresecse ates sissies eines =r | x x | |
53 suberenatum, Hantzsch. ......2..0.:.s0sensesen0 ee ey SR ee ee i xe
s subcontractum, W. and G. 8. West (P).........) x | |
Pe subprotumidum, Nordst. ........00.s.ceseneeneee ees | — x
e. subtumidum, Nordst., var. Klebsii (Gutw:)| x | x | x xi |x x
W. and G. 8. West (p) |
a SUbUNdULaLUmM NVA cn eeenmeesnanse cece beaci-e te ee x
ss subspectosum, NOvdst. ......ccsscesseccrereeeneneees |=) *
5 IT PHU VES, (GD) a5260q0070s0c000s9000RIG9000—5000 = Pes x —| x x
tetraophthalmum Uiciitz), Mieneghs (oo. o.creee. San x
Cosmocladium saconicum, De Bary ...seecccseeceeeeeeeeeen ees —|— x x
Xanthidium antilopeum (Bréb. 1 eRGta Sze SUNG ok Acetate at > x ~ || ss x
3 ‘ var. depauperatum, W.and G.S.| x x x x || 32
West (Py)
: = var. Hebridarum, W.and G.S.| x -— | x 4]
West (Pv) |
is 5 Varaleeven Schimidletceneres nso seece x | |
# eS var. polymazum, Nordst. ......... \Sacet |
. var. triquetrum, Lund. (p) ...... a = || x
5 armatum (Greb))> Rabenhteeceecercceeanceecees Be Sod me i ox
is 33 var. cervicorne, W.and G.S. West x | — | — x
5 ed PTAA ADP BRl}y, | ago odnbacnoe sno ac anonoa9 bod abAangCeD x > fit
Z Var umevnatum, (TED seen sane: | | x
3 fasciculatum, Whrenbs trbnssscsacaean sce sose sh etek
‘ subhastiferum, West (P)\ -.....022s0- ese sceee eens] x sarah 2s
A Were Mean Nis eRal (Em Shy) 23) = |
West (Pv) |
Bt controversum, W. and G. 8S. West, var. x |
planctonicum, W. and G. 8. West (Py) | |

1908. ]

The British Freshwater Phytoplankton.

181

| | | 3
(haga eta eet er
| ey
; Role ee Es ae eo
Species. pes Als | al |S iS ce
eles | Se | |S 2a
Bee | oe |e leis
e|fa/ 4|3|@| 24
an/O |— |F/a|A
Xanthidium tetracentrotum, Wolle, forma, W.and G. 8.| x |
West |
Arthrodesmus convergens, Ehrenb. ........c0cc0eeeeeceeseneen ee x — Ss sil se |
Fp crassus, W. and G. 8. West (P)...............| x = x = | 3 |
; Jipreins (BIE), IEEE, oonosonsocoocqoqoooence0ne9s80 x = x x | x
55 » var. longispinum, W. and G.S. West | x
(Py)
5 » var. Ralfsti, W.and G.S. West,forma! x = = x | x
a GHOEOFMES, WSLIN, ncncoccneocen7coucovotodoacee x = — x
- quiriferus, WV. and G.S. West (P) ......... x
53 SOMES, NOU: scodsasnoHboséheoceontepeceoonse x
35 triangulomis, Wagerh. 22. ....c--2-ee see see snes x x — x | x
a . var. subtriangularis (Borge), | x x x tars
W.andG.S. West (Pv) |
Staurastrum aculeatum (Ehrenb.), Menegh. .................. =| == — x
cs affine, W. and G. S. West.(B) ..........s.0c00-« Vr lhy t3<
as MEGAOHOS,, GAO, coc oacosesoovoonsanooesonapcopeencee|| == x |
5 anatinum, Cooke and Wills (p)_ ............... x x x Se x
a 5 var. grande, W.and G.S. West(p)) x = — x |
Pe i He Lagerheimii (Schmidle), nob. x |
es “s we) longibrachiatum, W. and| x
G. 8S. West (Pv)
sy fp var. pelagicum, W. and G. S.| x x
West (Pv)
3 var. truncatum, West (p) ......+-. So] == ee ee 8
5 angulatum, West, var. planctonicum, W.and| x
G. S. West (Pv)
is CP QUELITAT Op, LESLNS). aoacconsobunceoceacovesocceooncne |) a ce x = | = x
Rs Aira eh Mermvats a eactrieriec cians nose en daceeeC es x = x x
is 5 var. curvatum, W.andG.S. West | x
(Py)
5 Arctiscon (Khrenb.), Lund. (p).........-.....06+ x = x x | x
" anistiferums alts) Veccnemecccresceescecaesecereee =| = = x |
55 5} var. protuberans, W. and G.S.| x
West (Pv)
i COS AGU Oy 283210 Satan onpaceobond seounos cooacuobaEe: | x
BS FARICULOMD ED Ne mre ier ence eee CeCe eae ax |
5 55 var. subarcuatum (Wolle), West ...; x == x x
» GSTS, Wivtsl, ((9))sc0000000 bop anHeooAcoEbodoooAe LOS x = x x
a Bienianum, Tai Demy RPE RE AR RE Uae x == x
35 boreale, W. and G. 8. West (PB) ........0.0008 — x |
5 GROPCMIT, IRENE, sooonnosu Apovcooboubeescoonontes | x x x x x
i Brasiliense, Nordst., var. Lundellii, W. and | x — x Su Be
G.S. West (p) |
4 brevispinum, Bréb, (Pp) .csceececerecseese tener sees | x x x Seu CicTI 3c
3 es var. altum, W. and G. 8. West | hex — x :
(Pv) |
rf 55 var. obversum, W. and G. 8. | ‘x
West (Pv) |
Fi Cerastestilinim deepen mer tony ace ennine scieiie a aacents | — _- — x
a Clevei (Wittr.), Roy and Bissett ..... rsietalastie ex |
By conspicuum, W. and G.S. West (P) ......... | x
5 GUC By, NICS (G9) .s00c0s0godcqo0 990000 sboseocdquer | x x x Xone xy
- cuspidatum, TB Te iy avaionast weer celaertatei sae comahoeel —} — — |—|—!| x
i es yar. maximum, W. and G. 8.!| x x x eel e
West (p) |
VOL. LXXXI.—B. O

182 Mr. W. West and Dr. G. 8. West. [Oct. 30,

| 73)
| : a
| See Sen iz . | Ries
| : “| ag $\/M@]a.
Species. = | gd | eee
| 8S |22) = ee
2 | 23 | °3s |o alee
Sl 8m] -4] 3 | ®] 54
a|/O |— 1) Be
Staurastrum cuspidatum, var. divergens, Nordst. ......... pe tee
FS COMOEEFUD ny, VARI, osdercoqonas sanchtensiine iscoon aoa x
0 5 var. compactum, W. and G. §.| — a
West (Pv)
i SAGA Dy VES, (9) -cckobdqosne sot1a9p sscdeuadeaaueda) es x x fee x
a var. inflatum, West (Pp).......--..... x x x | == x
%) denticulatum (Gaga) Anche (po) eeeee eee —!| — x ea Ss
As Dickiet. Ralis s5..eh. ese eee ee x — x
Be dilatatum, Ehrenb., var. obtusilobum, De Not. x x x
4 dorsidentifer um, W. and G. S. West (P) .. —] — x
- ELASUIM, SBE (Pp), cca seeeiaeea cena aie x x — x x
forficulatum, THIGIE) Searecossanashaccusogboodaepec x |
eS Surcatum (Ehrenb.), Bréb. .............2. 2.50. x — x
% LPG Ey LIRSOF (19) See 360 e000) anc 00400 00 8o060 x | — x | eH
5 * forma eustephana (Khrenb.)....... — | — —_ <i US
~ 5 forma armigera (Bréb.)............ x |
i 3 var. reductum, W. and G. S.| — | — x
West (Pv)
3 gracile, Ralis 24th... cess eases hoe i]: iy xy) “|e a
i i van. neanwm,; Wallews: caters ne pexvel i=l} = Nee
6 » var.cyathiforme,W.and G.S. West,, x | — | x | |
forma
s grande bulma) specced ae eee eee Vie ea
5 GREPEVOSUTOy, ESUGES, Sogcc0 wwoooscosnonsedooaoencc. 224) —|;— x —|}— x
A, 3 yar. acutum (Bréb.), W. and} x | |
G. 8S. West | | | |
55 hexacerwm (Ehrenb.), Wittr. ...........:....5. — ee rh ual ie oe
| ss hirsucun; Breen matasnrtckin ee aes aioe ane | |
| a OREM Ry WIKI), sohodansonopecose0 OooSanGaooer eHudsy }— | — | — | = |) |
- inelegans, W. and G. S. West (P) ............ x | |
7 irregulare, W.and G. S. West.. Bs | x |
" jaculiferum, West (biradiate vertical view), Xe tore == 4 ye ||
43 (@eadiate vertical view) (p)—:.| x } «x | «x |= |x |
5 yar. excavatum, W. and G.S.| x = axa
West (Py) |
3 ay var. subexcavatum, W.andG.8.| x |
West (Pv) | |
ne (EAS OU PU Dy Wy pao nde sodactes co0050e5RAded— bc x | | |
‘ longispinum (Bail.), Arch. (Pp) ............00604. x = xieeilenae,-|) 3eanl
3 3 var. bidentatum (Wittr.), W. | x — prey ens. |
and G. S. West (p) | | |
Ay lunatum, Ralfs, var. planctonicum, W. and | x MMe ee eas. |||
G. S. West (Pv) | | |
‘ Maamense: PAv cheese ee eee er ee Conese ee — | — x |
ss Manfeldtiv, Delp. (p).-ceee sss eeeeeeeseeeeene- [=| = x |—]| x |
% megacanthum, Lund. (p) .2...-.60.2. 66 .60ees es iii a x xen
2p 5) var. Scoticum, W. and G.8.' x |
| West (Py) | |
3 monticulosum, Bréb., var. bifarium, Nordst. x |
3 mucronatum, Ralfs, var. subtriangulare, WV.| x —-),— eke
and G. 8. West (Pv) |
in PEEAETI Ty ABKHEIS | oonmg caddae das dacedsond ss aBAaeudes —| — Tht la eas
x Ophiurna and ((p) ee eereecee pete eee eee ee x — = |= | &
ie 5 yar. cambricum, W.and G.S. West — | — ae |e
A onbiculanemRalisie <eeeeeeen eee pecan eee x |
3 ne var. depresswm, Roy and Bissett | — <P

ean =

1908. |]

The British Freshwater Phytoplankton.

ee
co
(oe)

| ne}
le é
re | a ee a : m | ct
B)8e/% |2| 2).
Species. 7s giloetila|s ae
Si) Be | ga isle las
ae © o So p=} fs oR
S| 44 Se ey | See
o | 4m | | ae |) el
aor is WE Ves
Staurastrum paradoxwm, Meyen.................1:1eseseseeee ess] x Xi ex | x
A is yar. cingulum, W. and G. 8.| x x x x x
West (Pv)
var. longipes, Nordst. (p)......... x x x x | x x
i pelagicum, W. and G.S. West (P) ............ = x x = x
ee (RULOS WIN SHINA amen es srarvcbie, teeyscins etna scien ccs: \ See x
35 [DONMROFD HI, VRS d6ecencsen0s500000 000000000000 | x = Si I 88S
5 polytrichum, ae Boa slrar neue Sole eS EERT CE | x
Bs pseudopelagicum, W. and G. 8. West (P) ...| x x x x | Xx
~ [OUCTOMEIT Dy, VBIXNO:, <00 do0906000 008 000 ot0GeH0ONEDO OOF x x == | 4 88
Be SNCRODUCHTDy VHHLD, soogccocooshescnonacdgsbavosen ode aa x
a Sebaldi, Reinsch., var. productum, W. and | x == x
G. S. West (Pv).
| 5 var. ornatwm, Nordst.............-..--- (= |] = x | = x
- secangulare (Buln.), Rabenh. (P).--sseeeeeees. x = x x | x
. ; var. supernumerarium, W.and| x
G. S. West (Pv)
os SUT UCIT, IBOTHRE, .cacgeuegnescos soo endscoadusoondoeee x
I sublevispinum, W. and G. S. West ............ x
me subaudibrachiatum, W. and G. S. West (P) | x
| * subgracillimum, W. and G. 8. West.......---. x |
a SUUPY GIMEEUM NVIESb. crease na se eee ee suisec cee | x a Iie
H es Leliher um WALES) wa ase weeapesieesehs eh eee ate | x ST x x
& UCNKEGCFUM Oy ARENT .c00ogoc0G00080¢0 one nab eooPanoonbel| > 8 Xe a lea x
5 Tohopekaligense, Wolle, var. trifurcatum,| x == x
W. and G. S. West
- GUMUCUMEIB VG Ds) anc skesse cen COE ee oe AA eels | x = | = x
5 COROT Dy INO, ynasoononovsosos2.000cbo0ee coo x |
COSRATIZOG INENKES oa nonianh bad BooooDsdoRdoesaoseebiseL ee | x
Spondylosium pulchrum (Bail.), Arch., var. planum,| x = x = | 2%
Wolle (p)
| Spherozosma granulatum, Roy and Bissett .................. | x So it ea
53 Aubertianum, AWICEIED! Cantidad nononatacaacemtarae: tar =| = | x
* yar. Archerti (Gutw.), W. eal x — = <i ee |
G. 8. West (p) |
| * CROAT, UREN 5s spon voddgevooboseddnseosos0n0dones SS ae es
5 POTCIRULUT,, VANES ococopnscoeucod boooho obonnecdoee | x — | x x x |
| Desmidiwmn aptogonwm, Bred: ...6ic..c.cseccscdececcee suc eonsenes x == a a es |
| z coarctatum, Nordst., var. cambricum, West (p) | | x SS x |
| a graciliceps (Nordst.), JUBIEE NB ood boospeceacenandne x
| . occidentale, W. and G. 8. West ee) saauecunb0d8 x |
| ie Pseudostreptonema, W. and G.S. West (p).... — | — | x |
| ‘9 SODGTAEAIO I NERD ange baba ondoobe sesber DOaenO neces OO OS OE x = | = x ol
| Gymnozyga moniliformis, Wren wt anterascntaanaaeeecesscr cee ava| x — | x x | x
var. gracilescens, Nordst. ...... x
| Hyalotheca dissiliens (Shins) 1Bieeloy eanedacanecsedbensoneoadeaaan: | x = x x | x
| 53 3 forma tridentula, Nordst. ............ | x
a ina hxeas, “Mawiaay'(GD)) | soesondsonnecebavdoudoobane onob8e oes | x = x = || x
# TENCOSH,, VBAaeYEVAlDy, 5 45060 vovogd AeBudDHPgeE edd coHDEGOOOHO: x x x x x
i MAGMA, JREXCHID., (QD) onnopeaversonyaHeG0s6ann 9900000 x = x 83. || 8s
CUTIE, INOTGING, oc aces eyo non cbo00e doodoEb0K.oN8 06b0be (=|) = x x
Volvox CHRGIT Nah allo ssncanecorido vagocdaeh Ook Os moar ae UeOCLAOOE EG: =| = = x7 | — x
Pleodorina californica, Sha walccah ancy... nicrmictme octet x
LO Cie BEGGS, TOOT), a5. co0 coe .00.b0 cou conobo00d sou nHCbONodS x Stan hr ciae-¢ me || 8 x
Conniin, Aeon, WUBIN, searoocouconstasooecncvdceareoseenoob0bod x —- |) = x
Pandorina Morwm (Miill.), Bory. .........cccceeeeeveeeeeeseene x Xe]
Chlamydomonas pulvisculus, Ehrenb, .........:00.....s 0000000 —| — = |=} x

184 Mr. W. West and Dr. G. S. West. [ Oct. 30,

ie eat [Saal Zs
le. a Nee 5
: ie | 3 E s =
| | ).8.9 | <1 eye
Species. | = iene | st itis SA
| a=| 2 £ qd =I La) = A a
|'2 |) Be.| 6S | os | a ae
13 | 2a) o8 | 2 |e
acou ne Ele | 4
{
OLREEOCEUD GOSHG OSs LWIA, ononapaccesoqonceoonocee opaccacobooHDae SoD = x
Pediastrum Boryanum (Turp.), Menegh. ...........-......... x x x x | x
is a5 var, brevicorne, A. Br: .........---.-- — = So SS x
5 B var. granulatum, Ralfs............... — ee a x
5 4 var. longicorne, Reinsch. ............ — — a | x
5 dupler Mey enal-s-ceccasenscceeeetece eee tee eee x xe |) 2x —|— x
AC 5 VBS CAVDEOU Dy IN 1839) Sao seacossoousesocDee —{— | x —= | = x
5 var. clathratum, A. Br. (p) .........--. )—= |) = = | =—)|= x
< glandulifer 1m; Benn. |\>.cchea esuacr he aeaeececeeeeen | — x = |=) «
5 integrum, Nig. BoaREGHEBodososbaG Son vebabuigaodacdds Adc =| %
FF GU olay IMM Gilg, G5) Gaononecoooncoddasoqs e000 500086 x
Retras | (Hbrenb:)eRalis | acces. -en-se-eeceeeeeeees x — > | S| x
Sorastrum Americanum (Bohlin), Schmidle .........:........ x |
: SY DU OUKOSTUTIa IER, ocx mensceo asyecdoobacansesseos0e8hC50 907 | x
Celastrum cambricum, Arch. ((p)i2.-.c-:.-0eree---e seer ee ees | x — x )—] x
= MUCTOPOTUM Naan eee eerer ree cee ee eee ease cro cel —| — —_ x | — x
WOE, Nis coal (Er; Sh WEEE poonsacooscoussassbasnce x
a reticulatum (Dang.), Senn. (p) ...........0.e. 0.200 4 x —}|— x
35 SORES RICUT Bn NERS dhospesedacousdeesadpdes.an900700"300 306 x edie) m3
Crucigenia quadrata, Morren’ .....- 00s. .sccceceeeec eee eee neers =) = — |—}] — x
= rectangularis (Nag), Gaiy .....ec.cceceec eer eee =e | x — yj || x
forma irregulare (Wille)......... Bill) likes
ss Tetrapedia (Kirchn.), W. and G. 8S. West (P) | — |} — —— || — || — x
Scenedesmus acutiformis, Schréder, var. Brasiliensis| —| x |
(Bohlin), W. and G. 8. West |
a bijugatus (Turp:), Kaiba: <o.....seeneeeeeceeee x) a ex x
ss Bs forma arcuatus (Lemm.), W.and | — | — x
G. S. West (p) |
3 denticulatus, Lagerh., var. linearis, Hansg.| x | x
3 OUST BIS, URIRZAS sooosasaqonbaciyen seocd2qq0000290000 x | x
- JER PIB ALERTED, soo qopnnscaneranoosbe see 0500ceee" x | |
33 quadricauda (Turp.), Bréb. ...............2..05 Pex Bo | — x
95 _ var. abundans, Kirchn. ......... Bool Bo) x |= x
3 yar. horridus, ‘Kirchn. |... = x
Dimorphococcus UDLOOIBS Jhs UBIO pa soounauce on6 :onea0se00doKHsond x
Dictyocystis Hitchcockii, IWATE NS - SooqnanonsoonsagsoRO0D530n0 56 x
Hlakatothrix gelatinosa, Wille dodasaondagnaasocosashaobsasedoa609 —|; — = | —| &
Ankistrodesmus biplex (Reinsch.), G. 8. West ............... = = x
Pe faleatus (Corda), Ralls......................-5 eal eet ta lliaenes x | x x
5 3 var. acicularis (A. Br.),G.S.| x | x | x =) x x
West |
53 as var. mirabilis, G. 8. West... ....— | x Sparel are eae x
3 55 ene, yours (Guvme)) (Em Shi} || 5 = | = | = x
West (p)
ee Pa yar. spirilliformis, GS. West | x = = esnl| lh abs
Pfitzeri (Schroder), G. S. West (P) ...... x Soe EX ==) 23
Closteriopsis longissuma, We mms (Dp) Mesteseenere esse eee —-|) — x = | = x
3 53 yar. tropicum, W. and G. 8. | — xt) |
West |
Characium Debaryanum (Reinsch), De Toni.................. =| = x
Nelenastrum gracile; Weinsch. s....s2:s-sesescssese eee sees ae —| = Sj = | x
Kirchneriella lunaris (Kirchn.), M6b. ......-2.2....0.022+0- x
obesa, W. and G. 8. West (p) .........2.5...5.. x x x ceaeah (cama x
Oocystis apiculata, Weal ei otee, Bie Ananian mea mal eerith 4}, 28
Pe ee a7 Vengo) astoape na seborbanaepaenenGaacedéabeocuaneccda Higa
by REDS FAS, Cla ENG (1D) conatovosossneenendsdossonnsosvoeanr =) = aaiile 2
ho harssoni; Memmi GP) Meson heen reenact =) = x = | == x

1908.]

The British Freshwater Phytoplankton.

185

Species.
l
|
Oocystis parva, W. and G.S. West (p) .......:.ccecereee scenes
PES OULA TUG WAL Lie ain) mmeciac eae bibeaaklsriauiomeceeres
Nephrocytium Agardhianwm, N&g. .....0...ccceee vce eee tee eee ees
5 UR GTRT On, NIVGISS (QD). aocbouedoacedse son o00 080000000
Tetraédron minimum, Hamsg. .......06000 cece cece eee eee eee ees |
a cruciatum (Wolle), W. and G. 8S. West......... |
5 enorme (Ralfs), Hansg............0.c0cceceeceeeeeees |
5 Uimneticum, Borge (P) ..........cecenececes teense ees
regulare, Kiitz. rioce seb doneeeemoOsoes ano sErcoDaC ose
Dictyospherium pulchellum, Wood (p) .......c..ccrescceeceres |
Bhrenbergianum, Nag. ........cc0cececee scenes

Botryococeus Braunii, Kitz. {inclus. Ineffigiata neglecta] |

(p) |

3 protuberans, W. and G. 8S. West (P) .........

A SMAMENS, UAW, SootecosdoAcudosooooguoDdpuobooDE |

3 var. planctonicus, Lemm. .........

Syhzerocystis MARDI, (OCH (12) sro cnoasoocoa.oon5e6undnasa bon

Tetraspora lacustris, Wem. (P)) aces. ssnieavenensecnerncceene nes |

Gleocystis gigas (Kiitz.), IDRIS a, :soobanoaddobgodedddonananadood |
5 » var. planctonicum, W. and G.S. West

(Pr)

* infusionwm (Schrank), W. and G. 8. West

v BATIGIMOS Ty IEG, son tmnooscooecouds soandoacuds 800000900

Golenkinia paucispinosa, W. and G. 8. West (P) ......... |

Richteriella botryoides (Schmidle), Lemm., forma quad-
rata (Lemm.), Chodat (P)

HETEROKONTS.

Ophiocytium cochleare, A. Bri... ..ccccceccicee ete tee cee eee nee

S bieuspidatum, Wemm............0.c0c2c ssc sencneess

5 GSTAAD, \NOWG _snotcbsoadenbocdeoodgoapecocd0a0
Tribonema affine (Kiitz.), Gre Sip \A/CELA Neo nesernoaneedsdadacoe scan
Askenasyella conferta, W. and G. 8. West (P)............45.
Oodesmus Doederleinii, Schmidle (PB) ..........c0cceeeeeeeeeeues
Chlorobotrys regularis (West), Bohlin .............s0cceeee es

BaAcILLARIE®.

Melosina crenulata, KaitZ5 \eeeses ene. nenaiencmeseeeeeeetaenedes:
FP Bs var. tenuis (Kiitz.), Grun. ...............

x CRT DLLFU Ly NIG ONE) Sy doo aad oodsE booed ebuceaaeeRORoDOdG

5 granulata (Khrenb.), Ralfs (p) .......0.e seer
CUMAMSEALP I i ictev cba autnisentane scsjniiachi ns aaa seasiocse tor
Cyclotella COPGHG,, UV, (FD) poocooccseopodooapoodeseoneeoaq99009
hy a VEN, CUPUIS, (CRABB, poocannadcaqacupedodanooden

A Kittzingiana, CWauvin........0...recsscceereeseceseees
Meeneghiniand, Kite. 2.0.0... ccc ce ceecenceeeeecns ees

. OEHEM ete, UGA, | sg0eceooooos oun oK0uRdsdoobae0C00000

\, Stelbouciro,, Were, (0) soa coonedovgsudoscbnanbn60ncae
Coscinodiscus lacustris, Grun. (DP) .......cc0cc cee eee eve eeeeee ees
Stephanodiscus Astrea (Ehrenb.), Grun. (PB) ....-...c.600:
Riizosolenia eriensis, HW) 1. Sm. (B)) v2... cscceeessccrecesneeees
. engisBHeh, VEO, (LP) sopoaocopeeanedbogo.0sbvoonne:

o 5 var. stagnalis, Zach. ..........0...5+-+
morsa, W.and G. S. West (P) .........:0.0800es

Tetracyclus lacustris, Rallis) cass snessacaseuseareumas ce eee aaseipe

| ro
| A E
wn me} = | wm me
Be Ee cae ey
= a | od cal ot eS Pa
SU Sel Bel |e | cs ©
-— oo rt ro a) FA
SB | es F | a) | Ps
S 1 2 oi) g q 54
nm | O = 5 S| wa
x re —j}|}x x
x SS se fe fe x
x al ues } x | — 2
x — | x — |x
te x aes ay eee x
x |
Bag ete male
x
x
x — x x x x
28 as | |
x x | x lines x x
| |
| |
x | |
x | Va
x | |
x urea |e ae | x
BI pone g ee teee ee
x Sela x x x
fae; ein sy
x —_ x
x — <i |
way ea = |=) x
ibe pets x OR ates x
x | | |
x | |
x |
: |
x — | x — — x
UES aE i (est i
— — | x — reat x
mer
|
pais Seu x Bont ele 56
_— — — — | — x
— — x — | — x
x x x x x x
— — x |] eS x
x x x |—| x x
x x | x ex x x
ia | | xX
x | x |
x SS le ox |
ea See alien Nie
= = | SS | | x
| | x
x {
x — | ail
x | |
x — | x — | x
x se — | — x

186 Mr. W. West and Dr. G. S. West.

[Oct. 30,

| ze}
=I
: &
eile Sear | es | a |
2(/33/7 | 8) 8) 2.
Species. “ és & = g < a 2 =
‘| SS | SS | | = | oe
S|) aa | .5 | | =) se
jei6 |e eee
Tabellaria fenestrata (liyngb.), Kitz. ........0...cceeeeeee x tl ES oi 8
» var. asterionelloides, Grun. (Py) ...| x x x uf 8 x
s flocculosa (Rots) RGatzs sb aoe eeeeeneee eee x | es Daa
IDO MCE CADIS, UGTUPA, «ca coh son sononcdopseAdnoopeAsedeb4gacdDDd0n0 x =|, & — | — x
STROOD CORBET, 243, sap ¢0nonsc0008d02000000aNc0 son ceddadaoon4 =|) = — | — |] = x
ID WTR QUOMG MT, ENR s5098098019b00n000046a520050 29h sEdGSSonODEN x x x —| x x
5 Vee, CDOS (UNG) We LBL, saconesoaancser —| — — |—)/— x
| Fr agilaria Capucina, WesMaAznrennceseetee ime eer Renner | x x
| Ae COPSERUEDS, ERI, snoannsdooodsundes sno oooneacodoovade Mite tes — | — | — x
55 Crotonensis (A. M. Edw.), Kitton (p) ......... xa |P gx x =] * x
- A var. contorta, W. and G. West| x
(Py)
- Tae AHOS: (NAY terea)), (EGA, So, shsqsncdouer concn eas x | x x
LUDO HIRE (DAMM ETE GD, USF, sos oneaonnddegooosseosodonnennannans|| 2 | x x
Spyoaaine Alens (kati, Mave. so055n600-48000000s6 cod anpcooenaoc xx — il x x
ee op WE, CPGUSHISSOMIC, (ERA, 5 .cansnceassasa0q09 000 = | = x ‘
a » var. delicatissima (W.Sm.), V. H. (p) ...... | | x
Lemmermanni, W. and G. 8. West (P) ............ —)} — | x
Am TAC AAI Ts AGUAS aoe canoonHan cqocecnepranadonneSan add tee cds PSeilfe wiles slip 28 ==] x
oy RERIOTS (TACIZ,)), (ESAS 5o)-.csonaadoongodooagosoncosoanuc: |=) — x |x x
A JRADUMEDEIS, ILE, Goonnosoagemaocoos ono vGageabaNoADeCD (a x
m6 (Dihiey (SATO), IBV IMO), oocoustormacoo 300000 ssn cnsenN|| x = x ax x
y », var. longissima (W.Sm.), Grun. ............ | | x
a ar splendens (Keutz.),Guruns «ye eeeesederne =) = |) = | S| = x
| Aster HOLANIGD {OPPO IBINES, (GO) | sonagaconaosnonaareabeo9s000N000 Posse Hin eS x x | x x
5 gr acillima, Leib (p)teres Suaccsscoetias dane ooeeestd a es x Sax man |
| Ceratoneis Arcus (Ehrenb.), Keitzs) vary Amphiozys =< i ax
(Rabenh.), De Toni
Bunotia biceps: (Wi Sms), Ga Ss Wiest) fences ese eeeeee x SEIN acer eres |S
a IDGCHIATIG, IDNGWEND Ds snosadaogasocescadeus soasosdaccansnoans x
> gracilis (Ehrenb.), Rabenlis cenennnrenten een | x = Sy |
5 GET FOS (UBlaweESA})), (ESAWA, ace goo casas noe vesonacanasanaa|| x =a ill 23 Bo | 8S
. Taio (AW StiM,)),, IRENA, gag onscococonooasoatoans adouns | x = == || = | &
p maeranE( IS (UGTUZ,)), TRB) 5 3.cngo09 uo saocooocondoss | x = | & x | x
- i Vee, Duclos, CHAWA, — ssoassaadcaneanonnnse000 | x |
i 5 var. undulata, Ralfs......:........0...0000 | x |
= APR OP VBNGORBAVD, —h.~ sogoansonsoooshnnagsevcoonoonacds x |
Achnanthes coarctata, Bred. 00.0.2... .00.cc sce sen ese y ec see ese new ees = <a
a ERTS AULA Roce secctos OER ek enone On nace I) xi] |
" jewels, (URUVA))5 TERED, caasssagacndoeocoadeasa0 869080 | x mari esata esse || x
Staumnoneisianceps Mihten bigest ee eere ere eere rete eer ere | x |
“7 Phenicenteron, Whrenbs vre...csdess eee ccseceese sen |— x — —|x
Gomphonema acuminatum, Whrenb. .........66.020..c eee eee ees | x = = x
ss CONSiICLU SE hen byeeeee.eeeeeenece enero eee | = —| x
55 geminatum, NG) rw .ceaceeeean sank ou0 sso byoBBODAITT ex = x —= || x
5 OOUPUACEOT LO URDUZAS .pabosopb5ccnsde0575466c00K000s80 608 aa = x —— 1 x |
# var. Vibrio (Ehrenb.), V. H. — x |
9 olivaceum (Cay ANE!D,)), TRGB en050000n00680000980000 —— x = | =| & |
Cocconeis)Pediculus, Mhrenbay -an-se-ssee teres | x
AS Placentula, Mbrenbé ses gee hoa yaese Wate saenases | x — — —};— x
| Gyrosigma attenuatum (Kiitz.), Rabenh. .............00ee0 | eae x =) = x
Syeemecrae (Qwelis)), Os Bs sabnboncn 00 cab eos oocc00000 = | = — = | = x
Navicula alpina (Wi. Sm.), Ralis <2) ..0...cseceseeeceeceeeeeees x x
e IERAOUSSOM0 Ey UROM A sno 00G0000%0H bap soe vnannapHonnbon oo — x
oH divergens, Ralifis iso. cnasesesrenees sean | x |
Bin i UU DIS SV, Yo sctag ebeeeiia soadbbobocsdsinnon aiaadbe | | x
5 5 WER Zoom, Wo lls coacoodnobuase0cuanbe nee | x

1908. | The British Freshwater Phytoplankton. 187

hee |=
| : |
| a |g = a een ae
Pebsee ee sey] |
Species. = S| || yas) | el |) El eS
|e BE Re | a zie
ee ere, areolar enl sears. |
S\ aa | 4|s | | 24 |
oO (eI ee
|
I CRTUCULCONG UD DORM Zee ersten cee cane cla ote eee sca cle san eeae seh os % | SS i
a Tridis, Ehrenb., var. affinis (Ehrenb.), V.H. ... — | x - | |
5B. nz HULGNC Ry UTE Soccer coca eR een % i x ex
33 (raledles (WBleeesrelo)))., GHW, tecoo nad cdscodpeesds009 scdoee x.|
4 p. pvax. Dactylus) (Mhrenb,); Vi. Hi. °.....-..--. x ~
33 EUS ULUG INV Es SUE cota eel eiaehorte toa op eatiaeeis nice etapncrciyctoe — x
5 [RELISOSOS URIS. Semacenose don cao ppneeGeco oud Aenea cease ate: =) & x x | x
SOFOGTIS od CON ate Acoma sacbac oOo ORE REC OGG Cot GoD OOEEReore == | = |=) S|
Vanheurchia rhomboides, IB PED vegseisjnens satan oie tee sae apes | x | |
5 55 var. Saxonica, W. and G. 8.| x x x | x <a
West |
Cocconema cxspitosum (Kiitz.), GS. West .............2602. =| = x
5 cuspidatum (Kiitz.), G. S. West .................. x |
A Cistula, Khrenb., var. maculata, Kiitz. .......... x
5 COMIUGRUTPMED, UOMO coosaecboonocn ooncdonob oescoo nce A AL ok
P EBhrenbergii (Kiitz.), G. 8. West .................. x |
5 igastroides.(Ktutz.)s NODs merase ca sen see tneee- e en ee. x
33 GCE (GRENDEL), TOD, sap decseogsrbooreeogh saneceene =) = == x | x
a; lanceolate nrenb epee yee eeee eee eee x x x Sax
ventricosum (Ag.), MODS feiss eene eee held seeead = create | Noeaill| WE
Amphora OUGLUSRCLZ Ame ee emer pact iad Seselen ecu ieee asin <a x x == |] %
Epithemia turgida (@hrenbs) yRtitz sere eee eee ee | 3 x x = || 2 x
Rhopatodia gubba (Kaitz.),-O. Mull. «.. 2... eee eces ese scenes x x = =| — x
IN HESAUIGL, CACHOTIET FOS, \ Wig SEM ecosncopeceensapnashesan ene conheeune i x = |= x
| Be dissipata (Kiitz.), Grun., var. acuta V. H. ...... == x
| ay UGoGETFES (CN Mie S310 deocemnes scnbncupe selcobennseeeen x x x |
5 EP. al con (Ktutz3) a NVid SIME eee eee eee eee: x x tid | son he 3
. Siapiod (USHA) 5 Wo lS), coocnancocsecoronosuecodarcene (=|) = = |) 8 |e
- sigmoidea (Whrenb.); W.Sm. ......+..........---- lax Xiu | — x
Cymatopleura elliptica (Bréb.), W. Sm. ......0.0..0ee cece lala x x = | x
ie 5 var. Hibernica (W. Sm.) V. H. .... — | — — |— |) — x
ey 3 var. rhomboides, Grun...............- ;— — x
OLE EWI GSI ed jetscae es actecstadscatenactussscecs | x — x — x
Surirella biseriata, Bréb. (G0) tobe saab enchane toe eReAerRconcasere nes | & x Xe exe |) ox
5 linearis, W. Sm. Meraneeeehnse Taessee ater oreseec cally ONL ani — | x |
| Pr ovalis, Bréb. Bed Ioteiste Safety ae ee aise Sies.s cise edges | x | | | x
33 pr wan. pinnata@ (NVI Sm.) Vie Ee. s.2..---eeeee|) X |
oF op Welles CHa? (Uti) \Wo LBL; Copeseconsoerocoes eal
5 ry) Wales WaT (MGti77)) Wo 181s sconce coder sasvenb ode —|) — — | —|— x
6 POG iy ADIGA}, (GD) sncanneaeooaecc cbocabanonoHaeeedeene ie LX x x | x
x » var. splendida (Ehrenb.) V. H. (p)...... x Se tile Ell x
fh RT REERCLLES, USGTITZGS Po seaa ache anCet oe ea ER Reece ene sg) es ee | ng
CEG ttbiay, NW SS Doonenocodnoconneacoaeebeoc Mee nee eee =| = | & — | — x
Campylodiseus PG ber iicus eM Gren Diya ree eater ceeene dee cee x ew el GX — | — x
| MYXoOPpHYCE®. | |
Hapalosiphon Hibernicus, W. and G. 8. West............... x | |
Stigonema minutum, Hass. ...........000-0ce cece eee cree x a= xia |
INOStOCMMICTOSCOPLCUTI | OALIMey er ernee sere se ceae sane seeeeare see: | |
Anabena circinalis (Kiitz.), Hansg. (p)......-.....200 cesses x x |
a Hassallii (Kiitz.), AWARE one ans, Veen —| — x |
| ; Pa var. tenuis (W. and G. S. West),; —|; — x i
Lemm. |
pe -Mlos-aquee(laymeb>),, Breby (py) ces.-: seeeeeeeacees x) = x x | so |
Lemmermanni, P. Richter (P) ................-.... We sais fi ey is x ‘Ip Se x
Lyngbya bupunctaraemm: (OP) ees eee et) eee }—}| — | — | —)} x

188 Mr. W. West and Dr. G. S. West. [Oct. 30,

| | z
| 3
| @ 2 E . es 4
pe) eg) % | 8 ee
Species. cet hes 3 is 3 ce 3 Se
| fo | eS SS rc eens
SS qd. = a) ss eo .
| o|Mm] 4 | oe | 2) 34
alo |B |EF la}
Lyngby@ contorta, Wem) (EP) care. secre see seeeeee eee eee ae x
) Kiitzingii, Schmidle, var. distincta (Nordst.), | — — — —\— x
Lemm. |
* oogaiee, WesTatins (12) sognopcdoshacoscoesccosdcensnedc x — x —j— x
Bs Martensiana, Menegh. ©....-.2.....cc0: dscns eee econ —| — x
x CERTEOLI A, E@iNO ~ sannnaposodnscocroedoone sun s95e000° itex |
Oscullatoriaitenuis AG s.r se recon eeeee cone ee hee eee ex x x x | x x
3 limosa, BAI Uscncns a tals eseainls one eee ta eee eet neee =| = x =| = x
s Agardhii, Gomonti(p) eee tee reeeere ers | x x | x
- PAG MANIK AGE.S vases sone eente eee Rea ee — |) = a | |) se
Bs muvescens, De CM (R)\snonssaiineeocmeeoen eres x) — = |— | x
Phormidium tenue (Menegh.), Gomont ................00e.0 0+ =| = | & | x
Gleotrichia echinulata (Eng. Bot.), P. Richter (p)......... x |
GilaoveceimeanissiNaen vent keeecee choc teeseenaeteeeee cee x = | &
Synechococcus major, SCATOCH. ....0...0000.-c--0er see eenere seers sh x |
Merismopedia glauca (Ehrenb.), Nig. ........0.....seeeeeeeee allie Je x Sol
a nee 1B5RAD5 ((D)) oddn0nb0q000000000059009000000 x xa Px —|x
oy ELEGANS PAR IDES conan enecns diet seaeenestiee eter 0 i Sal
punctata, Meyen spa nbosooNnspEHoSDoNSaSqHsNE0000 x |
Mer ismopedia Lenuissvma, era Ty (Ey) eenelesieerne ste se eee i rane Silat x
Celospherium Kiitzingianum, INI -(()) odsoo0aacasncee~coc6ses x Pete fs —|x x
>» = © minutissumum, Viemm, (BP) ..5...-..-..e-ccen ee SM = x | — x
% WNdgelianum, Unger.* (P) ..............0...25 Seal: Pax aa mee x | — x
Ms natans, Lemm. (12h) ecannatonia sssnoecanconconeae —| = | &
Gomphosphera aponind, Kitz. ......00.ccc cee ece cess eer teens —|— | x == | == x
lacustris © hodat) (Ee) peeeerene saan teers x | | BPM iescae ax x
Aphanocapsa pulehra (Kiitz. Nj EMAC, saan oooneonsacesonuoecr —j| x |
Aphanothece saxicola, Nag. .........csceccseessereeeeeecnsees sees =| = | x
clathrate, W. and G. S. West (P) ............ —|/— | x — | — x
Dactylococcopsis rhaphidioides, Hansg. (P) .................. =| = x = |= x
Microcystis eruginosa, Kitz, (Pp)... ..ceececeeseeeecc ese eeeees =|} = x —|x x
. elabens) ((Bréb))y KOUbZ2 eriaciw eset sesee iene x x
3 WEAPON UGV, -2o5n9n59500 vod020000 200050505055000 = i x
3 HOC, IWesivin, (9) sadoatadasassoann6n9ce000n sane x | = x ei] SS
op marginata, Menegh. ..........000.....se0enessenseers 4 || |
oy Flos-aque (Wittr.), Kirchn. ............:6ssee0ee sa | 152
5 prasina (Wittr.), Lemm. (p) .....-.........5000 _ SC ex — | — x
pulverea (Wood), Migula ..........0.......00000 ;— | — — |—|-x
6 TOSEO=PETSUCINA, KUL. 10. .00sev renee ees eeeeee ener ;—| — — |—}]— x.
7 Siemans. Wrest, (9) sonengcecoosconssaacn5a99e0n6¢ x x x | =x x
Chroococcus cohwrens, Nag. ............-sseeseee sense sev eerececes x Etat iS ol
i JACI BGEICIIS, INES, .son.0c0000609000 900 907 2395000590500 —|/—) x |
es limneticus, Wemm. (PB) ...-..cc.c.eceee es esc ere eee x Nex x | — x
mM 5 var. subsalsus, Lemm. (P) .........| —/—)} x
¥ minimus (v. Keissler), Lemm. .................. | x x = =| x x
. VOCUS INIEV SS caps onaonnscopo5050 0c aco aAesobnndosD x x all
turgudus (Kiitz.), Nag. <o.ccs..ceseeenen-ere son ers x st ok —|x
|
PHROPHYCER. |
Stichoglea olivacea, Chodat (P) ...........0...00eecneereeeereees x |
Phzococcus planctonicus, W. and G. S. West (P) ......... x — x

* Tt seems probable that this “ species ” is merely a form of C. Kiitzingianum, Nig.

1908. | The British Freshwater Phytoplankton.

jo
(oe)
Ne}

io)
. a
Species. 4 ae “ @ [ee ie
@ | pe | 88 | a |e | am
SB di oO.) a a oo .
Sa | os |e |e leas
B/S eS Je ies
FLAGELLATA. | |
Dinobryon bavaricum, Imhof. (Pp) ..viceeeereeeccereeeecceetees | x ==) | & |
3 cylindricum, Tm oF (MS) hieccsescss pees ceed |= | x <a eS
A 4 var. pediforme, Lemm. ............| X — — x
35 Me var. palustre, emm. ............... | x = x
5 5 var. angulatum, Lemm. (p) ...... | x
a 6 var. divergey.s (Imhof.), Lemm.(p)| x Xiah ke ex se | & x
5 var. Schayxinslandii, Lemm. (p) | x an eet Xie
se elongatwm, Jeealavasen'( Goff aksneneopaseaoesaceeccdecawseoss x a eee ai eal.
: 35 var. undulatum, Lemm. (p) .........| x Xa || eexey | eeosn | X
i protuberams, Wemrn. (p).......2.ce-0essc2seeeneses ees ea ee tae eX ec elen xX
nA SentulaniaHhrenb eesessereeeeeees eee tee | x |
- a. var. thyrsoidewm (Chodat), Lemm.| — | — | x =|— | &
. SOCiGle pW MVE Maa. er cer sceete nastiest won eee i= | = x |
utriculus (Ehrenb.), Kilebsiteen enna scomesonenrsal|: 3 |
Halobryon Tauter born, Wem. |p) vererssseeesensea: soe cee ee x |
SUMOire, Weald, ABIGRETAD), socinee cobooeede poo LodeEsbobobbdoN CoDoBbOUaAE: ==] = 9) = |=] &
Mallomonas acaroides, 1etesdiay (Go) GeSapabee onan sodndeaododd scans lex x eal |
a QUEENA IINSENOIIE Gononoonossondusdnese noc sandeoecndce | x — x |
am POPROGWCHG, UT ETOGIE soc occocenoonnececroonbAeeosnooDo0be ;}—| — | x |
* longus, We, (TR) socococsncoboocccenonaoanaced I) 3 == |} == | ==} x
Diplosigopsis frequentissima, Lemm. (p) .......0......000000) X — me || Re
Biceca lacustris, J. Ol., var. longipes, Zach. (p) ............ {—}| — | x |x
OY DICPORES COG, IB TEIOO), 540 boace coco cooobecobenondonnebooeenboe — meg teat: | |
Lepocinclis ovwn (hrenb.), Lemm., var. punctato-| — | — |
striatum, Lemm. |
LY MWALE CGAINS,, WANED), SeeaeGarcosapececbo0dssd boo cocbooaoncndeed x Xoo lpn | le
PERIDINIER.
CHITOTOLUS UU DENS 8 son sco bobdre soc adeboDceuco ed ooeONaaBE NOR eORENO: } == | = |e
= poradoxum, Schilling (p) ........10---...00- x = | = ks
var. major, Lemm. (Py) ...... =| = | &
Glenodinium pulvisculus ¢Ehrenb))\ Stein (p)) aes see: x = x al = x
Ceratium cornutum (Ehrenb.), Clap. and Teaehin. (Dyas x x x S|) es
45 curoirostre, Hruitt.-Kaas, (PB) .t0.-.... ccs eeieeee ss. x |
hirundinella, ©, 1a, Wittig? (2) cecctbonondaceoucceed x ally Pe x | x x
Peridinium bipes, Sain ert, eine ee ate AeA er —|;— | x
i GQUuCHI TO, AB\skeexalle}, (79))oce000cn0e0 000 woo en ae0a00R0RRRAE =| = x = | == x
) inconspicuum, Demme eee ec oee e ioe ames ore x = — x x
- limbatum (Stokes), Lemm. (p)..........--::::00 =| = x
i 6) jousoiuizs Pskoawillsa¥s: (9) ccpemn dookeecenosocenodobeo ea x |
5 tabulatum (Bhrenb,), Clap. and Tachm. ...... x x | esl iess tiles x
of) OORT KEIN Se igo opoecacedeeanedocbecsenaedeses x |
0 Westii, Lemm. (CEA socttencte sotnaste sete statins x |
. Waller, Hruiti.-Kaas. (PB)... 0.0. .ce0r.ceee cee ree see x x ETO x x

The above list includes the species of the lake-plankton only, and the

Flagellata are very incompletely recorded.

Were the species of the helioplankton of large ponds, pools, and ditches
also included, many of the Protococcaceze (or Autosporaceze) would have to

190 Mr. W. West and Dr. G. S. West. [Oct. 30,

be added, such as sundry species of Chodatella, Lagerhewmia, Golenkinia, and
other genera.
The following summary is instructive :—

Species only.
| a | = Totals.
| | @ et al g Species and varieties
| = Syl Se of each group.
[ep netics ef) ea
| Reals 3 | = ie
=| se | TS S . a] o
Meio see | aie
6 ae es le eh ers
n |\o = = Bg | Species. | Varieties.
Ren sees 3 eS ee Nes
Chlorophycee (except Des- 54 | 31 37 | 20 | 24 33 81 18
midiacez ) |
Desmidiaces <...c.cc.0csesesen ee 176 85 | 103 | 101 96 22 236 68
Heterokontee ..........0...02c000e | 8 — 2 — — 3 | 7 (0)
Barcilllaniessaneeecec chess eeete 63 | 37 Alien east 41 | 94 22
Miyxoplayicerreretreeeeeeeeerice Sil i} ales 33 12 17 Ya | 58} 2
IPNRA OELS) 500 500 045000 99G005000 Boo) os 1 = | = = | 2 (0)
Flagellata (except Peridi-, 12 | 4 13 Gi 4 Se) as} a
nies) | |
EP eri Gini e eh se.t aumaecteswianaie ders TA? i ah 10 5 6 5 15 1
| inves l l
Totals of species for each | 354 | 178 | 246 | 162 | 188 | 128 506 118
| area, and grand total of | Gane
both species and varieties Grand total.
for entire British phyto- |
plankton | | | |
| |

The grand total of 506 species and 118 varieties is sufficient evidence in
itself that the British phytoplankton is exceedingly rich, and of this total
46 per cent. are species of the Desmidiacez.

Many of these constituents are, of course, adventitious or casual, consisting
of littoral or bog species which are carried into the plankton by the rains
and exist there only a short time before perishing. There are, however,
many constituents which are exclusively limnetic in habit, and also others
which are much more abundant in the plankton than in other situations.
These have been discriminated in the foregoing list.

XI. GENERAL SUMMARY AND DISCUSSION ON THE DOMINANCE OF DESMIDS.

The tabulated list gives a very adequate idea of the Algal constituents of
the British freshwater plankton, and also shows that many of these con-
stituents are common to the four British lake-areas.

The British lakes combine to some extent the characteristic features of the
Central European and Northern European lakes, but are on the whole more

MS

1908. | The British Freshwater Phytoplankton. 191

nearly akin to the latter. In addition they have peculiarities which tend to
mark them off from either of those groups; for instance, the relatively high
winter temperatures. Very many of these lakes never freeze, and most of
the others only rarely become covered with ice, and then for comparatively
brief periods. The summer temperatures are also comparatively low. The
highest temperature we have recorded in Windermere in the course of
twelve months’ observations (including one of our warmest summers) was
14°4 C. (58° F.), in Wastwater 17°-2 C. (63° F.), and in Ennerdale Water
15°5 C. (60° F.). The highest temperatures we have obtained in the Welsh
lakes were 17°5 C. (63°°5 F.) in Llyn Ogwen and 18° C. (64°°5 F.) in Llyn
Liydaw. The small sheet of water known as Llyn Elsie attained a summer
(August) temperature of 19°'7 C. (67°5 F.). The summer temperature of
the Irish lakes rarely exceeds 18°3 C. (65° F.). The average range of
temperature in the Scottish lochs is from about 5° C. to 13° C., and the
highest we have ourselves measured was 16°6 C. (62° F.) in Loch Earn.

In all the four British lake-areas the water is soft, with only small
quantities of dissolved lime, a peculiarity which accounts for the rarity of
snails at the lake margins.

The phytoplankton is never of very great bulk, and it is quite exceptional
for it to colour the water to any appreciable extent.

The periodicity of the phytoplankton is very variable in the different
lakes. In some it is conspicuous, but in others it is not very well marked.
Investigations at present in progress indicate that it is most conspicuous in
the shallower lakes, and particularly in those at considerable altitudes, but at
present our data are not nearly sufficient for generalisations to be made.

The Myxornycze& play quite a secondary part in the plankton of the
British lakes as compared with the Central European lakes. They are more
abundant in some of the Irish lakes than in any of the others, and some-
times the phenomenon of “water-bloom” makes its appearance. This
phenomenon, which is due to sudden and simultaneous maxima of a few of
the limnetic species of Blue-green Algz, is of very irregular occurrence in
the British lakes, and is practically confined to sporadic appearances in some
of the shallower-lakes.*

Oscillatoria tenwis is general but never abundant, and Anabenw Lemmer-
mannt should be specially mentioned, as its spores form deep blue-green
floating clusters, which sometimes give a decided colour to the surface water.

* The phenomenon of “ water-bloom” is better seen in some of the large pools and
meres of the lowland parts of Britain than in any of the lake-areas. A good description
of the “ breaking of the meres” has been given by Phillips in ‘ Trans. Shropshire Archeol.
and Nat. Hist. Soc.,’ 1883, and earlier records were by Greville, Dickie, and Drummond.

192 Mr. W. West and Dr. G. S. West. [Oct. 30,

Of the colonial unicells, Celospherium Kiitzingianum (and the form of it
known as C. Négelianum), Gomphospheria lacustris, and Chroococeus
limneticus are the most important. Microcystis (Clathrocystis) eruginosa
occurs more particularly in pools and small, shallow lakes, where the
temperature of the surface water becomes relatively high in the summer.

In the Scottish and the Welsh lakes the Blue-green Algee are decidedly
scarce. This scarcity is to be attributed to the Alpine character of so many
of these lakes, in which the maximum temperature of the surface water is
relatively low.

The FLAGELLATA are well represented by various Peridiniez, by Mallomonas,
and by several species of Dinobryon. In many of the larger British lakes
Dinobryon completely dominates the spring plankton, and a few colonies
generally persist through the summer and the early part of the autumn.
The most abundant species is D. cylindricum, and its var. divergens is equally
common. In the smaller lakes Dinobryon is fairly general, but does not
attain such great maxima as in the larger lakes. Mallomonas acaroides
sometimes occurs in prodigious abundance, but lasts only a few weeks.

Of the Peridiniez, Ceratiwm hirundinella is general, but it only occurs in
large maxima in the smaller lakes. The variations in this organism have
been well described and figured,* but there is one form in the lakes of the
Outer Hebrides and the west of Ireland} which is apparently unknown in
the plankton of the rest of Europe. In this curious form the first antapical
horn is very much deflected to one side (vide fig. 3). Species of Peridinium
often occur in very large quantity in the smaller lakes, and in the shallower
of the large lakes. P. tabulatum is frequent, but P. Wille: is general
throughout the Scottish, Irish, and English lakes It occurs most
abundantly in the small upland lakes (up to 1800 feet) with an Alpine
character. P. Westii is exclusively confined to the Scottish lakes, where it
appears to be frequent.

The BACILLARIEH are abundant in the British phytoplankton, but they
rarely occur in such great quantities as in the Central European lakes, and
scarcely ever form the vast maxima which periodically appear in so many of
those lakes. Diatoms are fewest in the plankton of the Welsh lakes,

* Lemmermann, in ‘ Archiv for Bot. utgifv. af K. Sv. Vet.-Akad.,’ Bd. 2, 1904, No. 2,
t. 2, f. 1—49; W. and G. S. West, in ‘ Roy. Soc. Edin. Trans.,’ vol. 41, 1905, p. 494 (c. fig.) ;
‘Roy. Irish Acad. Trans.,’ vol. 33, sect. B, part 2, 1906, pp. 98, 94 (c. figs. 1—9);
Bachmann in ‘ Archiv fiir Hydrobiologie und Planktonkunde,’ Bd. 3, 1907, pp. 55—58,
figs. I, Ia, II.

+ W. and G. S. West, in ‘ Roy. Irish Acad. Trans.,’ loc. cit., 1906, p. 94, f. 9.

t The distribution of Peridiniwm Willec, Huitf.-Kaas, extends from N. Italy, Ireland,
England, and Scotland to Norway, Finland, the Faeroes, and Iceland.

Hats

1908. | The British Freshwater Phytoplankton. 193

forming only an average of 11 per cent. of the entire phytoplankton. This
low percentage is probably due to the small. numbers of adventitious species
washed into the lakes from the mountain sides, and is possibly accentuated
by the stony character of the lake margins and lake bottoms. They occur in
the greatest variety in the Scottish and Irish lakes, probably owing to the
large number of adventitious species washed into the lakes by the rains.
The Pennate Diatoms are much the most numerous and conspicuous. |
Among the commonest forms are Asferionella (with a range of form and
size which embraces both A. formosa and A. gracillima) and the two species

Fre. 3.—Peculiar Form of Ceratiwm hirundinella, O. F. M., in which the first antapical
horn (at,) is greatly deflected to one side. x 200.

of Tabeilaria. T. fenestrata is much more abundant than 7. flocculosa, except
in the English lakes, where the reverse obtains. The chain-forms of
T. fenestrata are the most frequently observed, but the star-dispositions (var.
asterionelloides) are common except in the Welsh lakes. 7. fenestrata, var.
asterionelloides, is one of the dominating features of the late spring, the
summer, and the early autumn plankton of many British lakes, and it
exhibits great variability in the relative strength and breadth of the girdle
view of the cells.* That these differences are of no varietal importance is
proved by the occurrence of all intermediate stages.

We have not observed any star-dispositions of 7. flocculosa in any of the
British lakes, although such have been observed by Holmboe in Norway and
by Wesenberg-Lund in Denmark (7. flocculosa, var. pelagica, Holmboe).

The numerous plankton-forms of Asterionel/a almost convince one that
A. formosa and A. gracillima are merely states of the same species. No
chains of Asterionella were observed in any of the lakes.

The genus Fragilaria is somewhat rare, and of the species which occur
F. capucina is the commonest. YF. crotonensis is very general in Scotland

* Of. W. and G. S. West, in ‘ Roy. Soc. Edin. Trans.,’ vol. 41, 1905, Plate 2, figs. 1—3.

194 Mr. W. West and Dr. G. S. West. [Oct. 30,

and Ireland, but is always scarce. We have not observed it in the English*
or Welsh lakes. A variety of it—var. contorta—occurs in Loch Ruar,
Sutherland, which is unique in the curious twisting of its exceedingly short
filaments. This variety is not known from elsewhere.

Throughout all the British lake-areas, but more especially in the west of
Scotland and the west of Ireland, species of the genus Swrirel/a form a con-
siderable and consp¥cuous part of the phytoplankton. The most frequent is
Surirella robusta, var. splendida, which sometimes occurs in great abundance,
but S. biseriata and S. linearis are both general. In this respect the British
lakes compare with the lakes of Central Africa, in which several plankton-
species of Swrirella are abundant.{ In the Yan Yean Reservoir, Victoria,
S. robusta, var. splendida, is also a constituent of the plankton.§ Wesenberg-
Lund|| states that various species of the genera Swrirella and Cymatopleura
occur in the plankton of principally alpine or shallow lakes in level country,
and that they have been carried out by rivers and waves into the pelagic
region, where they vegetate but for a short period and then perish. We find
that in the British lakes, and also in those of Central Africa, the genus
Surirella is frequently a true plankton-genus, and the various species which
have been recorded both vegetate and multiply in the plankton to an extent
we have rarely noticed in other situations.

Several of the Naviculacez occur with considerable regularity.

Centric Diatoms are relatively few and insignificant in the British lakes.
Melosira is represented chiefly by JZ granulata and M. varians. The latter
is perennial in the plankton of British rivers. Species of Cyclotella are not
abundant, and only in Lough Corrib, Galway, have we observed the curious
gelatinous colonies which occur so frequently in some of the Central Huropean
lakes,

The genus fhizosolenia is represented by two (and if the record of
Rh. eriensis be correct, by three) species. &. longiseta is very rare, but
R. morsa occurs in some of the lakes of all the British lake-areas. We have
observed the restine-spores of this species in the June plankton of Thirlmere
in the English Lake District.

No species of Attheya has yet been observed in any of the British lakes.

The CHLOROPHYCE® are well represented in the British lakes, more

* his species occurs both in the plankton (helioplankton) and the benthos of the large
pools in the Midlands of England.

+ Consult W. and G. S. West, loc. cit., Plate 1, figs. 1—4, and Plate 2, fig. 6 (photos).

+ G.S. West, in ‘ Linn. Soe. Bot. Journ.,’ vol. 38, 1907, p. 85.

§ G. S. West, in ‘Linn. Soe. Bot. Journ.,’ vol. 39, 1909, p. 17.

|| Wesenberg-Lund, ‘Plankton Investigations of the Danish Lakes,’ Copenhagen, 1908,
p. 42.

1908. | The British Freshwater Phytoplankton. 195

especially by the Desmidiacee. Apart from the latter, Botryococcus Braunii
and Spherocystis Schroeteri are the most general and abundant. Species of
Oocystis are frequent, but never occur in quantity. Dictyospherium
pulchellum often occurs abundantly, but it also at times occurs in equal
abundance in bogs.

Ludorina elegans is fairly general, even in large lakes such as Lough Corrib,
although it reaches its maximum abundance in small lakes.

Pediastrum Boryanum and P. duplex are frequent in the plankton of the
shallower lakes, but P. simplex is very rare. Several species of Celastrum,
Scenedesmus, and Crucigenia occur in many of the lakes, but never in
quantity.

Species of Zygnema, Spirogyra, and Mougeotia occur in the plankton of
most of the lakes, principally in the late spring and summer. They are
usually the slender species of these genera, and are almost invariably sterile.
In the smaller alpine lakes JMJougeotia is often abundant, and forms no small
part of the phytoplankton.* The curious coiled Mougeotia-filaments of some
of the Scottish lochs have already been referred to. It would appear that
the coiling is a limnetic character,t developed to augment the floating-
capacity of the filament, and the fact of its presence is direct evidence that
some of these solitary filaments of Mougeotia are adapting themselves to a

_ life in the plankton.

THE MOST INTERESTING FEATURE OF THE BRITISH FRESHWATER PHYTO-
PLANKTON IS THE DOMINANCE OF Desmips. In 1903, and again in 1905, we
showed that in contrast to any previously known plankton that of the
Scottish lakes was unique in the abundance of its Desmids. Since then we
have found that this dominance of Desmids is not confined to the lochs of the
Scottish Highlands, but is a feature of the plankton of the four lake-areas of
the British Islands, and that the plankton of the western British lake-areas
differs markedly from all other European plankton in the abundance of its
Desmids.i

* Tn the alpine lakes of the Pike’s Peak Region, Colorado, Shantz states that species of
Spirogyra and W@dogonium form a large part of the summer plankton (wde ‘Amer.
Microscop. Soc. Trans.,’ March, 1907). Fragmentary filaments of various species of
G@dogonium axe also very frequent in the summer plankton of the British lakes.

+ Consult G. S. West in ‘ Linn. Soc. Bot. Journ.,’ vol. 38, 1907, pp. 85 and 86.

~ Among European lakes, only those of Norway and certain parts of Sweden approach
the British lakes in the possession of a conspicuous Desmid-flora in the plankton (consult
Huitfeldt-Kaas, loc. cit., 1906; and Lemmermann, in ‘ Archiv fér Bot. utgiv. af K. Sv.
Vet.-Akad.,’ Bd. 2, 1904), and it should be mentioned that many of these plankton
Desmids are identical with the British ones. Tanner-Fullemann has recorded the
occurrence of a number of Desmids in the plankton of the Schoenenbodensee (vide ‘ Bull.
de ’Herb. Boissier, 2me sér., t. 7, 1907), but the species which he records, when it is

196 Mr. W. West and Dr. G. S. West. [Oct. 30,

In discussing this phenomenon of the rich Desmid-flora of the British
freshwater plankton, it is necessary, in the first place, to briefly outline
the general distribution of the Desmidiaceze in the British Islands, quite
arrespective of the freshwater plankton. We have studied the distribution of
British Desmids in detail during the past 16 years, and the obvious fact,
patent to anyone who chooses to collect these plants over extensive areas,
is the much greater richness of the Desmid-flora in the western areas of the
country. The eastern districts of England are exceedingly poor, but on
passing from the newer Tertiary formations to the Older Paleozoic and
Precambrian formations the Desmid-flora gradually increases in richness,
attaining its maximum diversity in certain of the Precambrian areas.

The richest areas of all are the little boggy pools and smaller lakes of
the Lewisian Gneiss of North-west Scotland and the Outer Hebrides, and
similar areas on the Precambrian formations of Donegal, Mayo, and Galway.
There are also several very rich localities in the English Lake District and
North Wales, all on the Silurian and Ordovician with sundry Igneous
intrusions. There are, in addition, two rich localities in the south of
England, one on the Lower Greensand of Surrey (Thursley Common), and
the other on the Middle Eocene of Hampshire (the New Forest). In both
these localities there are deep, spongy bogs, with a fairly rich Desmid-flora,
but at the same time it is a flora which falls far short of the much richer
Desmid-floras of the Precambrian areas. We may add that these are not
statements based upon a few casual observations, but upon a detailed
examination of many thousands of collections made in all parts of the
country, from the Shetland to the Scilly Islands, and from the east of
England to the west of Ireland. It is also necessary to give some
explanation of what is meant by a “rich” area. We do not apply the term
“rich” to a mere abundance of Desmids, or even to the occurrence of a
great quantity of 30 or 40 species, but only to those areas in which 150
to 200 (or even 300) species can be found in more or less abundance,
including many of the rare species with a restricted distribution.

We have, therefore, as a foundation on which to base this discussion of

possible to be certain of his identifications, are those of shallow alpine and subalpine lakes,
and not in any way comparable to the characteristic plankton species of the western
British areas. Neither do we regard his records as constituting a “rich” Desmid-flora.

Among extra-European lakes, Victoria Nyanza has a conspicuous Desmid-flora in
the plankton, and the Yan Yean Reservoir, Victoria, possesses a very rich Desmid-
plankton, quite equal to the best of the British lakes, although with an entirely different
association of species. In the latter case the drainage water is mostly from Silurian and
Granitic outcrops, but it is not yet possible to make a definite statement concerning the
drainage into Victoria Nyanza.

1908. | The British Freshwater Phytoplankton. 197

the dominance of Desmids in the plankton of the British lakes, a fairly
complete and necessary knowledge of the general distribution of Desmids
in the bogs, pools, etc, throughout the whole of the British Islands. The
first point of importance is that the great majority of the British lakes
{those constituting the western and north-western lake-areas) are all
situated in the richest Desmid-areas in these Islands, or for that matter
in Europe. It is, therefore, not in the least surprising that the plankton
of these lakes should on the whole contain an abundance of Desmids.
That the Desmids of the plankton should differ considerably from those
of the bogs of the drainage areas—a matter discussed in a later part of
this paper—does not affect the main question, viz., that the phytoplankton of
these lakes possesses in many instances such an abundance of Desmids that vt cain
be correctly described as a Desnud-plankton.

Those facts which explain the abundance of Desmids in the bogs and
bog-pools, among the mosses of the dripping rocks, and among the leaves of
the submerged plants of the lake-margins, will likewise furnish the
explanation of the abundance of Desmids in the plankton, as the plankton-
Desmids have certainly originated from bog and swamp species, and others
are being constantly recruited from the same sources. In endeavouring to
discover the relationship between the conditions of environment and the
richness of the Desmid-flora, two facts stand out very clearly :—

1. The rich Desmid-areas correspond very accurately with the areas of the
old geological formations. They are mostly mountainous districts, with
considerable outcrops of Igneous rocks.

2. These areas also correspond, but with less accuracy, to the areas of
greatest rainfall.*

It is now necessary to enquire more closely into the relationships between
the geological nature of the drainage-area, the rainfall, and the richness of
the Desmid-flora.

We will first consider the rainfall of the areas in question. This is
relatively heavy, varying from about 45 to upwards of 100 inches, and is
due to two causes: first, to the fact that these areas are almost all near the
west coast, being districts in which large mountains are situated in close
proximity to the sea; and secondly, to the prevailing westerly and south-
westerly winds. Such conditions naturally result in wet, mossy hill-sides,
with numerous bogs. There is consequently much peaty water, rich in
humie and other organic acids, in which submerged plants, such ‘as
Utricularia minor, Sphagnum cuspidatum, S. subsecundum, and other

* Mr. James Murray (in ‘ Roy. Phys. Soc. Edin. Proc.,’ vol. 16, 1905, p. 58) also points
out that Sir John Murray had indicated this fact to him.

VOL. LXXXI.—B, IP

198 Mr. W. West and Dr. G. S: West. [ Oct. 30,

Y RAINFALL
OVER SO Ins.

ZY RAINFALL
G 40 TO So ins,

YZ RICH VE Z OLDER
Z DESMID AREAS. ESM AREAS. G PALROZOIC.
Fie. 5. Fie. 6.

Maps of British Islands, to show (fig. 4) areas with Rainfall over 40 inches ; fig. 5, distri-

_.bution of rich and very rich Desmid-areas, characterised by the western types
mentioned (pp. 201 and 202); fig. 6, distribution of Older Paleozoic and Archzan
rocks,

Note.—The Desmid-flora of the central and south-eastern counties of Ireland is very
imperfectly known. Notice the falling off in the Desmid-flora of Skye in fig. 5, corre-
sponding with the absence of Archzan rocks indicated in fig. 6.

1908.| | The British Freshwater Phytoplankton. — 199

aquatics thrive, and furnish all the requirements for the prolific growth of
Desmids.

This seems a natural explanation of the occurrence of a rich Desmid-
flora, and one which is accepted by Wesenberg-Lund* as the main cause of
the phenomenon.

A detailed study of the distribution of Desmids has shown us, however,
that the mere presence of suitable habitats is insufficient to account for the
ereat richness of the Desmidiacez in certain areas of these Islands. Among
the mountains of the Pennine Chain are some of the finest peat-bogs in
the British Islands—in all outward appearances ideal spots for the
occurrence of a rich Desmid-flora. Such are Cocket Moss, Austwick Moss,.
and others less well known. Again, on Thursley Common in Surrey, and
in the New Forest, are bogs which are unsurpassed in Britain as habitats for
the Desmidiaceze. In all these localities the bogs are deep and dangerous,
fed mostly by bottom springs, and furnish an ideal home for quantities of
submerged Sphagnum and Utricularia minor. Desmids occur in countless
millions among the larger aquatics; in fact, collections. made in these
localities would be regarded as “rich” or “very rich” by those who had no
experience of the western areas. Utricularia minor, which harbours some
of the best of British Desmids among its leaves, flowers profusely in the
localities mentioned, and no finer specimens can be obtained even in Mayo
and Galway. Yet the Desmid-flora which occurs among the Utricularia in
the above-mentioned localities is not to be compared with that which
occurs in precisely the same environment in the western British areas, and,
moreover, it contains none of thé real British rarities.

How is it that an ideal locality such as Cocket Moss, which would be
deseribed as “rich in Desmids,’ contains practically none of those species
which are both dominant and characteristic of the western areas? The
conditions are almost identical with those obtaining in the western bogs, and
the rainfall is from 50 to 60 inches.

From a consideration of the above remarks it is obvious that some factor,
other than mere abundance of rainfall and presence of ideal habitats, has a
profound influence on the distribution of Desmids. This at once causes us
to enquire into the statement that we have previously made,f viz., that the
rich Desmid-areas correspond geographically with the Precambrian and Older
Paleozoic outcrops (together with the intrusive Igneous material). (Consult

figs. 5 and 6.)

* Wesenberg-Lund, ‘ Plankton Investigations of the Danish Lakes,’ Copenhagen, 1908
p- 281.
+ Vide p. 197 of the present paper.
BY

200 Mr. W. West and Dr. G. 8S. West. [Oct. 30,

In the first place, it has been recently pointed out* that the association of
the rich Desmid-areas with the older strata is (in the British Islands) most
probably due in part to the antiquity and consequent hardness of the rocks.
The mountainous regions which have resulted from those changes in the
earth’s surface which have produced folding and contortion, and from the
resistance of these old, compressed rocks to subaerial denudation, are not
only directly responsible for the rainfall owing to their geographical position,
but are themselves most suitable for the formation of peat-bogs. In these
areas Desmids flourish, and therefore, so far as the British Islands are
concerned, the richness of the Desmid-flora bears a distinct relationship
to the antiquity of the geological formations of any area under consideration.
It seems probable that the determining factor is a chemical one. It is
certainly something more than mere suitability of habitat, otherwise how is
the relative poorness of the extensive bogs of the more recent formations to
be explained ?+

It is very probable that the chemical composition of the water plays an
important part in determining this distribution. That the chemical factors
are quite apart from the occurrence of brown, peaty water, is evident from
the poorness of the Desmid-flora of so many peat-bogs, and also from the
fact that the best and richest Desmid-floras only occur in clear water with
no obvious peaty characters. Wesenberg-Lund is incorrect when he states
that Desmids chiefly thrive in “ brown water rich in humic acids.”{ Some of
them certainly do, and often profusely, but these are generally the common,
ubiquitous species which have almost a world-wide distribution. The great
majority, including most of the western British types, prefer clear water
with little peat. An excess of the brown peaty material is distinctly
unfavourable. The plankton-Desmids also occur much more abundantly in
the clear lakes than in the brown peaty ones.

Although it appears so probable that chemical factors determine the
distribution of many Desmids, no definite information on this point has yet
been obtained. The drainage water which has percolated through the old
formations (rocks and soil) may possibly contain minute quantities of
something in solution which greatly favours the development of certain types
of Desmids, and may be directly responsible for their restricted distribution.)

* G.S. West, in ‘Linn. Soc. Bot. Journ.,’ 1909, vol. 39, p. 10.

+ How is it, also, that the peaty bogs and ditches of the fens of the east of England, such
as are left of them, are poorer in the Desmidiacez than any other part of the British Islands ?

{ Wesenberg-Lund, Joc. cit., 1908, pp. 280 and 281.

§ There would be nothing remarkable in this, as diatoms thrive and build up their

siliceous cell-walls in water containing silica in such minute quantities that ordinary
chemical analysis reveals no trace of it.

1908. | The British Freshwater Phytoplankton 201

In reference to the above remarks, James Murray* has stated that
“another theory is that the lochs which are richest in Desmids are only
found in the older geological formations, but this does not accord with facts,
as I find that such lochs occur in all the formations from the Lewisian to the
Tertiary, at least; and it will, I think, be found that some of these lochs lie
entirely in glacial deposits.” It should be pointed out that if glacial drift is
excluded none of the lake-areas are even near the British Tertiary formations.
Many of the lakes have doubtless been formed in Tertiary times, and if they
are spoken of as “Tertiary lakes” it must be distinctly understood that they
are situated in drainage-basins on the old formations. Those lakes which
Murray has termed “ Tertiary lakes” are certain of the Scottish lochs which
are surrounded by and rest upon more or less extensive sheets of glacial
drift. It must be distinctly borne in mind that most of this glacial drift
has been derived from the old rocks, and what is very much more important,
that the drainage into such lakes consists mostly of water which has partly
‘traversed the exposed outcrops of the old rocks of the surrounding hills and
mountains, and partly percolated through them. Therefore, any peculiarities
which may be characteristic of the drainage water from the old formations,
are also equally characteristic of the water of such lakes.

The point of primary importance is that the greater part of the drainage
water of such a lake has traversed the older rocks, and possesses those
peculiarities which so far as we can see account for the Desmid-flora not
only of its plankton, but of its littoral region and also of the surrounding
bogs. It is immaterial when the lake was formed, or whether its bed be one
of glacial drift or of old rocks.

IN THE BritIsH ISLANDS THE REALLY RICH DESMID-FLORAS, CONTAINING
MANY OF THE WESTERN BRITISH TYPES, ARE ONLY FOUND IN THOSE AREAS
WHICH COMBINE THE MOST SUITABLE HABITATS (such as are found on boggy
hill-sides with an abundant rainfall) WITH A DRAINAGE-WATER DERIVED FROM
GEOLOGICAL FORMATIONS OLDER THAN THE CARBONIFEROUS.

We would suggest a very exact chemical investigation of the water of bogs
and lakes in different areas as a possible means of throwing further light
upon this question. It has been suggested that the absence of lime is the
determining factor in the abundance of Desmids, and it may possibly have
much to do with the restricted distribution of the western British types.

There are a large number of western types of british Desmids, of which
the most important are included in the following list :—

Gonatozygon aculeatum, Hastings; Spirotenia trabeculata, A. Br.; Penium

* James Murray, loc. cit., 1905, p. 58.

202 Mr. W. West and Dr. G. 8. West. [Oct. 30,

adelochondrum, Elfv.; P. Clevei, Lund.; Tetmemorus Brébissonii (Menegh.),
Raifs, var. minor De Bary.

Micrasterias radiata, Hass.; M. conferta,'Lund.; I. pinnatifida (Kiitz.),
Ralfs; J£ apiculata (Ehrenb.), Menegh., and var. brachyptera (Lund.),
W. and G. S. West. .

Euastrum pictum, Borges. forma; HL. Turneri, West; HL. aboense, Elfv. ;
E. intermediwm, Cleve; E. pingue, Elfv.; #. validum, W. and G. 8S. West.

Docidium undulatum, Bail.; Pleurotenium eugeneum (Turn.), W. and
G. S. West.

Cosmarium bipunctatum, Borges.; C. capitulum, Roy and Biss., var.
grenlandicum, Borges.; C. Corribense, W. and G.S. West; C. commissurale,
Bréb., var. crasswm, Nordst., C. distichum, Nordst.; C. didymoprotupsum,
W. and G.S. West; C. entochondrum, W. and G. 8. West; C. monomazum,
Lund., var. polymazum, Nordst.; C. obsoletwm (Hantzsch), Reinsch; C. per-
foratum, Lund.; C. pseudexiguum, Racib.; C. pseudopyramidatwm, Lund., var.
stenonotum, Nordst. ; C. quadridentatum, W. and G. 8. West ; C. quadrifarium,
Lund.; C. retuswm (Perty), Rabenh.; C. retusiforme (Wille), Gutw. ;
C. Smolandicum, Lund., var. angustatum, West; C. sexnotatum, Gutw., var.
tristriatum (Liitkem.), Schmidle; C. subquadrans, W. and G. 8. West:
C. subretusiforme, W. and G.S. West; C. synthlibomenum, West; C. taxi-
chondriforme, Eichl. and Gutw.; C. tenwe, Arch.; C. tumidwmn, Lund..;
C. venustum, Bréb., var. hypohexagonum, West; C. viride (Corda), Josh. ;
C. zonatum, Lund.

Staurastrum Arctiscon (Ehrenb.), Lund. ; St. averswm, Lund.; St. bacillare,
Bréb.; St. Brébissonii, Arch.; St. Brasiliense, Nordst., var. Lundella, W. and
G.S. West; St. Cerastes, Lund. ; St. Clever (Wittr.), Roy and Biss.; Sé. con-
spicuum, W. and G. 8. West; S¢. corniculatum, Lund.; St. cwrvatum, West ;
St. dorsidentiferum, W. and G. 8. West; St. elongatum, Barker ; St. eraswm
Bréb.; St. forficulatum, Lund.; St. grande, Bulnh.; St. levispinwin, Biss. ;
St. longispinum (Bail.), Arch. ; St. maamense, Arch. ; St. megalonotum, Nordst. ;
St. natator, West; St. Ophiwra, Lund.; St. Picwm, W. and G. 8. West;
St. quadrangulare, Bréb.; St. setigerum, Cleve; St. sexangulare (Bulnh.),
Rabenh.; St. spiniferum, West; St. subgracillimum, W. and G. S. West;
St. cosmospinosum (Bérges.), W. and G. 8. West; St. Duacense, W. and
G.S. West; St. Hibernicum, West; St. gaculiferwm, West ; St. cornutum, Arch.

In addition to the above, there are a number of species which are
extremely rare in a few of the richest localities outside the western areas.
whereas in the latter they are generally distributed and many of them
prodigiously abundant. Such are :—

Spirotena acuta, Hilse; Netrivm oblongwum (De Bary), Liitkem., var.

1908. | The British Freshwater Phytoplankton. 203

cylindricum, W. and G.S. West; Peniwm exiguum, West; Clostervwin Ulna,
Focke; Letmemorus minutus, De Bary ; Micrasterias Sol, Khrenb.

HLuastrum crassum (Bréb.), Kiitz., var. scrobiculatum, Lund.; 2. crispulum
(Nordst.), W. and G: S. West; #. imerme, Lund.; #. pinnatum, Ralfs ;
LE. pulchellum, Bréb.; E. sublobatum, Bréb.; EH. ventricosum, Lund.

Cosmarium connatum, Bréb.; CO. Debaryi, Arch.; C. decedens, Reinsch ;
C. elegantissimum, Lund.; C. annulatum (Nag.), Arch., var. elegans, Nordst. ;
C. Hammeri, Reinsch ; C. isthmium, West ; C. Nymannianum, Grun.; C. ovale,
Ralfs ; C. parvulum, Bréb.; C. pseudamenum, Wille ; C. pseudopyramidatum,
Lund.; C. pseudoconnatum, Nordst.; C. spheroidewm, West; C. subundulatum,
Wille; C. variolatum, Lund.

Staurastrum aculeatum, Ehrenb.; St. anatinum, Cooke and Wills;
St. Arnellu, Boldt; St. furcatum, Ehrenb.; St. inconspicuum, Nordst. ;
St. arachne, Ralfs; St. lanceolatwm, Arch.; . St. oxyacanthum, Arch. ;
St. pungens, Bréb.; St. scabrum, Bréb.; St. aristiferum, Ralis; St. prleolatum,
Breéb.; St. pterosporwm, Lund.; St. subscabrum, Nordst.

Having discussed the most important facts concerning the general distri-
bution of Desmids in the British Islands, we can now return to the abundance
of the Desmids in the British freshwater phytoplankton.

We have shown which areas of these Islands possess the rich Desmid-
floras, and when one considers that the British lakes are almost all situated
in these western areas, it is not very surprising that they possess a plankton
containing numerous Desmids. Neither is it surprising that many of these
should be the western types, provided that these western types are capable
of withstanding the conditions of a limnetie life.

THEREFORE WE CONSIDER THAT THE DESMIDS OF THE BRITISH FRESHWATER
PHYTOPLANKTON ARE DUE LARGELY, AND THE WESTERN TYPES ENTIRELY, TO
THE SITUATION OF THE LAKES IN THE RICH DESMID-AREAS OF THE OLD
FORMATIONS. ‘

The antiquity of the geological formations is not a special factor in the
occurrence of the numerous plankton Desinids, but in the occurrence of
Desmids as a whole. The presence of numerous Desmids in the plankton of
the lakes follows as a matter of course.

One does not expect an abundance of Desmids in the plankton of the large
Swiss lakes. They are situated in poor Desmid-areas, and in North
Switzerland the geological formations are for the most part too recent.

Most of the Central European lakes are situated in areas relatively poor
in Desmidiacee. In Denmark the formations are Cretaceous and
Jurassic, largely overlain by drift, and similarly the lakes of Northern
Germany are situated on immense areas of drift, overlying comparatively

204 Mr. W. West and Dr. G. 8S. West. [Oct. 30,

recent formations. Hence the dearth of Desmids in the lakes. On the other
hand, the Scandinavian lakes are situated on the old formations, and contain
an abundance of Desmids, many of which are identical with those of the
British lake-areas.

The Desmids of the plankton have without doubt originated from the Desmid-
community of the surrounding area, although in most cases there is little
resemblance between the plankton-community and that which can be observed
in the surrounding drainage-basin. There is an almost complete absence from
the surrounding peat-bogs and dripping rocks of those species which are most
conspicuous and abundant in the plankton. The common Desmids of the
bogs are only found in the limnetic region of the lakes as casual or adven-
titious constituents, and therefore the great majority of the Desmids brought
by the rains into this limnetic region, with its new conditions of life, find it
impossible to maintain their further existence, and rapidly perish. The
plankton-community as a whole, as shown by Wesenberg-Lund, is a very
ancient one, and thisis further confirmed in the ease of the British lakes by
the existence of this distinct community of plankton-Desmids. We have
already stated* that many of the plankton-Desmids “have existed under
these pelagic conditions for a long time, as there is every indication of this
in the modifications some of them have undergone, and in the species and
varieties which are at present only known to occur in the plankton.”

During the vast period in which Desmids have been washed by the rains
from their bog habitats imto the lakes, a specific selection has taken place,
certain species having adapted themselves, with or without sight morpho-
logical changes, to a limnetic existence. Only those have survived which
were able to withstand the new conditions.

One of the principal conditions necessary for existence in the new life
would be the ability to float in the surface waters. In some species, more
especially in the discoid species of Micrasterias, this necessity has brought
about no morphological alteration ; and in others, ainongst which are certain
species of Cosmarium and Stawrastrum, there is again no change of external
form, but a copious development of surrounding mucilage. In many others
morphological changes have occurred, mostly in the further development of
those characters which have proved of most avail in the struggle against
sinking. It is thus that spines and processes have heen greatly increased in
length, so that many of the plankton-forms are the longest-spined forms
known.t Certain species of Stawrastrum and Arthrodesmus best exhibit this

* W.and G. 8. West, in ‘ Roy. Soe. Edin. Trans.,’ vol. 41, 1905, p. 512.
+ In the ordinary habitats of Desmids, and in the former habitats of plankton-species
spines are commonly found well developed. This armature has probably two functions,

1908. | The British Freshwater Phytoplankton. 205.

great development of spines. The species of Closteriwm found in the British
lake-plankton are mostly adventitious constituents, and are never abundant.

Nearly all the plankton-Desmids are summer and autumn constituents,
and the majority of them attain their maximum abundance in September and
October, during the slight fall after the maximum summer temperature.

Neither plankton-Desmids nor those which occur in other situations undergo
any seasonal form-variations. This we have conclusively proved by the
examination of large numbers of periodic collections of these plants. This
is merely what one would expect, as environmental form-changes in Desmids
occupy long periods of time. As regards the plankton, the variations in the
conditions of buoyancy during the year in the surface waters of a lake are
not so great as the environmental differences between the habitats in which
the same species of Desmid will thrive.

Large numbers of both the plankton and bog species survive the winter
in the vegetative condition, and the formation of zygospores appears to be
very rare.

In Diatoms it is known that the seasonal form-variation, when it occurs,
is in the colony and not in the individual, but colonial Desmids are much
fewer and much less abundant than colonial Diatoms.

Lastly, we would comment upon the cosmopolitanism of the freshwater
plankton-community. This is generally true except for the Desmids.
Wesenberg-Lund* states that the numerous plankton researches in the
Central European lakes have been unable to demonstrate any special,
geographically localised plankton-communities. He remarksf that the
“freshwater plankton-communities, in contrast to all other communities on
land or water, everywhere contain the same types, nearly everywhere the
same species.” As regards the Desmid-flora, however, these statements do.
not hold good. Wherever there are lakes with a rich Desmid-flora in the
plankton, there one also gets a more or less definitely localised plankton-
community. It has been statedt that the Desmidiacee show more decided
geographical peculiarities than any other group of Freshwater Algie,
notwithstanding the fact that a large number of them are cosmopolitan and
ubiquitous all the world over. These geographical peculiarities occur

one of anchoring the individual to its environment, and the other to serve as a protection
against Desmid-eating animals. Whether the more elongated spines of the plankton-
species likewise serve a similar function of protection against the depredations of the
plankton Rotifers and Entomostraca is a point which requires further investigation.

* Wesenberg-Lund, Joc. cit., 1908, p. 293.

+ Loe. cit., p. 318.

t G.S. West, in ‘ Linn. Soc. Bot. Journ., vol. 38, 1907, p. 82; W. and G. S. West, in
‘Ann. Roy. Bot. Gard., Calcutta,’ vol. 6, part 2, 1908, p. 176.

206 The British Freshwater Phytoplankton.

in the Desmid-community of the plankton quite as much as in the general
Desmid-community of the surrounding country. In fact, they appear to
be well marked.* i

Even with the meagreness of our present knowledge we can recognise
three distinct plankton-communities of Desmids, which can at once be
distinguished from each other, and which form a most interesting com-
parison. These are (1) the Desmids of the British (and to a certain extent
of the Scandinavian) plankton, (2) the Desmids of the plankton of Victoria
Nyanza, and (3) the Desmids of the Victorian plankton (as exemplified by
the Yan Yean Reservoir). There are doubtless several other distinct
plankton-communities of Desmids, notably in the South American and the
Indo-Malayan regions. There are marked geographical peculiarities in
the general Desmid-community of these regions, and should any of the
lakes be found on investigation to possess a Desmid-plankton, it is highly
probable that many of the species will possess their proper geographical
character.

* In considering this question it should be borne in mind that Desmids in the vegetative
condition cannot be blown about by the wind, as even partial desiccation is almost
invariably fatal. Also, that in most species zygospores are very rarely formed, and that
no zygospore has yet been observed of any of the typical plankton-species. The only
Desmids which appear to survive a partial desiccation are certain species of Oylindrocystis
and Pentium, and possibly of Mesotenium.

207

On the Presence of Ham-agglutinins, Hem-opsonins, and Hemo-
lysins in the Blood obtained from Infectious and Non-
Infectious Diseases in Man. (Second Report.)

By Lronarp 8. DupcGeon, F.R.C.P. Lond.

(Communicated by Dr. F. W. Mott, F.R.S. Received February 18,—Read
March 4, 1909.)

(From the Pathological Laboratories, St. Thomas’s Hospital.)

On July 31, 1908, my preliminary communication on this subject was
received by the Royal Society and was read on November 12, 1908.

In this report attention was drawn to certain phenomena occurring when
normal and immune human serum was allowed to act in the presence of
normal and immune human blood cells. The whole of the investigations
were carried out with human blood obtained from various infective and non-
infective diseases in man. The technique adopted in all experiments was
referred to in detail, and will not be described in the present communication.
The most important results were obtained in the examination of the agelu-
tinative properties of the blood when an interaction took place between
serum and red cells. It was shown that auto-agglutination was a rare
phenomenon, but iso-agglutination was common. In some instances hem-
agglutination occurred when the immune serum and normal red cells were
mixed together ; in other cases the effect was produced by the interaction of
normal serum and immune red cells. In many examples of this reaction the
agglutinated red cells were altered in shape and size, especially when the
clumps were exceptionally large. Attention was drawn to the distinction
between agglutination of red blood corpuscles and agglutination of rouleaux.
Saturation experiments were performed, and the specificity of the various
reactions was demonstrated. Immune serum from cases of infection with the
bacillus typhosus was rendered specifically inactive by saturation with suit-
able red cells, although the bacterial agelutinins remained.

Attention must be drawn to the fact that it was stated in the preliminary
report that agglutination was not observed when normal serum was added to
normal red cells, either of the same individual or from another healthy person,
with one exception. A considerable amount of further investigation along
this line has shown that the second part of this statement requires alteration,
as will be subsequently referred to.

Experiments on phagocytosis of red blood corpuscles were in the main
negative, although numerous methods were adopted. It was in only one case

208 Mr. L. 8. Dudgeon. On the Presence of [Feb. 18,

out of the entire number investigated that the phagocytosis was well marked.
In the majority of instances the experiments on hemolysis led to negative
results. In one case of acute poisoning, of unknown nature, physiological
salt solution (0°85-per-cent. pure sodium chloride) was found to be able to
hemolyse the immune red cells. In two cases of pneumonia evidence of
hemolysis was tested for im the blood examined im vitro. In one instance
well-marked auto-hemolysis occurred, in the other iso-hemolysis. Some
degree of iso-hemolysis was noted occasionally in the experiments with the
blood obtained from other diseases, and in one further case auto-hemolysis.

In the present communication many more interesting results have been
obtained in various experiments conducted along the same lines and much
more additional mformation acquired. It will be necessary at the commence-
ment to give a list of the various diseases that have been investigated, and to
point out that exactly the same care has been exercised in proving the
accuracy of the diagnosis.

14 cases of typhoid fever ; 9 cases of tuberculosis (mostly acute pulmonary) ;
3 cases of acute peritonitis due to appendicitis; 14 cases of anzemia (7 of
pernicious anzemia), myeleemia, congenital cholemia, and examples of anemia
secondary to various well recognised conditions; 7 cases of acute pneumonia ;
6 cases of acute streptococcus infection ; 2 cases of epilepsy ; 4 cases of syphilis ;
2 cases of diabetic coma; 3 cases of malignant disease; 5 cases of obscure
toxeemia, including one case of paroxysmal hemoglobinuria.

Teemolysins.

The technique adopted was exactly that already referred to in the pre-
liminary report, with the additional observations obtained by allowing the
mixture of serum and red cells to be in contact in ice for 1 hour previous to
incubation at 37° C. for a similar period, followed by exposure to ice for
several hours. These final results in all cases were similar, so that this.
method was abandoned,

In the previous paper, the following conclusion was arrived at:—‘“In all
these experiments on hemolysis, it was only occasionally that the hemolytic
action was distinctly shown; in the majority of instances no hemolysis
occurred.”

Typhoid Fever—No hemolysis was noted in any of the cases of typhoid
fever which were examined. The results obtained in this disease, as in many
others, were probably due to the fact that normal serwin was only occasionally
employed in these experiments. Out of the total number of cases investi-
gated in the present series—14—herolysis was demonstrated in nine
instances—a very high proportion.

1909. | Hem-agglutinins, etc., in the Blood. 209

It has been noted in these experiments that when the immune serwm
causes hemolysis of normal red cells that the cases are of the severe toxic type
and terminate fatally. It is true that the number of cases investigated is
too small to lay too much emphasis on this observation, but it is unquestion-
ably important.

In the first case, immune serum caused marked hemolysis in the presence
of normal red cells. A gradual diminution in the reaction occurred from the
mixture which contained 75:0 per cent. of serum down to that which had a
serum content of 25:0 per cent., and in one instance 12°5 per cent. In the
second case, which terminated fatally, an exactly similar result was noted ;
in the third case, the blood examined a few days before death and that
obtained at the post-mortem examination gave a similar result, with the
exception that the serum obtained during life had a slightly greater potency.

In the remaining cases in which hemolysis occurred, it was due to the
action of normal serum on the immune red cells, and the reaction took place
with about the same serum content as with the immune serum and normal
red cells in the fatal cases. In all the experiments immune serum and
immune red cells failed to react ; in fact, no other type of hemolysis occurred
beyond that referred to.

The blood of a case of paroxysmal hemoglobinuria has been examined
during the acute attack and during the interval. The blood drawn from the
finger in the usual way failed to show tinging of the serum with hemoglobin
when the usual precautions were adopted. It can be stated briefly that
during the height of an acute attack, which is caused by severe cold, that
the blood undergoes auto-hzmolysis, but when tested as the attack subsides,
although the man is obviously ill, no auto-hemolysis can be demonstrated.
The immune serum also fails to hemolyse normal red cells, although it
agelutinates them strongly, and the immune urine fails to hemolyse the
immune red cells. When, however, normal serum is added in definite
measured volumes to the immune red cells, hemolysis occurs, although not
to any very great extent, and hemolysing agg
demonstrated. It would seem that the red cells themselves are principally
affected—a, point still further illustrated by the phagocytic experiments to
be subsequently referred to. The blood of two cases of diabetic coma
obtained after death failed to heemolyse the immune red cells, but, as in the
fatal cases of typhoid fever, heemolysed normal red cells and agglutinated
them strongly ; in each instance hemolysis occurred down to a serum content

lutinins can be similarly

in the mixture of 37:5 per cent.
Splenic Haxtract—Owing to observations made on the splenic cells in
certain diseases in which much phagocytosis of red cells occurs, it suggested

210 Mr, L. 8. Dudgeon. On the Presence of — [Feb. 18,

itself that in the splenic juice might be found a substance or substances
intimately concerned with phagocytosis, hemolysis, and certain other
phenomena, although absent or present in an infinitesimal degree in the
blood serum. The spleen from one of the cases of diabetic coma was
extracted in a suitable machine, the thick lumpy extract filtered at high
speed, and the centrifugalisation repeated with the upper layer of extract
which separated during the first stage. This final sticky, but thin extract,
was used for testing its hemolytic value on normal red cells and the auto-
immune cells as in the case of serum investigation.

Four series of experiments were made by allowing the splenic extract to
act on auto-immune cells and normal cells, incubating the mixtures at 37° C.
for one hour, and then in the ice safe over night. A similar double series
of tubes prepared in an identical manner were first placed in ice overnight,
then at 37° C. for two hours, and finally in ice for several hours. All
results were identical. It must be pointed out here that the hemolytic
mixture was proved to be free from bacteria. Now while the immune blood
serum in this case failed to heemolyse the immune red cells, and only reacted
slightly in the presence of norinal cells, the action being limited to a serum
content of 37°5 per cent., yet the splenic extract was equally and strongly
hemolytic to normal and the auto-immune cells. The action was complete
with a splenic content of 25 per cent., and limited to a mixture containing
0:25 per cent. of splenic extract. In a case of pernicious anemia, on the
other hand, neither the immune serum nor splenic extract hemolysed the
immune red cells. It is impossible, on the results of two experiments, to
refer more fully to these investigations on the hemolysing action of the
splenic extracts on red blood cells, but from the work of Hédin and others
we know that the spleen does contain proteid enzymes of considerable
potency.

The blood was investigated very fully in seven cases of pernicious
anzemia, and of all diseases it might well be imagined that auto-hemolysis
would be demonstrated in this one. In the preliminary communication it
was pointed out that auto-hemolysis could not be proved. In this paper
a similar result must be recorded, neither could hemolysis be induced by
allowing the immune serum to act on normal red cells. It was found,
however, that the immune serum had the same power to hemolyse guinea-
pigs’ red cells as normal human serum possessed. In three instances
mormal serum was capable of hemolysing the pernicious red cells to a
marked degree: in each example hemolysing agglutinins were present, while
in two out of the three cases phagocytosis of the immune red cells occurred
to a striking degree in the presence of normal serum and normal leucocytes.

es

1909. | Hem-agglutinins, etc., in the Blood. 212

In one instance, although normal serum hemolysed the immune red cells
during the patient’s life, yet the action failed to take place with the immune
cells after death. In every case the serum had the same striking greenish
yellow coloration previously referred to.

In a case of acute primary syphilis, normal human serum was found to
cause a high degree of hemolysis in the presence of immune red cells, and
the immune serum had a somewhat similar although less marked action on
normal red cells. A similar result occurred in a case of acute pneumonia.
The immune serum, when acting on normal red cells and the normal serum
on immune cells, showed a high and equal degree of hemolysis, while the
immune serum and immune red cells failed to react.

These are the most important and striking results in the hemolytic
experiments, but it is necessary to emphasise the fact that many more positive

- results have been obtained than are quoted in the previous report, and those

which are of special importance occurred in the blood reactions of the typhoid
fever patients.

In many of these experiments on hemolysis, and similarly in the
agglutination and phagocytic investigations, the chief interest centres around
the ummune red cells. It is especially concerning these bodies that further
research is being conducted.

Hem-agglutinins.

Attention has already been drawn to the fact that the same technique has
been adopted in all these experiments as previously given in detail in the
preliminary communication.

I must again emphasise the rarity of auto-agelutination of red blood
corpuscles, With. one striking exception—that of a black man—no true
example has been met with. This case will be subsequently referred to.
in detail.

In every instance, without exception, iso-agglutination occurred whenever

iso-hemolysis was demonstrated. While the agglutination experiments were

being examined microscopically it was generally possible to detect the
hemolytic agglutinins from the pure agglutinins. In the former, many red
cells at the margin of the clumps were smaller than those centrally placed
and greatly resembled oil droplets. In the most striking instances, free
hemoglobin could be seen and numerous ghosts.

Iso-agglutination was of common occurrence in many diseases, as stated in

the preliminary communication. ‘This was especially so in typhoid fever and
tuberculosis.

212 Mr. L. 8. Dudgeon. On the Presence of — [Feb. 18,

Hem-agglutinins in Normal Blood.

Attention has already been drawn to a statement made in the preliminary
communication on this subject. Since then a wide study of hem-agglutinins
in normal blood has been made. It has now been proved that although a
certain sample of normal serum may fail to react with one or two specimens
of washed normal red cells, yet, if sufficient examples of normal red blood
cells are presented to that specimen of normal serum, agglutination will be
found to occur in a certain proportion of instances, while some samples of
normal serum agglutinate most specimens of normal red cells presented to
them. With our wider knowledge we can state that auto-agglutination does
not occur, and that the hemolytic agglutinins have not met with, that type
of agglutination which is of such interest in typhoid and certain other
infections. These results are important because they compel one to realise
that the demonstration of hem-agglutination is not necessarily a reaction
of pathological significance.

Auto-agglutination.

A negro from the West Indies, who had not been out of England for over
ten years, showed a blood possessing remarkable properties. He was
considered to be a case of tertiary hepatic syphilis. When blood escaped
from his tissues from a single puncture, the red cells could be seen to be
clumped in the plasma, and when the bleeding was continued into citrated
-saline the red cells fell to the bottom of the tube in enormous clumps. This
is the only instance met with of a blood showing spontaneous agglutination,
and auto-agglutination of such a high degree. In the preliminary com-
munication, attention was called to the condition of the blood in a ease of
long standing epilepsy : here well-marked auto-agglutination was present, but
not spontaneous agglutination, and the degree of agglutination was nothing
like to the same extent as was obtained with the blood of the negro. On
the addition of the immune serum to normal red cells a high degree of
agglutination occurred, and it was the true hemolytic type.

It had previously been shown that if an immune serum is diluted with
normal saline, it rapidly loses its power of agglutinating red cells, quite out
of proportion to what is observed in the case of the bacterial agglutinins.
This sample of immune serum, however, clumped normal red cells when
diluted to the extent of 1 in 10 almost as well as the undiluted serum, and
some clumping occurred in a dilution of 1 in 500. This is the only instance
out of the whole series of sera which have been examined in which such
a phenomenon was noted. When the negro’s blood was examined about

1909. | Hem-agglutinins, etc., in the Blood. 2138

a month later, neither spontaneous nor auto-agglutination was present, and
normal serum failed to react with these cells, while when the immune serum
and normal red cells were mixed hemolytic agglutination occurred as:
before.

Typhoid Fever.—Auto-ageglutination was never found to occur. The
iso-agglutinins are of great interest. In every instance marked agglutination
of red cells occurred when either the immune cells were added to normal
serum or normal cells to immune serum. In a certain number of experi-
ments both phenomena occurred. The fact that there was absence of
agglutination when immune serum was added to normal red cells, although
the serum clumped typhoid bacilli, can readily be explained by the absence
of any relationship between bacterial and hem-agglutinins. It was
especially in these experiments that the hemolytic agelutinins were noted
after serum and red cells had been in contact for one hour at 37° C.

Attempts were made to extract “hypothetical agglutinins” from the red
blood corpuscles in those diseases in which a marked reaction took place
between the immune red cells and normal serum, but without success. The
red cells were powdered in a mortar with finely broken glass, and the
mixture centrifuged at high speed. The fluid resulting from this procedure
was added to suitable red cells, but in no instance did agglutination occur.
Other methods with a similar object in view were found to be equally futile.

Specific Agglutinins.

A considerable amount of further work has been completed to emphasise
the specificity of the hem-agglutinins. The same technique has been
adopted, except that it has been shown that it is unnecessary to incubate the
active mixtures of red cells and serum for several hours as on previous
occasions. Two or three hours has since proved to be sufficient, while it has
been shown that a considerable degree of this special reaction can be
removed when the active red cells and serum are mixed well together for two
or three minutes, not incubated, but immediately centrifuged, and the
resulting fluid tested in the usual manner. It was found that although
slight agglutination with the clear fiuid and suitable red cells could be
demonstrated, yet the greater proportion of it had been removed.

This experiment will serve to show the effect of a temperature of 37° C.
for one and a quarter hours on the specific agglutinins:

Immune Serum (Typhoid) + Normal red cells.
Extreme degree of agglutination present.

VOL, LXXXI.—B. Q

214 Mr. L. 8. Dudgeon. On the Presence of — [Feb. 18, —

Same serum saturated with same undiluted normal red cells for one and
a quarter hours at 37° C.; mixture centrifuged.

Clear fluid + Normal red cells.
No agglutination.

When the immune typhoid serum was saturated with the typhoid red cells
an a similar manner to the above, the clear fluid agglutinated normal red
cells as before. It must be pointed out here that although saturation of
a serum with suitable red cells will render the serum specifically inactive,
and that saturation with inactive red cells will have no effect, yet, if the
Serum is capable of agglutinating the active red cells from two different
diseases, saturation with one class of red cell may fail to render the serum
inactive for the other red cells. Further work is being done on this
important point at the present time.

The Effect of Saturating an Immune Serum with Melanin.

It has been shown conclusively in this paper that if you saturate typhoid
serum with suitable red cells you remove the hem-ageglutinins, but leave the
bacterial, and vice versd.

Other experiments were undertaken for the purpose of ascertaining whether
it would be possible to remove or greatly diminish the hem- or bacterial-
agglutinative action of the serum by means of melanin as Mr. Shattock and
I have found to occur in experiments on phagocytosis.* Immune serum
(typhoid) was saturated with sterile melanin for 12 hours at 37°C. The
resulting fluid obtained after centrifugalisation was still capable of agglutinating
typhoid bacilli and suitable red cells as the unsaturated serum, and also to
the same degree.

The Effect of Saturating a Serum with Heated Red Cells.

In the experiments about to be referred to, it has been shown that it is
possible to remove the agglutinative properties of a serum by saturating it
with heated red cells. The red cells for this purpose are thoroughly washed
in the usual manner, and definite proportions from the deposit of red cells
obtained at the end of the final process of centrifugalisation are put in suitable
tubes with a small quantity of physiological salt solution ; the tubes are sealed,
placed in a tube of water at a definite temperature, and then put into a water
bath at the same temperature as the tube of water. By this means, and this

* §. G. Shattock and L. 8S. Dudgeon, ‘ Roy. Soc. Proc.,’ B, vol. 80, 1908, “ Observations
on Phagocytosis by means of Melanin and the Comparison of the Opsonic Index with the
Hemo-phagocytic Index.”

215

mm the Blood.

mins, etc., mt

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216 Mr. L. 8. Dudgeon. On the Presence of — [Feb. 18,

only, is it possible for the cells to reach the temperature indicated by the
thermometer. We know that it is stated that if a serum is heated to 65° C.,
it loses certain properties which the unheated serum possesses; as a matter of
fact, if human serum is kept at the temperature of 65° C. for 15 minutes in
the manner indicated, it is rendered useless for serum investigation owing to
the fact that it is coagulated.

In the experiment about to be referred to, certain immune red cells were
heated at 60° C. in the manner indicated for 50 minutes, and at the end of
that time formed a thick dark mass. Pneumonic serum and these red cells
gave a high degree of agglutination before saturation, but after the serum
had been saturated with the heated red cells for one and a quarter hours at
37° C., it was rendered inactive. This shows that, although the red cells
were physically so altered, yet they still possessed a specific function. Similar
results were obtained in another series of experiments in which the red cells
were heated to 60° C. for one hour.

The Relation of the Bacterial and Hem-agglutinins.

Several series of experiments have been completed, and the results clearly
demonstrate that the bacterial and hem-agglutinins are distinct. Typhoid
serum, especially a serum possessing high agglutinative properties, is
especially suitable for the purpose.

As will be seen from the accompanying table, the highest degree of
specificity exists, and every experiment gave similar results. The saturation
experiments were carried out in every detail in an identical manner.

Phagocytosis.

In summarising the technique and the results of the experiments on
phagocytosis in my preliminary paper, it was stated that “ It is unnecessary
to refer at great length to the very large number of experiments made, as it
was only in a few instances that phagocytosis was pronounced. In quite
a number of instances, whether the serum was unheated, or diluted, or heated,
or whether normal or immune leucocytes were employed, the degree of phago-
cytosis was infinitesimal.’ There was only one instance out of the total
number investigated in which the phagocytosis of red blood corpuscles was
a conspicuous feature. In the present communication striking instances of
red cell phagocytosis have been recorded on several occasions, these different
results mainly depending upon the nature of the diseases which are now
being investigated. It is especially marked in such acute diseases as typhoid
fever, while in pernicious anemia, where it might be expected to occur, it is

1909. | Hem-agglutinns, etc., in the Blood. 217

usually absent. Care must always be exercised in arriving at conclusions on
this subject, because, unless the film preparations are made with special
precautions, red cells which are apparently engulfed are really extra-cellular,
and also, if the hemolysins are active in the samples of serum employed, the
red cells which are engulfed may be either pale or difficult to recognise, and,
therefore, phagocytosis may be unknown. In a case of typhoid fever with
typhoid pyuria a high degree of phagocytosis was noted. Normal leucocytes
+immune red cells+ normal serum showed no less than 37 red cells engulfed
by 50 leucocytes, while when the normal serum was diluted with immune
serum the phagocytosis was correspondingly reduced. In this instance
normal serum caused marked agglutination of immune red cells and also
heemolysed them; in another case of typhoid the phagocytic test carried out
in an identical manner showed 26 red cells engulfed by 50 leucocytes, the
normal serum in this case also hemolysed and agglutinated the immune red
cells, while in a still further example the immune serum+normal red cells
and immune leucocytes gave an active result—50 leucocytes contained 39 red
cells; when normal serum was added to the immune red cells the phagocytosis
was well marked, but very much less so; here also the immune serum
strongly agglutinated normal red cells, but did not hemolyse them. In
certain other instances of typhoid infection some degree of phagocytosis was
noted, but only of an indifferent type. In the case of paroxysmal hemo-
globinuria the immune red cells in the presence of normal serum and normal
leucocytes showed a high degree of phagocytosis, 50 leucocytes contained
39 red cells. Experiments conducted with other variations in the cells and
serum gave negative results.

The normal serum in these experiments, hemolysed, agglutinated and
incited phagocytosis of the immune red cells, but in different degrees, the
most marked results occurring in the agglutination experiments, the least
striking were in the hemolytic. Ina case of untreated secondary syphilis,
experimental results showed absence of hemolysis and agglutination, but
complemented immune serum incited a high degree of phagocytosis of the
immune red cells. Perhaps the most striking example of phagocytosis of the
red blood corpuscles occurred when the serum from a case of epilepsy was
added to normal red cells in the presence of normal leucocytes, 50 white cells
contained 46 red blood corpuscles. The immune serum also strongly
agglutinated these cells. Many more examples of this variety of phagocytosis
have occurred in the numerous experiments which have been made, but the
most interesting and striking results are those which I have recorded.

All instances in which hemolysis occurs when the red cells and serum
interact must tend to limit the accuracy of the phagocytic experiments,

218 On the Presence of Hem-agglutinims, etc., in the Blood.

because so many red cells which have been engulfed by the leucocytes are
ghosts and, no doubt, have been too severely heemolysed to be recognisable.

In the concluding remarks in the preliminary paper on hzem-opsonins, it
was stated that “ The experiments referred to in this communication entirely
agree with the observations of Barratt and Keith, conducted with the blood
sera and cells from the lower animals. There was nothing to show that the
agglutination, opsonic, or hemolysing properties of normal or immune sera on
red blood corpuscles have any direct relation to one another.”

From the investigations which have been made for this report, some of
which have been referred to, it has been noted that a serum may hemolyse,
agglutinate, and incite phagocytosis of certain red cells, but although it
may be capable of agglutinating and hemolysing certain red cells, yet the
degree of phagocytosis which is present may be negligible, or phagocytosis may
be present without the other phenomena; on the other hand, a serum which
has a high agglutinative value is more likely to give a somewhat similar
incitor reaction than otherwise, although not necessarily.

I have again to thank Mr. H. A. F. Wilson for his invaluable assistance,
and also Dr. Athole Ross and Mr. Irvine, all co-workers in my laboratories.

219

The Theory of Ancestral Contributions in Heredity.
By Karu Pearson, F.R.S.

(Received March 19,—Read April 22, 1909.)

Under the above title a paper has recently appeared by Mr. A. D.
Darbishire in the ‘ Roy. Soc. Proc., vol. 81, B, p. 61 ef seqg., giving further
experimental evidence with regard to the inheritance of certain characters
in peas. The paper is an interesting one, but the method adopted is not,
I venture to think, capable of answering the problem which the author set
himself. It has been supposed by some Mendelians that the theory of
inheritance summed up in the “law of ancestral heredity ” was in some way
invalidated by investigations such as Mr. Darbishire’s, and that opinion
consciously or unconsciously seems to be expressed in the paper just
referred to. The law of ancestral heredity is embraced in the following
statements :—

(i) In a population breeding without assortative mating the regression line
for offspring on any ancestor is linear.

(ii) The correlations between offspring and the successive grades of
ancestry form a progression diminishing geometrically as we ascend to distant
grades ; and

(1) The general relation of an individual to his ancestry can be closely
expressed by the multiple correlation formula.

In a memoir published in the ‘ Phil. Trans., vol. 203, pp. 53-86, I showed
that these principles held for material obeying Mendel’s laws—in particular
(i) and (11) hold for the simple case of alternative characters such as are
said to oceur in the case of peas.

The only instance that I am aware of in which ancestry does not matter
is that in which the geometrical progression is of the form :

PyPeni Boia:

I treated this case at length in the ‘ Phil. Trans.,’ vol. 187, A, pp. 304-6
(1896), remarking that the grandparents were quite indifferent, when the
parents had been selected. Unfortunately, this is not true when the
correlation coefficients are

x i 3 x a x L ete

Se AOS Bis 8 BY i
as is the case with the somatic correlations on the Mendelian theory. In other
words, ancestry does matter in the latter theory. What is the explanation,

220 Prof. K. Pearson, [ Mar. 19,

therefore, of the apparent contradiction between such experiments as those
of Mr. Darbishire and the theoretical development of the Mendelism which
they profess to establish ?

It does not seem hard to account for the divergence. Experiments such
as those of Mr. Darbishire do not deal with a population as a whole, and
consider the contributions to the next generation of all its components
supposed to be mated at random. I feel quite certain that if Mr. Darbishire
makes the requisite crosses in due proportions, and does not weight with
differential fertility, he will find that ancestry does matter. That it does
matter is just as good a proof of Mendelism as Mr. Darbishire’s proof in the
simpler case that it has not any effect. If he fails to find its influence, then
he will have refuted Mendelian theory.

To illustrate my point, take a population distribution which would follow
from crossing two pure races with respectively dominant and recessive
characters represented by the letters D and R. Suppose the hybrids to
eross at random, then the population will remain absolutely stable with the
permanent formula

(DD)+2(DR)+(RR).

Now suppose this to cross with itself or with
(DD)+ 2 (DR) +(RR).

Table I gives the scheme of offspring with their parents. This population
of 16 individuals of 6 different types of parentage now crosses with itself.
The result is a population of 256 individuals showing 15 types of grand-
parentage. This is exhibited in Table II. If Mr. Darbishire’s principle
that ancestry is of no importance were correct, then the differences in type
of these grandparents would not be of any significance.

Table I.

| Offspring.

Parents. | = |

| le] we

1909.| The Theory of Ancestral Contributions m Heredity. 221

Table II.

Grandparents. Offspring.

RR. DR. RR.

S)
=)
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Now Table Il may be examined from several standpoints. We may
first consider the gametic constitutions of the grandparents and of the
offspring. Thus we have :—

No. of DD’s in grand- | Percentage of DD’s in
parentage. | offspring.
|
4 100
3 67
2 41
1 22
0 11

In other words, the constitution of the grandparentage substantially
modifies the offspring ; there exists in this sense an “ ancestral contribution ”
to the heritage.

Of course the gametic constitution RR follows precisely the same system
of percentages, and is again influenced by ancestry.

If, however, we take the gametic constitution DR in such a population
we find—

No. of DR’s in grand- Percentage of DR’s in
parentage. offspring.

OrFNnNwe
OI
(=)

222 Prof. K. Pearson. [Mar. 19,

At first sight this seems to indicate that for this case there is no ancestral
influence, where we should expect by increasing the number of DR’s in the
grandparentage to increase the number in the offspring. But this criticism
is not valid, for, in the population we are dealing with, it is clear that DR is
the modal or mean group, and that, accordingly, it is perfectly neutral in
determining the regression or correlation of the gametie character. In
other words, the deviations of the DR ancestry from the mean population
gametic character are all zero and accordingly they have no weight in
causing the offspring to deviate from the population norm. They have,
in fact, no more effect on the offspring than, in the case of stature, a number
of mediocre ancestors have in raising or lowering the average deviation of
the offspring from the general population mean.

Lastly, turning from the gametic constitution to the somatic character,
I have represented in the fourth and fifth columns of Table II the extent
to which the dominant character is present in the ancestry, and in the
accompanying table one sees the effect on the offspring :—

No. of grandparents with | Percentage of offsprimg with
dominant character. dominant character.
4 89
3 78
2 59
1 33
O (0)

It will thus be obvious that, judging solely by the patent, that is the
somatic character of the grandparentage, there is a very marked influence
of the ancestry on the heritage; that, if we select ancestry by somatic
character only, we shall expect an influence on the offspring varying from
0 to 90 per cent. in intensity, according to the nature of the selection.

I think, therefore, that to deny the influence of ancestry—at any rate
that influence in the sense in which the biometrician uses the term—is to
deny the application of Mendelism to populations mating at random.

If we start with a population in which the proportions of DD’s, DR’s, and
RR’s are not those of a simple hybridisation, but given by

p(DD) + 29 (DR)+5(RR),
then after the first generation of random mating the population will be

(p+ 29¢+8) (p+)? (DD)+2 (p+) (s+9q) (p+ 2¢+5)? (DR)
+(p+2q+s)?(s+¢)? (RR),

1909.| The Theory of Ancestral Contributions in Heredity. 223
_ or its constituents will be proportional to

(p+q?(DD)+2(p+q)(s+@ (DR)+(s+9)? (RR),

and this ratio is maintained ever afterwards.*

A little consideration will show that our Table II is obtamed by a
symbolic process which will not be affected if we replace D by (p+q) D
and R by (s+q) R, so that to exhibit the results for a Mendelian population
of any constituent proportions we have only to multiply all the numbers
in any row of offspring of Table Il by (p+g) for a DD grandparent, by
(p+q) (s+q) for a DR grandparent, and by (s+g)? for an RR grandparent,
starting with the stable population which arises after the first random
mating. We then reach the following table for the case of classification
by somatic characters, where for brevity I write: p+qg = 7,s+q=kx, and
m?/x? = n = ratio of pure dominants to recessives in the stable population.
It will be seen that whatever be the proportions of the Mendelian com-
ponents in the original population, then a selection of grandparents influences
widely the somatic characters of the offspring.

Whether, therefore, Mendelism be or be not the final word as to inheritance
(and I personally, especially in the case of human characters, must continue
to suspend my judgment), it is clear that ancestral influence cannot be
denied in the case of any population mating at random and inheriting on
Mendelian lines.

Table III.
| No. of grandparents with | Percentage of offspring
dominant character. | with dominant character. |
4 | 100 (7+1)(2+8)
| | (n+2)? }
| n+1)(22+5)
3 Tog Masia)
2(n+2)?
5 100 (52+11)(~+1)
mi 6 (a+2)?
1 (w#+1)
2(n+2)
0 0

* The stability after the first generation is very obvious, but, as far as I know, was:
first stated in print by G. H. Hardy, ‘Science, vol. 28, p. 49.

224 The Theory of Ancestral Contributions in Heredity.

We have the following table for various values of ”:—

Novok eeandpaente Percentages of offspring with dominant character.
with dominant
character. 3 S10 La 2. ils. eat = =i.

4 99 3 | 97 94. 89 84. 80 77
3 95 | 90 84 78 72 68 65
2 78 72 66 59 54 50 48
i 46 42 37 °5 33 30 28 26
10) 0) 0) 0 0 0) 0) (0)

L 2 ! | * '

When experimental work is adduced to demonstrate that ancestry has no
influence, it will on investigation be found that the writer is:

(i) Confining his attention, as Mr. Darbishire, to isolated lines of inheritance,
with restricted matings ;

(ii) Asserting that a gametic knowledge of parents is equivalent to a
gametic knowledge of ancestry.

In neither case does the argument touch the ancestral position, which is
summed up in the assertions that if we measure inheritance by the resem-
blance of somatic characters between offspring and ancestry, then, in a
population mating at random :

The more ancestors of any grade with a given somatic character the more
offspring with that character.

For ancestry of different grades the influence is diminished in geometrical
progression at each stage.

These principles were first deduced empirically from observations and
records without any theory as to the mechanism of heredity. If Mendelism
be true for any characters in cross-fertilised plants, then these principles
hold also for heredity in that plant-population, for they are essential features
of the Mendelian theory (and, as a matter of fact, of a good many other
determinantal theories). No proof or disproof of them can be directly
deduced from Mr. Darbishire’s memoir, but since that memoir brings evidence
for the truth of Mendelian theory, it indirectly asserts the truth that
ancestry is influential, at least in the field where the biometrician expects
and asserts it to play a part. This paper contains only another aspect of
the results reached in 1904, but it provides in the simple case—the grand-
parentage—the actual percentage measures of , the influence of ancestry
according to Mendel. Its justification is the misinterpretation which is likely
to be placed on the statement that “there is nothing like ancestral
contributions witbin the limits of a single unit-character.”*

* Darbishire, ‘Roy. Soc. Proc.,’ B, vol. 81, p. 71.

bo
bo
ou

On the Ancestral Gametie Correlations of a Mendelian Population
mating at Random.

By Karu Pearson, F-.R.S.
(Received April 2,—Read April 22, 1909.)

(1) The population to be considered in this paper is supposed to be
initiated by a group of s; individuals with the protogenic constitution (AA),
s, individuals with the allogenic constitution (aa), and s3 individuals with
the hybrid constitution (Aa), where the mating is given by the simple
Mendelian formula: (AA) x(aa) =4(Aa). I do not assume at this stage
any relation between the gametic constitution of an individual and its
somatic character. I propose first to consider the correlation between any
ancestor and the resulting array of offspring, when we regard only their
gametie constitutions. JI assume that all mating in the population is.
random, 2.c. that every possible mating occurs simply in the proportions of
the frequency of individuals of given gametic constitution in the population,
and that there is no differential fertility or selective death-rate.

In a paper published in the ‘ Phil. Trans.,’ vol. 203, A, 1904, p. 53 e¢ seq.,
I have dealt with the correlation between the somatic characters of the
aneestry and the offspring in a population of a Mendelian character, more
general in that I supposed the character to depend upon » couplets, and not.
a single Mendelian couplet, less general in that I supposed the population
to have arisen from a series of initial hybridisations, and not from a
mixture as in the present case of hybrids and members of two pure races.
in any proportions. In that paper I showed (a) that there was correlation
between any ancestor and the offspring, (6) that the regression for any
ancestor and the offspring was linear, and (c) that the correlations decreased
in geometrical progression. These are the chief characteristics of the Law of
Ancestral Heredity. It was clear that, judged by somatic characters only,
ancestry was of importance. The result depended on Mendel’s first principle
of dominance being absolutely true. The values of the correlations were, how-
ever, less than those with which biometric work had made us familiar.

(2) In the present paper I start with a more general population, and
investigate the correlation of the gametic not the somatic characters.

The general formula for the population before the first mating is

5 (AA) -+ 283 (Aa) + 82 (aa). (i)
After the first random mating it is

(81 +83)? (AA) -+ 2 (s1 + 53) (So+53) (Aa) + (52+ 53)? (aa).

226 Prof. K. Pearson. On the Ancestral Gametic [Apr. 2,

I write this for brevity
p’ (AA) + 2pq (Aa) + 9? (aa), (il)

and this constitution remains permanent in all successive matings. Hence
the standard deviations of the gametic constitutions remain the same
generation after generation, and the correlation coefficient is in every case
equal to the slope of the regression line. I shall determine the slope of this
line which will give the correlation and show that the regression is truly
linear in each case.

(3) I consider first the effect of individuals of each special type mating
with the general population (11).

(a) Type (AA): the array of offspring is (p+ 4) [p(AA)+ 4 (Aa)],
(4) Type (aa): (p+) lp (Aa) +¢ (a0)],
(c) Type (Aa): » 3(p+Q)[P(AA)+(p+9)(Aa)+9(ae)].

Thus, in seeking what any differentiated group
tf) (AA)+¢s (Aa) + ts (aa)

produces when mated with the general population, 7e. when mated at
random, all we have to do is to replace (AA), (Aa) and (aa) by the above
three expressions respectively.

In this manner I obtained the array of offspring due to any parent, any
grandparent and any great grandparent. These at once allowed me to
reach the general law of distribution, and, assuming this, one multiplication
by the general population (ii) demonstrated by induction the validity of the
results reached. These are as follows :—

I term nth parent any individual x generations back in the direct ancestry :
thus a lst parent is the father or mother; a 2nd parent, a grandparent;
a 3rd parent, a great grandparent, and so on.

(i) If the mth parent be an (AA), then the array of offspring due to random
matings is
p(ptgre Gy *{(2""* p+q)p(AA)+[(2"— 1) p+9)q (Aa)

+(2"-1—1) 9? (aa)}.

(ui) If the nth parent be an (Aq), then the array of offspring is
(pq) (p+ gr (By? {[2"—D) p+) p (AA) + [p? + 2(2"—1) pg +?) (Aa)
+[p+(2"—1) 9] 9 (a@)}.
(au) If the nth parent be an (aa), then the array of offspring is

Q (p+gyre (a) > {(2" *—1)p? (AA)+[(2"—=1)¢+-plp (Aa)
+(2"-19 +p) q (aa)}.

1909. | Correlations of a Mendehan Population. 227

(4) These distributions correspond to the cases of 2, 1 and 0 A elements
in the gametic constitution of the nth parent. And we have at once the.
following result :—

Number of protogenic elements Average number of same
in nth parent. elements in array of offspring.

Dnt 1, 9

2 See i ei ieee a lie cea => Y2,
2" (p+)

Dn ahaa

OUR EE AM anette e Qt =)pt+g = Th,
2" (p+4)
Qrt1_9 t

Opie cenit a vea sis os es) p = Y.-
2"(p+q)

Accordingly, the average number of protogenic elements in the array of
offspring decreases uniformly with the decrease in number of the like
elements in the nth parent, 2.¢.

y—y = (3) = A—Jo.

Thus the regression between the nth parent and the offspring is linear,
and the correlation coefficients form a geometrical series of ratio }, and
first term 3. Further, the exact constitution of the population, as far as
the number of protogenic, allogenic or heterogenic individuals is concerned,
is of no influence on the result at all. For all mixtures following the simple
Mendelian rule: (AA) x (aa) = 4(Aqa), the ancestral correlations for gametic
constitution are :

iRarentalycorrelationv ser ..see asec: 0-500
Grandparental correlation ............ 0-250
Great grandparental correlation...... 0°125 and so on.

It will be seen at once that these correlations are of the type p, p?, p®, etc.,
for which, in my memoir of 1896, I worked out the multiple regression
formula, and showed that the ancestors were quite indifferent. “A know-
ledge of the ancestry beyond the parents in no way alters our judgment as
to the size of organ or degree of characteristic probable in the offspring nor
its variability."* This remark and the proof apply equally of course to
gametic and to somatic characters if the correlation be of the above form.

(5) Accordingly there remains not the least antinomy between the
Mendelian theory and the Law of Ancestral Heredity, if we confine our
attention to gametic constitution. The Mendelian ancestry is correlated
with the offspring in a series descending in a geometrical progression, and
the regression is linear. The values of the correlation coefficients are

* “ Regression, Heredity, and Panmixia,” ‘ Phil, Trans.,’ A, vol. 187, 1896, p. 306.

228 Prof. K. Pearson. On the Ancestral Gametic [Apr. 2,

precisely those which it was pointed out in 1896 would lead to a knowledge
of the parental constitution* replacing that of the ancestry.

(6) The striking point, however, of the present investigation is that the
values now shown theoretically to exist for the ancestral gametic correlations
in a simple Mendelian mixture are very close to those determined for
somatie characters in biometric investigations, whereas the somatic correla-
tions for a Mendelian population, if we maintain intact the principle
of absolute dominance, appear theoretically to be too low.

Thus the value for parental correlation in man, horse, dog and cattle is
about 0:5, and for the grandparental correlation lies between 0°25 and 0°50;
but this tendency in the grandparent to some shght excess on the Mendelian
gametic value must not be given too much weight.

(7) It seems desirable to consider how far the results in my paper of
1904 for the somatic correlations are modified if we assume for our popu-
lation

p’ (AA) + 2pq (Aa) +9? (aa),
and do not make p = gq.

Assuming the principle of dominance to be absolute, I enquire what is
the proportion of offspring possessing the dominant charactert (7.e. (AA)
or (Aa) ) supposing the xth parent to possess it (ze. to be (AA) or (Aa) );
and again, what is the proportion possessing the dominant character,
supposing the zth parent does not possess it (7c. to be aa).

Percentage of dominant offspring.
(100 x eae
27" (p+ 9) (p+ 29)
5100 x 2 PPh 29 aie
2" (p+ ay (P+ 29)
From this it follows that the correlation which is equal to the regression is

nth parent dominant in somatic character.

nth parent recessive in somatic character .

e-scatel
Qn-1 p 429°

; hE gl hh Meta ‘ :
If p= 4, this is 3 Jnr» In agreement with the conclusion of my memoir

of 1904. But unless ¢/ (p+2q)=}, we. the number of pure dominants in the
population be vanishingly small (as well, of course, as the number of impure
dominants !), this is not a series to which the form p, p*, p? . . . applies, and
when we judge (as we must in most instances in man) by the somatic and
not the unknown gametic constitution, the ancestry does matter.

* Asa matter of fact, a knowledge of the gametic constitution of the ancestry in any
generation would be equally sufficient with that of the parents.
+ It is assumed that A is dominant over a.

1909. | Correlations of a Mendelian Population. 229

The following table illustrates the percentages of dominant charactered
offspring when we selected an ancestor of given character :—

Percentage of dominants in offspring.

Ancestor. p= 24. Pp=4. q = 2p.
Dominant. | Recessive. | Dominant. | Recessive. | Dominant. | Recessive.
|
(Parenti wre teanecacahcavest 91°7 66 ‘7 83 °3 , 50°0 57°8 33 °3
Grandparent ............ 90 °3 77°8 79 2 62 °5 56 °7 44 °4,
ord parent’ ............... 89 6 83 °3 77:1 68 -7 56:1 50 ‘0
4th parent ............... 89-2 86:1 | 76-0 71°9 55 °8 52°8
5th parent ............... | 89-1 87 °5 75 °5 73 °4 55-7 54°2
6th parent ............... | 89-0 88 °2 75 °3 74-2 55 °6 54:9
oth parent............... | 88:9 88 °9 75 °0 75-0 55 6 55 °6

It will be clear that the difference of the percentage of dominants in the
offspring according as a parent, grandparent or great grandparent was
dominant or recessive in somatic character is quite marked ; and only as we
approach the higher ancestry, where the correlation is growing very weak,
does the percentage difference grow imperceptible.

(8) That ancestry does not matter if we know the gametic constitution of
the parents, that it does matter if we only know the somatic character of
the parents appears to be the solution of one of the difficulties which some
have found between the Mendelian and biometric methods of approaching
the subject.

There is, however, I venture to think, another aspect of these results
which is worthy of fuller consideration. Namely, the fairly close accordance
now shown for the first time to exist between the ancestral gametic
correlations in a Mendelian population and the observed ancestral somatic
correlations suggests that the accordance between gametic and somatic
constitutions is for at least certain characters possibly more intimate than
is expressed by an absolute law of dominance. If (Aa) were a class, or
possibly on a wider determinantal theory a group of several classes, marked
by an individual somatic character—not invariably identical with the
somatic character of (AA)—there would be little left of contradiction
between biometric and Mendelian results as judged by populations sensibly
mating at random. It is the unqualified assertion of the principle of
dominance which appears at present as the stumbling block.

VOL. LXXXI.—B. R

930

The Origin and Destiny of Cholesterol in the Animal Organism.
Part V.—On the Inhibitory Action of the Sera of Rabbits
fed on Diets containng Varying Amounts of Cholesterol on
the Haemolysis of Blood by Saponn.

By Mary T. FRASER and J. A. GARDNER.

(Communicated by Dr. A. D. Waller, F.R.S. Received April 3,—Read
May 6, 1909.)

(From the Physiological Laboratory, University of London, South Kensington.)

In an earlier paper* of this series it was shown that cholesterol is not
excreted in the feces of herbivorous animals, and that when rabbits are fed
on a diet free from phytosterol but containing measured quantities of
cholesterol, a portion of the latter substance is absorbed. The hypothesis
was put forward that cholesterol is a substance which is strictly conserved in
the animal economy; that when the destruction of the red blood corpuscles
and possibly other cells takes place in the liver, their cholesterol is excreted
in the bile, and that the cholesterol of the bile is reabsorbed in the intestine
along with the bile salts, and finds its way into the blood stream to be used
in cell-anabolism. It was also suggested that any waste of cholesterol might
possibly be made up from that taken in with the food. In order to test this
view, comparative estimations were made of the total cholesterol content
of the blood of rabbits that had been respectively fed on bran which had
previously been thoroughly extracted with ether, and on the same extracted
bran with the addition of known amounts of cholesterol, care being taken
that the animals were otherwise identically treated and were kept in good
health. The results of the experiments showed that some, at any rate, of the
cholesterol absorbed found its way into the blood stream. It seemed to us.
desirable to ascertain next whether the cholesterol was absorbed into the
blood stream as such or in the form of esters or in both states, and also
whether the phytosterol of vegetable food can be utilised for the formation
of cholesterol in the organism. Owing to the small percentage of these
substances in the blood, it did not seem probable that the chemical methods
hitherto available for their estimation were sufficiently accurate to give
reliable information unless in each experiment a larger number of animals
was taken than we could conveniently attend to. It seemed likely, however,
that a comparative study of the inhibitory effects of the sera of rabbits fed

* “Origin and Destiny of Cholesterol,” Part III, ‘Roy. Soe. Proc.,’ B, vol. 81, 1909
p- 109.

Origin and Destiny of Cholesterol in the Animal Organism. 231

on different diets on the hemolytic action of saponin on blood might throw
light on the points mentioned.

The experiments of Hausmann,* Abderhalden and Le Count} have proved
that whereas cholesterol and phytosterol inhibit the action of saponin, their
esters do not do so. More recently Windaus{ has shown that the inhibitory
action of cholesterol and phytosterol is due to the fact that they form
pharmacologically inactive compounds with saponin. We therefore decided
to make a comparative study of the inhibitory action of the sera of rabbits
fed respectively on ether-extracted bran and extracted bran with cholesterol,
extracted bran and extracted bran with cholesterol esters, and, finally,
extracted bran and extracted bran with phytosterol.

In the present paper an account is given of these experiments.

Method of Feeding the Animals under Experiment.—In each experiment,
two large healthy rabbits, A and B, were fed for nine or ten days on bran
which had been thoroughly extracted with ether. Excess of food was placed
in the cages so that the animals could eat as much as they wished. In the
ease of rabbit A, after the third day a weighed amount of cholesterol mixed
with a small quantity of moist extracted bran was given daily in addition,
care being taken that the animal ate the whole of it. The rabbit was killed
three or four hours after the last cholesterol meal, and the blood collected in
a sterile vessel. The blood was allowed to clot and was placed in the
refrigerator until the serum separated. The rabbit B was killed at the same
time and its blood collected and treated in a similar manner. Great care was
taken to keep the animals in good health and as far as possible under the
same conditions. It is also desirable not to extend the experiment over too
long a period lest the continued sameness of the diet should have a deleterious
effect. If the animals are in bad health, more especially if they waste away
and become emaciated, the results are entirely vitiated. This is well illus-
trated in the following experiment, which was commenced for another
purpose and only continued as a matter of curiosity. Six medium-sized
rabbits, weighing respectively 1°3, 1-4, 1, 1:2, 1:5, 2:3 kilogrammes, which
were, to begin with, in very poor condition, were fed for three weeks
on extracted bran. They continued im poor condition and became very
emaciated, and had the experiment been further prolonged would no doubt
have died. They yielded altogether only 190 grammes of blood. The blood
was analysed by the method described in an earlier paper.§ 0-1799 gramme

* ‘ Hofmeister’s Beitr.,’ vol. 6, p. 567.

+ * Ztschy. fiir exper. Path. und Ther.,’ vol. 2, p. 199.

{ ‘Ber. d. deut. chem. Ges.,’ 1909, vol]. 42, No. 1, p. 288.
§ Lbid.

232 Miss Fraser and Mr. Gardner. Origin and [ Apr. 3,

of pure cholesterol benzoate was obtained, corresponding to 0°1423 gramme of
cholesterol, or 0:0745 per cent. This is a higher percentage than we have
ever found in healthy animals, even in the case of those fed on diets rich in
cholesterol. We have not yet had an opportunity of making further
experiments in this direction, but we do not think the result is in disagree-
ment with the hypothesis referred to at the beginning of this paper.

Method of Carrying out the Hemolytic Experiments——The method employed
is to mix together a suspension of blood corpuscles, a solution of saponin
and the serum, making up to a constant volume with physiological salt.

For this purpose the following solutions are used :—

(1) Physiological salt—a 0°85-per-cent. solution of NaCl (specially
purified) in distilled water.

(2) Saponin (Merck)—a 0-01-per-cent. solution in the physiological salt.

(3) Rabbit’s blood corpuscles—a 5-per-cent. suspension in physiological
salt.

In carrying out the experiments, the mixtures of blood, saponin, serum, and
physiological salt are very carefully measured into glass tubes with accurately
ground glass stoppers. The tubes are then placed in clamps fitted on to
a circular plate of wood in such a manner that they radiate from the centre
to the circumference. This disc, with the tubes attached, is slowly revolved
in a vertical plane by means of a clockwork drum, the whole apparatus being
kept at a constant temperature of 37° by placing it in an incubator.

As the tubes are completely inverted during the revolution of the disc, the
corpuscles are kept equally distributed throughout the mixture without
violently agitating the contents.

Elastic bands hold the stoppers securely in position to prevent their
coming out of the tubes as they expand with the heat of incubation. The
most favourable period of incubation was found to be three hours. After
this the tubes are placed on ice and the corpuscles allowed to settle, or they
may be at once centrifugalised. In either case the amount of hemolysis is
judged from the colour of the clear supernatant liquid.

By taking readings of a tintometer [Michael], on consecutive days, it was
found that the tint of the tubes kept on ice did not vary, this showing that
further hemolysis than that taking place during incubation was prevented
entirely by placing on ice. The ice method was adopted generally.

Experiments to ascertain whether Cholesterol administered with the Food and
absorbed by the Animal appears in the Blood Stream as such.

Experiment I.—It was first necessary to ascertain the least amount of
saponin solution which would produce complete hemolysis in a given

1909.| Destiny of Cholesterol in the Animal Organism.

volume of a 5-per-cent. suspension of rabbit’s blood corpuscles.

é

233

The results

of the experiment are embodied in the following table :—

Table I.
Amount of blood, Amount of saponin, | Amount of NaCl, Bosrilh
5-per-cent. suspension. 0-O0L per cent. 0°85 per cent. ete
C.c. C.c. CG,
2 Orl 3°9 Incomplete hemolysis
2 | 0-25 3°75 | 7 %
2 0°5 3°5 | ” ”
|
0 :01-per-cent. sol.
2 0-1 3°9 ) ”
2 0-25 3°75 | ik bi
2 0°5 3°5 | ” ”
2 1 3 | Complete hsemolysis
2 2 2 ) ”

Incubated for three hours at 37° C.

Thus 1 e.c. of a 0-01-per-cent. solution of saponin in a 0°85-per-cent.
solution of NaCl completely hemolyses 2 c.c. of a 5-per-cent. blood suspension
in a total volume of 6 c.c.

It was found, however, in the course of the experiments, that the least
amount of saponin needed to cause hemolysis varied slightly with different
specimens of rabbit’s blood. For this reason it was necessary always to have
the serum of a rabbit fed on extracted bran alone to compare with the
serum of a rabbit fed on extracted bran and cholesterol, etc., so that the
comparative experiments were carried out on the corpuscles of the same
rabbit.

Experiment 11—Rabbit 1; fed on extracted bran for 10 days; weight of
rabbit without blood = 1-7 kilogrammes.

Rabbit 2; fed for 10 days on extracted bran, during the last six days
given 4 gramme cholesterol per day. Weight of rabbit without blood
= 2°5 kilogrammes.

Rabbit 3; fed for 10 days on extracted bran, during the last six days
given 4+ gramme cholesterol per day for the first three days, 1 gramme
per day for the last three days. Total, 3¢ grammes cholesterol.

Animals kept under the same conditions and killed at the same time.
Sera collected 36 hours after death.

Results of experiment are given in the following tables :—

234 Miss Fraser and Mr. Gardner. Origin and |. [Apr. 3,

Table Il —Inhibitory Action of Serum of Extracted Bran-fed Rabbit

(Rabbit 1).
aot ob eed, | Amount of | Amount of NaCl, | Amount of saponin, Resale
I paeoat serum. 0°85 per cent. 0-01 per cent. ohn
suspension.
c.c. OG: Oe Cc.
2 0-025 2-975 1 Almost complete
| hemolysis
2 0-05 2°95 1 Slight hxmolysis
2 @cil BQ. iL Trace hemolysis
2 0°5 2°5 1 No hemolysis
2 it 2 al | ”

Table I1I.—Inhibitory Action of Serum of Rabbit fed on Extracted Bran
+ Cholesterol (Rabbit 2).

|
rs OF lees)! Amount of || Amount of NaCl, | Amount of saponin, |
5-per-cent. x : 4 Result.
Lapa serum. | 0-85 per cent. 0:01 percent. |
suspension. |
exc: CrCe C.c. Cie: |
2 0 025 2-975 1 No hemolysis |
2 0-05 2-95 1 ‘
2 O01 2:9 1 “
2 0°5 2°5 1 2 |
2 1 2 1 * |
| |

Table ITV.—Inhibitory Effect of Serum of Rabbit fed on Extracted Bran
+ Cholesterol (Rabbit 3).

| | | |
Amountot blood, Amount of Amount of NaCl, | Amount of saponin,
5-per-cent. | ‘ | 3 Result.
5 serum. 0°85 per cent. 0-01 per cent.
suspension.
cies C.c. | c.c. c.c.
2 0 025 2-975 1 No hemolysis
2 0-05 2-95 1 i:
2 O01 2-9 iL ”
2 0°5 2°5 1 »”
2 il 2 1 es

Incubated for three hours at 37° C.

In order to control these figures, the experiments were repeated the
following day, with identical results. It was found, however, that when
kept over a period of several days the sera lost their inhibitory effect to
some extent, but even then the relative strengths remain in the same
proportion.

1909.| Destiny of Cholesterol in the Animal Organism. 235

_ Hxpervment I1l.—Rabbit 4; fed on extracted bran for 14 days, weight
= 15 kilogrammes.

Rabbit 5; fed on extracted bran for 14 days, the last eight days with
cholesterol in addition; } gramme the two first, } gramme the next four,
and 1 gramme the last two days. ‘Total cholesterol, 44 erammes ; weight
= 2 kilogrammes.

Animals kept under similar conditions and killed at the same time.
Serum collected day after death.

The results of experiments are tabulated below :—

Table V.—Inhibitory Effect of Serum of Rabbit fed on Extracted Bran
(Rabbit 4).

ee Amount | Amount of NaCl,| Amount of saponin, | Result |
P Gy of serum. | 0°85 per cent. 0-01 per cent. sre
suspension.
C.c. C.c. CL; | CL, |
2 0 025 2975 | iL | Almost complete heemo- |
| lysis

2 0°05 2°95 1 Considerable hemolysis |
2 Orl 2°9 1 A trace hemolysis |
2 0°5 2°5 1 No hemolysis

Table VI.—Inhibitory Effect of Serum of Rabbit fed on Extracted Bran
+ Cholesterol (Rabbit 5).

| |

aan oe vere, Amount | Amount of NaCl,| Amount of saponin, | Resale |

percen’- | of serum. | 0°85 per cent. 0-01 per cent. | gaat |
suspension, | |
}
C.C. C.c. C.c. | cic: |

2 0-025 2-975 1 | Considerable haemolysis

2 0°05 2°95 1 A trace hemolysis |

2 0-1 2°9 1 | No hemolysis |

2 0°5 2°5 1 | . |
|

Exactly similar results were given by the sera heated for over an hour to:
56° C., as the following table shows. The heating was performed with the
object of showing that the inhibitory action of the sera on hemolysis is not
due to an organic enzyme.

236 Miss Fraser and Mr. Gardner. Origin and [Apr. 8,

Table VII.—Inhibitory Action of Heated Serum of Rabbit fed on Extracted
Bran alone (Rabbit +).

|
Amount of blood, | Amount | Amount of NaCl,| Amount of saponin |
S-per-cent. | of serum 0-85 per cent 0-01 per cent. — Bevel.
suspension. | au P : P :
c.c. c.c. €.c. C.c.
2 0-025 2-975 1 Almost complete hxmo-
lysis
2 0:05 2-95 1 Hemolysis
2 01 | 2°9 1 Slight hemolysis
2 0°5 2°5 il No hemolysis

Table VIII.—Inhibitory Action of Heated Serum of Rabbit fed on Extracted
Bran + Cholesterol (Rabbit 5).

eae unt of eee Amount | Amount of NaCl, | Amount of saponin, Result
aBer cen | of serum. | 0°85 percent. | 0-01 percent. | ane
suspension. | | |
c.c. c.c. C.c. C.c.
2 0 °025 2:975 | 1 Hemolysed
| 2 0:05 =| 2-95 | 1 A trace hsmolysis
2 Opal 2°9 | 1 No hemoylsis
2 OB | 2°5 | 1 x
|

A third series of observations was made the same day, with the sera in a
10-per-cent. solution of physiological salt. The results are given below :—

Table [X.—Inhibitory Action of 10-per-cent. Solution of Serum of Rabbit fed
on Extracted Bran (Rabbit 4).

Amount of blood,| Amount | : ;
5-per-cent. of serum, oo aan a MleXCh, Asaomalh o ie ame Result.
; per cent. 0-01 per cent.
suspension. 10 per cent.
exc! C.c. C.c. rcs
2 0°25 2°75 1 | Almost complete hemo-
| Lysis
2 0°5 2°5 1 Hemolysis
2 1:0 2°0 1 Slight hemolysis
2 1:5 1:5 1 | No hemolysis

1909.] Destiny of Cholesterol in the Animal Organism. 237

Table X.—Inhibitory Action of 10-per-cent. Solution of Serum of Rabbit fed
on Extracted Bran + Cholesterol (Rabbit 5).

| |
DEEL: TAGES) ae | Amount of NaCl, | Amount of saponin, |

|
5-per-cent. of serum, | 7 .o~ f | Result.
suspension. [10 per an 0°85 per cent. 0 -O1 per cent.
| 1
| |
c.c. c.c. EG. CC
2 0-25 2°75 1 Hemolysed
2 0°5 2-5 1 A trace hemolysis
2 1:0 2°0 1 | No hemolysis
2 1°5 1°5 Ui >»

We repeated these experiments with three other pairs of rabbits, and the
results were so similar that we need not recapitulate them. It is obvious,
therefore, that the cholesterol absorbed from the food appeared in the blood
stream in the free state. Whether any entered in the form of esters it is
impossible to say, but experiments are in progress which we hope will throw
light on this subject.

It now seemed of interest to ascertain whether, the animals being fed on
esters of cholesterol in addition to extracted bran, the esters were absorbed
entirely as such, or whether they undergo hydrolysis in the process of
digestion, and find their way into the blood stream as free cholesterol, in
part, at any rate.

For this purpose we made use of pure cholesterol oleate and cholesterol
stearate for feeding the animals.

Experiment TV.—Rabbit 6; fed on extracted bran for 18 days; weighed
2-2 kilogrammes at beginning, 2°3 kilogrammes at end of experiment.

Rabbit 7; fed on extracted bran for eight days, the last six days fed with
cholesterol oleate in addition ; } gramme for four days, 1 gramme per day
for two days; weight of animal = 2 kilogrammes at beginning and end of
feeding.

Rabbit 8; fed on extracted bran for eight days, the last six days fed with
cholesterol stearate in addition ; + gramme for four days, 1 gramme per day
for two days; weight of animal = 2 kilogrammes at beginning and end of
feeding.

Animals were kept under the same conditions and killed at the same time.
Serum collected next day.

Results of experiment are tabulated below :—

238

Miss Fraser and Mr. Gardner.

Origin and

[Apr. 3;

Table XI.—Inhibitory Action of Serum of Rabbit fed on Extracted Bran
. (Rabbit 6).

Amount of blood,

Amount | Amount of NaCl,| Amount of saponin
5-per-cent, Z y 3 y Result. |
suspension. of serum. | 0°85 per cent. 0 ‘01 per cent. ;

|
€.c. OL, c:c! cre
2 0 025 2-725 1-25 Almost complete heemo-
lysis
2 0:05 , 2:7 1°25 Considerable hemolysis
2 0-1 2 +65 1°25 in .
2 0°25 2°5 1°25 Slight heemolysis

Table XII—Inhibitory Action of Serum of Rabbit fed on Cholesterol Oleate
in addition to Extracted Bran (Rabbit 7).

seer? ot Blows, Amount | Amount of NaCl, | Amount of saponin i |
ODER COR of serum. | 0°85 percent. | 0-01 per cent j Hehe |
suspension. cal Re ei | F ret
2 \
cles C.c. C.c. C.c.

2 0 °025 2°725 1°25 Hemolysis

2 0:05 2-7 1-25

2 0:1 2 65 1-25 Slight heemolysis

2 0°25 2°5 1°25 No hemolysis

Table XI1I.—Inhibitory Action of Serum of Rabbit fed on Cholesterol
Stearate in addition to Extracted Bran (Rabbit 8).

|
see oftblood, | Amount | Amount of NaCl,| Amount of saponin, | 3
-per-cent. : 5 ‘ f Result.
é : of serum. | 0°85 per cent. 0-01 per cent.
suspension. |
(XE CHO, Ge: c.c.
2 0-025 2-725 1°25 Hemolysis
2 0-05 2nG 1°25 if
2 orl 2 65 1:25 Slight hemolysis
2 0°25 2°5 1°25 No hemolysis

The experiments were again performed with the sera after heating them to

56° C. with the same object as in the case of the previous experiments.

following tables give the results :—

The

1909.] Destiny of Cholesterol mm the Animal Organism. 239

Table XIV.—Inhibitory Action of Serum of Rabbit fed on Extracted Bran
(Heated) (Rabbit 6).

Amount of blood, :
Amount | Amount of NaCl,.| Amount of saponin,
S-per-cent. | of serum. | 0°85 per cent. 0 ‘Ol per cent. Hose.
suspension.
C.c. C.c. cic OL,
2 0 .:025 2 °725 1°25 Almost complete hemo-
lysis
2 0:05 2:7 1°25 Considerable hemolysis
2 0-1 2-65 1°25 Bi a
2 0°25 25 1°25 Slight hemolysis

Table XV.—Inhibitory Action of Heated Serum of Rabbit fed on Extracted
Bran + Cholesterol Oleate (Rabbit 7).

eee oe Amount | Amount of NaCl, | Amount of saponin, Teel
La ee of serum. | 0°85 percent. | 0-01 per cent. seat:
suspension.
| | a
c.c. C.c. C.c. c.c.
| 2 0 025 2 °725 1:25 Hemolysis
| 2 0:05. | 2°7 1-25 5
| 2 Niger Olle 2-65 | 1-25 Slight hemolysis
2 | OF | 2°5 1°25 No hemolysis

Table XVI.—Inhibitory Action of Heated Serum of Rabbit fed on Extracted
Bran + Cholesterol Stearate (Rabbit 8).

&

| |

enn eeplood,| Amount | Amount of NaCl, | Amount of saponin |
D-pSeaetate, | of serum. | 085 per cent.: 0-01 per cent. : Result. |
suspension. | |

C.c. C.c. c.c. Cie: |

| 2 0 025 2725 | 1-25 Hemolysis |
| 2 0-05 DH | 1-25 ‘ |
2 O01 2°65 | 1-25 | Slight hemolysis |

2 | 0 +25 2°5 1:25 | No hemolysis |

They were also carried out with 10-per-cent. solution of the sera in physio-
logical salt.

240 Miss Fraser and Mr. Gardner. Origin and [Apr. 3,

Table XVII.—Inhibitory Action of Serum in 10-per-cent. Solution.
Extracted Bran-fed Rabbit (Rabbit 6).

Amount of blood,| Amount = 2
5-per-cent. "| of serum, ee OE ae eee & sa me Result.
suspension. 10 per cent. Saeco pen Ce
cic: Oo. c.c. cles
2 0:25 = | 2°5 1°25 Almost complete hremo-
lysis
2 0-5 2°25 1°25 Considerable hemolysis
2 1-0 1°75 1-25 S
2 2°5 | 0:25 1°25 Slight hemolysis
|

Table XVIII.—Inhibitory Action of Serum of Rabbit fed on Extracted Bran
+ Cholesterol Oleate in 10-per-cent. Solution (Rabbit 7).

sanguin) OF (aloe, Amount | Amount of NaCl, | Amount of saponin
Pape geay, of serum. | 0°85 per cent. 0-01 per cent. a Resale
suspension.
|
cic: 6.0: | Cicer Oxo.

2 0:25 | 2°5 1°25 Hemolysis

2 0°5 2°25 1°25 x

2 1:0 1°75 1-25 Slight hemolysis

2 2°5 0°25 1°25 No hemolysis

i |

Table XIX.—Inhibitory Action of Serum of Rabbit fed on Extracted Bran
+ Cholesterol Stearate in 10-per-cent. Solution (Rabbit 8).

ee of Pleas | asmetiat Amount of NaCl, | Amount of saponin, |
-per-cent. of serum, 2 : ; Result.
: 0°85 per cent. 0-01 per cent.
suspension. 10 per cent. . |
| |
|
| c.c. G.c. | C.c. C.c.
| 2 0°25 | 2°5 1-25 Hemolysis
| 2 0°5 2°25 1°25 59
2 1-0 1-75 1°25 Slight hemolysis
2 2°5 0-25 1-25 No hemolysis

These experiments clearly show that the esters of cholesterol undergo
hydrolysis during the digestive process and appear, partially at any rate, in
the blood stream as free cholesterol.

As cholesterol is not a normal constituent of the food of rabbits, it
appeared interesting to find out (1) whether vegetable phytosterol is
absorbed by the animal if given in the food; (2) whether this can be
utilised by the animal.

Experiment V.—To ascertain whether the phytosterol of the food in the

1909.] Destiny of Cholesterol in the Animal Organism. 241

case of rabbits is excreted unchanged or whether any is absorbed or
destroyed during digestion.

A large healthy rabbit was fed for eight days on a mixture of equal parts
of extracted bran and extracted wheat germ, and the feces were collected
during the last seven days. The feces, which when dried weighed
60 grammes, were extracted with ether and the ethereal solution saponified
with sodium ethylate. The unsaponifiable matter was obtained in the form
of a clear stiff oil, weighing 04115 gramme. This was dissolved in
5 cc. absolute alcohol and left to crystallise. A small quantity of greasy
crystalline matter separated, too small to be readily purified. It was not
cholesterol, but consisted of the crystalline “phytosterol” of bran, from
which it is difficult to free completely the original bran by simple extraction.

The animal was then fed for one day on the same diet, the feces being
discarded, and for the following eight days on a daily ration of ? gramme of
phytosterol (from wheat-germ), 30 to 40 grammes of extracted bran and an
equal quantity of extracted germs of wheat, care being taken that the animal
ate the whole of the phytosterol—2 grammes in all. It was then fed for
four more days on the same diet, but without phytosterol. The feces
collected during the 12 days weighed, after drying, 189 grammes. The
animal remained in good health and its weight was constant all through the
experiment. The feces were treated as before and yielded 2:3965 grammes
of greasy unsaponifiable matter. This was repeatedly recrystallised from
alcohol and two crops of pure phytosterol were obtained, the weights and
melting points of which were: crop 1, 06465 gramme, M.P. = 132°C.;
crop 2, 0603 gramme, M.P. = 132° C. The mother liquors and residues
were then evaporated to dryness and heated for a few minutes at a
temperature of 180°—200° C. with benzoyl chloride. The product was then
poured into a suitable quantity of alcohol and allowed to stand. The
difficultly soluble crystalline matter which separated was recrystallised from
hot alcohol. In this manner 07149 of white phytosterol benzoate was
obtained which on recrystallisation from ethyl acetate was obtained in
characteristic crystalline form and melted at 141° C. 1:3675 grammes
of pure phytosterol were therefore recovered out of the 2 grammes
administered.

A consideration of the quantity of phytosterol recovered and also of the
relative quantities of unsaponifiable matter obtained in the two parts of the
experiment makes it clear that some of the phytosterol of the food was
either destroyed or absorbed, but most probably absorbed.

Laperiment VI.—In order to ascertain whether phytosterol given in the
food has the same effect as cholesterol.

242 Miss Fraser and Mr. Gardner. Origin and [Apr. 3,

For this purpose animals were fed with actual bran and wheat-germ, which
is rich in phytosterol, and other animals were fed on extracted bran with the
addition of measured quantities of pure phytosterol.

Rabbit 9; fed on extracted bran for 10 days; weight of animal
= 1-4 kilogrammes.

Rabbit 10; fed on extracted bran for three days, then ordinary bran and
wheat-germ for six days; weight of animal = 1:7 kilogrammes. The
following table gives the result of experiment :— .

Table XX.—Inhibitory Effect of Serum of Rabbit fed on Extracted Bran

(Rabbit 9).
| £ blood Hy
| Amount of blood,| amount | Amount of NaCl, | Amount of saponin, |
5-per-cent. ; Ss : | Result.
Z | of serum. | 0°85 per cent. 0-01 per cent. |
suspension. |
|
C0, c.c. c.c. c.c.
2 0-025 | 2975 1 Almost complete heemo-
| lysis
| 2 OOS 2-95 1 Slight hemolysis
| 2 orl 29 il A trace hemolysis
| 2 0°5 2°5 1 No hemolysis

Table XXI.—Inhibitory Effect of Serum of Rabbit fed on Ordinary Bran and

Wheat-germ (Rabbit 10).
| | | 2
x | |
eae a blogs, Amount | Amount of NaCl, | Amount of saponin, | Result
“per-cen’- | of serum. | 085 per cent. 0-01 per cent. ;
suspension. | |
| | io oe
ee Gre: Cx. | C.c
2 0 °025 2-975 1 Hemolysis
2 0:05 | 2-95 1 Slight hemolysis
2 0-1 2:9 1 No hemolysis
2 05 | 2°5 1 4

This experiment was repeated the following day with identical results.

Rabbit 11; fed on extracted bran for 11 days; weight of animal
= 2 kilogrammes.

Rabbit 12; fed on ordinary bran and wheat-germ for nine days, having
been fed with extracted bran for two days. previously ; weight of animal
= 2 kilogrammes.

The animals were kept under exactly similar conditions and killed at the
same time. Serum collected day after death. The results are tabulated
below :—

1909.|. Destiny of Cholesterol in the Animal Organism. 243

Table XXII.—Inhibitory Action of Serum of Rabbit fed on Extracted Bran
(Rabbit 11).

yaa ae eee Amount | Amount of NaCl,| Amount of saponin, Recele
geen se) | hofserume | OUSs per cent. 0-01 per cent. | isa |
suspension. | | | |

c.c. Ges c.c. C.C. |

2 0 025 2°975 1 Almost complete hemo- |
lysis |
| 2 0-05 2°95 il | Hemolysis |
2 Ls Oa 748) 1 Slight hemolysis |
2 ee Oo 2°5 1 No hemolysis |
|

Table XXIJI.—Inhibitory Action of Serum of Rabbit fed on Ordinary Bran
: and Wheat-germ \(Rabbit 12).

| | | }
ae of blood, Amount | Amount of NaCl,| Amount of saponin, |
-per-cent. | 3 | : Result.
: of serum. | 0°85 percent. | 0-01 per cent.
suspension. | | |
| |
C.c. | ee C.c. c.c |
2 | 0-025 2-975 1 | Hemolysis
| 2 0-05 2°95 it | Slight hemolysis
2 O-1 2:9 1 | A trace hemolysis
2 | 0°5 2°5 1 | No hemolysis
|

Experiments were also carried out with the sera after heating for over an
hour to 55° C., and in 10-per-cent. solutions made up with physiological salt.
The results agreed entirely with the above tables.

The serum of the rabbits fed on ordinary bran and wheat-germ shows
a slightly greater inhibitory power than the serum of the rabbit fed on
extracted bran. This seems to indicate (1) that some of the phytosterol of
the wheat-germ found its way into the blood stream, or (2) possibly caused
an increase of cholesterol in the blood. Rabbits experimented on disliked
the wheat-germ so that it was often neglected, the extracted bran being
always given the preference. We therefore resolved to give, in subsequent
experiments, weighed quantities of pure phytosterol.

Rabbit 13 ; fed on extracted bran for seven days.

Rabbit 14; fed on extracted bran for 12 days with phytosterol im addition,
eight days with ¢ gramme per day, then four days with } gramme per day.

Animals kept under similar conditions.

244 Miss Fraser and Mr. Gardner. Origin and [Apr. 3,

Table XX1IV.—Inhibitory Action of Serum of Rabbit fed with Extracted
Bran (Rabbit 13).

sane of blbod: Amount | Amount of NaCl, | Amount of saponin,
-per-cent. 5 : Result.
: of serum. | 0°85 per cent. 0 -O1 per cent.
suspension.
C.c. ce cies OO:
2 0-025 2-975 1 Complete hemolysis
2 0-05 2°95 il Considerable hemolysis
2 O'l 2°9 1 Slight hemolysis
2 0°5 2°5 1 No hemolysis

Table XX V.—Inhibitory Action of Serum of Rabbit fed with Extracted Bran
+ Phytosterol (Rabbit 14).

aed a Pees Amount | Amount of NaCl, | Amount of saponin, Tecrilt
clan care of serum. | 0°85 per cent. 0-01 per cent. ata
suspension.
c.c. c.c. c.c. C.c.
2 0-025 2°975 il Hemolysis not complete
2 0°05 2°95 1 A trace hemolysis
2 Ol 2°9 1 No hemolysis
2 0°5 2°5 1 ip

These experiments were repeated with the same sera, on consecutive days,
three times altogether, and in every case the result was identical.

Further, it was found that on addition of greater quantities of saponin to
the same volume of blood, if the serum were added in proportional
quantities, hemolysis was in every case prevented, if the ratio between
saponin and serum were the same as that which prevented hemolysis in the
above experiment.

The table is given below :—

Table XX VI.—Inhibitory Effect of Serum of Rabbit fed on Extracted Bran
with Phytosterol in addition (Rabbit 14).

Amount of blood,) 4 mount | Amount of NaCl, | Amount of saponin, Paevilt
Dg Coat of serum. | 0°85 per cent. 0-01 per cent. alate
suspension.

c.c. c.c. C.c: OW,
2 0 025 2°975 1 Hemolysis
2 0:05 2°95 1 A trace hemolysis
2 O'l 2°9 1 No hemolysis
2 0°15 2°35 1°5 ih
2 0:2 1°8 2 i
2 0 ‘25 1°25 2°5 is
\

1909.] Destiny of Cholesterol in the Animal Organism. 245

This is interesting as giving an indication of the quantitative reaction
between the serum and saponin, and therefore of its chemical nature.

In order to ascertain whether the result would be affected by heating or
dilution, however, another pair of rabbits was used.

Rabbit 15; fed for 14 days on extracted bran. Weight of animal
= 1) fedlosrammnes

Rabbit 16; fed on ordinary bran and wheat-germ for 13 days, the eal
eight days with phytosterol in addition; for six days } gramme per day, then
2 gramme the next day, and 1 gramme ine last day.,

Animals kept under similar conditions and killed! at the same time. The

results of experiments are given below :—

Table XX VII.—Inhibitory Effect of Serum of Rabbit fed on Extracted Bran
(Rabbit 15).

|
apr canpt blood, Amount | Amount of NaCl, | Amount of saponin, Recult
eae of serum. | 0°85 per cent. 0-01 per cent. si
suspension.
CL, CM, C.c. C.c.
2 0 025 2°S75 1 ‘| Hemolysis not quite
complete
2 0-05 2-95 1 Considerable hemolysis
2 O11 2-9 1 Slight hemolysis
2 0°5 2°5 1 No hemolysis

Table XXVIII.—Inhibitory Effect of Serum of Rabbit fed with Phytosterol
in addition to Ordinary Bran and Wheat-germ (Rabbit 16).

eguiN GE Diath | Amount | Amount of NaCl,} Amount of saponin, ;
B-per-cent. | of serum. | 0-85 per cent: 0-01 per cent. Teac
suspension. a
|

@.e; 1 Oe c.c. Cie:

2 0-025 2-975 1 Hemolysed

2 0-05 2°95 1 A trace of hemolysis

2 0-1 2:9 1 No hemolysis

2 | 0-5 2°5 1 .

VOL, LXXXI.—B. s

246

_ Miss Fraser and Mr. Gardner,

Origin and

[Apr, 3,

Table XXIX.—Inhibitory Effect of Rabbit fed on Extracted Bran heated
to 56° C. for an hour (Rabbit 15).

ep ouneN pico Amount | Amount of NaCl, | Amount of saponin
Dap Se PERe, of serum, | 0°85 per cent, : 0-01 per cent. : Sur
suspension,
ice CO; C.c. C.c.
2 0-025 2°975 1 Hemolysed (not quite
complete)
2 0:05 2°95 if Considerable hemolysis
2 Or1 2:9 1 Slight hemolysis
2 0°5 2°5 iL No hemolysis

Table XXX.—Inhibitory Effect of Serum of Rabbit fed on Phytosterol in

addition to Ordinary Bran and Wheat-germ.

an hour (Rabbit 16).

Heated to 56° C. for

Scien of blood Asaeukt
~per-cent. | of serum, | 0°85 per cent,
suspension.

C.c. C.c. c.c.

2 0 °025 2-975

2 0-05 2°95

2 al 2°9

2 0°5 2°5

Amount of NaCl, | Amount of saponin,

0°01 per cent.

ie)

Hee S

Result,

Hemolysed
A trace hemolysis
No hemolysis.

”

Table XX XI,—Inhibitory Effect of Serum of Rabbit fed on Extracted Bran
in 10-per-cent. Solution made up with Physiological Salt (Rabbit 15).

|| Amount of blood,
5-per-cent.
suspension.

ie}
wa

bo be bo

Amount
of serum,
10 percent. |
cies cic:
0°25 © | 2°75
}
Os Sigma 25
1-0 | 2
1°5 | 1°5

| Amount of NaCl, | Amount of saponin, |
0°85 per cent. | 0-01 percent. |

Result.

eee

Hemolysis almost com-
plete

Considerable hemolysis

Slight hemolysis

No hemolysis

1909.| Destiny of Cholesterol in the Animal Organism. 247

Table XXXII.—Inhibitory Effect of Serum of Rabbit fed on Phytosterol in
addition to Ordinary Bran and Wheat-germ in 10-per-cent. Solution of
Physiological Salt (Rabbit 16).

ai o plced, Amount | Amount of NaCl, | Amount of saponin, Reenle |
nue at of serum, | 0°85 per cent. 0-01 per cent. : |
suspension,

|

C.c. C.c. C.c. C.C. |

2 0:25 (| 2-75 1 Hemolysed

2 0°5 2°5 1 A trace hemolysis |

2 al 2 1 No hemolysis |

2 oped 1‘5 1 : |

|

These experiments fully confirm the conclusion arrived at from a com-
parison of the sera fed on extracted bran and on ordinary bran + wheat-germ.

Conclusions.

1. When cholesterol is given with the food of rabbits, some is absorbed
and finds its way into the blood stream as free cholesterol, only a portion of
the total cholesterol given in the food is absorbed, the rest being excreted
unchanged, The amount of cholesterol which finds its way into the blood
stream was not increased in our experiments by increasing the amount given
in the food, It would appear probable, therefore, that the animals only take
up such an amount of cholesterol as they can utilise.

2. Cholesterol when in the form of esters undergoes hydrolysis in part, at
any rate, during digestion, and appears in the blood stream as free
cholesterol.

3. When animals are fed on phytosterol, this substance is in part absorbed,
just as in the case of cholesterol, and appears in the blood stream either
itself or in the form of cholesterol. The latter point can, however, only be
decided by the examination of very large quantities of the blood of animals
fed on phytosterol, |

Experiments are now in progress which we hope will decide this question.

We take this opportunity of expressing our thanks to the Government
Grant Committee of the Royal Society for assistance in carrying out this
work.

248

The Influence of Glucosides on the. Growth of Acid-fast Bacilli,
with a New Method of Isolating Human Tubercle Bacilli
directly from Tuberculous Material contaminated with other,
Micro-organisms. (Preliminary Note.) | we

| «By F. W. Tworr.

|

|

| pedis |
(Communicated by Leonard Hill, F.R.S. Received February 18,—Read |
March 4, 1909.)

This investigation was undertaken to test the action of acid-fast bacilli on
the glucosides, and to see how far any fermentation reactions obtained would
differ with the various strains of human and bovine tubercle bacilli tested,
and also to obtain if possible a better medium on which to isolate and grow
tubercle bacilli. In all, 43 glucosides were tested with acid-fast bacilli,
including human and bovine tubercle bacilli, but there was no evidence of
fermentation with any of the glucosides:

One glucoside, ericolin, was found to kill off a large number of species of
micro-organisms, especially bacilli of the colori group and various cocci, but
had very little effect on the acid-fast group of bacilli.

By means of this glucoside the isolation of tubercle bacilli directly from
human sputum contaminated with other organisms becomes quite easy. The
glucoside should be made up with distilled water in a 2-per-cent: solution; a
lump of sputum is then placed into a test-tube containing the ericolin and
placed at 38°C. for ? hour to 1 hour; sub-cultures are then madé on to
Dorset’s egg medium, and pure growths of tubercle bacilli will be obtained in
1428 days; the tubes are sometimes contaminated with a few other
organisms, chiefly tiny colonies of streptococci and slow-growing colonies of
organisins of the streptothrix group, but they are so few that they in no way
interfere with the tubercle colonies, which can be easily sub-cultured.

aL

249

Reciprocal Innervation of Antagonistic Muscles. Fourteenth
Note.—On Double Reciprocal Innervation.
By C. 8. SHERRINGTON, D.Sce., F.R.S.

(Received March 25,—Read May 6, 1909.)
From the Physiology Laboratory, University of Liverpool.)
J SY i, Yi Pp

I.—Reflex excitation and inhibition when brought to play simultaneously on
the motoneurones of an extensor muscle can be so balanced that there results
in the muscle a contraction, the degree of which evidences algebraic summa-
tion of the two opposed influences, the inhibitory and the excitatory.* This
summation is obtainable not only in the decerebrate animal but also in the
purely spinal (fig. 1). In the latter it proves obtainable in preparations quite
recently made spinal, for instance, after decapitation. This circumstance
much facilitates the physiological study of the phenomenon. In other words,
the grading (fig. 1) of reflex contraction of the extensor by varying intensity
of inhibition acting along with reflex excitation can be studied in the animal
freshly made spinal as well as in the animal in the decerebrate condition.
The muscle which I have chiefly employed in the purely spinal preparation is
the isolated extensor of the knee in the decapitated cat.

A difference doés indeed exist between the reactions of the preparation
in the decapitated and in the decerebrate condition. In the decapitated
preparation, as in the decerebrate, the reflex effect of any inhibitory afferent
is easily seen if stimulation of that afferent is employed concurrently with
stimulation of an excitatory afferent. If however the inhibitory afferent is
stimulated during the ordinary resting condition of the preparation, there is
- usually no change in the muscle to show that the inhibitory afferent is pro-
ducing any effect at all (fig. 2). This is because the extensor muscle in the
decapitated preparation is not exhibiting tonus and lies relaxed, therefore
affording no background of contraction against which an inhibitory reflex cam
reveal itself by causing relaxation. Even in this condition it can, however,
easily be shown that the inhibitory afferent is then really producing a state of
inhibition in the preparation, although that state is not made evident by any
further relaxation. If a stimulus sufficient to cause reflex contraction of the
muscle be applied to the excitatory afferent while the inhibitory afferent,,
although apparently without effect, is being stimulated, the excitatory
afferent is found to be ineffective (or only partially effective) then; but it,

* Sherrington, ‘ Roy. Soc. Proc.,’ Note XIII, Nov., 1908.
VOL. LXXXI.—B. T

250 Prof. C. 8. Sherrington. Reciprocal [ Mar. 25,

immediately becomes effective (or more effective) on withdrawal of the con-
current inhibitory stimulation (fig. 2).

Similarly with flexor muscles. The flexors of the knee can be suitably
studied with semitendinosus or biceps femoris. With the latter, the posterior
part only of the muscle need be used, the anterior part not being a true knee

Pea Cae SN NON SST MES NN NG NNN NNN NNN

Fie. 1.—Reflex contraction of Vastocrureus in spinal mammal (decapitated cat), excited
by faradic stimulation of contralateral popliteal nerve (C). During this contraction
the ipselateral peroneal nerve was faradised for the time shown by the lower signal (1),
and produced a lessening of the reflex contraction ; this inhibitory effect is greater
according as the intensity of the stimulation of the ipselateral nerve is increased. In
the second observation it reduces the reflex contraction to less than half ; in the third
observation it suppresses it altogether for so long as the inhibitory stimulus endures.
In the third observation, with the strong inhibition, so abrupt and quick is the
suppression of the contraction that the writing lever gives a vibratory shake at the
end of its fall. The tremor seen to occur in the second observation during the period
of concurrent excitation and inhibition is usual, and is more marked in the decapitated
preparation than in the decerebrate. Time in seconds above.

flexor (cat, dog). The muscle, e.., semitendinosus, is isolated by detachment
from tibia and by severance in both hind limbs of all nerves other than its
own. The reflex contraction or relaxation of the muscle can then be studied
by the myograph. Reflex contraction is easily obtained by stimulation

1909. | Innervation of Antagonistic Muscles. 251

(mechanical or electrical) of the central stump of almost any afferent nerve
of the ipselateral hmb. If while this reflex contraction is in progress the
central stump of a nerve, ¢.g., popliteal, of the contralateral fellow limb be
suitably faradised, there ensues immediate relaxation of the contracting
semitendinosus (fig. 3). But if the contralateral afferent be stimulated while
the preparation is at rest there may be no evidence of inhibition or of any other

Fie. 2.

Fic. 2.—Reflex contraction of Vastocrureus muscle evoked by stimulation of central
end of contralateral peroneal nerve (C). Before this stimulation a stimulation of
the ipselateral peroneal (1) was commenced. This latter stimulation produced no
visible effect on the muscle, but it is only on its cessation that the contralateral
stimulation is able to produce a contraction. Cat, decapitated preparation. Time in
seconds above.

Fig. 3.—Reflex contraction of Semitendinosus, a knee-flexor, evoked by stimulation of
central end of ipselateral peroneal nerve (I), and inhibited during concurrent
stimulation of contralateral popliteal nerve (C). Cat, decapitated preparation.
Time in seconds above.

effect. The reflex stimulus might then be supposed not to be exerting any

influence whatever on the flexor muscle. That is because there is then in

the muscle no tonic or other contraction against which the reflex inhibition

ean show. That the reflex inhibitory influence is, however, really at work
i 2

252 Prof. C. 8. Sherrington. Reczprocal [Mar. 25,

can be shown by the same device as that employed above to reveal it under
similar circumstances in the extensor muscle.

Another way in which the hidden inhibition can be revealed is by
administering a small dose of strychnine. It was pointed out previously
that strychnine converts reflex inhibition of the extensor and certain other
muscles into reflex excitation. I find this hold good also in regard to reflex
inhibition of flexors. Intravenous injection of 0:15 milligramme strych.
hydrochl. per kilogramme (cat) almost at once converts the inhibitory effect
of a contralateral afferent on semitendinosus into an excitatory effect (fig. 4).

Fie. 4.—Effect of stimulation of contralateral peroneal on Semitendinosus ; A, before:
strychnine ; B, after. In B the contraction of vastocrureus is not included, but
semitendinosus (ST), which had shown no contraction in A, because the stimulus was
inhibitory to it, in B under the same stimulus exhibits the marked contraction
shown, the small dose of strychnine having converted reflex inhibition into reflex
excitation. The contraction long outlasts the period of stimulation in B.

Stimulation of the afferent then, in the resting preparation, instead of
leaving the muscle apparently untouched, produces reflex contraction of it..
The influence on the flexor motoneurones, which was previously unable to-
show itself in the relaxed muscle, because inhibitory, at once reveals its
existence, because under strychnine it has become an excitatory influence..

1909. ] Innervation of Antagonistic Muscles. 253

And this holds good with the flexor muscle both in the decapitated and in
the decerebrate preparation. The view that strychnine, by lessening an
intracentral resistance, so allows the nervous impulse access where it
previously had none, is therefore hardly applicable to the flexor any more
than to the extensor: in both cases the effect is well explained by the
strychnine converting reflex inhibition into reflex excitation.

IIl.—The algebraic summation of reflex excitatory and inhibitory effect
shows itself in the flexor muscle as well as in the extensor. Suppose reflex
contraction of semitendinosus to be in progress under faradisation of the
central stump of an afferent nerve, eg., peroneal, of the ipselateral limb. If
then the central stump of an afferent nerve, ¢.g., popliteal, of the contralateral
fellow limb be suitably faradised, there ensues an inhibitory relaxation of the
contracting muscle. When the relative intensities of the stimulations of the
two-nerves are appropriately adjusted, the contraction of the muscle is not
wholly abolished but is merely lessened in degree. The inhibitory stimulus
under these circumstances causes first a rapid partial lengthening of the
muscle, indicated by a descent of the myogram line; this rapid descent
reduces the contraction to a certain level of diminished contraction, which
is then equably maintained during the further continuance of the inhibitory
stimulus, giving a plateau in the myogram. If, then, the inhibitory stimulus
be withdrawn, the excitatory still continuing as before, the reflex contraction
increases again forthwith, regains a higher level, and, subject to fatigue,
continues until finally the excitatory stimulus is itself withdrawn. That
this grading which involves inhibition as one factor can be accomplished in
either of two ways is proved by observation as follows :—Throughout a (fig. 5)
consecutive series of reflexes the excitatory stimulus can be kept of constant
intensity, while the inhibitory is varied in intensity in the sequent reflexes.
When this is done, the amount which inhibition can subtract from the reflex
contraction is found to be graduable from the hardly perceptible up to
complete cancelling of all contraction for the time being. Conversely,
a series of reflexes (fig. 6), in which the inhibitory stimulus remains constant
in intensity but the excitatory is varied from reflex to reflex, produces degrees
of reflex contraction extending from hardly perceptible lessening of the reflex
contraction even to complete relaxation of the contracted muscle.

With flexor motoneurones, therefore, as with extensor, a grading of reflex
contraction is obtainable by employing concurrently with reflex excitation
an appropriate reflex inhibition, The algebraic summation of these two
opposed influences results in an infinite series of grades of intensity of
contraction both in the extensor and in the flexor muscle.

IJI—The records show that the combining of any particular value of

[Mar. 25,

Reciprocal

Prof. C. 8. Sherrington.

254

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255

of Antagomstic Muscles.

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256 Prof. C. 8. Sherrington. Leczprocal [Mar. 25,

excitation with any particular value of inhibition results in a grade of con-
traction less than that which the excitatory stimulus without the inhibitory
would give, and that this grade of contraction is fairly quickly reached, and
that then this grade continues unaltered provided there is no alteration in
the antagonistic stimuli. In other words, the myograph traces under the
combined stimulation a plateau (figs. 5 and 6) following on a brief descent.
The same plateau is seen when combined excitatory and inhibitory stimulation
follows on purely inhibitory stimulation (fig. 7). This plateau indicates that
under the combined stimuli a condition of equilibrium is reached, the two
opposed reflex influences of excitation and inhibition balancing at a certain
level of contraction or relaxation, the length of the muscle then remaining

Fia. 7.—Reflex contraction of Vastocrureus. The contraction is of two grades in each
observation, aceording as there is concurrent stimulation of inhibitory and excitatory
afferent, and of excitatory afferent alone. C denotes time of stimulation of contra-
lateral peroneal nerve, I of ipselateral peroneal. Cat, decapitated preparation. Time
in seconds.

constant. This result is such as might be expected. It is a result, however,
which in my experience is not obtained when reflex inhibition, however
feeble, is pitted against that kind of reflex excitation which the muscle
(extensor) expresses as tonus. A tonus preparation of the extensor muscle
can be obtained by decerebration producing decerebrate rigidity. -The
relaxation caused in such a preparation by reflex inhibition progresses so
long as the inhibitory stimulus continues (fig. 8). The weaker the inhibitory
stimulus the slower the rate of relaxation, but the relaxing of the muscle
progresses, so long as the inhibitory stimulus continues, until the full degree
of relaxation and the post-mortem length of the muscle is gradually reached

1909. | Innervation of Antagonistic Muscles, 257

(fig. 8). In other words,a balance between the reflex inhibition and the
reflex excitation which tonus expresses is never attained.

This state of tonus appears due to a natural stimulation of mild steady
quality, and in decerebrate rigidity it is, as has been shown, autogenous
in the sense that its afferent path is constituted by proprioceptive nerve-

Fig. 8.—Vastocrureus preparation in Decerebrate Rigidity. Reflex inhibition is pro-
duced by stimulaticn of the central end of the severed ipselateral peroneal nerve
below knee, the stimulus being a series of slowly repeated break induction shocks,
shown by the signal lines SA and SB. Time is marked (TA, TB) in fifths of
seconds. In observation A the break-induced currents were more intense (100 units
of Kronecker scale) than in observation B (20 units of Kronecker scale). Cat, decere-
brate preparation. In both the observations the relaxation of the muscle proceeds
to the full resting length of the muscle, but in observation A the fully relaxed
condition is obtained speedily in the course of nine repetitions of the stimulus ;
whereas in observation B the same degree of relaxation is reached only gradually in
the course of eighty-six repetitions of the (weaker) stimulus. The signal line at the
foot of the figure marks the duration of the stimulation in observation B.

fibres arising in the muscle itself.* This autogenous tonus no artificial
stimulus such as we apply to afferent channels experimentally for evoking
reflexes has as yet succeeded in giving. The failure to exactly balance the
natural reflex excitation which maintains this tonus by any artificial

* Sherrington, ‘Quart. Journ. of Expt. Physiol.,’ vol. 2, p. 109.

258 Prof. C. 8. Sherrington. Reciprocal [ Mar. 25,

stimulation applied to inhibitory afferents may simply mean that we fail by
artificial inhibitory stimuli to induce a reflex inhibition of such a kind as
a natural inhibitory tonus would be, just as we fail by artificial excitatory
stimuli to induce a reflex contraction of such a grade as is the natural
excitatory tonus. In light of this consideration the probability appears that
just as there exists a natural excitatory tonus, eg., the state of the extensors
and of some adductors in decerebrate rigidity, so likewise there is a natural
reflex inhibitory tonus, but that artificial stimuli such as we have at command
at present fail to reproduce it. It may be that the relative slackness of the
flexor muscles, in other words their exclusion from excitatory tonus, in
decerebrate rigidity is the expression of an inhibitory tonus. Decerebrate
rigidity is, as I have shown elsewhere,* to be regarded as a postural reflex,
namely, the reflex of standing. If the flexors are under an inhibitory tonus
in that reflex condition while the extensors are under an excitatory tonus, it
becomes clear that reciprocal innervation applies in this static postural reflex
as well as in the ordinary reflexes which execute movements and changes of
posture.

In the myograph records (fig. 8) which illustrate failure to obtain balance
between the natural reflex tonus of the extensor and the artificially excited
reflex excitation there occurs a further feature. The rate of elongation of
the tonic muscle under the weak artificially excited inhibition continues
about the same from start to finish, even when the starting point is with the
muscle fairly fully shortened and the end point is with the muscle fully
relaxed. In other words, the degree to which the reflex inhibition over-
balances the natural excitation causing the tonic contraction seems practically
the same throughout. But the intensity of the stimulation of the inhibitory
afferent in these observations was kept the same from start to finish ; hence
the inference is that the intensity of the natural reflex excitation causing the
tonic contraction is about the same throughout. That is to say, the intensity
of the excitatory tonus is practically the same when it keeps the extensor
muscle so shortened as to maintain the knee fully extended as when the
muscle under the tonus maintains the knee at an angle of eg. 60°. In other
words, the intensity of the reflex tonus of the extensor may be practically
the same while the muscle maintains postural lengths widely different.
Thus the same conclusion is reached as has been arrived at by other
observations which I have published before.

IV.—The extensor muscle of the knee (vastocrureus) and the flexor muscle

* ‘Integrative Action of the Nervous System,’ London and New York, 1906.
+ Sherrington, ‘Roy. Soc. Proc.,’ Note XII, August, 1908 ; and ‘ Quart. Journ. of Expt.
Physiol.,’ loc. cit.

1909. | Innervation of Antagonistic Muscles. 259

(e.g. semitendinosus) of that joint being suitably isolated, the myograph can be
arranged to register simultaneously the state of contraction or relaxation of
both muscles. If, then, excitatory and inhibitory afferents for each of these
muscles be stimulated concurrently, summation of the opposed influences of
the antagonistic nerves is found to be exhibited concurrently by both
muscles. The results of double (excitatory together with inhibitory)
stimulation on the antagonist muscles can be grouped into cases belonging to
three types. Which particular one of these three types occurs in any
particular case depends on the relative intensity of the stimulation of the
two afferent nerves. In my experiments usually one of the two nerves has
been ipselateral, that is, belonging to the limb to which the observed muscles
belong; the other contralateral, that is, belonging to the opposite fellow limb.
The ipselateral nerve excites the knee flexor and inhibits the knee extensor
(fig. 94, Obs. I and II). The contralateral nerve excites the knee extensor
and inhibits the knee flexor (fig. 94).

Under concurrent stimulation of both ipselateral and contralateral nerve
one type of result is that the knee flexor contracts and the knee extensor is
relaxed apparently fully (fig. 98, Obs. V; fig. 11, Obs.I and II). This is the
result when the stimulation of the ipselateral afferent is strong and that of
contralateral relatively weak, though yet efficient to cause contraction of
extensor muscle were not the ipselateral stimulus concurrently at work. _

In a second type of case the result of the concurrent stimulation of the
opposed afferents is that knee extensor contracts and knee flexor is relaxed
apparently to the full (fig. 5, Obs. IV). For this result the stimulation of
the contralateral afferent has to be strong and that of the ipselateral quite
weak, though strong enough to relax the extensor and contract the flexor
were not the contralateral stimulus in action at the time.

In combinations belonging to the above two types, one of the antagonist
muscles contracts while its opponent, if previously at rest, does not enter into
contraction, or, if previously contracting, is thrown out of contraction and
relaxed, exhibiting no obvious contraction either to visual inspection or in the
myograph record. The result in these two types of instance, although
obtained under concurrent stimulation of both afferents, resembles to outward
appearance that of stimulation of either the one or the other of the antagonist
afferents alone.

If, however, the relative intensity of stimulation of the two afferent nerves
differs less than in the two sets of cases mentioned, the result of the concurrent.
stimulation of the two nerves falls out differently. In this third type of case
the antagonistic muscles, flexor and extensor, both exhibit obvious contraction
concurrently (fig. 9, Obs. II and IV; and fig. 10). Thus the Observation II,

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1909. | Innervation of Antagonistic Muscles.

262

Prof. C. 8. Sherrington. Reciprocal [ Mar. 25,

fig. 94, opens with stimulation of the ipselateral afferent. This causes con-
traction of the flexor and, at the same time, relaxation of the extensor. Some-

Fic.

10.—Reflex contraction of Semitendinosus, Fl, and Vastocrureus, Et, recorded with
double-myograph. The lever attached to vastocrureus (Et) writes not directly below
but about a centimetre to the right of that attached to semitendinosus (Fl); control
ares, cut when the recording surface was not moving, indicate the moment of
commencement of stimulus on each myogram line. The ipselateral peroneal nerve
(afferent end) is stimulated as shown by the lower signal line Fl’, and causes contrac-
tion of semitendinosus and relaxation of vastocrureus. Then follows stimulation of
contralateral peroneal nerve as shown by the upper signal Et’. This combined
stimulation lessens, but not to extinction, the contraction of semitendinosus and at
the same time brings into contraction the antagonistic muscle, vastocrureus. On
reverting to the single stimulation again, the contraction of semitendinosus increases
once more and that of vastocrureus disappears. Finally, on cessation of the single
stimulation FI’, a rebound, 7, from inhibitory relaxation to increased tonic contraction
occurs in vastocrureus synchronously with the relaxation of the flexor. Time above
in seconds. Decerebrate cat.

what more than a second later stimulation of the contralateral afferent was

commenced, the stimulation of the ipselateral afferent continuing unaltered.

1909. | Innervation of Antagonistic Muscles. 263

Under this concurrent stimulation of both nerves the extensor muscle at once
contracts and the contraction of the flexor muscle continues, but in much
diminished degree. About two seconds later the stimulation of the contra-
lateral afferent is discontinued, that of the ipselateral remaining as before. The
contraction of the extensor at once dies out, and the flexor resumes the higher
degree of contraction, approximately, which it exhibited before the stimulation
of the contralateral nerve set in. Finally the stimulation of the ipselateral
afferent itself 1s withdrawn; the contraction of the flexor ceases forthwith,
and the relaxed extensor then exhibits a rebound contraction, regaining its
tonus, and exhibiting, indeed, a greater tonic shortening than it possessed
before it was subjected at all to inhibition.* It is obvious from the record
that the flexor muscle and the extensor, during the concurrent stimulation,
are in contraction both of them at the same time, and that the height of con-
traction of the flexor is less under the twofold stimulation than under that
-of the ipselateral afferent alone. That is evident not only from comparison
with the flexor contraction in the first and third parts of Obs. II itself, but
from comparison with Obs. I, where the behaviour of the two muscles under
the stimulation of ipselateral afferent alone with the same intensity of
stimulus as in Obs. II is seen. Observations I, II, and III are succes-
sive observations taken at one minute interval. In Obs. I the ipselateral
afferent alone was stimulated, and in Obs. III the contralateral afferent
alone. Returning to Obs. II, although there is clear evidence from it that
the contraction of the flexor during the concurrent stimulation of the
antagonist nerves is an algebraic resultant of excitation and inhibition values,
what is not so evident is that the concurrent contraction of the extensor
‘similarly exhibits an inhibitory component. That this is, however, really the
case is quite clear on comparing Obs. II with Obs. III. In Obs. III the
contralateral afferent alone was stimulated. This stimulation has no obvious
effect on the flexor muscle (see above, Section I); on the extensor it has the
-effect of exciting a contraction. The intensity of stimulus applied to the nerve
was unaltered from that of Obs. II. But the height of contraction evoked is
much greater in Obs. III than in Obs. II. The height of contraction has, of
-course—this being a tonus preparation of the extensor—to be reckoned, not
from the particular grade of contracted state which the tonic muscle
happens to be exhibiting at the moment of excitation, but from the base line
of full relaxation, such as is revealed under the inhibitions in Obs. I and II.
The greater intensity of the extensor’s contraction in III than in
Obs. II proves that in Obs. II the extensor’s contraction is less than it is under
the same intensity of stimulus applied to the contralateral afferent alone and

* Sherrington, ‘Roy. Soc. Proc.,’ Note IX, 1906.

264 Prof. C. 8. Sherrington. Reciprocal [Mar. 25,

that it is less by an amount represented by the difference between the height
of contraction in Obs. II and Obs. III respectively. Comparison of Obs. II
with Obs. III furnishes, therefore, one proof that under the concurrent stimu-
lation the contraction of the extensor like that of the flexor shows algebraic
summation of neural states of unlike sign. And comparison of Obs. II with
Obs. IV (fig. 98) furnishes further proof of the same thing. Obs. IV is a
repetition of Obs. II, the intensities of the stimuli being the same in Obs. II,
but the sequence of the stimuli in Obs. IV is in reverse order to their
sequence in Obs. II. Obs. IV opens with stimulation of the contralateral
afferent; this stimulation produces contraction of the extensor with no
obvious effect on the flexor, the preparation not being a tonus preparation as
regards the flexors. After the contralateral stimulus has been in operation
for rather more than a second the ipselateral afferent is stimulated, the
stimulation of the contralateral continuing as before. The effect of the
concurrent stimulation of the two nerves is that the contraction of the
extensor is considerably reduced in height, and that a contraction of the
flexor occurs, but this contraction of the flexor is much less than that given
by the ipselateral stimulus acting alone as in Obs. I and Obs. II. After
about 2 seconds’ duration the ipselateral stimulus is withdrawn, there then
ensues Immediately a disappearance of the flexor’s contraction and a resuimp-
tion by the extensor of the full height of contraction it showed originally
under contralateral stimulus alone. Finally the contralateral stimulus is
withdrawn and the contraction of extensor soon begins to decline and
somewhat gradually passes off. The difference between the abrupt reduction
of the extensor’s contraction directly the ipselateral stimulus (inhibitory)
joined the contralateral and the delayed and gradual dying out of the
contraction on mere withdrawal of the exciting stimulus (contralateral) is
characteristic of the difference between onset of inhibition and mere
cessation of excitation in a tonus preparation.

Fig. 9B, Obs. V, taken a minute later than Obs. IV, supplies a repetition
of the latter with increased strength of the ipselateral stimulus. ‘The result
then given during the concurrent stimulation of the opposed afferents is
different. The simple change in the intensity of stimulation of one of the
afferents removes the result from the type 3 to type 1. Under the con-
current stimulation in this observation the flexor contracts while simul-
taneously the contraction of the extensor is apparently completely
suppressed, whereas under the concurrent stimulation in Obs. II and IV
both the flexor and extensor are synchronously in active contraction.

In this third type (fig. 9, Obs. II, Obs. IV, and fig. 10) of result,
therefore, concurrent stimulation of the two antagonistic afferents produces

1909. ] Innervation of Antagomstic Muscles. 265

in the antagonistic muscles obvious contraction concurrently in both.
But though both the muscles are contracting the contraction of each is
seen to be less than it would be were there not then in action the reflex
which produces contraction of its antagonist. And this is so because
some reflex inhibition of the motoneurones of each accompanies the reflex
excitation of the motoneurones of the other. Thus the contraction of
semitendinosus is less than it would be were not the contralateral afferent
being concurrently stimulated, and the contraction of vastocrureus is less
than if the ipselateral afferent were not concurrently stimulated. As to
which of the two antagonists exhibits the greater contraction, that feature
is determined by the relative intensity of the stimulation of the two
antagonistic afferents. To obtain the type 3 of result the effects of the two
opposed afferents have, of course, to be sufficiently nearly balanced, and the
intensity of the one must not wholly overbalance that of the other. Within
these limits, however, all degrees of grading of combination of the two seem
obtainable by simple adjustment of the relative intensity of the stimulation
‘of the opposed nerves, In short, a delicate grading of the intensity of
contraction is effected by bringing into play a double reciprocal innervation.
‘The result of the double reciprocal action is that each of the two muscles
-displays simultaneously with its antagonist an algebraic summation of reflex
anhibition and reflex excitation. In other words, antagonistic reflexes add
themselves algebraically with unlike signs.

To obtain this result with spinal reflexes, ¢.g. in the decapitated mammal,
‘or with reflexes produced by stimulation of afferent nerves in the decerebrate
animal, it is necessary to stimulate synchronously two or more afferents of
‘opposed effect as regards the antagonist muscles selected. Stimulation of
-either of such opposed afferents. taken alone never suffices, in my experience,
‘to produce simultaneous contraction, e.g. of the knee flexor and extensor. In
‘short, concurrent contraction of the antagonists denotes that two antago-
mistic reflexes are synchronously at work and are operating with intensities
not far removed from balancing one another. In this respect there
is a significant resemblance between spinal reflex co-ordination and that
exercised by the motor cortex cerebri. Electric stimulation at one cortical
{point giving contraction of, eg. flexor of elbow with relaxation of the
.antagonist extensor, and stimulation at another cortical point giving contrac-
tion of the antagonist extensor with relaxation of the flexor, synchronous
‘Stimulation of the two cortical points together does often, I find, give,
if suitably graded, a concurrent contraction of both flexor and antagonist
.extensor together.

V.—Apart from the intensity of the stimuli applied to the opposed afferent

VOL. LXXXI.—B. U

266 Prof. C. 8. Sherrmgton. Reciprocal [Mar. 25,

nerves there is, however, another factor controlling the balance of reflex
result on the antagonistic muscles. It was shown previously that the
ipselateral reflex on the knee muscles is, as compared with the crossed reflex,
the prepotent one. Remembering that the influence of the ipselateral
afferent is excitation of the flexor and inhibition of the extensor, while that °
of the contralateral afferent is inhibition of the flexor and excitation of the
extensor, it has been shown that each of these afferent nerves can be made
to preponderate in reflex result over the other. This preponderance in effect
of the one over the other can be impressed on either the one or the other of
the two by simply stressing the intensity of the stimulus to the one it 1s desired
should preponderate. But it is less easy to secure prepotency for the
contralateral nerve than for the ipselateral. The ipselateral reflex seems to.
function with an inherent greater intensity than the contralateral. Fig. 11.

Fig. 11.—Double-myograph Observation. E marks vastocrureus, F semitendinosus,,.
C contralateral stimulus, I ipselateral stimulus. Cat, decerebrate preparation. Time:
in seconds. Description in text. The myograph lever attached to vastocrureus
writes not directly above that attached to semitendinosus, but about a half centimetre-
to right of it; ares cut by the levers on the recording surface when stationary mark
the moment of application of stimulus on each myograph line.

exemplifies the effect that this has on the experimental adjustment of the-

stimuli required for algebraic summation of excitation and inhibition. The

observation opens with stimulation of the contralateral afferent which.
produces contraction of the extensor muscle and no obvious effect on the
flexor, the preparation not being a tonus preparation as regards the flexor.

Two seconds later there ensues stimulation of the ipselateral afferent,

stimulation of the contralateral continuing unaltered; the flexor at once

contracts, and the contracting extensor is at once relaxed, and so speedily
and fully that its lever gives a vibratory shake after the fall. A second and

a half later the ipselateral stimulus is withdrawn; the contraction of the

flexor then dies off, and the contraction of extensor reappears and regains its .

1909. | Innervation of Antagonistic Muscles. 267

old prominence. Finally, the contralateral stimulus is withdrawn and the
extensor’s contraction gradually passes away. After a minute’s pause the
observation was repeated with unaltered intensity of the stimuli, but with
reversal of their sequence. The ipselateral stimulus precedes and produces
its unopposed effect ; this stimulus still remaining in operation, tlhe contra-
lateral stimulus is now combined with it and remains without visible
influence on the myogram, although what its unopposed effect would be
is guaranteed by the just preceding observation. This comparatively easier
suppression of the contralateral reflex by the ipselateral than of the
ipselateral by the contralateral explains how it is that when similar afferent
nerves of both hind limbs or similar points of skin in the limbs are
simultaneously and similarly stimulated extension occurs in neither limb and
flexion oecurs in both.*

The effect of fairly strong and equal concurrent stimulation of symmetrical
afferents of the two limbs in giving flexor effect at both knees, whereas
stimulation of one of the afferents gives flexion at ipselateral knee and
extension at contralateral, recalls strikingly a result obtained by Mott
and Schafert on the eyeball movements under concurrent stimulation of
balanced points of the two cerebral hemispheres right with left. Stimulation
of the cortex of one hemisphere gives lateral deviation of both globi to the
opposite side. It has been shown that in this movement reciprocal inner-
vation is at work producing inhibition of one muscle with excitation of its
antagonist. Mott and Schafer found concurrent stimulation of points in the
two hemispheres produce, when appropriately graded, a convergence of the
two globi. Here, obviously, of the two antagonistic influences, that on the
ipselateral globus preponderates over that on the contralateral, just as in the
case of the limbs the ipselateral reflex preponderates over its antagonistic
contralateral. And in the eyeball result, just as in the result on the limbs, I
imagine that appropriate analysis would reveal in this case an algebraic
summation of excitation and inhibition just as with the flexor and extensor
knee muscles. In short, in both cases antagonistic reactions are adding them-
selves with plus and minus signs.

VI—It will be noted that the ipselateral afferent, just as 1t is prepotent in
excitation, so also is prepotent in inhibition. This supports the view put forward
in these Notes§ that the inhibition and the excitation are complemental

* Sherrington, article “Spinal Cord,” ‘Schafer’s Text-book of Physiology,’ vol. 2, p. 840,
1899 ; also ‘ Integrative Action of Nervous System,’ p. 225, 1906.

+ ‘Brain,’ vol. 13, p. 170, 1894.

t Sherrington, ‘ Roy. Soc. Proc.,’ Note II, vol. 53, p. 411, 1893.

§ Sherrington, ‘ Roy. Soc. Proc.,’ Note VIII, vol. B 76, p. 277, 1905.

268 Reciprocal Innervation of Antagonistic Muscles.

parts of one and the same reflex. In harmony with this stands also
the fact that when the one or the other afferent is made prepotent by suitably
increasing the intensity of its stimulus, the prepotency obtains both for exci-
tatory influence and inhibitory influence alike. The reflex produced by either
afferent is, in fact, a reflex of simultaneous double sign (+ reflex). And in
the observations furnished there is a good deal of evidence suggesting that
the relation, between the intensity of the excitatory part of the reflex and the
inhibitory is such that the intensity of the one increases proportionally with
that of the other. The rhythm of the inhibitory effect in the motoneurones
of one muscle appears also to be synchronous with that of the excitatory
effect in the motoneurones of the antagonist muscle, to judge by the rhythmic
tremor not unfrequently evident on the myograph tracings. Observation IV
of fig. 9B furnishes an illustration of this.

VII.—It was shown previously that after reflex inhibition of the moto-
neurones of the extensor muscle there ensues, in decerebrate rigidity, on
withdrawal of the inhibitory stimulus, a contraction of the muscle. This is
the rebound contraction which [ attribute to “successive spinal induction.” It
may far exceed in intensity the tonic contraction which existed prior to the
inhibition. The question arises as to what state obtains in the flexor muscle
when this rebound contraction of its antagonist occurs. With the double
myograph recording both extensor and flexor simultaneously, this relation can
be studied. Fig. 94, Obs. I and II, and fig. 10, 7, give instances of the time
relations observable. On withdrawal of the ipselateral stimulus, the flexor
contraction passes off, and at the same time there takes place the rebound
contraction of the extensor. Often a relatively long latency period intervenes
between the cessation of the stimulus and the onset of the extensor’s rebound
contraction. Similarly it is not unusual for the contraction of the flexor to
outlive the duration of the excitatory stimulus for a certain time, the flexor
motoneurones exhibiting after discharge. The simultaneous record of the two
muscles shows that the rebound contraction of extensor synchronises with the
dying out of the after-contraction of the flexor; the after discharge (rebound)
of the extensor motoneurones comes into action as that of the flexor moto-
neurones passes off. Therefore, in the after-action shown by these antago-
nistic centres, there is evident the same kind of reciprocal activity as is seen
in their reaction under stimuli in application at the time. In after-discharge
and in tonus, as also when reacting to ordinary reflex stimulation, reciprocal
innervation seems to hold good in regard to the mutual relation between the
motor centres of the antagonistic muscles.

269

The Properties of Colloidal Systems. 1.—The Osmotic Pressure
of Congo-red and of some other Dyes.

By W. M. Baytiss, F.R.S., Physiological Laboratory, University College,
London.

(Received April 19,—Read May 6, 1909.)

Experiments made in 1895 by Linder and Picton* upon solutions of
arsenious sulphide indicated that colloidal solutions possess a real osmotic
pressure, although the authors themselves claim no quantitative value for
their results. In 1905} further experiments were made, but again great diffi-
culties were met with, and, although it seemed evident that osmotic pressure
was present, the numerical values obtained were irregular and small.

The first definite proof that certain colloidal solutions are able to exert a
not inconsiderable osmotic pressure was given by Starling? in the case of the
colloids of blood-serum. When separated by a gelatin membrane from a
solution obtained by filtration of some of the same serum through Martin’s
gelatin filter, the pressure rose to about 30 mm. of mercury.

Waymouth Reid§ found that solutions of carefully purified hemoglobin
gave an undoubted osmotic pressure when separated from water by a
membrane of parchment-paper, but regards this fact as evidence that
hemoglobin forms a true solution.

Moore and Parker|| determined the osmotic pressures of the colloids of
white of egg, of serum, and of soap solutions, while Moore and Roaf{l made
measurements of those of serum-proteins, of gelatin, and of gum-acacia.

Hiifner and Gansser** and, somewhat later, Roaf,++ independently of one
another, made careful determinations of the osmotic pressure of heemoglobin
solutions, to which reference will be made in a subsequent page.

It will be noticed that, in all these cases, with the exception of arsenious
sulphide and soaps, the chemical constitution of the body investigated is
uncertain, although the molecular weight of hemoglobin has been calculated
from its content in iron. The experience of those who had worked with
arsenious sulphide and with soaps was not encouraging for further research,

* “Chem. Soe. Trans.,’ vol. 67, p. 72, 1895.

+ ‘Chem. Soc. Tene vol. 87, p. 1909, 1905.

{ ‘Journ. Physiol.,’ vol. 19, p. 322, 1896, and vol. 24, p. 318, 1899.

g ‘Journ. Physiol.,’ vol. 33, p. 12, 1905.

|| ‘Amer. Journ. Physiol., vol. 7, p. 261, 1902.

4] ‘Biochem. Journ.,’ vol. 2, p. 34, 1907.

** © Archiv f. Physiol.’ (Engelmann), 1907, p. 209.

tt ‘Physiol. Soc. Proc.,’ 1908, p. i, in ‘Journ. Physiol.,’ vol. 39, 1909.

270 Dr. W. M. Bayliss. [Apr. 19,

so that it seemed desirable to investigate the behaviour of colloids of known
chemical constitution and molecular weight, the latter to be as small as
possible, in order that the osmotic pressure should be sufficiently great. By
this means it might be possible to estimate the number of molecules taking
part in the formation of “ solution-aggregates ” or colloidal elements, and also
to obtain more definite information as to the effect of electrolytes on the
osmotic pressure.

Certain of the aniline dyes form colloidal solutions, if we may take
Graham’s criterion of non-diffusibility through parchment-paper as decisive.
One of these dyes is congo-red, whose constitution and molecular weight are
well known. My attention was first called to the fact that solutions of this
body have a considerable osmotic pressure by phenomena met with in purify-
ing it by dialysis. It was striking and, in fact, a matter of some
inconvenience, to find that the contents of the parchment-paper tubes rapidly
increased in volume by taking up water and, unless some of the fluid was
removed, continuously overflowed. Congo-red, therefore, formed the starting-
point of the observations to be recorded in the present paper.

Owing to their high colouring power, the aniline dyes present many
advantages for the study of colloidal properties. In the investigation of
osmotic pressure, for example, the slightest leak in the membrane of the
osmometer is detected at once.

The particular form of osmometer used was that of Moore and Roaf,*
modified in order to change at will the fluid on the side of the membrane
opposite to the solution under investigation. Repeated changes of distilled
water could be made until no further change in the osmotic pressure
occurred, while the effect of the presence of various electrolytes or other
bodies could be examined. A diagram of the apparatus is given in fig. 1.

Congo-red, as obtained from Kahlbaum, was found to contain an
appreciable amount of sodium chloride. In order to remove this, hydro-
chloric acid was added until the red colour had vanished and the free acid,
precipitated, was washed on a filter with distilled water. It was soon found
that the free acid went into a beautiful deep blue colloidal solution, which
passed through the filter. (This observation has been published by Pelet-
Jolivet and Wild,t since my experiments were made.) I was obliged,
therefore, to resort to prolonged dialysis against distilled water. This
dialysed solution was placed in the osmometer. It gave a very small
osmotic pressure, about 6 mm. Hg. Subsequent experiments, to be described
below, were made to determine the osmotic pressure of this blue colloid

* Loe. cit.
+ ‘ Kolloid-Zeitsch.,’ vol. 3, p. 175, 1908.

1909. ] The Properties of Colloidal Systems. 271

more accurately. Dilute solution of sodium hydroxide was then run
through the lower chamber in order to convert the free acid into the sodium
salt. This solution was replaced at intervals of 24 hours until the outside
solution remained permanently slightly alkaline. During this process the
osmotic pressure rose gradually to about 40 mm. Hg. Repeated changes of

Fie. 1.

A, Osmometer of Moore and Roaf.

B, Mercury manometer, read by means of a reading microscope.

C, T-tube, with inner smaller tube, to allow a current of water, or other fluid, through
the outer, lower, chamber of the osmometer.

D, Tube, connecting the two chambers when the stopcock is opened. This is done in
order to control the zero of the manometer at any time.

EK, Inlet tube to the lower chamber. F, Outflow.

G, Soda-lime tube to exclude atmospheric CO,.

H, Tube from flask of distilled water or other fluid.

The osmometer was immersed in a thermostat.

distilled water were then similarly run through as long as the pressure
continued to rise. After ten days, equilibrium was attained with a pressure
of 79°3 mm. Hg at a temperature of 30°2 C.

The molecular weight of congo-red (the di-sodium salt of benzidine-
tetrazo-di-naphthylamine-di-sulphonic acid) is 696°47. On the basis of an
osmotic pressure of 22:4 atmospheres for a molar solution (as true solution)
at 0°, a 1-per-cent. (= 10 grammes per litre) solution at 30°2 should have a
pressure of
273 + 30:2 10

22°4 x 760 x 373 69647

= 271-4 mm. Hg.

22 Dr. W. M. Bayliss. [Apr. 19,

At the end of the above experiment, the solution was pipetted out of the
osmometer and its concentration determined by evaporating to dryness a
known volume and drying the residue to constant weight in a toluene
bath in the usual way. It was found to be 0°30 per cent., so that, if the
dye had been present as separate molecules, the osmotic pressure should
have been 0°30/1 x 2714 = 81:4 mm. Hg. The actual value found was
79°3 mm. Hg, or 97 per cent.

In other experiments the agreement with theory was not so good, eg.
207 mm., instead of a theoretical 228 mm., or 91 per cent., for a 0°84-per-
cent. solution at 30°2; 77-4 instead of 84, or 92 per cent., for a 0°309-per-
cent. solution at 30°77; and 128 instead of 146, or 88 per cent., for
0:58-per-cent. solution at 10°.

It is obvious that these values could only be obtained if the greater part
of the elements responsible for the production of osmotic pressure were
present as single molecules, since any value greater than one-half the
theoretical implies that a part of the active elements consists of single
molecules.

When the molecular weight of hemoglobin is calculated from the content
in iron, a value of about 12,000 to 14,000 is obtained.* Now this is the
same number obtained from the osmotic pressure determinations of Hiifner
and Gansser,t and of Roaf.t Hemoglobin, therefore, exists in solution in
single molecules, although, like congo-red, it does not pass through parch-
ment-paper. The molecular weight of congo-red, however, is very much less
than that of hemoglobin, only about one-twentieth in fact, so that it is more
surprising to find it to behave as a colloid.

On this account it is advisable to examine how far congo-red exhibits
other properties associated with colloids. To what degree does a molecule
of such dimensions show the characteristics of matter in mass, possessing
surfaces ?

In the first place, what appearance does a solution of congo-red show in
the ultra-microscope ? According to Michaelis§ the particles present are
sub-microscopic, that is, resolvable into separate bright points. The same
statement is made by Pelet-Jolivet and Wild.|| My observations are not
entirely in agreement with those of the investigators mentioned. There are
undoubtedly, a few scattered bright points to be seen, but these only

* See Schultz, ‘Die Grésse des Eiweissmolekiils, p. 31, Jena, 1903.
+ Loe. cit.

t Loe. cit.

§ ‘Deutsche Med. Wochensch.,’ No. 42, 1904.

|| Loc. cté.

1909. | The Properties of Colloidal Systems. 273

account for a very small part of the total quantity of the dye present in the
solution, as can easily be shown as follows: The blue colloidal free acid of
congo-red, even in extreme dilution, shows the track of the beam of light
filled with shining points of a beautiful copper colour and of nearly equal
size, so far as their diffraction images enable one to judge. If a drop
of dilute alkali be added to this solution, the track of the beam suddenly
vanishes, occasionally a bright poimt moves into the field and back again.
These few particles seem to be slightly larger than those of the acid. When
the illumination is carefully adjusted and made as brilliant as possible, close
attention shows that the track of the beam is very faintly visible as a bluish
grey haze, not resolvable into separate points, at all events not with the
means at my disposal, viz., are light, Zeiss D* objective. As I am inclined
to interpret the phenomena, the faint haze is the optical expression of the
part of the dye present in the molecular state, and the rare bright points are
due to aggregates of a number of molecules, produced by the action of traces
of electrolytes, to which congo-red is enormously sensitive, as will be shown
below. The’ solutions described by previous observers as being resolvable
into particles by the ultra-microscope were, in all probability, not sufficiently
free from electrolytes.

The ultra-microscope, then, does not throw much light on the nature of
solutions of congo-red, since, although it does not contain particles large
enough to be visible by means of this instrument, other undoubted colloids,
such as ferric hydroxide, are similarly non-resolvable, but show a faint haze.*
Moreover, the phenomena described above in the case of congo-red are very
like those seen by Michaelis} in certain protein solutions, namely, a part
visible as granules and the rest not so resolvable.

The property of carrying an electric charge, not as an ion, but on
undissociated molecules, is shared by congo-red with matter in mass. In
Whetham’s boundary apparatus the dye moves as a whole towards the anode
and is, therefore, negatively charged. The origin of the charge is obscure,
but is, perhaps, derived from electrolytic dissociation.t{ In accordance with
its nature as an electro-negative colloid, congo-red is aggregated or pre-
cipitated by cations, especially powerfully by bi- and tri-valent ions. It is
also precipitated by an electro-positive colloid, such as toluidine-blue or
ferric hydroxide. The precipitate has the properties of an adsorption-

* Zsigmondy, ‘ Zur Erkenntniss der Kolloide, Jena, 1905, p. 148.

+ ‘Virchow’s Archiv,’ vol. 179, pp. 205—208, 1905.

{ The phenomena seen in the boundary apparatus are of some complexity, being accom-
panied by slight electrolysis. These will more properly form the subject of a separate

paper.

274 Dr. W. M. Bayliss. [Apr. 19,

compound, in that its composition varies with the relative concentrations of
the two colloids present in the solution. As I have shown elsewhere,* the
behaviour of congo-red in respect of adsorption by cotton and other
materials is that of an electro-negative colloid.

The statement is sometimes made that colloids have no definite point of
saturation. Congo-red, on the contrary, has, in a certain sense, a somewhat
indefinite limit of solubility. It appears, however, that many colloids,
especially the inorganic ones, tend to aggregate and deposit when their
particles are brought into too close apposition; it may be that traces of
electrolytes are responsible for this behaviour, which is thus not the same
thing as the crystallisation of a super-saturated true solution.

Recent research tends to show that there is no real line of demarcation to
be drawn between colloids and crystalloids. Congo-red is evidently one of
those interesting cases which have some of the properties of both classes. In
any case, it does not seem reasonable to expect fundamental differences as
regards properties dependent on dimensions of the active elements between
a large molecule such as congo-red, containing some 70 atoms, and a particle
of colloidal gold containing a similar number of atoms. The properties
referred to are those dependent on diffusion, such as osmotic pressure and
those dependent on surface development.

The fact that, in the case before us, true solution and colloidal solution are
one and the same thing suggests several interesting questions. At what
molecular size do bodies begin to show properties due to surface develop-
ment, although still in the condition of single molecules? Again, why
should we not be able to reduce the number of molecules in the aggregates
of colloidal gold until they consist of single molecules? In this case we
should have a true solution of gold in the metallic state, not in the ionised
condition. It is possible that differences of electric potential and surface-
tension oppose obstacles to such a phenomenon.

The great sensitiveness of congo-red to traces of electrolytes has already
been incidentally referred to and this fact makes it a matter of considerable
difficulty to obtain the maximum readings of the osmotic pressure as given
above. For example, a solution of congo-red, containing 0°84 per cent. of
the dye, dialysed against repeated changes of ordinary distilled water until
no further rise of pressure took place, gave an osmotic pressure of only
118 mm. Hg, whereas on using water which had been distilled after the
addition of potassium permanganate and sulphuric acid and a second time after
the addition of barium hydroxide, being kept from contact with air by soda-
lime tubes, the pressure rose to 207 mm. Hg. The ordinary distilled water

* ‘Biochem. Journ.,’ vol. 1, pp. 175—232, 1906.

1909. | The Properties of Colloidal Systems. 275

used above was a fairly good sample, having a conductivity of not more than
5 gemmhos at 18° while the purer water had a conductivity of 1:8 gemmhos.
Tf still better water had been used, no doubt the full theoretical value of
228 mm. for the osmotic pressure would have been reached.

Since the ordinary distilled water presumably contained carbonic acid,
I tried the effect of water through which carbon dioxide gas had been passed
until its conductivity was 23 gemmhos. By titration, this solution was
found to contain 0°19 gramme CO: per litre. The osmotic pressure fell from
207 to 120 mm. He.

The powerful action of so weak an acid as carbonic is rather surprising,
and makes it unnecessary to subject stronger acids to detailed investigation.

When the solution of carbonic acid in the above experiment was replaced
by a decinormal solution of sodium chloride, the osmotic pressure fell in the
course of 24 hours to 15 mm. Hg. When equilibrium was established, the
concentration of sodium chloride on both sides of the membrane would be
about one-twentieth normal.

Linder and Picton* found that when aggregation of arsenious sulphide was
brought about by an electrolyte, it was impossible to reverse the process by
washing with distilled water. Similarly, although repeated changes of
distilled water were passed through the osmometer after the sodium chloride,
until the issuing water gave no reaction with silver nitrate, the osmotic
pressure only rose to about three-quarters of its initial value. It is possible
that very much more prolonged dialysis might have produced further effect,
but it seemed more important to use the apparatus for other experiments,
since all these experiments are of necessity of long duration. This washing
with water was, in one case, continued for three weeks, and, although after
this process the osmotic pressure had risen only to three-quarters of what it
was before the action of sodium chloride, no further rise was to be detected
on the mercurial manometer when a fresh change of water was added. If
a more delicate manometer had been used, it is quite possible that the
pressure would have been found to be still rising very slowly, since the
extreme slowness of removal of the last traces of electrolyte is characteristic
of adsorption phenomena.t

In order to test the action of alkali, a solution of the blue free acid was
placed in the osmometer. Only a small pressure of a few millimetres was
obtained, too small to read accurately on the mercurial manometer. When
dilute sodium or ammonium hydroxide was run into the lower chamber, the
pressure rose rapidly as long as the alkali combined with the dye-acid.

* “Chem. Soc. Trans.,’ vol. 87, p. 1911, 1905.
+ Bayliss, ‘Biochem. Journ.,’ vol. 1, p. 182, 1906.

276 Dr. W. M. Bayliss. | Apres

When excess was present, as shown by the permanent alkaline reaction
of the solution, the pressure fell again. The explanation is, no doubt, that
the aggregating action of the cation made itself felt. In view of the results
of Moore and Roaf* on the augmentation of the osmotic pressure of protein
solutions under the action of alkali, the fact noted by me is of some interest.
It seems probable that, in the case of proteins, the rise of osmotic pressure
is the result of the formation of new colloids by chemical action, these
“salts ” having smaller “ solution-aggregates ” than the original colloid.+

If we were unaware of the chemical nature of congo-red and observations
were being made of the osmotic pressure of the solution of the free acid, we
might imagine that alkali caused a large increase in the osmotic pressure of
this body. The fact is that the blue colloidal solution of the free acid,
as will be shown later, consists of large particles, easily resolvable by the
ultra-microscope, and producing only a small osmotic pressure. When
alkali is added, the salt, ordinary congo-red dye, is produced, and this, as.
shown above, exists in solution in single molecules with high osmotic pressure.

The fact that, as an electrolyte is added in stages, pausing sufficiently long
between each addition to allow equilibrium to be established, there is
a definite osmotic pressure for each step, so that a continuous smooth curve is
obtained, shows that the action of a low concentration of electrolyte must be
exerted on a part only of the molecules present. For example, it is not
every molecule that unites with another one, since, if so, there would be no
intermediate stages between full and half osmotic pressure. From the fact
that these stages do exist it follows that a number of molecules are left
single. The process is analogous to the association which takes place in
ethyl alcohol when dissolved in benzene, where the apparent molecular
weight of the alcohol rises from 50 to 208 in regular gradation as the
concentration rises from 0:494 to 14°63 grammes to 100 of benzene.t This.
can only be explained by the assumption of a steadily increasing number of
molecules becoming associated with others, while the rest remain free.

Ultra-microscopic observations of the actions of electrolytes on congo-red,
although somewhat difficult to interpret, confirm the results given by measure-
ments of osmotic pressure A dilute solution, showing only a very few
scattered bright points, on the addition of a solution of carbonic acid or a
natural salt contains a greatly increased number of these bright particles,
which vary considerably in size. In this respect they contrast with the
particles seen in the colloidal solution of the free acid, which are strikingly

* Loe. cit., p. 66.

+ See also Lillie, ‘Amer. Journ. of Physiol.,’ vol. 20, pp. 127—169, 1907.
t Walker, ‘ Introduction to Physical Chemistry,’ 4th ed., p. 205, 1907.

1909. | The Properties of Colloidal Systems. 277

uniform in size. It appears that the action of an electrolyte is, so to
speak, selective, leaving some molecules free, while causing others to aggregate
into particles, consisting themselves of very different numbers of molecules.

Faraday showed, more than 50 years ago,* that the ruby-red solutions of
gold which he prepared by reduction of gold chloride were suspensions of
minute particles of metallic gold. He also noticed that the colour of the
solution became blue under the action of sodium chloride in dilute solution,
and was precipitated by stronger solutions. Both these effects were absent if
a small amount of “jelly” had previously been added to the gold solution.
This latter “protecting” action of “stable” colloids is now well known,
forming the basis of Zsigmondy’s “ gold number ” as a characteristic of proteins
Congo-red behaves, as regards this protection from precipitation by electro-
lytes when a stable colloid is present, in the same way as the inorganic
colloids. It may be noted, in passing, that this is a phenomenon usually
ascribed to surface properties. The dye also is protected from adsorption
by paper, under the influence of electrolytes, when a trace of gelatin is
present.— It was, therefore, of interest to examine the influence of stable
colloids on the reduction of osmotic pressure produced by electrolytes.

Since my experiments on adsorption above referred to indicated that this
protective action was greater when the stable colloid had an electric charge of
the same sign as that of the dye, or of opposite sign to that of the precipi-
tating ion, I chose for the present experiments a dialysed solution of Griibler’s
serum-albumin, to which a trace of ammonium hydroxide was added in the
first experiment. The solution in the osmometer contained about 0°18 per
cent. of the dye and 0:25 per cent. of serum-albumin. When dialysed against
distilled water, the osmotic pressure rose to 42 mm. of mercury. The water
was then displaced by a decinormal solution of sodium chloride. To my
surprise, the pressure fell to zero in about 14 hours. Moreover, as in the experi-
ments without stable colloid, it was found that after 14 days’ changes of
distilled water the pressure could only be brought back to three-quarters of
its original value. Measurements of the electrical conductivity of the water
after interchange with the colloidal solution showed that the latter parted
with its electrolytes very slowly. They were probably held in a state of
adsorption by the protein as well as by the dye.

In another experiment I first tested the particular solution of serum-
albumin used and found that in the proportion of 5 c.c. to 50 cc. of one-
thousandth normal congo-red solution, precipitation by one-hundredth

* ©Phil. Trans.,’ vol. 147, 1858: As to the Nature of the Solutions, see pp. 160 and 172;
Precipitation by Salt, p. 165; Protection from Action of Salt by “ Jelly,” p. 175.
+ ‘Biochem. Journ.,’ vol. 1, p. 201, 1906.

278 Dr. W. M. Bayliss. [Aprin9;

normal calcium sulphate was prevented, although in the absence of the
protein complete precipitation occurred. In order to be quite certain of
adequate protection, I added 10 c.c. of the albumin solution to 50 cc. of the
dye and placed the mixture in a Schleicher and Schiill diffusion thimble of
parchment-paper tied on to a glass tube fitted with a cork and a long
narrow tube to act as manometer. This was placed in water contained in a
large test-tube and then immersed in a thermostat at 30°'7.C. The osmotic
pressure rose to 200 mm. of water in the course of 30 hours, being 94 per
cent. of the theoretical value. Calcium sulphate in one-hundredth normal
solution was then put into the outer tube instead of the water. In about
24 hours the pressure had fallen to 50 mm. and no further fall took place.
In this case a certain protection was shown, since, without the albumin, the
pressure would have gone down nearly to zero.

The apparent disagreement between the results of osmotic pressure
measurements and the naked eye appearances are, I think, to be explained
in the following way. When the contents of the osmometer in the last
experiment were poured into a glass vessel and observed carefully, it was
obvious that, although no precipitation had taken place, the solution was
distinctly more turbid than a similar one to which no calcium sulphate had
been added. On examination under the ultfa-microscope, the former was
resolvable into a multitude of distinct, but not brilliant, particles; whereas
solutions of congo-red itself, as already shown, are not resolvable. It is
clear, therefore, that a certain degree of aggregation had in reality taken
place, although the particles formed are much smaller than those formed
when calcium sulphate acts upon congo-red in the absence of a protecting
colloid. In this latter case, they are large enough to fall as a precipitate.
The actual values of the osmotic pressure observed, 200 mm. and 50 mm.,
show that, under the conditions of this experiment, aggregates of four
molecules are formed. It appears that, so far as congo-red is concerned, the
mode of action of a stable colloid is to form, under the influence of an
electrolyte, a colloidal complex with the dye, which complex, although
consisting of several molecules and therefore, when formed, causing a large
fall in the osmotic pressure of the solution, is yet in particles sufficiently
minute not to fall as a precipitate. The reason why the aggregates formed
are small is, no doubt, connected with the lowering of surface-tension caused
by the protein. It is to be noted that, unless the solution of dye+albumin
+electrolyte had been compared with a similar solution without the
electrolyte, it would have been supposed that complete protection from
the action of the electrolyte had been brought about, since no true precipi-

tation occurs.

1909. | The Properties of Colloidal Systems. 279

Results similar to the above have been obtained in the case of arsenious
sulphide, aggregation without precipitation also occurs here. In order that
the albumin shall be efficient as a protecting colloid it is necessary that it be
of the same sign as regards its electric charge as the arsenious sulphide,
that is, electro-negative; if electro-positive, it tends to aid the action of
the electrolyte.

I am unable, at present, to state definitely whether the same con-
siderations apply to the action of electrolytes and stable colloids on gold
hydrosols, the “ gold number” in fact. I cannot make out any difference in
the appearances under the ultra-microscope of mixtures of gold hydrosols
and serum-albumin, with and without calcium sulphate. To the naked eye,
there is perhaps a slight tendency to a more purple colour in the case of the
former, but nothing approaching the blue of the mixture of gold and
electrolyte in the absence of the albumin. The ultra-microscope shows also
that the particles in the blue solution are larger and less numerous than in
the ruby-coloured ones. It is possible that complexes of gold particles and
protective colloid, of the kind described by Zsigmondy,* may be formed
without change of colour of the gold. The whole series of phenomena in
these cases of protection are of much complexity and in need of further
investigation.

It is somewhat remarkable that congo-red is the only dye which I have
been able to make use of which shows this combination of non-diffusibility
through parchment-paper with existence of single molecules in solution.
The greater number pass through parchment-paper with more or less
rapidity, although showing many colloidal properties, so that no permanent
osmotic pressure can be obtained. Aniline-blue, with a molecular weight of
734, does not pass through. A solution containing 0°266 per cent. gave an
osmotic pressure of 30-4 mm. Hg instead of the theoretical one of
68°8 mm. Hg, indicating a mean “solution-aggregate ” of two molecules. It
must be stated, however, that this solution was apparently not free from
electrolyte, since, although when the experiment ceased no further permanent
rise in osmotic pressure was produced by change of water, there was still
a certain amount of “initial osmosis.” The displaced water, also, showed
a fairly high conductivity, 40 gemmhos, which did not diminish during the
last five days. This outer solution was always stained faintly blue, due, as I
shall show in another paper, to dissociation of the dye and permeability of
the membrane to the products of this dissociation. In a similar way, the
dialysate of congo-red is very faintly red. In the ultra-microscope the
appearance of the solution removed from the osmometer was very like that

* ‘Zur Erkenntnis der Kolloide,’ p. 116, Jena, 1905.

280 Dr. W. M. Bayliss. [Apr. 19,

of congo-red, as described above; a faint haze, with bright particles here
and there, these bright points being much more numerous than in congo-red.
I am inclined, therefore, to attribute the low osmotic pressure found to
incomplete removal of electrolytes, the solution really being one of single
molecules, but, owing to the electrolytes, a large number of these are
ageregated to particles, the rest being single.

The free acid of congo-red, as already stated, forms a deep blue colloidal
solution, with comparatively large particles, easily resolved bv the ultra-
microscope. These particles are of a very nearly uniform size. In view of
the debated question as to the source of osmotic pressure, it 1s of interest to
see whether an unquestionable suspension, such as this is, has an appreciable
osmotic pressure and, at the same time, to determine the number of particles
present in unit volume. Since, as shown above, the salts of this acid have
so considerable an osmotic pressure, while that of the particles of the acid
would probably only be small, it is obviously of the greatest importance to
ensure the absence of traces of alkali, whether in the water used or from
glass vessels. The apparatus shown diagrammatically in fig. 2 was devised
for this purpose. It will probably be found of use in the investigation of
other colloids sensitive to traces of electrolytes, such as arsenious sulphide.
All the tubes in contact with the fluids in the osmometer are of quartz and
the containing vessel of glass is thickly coated with paraffin inside. The
membrane is a Schleicher and Schiill parchment-paper thimble, as used by
Waymouth Reid and by Hiifner. It is necessary to test these thimbles very
carefully for holes and, for my purpose, to extract thoroughly before use
with hydrochloric acid and distilled water to remove soluble salts. In future
experiments it would perhaps be better to use membranes of collodion.

The distilled water must be carefully prepared. I found it best to distil
tap water (New River) after adding potassium permanganate and sulphuric
acid, and again after the addition of barium hydroxide. When essential to
exclude carbon dioxide entirely, the water from the quartz condenser drops
directly into the osmometer, as shown in the figure.

In an actual experiment with congo-red acid, the outer water was at first
made acid with hydrochloric acid to ensure the absence of any salt of congo-
red in the contents of the osmometer. The solution in the inside had been
dialysed for a long time against distilled water. The concentration at the
end of the experiment was 0°465 per cent. of the free acid. At the beginning
of the experiment no measurable osmotic pressure was obtained; the acid
was in the form of a precipitate, due to the presence of hydrochloric acid.
Distilled water, from potassium permanganate and sulphuric acid, was then
allowed to flow from the condenser, gradually displacing the hydrochloric

1909. | The Properties of Colloidal Systems. 281

acid. It is interesting to note that the osmotic pressure rose distinctly,
while the outflowing water still gave reactions with silver nitrate and with

a, Tube on to which the parchment-paper thimble is tied. This tube has aside branch,
connected by a short piece of rubber tubing to 6, and thus, when the clip on the
tubing is released, the inner and outer chambers are put in connection. This is of
importance to control the zero point.

c, Narrow bore tube, serving as manometer. d, Outlet tube from outer chamber.

e, Tube conducting water from the quartz condenser, f.

g, Flask to collect the outflowing water, apart from contact with the atmosphere.

h, Tower containing dilute sulphuric acid. #, Soda-lime tower.

J, Flask for boiling water. m, Trap to collect spray. n, Glass wool.

o, Outlet for water condensed in m.

The containing vessel is thickly coated with paraffin and kept ina thermostat. The
tubes @, c, d, e, and f are of quartz.

Giinzberg’s test, and, in fact, the pressure had risen to 60 mm. of water when

60 c.c. of the water neutralised 1:4 c.c. of decinormal ammonia. The distilled

water, containing carbonic acid but no basic substance, was run in at intervals
Vin Exes ——B. Xi

282 Dr. W. M. Bayliss. | Ajo ios

of 24 hours until no further rise in the osmotic pressure took place. The
value attained was 91 mm. of water, or 6°8 mm. Hg at 30°-9 C. There seems
no possibility whatever of the presence of a salt of the dye in the above con-
ditions. In any case, there was a fairly large pressure when free hydrochloric
acid was detected in the outer fluid. A further reason for denying the
presence of alkaline bodies in the water used is that, if this had been so, the
pressure would have continued to rise as long as fresh water was run in,
until finally the great pressure of the salts of congo-red would have been
reached and the contents of the osmometer converted entirely into the salt.

The conclusion must be that a definite osmotic pressure can be exerted by
a solution consisting of an undoubted suspension of particles, resolvable under
the ultra-microscope. When water distilled from over barium hydroxide was
run in, there was not much further rise in the osmotic pressure, the maximum
being only a few millimetres higher. It appears, therefore, that this colloid
is not particularly sensitive to traces of free acid.

If now we proceed to calculate, from the concentration of such a colloidal
solution as that of the above experiment, what the osmotic pressure should be
if the dye were present in single molecules, we find that it is 20 times that
actually found. Assuming for the present that the kinetic theory of the
osmotic pressure of colloids is correct, this means that the average number of
molecules forming a colloidal particle of the free acid of congo-red is 20.

This being so, and the separate particles being easily seen in the ultra-
microscope, it seemed to be a point of interest to attempt to estimate the
dimensions of the particles in the manner described by Siedentopf and
Zsigmondy.* For this purpose a part of the contents of the osmometer
at the end of the experiment was diluted 1300 times, so that the ‘particles
might be sufficiently far apart to be counted. This counting was somewhat
difficult, owing to the rapid movement of the particles. The mean of
a number of determinations was between 8 and 9 in a volume of
56x10-6 cubic millimetre. The undiluted solution, therefore, contained
2x10" in 1 ec. There is, however, a possibility not to be lost sight of.
As I shall show in a later paper, this acid of congo-red appears to be very
slightly soluble in water at 100°, in true solution, ionised. Although the
amount dissolved at room temperature is infinitesimal, it may be sufficient.
to vitiate conclusions drawn from solutions necessarily very dilute. Accord-
ingly, I have made determinations, similar to the above, with solutions of
one and a-half and of twice the dilution of that one. The three values are
placed together in the table for comparison. It will be seen that the values
are, within the limits of error, proportional to the dilution.

* “Dyrude’s Ann.,’ vol. 10, pp. 17, 21, and 22, 1903.

1909. | The Properties of Colloidal Systems. 283

IDaliptaions (iss attack Oe Number of particles
litres containing Fai lae
1 gramme). sie
280 14—16 x 107
420 10 -‘7—12 °5 x 107
560 8-9 x 107

Now, the total weight present in 1 cc. of the original solution is
465 milligrammes, so that the weight of each particle is
4:65
2x10"
Further, the specific gravity of the solid acid is 146, determined by
weighing under toluene in a pyknometer.* Hence, the diameter of each

= 2:3 x10" milligramme.

particle is 310 py.

We may, perhaps, go even further still. According to the osmotic pressure
measurements and assuming the kinetic origin of this pressure, each particle
contains on the average some 20 molecules; so that, if this theory be
correct, we ought to be able to obtain an approximate value for the molecular
dimensions of this body. When calculated from the data given, the weight
of a molecule of congo-red acid comes out to be

1:16 x 10~” milligramme,
or nearly 10° times that of hydrogen.t And the diameter
WILY jaye.

The molecular weight being 652:372, the number of molecules contained in
1 gramme-molecule comes out as
652372
TNGEXOs 25

The number of molecules in a gramme-molecule of a perfect gas is usually

= 19 9¢ ILO

estimated at about
Org O2en

Considering the many sources of error, the result obtained for the
molecular dimensions of our colloid does not seem very far out. This being
so, the hypothesis of the kinetic origin of osmotic pressure is, so far,
supported.

The chief difficulty in the estimation of the number of particles under the
ultra-microscope is, in the case before us, the lively movements which they

* Ostwald-Luther, ‘Phys.-Chem. Mess.,’ 2te Aufi., p. 147, 1902.

+ Walker, ‘Introd. to Phys. Chem.,’ 4th ed., p. 217.
{ See Perrin, ‘Comptes Rendus,’ vol. 147, p. 531, 1908.

284 Dr. W. M. Bayliss. [ Agoreoe

manifest. In order to stop this movement, I mixed a part of the diluted
solution, as used for the previous measurements, with an equal volume of a
2-per-cent. solution of gelatin, warmed just sufficiently to liquefy it. This
method was used by J. Duclaux,* in his investigations of ferric hydroxide.
Although the gelatin used by me had been soaked in repeated changes of
toluene-water, it retained a certain amount of its adsorbed electrolytes ;
so that on adding the blue colloid to it, a distinct change of colour towards
purple resulted. Although the particles were seen to be immobilised, it
did not seem worth while to proceed further with the laborious deter-
minations, since the change of colour indicated a change in the colloid.

The determinations of molecular dimensions given above are intended to
show the possibilities of the method. The exact numerical data are, no
doubt, capable of correction when a more satisfactory means of immobilising
the particles has been found. The values obtained appear high, even for
a molecule containing 70 atoms, such as the one in question. From
Zsigmondy’s observations with colloidal gold it would seem that particles of
these dimensions should be resolved by the ultra-microscope. It is true
that the impression given to the observer is that the solutions of congo-red
are just on the limit. Moreover, the fact that molecules of congo-red are
unable to pass through parchment-paper shows that they far exceed in
dimensions those of crystalloid bodies.+

Further experiments are in progress, as also others in the manner of those
of Perrint with suspensions of gamboge. It would be premature to draw
conclusions from the results of the preceding pages as to whether the
particles as wholes are responsible for the osmotic pressure, or whether only
a part of each one, such as adsorbed ions, alone is active in this respect.
So much may be said, that my observations speak decidedly in favour of the
kinetic theory of the osmotic pressure of colloids. According to this theory
the “ Brownian movement” of the particles corresponds to the molecular
movement assumed in the kinetic theory of gases.

Important recent confirmation of this view is to be found in the experi-
ments of Perrin already alluded to, which show that the kinetic energy of
a colloidal particle is identical with that of a molecule. This observer shows
that, if we take the number of molecules contained in one gramme-molecule
of a perfect gas to be

6 or 7 x 103,

* ‘Comptes Rendus,’ vol. 147, pp. 131—138, 1908.

+ As regards size of pores in parchment-paper, see Bechhold, ‘ Zeit. f. Phys. Chem.,’
vol. 64, pp. 328—342, 1908.

t ‘Comptes Rendus,’ vol. 146, pp. 967—970, and vol. 147, pp. 530—532, 1908.

1909. } The Properties of Colloidal Systems. 285.

as given by the kinetic theory, the osmotic pressure of a solution containing
# molecules per unit volume is

nx 40 x 107 atmos.

When deduced from the rate of fall of the particles in a gamboge suspen-
sion, assuming Stokes’ formula to apply, and taking n to refer to particles,
the osmotic pressure works out to be

nx 36x 107 atmos.

When deduced from the distribution of particles in a vertical column, after
attainment of equilibrium, the formula becomes

nx 42x 107) atmos.

From my observations, determining the concentration of particles by direct
enumeration under the ultra-microscope, the formula becomes

nx44x 107 atmos.

Such close approximations to the theoretical value must be more than mere
coincidence.

Ramsay and Senter* also concluded, from experiments on the density of
arsenious sulphide solution taken by different methods, that the particles
behave as if in true solution.

On the other hand, it is evident that my experiments lend no support to
the theory according to which the osmotic pressure of a colloidal solution is
due, in some way not very clear, to ions associated with the colloidal
particles. It is difficult to understand how these ions can still exert their
osmotic pressure when forming part of a complex system, which must move
and act as a whole. This much may be said, congo-red gives an osmotic
pressure which is at its highest when foreign electrolytes are most effectively
excluded. This must be understood as in no way excluding, as the ultimate
source of the negative charge, electrolytic dissociation of the colloid itself.

It is very doubtful whether electrolytes in the state of adsorption are
ionised at all. Ruert finds that the chlorine present in colloidal zirconium
hydroxide gives no reaction with silver nitrate. Similarly in the case of
ferric hydroxide, the chlorine can only be detected after destruction of the
colloid by nitric acid.

The general conclusion to be drawn is, I think, that whether a body
present in solution be in the form of particles, molecules or ions, each of

* B. A. Reports, 1901. In 1905 (Journ. Phys. Chem.,’ vol. 9, p. 319) Senter also made
the suggestion that Brownian movement in colloids is equivalent to molecular movement
in true solutions.

+ ‘Zeits. f. Anor . Chem.,’ vol. 43, pp. 88—93, 1905.

286 The Properties of Colloidal Systems.

these acts as an individual and equivalent element in the production of
osmotic pressure.
Summary.

Congo-red, although a colloid in the sense of not being diffusible through
parchment-paper and exhibiting certain other colloidal properties, has an
osmotic pressure equal to that which would be given if it were present in
true solution in single molecules.

The solutions are not resolvable into particles by the ultra-microscope.

The theoretical osmotic pressure is only to be obtained in the complete
absence of extraneous electrolytes. Even the carbonic acid present in
ordinary distilled water is sufficient to cause a marked fall in the osmotic
pressure.

The manner in which electrolytes produce this fall is by causing aggrega-
tion of molecules to particles. This is the same whether acid, alkali, or
neutral salt be in question.

The action of a stable colloid in protecting against the effect of electrolytes
is shown to consist, in the cases of congo-red and arsenious sulphide, in the
production of minute aggregates, which, although causing fall in osmotic
pressure by diminution of effective concentration, are not of sufficient size to
precipitate. Hence the protective power can only be regarded as a limited
one, due probably to the formation of complex colloids.

The free acid of congo-red forms a blue colloidal solution when dialysed.
This is easily resolvable under the ultra-microscope, but gives a definite and
measurable, though small, osmotic pressure, about 14 mm. Hg for a 1-per-cent.
solution. Assuming the kinetic theory to be correct, this means that the
aggregates contain, on an average, 20 molecules.

Estimation of molecular dimensions are given on the basis of enumeration
of the number of particles in unit volume by means of the ultra-microscope.
The values found are considerably larger than the accepted ones for water, ete.
The whole of the results are capable of explanation on the assumption that
colloidal particles possess the kinetic energy of molecules, but do not lend
support to any view which postulates the necessary presence of foreign
electrolytes.

(The expenses were defrayed by a grant from the Government Grant
Committee of the Royal Society.)

287

Some Hffects of Nitrogen-fixing Bacteria on the Growth of Non-—

Leguminous Plants.

By W. B. Bortomugy, M.A., Professor of Botany in King’s College, London.

(Communicated by Prof. J. Reynolds Green, F.R.S. Received April 20,—Read
May 6, 1909.)

During the course of an investigation on “The Cross-inoculation of the
nodule-forming bacteria from Leguminous and non-Leguminous plants,” it
was noticed that in all the bacterial cultures prepared from the algal zone of
the root-tubercles of cycads taken from below the surface of the soil,
Pseudomonas radicicola was associated with a species of Azotobacter.

In order to determine to what extent, if any, this association gave an
increased power of assimilating free nitrogen, the two forms were obtained as
pure cultures by successive platings on a medium composed of maltose
20 grammes, monobasic potassium phosphate 0°5 gramme, sodium chloride
0°5 gramme, calcium carbonate 0°5 gramme, ferrous sulphate 0-1 gramme,
agar 15 grammes, and distilled water 1000 cc. Separate cultures of each
and a mixed culture were then grown in fluid media in duplicate 500 cc.
Erlenmeyer flasks containing 250 c.c. of the above medium, omitting the
agar but adding 10 grammes of mannite. Control flasks were kept side by
side with the inoculated flasks.

All the flasks were incubated at 24° C. for 15 days, care being taken to
renew the air in the flasks at intervals, then a nitrogen determination was
made of the contents of each flask. The results of these analyses gave the
following averages :—

Comtrolearscsarciavecea cas 0:48 milligramme N per 100 c.c.
Pseudomonas alone............ 0:91 5 a 90
Pseudomonas + Azotobacter... 1:24 HF in 5)

Hence Pseudomonas and Azotobacter together make a powerful combination
for the fixation of free nitrogen.

The bacteria in the root-tubercles of cycads appear to live imbedded in the
slime they produce outside the cortical cells in the open spaces of the so-called
algal zone. The cortical cells which project into this zone presumably absorb
the nitrogenous products of the bacterial activity, and thus the cycad is
benefited. If, therefore, this combination of bacteria outside the cortical
cells is a direct benefit to the cycas plant, the possibility presented itself that

* Report British Association, 1907.

288 Prof. Bottomley. Hffects of Nitrogen-fixing [Apr. 20,

a mixed culture of Pseudomonas and Azotobacter applied directly to the roots
of other non-leguminous plants might benefit their growth also.

The first experiments were made on oats (Avena sativa). Four 5-inch pots
were filled with sand freed as far as possible from organic matter and nitrates,
and given a sufficient dressing of phosphates, potash, and lime. Twenty oat
seeds were planted in each pot, and as soon as the young plants were about
1 inch high two of the pots were watered with a mixed culture of Pseudo-
monas and Azotobacter grown in the previously mentioned medium. The
plants were watered regularly with distilled water and allowed to grow until
the untreated plants exhibited signs of drooping. Each plant was then
carefully air dried and weighed, with the following result :—

Untreated, average weight per plant ...... 0-42 gramme
AREA TEC ier ccte bce visions Rint Meiccteeke er MECC REE 0°74 sé

an increase of 0°32 gramme, or 76 per cent.

In 1908, field experiments were made on barley. Two plots, each having
an area of 484 square yards, were planted, one with seed moistened with the
mixed culture, the other with seed untreated. The land was very poor and
low in organic nitrogen, having carried oats in 1906 and barley in 1907. The
yield of grain per plot at harvest was :—

lWinineatcdess sess eer 608 lb.
Mreatedissatune mst: 691 ,,

an increase of 83 lb., or 13°6 per cent.

On the same farm a strip of land was sown with treated seed through the
centre of a 34-acre barley field. It was found impossible at harvest to keep
the yield of treated seed separate from the rest, but samples of grain from the
treated and untreated parts were taken and analysed, and it was found that
the barley from the treated seed had the higher nitrogen-content.

Milligrammes Weightof Miulligramme
of N per cent. 1000 corns. N per corn.

Untreated Sans 15) 48°5 0:75
AREA! -cooss50n 1:76 49°5 0°87

Experiments on hyacinths (Galtonia candicans) grown in sandy soil. The
soil was dressed with lime in August, 1907, and remained fallow until April,
1908, when it was manured with cow manure—10 tons to the acre. Bulbs
of equal size were then planted, 250 in each bed, a path of 14 inches dividing
the two beds. The treated bulbs were twice watered with mixed culture
solution, once in May and once in June, the control bed being watered with
pure water at the same time. The difference between the treated and

1909.| Bacteria on the Growth of non-Leguminous Plants. 289

untreated bulbs was very noticeable during growth, the treated being more
vigorous. The treated bulbs, when lifted, were noticeably larger than the
untreated. After the bulbs were dry they were carefully weighed, and

yielded
Untreated......... 69 lb. 3 oz.

WreeeClL° sccacease S2Eee as

an increase of 12 lb. 144 oz., or 18°6 per cent.

Experiments on parsnips grown in ordinary garden soil. In January,
1908, the ground was deeply dug and given a medium dressing of London
dung followed by a dressing of powdered chalk. The seeds were sown early
in February in rows running north and south. A fortnight after the north
half of each row was watered with a mixed culture of Pseudomonas and
Azotobacter. In January, 1909, the roots were harvested, every root being
weighed, with the following results :—

Untreated ...... 68 roots 22 lb. 14 oz. 5°38 0Z., average per root
Mlereated sk: :.c..cs. OD) gn AD 5 iO, Odd) 5, es

an increase of 21-7 per cent.

For the fixation of free nitrogen in laboratory cultures of Pseudomonas
and Azotobacter the presence of carbonate of lime in the medium is necessary.
In all the above experiments care was taken that a sufficiency of carbonate
of lime was present in the soil to enable the bacteria to do their work
effectively.

290

The Intracramal Vascular System of Sphenodon.
By Artaur DEnpy, F.RS.

(Received March 30,—Read April 22, 1909.)

(Abstract. )

This memoir contains a detailed description, with illustrations, of the
intracranial blood-vessels of the Tuatara, of which no account has hitherto
been published. The description is believed to be more complete than any
hitherto given for any reptile, and a considerable number of vessels are
described which have not hitherto been noted in Lacertilia. This compara-
tive completeness of detail is largely due to the employment of a special
method of investigation. By this method the entire contents of the cranial
cavity are fixed and hardened in situ, and are then in excellent condition
either for dissection or for histological purposes. The brain does not occupy
nearly the whole of the cranial cavity, there being a very large subdural space
(especially above the brain), across which many of the blood-vessels run,
together with delicate strands of connective tissue which connect the dura
mater with the pia. The eyeballs are removed and an incision is made on
each side in the cartilaginous wall which separates the cranial cavity from the
orbit. Acetic bichromate of potash (made up according to the formula given
by Bolles Lee) is injected into the cranial cavity through these incisions, and
the entire animal, after opening the body cavity, is suspended in a large
volume of the same fluid for about five days, and then graded up to 70 per
cent. alcohol. When the cranial cavity is now opened up the cerebral vessels
are seen with extraordinary distinctness, although they have not been arti-
ficially injected.

Further details were made out by means of serial sections, both transverse
and longitudinal, and both of the adult and of advanced. embryos (Stage 8).
In most respects the arrangement of the intracranial blood-vessels agrees
with that found in the Lacertilia, so far as these have been investigated, but
there is an important difference in the fact that the posterior cephalic
vein leaves the cranial cavity through the foramen jugulare and not
through the foramen magnum, while a slightly more primitive condition
is shown in the less complete union of the right and left halves of
the basilar artery. Sphenodon makes some approach to. the condition
of the Chelonia in this latter respect, but differs conspicuously from
this group in the fact that the circle of Willis is not completed
anteriorly, as well as in the fact that no branch of the posterior cephalic

Variations in Pressure and Composition of Blood in Cholera. 291

vein leaves the cranial cavity through the foramen magnum. A very
characteristic feature of Sphenodon is the development of large transverse
sinuses resembling those of the crocodile, but these communicate with the
extracranial vascular system in quite a different manner from that described
by Rathke in the latter animal.

The Variations in the Pressure and Composition of the Blood in
Cholera; and their Bearing on the Success of Hypertonic
Saline Transfusion in its Treatment.

By Lzonarp Roaurs, M.D., F.R.C.P., F.R.C.S., L.M.S., Professor of Pathology,
Calcutta.

(Communicated by Sir T. Lauder Brunton, Bart., F.R.S. Received
December 4, 1908,—Read January 28, 1909.)

During the quarter of a century which has elapsed since the discovery by
Prof. Koch of the comma bacillus of cholera, research work has been almost
‘confined to the bacteriology of the subject. Unfortunately, with the exception
of M. Haffkine’s prophylactic inoculations, which are now very little used
even in India, this line of work has done little or nothing to help the
practitioner who is confronted with the treatment of this terrible disease. No
powerful antitoxic serum of practical value has been produced, and even if
‘such should still be obtained, many patients come under treatment in such a
state of collapse that no medicine can be absorbed, even if retained.

The old controversy between the evacuant and conservative methods of
treatment has long since ended in the practically universal adoption of the
latter, although as late as 1866 Dr. George Johnson advocated castor oil,
‘denying that there was any relationship between the amount of fluid lost from
the body and the mortality, while he strenuously opposed the use of
intravenous saline injections to replace it. There is still much difference of
opinion about the latter treatment, for although all who have used transfusions
testify to the remarkable immediate improvement in the pulse and general
condition, yet this is commonly of such brief duration that many think it only
serves to needlessly prolong the agony of the patient, so that of recent years
it has been only exceptionally resorted to in India.

For a long time I have been investigating the blood changes in cholera (1), in
the hope of finding some indication for a more rational and successful line of

292 Prof. L. Rogers. Variations in Pressure and {[Dee. 4,

treatment, than the mere administration of more or less useful drugs, into a
gastro-intestinal tract, which is too congested to retain any appreciable powers
of absorption. The great loss of fluid from the blood was shown by marked
increase of the number of red corpuscles per cubic millimetre, which
sometimes rose from 5,000,000 to over 8,000,000, while, like others, I found
leucocytosis to be constantly present, and when of a marked degree to be of
bad prognostic value. Differential counts showed a great decrease of the
lymphocytes, corresponding with their accumulation in the lymphoid tissue of
the alimentary tract, while the large mononuclears were markedly increased,
both relatively and absolutely, this change being in proportion to the
mortality, indicating its probable relationship to some specific toxin of the
disease. These changes were often of great value in the difficult diagnosis of
cholera from ptomaine poisoning, as I have not found them in the latter
affection.

More recently I have studied the blood-pressure in cholera as an indication
for the necessity of transfusion and the quantities to be injected. My first
experiments in this line were carried out in conjunction with Captain
J. W. D. Megaw, I.M.S. (2), who was in charge of the cholera patients at the
Calcutta Medical College, normal saline solution (1 drachm to the pint)
being used in accordance with the general custom. The patients were nearly
all natives of India, whose normal blood-pressure averages only about
100 mm., being thus considerably lower than that of Europeans. In cholera,
the pressure was rarely over 50 mm. on the admission of the patients, while
in many it was too low to be estimated in the radial artery, owing to absence
of any pulse at the wrist. We found that 1 pint of saline solution injected
into a vein usually had very little effect in raising it, but a second pint
generally increased it to 90 mm. or more. The immediate effects of this
treatment were little short of marvellous. The terrible restlessness and
cramps disappeared, and the worn out patient often fell asleep before its
completion. The pulse was fully restored, and the blueness and coldness
were replaced by the normal pink hue of the lipsand nails and warmth of the
extremities. A few improved steadily from that time, but only too frequently
within 2 to 12 hours the copious rice water stools and vomiting recurred, and
the patient relapsed into as bad a condition as before, with complete loss of
the improvement in the blood-pressure. Repeated transfusions usually failed
to save such patients, and although the mortality did show some reduction
during the year Captain Megaw continued them, yet the disappointments.
were so many that the method soon came to be adopted only in a few
desperate cases, and was eventually almost entirely abandoned once more, as.
it had been by so many earlier enthusiasts.

1908. | Composition of the Blood im Cholera. 293

On thinking over the causes of this comparative failure, the following
possible explanation occurred to me. The great loss of fluid through the
stomach and bowels produces a concentration of the blood, which might be
expected to increase the proportion of salts it contains, and therefore present
a greater osmotic resistance to further draining off of fluid through the
damaged intestinal mucous membrane. Thus, a conservative process, tending
to check the diarrhoea, would come into action, which would be interfered
with by the injection into the blood stream of large quantities of normal salt
solution of lower salt content than the now concentrated hypertonic blood-
serum. The drain through the bowel would therefore be restarted, and the
restored fluid and blood-pressure rapidly lost again, as is so commonly seen in
actual practice. If this view is correct, the indication would be to inject
hypertonic salt solutions, so as to supply sufficient fluid to restore the
circulation, and at the same time maintain the conservative beneficial
hypertonicity of the blood, which would tend to carry more fluid into the
circulation, instead of removing it through the damaged bowel wall. In 1907
I had an opportunity of discussing this point with both Sir Lauder Brunton,
Bart., F.R.S., and Prof. Buckmaster, who were of the opinion that moderately
aypertonic salt solutions might safely be injected intravenously.

The unusually great prevalence of cholera during the first half of 1908 in
Calcutta furnished abundant opportunities for testing the value of intravenous
transfusions of hypertonic salt solutions, for a trial of which I am greatly
indebted to Captain Maxwell Mackelvie, I.M.S., who had in the meantime
succeeded to the charge of the cholera wards. At first, 0:95-per-cent. sodium
chloride solution was tried, and, as the results appeared to be distinctly
more favourable than with the previously used 0°65-per-cent. one, it was soon
raised to 1°35, or 2 drachms to the pint. The quantities injected were also
raised by Captain Mackelvie to 3 or 4 pints ata time. Ina note(3) published
in the ‘Indian Medical Gazette’ in May, 1908, we recorded the results in
72 consecutive cases, in which transfusion with the above strength was used
in all who required it, with a mortality of only 27°8 per cent:, as compared
with an average during the five years preceding the revival of transfusion at
the hospital of 61-2 per cent. Moreover, at a neighbouring hospital, during
the same period as our cases were treated, the mortality was just twice as
great, intravenous injections not being used there, although their previous
mortality during a series of years was practically the same as at the Medical
College Hospital, namely, 63 against 61 per cent. At the present time our
cases number 175, with a death-rate of 33 per cent., including moribund and
complicated ones, which is but a very little over half that of the pre-
transfusion period, although the mortality is always exceptionally high in

im Pressure and [Deec. 4,

rations

Var

Prof. L. Rogers.

294

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1908. | Composition of the Blood i Cholera. 295

such epidemic years as the present one, and the patients were frequently too
numerous to allow of as many being transfused as we should have liked.
Very favourable reports have also reached me from two other medical officers
who have tried hypertonic solutions in cholera.

During the last few months I have made a number of observations on the
blood changes before and after transfusion in cholera, which appear to furnish
a rational basis for the regular use of large hypertonic transfusions in the
treatment of the disease, and propose to deal with them in the present
communication. The data have all been tabulated for convenience of
reference.

The Relationship of the Amount of Fluid lost from the Blood in Cholera to the
Degree of Collapse and the Death-rate.

As has been pointed out by Macnamara, Wall, and others, the amount of
the watery evacuations in cholera will not furnish accurate data for estimating
the degree to which the blood is drained of fluid, for the very rapid escape of
2 or 3 pints will have a far greater effect on the circulation than much
more considerable loss spread over a longer time. Thus, in the so-called
cholera sicca, in which death takes place without actual diarrhcea, the small
intestines always contain several pints of liquid, the rapid draining of which
trom the blood has caused fatal circulatory failure. In order to ascertain the
real loss of fluid from the blood in cholera, and the effects of various quantities.
of intravenous saline injections required to replace it, I have taken blood
immediately before and after transfusion, defibrinated it at once, and
measured the relative volumes of corpuscles and serum by centrifuging in
the hemocrite. As the red corpuscles are not lost to the circulation, but
only the fluids of the blood, the reduction in the volume of serum can be
readily calculated, and the actual loss of fluid from the serum of the blood
estimated from the total volume of the blood in the body. According to
some observations on healthy Bengalis by Captain McCay, I.M.S., the average
proportions of corpuscles and serum respectively were 45 and 55 per cent.,
showing a slightly smaller proportion of corpuscles than in Europeans.
Assuming that the red corpuscles are not lost from the circulation, the
diminution in the serum can be calculated by the following formula from the
hemocrite figures. As the percentage volume of corpuscles found is to
the normal value (45 per cent.), so is the volume of serum found to X. For
example, if in a case of cholera the volume of corpuscles is 71 per cent. and
of serum 29 per cent., then as

els Se -57, Oy XGA ke — OF

296 Prof. L. Rogers. Variations in Pressure and [Dee. 4,

That is, out of 55 original volumes of serum in the normal native blood only
16 remain, so the loss is 39 out of 55 volumes, or 71 per cent. of the total
serum of the blood. In this way the figures given in column 2 have been
arrived at. Further, taking the total circulating blood as one-thirteenth of
the body weight (the exact amount is still disputed, but as my figures have
but a relative value, this is immaterial in the present instance), I estimate
the amount of blood in the body of a native of average weight as 6 pints, or
120 ounces. The percentage of serum being 55, this would give 66 ounces
of blood serum in the circulation. The percentage loss in a given case
having been calculated as above, the absolute loss in ounces is easily obtained,
and the figures are given in column 3 of the table.

The cases have been subdivided, in accordance with the severity of the
cases, into the three following classes. Firstly, those examined in the acute
stage of the disease, which proved fatal from its direct effects. (Four cases
in which the patients recovered from the collapse stage to die of late com-
plications have been omitted, as the deaths were due to such affections as
dysentery and parotid abscess, which afford no indication of the original
severity of the cholera.) Secondly, those who showed such well-marked
collapse as to require intravenous transfusion, but ultimately made a good
recovery. Thirdly, milder, though well-marked, cases of cholera, in which
transfusion was not necessary. The disease in most of the cases was confirmed
bacteriologically, although the diagnosis was left to the physician in charge,
so as to be strictly comparable with earlier series.

The average loss of fluid from the blood in each class is given in the table,
and clearly demonstrates that the urgency and fatality of the cases was in
direct proportion to the diminution in the volume of the blood-serum. Thus,
among those in which the disease proved directly fatal, the loss of serum
averaged no less than 64 per cent., or almost two-thirds, the total loss being
42 out of 66 ounces. In the second class of recoveries after transfusion there
was a loss of 52 per cent. of the serum, or 34 ounces, while in the third series
of recoveries without transfusion the losses averaged only 35 per cent., or
23°3 ounces.

The Specific Gravity of the Blood i Cholera.—The above observations
furnish accurate estimations of the concentration of the blood due to loss of
fluid in cholera, and demonstrate a definite relationship between its degree
and the severity of the disease, while they indicate the necessity of replacing
the loss in severe cases by some form of transfusion. For clinical purposes,
however, some simpler method of rapidly ascertaining the required informa-
tion is essential, especially in Indian hospitals far removed from laboratory
conveniences. An easy, and sufficiently accurate, way is to estimate the

1908. | Composition of the Blood in Cholera. 297

specific gravity of the blood by the chloroform and benzene method, or more
conveniently in a hot climate by Lloyd Jones’ plan of using a series of small
bottles containing solutions of glycerine in water of different specific
gravities, into several of which a drop of blood can be placed until the one
in which it neither sinks nor rises is found. I have made a number of
observations in this manner, and find that in bad cases of cholera the specific
gravity usually rises over 1070, while in those which did not require
transfusion it only reached up to about 1065, so that a clinically valuable.
approximate estimation of the concentration of the blood in cholera can thus:
be very rapidly obtained at the bedside. Moreover, this test can be repeated
during transfusion so as to ascertain when the blood has been diluted down
to its normal point, or better, a little below it, and the quantity of fluid
injected can be so regulated.

The Quantity of Fluid required to replace the Loss in Cholera.

Columns 1 and 5 in the table show the percentage volume of serum in the
blood before and after intravenous transfusion with 1°35 sodium chloride
solution in the quantities indicated in column 4. The usual amount was
4 pints, and in several successful cases this quantity was repeated a second
time. These are larger amounts than are usually recommended for intra-
venous use in cholera, but have given very good results. Moreover, column 5
of the table shows that they did not increase the volume of serum very
materially above the normal level of 55 per cent., while any excess will pass
rapidly into the greatly drained tissues, and some allowance must be made
for further losses through the bowel before the disease completely subsides.
In several cases the blood was found to have again become so concentrated
by the following day as to necessitate a second injection, with ultimate
recovery, so that the amounts used were certainly not excessive. At the
same time there must be a limit to the quantities that can be rapidly
administered intravenously at one time, and the fact that in several cases the
volume of serum was raised to between 65 and 70 per cent. appears to
indicate that 4 pints is sufficient in most cases. In the very exceptional
concentration of the blood in case 5, however, even this amount failed to
increase the serum even to the normal point, the specific gravity after the
injection being also 1064, or considerably above the normal, and the patient
ultimately died.

In the less severe types much fluid can be got into the system by rectal
injections, which are of great value. Subcutaneous injections are often used,
but act much more slowly than intravenous ones in raising the blood-
pressure, and are very liable to be followed by abscess in the low state of

VOL. LXXXI.—B. Ns

298 Prof. L. Rogers. Variations in Pressure and {[Dee. 4,

vitality of the tissues in cholera. I have recently had a simple cannula
constructed with circular end sharpened like a cork borer, which can be safely
passed through the abdominal wall after incising the skin and fascia. By
this means saline fluid can be rapidly run directly into the abdominal cavity,
and it has been successfully used in a number of cholera cases in one of the
Calcutta hospitals. It can also be carried out in a much shorter time than
the more difficult operation of tying a cannula into the collapsed vein of a
cholera patient, but it takes several hours to be absorbed, so is less efficient
in urgent cases than the intravenous injections. The hypertonic solutions
have been found to give better results than normal saline by all methods of
administration.

The Loss of Chlorides from the Blood in Relation to Hypertonic Transfusions.

Edmund Parkes, in 1849, showed that the rice-water stools of cholera
contain very little albumen, but from $ to 1 per cent. of salts, so that for
every hundred ounces of fluid evacuations from the bowel nearly an ounce of
salt is lost from the blood and tissues. The main bulk of the salts consists
of chlorides, which may be taken as a guide to the amount in the blood at
any time. Some recent estimations made by me gave an average of 0°53 per
cent. of chlorides in cholera stools. The vomited matter contains much less
salt than the bowel discharges, apparently owing to the gastric mucous
membrane being less involved in the disease processes than the intestinal.
Different observers have obtained very varying results as regards the
percentage of salts found in the blood in cholera, some maintaining that
they are higher, and others lower, than normal, but the observations on this
point in the very limited literature available in Calcutta were all made many
years ago. I have, therefore, estimated by the silver nitrate method the
amount of chlorides in the blood in a number of cases both before and after
transfusion with the 1:35-per-cent. sodium chloride solution. The results are
shown in columns 9 and 10 of the table.

Taking first cases 1—7, which were fatal in the acute stage, we must bear
in mind that on the average two-thirds of the fluid had been lost from their
blood. If only the water had been removed without any saline constituents,
then the chlorides should have been present in three times the normal
amounts. The percentage of salts in the blood in Bengalis has been found by
Captain McCay to be somewhat higher than in Europeans, namely about
1 per cent., about 0°8 per cent. of which are chlorides. Yet in the fatal
cholera cases, in spite of the great concentration of the blood, the chlorides
were below the normal in four out of five, the single exception being a very
old man who died of heart failure. Fully two-thirds of the total chlorides of

1908. ] Composition of the Blood in Cholera. 299

the blood must, therefore, have been lost. In case 5, with only 0°67 per cent.
of chlorides, the serum showed actual commencing hemolysis, which com-
pletely disappeared when they had been raised to 0°88 by hypertonic
transfusion, while similar results have been noted in a few other cases with
very low salt content. Column 10 shows that a material rise in the
percentage of chlorides in the blood was obtained by transfusion of 3 to 4 pints
of 1°35-per-cent. sodium chloride solution, but the average amount after it
was still only 0:95 per cent.

On turning to the recovering cases 8 to 24, we find the average percentage
of chlorides in the blood before transfusion was 0°90, or considerably higher
than in the fatal series, and the same remark applies to the figures obtained
after transfusion, when the average was 1:07 per cent. Moreover, in cases 10
and 16, which showed a low amount of chlorides after the injection, the
disease was very severe, and in one a second transfusion was necessary to
save the patient. It will be observed that several of the cases, which
recovered after the injections, showed very low chlorides in the blood at the
first estimation, but when they were raised to about 1 per cent. or over by
the hypertonic solutions, the patients almost invariably did well as far as the
collapse stage of the disease was concerned, which is the most dangerous
period in cholera.

Another striking feature was the far less tendency of the hypertonic
solutions to restart the copious rice-water stools, which so commonly renders
the use of normal salines of such very temporary value. It is not too much
to say that at the Medical College Hospital, where cholera patients are
usually brought in an advanced stage of collapse, the simple substitution of
2 drachms of salt to the pint instead of one, for transfusion, has so
revolutionised the treatment, that, whereas formerly it was considered a
matter for surprise when a severe case of cholera recovered, it is now a great
disappointment when such a case is lost in the collapse stage.

The Coagulability of the Blood i Cholera.

A few observations on the clotting power of the blood have been made by
Sir Almroth Wright’s method. The results were very variable, the time
being normal in some, slightly reduced in most, and very markedly in a few.
It is noteworthy that in several of the worst cases, with a specially low
percentage of chlorides, blood taken in glass tubes remained quite uncoagu-
lated after several hours, and in one such case there were hemorrhagic stools,
found post mortem to be dependent on extensive petechial hemorrhages in
the cecum. After transfusion with hypertonic solutions the blood in such
cases clotted firmly and gave a clear serum. In view, however, of the

Y 2

300 Prof. L. Rogers. Variations in Pressure and {Dee. 4,

frequency of reduction of the clotting power of the blood in cholera, I now
add 3 grains of calcium chloride to each pint of salt solution, and have seen
no hemorrhagic stools in the few cases since treated.

Strength of the Hypertonic Solutions.

In a few cases, 1:65-per-cent. solution (24 drachms to the pint) were used
with good results, but as the 1°35 per cent. has proved successful in so
many cases, and, moreover, suffices to raise the chlorides in the blood well
above the normal, it is recommended for routine use. The higher strength
may be reserved for second transfusions in very severe cases, or when the
lower strength has not increased the percentage of chlorides in the blood
to the desired point of 1 per cent. or over, and especially if watery diarrhea
continues. We have never seen any harm from running these solutions
rapidly into a vein in cholera, 4 pints having frequently been administered
in 20 minutes. The specific gravity of the 1°35 solution is 1006, and of 1°65 is
1008, a knowledge of which allows the strength of the solution to be rapidly
verified.

Lffects of Intravenous Saline Injections on the Blood-pressure.

The blood-pressures immediately before and after transfusions are shown
in columus 6 and 7 of the table. Nil means that there was absolutely
no pulse at the wrist, so it could not be recorded in the radial artery, which
was the site of the other observations. It usually required about three pints
to raise the blood-pressure to 100 mm., which is the normal for Hindus.
Experience showed that the best results were obtained by continuing the
injections until the pressure rose somewhat above that point, for which
purpose 4 pints were generally necessary, and allow some reserve. In
some of the worst cases it was impossible to get it above 80 mm., as in
numbers 1 and 2 in the table, for it remained at that point in spite of another
pint being run in. One instance was met with in which even 63 pints failed
to raise the pressure above 65 mm. In such there appears to be some
vasomotor paralysis present, possibly due to absorption of albumoses through
the damaged intestinal mucous membrane. Adrenalin forces up the pressure
in them, but its effects are only very temporary, as I formerly found it to be in
the vasomotor paralysis of viperine snake poisons (4). The blood-pressure, then,
is a valuable guide to the amount of saline solution to be run in, a pressure
slightly above the normal being aimed at; but if 4 or more pints have been
given, and the last 20 ounces or so have produced no further improvement,
it is useless to continue it at that time. Fortunately a degree of vasomotor
paralysis which prevents the pressure being raised to 90 or 100 mm. in natives
of India is rare, as such have always terminated fatally in my experience.

1908. | Composition of the Blood in Cholera. 301

The Relationship of Blood-pressure to Post-choleraie Ureemia.

One result of tiding so many severe cases of cholera over the collapse stage
by large hypertonic saline transfusions, is to accentuate the importance of
uremia in the later stages of the disease. This very serious complication is
especially seen in two distinct classes of cases :—Firstly, in patients brought
to hospital more than 48 hours after the onset of the symptoms, which not
infrequently have been comparatively mild at the onset, and whose treatment
has been neglected. Secondly, the other extreme of patients admitted early
on account of the great severity of the affection, who have been supported
by repeated transfusions to the stage of reaction, when it is found to be
extremely difficult to restore the renal secretory activity, which has been in
abeyance during the prolonged period of very low blood-pressure.

On cutting sections of the kidneys of patients who had died in the uremic
stage of cholera, 1 was much struck by the amount of effused blood in and
around the convoluted and straight tubules and the tense state of the capsule
enclosing the extremely congested organ: all suggesting an actual mechanical
difficulty in the re-establishment of an efficient circulation through the organ.
In order to test if this was the case or not, I tried perfusion of normal saline
solution through the renal artery from different heights, so as to measure
the actual pressure required to obtain a fairly full outflow from the renal
veins. For this purpose I used both healthy kidneys, got from the bodies
of patients dying of other diseases, and also several from those who had
succumbed to the ureemia of cholera. In the former, a pressure equivalent
to 20 or 30 mm. of mercury sufficed to obtain a good flow through the
kidney circulation. On the other hand, in the cholera ones no flow at all
was got under a pressure of about 60 mm., and then only drop by drop, and
it was not until 90 or 100 mm. was reached that anything like a full stream
was observed. In one experiment, subsequent slitting of the capsule of the
organ reduced the pressure required by about 20 mm. In a case of cholera
in which the patient died of late complication with empyema, after the
secretion of the urine had been freely established a pressure equivalent to
30 mm. of mercury sufficed for the free perfusion of normal saline through
the kidneys, showing that it was only in uremic cases that obstruction of
the renal circulation existed in such a marked degree.

Since the above observations were made, the blood-pressure has been
carefully watched day by day after the termination of the collapse stage, and
it has been found that the uremic symptoms are more common in those
whose blood-pressures do not rise above 100 mm. For example, two very
severe cases of cholera were admitted to hospital at about the same time,
both of whom received two saline transfusions of 4 pints each, and

302 Variationsin Pressure and Composition of Blood in Cholera.

recovered from the collapse stage. On the fifth day both had passed little
or no urine and showed definite uremic symptoms. The blood-pressure of
one was now only just 100 mm. and he died the following night of uremia.
That of the other was 120 mm., and he began to pass urine that day, and
made a good recovery. Soon after another patient developed well-marked
uremia, his respirations being over 40 per minute and very laboured, while
he was practically unconscious, and apparently in a hopeless state. As his
blood-pressure was found to be only just 100 mm., adrenalin and digitalis
were administered subcutaneously, and on the following morning his pressure
was 110 mm., he had recovered consciousness, was passing urine freely, and
got well from that time. Thus, the indications derived from the post-mortem
kidney perfusion experiments have been borne out in practice, and it is clear
that the blood-pressure is the most important factor to be attended to in the
treatment of the deficient renal secretion, which ensues in so many severe
cholera cases, after the danger-of death from collapse has been averted by
saline transfusions.
Conclusions.

1. In cholera there is a very definite relationship between the amount of
fluid lost from the blood and the severity and mortality of the disease.

2. This loss is usually so great as to indicate saline transfusions to restore
the circulation.

3. Injections of normal salt solutions are commonly of only very temporary
benefit.

4. Hypertonic salt solutions (1°35-per-cent. sodium chloride, or 2 drachms
to the pint) are much more effective, their use having reduced the mortality
by about one-half.

5. A great loss of chlorides from the blood occurs in cholera, most marked
in the worst cases. If the percentage of chloride is considerably raised by
the intravenous injection of hypertonic salt solutions recovery usually
ensues.

6. The development of uremia in the reaction stage of cholera is
associated with a comparatively low blood-pressure, measures to raise which
are indicated for the prevention and treatment of this very serious
complication.

REFERENCES.

‘Lancet,’ 1902, vol. 2.

“Indian Medical Gazette,’ March, 1908.
Lbid., May, 1908.

‘Phil. Trans.,’ B, vol. 197 (1904), p. 123.

Boe to

303

The Effect of Heat upon the Electrical State of Living Tissues.
By A. D. Water, M.D., F.R.S.

(Received February 20,—Read March 4, 1909.)

I. Of Muscle.

The question whether the sudden application of heat acts as a physiological
stimulus to nerve and muscle naturally leads on to a study of the effect of
local heat upon the electrical state of living tissues. I have done this
(1) upon muscle, (2) upon nerve, and (3) upon the skin. As far as I know,
the only tissue hitherto tested in this respect is muscle.*

Du Bois-Reymond first,f then Worm-Miiller{ in more detail, observed that:
a muscle dipping in an indifferent fluid that was gradually heated and led oft
to the galvanometer from the fluid and from the undipped muscle exhibited,
with rise of temperature, positivity followed by negativity of the undipped
portion. Hermann, in 1870,§ flatly contradicts this statement, but in the
following year|| gives an account of experiments from which he concludes
that warmer portions of living muscle are positive in relation to cooler
portions, ze. that differences of temperature in the muscle give rise to
a special electromotive force (eine besondere elektromotorische Kraft). But
the protocols of his experiments, especially when they are plotted as curves,
are not very convincing. The question is one that requires to be carefully
re-tested. None of the experiments quoted affords any conclusive proof
that the influence of rise of temperature upon muscle currents were true
physiological effects apart from physical (thermo-electric) changes.

The method employed in the present series of observations was as indicated
in the following representative experiment.

* J learn that Engelmann, in 1872, examined the influence of temperature upon skin-
currents, obtaining with rise of temperature a negative variation of the normal current,
‘ Pfliiger’s Archiv,’ vol. 6, p. 138.

+ Du Bois-Reymond, ‘Untersuchungen iiber Thierische Elektricitit,’ vol. 2, p. 178;
‘Gesammelte Abhandlungen,’ etc., vol. 2, p. 202.

t ‘Worm-Miiller, ‘Versuche tiber die Einflusse der Warme auf die elektromotorischen
Krafte der Muskeln und Nerven, Wiirzburg, 1868.

§ Hermann, “Versuche iiber den Verlauf der Stromentwicklung beim Absterben.”
‘Pfliiger’s Archiv,’ vol. 3, p. 48, 1870: “Genau bei 40°... . entwickelt sich ein im
lebenden Muskel aufsteigender Strom. Die absterbende Muskelsubstanz erlangt also ihre
Negativitat gegen die lebende in dem Momente der Erstarrung..... Hine (nicht.
thermoelektrische) Positivitiit der erwarmten Substanz im Sinne Worm-Miiller’s existirt
nicht.”

|| Hermann, same title, ‘ Pliiger’s Archiv,’ vol. 4, p. 163, 1871.

304 Dr. A. D. Waller. Effect of Heat upon the [Feb. 20,

A sartorius muscle led off to the galvanometer in the usual way. A
platinum wire adjusted transversely under one of the electrodes A or B
of an accumulator, a metronome key and a hand-key K, all in the same
circuit, so that the metronome gives regular brief periods of closure (when
the hand-key is also closed) that raise the platinum wire to a red heat for
a fraction of a second. The glowing wire heats the muscle locally at A by

A oe)
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: S : AB.

radiation and by the ascending current of heated air. The heat is graduated
by bringing the wire nearer or farther from the muscle, or by varying the
number of effective closures, or by varying a resistance in the accumulator
circuit. It can be calculated if required. But the important condition to be
secured is not so much an accurately known amount of heat as an accurately
repeated application of a definite regular amount of heat. In these experi-
ments the heat at each glow of the wire amounted to 0°08 calorie.

The movements of the muscle are recorded by a lever on the smoked
cylinder or plate. The movements of the galvanometer mirror indicative of
the electrical changes are at the same time recorded photographically.

Result :—

Moderate heat at B gives current in the muscle from A to B.

Excessive heat at B gives current in the muscle from B to A (= acurrent
of injury).

After which moderate heat at B gives hardly any effect, because B is
injured.

Dead (i.e. heat-rigored) muscle gives small effects in the same direction,
that are ordinary physical (thermo-electric) effects.

The results on muscle are nearly quite satisfactory, the only drawback to
their absolutely conclusive character is this small residual positive effect in
the same direction as the ordinary thermo-electric currents from the heated
side. But as will be understood from the consideration of skin effects, even

1909. | Electrical State of Inving Tissues. 305

this doubtful feature is eliminated when muscle-effects and skin-effects are
studied comparatively.

A B
I. Muscle—
Cabinet acs ocnssoicsuaeas) Spero: wl) Tae
Excitation or injury ............ Spiers tie Segre
ite Nerve—
IEIGMT coogoasbdoas dagannapspe0600000 a,
Excitation or injury ............ Ee
III. Skin—
TEIGAIS ooeoucsanoooenaoogs0 bes bade SS SS
TODGUEKTON, Goodacononaaccnseadco dos ees ——

The arrows under A and B indicate the direction of currents in the tissue
in response to local warmth or local excitation at A and at B respectively :
2g., if muscle, led off at A and B to the galvanometer, is heated at B, there is
current in the galvanometer from B to A, in the muscle from A to B as
indicated by the first arrow under B. Following the usual phraseology, we
say that B becomes “ positive.” The ‘second arrow under B indicates that
there is current from B to A in the muscle when B is rendered active by
injury or excitation, or, according to usual phraseology, B becomes “ negative.”

The arrows opposite the nerve indicate the analogous currents by local
warmth or by local excitation, identical in direction with those of muscle.

The local skin currents both to heat and to excitation are of reverse
direction to those of muscle (and of nerve), ¢.g., if skin led off by electrodes
A and B applied to its external surface is warmed at B, there is current in
the galvanometer from A to B (“ingoing” current at B, or B “negative ” to
A). If it is excited at B, there is current in the galvanometer from B to A
(“ outgoing” current at B, or B “ positive” to A).

In the foregoing description the ordinary terms “negative” and “ positive ”
have been used. For my own part I find it conducive to clearness to think
of the active spot B as “electro-positive,” giving current from B to A in the
tissue, rather than as “ negative,” giving current from B to A through the
galvanometer, and I call B “ zincative.” But the least ambiguous description
of direction of current between A and B is afforded by the arrows.

MEO; Nerve:

A nerve disposed in a similar manner gives similar results, with this
difference, that the positive response to heat is relatively more evanescent
and more easily replaced by the negative response to injury. This
disappearance may occur so rapidly that a photograph taken after a few
trial deflections may exhibit only a series of injury responses (negative with
increasing negativity), the heat responses (positive with increasing negativity)
having occurred with the trial deflections. It is advisable, therefore, to take

306 Dr. A. D. Waller. Effect of Heat upon the [Feb. 20,

nerve-photographs without any preliminary trials, as will be obvious on
consideration of the record given herewith.

III. Of Skin.

Frog’s skin, which, according to my previous observations,* invariably
responds to local excitation by an outgoing current when led off by its
external, but not by its internal, surface, is a particularly satisfactory tissue
upon which to study the electrical effects of heat, for the electrical sign of
the local effect of excitation is the reverse of that of muscle or nerve, and it
possesses an effective (external) surface and an ineffective (internal) surface
that can be separately tested.

Experiment.—Two unpolarisable electrodes A and B, in contact with the
external or effective surface of the skin to a galvanometer. A stirrup of
platinum wire in an accumulator and metronome circuit as for muscle.

Heat under B gives a large effect in the negative direction, indicating
current in the skin from B to A (=B zincative), i.e. in the contrary
direction to that of the current aroused by electrical or mechanical excitation.

Repeating the trial with the skin turned round so that the electrodes A B
are in contact with its inner ineffective surface. Warmth applied as before
to B gives little or no deflection; the deflection, if any, is in the opposite
(negative) direction.

A killed piece of skin gives little or no deflection from the warmed
spot B; the deflection, if any, is small and in the positive direction.

_ Thus in the living skin as in living muscle a current is aroused by warmth
which is antidrome to the current aroused by electrical excitation ; the facts.
in the two cases are as follows :—

|
| Local excitation of A. Local warmth to A.
|
MIHWSEE — cooaoosoapse A negative A positive
SIESTA, | Gsdanadabavonbd A positive A negative

|

Photographic records of the electrical effects of heat upon muscle, nerve,
and skin. The connections in the three cases are with two points, A and B,
as given above, heat being in each case applied at A or at B, as indicated
by the signal marks h, h, giving in the case of muscle and in that of nerve
heated at B response in the direction from A to B, and in that of the skin
from B to A.

I. The muscle record consists of three successive positive responses to heat.

* Waller, ‘Roy. Soc. Proc.,’ vol. 68, p. 480, 1901, “ Signs of Life.”

1909); Electrical State of Living Tissues. 307
at A (A =antizn.); the general after-effect is in the negative direction,
followed by four positive responses to heat at B (B = antizn.); the general

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308 Dr. A. D. Waller. Effect of Heat upon the [Feb. 20,

after-effect is negative. The muscle is then scalded, and submitted to five
successive glows as before; when a smaller deflection in the positive direction
occurs at each glow. It is presumably a physical (thermo-electric) effect.

II. The nerve record consists of three successive effects of three successive
glows under B, followed by four successive effects of four successive glows
under A. In the B group the first response is positive, the second is small
positive followed by large negative, and the third is negative. In the A group
the first (positive) response is the largest, the second (positive) is smaller, the
third is smallest and gives place to a deflection in the negative direction, due
to injury, the fourth is a small negative deflection.

III. The skin record consists of two groups of four successive responses ; in
the first group the external surface, in the second group the internal surface,
of the skin is led off to the galvanometer. In the first group, each response
at Bis relatively large and the current is directed in the skin from B to A

“jngoing ” current, or B zincative). In the second group the responses are
hardly perceptible.

In muscle (and in nerve), where the electrical effect of local excitation is
“negative,” the effect of moderate heat is “ positive.”

In the skin, where the electrical effect of local excitation is “ positive,” the
effect of moderate heat is “ negative.”

Excessive heat, producing injury, gives a “ negative” effect in muscle (and
nerve), a “positive ” effect in the skin.

N.B.—« Negative” = “ Zincative.” “Positive” = “ Antizincative.”

To my understanding, the expressions “positive” and “negative” are
ambiguous without this specification, and the description given in the text in
terms of “positive” and “ negative,’ while correct, is very confusing.

The general conclusion from all these experiments is that the first electrical
effect of moderate local heat is of opposite direction to that of local excita-
tion and of local excessive heat, ze. that the effect of moderate heat is
“anti-excitatory.”

[Postscript (added May 27)—In consequence of a question put to me at
the conclusion of the demonstration that accompanied the above com-
munication, I have taken observations on the effects of gradual rise and fall
of temperature upon the normal (ingoing) current as well as upon the
electrical response of the frog’s skin. The alterations of temperature were
brought about by gradually warming and cooling a metal box containing
the skin and electrodes, the thermometer giving the temperature of the air
in the box. .

With rising temperature the electrical response of the skin increased at

1909. | Electrical State of Living Tissues. 309

first and subsequently diminished, disappearing at a box temperature of
between 45° and 50°. The normal (ingoing) current at first increased and
subsequently diminished.

With falling temperature the electrical response diminished progressively,
being altogether abolished at a box temperature of between —4° and —5°.
The normal ingoing current diminished progressively with the fall of
temperature. At the box temperature of 4° to 5° a sudden diminution of
the ingoing current (7.c. an outgoing effect) was sometimes witnessed, which
I attributed to an excitation occurring at the point of congelation. But in
other cases mere irregular deflections were seen at this point.

Thus, as may be seen from the accompanying plotted curves, the effect of
heat and of cold was in the same (outgoing) direction; the increased ingoing
effect at the first application of heat being no greater than the spontaneous
increase that takes place without alteration of temperature. |

VOLT
0-00!
0-002
0-003

0-004 VOLT

0-005 0-00!

60° 0-002
50°

40° 0°003
30°

20 20°

10° 10°

0 oO

MIN 5 10 [Sin cOmee5n 50) eso 40

Effect of gradual rise of temperature upon the normal Effect of gradual fall of temperature upon the
skin current. Upper line = E.M.F. readings by normal skin current. Upper line = E.M.F.
compensator ; lower line = temperature curve. readings by compensator ; lower line = tem-

perature curve.

310

The Incidence of Cancer in Mice of Known Age.

By E. F. BasurorpD, M.D., and J. A. Murray, M.D., B.Sc., Imperial Cancer
Research Fund.

(Communicated by Prof. J. Rose Bradford, Sec. R.S. Received April 30,—
Read May 20, 1909.)

The opportunity of obtaining accurate information of the frequency of
spontaneous cancer in mice at different age-periods has presented itself
in the course of a prolonged inquiry into the possibility of hereditary trans-
mission of a liability to cancer. We have approached the question of heredity
experimentally by breeding systematically from mice spontaneously affected
with malignant new growths, and propose to determine the frequency of
‘spontaneous cancer in mice in whose ancestry the disease has occurred with
varying frequency. This investigation is still in progress and cannot
be reviewed profitably for several years, but the data which have so far
accumulated are of sufficient interest, in their bearing upon the statistical
and biological importance of the age-incidence of the disease, to warrant
a preliminary account being published; although the small numbers at
present available still render the greatest caution necessary.

The method by which the data have been obtained is as follows: Mice
spontaneously affected with cancer are not killed when brought into the labora-
tory, but the tumours are excised and used for transplantation. The clinical
course, the microscopical examination, and the results of transplantation of the
tumours, together with the post-mortem examination of the animals, give the
best security for the correctness of the diagnosis of cancer. It is only under
these precautions: that the breeding experiments have proceeded. Each
spontaneously affected mouse, or pair mated for breeding, has been housed
in a separate cage. The cages have been sterilised and changed at regular
intervals. In the first instance, the males mated with these spontaneously
affected females were the offspring of spontaneously affected animals received
pregnant. Later, males bred in the laboratory from cancerous parents were
used, so that the pedigrees constructed for the later litters show some strains
with a relatively enormous preponderance of cancerous ancestors. When
a litter is born each young mouse receives a number, the date of birth is
entered in a list, and the sex and colour or other distinguishing marks
noted against each. So soon as they are able to look after themselves the
litters are separated from the mother, and the males and females segregated
in fresh cages. It is thus possible to distinguish each animal born in the

The Incidence of Cancer in Mice of Known Age. 311

laboratory by reference to an index, which at once gives the ancestry, the
date of birth, and the age of the animal in question. The mice have been
systematically examined daily.

There is difficulty in obtaining offspring from mice suffering naturally
from cancer, and the breeding experiments began to be regularly successful
in January, 1907. The first case of cancer was found in a female mouse in
March, 1908, the animal being nine months old. Since that date 18
additional spontaneous malignant new growths have been obtained. Every
ease has been subjected to careful microscopical examination, and only
undoubted cases of malignant new growth are reckoned. So far all have
occurred in female mice, and with two exceptions have affected the mamma.
The exceptional cases were one of generalised malignant lymphoma, and one
of melanoma or melanotic sarcoma of the external ear. The remainder were
carcinomata of the mamma in which the adenomatous character was present
in varying degree; in two of them small areas of keratinisation were found
in the sections examined.

It was of interest to determine at what age the tumours were /irst observed,
and to determine the number of animals of the same age under observation.
As no cases of new growth have yet occurred in the males* bred, these are
excluded from the present purview. The females were distributed by means
of a card index into five groups differing in age from each other by intervals
of three months. Animals under six months old have been excluded
because of the high mortality in the first six months of life from infectious
diseases of all kinds (pneumonia, enteritis, septicemia), and because the
youngest mouse in which a true malignant new growth occurred was
exactly six months old. It will be noted from the table given below that
no female animal attained the age of two years. On April 26, 1909,
a census of the females was taken, and all those which had died over
six months old, since the beginning of the experiment, were added to the
corresponding age-groups. The mice which were then still living, after
developing cancer, and those which had died from the disease, appear in the
age-group corresponding to the age at which the disease was first discovered
in them. The percentages of the following table are therefore not strictly
comparable with death-rates, but are to be read as giving the liability to
cancer at different age-periods.

The progressive increase shown in the table presents a remarkable corre-
spondence with the facts long familiar to students of the incidence of
cancer in the human subject. It furnishes a striking confirmation in the

* Of the first 1145 mice bred, 588 were males and 557 females. The preponderance
of cancer in the female is due to the great liability of the female to cancer of the mamma.

312

Drs. E. F. Bashford and J. A. Murray.

[Apr. 30,

mouse, of the conclusion we advanced in ‘ Roy. Soe. Proc.,’ vol. 73, January,
1904, and in the First and Second Scientific Reports of the Imperial Cancer

ae 6—9 —12 =i —18 21 | eee
i months. months. months. months. months.

and over.
Motalll “anecsaecseseemnetioes 135 110 | 94, 21 6 —
@ancer® Jhncscmpossseceh 3 A 7 B} 2 ee
IRenicenterecerceeseeee 2:2 3°5 | 7:4 14-2 33 °3 —

Research Fund, that, in animals as in man, the recorded frequency of cancer
varies with the opportunities for examining a large number of adult and aged
individuals.

Account was taken of the age-incidence of cancer in the human subject in
the hypotheses of Thiersch* and of Cohnheim,+ which were formulated for
man only, and are untenable to-day. The general biological significance of
the age-incidence of cancer, for which we have so often argued, has been
ignored, or, when mentioned, minimised by most pathologists, and, in recent
years, also by those engaged in the experimental study of the disease. It
is, perhaps, not too much to hope that the foregoing presentation of the
facts will henceforward impress on those engaged in the investigation of
precise knowledge of the ages of men or
animals in whom the incidence of cancer is being studied. In particular,
the difference between mice 15 months old and 21 months old in their
liability to cancer at once invalidates completely all statements of the
relatively greater frequency of cancer in one group of mice than another,
when the exact age of the animals is not known. The same objection must be
raised to assertions of the occurrence of epidemics in other animals. Such
statements have been frequently made, and have received wide currency
since experiment demonstrated the possibility of the artificial transmission
of cancer from one animal to another of the same species, but only, however,
by implanting living cancer-cells, and also demonstrated that this form of
transmission could not be made responsible for the great frequency of
malignant disease. The above criticism therefore applies with destructive
force to all statements which have appeared up to the present on the occur-
rence of epidemics of cancer in mice and rats. Until it can be shown that
the conditions of experiment have altered the normal age-incidence of the

cancer the urgent necessity for

* Thiersch, ‘ Der Epithelialkrebs, namentlich der Haut,’ Leipzig, 1865.
+ Cohnheim, ‘ Vorlesungen iiber allgemeine Pathologie,’ Berlin, 1877, 2nd Edition,

1882.

1909.| The Incidence of Cancer iv Mice of Known Age. 313

disease, the theses which have found such ready acceptance must be regarded
as not proven.

As in the case of other communities of mice in outside breeding establish-
ments, our stock, at present under consideration, is a highly in-bred one. It
is not profitable at present, considering the small number of tumours which
have been obtained, to analyse the cancerous and non-cancerous individuals
with reference to this factor or to the ancestry.

The positive value of these observations lies in the statistical confirmation
they bring to the results of the comparative histological and biological studies
of the Imperial Cancer Research Fund, which have shown the close parallel,
amounting in many particulars to complete identity between malignant
new growths in man and other vertebrates. They demonstrate that the law
of the age-incidence of cancer holds also for the shortest-lived mammals as it
holds for man. Since the facts accord with the imperfect data we have
elicited for other vertebrates, they make the general applicability of the law
of age-incidence probable, and therefore any explanation of the etiology of
cancer must accord with the circumstance that, when considered statistically,
cancer is a function of age, and when considered biologically is a function of

senescence.

VOL, LXXXI,—5, Z

314

The Electrical Reactions of certain Bacteria, and an Application
wm the Detection of Tubercle Bacilli in Urine by means of
an Electric Current.

By CHarLes Russ, M.B.

(Communicated by A. D. Waller, M.D., F.R.S. Received May 21,—Read
June 24, 1909.)

Introduction.

The aim of the following experiments was to ascertain whether bacteria
suspended in an electrolyte through which a current passes are transmitted
to either electrode, and if so, whether pathogenic organisms could be collected
and extracted by such means from pathological liquids.

Method of Experiment.

The first observations as to a possible migration of bacteria under the action
of an electric current were made in the following way :—

A cover square was fitted with two platinum foil terminals, separated
about 6 mm. from each other. A drop of weak bacterial emulsion
made electrical connection between these two terminals, and was prevented
from evaporating by another cover square resting on the top of the first one,
the edges of which were greased; the “glass cell,” as it may be called, was
then mounted on a stand (fig. 1), which rested on the stage of a microscope,
and a current of about 1 milliampere sent through it.

——/

Fie. 1.

It was at once found that the bacteria, viz., Staphylococcus aureus,
Streptococci, M. melitensis, B. tuberculosis, B. coli, B. typhosus, B. of Gaertner,
B. pyocyaneus, and Hoffmann’s bacillus, moved towards one electrode, their
direction of movement being reversed on reversal of the current; the
velocity of transmission was approximately estimated at about 1/100 mm.
per minute (under the particular conditions of pressure and sectional area).

The Klectrical Reactions of certain Bacteria, etc. 315

It was a matter of considerable difficulty to-keep a particular bacillus
continuously in view and thus watch its migration; to obviate this, and in
view of the possibility of the observed movement being due to some variation
of surface tension of the fluid under the influence of the current, the experi-
mental arrangements were altered and observations made in the following
way :—

An emulsion of the Bacillus coli was made by pouring normal saline upon
a fresh agar culture of the organism, sweeping the growth by a platinum loop
into the saline, and pouring it into a test-tube, which was then sealed in the
blow-pipe; by thorough agitation an even emulsion was made; some of this
emulsion was then poured into a glass U-tube, fitted with platinum terminals
which just dipped beneath the liquid; a current from Leclanché cells was
sent through the emulsion. Observations at frequent intervals showed that
the bacilli were accumulating under the anode in one limb of the U-tube ;
after several hours a dense column had formed, the fluid in the opposite limb
becoming clear; reversing the current reversed the direction of motion of
the bacteria.

A systematic series of tests was next undertaken with different bacteria
in various electrolytes. To expedite the work, four similar U-tubes were
mounted ona stand as seen in fig. 2, and observations made simultaneously
of the behaviour of the bacteria when suspended in different electrolytes, the
same current traversing the four solutions in series. The current was

Kia. 2.

measured by a tangent galvanometer in circuit, and the voltage between the
terminals of the U-tubes by a voltmeter when required. Usually 4-per-cent.
solutions of the electrolytes were used ; with weaker solutions, e.g., 4 per cent.,
gravitation of the bacteria to the bottom of the U-tubes occurred before the
electrical effect was evident.

316 Mr. C. Russ. [May 21,

It was thus found that :—
(1) The accumulation at an electrode varies in degree with different species

in the same electrolyte.
(2) The accumulation at an electrode varies for the same species in different

electrolytes.
Table I.
— + - ‘+ = + = +
Nas. SO,. Na NO. Na, HPO,.| Na Cl.
B. coli communis .......2..+. | ‘tne a “3 -
TB} HYPOOOSS “nn noasonecen7081 a 8 a “3
Staphylococcus aureus... Feeble | Feeble Not tested *
* | *
Tubercle bacillus............ | Nil | Nil Nil Feeble
| | *
| Hoffmann’s bacillus ...... - fs | a
Pyocyaneus, B. ......... 00 ad | ie *
WEIMEUILEN STS We veclee cesses: * | * | | *
Streptococcus ............... Nil | Nottested | * | nia
| Gaertner’s bacillus ......... * % * *

The acid radicle column = anode.
base S = cathode.
* indicates aggregation.

*< = marked aggregation.

Table I contains the results of a number of observations made with different
bacteria in four electrolytes which all contained the same basic element
(sodium), but different acidic elements.

A similar set of observations was carried out with four electrolytes having
the same acid radicle (SO,), but different basic elements, and the results
collected in Table IT.

Table II.
- + - + - + = +
Zn. SO,. Ks. SO,. Mg. SO,. | (NHy)2. SO.
B. coli communis....i....1. | Nil * Nil Nil
18}, (PO WOSLIS. camconocaanccoscns * EJ 5; ‘5
Staphylococcus aureus ... Si * Bo Feeble
: | *
Tubercle bacillus............ 53 Nil D2 x
B. PyOcyAneus ......0.0reeees a z Nil Nil
BIG GETENET in. ussseneeorese Bs * ss Feeble
*
MM, melitensis ....ccccceecres Feeble a ” *
*

The streptococcus in Table I died before Table II was begun.
The ? was necessitated by oxide formations referred to under choice of electrolytes.

1909.| The Electrical Reactions of certain Bacteria, ete. Bly

It was important to decide the relationship of these bacterial movements to
the bacterial vitality. In point of fact the accumulation occurs in boiled, 2.¢.
dead, as well as in living cultures. As regards the probable nature of the
movement of the bacteria under the action of the current, we are in the
presence of the following alternatives :—

(1) If bacteria possess electric charges, they might be attracted to the
electrode of opposite sign, and migration of the bacilli would ensue.

(2) Minute particles suspended in fluids, through which a current is sent,
are known to be directed to the electric terminals. During the course of this
work solutions have been found in which no movement of the bacilli was
effected by the current. It will be seen from some experiments to be
described, that the phenomena are hardly to be explained on the lines
indicated above.

(3) When a current passes through an electrolyte, the usually accepted view
of the main processes therein involved is that there is a movement through
the solution of the ions of the solute in opposite directions towards the
electrodes. If we suppose that some chemical affinity exists between the
ions and the bacteria, there is at once the possibility of migration of the
bacteria towards the electrode; the transmission of the bacteria to the anode
in one electrolyte, and the cathode in another (eg., the tubercle bacillus
proceeds to the anode in sodium chloride, and to the cathode in ammonium
sulphate) can be attributed to a chemical affinity between them and the ions
a the electrolyte.

The migration of bacteria in electrolytes having been established, the next
step was to ascertain whether bacteria present in very small numbers in a
pathological liquid would be transmitted to either electrode in an electrolyte,
and thus concentrated in a small volume of liquid for further examination.

With this object in view, the tubercle bacillus in urine was chosen because
of the convenience in recognising the organisms without there being any
necessity of undertaking cultivation work. The chief conditions required for
the purposes of these experiments are that the electrolyte (1) should conduct
electricity well, (2) should not be destructive of organic matter, (3) should
form a colourless fluid in aqueous solution, (4) should yield no metal or
metallic oxide as a result of electrolysis; for such action has been found to
mask bacterial aggregation.

The search for a suitable electrolyte for the tubercle bacillus proved a long
and laborious one. The general method of experimenting was practically
identical with that described in which the U-tubes were used. The tubercle
bacilli from glycerine agar cultures were ground in a mortar, made into an
emulsion with water and added to the electrolytes under test. Out of

318 Mr. C. Russ. [May 21,

43 substances (a list, of which is appended) tried in this way, a decided
aggregation was observed with ethylamine, ammonium sulphate, acetamide,
and iodide of potash. The first three will be noticed to contain an NH» or
NH, group. The aggregation in acetamide or potassium iodide is anodic, but
weaker than the cathodic aggregation in ethylamine, or ammonium sulphate.

A curious effect was noticed with ammonium sulphate, viz., an early
aggregation at the cathode, soon followed by a sinking away of the massed
organisms, resulting in the appearance of a clear zone between the bacterial
cloud and the platinum foil. This was probably the result of some
secondary electrolysis at the terminal in question ; this disturbing action led
to the abandonment of ammonium sulphate for the purposes of these
experiments.

A good accumulation was found to occur with a mixture of ethylamine and
lactic acid, the migration of the bacilli being towards the cathode; separate
tests indicated no reaction in lactic acid, and a moderate one with ethyl-
amine alone. The movement of the tubercle bacillus in normal urine was
found to be slightly anodic. The addition of urine to the lactic acid and
ethylamine did not interfere with the agereeation of the tubercle bacilli at
the cathode. At the end of the test the presence of the bacilli in large
numbers at the cathode, and their absence at the anode, was confirmed by
making stained films from the cloudy and clear fluids round the respective
poles.

It was, however, noticed in these films that the bacilli stained feebly after
several hours of electrolysis in the ethylamine lactic acid and urine mixture ;
this difficulty was overcome by the addition of bromic acid to the mixture.

The proportions of these substances found to be most satisfactory were :—

Ethylamine, 5-per-cent. solution......... One part.

Lactic acid, 10 Agia hE aa etn med Four parts.
Bromic acid, 5 et Bei se AR Two parts.

Urine: fi iacdteh o cesbeu donc cotaeemeanaee rec One or two parts.

It may be useful to mention here that when electrolytes are added to
urine, and the mixture electrolysed, there is formed at the cathode a white
floceulent substance which is insoluble in the fluid, but soluble in mineral
and organic acids. As this substance is easily mistaken (in aggregation tests
in urine) for the bacterial cloud expected at the electrode, the solubility in
acid should be tested for. It may further be distinguished by its rapidity of
appearance and buikiness of the cloud, in which particulars it contrasts with
the true bacterial massing at the foil; the lactic acid used in the mixture
above mentioned prevents its formation.

1909.] The Electrical Reactions of certain Bacteria, ete. 319

In order to apply the observed aggregations of tubercle bacillus as
a method of their extraction from tuberculous urine, the experimental
arrangements were altered as follows :—

The modified U-tube (fig. 3) was filled with a mixture of tuberculous

Fic. 3.

urine, ethylamine, lactic and bromic acids, thoroughly shaken previously.
In the narrow limb of the vessel a platinum foil strip was submerged, and
served for the transmission of current through the main column of fluid.
The gases liberated at this foil escaped by the narrow limb, and were thus
prevented from traversing the bulk of the fluid. In the broad lmb a glass
tube was submerged slightly and traversed by a platinum wire, which just
touched the fluid surface. The lower end of the glass tube formed a
bacterial trap by arranging the circuit so that the platinum wire was the
cathode. On passing a current the tubercle bacilli contained in the
tuberculous urine in the vessel are conveyed to the cathode as in the U-tube
experiments; they eventually enter the fluid enveloped by the submerged
end of the tube, and remain close to the platinum wire. After sufficient
time the wire was carefully lifted out, the glass tube slightly lowered in the
vessel, the top of the tube closed by the wet or greased finger, and lifted
away to a glass slide, and examined for tubercle bacilli. The fluid contained
in the trap was 2 or 3 minims in volume, and strongly alkaline at the end of
the process, the bulk of the mixture remaining acid. This alkaline fluid was
acidified with a 10-per-cent. solution of acetic acid on the slide previously
smeared with albumen fixative; it was fixed by heat and stained by the
ordinary Z N carbol fuchsin method. Tubercle bacilli were found in small
numbers in the early attempts.

The following experiment, in which the centrifuge and the electric current
were compared in the detection of tubercle bacilli placed in very small
numbers in a test fluid, is given in illustration of the power of the electrical
method :—

320 Mr. C. Russ. [May 21,

My colleague, Dr, Fletcher, prepared an emulsion of tubercle bacilli in
water, and standardised to contain 1000 bacilli per cubic centimetre; he
thoroughly mixed 3 cc. (ze. 500 “T.B.”) of this emulsion with 100 c.e. of
normal urine. Allowing this mixture to stand for 24 hours in a conical
vessel to assist sedimentation of the bacilli, he then siphoned off the super-
natant three-quarters of its volume; the remainder was centrifugalised
(1/8 H.P. electric centrifuge) three times. In a stained preparation from the
final deposit he found no tubercle bacilh. I added 4 cc. (2c. 500 tubercle
bacilli) of the same emulsion to 50 cc. of normal urine, and made up the
volume to 100 e.c. by adding bromic acid, lactic acid, and ethylamine, and
thoroughly shaking the mixture. This mixture was electrolysed in the
vessel illustrated in fig. 3. After 21 hours’ electrolysis 128 tubercle bacilli
were counted in the stained preparations made from the trap contents.

Detection of “T. B.” in Tuberculous Urines by the Centrifuge and Electric

Current.
|
Centrifuge. | Electric current.

Case. | f | Description of | Voluiz No. of | No. of “T. B.” seen
Volume WE | SOBEL scan tia | 2 aan times | in film but which
urine used. films. NBEO: diluted. | is not a total count.

|
| C.c. | G.c. bacilli.
1 10 Moderate numbers ...' 10 15 59
2 30 a eae HO 5 38
3 repeat 300 | 7 6 205
4 20 Fairly numerous ...... 6 U 84,
5 | 20 6 7 149
6 20 6 U 103
7 | 20 bas 2 21 15
8 | 10 Fairly numerous ...... 5 9 22
9 10 5 9 68
10 10 5 9 35
11 10 5 9 40
12 10 10 10 65

The volume used by centrifuge = 10 c.c. once, twice or thrice repeated ; 7.e. the capacity of the

tube = 10 c.c.
The film forms a permanent record of the experiment in each case.

These experiments concluded by repetition of this process in 12 cases,
using always tuberculous urine, in which the bacilli had been previously
found by the centrifuge. During these cases, variations of the time the
current passed and of its strength were introduced. The results are
collected in Table III, the main conclusions being :—

(1) Tubercle bacilli were obtained in the trap upon the conclusion of
every test.

1909.| The Klectrical Reactions of certain Bacteria, ete. B2il

(2) They were present in larger numbers than would have been obtained
in 2 or 3 minims of the original urine.

(3) The transmission of the bacilli by the current is emphasised by the
relatively large number of bacilli obtained in the trap when working with
very much diluted solutions of the original urine.

(4) The evidence is not complete that ‘all the bacilli present in the urine
used were conveyed into the trap (though examinations of the “catch” were
made at successive stages of the electrolysis).

The appearance of the bacilli in the films made from the trap is similar to
that in a film from centrifuged urine, though the pus cells undergo
dissolution with increasing alkalinity of the trap contents. If the electro-
lysis was carried on for 24 to 36 hours the bacilli failed to take the stain
properly ; such exposure, however, is unnecessary.

I am indebted to Mr. S. Russ, Demonstrator of Physics, Manchester
University, for assistance with the electrical technique and measurements ;
to Dr. Hastes for the use of his laboratory during the earlier part of the
experimental work, and to Mr. Pardoe for tuberculous urine from St. Peter’s
Hospital.

In conclusion, the results of the present preliminary investigation may be
summarised as follows :—

Certain bacteria under the influence of a suitable current aggregate at one
or other electrode. The aggregation varies with the nature of the
electrolyte, and is probably due to affinity between the products of
electrolysis and the bacteria. It occurs with killed as well as with living
bacteria. The aggregation by electrical currents affords a means of
collection and examination. The differences in behaviour of various
bacteria are such as to suggest the possibility of utilising the method for
purposes of specific discrimination ; but in this particular the data hitherto
obtained are not sufficient to warrant definite statements.

List of Electrolytes in which Reaction of Tubercle Bacilli was sought.

Inorganic.—Potassium iodide, iodic acid, sodium sulphite, sodium carbonate,
sodium bicarbonate, sodium nitrite, ammonium magnesium phosphate,
potassium chlorate, microcosmic salt, ammonium magnesium sulphate,
potassium cyanide, and copper sulphate.

Organic—Sodium tartrate, sodium acetate, sodium citrate, maltose,
glucose, lactose, cane sugar, ethyl alcohol, methyl alcohol, acet-aldehyde,
glycerin, formalin, formic acid, amido-acetic acid, urea, uric acid, sodium
urate, oxalic acid, urea nitrate, lactic acid, acetone, chloral hydrate,

VOL. LXXXI.—B. 2A

322 The Electrical Reactions of certain Bacteria, ete.

phenylene-di-amine, sulphanilic acid, picric acid, earbol fuchsin, chloroform,
carbolic acid, acetic acid, and acetamide.

[ Vote.—Since completing these experiments I have searched the literature
of the subject with the following results :—

Abbot and Life (‘ American Journ. Physiol.,” 1908, p. 202) tested micro-
scopic quantities of motile bacteria suspended in distilled water, and
observed to-and-fro movements in a glass trough. They used excessively
minute currents, and concluded that variations in the observed reactions of
the organisms depended upon their cultivation in acid or alkaline media.
They found no effects with dead or non-motile (living) species ; they avoided
currents large enough to cause electrolysis, and concluded that their results
were galvanotropic.

Apostoli and Laquerriere (‘ Compt. Rend.,’ 1901, vol. 133, p. 186) con-
cluded that constant currents are able to sterilise cultures or attenuate their
virulence. They studied especially the conditions necessary to produce such
effects.

S. Kriiger, “ Ueber den Einfluss des constanten electrischen Stromes auf
Wachsthum und Virulenz der Bacterien” (‘ Zeitschrift fiir Klinische
Medicin, 1893), contributes an account of the lethal effects of electricity
applied to bacteria.

Other references are appended to papers I have not yet been able to
consult, viz. :—

Friedenthal, H. “Ueber den Einfluss des elektrischen Stromes auf
Bakterien,” ‘ Centralblatt f. Bakteriol.,’ 1. Abt., Jena, 1896 (vol. 19, pp. 319—
324); “Ueber den Einfluss der Inductions-Electricitat auf Bacterien,”
abid. (vol. 20, p. 505).

Jennings, H.S. Papers on reactions to electricity in unicellular organisms.
‘J. Comp. Neurol. and Psychol.” Granville, vol. 15, 1905, 528—534.

Bang, 8. “Wirkungen des elektrischen Bogenlichtes auf Tuberkel-
bazillen in Rein-Kultur,’ ‘Mitt. Finsens Lysinst. Kopenhagen, Leipzig,
H. 3, 1903 (97—112).]

323

Trypanosoma mgens, n. sp.

By Colonel Sir Davip Brucs, C.B., F.R.S., Army Medical Service ; Captains
A. E. Hamerton, D.S.O., and H. R. BaTEmMan, Royal Army Medical
Corps; and Captain F. P. Macxig, Indian Medical Service.

(Received April 30,—Read May 20, 1909.) u

(Sleeping Sickness Commission of the Royal Society, 1908—09.)
[PLATE 7.]

This is such an extraordinary looking parasite that the Commission thinks
it deserves a short preliminary note, a name, and to be figured.

The name is taken from Virgil’s description of the Cyclops, informe, ingens.
It was first discovered in the blood of a reed-buck on February 13, 1909, at
Namukekera, Uganda (lat. 0° 40’ N.; long. 32° 15’ E.), the estate of the
Uganda Company, Limited ; then in a bush-buck, and lastly in an ox. The
wild animals and the cattle feed in the same pastures, so that it is not
remarkable that the oxen should become infected.

At present it is not known what the carrier is, and this will probably be
a difficult thing to determine. Collections of the blood-sucking flies and
ticks are being made on the Namukekera Estate, and this may lead in time
to the discovery of the carrier. Up to the present the following list includes
all the blood-suckers found in this particular district :—

Chrysops distinctipennis, Austen. Hematopota unicolor, Ricardo.
Stomoxys calcitrans, Linn, Heematopota, sp. nov.
Stomoxys nigra, Macq. Hematopota brunnescens, Ricardo.

Tabanus teniola, Pal. de Beauv.

Trypanosoma vngens, when seen alive in a fresh preparation, moves slowly
and deliberately across the field of the microscope, with a fine rippling, or at
times a broader undulating movement.

In stained preparations this huge trypanosome may measure as much as
122 microns, and even then it is lying in such a formless huddled-up way
among the red blood corpuscles that it looks capable of stretching out to
a much greater length. The other specimens figured measure 72, 77, 88,
and 82 microns. The breadth is 7 to 10 microns.

The micronucleus is small and round. It measures about a micron in
diameter. It lies posterior to, and quite close to, the nucleus. From it, in
well-stained specimens, a well-marked, though narrow, undulating membrane
arises, which runs to the anterior extremity and ends in a free flagellum.

The nucleus is oval in form, and lies across the body. It is situated

VOL. LXXXI.—B. 2 8B

324 Trypanosoma wmgens, n. sp.

nearer the posterior end than the anterior, and in our specimens has stained
a pale pink.
The body substance is markedly granular behind the nucleus, while in
front the structure described as myonemes is particularly well marked.
More minute measurements of one of these trypanosomes are as

follows :—
microns.

iIRosterior end! to micronucleus ieeceeseeeeseee eee ee tee eeeee ace 18
rom: micronucleus to miucleus) eeccssesceestenessneceeneeee seeeeeee 4
Nucleus: long diameter, 8 microns; short diameter ...... +
Nucleus to:amterior/end! Sei, eeseosae ccsn cee teeeeeecneeseeneceee 40
Hree flagella .2.éjgaseeescuinsmepicmestisas se aieereeteanconcnee dees aese es 17

Total ecto tete 83

It is unnecessary in this preliminary note to go more fully into the
structure of this trypanosome, or to describe it at greater length. An
examination of the coloured drawings reproduced in Plate 7 will give a
more distinct idea of its appearance than any written description.

The drawings were made by Lady Bruce, R.R.C. Figs. 1, 3, and 4 are
from reed-buck, fig. 2 from the ox. All are magnified 2000 and stained

Giemsa.

Roy. SOC 0G VOlIOL, Plate i.

The Effect of the Injection of Intracellular Constituents of
Bacteria (Bacterial Endotorins) on the Opsonising Action
of the Serum of Healthy Rabbits.

By R. TANNER HEWLETT, M.D.

(Communicated by Prof. W. D. Halliburton, F.R.S. Received June 5,—Read
June 24, 1909.)

In a series of researches the late Dr. Allan Macfadyen studied the
properties of the intracellular constituents of bacteria and other organisms
obtained by mechanical trituration of the organisms in the presence of liquid
air. He showed that the cell juices thus obtained are :—

(1) Toxic on injection into animals (e29., B. typhosus,* Spirillum cholere,t
B. suisepticus,t pneumococcus,§ and others).

(2) Capable of inducing the formation of anti-endotoxins on injection into
animals (¢.9., B. typhosus,| Spirillum cholereS) which possess protective and
curative properties im vivo, and bacteriolytic properties in vitro.

(3) Cause the development of agglutinins (¢.9., B. typhosus** and yeastt+).
It was thought that it might be of interest to investigate whether the intra-
cellular bacterial constituents are capable of inducing alteration in the
opsonising action of the serum of normal rabbits.

The organisms selected were the B. typhosus, the B. tuberculosis, and the
M. pyogenes, var. aureus (Staphylococcus pyogenes aureus). The intracellular
constituents of these organisms were obtained by the Macfadyen method,ij
viz., by growing the organism on surface agar in Roux bottles, scraping off
the growth, suspending this in sterile water, centrifugalising and collecting
the bacterial paste on the walls of the centrifuge. The bacterial paste is
weighed so as to ascertain the amount, and then ground in the machine after
freezing.

After grinding, the ground material is made up with distilled water or
with 0:1-per-cent. sodium hydrate, so as to form a 10-per-cent. solution

* ‘Roy. Soc. Proc.,’ vol. 71, 1903, p. 77 (with Sydney Rowland).
+ ‘Lancet,’ 1906, vol. 2, p. 494.
£ ‘Centralbl. f. Bakt.,’ Abt. I (Originale), vol. 43, 1907, p. 148.
§ ‘Brit. Med. Journ., 1906, vol. 2, p. 776.
|| ‘Roy. Soc. Proc.,’ vol. 71, 1903, p. 351, and vol. 77, 1906, p. 548.
“i ‘ Lancet,’ 1906, vol. 2, p. 494.
** “Vancet,’ 1906, vol. 1, p. 373.
+t ‘Centralbl. fiir Bakteriol.,’ Abt. I, vol. 30, 1901, p. 368.
ti See ‘The Cell as the Unit of Life’ (Churchill, 1908), p. 274.

He, Vi) Ge

326 Dr. R. T. Hewlett. The Effect of the Injection of [June 5,

(calculated on the original weight of the moist bacterial paste), and filtered
through a sterile Berkefeld filter. One to three cubic centimetres of the
filtered solution are then dried im vacuo over sulphuric acid and weighed, so
as to ascertain the weight of material contained in the endotoxin solution.
This weight is regarded as the weight of endotoxin; actually the endotoxin
is slightly less than is represented by this weight, in consequence of the
presence of traces of salts. The amount of endotoxin having been thus
ascertained, sufficient sterile 0°8-per-cent. sodium chloride solution is added
to the filtered solution of endotoxin so as to form a 1-per-mille solution.
All the operations are performed aseptically, in order to obtain a sterile
preparation.

The rabbits were all large healthy animals, and blood was obtained in
Wiight’s capsules from an ear vein. In all instances, the blood used as the
control was taken at the same time as the samples from the inoculated
animals, and the specimens for counting the number of bacteria ingested by
the polymorphonuclear leucocytes were made in the usual manner within
two to three hours after bleeding the animals. The leucocytes employed
were human leucocytes, as rabbit’s leucocytes were found to be less satis-
factory for making the stained films, and the counts were made on 50 cells.
All the inoculations of endotoxin, tuberculin, and vaccine were made
subcutaneously in the back.

RESULTS OBTAINED.

A. Typhoid endotoxin.—The determination of the effect of injections of
typhoid endotoxin on the opsonising action of rabbit's serum is complicated
by the fact that agglutinins and bacteriolytic substances are formed which
cause agglutination and solution of the organisms (typhoid bacilli) in the
mixtures of serum, leucocytes, and organisms employed for preparing the
films with which the counts are made. The results, therefore, in this case
must be regarded as approximate only. The endotoxin was prepared from
an avirulent strain of the typhoid bacillus. Three rabbits were taken, one
being kept as a control, the two others each receiving a dose of 0°1 milligramme
of endotoxin.

In addition to determining the opsonising action of the undiluted serum,
the effect of dilution was also studied, for Klien*shas shown that dilution
up to a certain point increases the opsonising action of human typhoid
serum.

The following results were obtained :—

* ‘Bull. of the Johns Hopkins Hospital,’ vol. 18, Nos. 195 and 196, 1907, p. 245.

tt

1909. | Intracellular Constituents of Bacteria, ete. 327

Table I—Number of Typhoid Bacilli ingested by 50 Polymorphonuclear

Leucocytes.
|
tea Control Beene | Aes
Fovod | DUOC | oposite mado || Rept, | C222 Viegas ia,| Oxeee
of serum. = 11-0); index. | | index,
Control undiluted 96 82 0°85 | 88 0°9
before lin 5 60 | 54 0':9 54: 0'9
inoculation 1 10 38 | 42 11 36 1°‘0
|
24hours | undiluted 112 |. 20 0:17 28 0:25
after lin 5 86 | 12 0°13 | 14 0°16
inoculation i © 54 (0) _— 10)
48 hours undiluted 122 | 166 1-4 158 | LB
after lin 5 74 398 5-4 336 45
inoculation lO) 43 | 154 3°6 | 166 3°8
3days | undiluted 112 | 857 3-2 369 3°3
after lin 5 70 | .204, 3°0 254 3°6
inoculation 1 10 35 | 114 3°3 116 3°3
1 20 _ | 70 —_ 84
5 days | undiluted 196 | 392 2-0 SPN ety al ikke
after lin 5 48 | 548 11 °4 | 496 10°3
inoculation 1 10 33 || 615 15 °6 448 13 6
1 20 14 | 825 23 0 857 25°5
i §0 — ator _ 48
6 days undiluted 135 || 103 0°8 131 1°0
after lin 5 64 | 270 4 +2 160 | A
inoculation ee 33 | 124 3°7 135 4:0
i 0) — 98 — om
1 50 — 83 — 80
1 100 — 66 — 29
\]
7 days undiluted 114 155 14 125 ral
after lin 5 49 110 2°3 71 1°4
inoculation 1 10 28 71 2 +4, 42 1°5
fein 0 a ee 50) — 60
i A | —- 35 — 29
i 1@0) || — || 29 — 22
8 days undiluted 121 | 202 17 ea I EO
after lin 5 110 | 240 2:2 145 1°3
inoculation La elOe sf 51 | 168 3°3 198 3°9
TBO. | = | 164 BE 126
iL fo) — 2 CS _ 45
12 days undiluted | 118 | = 112 0:95
after lin 5 60. — 112 1°9
inoculation Oia 23 — 180 78
il = 2 | — — 164,
50 — — | 98
1 100 = ae AS
| \| |

It is not suggested either in Table I or in Table II that the index is correct to the second
decimal, The figure in the second decimal place is given only to indicate the frend of the index.

328 Dr. R. T. Hewlett. The Effect of the Injection of [June 5,

RABBIT II

Index

RABBIT I

Nm WwW

QWE WO OMS OoOms 6a es fe

Seo So oF KF ee ew NHD

24 hours after
48 hours after
72 hours after
Vv days after
VI days after
Vil days after
Vill days after

¢
2
=
=
[3]
cS
c
i)
=
é
7)
[<2]

48 hours after
72 hours after
V days after
VI days after
Vil days after
Vill days alter
Xt days after

&
Q
=
a
Pp
~
3
o
<=
+
N

Before Inoculation

Cuart IT.—Opsonic Index after Inoculation

Cuart J.—Opsonic Index after Inoculation
with 0°1 mgrm. Typhoid Endotoxin.

with 0°71 mgrm. Typhoid Endotoxin.
From the foregoing table it is evident that an injection of 0-1 milligramme
of typhoid endotoxin produces, 24 hours after inoculation, a considerable
decrease in the opsonising action of the serum, that is a marked “ negative

phase ” (Wright), followed by a considerable rise in the opsonising action of
The opsonic index yielded by the

the serum which persists for some days.
The dilution

undiluted serum is given in graphic form in Charts I and II.
of the normal serum produces a decrease in its opsonising action, whereas a
dilution of the serum of the inoculated rabbits produces an apparent increase
in the opsonising action.

B. Staphylococcus endotoxin—Three sets of experiments were carried out
with the endotoxin of the JL. pyogenes, var. aureus (Staphylococcus pyogenes
aureus), Viz., a comparison of the effects of the endotoxin derived from
(a) an ordinary old laboratory strain of the organism, (0) a recently isolated
strain, and (c) the effect of a vaccine prepared from the strain used for a,
on the opsonising action of the serum of normal rabbits. Equivalent
quantities (0-1 milligramme solid matter) both of vaccine and of endotoxin
were given, and the endotoxin solution was prepared with 0:1-per-cent.
sodium hydrate solution. The opsonising action of each serum, some time
alter inoculation, was also tested with both strains of organisms. The
results obtained are given graphically in Chart III of the opsonic indexes.

1909. } Intracellular Constituents of Bacteria, ete. 329

RABBIT Ul ‘RABBIT IV RABBIT V

J
=}
Qa
o
w

SOs td On KOMTOM ON KONTO
NDOSCHENHOKkKENADDOOCH HNO %

er
er

Vil days after
Xvi days after
5 weeks after
7 weeks after
72 hours after
Vit days af
XVI days after
5 weeks after
7 weeks after
24 hours after
48 hours af
72 hours after
Vil days after
XVI days aft

5 weeks after
7 weeks after

48 hours aft
Before Inoculation

S
6
ess
aos
i]
[3]
8
&
&
vo
a
2
VY
mx -

24 hours after

48 hours after

72 hours after
‘Before Inoculation

24 hours after

Cart III.—Staphylococcus Vaccine and Endotoxins.

Rabbit III received 1 ¢.c. (= 1000 x 10* = 0'1 milligramme) Staphylococcus Vaccine.
Rabbit IV received 01 milligramme Staphylococcus Endotoxin, old strain.
Rabbit V received 0:1 milligramme Staphylococcus Endotoxin, ew strain.

Main trace = index determined with old strain of Staphylococcus.
Small upper trace = index determined with new strain of Staphylococcus.

It will be seen from these experiments that the endotoxin prepared from
the old laboratory strain (Rabbit IV) gave nearly as marked a rise in the
opsonic index as the vaccine (Rabbit III), but that the former seems to
induce less negative phase than the latter, and its effect is more per-
sistent. The endotoxin prepared from the recently isolated strain (Rabbit V)
induced a rise in the opsonic index much more marked than that induced by
either the vaccine or the endotoxin prepared from the old laboratory strain.
The sera, some time after inoculation (two to seven weeks), tested against the
recently isolated strain, gave an opsonic index slightly higher in the case of
the vaccine (Rabbit III) and much higher in the case of the endotoxins
(Rabbits IV and V) than that obtained when tested against the old laboratory
strain.

The effects of different amounts (0:1, 0:01, 0:001 miligramme) of another
freshly prepared staphylococcus endotoxin solution were also tested, and the
results are given graphically in Chart IV of the opsonic indexes, and are

330 Dr. R. T. Hewlett. The Effect of the Inyection of [June 5,

there compared with the injection of an ordinary dose of staphylococcus
vaccine (1:0 c.c. = 1000 x 10® cocci = 0-1 milligramme).

From this chart (IV) it will be seen that a marked rise in the opsonic
index results from the injection of staphylococcus endotoxin, and that the
rise corresponds with the dose of endotoxin given. Even the smallest dose
of endotoxin (0:001 milligramme, Rabbit IX), produced a considerable and
lasting rise in the index, a rise more marked than in the case of the vaccine

(Rabbit VD).

Index RABBIT VI RABBIT VII RABBIT Vit RABBIT IX

N
.
an

.

DOSC=NWANANHBD GOOCH NWHen

‘
‘
\

Seno SSeS oo e Sew OS woe

eo

er
er
er

96 hours afte
XX! days after

IX days after
XIVdays after
Before Inoculation
IX days after
XIV days after
XXVIII days after

IX days after
XlVdays aft
XXidays a
48 hours after
72 hours after
96hours after
IX days after
x!V days after
Xx! days after
Before Inoculation
2thours after
48hours after
72hours after
96 hours after
XI days after
xxvilldays after
24 hours after
48 hours after
72 hours after
96 hours after

XXVilidays after
Before Inoculation

Before Inoculation
24 hours after

2 4 hours after
Xxvill days alter

48 hours aft
7 2-hours a

Cuart 1V.—Staphylococcus Vaccine and varying doses of Staphylococcus Endotoxin.

Rabbit VI received 1:0 c.c. (1000 X 10° cocci = 0'1 milligramme) Staphylococcus vaccine.
Rabbit VII received 0:1 milligramme Staphylococcus endotoxin.

Rabbit VIII received 0:01 milligramme Staphylococcus endotoxin.

Rabbit 1X received 0-001 milligramme Staphylococcus endotoxin.

A few experiments on the effect of dilution on the opsonic action of a
vaccine serum and of an endotoxin serum were also made, and the results
obtained are given in Table II.

From Table IT it will again be seen that the endotoxin produces a greater
rise in the opsonic index than the vaccine does. In this case dilution does
not affect the index in the same way as dilution of typhoid serum; on the
whole the index remains much the same in the undiluted and the diluted
serum, though with dilutions of 1 in 5 and 1 in 10 more cocci are ingested

1909. ] Intracellular Constituents of Bacteria, ete. 331

by the leucocytes than when the serum is undiluted, and this applies to the
serum both of the inoculated, and of the uninoculated, animals.

Table Il—Effects of Dilution on Staphylococcus Vaccine and Endotoxin
Sera. Number of Cocci ingested by 50 Polymorphonuclear Leucocytes.

Dilution Control | Vaecine Onan Endotoxin Onweee
Period. of (opsonic index | serum hae ab serum se ae
serum. =1). (1e.c. vaccine). eae \(O'1 mgrm.). re
Control undiluted 210 235 1:1 199 0°95
before rom, 5 835 356 1-06 315 0:94
inoculation i 30) 432 410 0°95 397 0:92
24 hours undiluted 233 183 0°8 400 1:3
after iron 286 . 256 0:9 241 0°85
inoculation i 110) 295 190 0 64 214 0°7
1 20 108 121 1:1 144 1:3
48 hours undiluted eco 131 i 326 3°8
after 1in10 100 169 197 358 3°6
inoculation
72 hours undiluted 58 92 1°6 156 PACT
after 1in 10 80 133 1-66 131 1°6
inoculation
6 days undiluted 125 186 1°5 | 306 2°4
after 1 in 10 185 211 1:14 365 2-0
inoculation :
12 days | undiluted 102 131 1:3 Wi7 1°75
after
inoculation

(See Note at end of Table I.)

C. Bacillus tuberculosis—Two preparations of tubercle endotoxin were
used, one prepared from untreated tubercle bacilli, the other prepared from
tubercle bacilli previously extracted with ether. The effect produced by the
endotoxins was compared with that produced by a small dose of German
Tuberculin R. The results obtained are charted graphically in Chart V of
the opsonic indexes.

From Chart V it will be seen that the German Tuberculin R (dose
0:002 milligramme) produced little effect (Rabbit X). A corresponding dose
of tubercle endotoxin (prepared with wneatracted bacilli), on the other hand,
induced a marked and prolonged rise in the opsonic index, which was
preceded by a slight negative phase (Rabbit XI). A relatively huge dose of
the same endotoxin (1 milligramme) produced a decided negative phase
followed by a rise in the opsonic index of approximately the same amount as
that produced by the smaller dose (Rabbit XII). A similar dose (1 milli-
gramme) of the endotoxin prepared with ether-extracted tubercle bacilli

332 Dr. R. T. Hewlett. The Effect of the Injection of [June 5,

produced an alteration (negative phase and subsequent rise) less marked than
with the endotoxin prepared from the unextracted bacilli (Rabbit XIII).

RABBIT xX RABBIT XI RABBIT XII RABBIT Xill

r
i

'
‘
-<

ilation
er
er
er
er
er
er
er
[ter
er
er
ter
cr
er
lation
er
er
er
er
er

Before Inoculation
Vil days a
XVI days afi
Vildays afte
XVidays a
5 weeks afte
7 weeks afte
24hours aft
48 hours aft
72 hours afi
Vil days after
XVidays aft
5 weeks after
Z weeks after

24 hours after

J
S
=
8
So
c
~
2
9
ina)

-
7
a
o
=
=
£
+
cot

72 hours afte
Before Inoc

72hours after
48 hours a

Before Inoc
48 hours afte
Vil days aft
XVidays atte
5 weeks afte
7 weeks aft
48hours aft
7 weeks af

CuHart V.—Tuberculin and Tubercle Endotoxin.

Rabbit X received 0:002 milligramme Tuberculin R.

Rabbit XI received 0:002 milligramme tubercle endotoxin.

Rabbit XII received 1:0 milligramme tubercle endotoxin.

Rabbit XIII received 1:0 milligramme ether-extracted tubercle endotoxin.

D. Effect of keeping on the Activity of the Endotoxin Solutions—In view
of the possible use of endotoxin solutions for vaccine treatment, it was
thought desirable to make tests on their activity after they had been kept
for a period. The tubercle endotoxin was prepared on March 9, 1908, and
the staphylococcus endotoxin was prepared on March 10,1908. They were
the same solutions as those employed in the experiments detailed in
Sections B and C above, were kept in an ice-safe and were injected on
May 1, 1908, into fresh rabbits, 2.e. approximately seven weeks after pre-
paration. The results are given graphically in Charts VI and VII of the
opsonic indexes.

In the case of the tubercle endotoxin, doses similar to those given in the
experiments in Section C were administered. It will be seen from Chart VI
of the opsonic indexes that the endotoxin solutions were quite as active as
previously. Rabbit XV, receiving the large dose of endotoxin prepared from
unextracted bacilli, became unwell on the twelfth day, and died on the

thirty-sixth day, after injection.

1909. | Intracellular Constituents of Bacteria, ete. 333

RABBIT XIV | RABBIT XV RABBIT XVI

Qa

NOU OCKF NW AUYNANA DOCK NH WwW AO

x

he
S
g
2
=)
2
+
N

i — i — i oe oe oe OS

72 hours after

XVI days after
5 weeks after
7 weeks after
72 hours after
VII days after
XVI days after
5 weeks after
24 hours after
VII days after
XVI days after
5 weeks after
7 weeks after

Vildays after
Before Inoculation

Before Inoculation
Before Inoculation

é

24 hours after
72 hours after

|

Cuart VI.—Old Tubercle Endotoxin Solutions (7 weeks old).

Rabbit XIV received 0:002 milligramme tubercle endotoxin.
Rabbit XV received 1:0 milligramme tubercle endotoxin.
Rabbit XVI received 1:0 milligramme ether-extracted tubercle endotoxin.

In the case of the staphylococcus endotoxin, in view of possible
deterioration on account of keeping, 10 times the dose (1 milligramme)
previously given was injected. It will be seen from Chart VII of the opsonic
indexes that a marked effect on the opsonic index was induced, greater, as
before, in the case of the endotoxin prepared from the recently isolated
strain (Rabbit XVIII).

Another experiment with the latter endotoxin (prepared on March 9, 1908)
was performed on November 3, 1908, approximately eight months after
preparation, the dose being 0'1 milligramme. Again it will be seen from
Chart VIII of the opsonic indexes that a marked effect was produced
(Rabbit XIX). The staphylococcus endotoxin employed in the experiment
detailed in Section E below was the same preparation and was then
10 months old, and from Chart IX it will be seen that it was still very
active.

These experiments indicate that the endotoxin solutions deteriorate but
slowly, and retain a considerable proportion of their activity for at least
three to six months.

E. Production of “ Negative Phase” by Injection of Endotoxin.—It was

334 Dr. R. T. Hewlett. The Effect of the Injection of [June 5,

Index RABBIT XVII RABBIT XVIII

Sesame ooo Se) Ae BR) DD)
CORMNWANANDODH NWA QAY

er

Before Inoculation
vil days af
Xvi days after
5 weeks after
7 weeks aft
Before Inoculation
2¢hours after
72 hours after
vil days after
Xvi days after
5 weeks after
7 wecks after

2+thours after
72hours after

Cuart VII.—Old Staphylococcus Endotoxin Solutions (7 weeks old).

Rabbit XVIT received 1:0 milligramme Staphylococcus endotoxin, old strain.
Rabbit XVIII received 1:0 milligramme Staphylococcus endotoxin, new strain.

2-0 ' RABBIT XIX

com me re re ee Ee
CweotTNwWKtnNanxnao

24 hours after
48hours after
72hours after
96hours after
IX days after
XIV days after
XXI days after
xxvilidays after 6”

i=]
aS
=
i]
7)
fe}
Cc
)
z
2
vo
f=)

Cuart VIII.—Old Staphylococcus Endotoxin Solution (8 months old). (Prepared from
new strain of Staphylococcus.) Rabbit XIX received 071 milligramme endotoxin.

considered desirable to attempt to ascertain whether the endotoxin
produces a negative phase comparable to that produced by a vaccine. For
this purpose relatively large doses of staphylococcus vaccine (15,000 x 10°
cocci = 1°5 cc. vaccine), and of fresh staphylococcus endotoxin (1 milli-

1909. } Intracellular Constituents of Bacteria, ete. 335

gramme) were injected, and the opsonic index was determined 15 hours,
20 hours, 24 hours, 48 hours, and 72 hours, and five days and seven days
after inoculation. The results are given graphically in Chart IX of the
opsonic indexes.

Linaless RABBIT XX RABBIT XX!

o
an

coo
an

er

cr

er
er
er
er
er

20 hours after
V days a
X days aft
Before Inoculation
15 hours after
V days aft

15 hours aft
2¢hours a

48 hours aft
72 hours aft
24hours afi
72 hours aft

S
3
“sS
pes
3s
Qo
e)
Gi
=
o
=
&)
vo
[==]

Cuart IX.—To ascertain extent of “ Negative Phase” with large doses of Staphylococcus
Vaccine and Endotoxin.
Rabbit XX received 1°5 c.c. (= 15 x 108 cocci = 0:15 milligramme) vaccine.
Rabbit X XI received 1:0 milligramme endotoxin.

From these experiments (Chart IX) it would appear that the vaccine
(Rabbit XX) produces a decidedly greater negative phase at the twentieth
hour after injection than the endotoxin does (Rabbit XXI), although, weight
for weight, six and a half times as much active material was administered
in the case of the endotoxin than in that of the vaccine. The results of
this experiment (and also of those detailed in Sections B, C, and D) suggest
that the endotoxin induces decidedly less negative phase than a vaccine.

I have to express my best thanks to Mr. Wellcome for the facilities he has
afforded me at the Wellcome Physiological Research Laboratories for carrying
out the greater part of this work, and to Mr. E. Thompson, Laboratory
Assistant, on whom much of the labour of making the counts of the opsonic
determinations has fallen.

(Part of the expense incurred in this work has been defrayed by a grant
from the British Medical Association.)

336

The Alcoholic Ferment of Yeast-juice. Part IV.—The Fer-
mentation of Glucose, Mannose, and Fructose by Yeast-juice.

By ARTHUR HARDEN, F.R.S., and W. J. YouNG.
(Received June 10,—Read June 24, 1909.)

The results previously communicated by the authors* were obtained
exclusively with glucose, and in the present paper an account is given of
‘the behaviour of mannose and fructose towards yeast-juice both in the
presence and absence of added phosphate.

Buchner examined the fermentation of fructose by yeast-juice and found
that it proceeded at precisely the same rate as that of glucose.t No experi-
ments with mannose appear to have been previously performed.

The fructose employed throughout these experiments was Kahlbaum’s
crystallised fructose prepared from inulin. The mannose was prepared by
the hydrolysis of ivory-nut and was purified by conversion into the phenyl-
hydrazone, which was recrystallised from hot water and was finally decomposed
by benzaldehyde in the usual manner.{ All the experiments were performed
at 25° in the presence of toluene.

I. Relative Rates of Fermentation of Glucose, Mannose, and Fructose.

Both mannose and fructose are freely fermented by yeast-juice, as they are
also by living yeast. The relative rates of fermentation of these three sugars
by yeast-juice vary somewhat in different experiments, but on the average of
the experiments performed the fructose appears to be fermented rather more
quickly than either mannose or glucose, whilst the mannose is also fermented
slightly more rapidly than glucose.

Table I gives the experimental results, 25 c.c. of yeast-juice being employed
in each case.

In experiments 1, 2, and 4 the rates were taken after the mixture had
been incubated for about half an hour. In experiment 3, observations of
rate were made during three different intervals, and the time during which
the mixture had been incubated before the commencement of each of these
was—(a) 25 minutes, (6) 70 minutes, (c) 130 minutes.

* “Roy. Soc. Proc.,’ B, 1906, vol. 77, p. 405 ; 1908, vol. 80, p. 299.
+ “Die Zymasegirung,’ p. 100. '
{ Herzfeld, ‘Ber. Deutsch. Chem. Ges.,’ 1895, vol. 28, p. 440.

The Alcoholic Ferment of Yeast-juice. 337

Table IL—Relative Rates of Fermentation of Glucose, Mannose, and
Fructose by Yeast-juice.

| Rate of fermentation.
Noa Anhount | | Time Cubic centimetres evolved Ratio of rates.
, Votal | ~- in the time given. |
of of See. |) ee
exp. | sugar. j | mins. | ape aS |
| Glucose.| Mannose. | Fructose. | Glucose.) Mannose. | Fructose.
|
| | Pee
al il 25°6 45 Oia 3 5°3 1 | 0°94 | 1-66
2 1 25 6 | 60 24°8 | 28 29 il iL83 || 1°17
3a 4 45 35 6:8 | 7-2 8-7 1 1-06 1-28
b 4 45 | 60 | 10-4 | 11 14-1 1 1:06 | 1:36
e 4 45 | 265 40:2 | 38°6 46 9 it 0°96 Waly
4 1°5 32 °5 | 135} 518 55-9 55 °7 il 1:08 1-08
| |

Il. Votal Fermentation.

The total weight of carbon dioxide evolved from an excess of the sugar by
a given volume of yeast-juice was also found to be slightly greater with
fructose than with glucose, whilst that evolved from mannose in the only two
experiments made was considerably less than from glucose.

The following are the experimental results, 25 c.c. of yeast-juice being
employed, and the incubation continued until fermentation had ceased :—

Table II.—Total Fermentation of Glucose, Mannose, and Fructose.

N | | Total carbon dioxide in grammes. | Ratio of totals.
oi | Sugar | Total ie
to) added. | volume. | | | |
satel Glucose. | Mannose. | Fructose. | Glucose. | Mannose. Fructose. |
5 4 | 32°5 | 0-6556 | 0-4452 | 0-7436 1 OS | 1:13
| | | | |
6 4, | 32°5 | 0°7405 0 6226 0 :8624 1 | 0°67 | 1°16

Ill. Fermentation of Mannose by Yeast-juice vn presence of Phosphate.

Mannose behaves towards phosphates in the presence of yeast-juice in
precisely the same manner as glucose.* A rapid rise in the rate of fermenta-
tion occurs; an extra amount of carbon dioxide and alcohol are produced
which are equivalent to the phosphate added, and the phosphate is converted
into a hexosephosphate which is not precipitable by magnesium citrate
mixture and can be isolated in the form of a lead salt. As in the case of
glucose, an optimum concentration of phosphate exists at which a maximum
rate of fermentation occurs. Beyond this optimum, increase of concentration

* Harden and Young, loc. cit.

338 Messrs. A. Harden and W. J. Young. [June 10,

of phosphate lowers the rate of fermentation. The rates obtained with
mannose and glucose in comparative experiments are approximately equal.

These phenomena are illustrated by the following experiments :—

Experiment 7.—Two quantities of 25 c.c. of yeast-juice+ 5 c.c. of a solution
containing 1 gramme of the sugar were incubated until a constant rate had
been attained, and 5 c.c. of an approximately 0°3 molar solution of sodium
phosphate were then added.

Experiment 8.—Two quantities of 25 c.c. of a different sample of yeast-
juice+ 1 gramme of the sugar were incubated as above and 10 c.c. of the same
sodium phosphate solution (0°3 molar) were added.

The readings made after the addition of the phosphate are tabulated below,
the numbers expressing the volume of carbon dioxide evolved in the
five minutes preceding the time given in the first column :—

Table [1].—Fermentation of Glucose and Mannose in presence of Phosphate.

Carbon dioxide evolved in preceding 5 minutes.
| Time after addition. Experiment 7. Experiment 8.
Glucose. Mannose. Glucose. Mannose.
|
5 4 *4. 6°4 22-4 22:5

10 8-2 6-0 23 8 26-7
| 15 91 6°8 22 2 22 °1
| 20 9°6 7:0 16°8 14°5

25 “a 6-0

30 4°90 4°8

35 1°56 2:7

40 1-0 1:0

45 io 1°0

Experiment 9. ormation of a Hexosephosphate—Twenty-five cubic
centimetres of yeast-juice were incubated until the rate of fermentation
became constant at 0°8 cc. in five minutes. Ten cubic centimetres of a
0-3 molar solution of sodium phosphate were then added. The rate rose to
71 cc. in five minutes, and incubation was continued until it had again
fallen. The liquid was then boiled and filtered, and the amount of free
phosphate estimated as Mg:P:0;. The whole solution was found to yield
0:0567 gramme of MgsP:0; The phosphate added corresponded to
0°3263 gramme Mg»P.0;, and hence the difference between these quantities,
0°3263—0-0567 = 0°2696, corresponds to the minimum amount of phosphate
rendered non-precipitable by magnesium citrate mixture.

Experiment 10. Hquivalence of extra Carbon Dioxide evolved to the

19093) The Alcoholic Ferment of Yeast-juice. 339

Phosphate added.—Parallel experiments with glucose, mannose, and fructose
were made with yeast-juice from the same preparation, the method being
that previously described.* Three quantities of 25 c.c. of yeast-juice+9 c.c.
of a solution containing 1 gramme of the sugar to be examined were incubated
with toluene at 25° for one hour, and to each were then added 5 c.c. of
a solution of sodium phosphate corresponding to 0°1632 gramme of Mg.P20;
and equivalent to 32°6 cc. of carbon dioxide at N.T.P. The rates of
fermentation were then observed until they had again fallen and attained
a steady value, the gases being measured moist at 19°°3 and 760'15 mm.

Table 1V.—Relation of Carbon Dioxide evolved to Phosphate added for
Glucose, Mannose, and Fructose.

Glucose. Mannose. | Fructose.

Maximum) attarme divs. rs.ceestasnseiacsnsesstedesargencceeeeceees cere: 96 a 11°3
Final rate of juice, cubic centimetres in 5 minutes ......... 1‘1 0 ‘96 1:08
Total evolved in 55 minutes atiter addition of phosphate...) 49 °7 AT ‘8 47 “6
Correction for rate of juice in absence of phosphate ...... 12:1 10 ‘6 11°9
Total equivalent to phosphate ............s.:secsseee eee eee eee ees 37 6 37 °2 35-7
S ;, ALAN DER ait ee euenetay ates 34-4 34:0 326

These numbers agree well with the value calculated from the phosphate
added, viz., 32°6 c.c.

Experiment 11. Effect of an Excess of Phosphate-—Two quantities of
15 c.c. of yeast-juice+7 c.c. of a solution of mannose containing 1 gramme
of the sugar were treated with 10 and 15 cc. respectively of a 0°6 molar
solution of potassium phosphate, K2HPO,. The following readings were
obtained, showing that in presence of 15 c.c. of the phosphate solution the
rate is less than in presence of 10 c.c., and that in neither case is a high
maximum rate attained :—

Table V.—Effect of an Excess of Phosphate on the Fermentation of

Mannose.

Pian he Rate in preceding 5 minutes. |
addition in |
TUES, 10 c.c. phosphate. 15 c.c. phosphate.

|
10 2-2 2:1
20 19 ae
30 2-4 Az
40 | 30 1°5
60 | 4-2 17

* “Roy. Soc. Proc.,’ B, 1906, vol. 77, p. 414.
VOL. LXXXI.—B. Zi

340 Messrs. A. Harden and W. J. Young. [June 10,

IV. Fermentation of Fructose by Yeast-juice in the presence of Phosphate.

Fructose, like mannose, agrees qualitatively with glucose in its behaviour
towards phosphates, but it differs quantitatively from both these sugars in
two important respects: (1) The optimum concentration of phosphate is
much greater; (2) the maximum rate of fermentation attainable is much
higher.

These points of resemblance and dissimilarity are brought out by the
following experiments :—

(a) When a phosphate is added to yeast-juice containing fructose the
rate of fermentation rises to a maximum and then falls to a rate which is
usually slightly higher than the original rate of fermentation.

Experiment 12.—Ten cubic centimetres of a 0°3 molar solution of sodium
phosphate were added to a mixture of 25 c.c. of yeast-juice and 1 gramme of
fructose, the original rate of fermentation of which was 0°8 ec. in five
minutes. The total volume of the mixture was 35°6 c.c. The readings were

as follows :—

Table VI.—Fermentation of Fructose in presence of Phosphate.

Time after addition | Cubic centimetre of
of phosphate in CO, evolved in

minutes. preceding 5 minutes.
5 14°9
10 21°3
15 21°7
20 173
25 10°9
30 2°0
| 35 15
| 40 1:0
45 1:2

The maximum rate attained varies very considerably with different samples
of yeast-juice, as is shown by the following numbers (see Table VII), which
refer in each case to 25 c.c. of yeast-juice.

It is interesting to note that the two high rates, 80 and 76:2 cc. per
five minutes, are equal to about half the rate obtainable with an amount of
living yeast corresponding to 25 c.c. of yeast-juice, assuming that about
40 grammes of yeast are required to yield this amount of juice, and that
this amount of yeast would give about 140 cc. of carbon dioxide per
five minutes at 25°, which has been found experimentally to be the average
rate obtainable with the top yeast employed for these experiments.

1909. | The Alcoholic Ferment of Yeast-juice. 341

Table VII.—Maxima attained by the Fermentation of Fructose in presence
of Phosphate.

Number Volume of ‘0°6 molar Wiesorana, wake
: Total | attained, cubic
of phosphate solution | | : F
F volume. centimetres CO, in
experiment. added. Biainites
C.c. cle
13 12°53 75 80
14 12°5 50 27:1
15 10 50 31°2
16 20 55 76 °2
| |

(6) Equivalence of the extra carbon dioxide evolved to the phosphate
added. One example of this has already been given in Experiment 10,
p. 338.

Experiment 17.—In another case the phosphate added was equivalent to
65:2 c.c. of COz at N.T.P.

Cie
Gas evolved in 45 minutes ...............s002000: 82 -2
Correction for rate of juice 1°2 c.c. per | 10 ‘8
5 minutes for a period of 45 minutes |
|
Gas evolved at 19 and 747 °°75 oo... ...cseeeeee | 71-4.
Wiolumes at NIB See eeaecelenctacrennns-sden- od | 64 -2

(c) Production of a hexosephosphate non-precipitable by magnesium
citrate mixture.

Experiment 18.—The experiment was carried out precisely as Experiment 9.
The amounts of phosphate are expressed as Mg2P20; :—

|
Ph osplaterad dle dies teresa seater seeeeecere sees 0 -3263
Free phosphate after fermentation ............ 0 0426
|
| Phosphate rendered non-precipitable............ | 0 -2837

The solution after boiling was found to contain a hexosephosphate which
has been isolated in the form of a lead salt and is at present undergoiny
investigation.

(d) Existence of an optimum concentration of phosphate.

The following table shows the maximum rates. produced by the addition

OP

340 Messrs. A. Harden and W. J. Young. [June 10,

of varying volumes of a 0°6 molar solution of potassium phosphate, K2zHPQu,
to yeast-juice and fructose. In all comparable experiments the total
volumes were kept equal by the addition of a solution of potassium
bicarbonate as previously explained for glucose ;* in each case 2 grammes of
fructose were employed. The maximum obtained and the optimum
concentration are printed in thick type :—

Table VIII.—Maximum Rates of Fermentation and Optimum Concen-
trations of Phosphate for Fructose.

|e Noor Valera: fi Cubic centimetres of ‘Mastin eae
a sate otal volume. 0°6 molar solution F
experiment. yeast-juice. of K,HPO, added. per 5 minutes.
Cics c.c (CAG,
19a 5 25 (@) 0°5
b 5 25 5 14°2
ce 5 25 10 5°5
d 5 25 15 1°8
20a 15 40 3 22°5
b 15 40 | 75 25°4
c 15 40 10 20°7
d 15 40 15 11°3
e 15 40 20 74
21a 10 35 3 31°5
b 10 35 5 32:2
c 10 35 i) 28 °5
d 10 35 10 20-2
e 10 35 15 9-2
i 10 35 | 20 5-7
|

It thus appears that, precisely as in the case of glucose, progressive increase
in the concentration of phosphate beyond the optimum produces a corre-
sponding decrease in the rate of fermentation, and at a high concentration
the rate becomes extremely slow.

(e) Comparison of the optimum concentrations of phosphate and of the
maximum rates produced at those concentrations for fructose and glucose.

The following results, which all refer to 10 cc. of yeast-juice, clearly
show that the optimum concentration of phosphate for the fermentation of
fructose is from 1°5 to 10 times that for glucose, and that the maximum rate
of fermentation for fructose is two to six times that of glucose.

* Roy. Soc. Proc.,’ B, 1908, vol. 80, p. 307.

1909. | The Alcoholic Ferment of Yeast-juice. 343

Table IX.—Optimum Concentrations of Phosphate and Maximum Rates
of Fermentation for Fructose and Glucose.

| Optimum volume of Maximum rate in cubic

é centimetres of CO, per
No. of Sugar, in Total | DAE Oe [pao pane 5 minutes.
experiment. | grammes. volume. |

Glucose. | Fructose. Glucose. Fructose.

22 2 35 2 eae 75 32-2

23 4 50 1 10 5°4 28 °*4

24 1°6 23 2 5 8 17

25 1 25 1°75 5 5°2 25 °9 |

26 2 25 5 15 16-2 31 °2 |

27 2 20 2 | 3°5 7:9 22 °6

28 2 22 °5 O75 | 2 34 22 °2

V. Effect of the Addition of Fructose on the Fermentation of Glucose or
Mannose in presence of a large Excess of Phosphate.

When the rate of fermentation of glucose or mannose by yeast-juice is
greatly lowered by the presence of a large excess of phosphate, the addition
of a relatively small amount of fructose causes rapid fermentation to occur.
This induced activity is not due solely to the fermentation of the added
fructose, since the amount of this sugar may be insufficient to yield the gas
evolved.

The general nature of this phenomenon may be gathered from the following
experiment :—

Experiment 29.—Two quantities of 25 cc. of yeast-juice + 2 grammes
glucose + 20 c.c. of 0°6 molar K2HPO, solution + toluene were incubated at
25°. The amount of phosphate was largely in excess of the optimum, and
the rate of fermentation was found to be 1°8 c.c. per five minutes.

A. To one of these were added 1 cc. of glucose solution containing
0:2 sramme of the sugar, and 4 cc. of the phosphate solution. The rate of
fermentation fell to 1°5 and continued at this value.

B. To the other were added 1 cc. of a solution containing 0'2 gramme of
fructose and 4 c.c. of the phosphate solution. The rate of fermentation at
once rose, as shown by the following readings.

As the evolution of carbon dioxide proceeds the phosphate is converted
into hexosephosphate, and its effect on the fermentation of the glucose
lessened, and hence in order to maintain the original concentration of
phosphate it is necessary to add a fresh quantity at intervals to B, the
amount required being calculated from the gas evolved, 13°5 cc. of CO2 at

344 Messrs. A. Harden and W. J. Young. [June 10,

CO, evolved in preceding 5 minutes.

; hr, Substances added.
Time after addition.

A. B.
Glucose and phosphate. | Fructose and phosphate.

5 minutes i055 2°6
IO ig¢ 1°8 6°9
ise, 1h | 13°8
Bs 1°5 19 °4.
25 155 25-1

17°2 and 762-4 mm. being equivalent to 2 ¢.c. of the-phosphate solution.
The further course of the experiment was as follows :—

13)
Ti te Gas evolved since if |
ime after addition = oe Phosphate added_ | Rate per
of phosphat | last addition of 0-6 molar solution. | 5 minut
ae ae | phosphate. ; ae mg ex
minutes. c.c. (Gx, Cre:
25 67 °8 4 25a
30 — -- Z0ek
35 53 °9 10 26°8
40 — — 22°1
45 = — 19-7
50 — — 18-5
55 — | -- 19-8
60 — — 20°5
65 1205 10 19°9
70 — — 17°3
75 — | —_ 18 -2
80 = = 17-4
85 — — 15°8
90 i = } — 16°8
95 | 97 °9 | = 12 °4
|
Total gas evolved ...... 340 *1

Hence, although the concentration of the phosphate was never allowed to
fall much below. the original value and was generally considerably above it,
the addition of 0°2 gramme of fructose to 2 grammes of glucose produced
a total evolution of 340-1 ¢.c. of COs, corresponding to the total fermentation
of 13 grammes of sugar (in the ratio CgHi20g: 2 CO2), whereas in the
absence of fructose the total fermentation would have been only 285 e.c.,
corresponding to 0°22 gramme of sugar. As the fructose added was only
0:2 gramme and the subsequent evolution of carbon dioxide corresponded to

1909. | The Alcoholic Ferment of Yeast-jwice. 345

the total fermentation of 13 grammes of sugar, it is obvious that the
addition of the fructose must have induced the fermentation of the glucose.

Experiment 30.—Similar results were obtained in another way by
employing so large an excess of phosphate that the fermentation observed
did not reduce the concentration of phosphate to the limit at which the rapid
fermentation of glucose in the absence of added fructose became possible.

Four quantities of 15 cc. of yeast-juice +5 c.c. of a solution containing
2 grammes of glucose were employed—

1. 5 cc. of 0°6 molar potassium phosphate solution were added.

2. 75 ec. of phosphate solution were added.

3. 15 cc. of phosphate solution were added.

4. 15 ce. of phosphate solution and 0°5 cc. of a solution containing
0:05 gramme of fructose were added.

The following observations were then made :—

Rate for 5 minutes.
Time. Gane ee See |
ales 2. 3 A. |
| i

5 minutes 2:0 19 | 0°8 | 4:2

1 2-6 AE-Gur ual hea 4-0

15 7 B 7 2-2 06 6°6

20) op B37 PAL | 0°8 | 8°9

25 4 5 °2 2°6 0-7 11°6

30° 6-4 2°9 0°6 14:2

35 a 9°6 3°5 0°7 14°8

a | : aa
Total evolved ...... 33 °2 16°8 5S} | 64 °3

In this case the amount of gas evolved in No. 4 is equivalent only to
48 c.c. of the phosphate solution, and the final concentration is therefore
10:2 c.c. of phosphate in 35 cc. The concentration of phosphate, therefore,
never falls as low as that present in No. 2 (7°5 in 27°5 or 9°5 in 35), and yet
the fermentation is much more rapid than in this flask, which itself contains
a concentration of phosphate greatly in excess of the optimum, as shown by
a comparison with No.1. The amount of carbon dioxide yielded by the
complete fermentation of 0°05 gramme of fructose is only 13°5 c.c., so that
there can be no doubt that most of the gas evolved was derived from the
glucose.

(Note——Experiments 1, 2, and 4 are not strictly comparable, since the
contents of the flasks were not made up to the same volume, but the
difference in rate due to this is negligible.)

346 Messrs: A. Harden and W. J. Young. [June 10,

VI. Specific Character of the Inductive Action of Fructose.

This inductive effect is specific to fructose and is not produced when
glucose is added to mannose or fructose, or by mannose when added to
glucose or fructose, under the proper conditions of concentration of phosphate
in each case.

The experiments on this point were carried out by ascertaining in each
case the excess of phosphate necessary to produce a slow rate of fermenta-
tion and then making two parallel experiments, one with, and the other
without, the addition of the small quantity of the sugar to be tested for
inductive power. All the observations were made with 15 c.c. of yeast-
juice.

Table X.—Specific Character of the Inductive Effect of Fructose.

Cubic | Grammes of sugar present. |
No. of centimetres | Total | Gas Tinie
experiment.| O°6 molar | volume. | evolved. i
phosphate. | Mannose. | Fructose. | Glucose.
| c.c. C.c. | mins.
3la 15 » BY 1 0 0 8:7 30
b iB} 37 1 | 071 (e) 43-3 | 30
c 15 37 1 (0) 071 10°99 | 380
32a 10 32 1 0 0 189 | 30
b 10 32 1 O'l 0 76°3 30
38a 60 82°5 0 i 0 13 °6 20
b 60 82°5 0°15 il 0 7:9 20
34a 10 30 0 0 1 11 *4 30
b 10 30 0-1 0) 1 14:1 80
35a 75 90 0 2 0 18 *4 30
b 75 90 0 2 01 16-1 80

This remarkable property of fructose, taken in connection with the facts
that this sugar in presence of phosphate is much more rapidly fermented
than glucose or mannose, and that the optimum concentration of phosphate
for fructose is much higher than for glucose or mannose, appears to indicate
that fructose when added to yeast-juice does not merely act as a substance to
be fermented, but, in addition, bears some specific relation to the fermenting
complex.

All the phenomena observed are, indeed, consistent with the supposition that
fructose actually forms a permanent part of the fermenting complex, and that,
when the concentration of this sugar in the yeast-juice is increased, a greater
quantity of the complex is formed. As the result of this merease in the

1909. ] The Alcoholic Ferment of Yeast-jwee. 347

concentration of the active catalytic agent, the yeast-juice would be capable
of bringing about the reaction with sugar in presence of phosphate at a
higher rate, and at the same time the optimum concentration of phosphate
would become greater, exactly as is observed. The question whether, as
suggested above, fructose actually forms part of the fermenting complex, and
the further questions, whether, if so, it is an essential constituent, or whether
it can be replaced by glucose or mannose with formation of a less active
complex, remain at present undecided, and cannot profitably be more fuily
discussed until the results of experiments now in progress are available.

It must, moreover, be remembered that different samples of yeast-juice
vary to a considerable extent in their relative behaviour to glucose and
fructose, so that the phenomena under discussion may be expected to vary
with the nature and past history of the yeast employed.

Summary.

1. Mannose behaves towards yeast-juice both in the presence and in the
absence of added phosphates in the same manner as glucose.

2. Fructose resembles both glucose and mannose in its behaviour towards
yeast-juice, but in the presence of phosphates is much more rapidly
fermented than the other sugars, and the optimum concentration of phosphate
is much higher.

3. Fructose has the property of inducing rapid fermentation in presence
of yeast-juice in solutions of glucose and mannose containing such an excess
of phosphate that fermentation is only proceeding very slowly. No similar
property is possessed by glucose or mannose.

348

The Discovery of a Remedy for Malignant Jaundice in the Dog
and for Redwater in Cattle.

By Gerorce H. F. Nutra, M.D., Ph.D., Se.D., F.R.S., Quick Professor of
Biology in the University of. Cambridge; and SryYmMourR HaDWEN,
D.V.Sci. (McGill) of the Department of Agriculture, Canada.

(Received June 22,—Read June 24, 1909.)

Judging from the literature relating to piroplasmosis, no drug is known
which exerts any curative action on either canine or bovine piroplasmosis.
The canine disease is exceedingly fatal, and, in certain localities, especially
in South Africa, it is almost impossible to keep dogs. On the other hand, the
disease in cattle causes enormous financial losses, especially in America,
Australia, and Africa, not only by causing a considerable mortality, but
also by producing a long-lasting anemia in many of the affected animals.

The discovery of a drug which will bring about a cure of piroplasmosis is
therefore a matter of practical importance. Our object in publishing this
communication is to announce the discovery of such a remedy. Whilst a
full description of our experiments will shortly be published in ‘ Parasitology,’
we desire to place the main facts on record. |

Canine Piroplasmosis.

We have discovered that trypanblau and trypanrot are highly efficient
remedies in the treatment of canine piroplasmosis. The drugs exert a direct
and observable effect upon the parasites by causing the pyriform parasites to
quickly disappear ; in most cases causing the total disappearance of the
parasites from microscopic observation in the peripheral blood. The disap-
pearance of the parasite is usually temporary, since they may reappear in
small numbers after an interval of 9 to 12 days; the treated dogs, as a rule,
show no symptoms and gradually progress towards complete recovery. In
our experience the treated dogs show little or no loss of weight; this being in
marked contrast to what is usually observed in the dogs which chance to
recover naturally.

Our experiments were carried out upon 20 dogs of various breeds and ages,
the majority being highly susceptible puppies. We experimented witha very
virulent strain of Piroplasma canis from Cape Colony. All of the 7 control
(untreated) dogs died of piroplasmosis: 6 died within 7 to 13 days, and 1 on
the 36th day after inoculation with virulent blood.

The remaining dogs, 13 in number, were treated as follows :—

The Discovery of a Remedy for Malignant Jaundice, etc. 349

Results of Treatment with Trypanblau (up to June 17, 1909).

. | Control dogs.
Dog. Treated dogs. (Inoculated at the same time as
the treated dogs.)
| iL Kept under observation 90 days................0++++ Control died after 7 days.
2 5p = SS ie smareacececear sees sss 5 Bes aba Lee
3 . 5 GO) see desteuececuscup sues fis a oe
4 is % (Gis 6A Nene ncaa a tel s a Arata
5 ‘ aS ADM Eis cctisineaceze he obo. 3 lye SO pass
6 re 35 LE ory Bono bsaenuneS podéedbeo . i Sie.
7 " Fy Bye oo somnca ne bncnaeABOOaCe Ss ‘ do
8 DiedMotprelapseratter Ss yee. scceecseamscee: 4 "5 (hee
9 6 9 116%. ah bee ncatedecduabedacoc 3 tifenes
10 Treated when moribund, the dog died three
hours later, but the drug obviously affected the
parasites
| 11 Treated 24 hours after inoculation (i.e. before | 5 y De tess
the parasites had appeared), dog alive and well
after 65 days; it never showed parasites
|
Results of Treatment with Trypanrot.
Dog. Treated dogs. | Control dogs.
12 Aliveiand well/after 1VL days secee2scencn2- se. Control dog died after 9 days.
13 Treated in an advanced stage of the disease, | | BA lathes

the dog died 20 days after inoculation, but |
no parasites could be found in the blood. |
The dog apparently died from the after

effects of piroplasmosis

The above table speaks for itself, it scarcely requires any comment. We
very much regret that we have lost four of our treated dogs from inter-current
disease. Two of these dogs died of distemper (42 and 43 days respectively
after inoculation), one died of distemper and mange combined (69 days after
inoculation), and one died of severe generalised mange due to Demodex
folliculorum (52 days after inoculation). Dog 13 was treated in an advanced
stage of the disease, but its life was prolonged ; it is worthy of note that no
parasites could be found in the animal at autopsy. Dog 10 was treated only
three hours before death; the drug markedly affected the parasites, but it
was too late to save the dog’s life. In only two cases did we see a relapse
follow five days after apparently successful treatment; this was in two very
small and poorly developed puppies. The remaining dogs are alive and well
to-day and show no parasites microscopically in their blood.

As previously stated, the drugs exert a direct effect upon the parasites.
The percentage of infected corpuscles is decreased, the pyriform parasites
disappear, rounded and degenerated parasites are seen for a time, and, after

350 The Discovery of a Remedy for Malignant Jaundice, ete.

a while, all parasites are lost to view. When, after an interval, the parasites
reappear, they do so in exceedingly small numbers, and, after a while, they
disappear completely and finally.
All of our dogs were treated by subcutaneous injections of saturated
solutions of the dyes.
Bovine Piroplasmosis.

With regard to redwater, we are in a position to state that trypanblau
exerts a very prompt effect upon the parasite. The effect is precisely
similar to that observed in Piroplasma canis, Our experiments upon the
bovine disease are still in progress, but we feel that they are sufficiently
advanced to warrant the trial of the remedy in the field. We shall report
upon our results in a future communication.

The results of these experiments are of considerable interest, since they
throw additional light upon the biology of the parasites and entirely confirm
the observations of Nuttall and Graham-Smith upon the usual mode of
multiplication of the parasites in the circulating blood. The striking effects
of the drugs upon the parasites led us directly to make enumerations of the
different forms of parasites occurring in the blood of treated and untreated
animals. The result of these observations has been to bring to light several
interesting facts regarding the life-history of the parasites.

East Coast Fever.

Incidentally we may mention that in one experiment which we have tried,
trypanblau exerted no effect whatsoever on the parasite of East Coast Fever
in cattle. This does not appear to us surprising, since the parasite is very
different from Piroplasma, although most writers still persist in retaining it
in the genus. For reasons stated elsewhere by Nuttall (1908), the parasite
of East Coast Fever should be named Theileria parva.

Conclusions.

The obvious practical conclusions to be drawn from our results is that the
remedies will prove of value in practice. It is highly probable that they will
act in a similar manner in relation to equine and ovine piroplasmosis. The
mere fact that remedial agents have been found for diseases which have
hitherto run their course, in spite of all treatment, is encouraging, since with
time we may reasonably hope to cope with these maladies in an efficient
manner.

301

The Vacuolation of the Blood-platelets: an Expervmental Proof
of ther Cellular Nature.

By H. C. Ross, late Surgeon Royal Navy.
(Communicated by Prof. C. 8. Sherrington, F.R.S. Received May 12, 1909.)

On July 27, 1907, a paper appeared in the ‘ Lancet’ by Ronald Ross,
S. Moore, and C. E. Walker, entitled “A New Microscopical Diagnostic
Method and some Simple Methods for Staining Liquid Blood.” It described
new methods for the staining of blood-cells im vitro—notably the agar-jelly
method—by mixing polychrome methylene blue with agar and preparing a
film: blood-cells spread upon this will absorb the stain, and if the jelly is
suitably prepared the different morphological elements of the cells can be
readily distinguished. It also described how the leucocytes, after they had
been resting on the jelly for a short time, developed bright red spots in their
cytoplasm ; and it was suggested that the spots might be centrosomes.

The spots appear as bright scarlet points, and resemble closely what one
would imagine that centrosomes would look like if they could be seen in
polymorphonuclear leucocytes. The “red spots,” however, could not be found
in the blood-platelets, and believing them to be centrosomes, the authors
suggested in their paper in the ‘ Lancet’ that if they could be demonstrated
in the blood-platelets, it would settle the nature of these bodies, and close an
old existing controversy.

About two months ago I was fortunate enough to observe these “red
spots” in the blood-platelets, and if the following experiment is repeated
the spots can be produced in them in every specimen.

Prepare a neutral solution which contains 3 per cent. sodium citrate,
1 per cent. sodium chloride, and 1 per cent. morphine hydrochloride.
Draw up into a capillary tube a volume of this solution, and add to it an
equal volume of blood drawn freshly from the finger. After allowing the
two fluids to mix, incubate the tube at 37° C. for five hours. A film of agar-
jelly must now be prepared, which contains a sufficiency of Unna’s stain to
stain deeply the granules of polymorphonuclear leucocytes when they are
spread upon it. One suitable formula for preparing this jelly is given in
the ‘Lancet’ for January 16 and February 6, 1909, and another suitable
formula will be found in the first equation in a paper recently published in
the ‘ Proceedings of the Royal Society,’ B, vol. 81, 1909, p. 102. Place a
drop of the incubated blood and morphine solution on to a cover glass, which
should be inverted and allowed to fall flat on to the agar film. In about

352 Mr. H. C. Ross. [May 12,

15 minutes’ time it will be seen that probably all the blood-platelets will
show one or more bright red spots in them, and these spots are identical in
appearance (except that they are usually smaller) with those seen in the
cytoplasm of leucocytes when blood is examined by this in witro method
by which the spots in the larger cells were observed by R. Ross, Moore, and
Walker.

It is, however, necessary to state that the spots are not centrosomes. At
first I believed them to be such, but further investigation showed the
suggestion to be erroneous. They are diffusion vacuoles. If the diffusion of
stain into leucocytes is accelerated by heat or alkali so as to cause maximum
staining without actually killing the cells, then stained liquid also passes into
the colloid cytoplasm and remains suspended in it as a “red spot.”
A description of the nature of these spots was published in the ‘ Journal of
Physiology, September, 1908. These vacuoles get gradually larger, and when
the leucocytes die, the spots suddenly disperse owing to the liquefaction of the
cytoplasm.

The suggestion of R. Ross, Moore, and Walker, that if the spots could be
seen in the blood-platelets it would be proof of their cellular nature, was
evidently based on the assumption that the spots were centrosomes. Since
they are not centrosomes, it may be said that their appearance in the blood-
platelets now falls into insignificance. But the spots in the platelets also get
larger and then disperse after an interval, and their disappearance is practi-
cally coincident with the disappearance of the vacuoles in the leucocytes,
which is caused by the liquefaction of the cytoplasm occurring at death.
Morphia also causes extreme vacuolation of the leucocytes.

As a matter of fact, studying the question from another aspect also shows
that their vacuolation is proof that the blood-platelets are cells. When fresh
blood is placed on the jelly, and no morphia is employed, the leucocytes do
not become extremely vacuolated, nor do the platelets develop the spots.
Why should morphia have this effect ?

It was shown in a former paper* that different cells have different
coefficients of diffusion, and that, in blood-cells, approaching death causes a
lowered coefficient. It is obvious that the vacuoles will appear more readily,
that is, liquid will diffuse more readily into cells which have a lowered
coefficient of diffusion.

It is apparent, therefore, that the blood-platelets must be cells, or rather
must be composed of living protoplasm, for their vacuolation is produced
experimentally by the action of the poison, as morphia, in causing the gradual
approach of death, also lowers the coefficient of diffusion. Morphia lowers

* “Roy. Soc. Proc.,’ B, vol. 81, 1909.

1909. | The Vacuolation of the Blood-platelets. 353

the coefficient more than any substance that has been tried as yet, and it
appears to have a greater effect on the cytoplasm than other poisons. As all
the platelets in a specimen usually become vacuolated, I believe that they all
belong to one class of cell, derived from one source.

In view of the vacuolation, the precipitate theory of the blood-platelets
becomes untenable. Another theory, that they are the extruded nuclei of
red cells, can also, I think, be dimissed. Former researches have shown that
the nucleus of a cell is its “higher centre.” It is difficult to believe that a
cell can thrust out its nucleus. Moreover, in the paper in ‘The Journal of
Physiology, already referred to, it was stated that the spots in leucocytes
were never seen to be connected with the nucleus. The platelets, therefore,
can hardly be nuclei. The nuclear theory is, I believe, the outcome of the
examination of dead structures. Blood-platelets are never seen emerging
from red-cells when 7m vitro methods are employed.

I believe that the theory that the blood-platelets are fragments of
leucocytes is the correct one ; they have the same coefficient of diffusion as
those cells, and usually contain a few granules which have the same staining
rate as those of the leucocytes. When blood is placed on jelly which excites
amceboid movement in leucocytes,* the platelets have been seen to extrude
and retract pseudopodia. On a few occasions, a pseudopodium has been seen
to become separated, and the fragment appears to be identical with the
other platelets seen in the specimen. It may contain clear cytoplasm, a few
granules, or even a lobe of the nucleus.

* “The Lancet,’ January 16, 1909.

304

Further Results of the Experimental Treatment of Trypano-
somiasis : being a Progress Report to a Committee of the
Royal Society.

By H. G. Piuvmer, F.L.S8., and W. B. Fry, Captain R.A.M.C.

(Communicated by J. Rose Bradford, M.D., Sec. R.S. Received June 28, 1909.)

The following results are a continuation of the work of which summaries
have already appeared in the ‘ Proceedings of the Royal Society.’*

These experiments have been carried out, with the same strain of Surra as
was used before, at the Brown Institution and the Lister Institute.

A.—Condition of the Animals living at the Date of the Completion of the Tables
in the last Paper.
Rats treated with Sodium Antimonyl Tartrate, 1 per cent. (p. 478).

No. 7 died 428 days after inoculation. -
2 32 ”? 409 ”

Rats treated with Sodium Antimonyl Tartrate, 5 per cent., in Colonel Lambkin’s
Medium (p. 482).

No. 13 died 216 days after inoculation.

Rats treated with Antimony (metal), 5 per cent., in Colonel Lambkin’s Medium

(p. 483).
No. 10 died 205 days after inoculation.
Dede aes hol 3
SOME OOO i
Peat ay Oy) BS
p20 00 is

Rats treated with Lithium Antimonyl Tartrate, 0°25 per cent. (p. 485).
No. 4 died 145 days after inoculation.

ep ace)
ty One Com, 2
yrs Kveaess 74a e
Oss AY) i;

* B, vol. 79, 1907, pp. 500—516 ; and B, vol. 80, 1908, pp. 1—12 and 477—487.

Results of the Experimental Treatment of Trypanosomiasis. 355

Most of the above rats died from cold; none of them died from the
disease, and no trypanosomes were found in their blood or organs, and
inoculations made therefrom were entirely negative.

B.—Further Experiments.

Rats treated with Lithium Antimonyl Tartrate, 1 per cent.

A further series of experiments has been carried out with this substance
on a large number of rats, giving four doses of 4 to 5 minims (according to
weight) of a 1 per cent. solution subcutaneously, a dose being given every
other day. Practically by this method every rat can be cured. They have
lived for varying periods, up to 249 days, and in no case have trypanosomes
been found after death in the blood or in the organs. No rat has died of
the disease, and in no case thus treated has there been a recurrence. The
results have therefore been more constant than those attained with sodium
or potassium antimony! tartrates. The treatment was begun on the third or
fourth day after inoculation; it will be seen below that when it is left until
the number of trypanosomes in the peripheral blood is very great, although
they may be driven out of the blood, it does not cure: so that the time at
which treatment is commenced is of considerable importance.

It has also been given intravenously in rabbits, but with far less effect
than when given subcutaneously. The elimination in this case is very rapid,
to which fact we attribute its comparatively feeble action.

Rats treated with Lithium Antimonyl Tartrate on the Fifth or Sixth Day of
the Disease.

The blood at this period of the disease is swarming with trypanosomes,
and experiments were made in order to see what effect this salt of antimony
would have upon the disease at this period. If one dose of 5 minims
of a 1 per cent. solution be given the rats die on the seventh day, so that
little or no effect is produced. If two such doses be given, one on the fifth
and one on the sixth day, the average time of death in 10 rats was 194 days,
and living trypanosomes were found in the blood at death. When four doses
were given, one on each day from the fifth to the eighth, the time in three
rats was lengthened to 81 to 86 days: in one of these even living trypano-
somes were found in the blood after death. By comparing these results with
those mentioned in the former section it will be seen that the time at which
the administration of the drug is begun is of importance, as well as the
number of doses. The animals stand the best chance of cure when no
recurrences take place, and this is best ensured by the method described in
the previous section.

VOL. LXXXL—B. 2D

356 Mr. Plimmer and Capt. Fry. Further Results of [June 28,

Further experiments made with Rats treated with Antimony in order to find
out in what Organs the Trypanosomes are latent.

Following on the experiments made on rats treated with sodium antimonyl
tartrate, with the view of finding out where the trypanosomes are latent, and
recorded in the last paper,* a further series of experiments has been made
on rats inoculated with Surra, which is more amenable to treatment with
antimony than the Nagana used in the former series, and completely
treated (that is, given a curative series of doses) with lthium antimonyl
tartrate: this, as stated in the paper referred to, appears to be the most
active of this variety of salt.

Seven rats were treated with four doses of 5 minims of a 1 per cent.
solution of lithium antimonyl tartrate, and they were killed in succession,
one on the 6th, 7th, 10th, 14th, 16th, 22nd, and 30th days after the last
dose. The livers and bone-marrows were made into an emulsion with the
minimum quantity of 0°89 per cent. salt solution, and 1 cc. of the emulsions
of these organs and 1 ¢.c. of heart’s blood was injected separately into other
rats. The results were entirely negative. Microscopic preparations were
made of the material injected and no organisms were seen, and none of the
sub-inoculations gave a positive result.

Experiments made in order to see if any Protection was afforded by Initial
Treatment with Antimony.

re

A series of six rats was treated with four doses of 5 minims of a
1 per cent. solution of lithium antimony] tartrate, one dose every other day
in the same manner as when given for curative purposes. They were then
inoculated with Surra, one on the first day after the completion of the treat-
ment, and one on the 2nd, 4th, 5th, 9th, and 10th days after. They all died
on the 5th or 6th days after inoculation, just as untreated rats would have
done, so that antimony in this very soluble form is of no protective use in
rats, owing most probably to its rapid elimination.

The blood of an uninfected rat treated as above has also been used in the
in vitro experiments recorded below.

Rats treated with Sodium Antimony Lactate and with Antimony Sodium
Calewwm Lactate.

Through the kindness of Messrs. von Heyden we have been enabled to
make some experiments with the above compounds. The sodium antimony
lactate contains 26 per cent. of antimony, and the antimony sodium calcium

* ©Roy. Soc. Proc.,’ B, vol. 80, p. 487.

1909.]| Haperimental Treatment of Trypanosomasis, 357

lactate 17 per cent., so they are both much weaker in antimony than the
tartrates which we have used. By the addition of a small quantity of lactic
acid we were able to get a 1 per cent, solution of both salts, and in this
strength the solutions were not very irritating, but neither with rats nor with
larger animals are they as effective as the tartrates or the metal.

The following table shows the results obtained with sodium antimony
lactate 1 per cent.

Average duration of untreated disease 6°9 days :—

Rats of 150 to Nn oeidce
200 grammes umber of Goses, | Recurrences. Lived, Remarks,
: and: quantity.
weight,
1 2 of 4 minims 0 9days |. Died from enteritis,
2 4of4 ,, 0 20 ,, | Died from retained fcetus,
3 A ofdn 1 Saree
A 5of4 ,, 1 AGM Mi Se
5 Gio 4h 5, 2 100 ,, | (No trypanosomes found
6 40f5 ,, 0 TA 5. | in any of these rats after |
a Biol Si, 55 il 48, | death.)

The following table shows the results obtained with antimony sodium calcium
lactate 1 per cent.

Average duration of untreated disease 6°9 days :—

Rats of 150 to | Number of R
200 grammes | _ doses, and on Lived. | Remarks,
weight. quantity. aie |

1 3 of 4 minims | 1 | 83 days | No trypanosomes found post mortem.
2 4of4 ,, | (0) 25 ,, | Living trypanosomes found post mortem.
3 5of4 ,, 2 63. ,, | No trypanosomes found post mortem,
1 Gof4 Dab Aves BH os : ‘
5 Sof5 ,, PA Oe oy | 5 % 9
6 6 of 5 ” 2 68 9) ” ” bb}
7 8 of 5 ” 2 57 ” | ” ” ”
8 Aok7 On tsi | ; i

On dogs the effect was very much less marked than on rats, and an
effective dose became inconveniently large.

The following experiments show the relatively greater time taken for these
salts to act, as compared with the sodium or lithium antimonyl tartrates,
which drive all the trypanosomes from the peripheral blood in about an hour
after the dose.

A Surra rat was taken on the fourth day, when the trypanosomes are
numerous in the blood, and 5 minims of a 1 per cent. solution of sodium
antimony lactate were injected.

2D 2

358 Mr. Plimmerand Capt. Fry. Further Results of [June 28,

Blood was taken - and showed the following :
% hour after injection... Trypanosomes affected by the drug: are extremely
active, and show a tendency to swell.
alt - i. .... Very few normal trypanosomes to be seen: nearly
all are swollen and spherical in shape
(= “hattledores”). Still large numbers.
14 hours 5 ... Much smaller number of trypanosomes to be seen:

a few “battledores”: a few motionless ones,
and one or two normal forms.

2 x i “ Battledores” have all disappeared: one or two
slowly moving normal forms seen.

Deis Be coq ADH OY

oe ... No trypanosomes found.

A similar experiment made with a rat treated with antimony sodium
calcium lactate yielded practically the same result. Further experiments
made with these drugs in vitro will be mentioned later.

Experimen made with Antimony (Metal in state of finest Division) suspended
in various Orly Media.

Since the curative results following treatment with the metal antimony*
suspended in Colonel Lambkin’s medium seemed promising, many trials have
been made with the metal suspended in other oily media, such as olive oil,
eod liver oil, lanolin, egg-yolk, ete., in order, if possible, to obviate, or at any
rate reduce, the extremely irritating properties of the metal, which seriously
interfere with its practical use.

In olive oil a 5 per cent. suspension was used: with one dose of 3 minims
Surra rats lived for 15 days, and died with living trypanosomes in their
blood. Seventeen Surra rats were given one dose of 5 minims on the fourth
day of the disease, and they lived from 41 to 133 days: in these there were
mo recurrences, nor were trypanosomes found after death, and sub-inoculations
were in every case negative. Six Surra rats were treated with the same
dose in order to observe the time taken for the complete disappearance of
the trypanosomes from the blood.

Blood was taken and showed the following :
4 hour after injection... Trypanosomes very active.
1 v: i ... As numerous: show evidences of swelling.
14 hours y ... Stillnumerous: nearly all swollen: some “ battle-
dores.”
2 4 4 Very few forms found: all “ battledores.”
Diners 4 No trypanosomes seen.

we % Roy. Soc. Proc.,’ B, vol. 80, p. 483.

1909.] Experimental Treatment of Trypanosomasis. 359

Two Surra rats were taken on the fifth day, when the blood was swarming
with trypanosomes, and 6 minims were given. Two and a-half hours after the
rats were killed, and smears were made from the lungs, liver, spleen, kidney,
bone-marrow, heart’s blood, and brain. In none of the specimens could a
trypanosome be found after prolonged examination.

This oil was also given to several rats upon recurrences after treatment
with small doses of the lactates mentioned above: in these cases the effect
was much less marked, even although the number of trypanosomes in the
blood was much less than in the rats treated for the first time. This accords
with our general experience that recurrences are much more difficult to deal
with than the initial infection, and this applies to all the drugs we have
tried.

A suspension in cod liver oil took four hours to drive the trypanosomes
out of the peripheral blood.

The suspension in egg-yolk appeared to act in rats better than any other;
in dogs, however, the results were variable ; sometimes strikingly good, at
others no better than the other mixtures: sometimes causing great irritation
and sloughing, sometimes not causing any irritation at all. We have rats alive
for more than 120 days after inoculation, with no recurrences, after one dose.

An experiment was made to see how long one dose took to drive the
trypanosomes out of the blood. A Surra rat on the fourth day was treated
with 5 minims of a 5 per cent. suspension.

Blood was taken and showed the following :
~ hour after injection... Trypanosomes much affected, but not decreased.
Many “ battledore ” forms.
14 hours 53 ... Trypanosomes reduced in numbers: all swollen

and “ battledore” forms: very little movement.
Ze, i ... No trypanosomes found.

Experiments with Quassia.

Dr. Guillemard, of Cambridge, suggested that quassia, on account of its
known poisonous effects on some of the lower forms of life, should be tested
for its trypanocidal qualities. A series of experiments was therefore under-
taken on rats.

Six Surra rats were treated on the third and following days of the
disease with a 5 per cent. solution of the pharmacopceal extract of quassia :
they were given three doses subcutaneously—5 minims on the third day,
10 minims on the fourth, and 10 minims on the fifth day. The trypanosomes
were entirely unaffected, and the animals died on the sixth—seventh day.

360 Mr. Plimmer and Capt. Fry. further Results of [June 28,

Another series of 12 Surra rats was treated with a two hours’ decoction of
quassia-wood made with the minimum amount of water. Of this three doses
were given—5 minims on the third day and 10 minims on the fourth and
fifth days. The trypanosomes in these rats were also entirely unaffected, and
the animals died on the sixth—seventh day. It was also tried intravenously
in rabbits in doses of 20 minims of the decoction: no effect was produced, and
the rabbits died on or about the 42nd day.

Experiments made in vitro correspond with these results, and will be
described later,

Experiments with Arsenophenylglycin.

Professor Ehrlich kindly sent some of this substance to Dr. Bagshawe, the
Director of the Sleeping Sickness Bureau, with which we have made some
initial experiments upon rats. Ehrlich found that Nagana mice could be
cured, in practically every case, with this substance. But the effects on
larger animals, so far as we have gone, are not quite so satisfactory, and it
compares in this undesirable manner very well with the antimony tartrates,
with which we can cure practically every case of Surra in rats, but which do
not have anything like the corresponding effects on rabbits, guinea-pigs, and
dogs. It is not only in the question of practical dosage that difficulties arise :
each kind of animal has a personal equation, and their reaction to a given
drug is not similar. This, and the relatively larger dosage in bigger animals,
present considerable practical difficulties in the treatment of trypanosomiasis.

Our experiments have given the following results. Out of eight Surra
rats of 180 to 200 grammes weight which were given one dose of
25 minims of a 1 in 80 solution of arsenophenylelycin, four died on the
19th day with living trypanosomes in their blood, the recurrences having
taken place on the 16th—17th day. Two were given three and five doses
respectively of 5 minims of a 1 per cent. solution of lithium antimonyl
tartrate on the 17th and following days, and they lived 59 and 51 days. Of
the two which are still living (95 days), one has had five doses of 5 minims
of a 1 per cent. solution of lithium antimony] tartrate, beginning on the
17th day, and the other had one similar dose given on the day before the
recurrences occurred in the other rats.

The following experiment shows the effect of this substance upon the
trypanosomes in the blood, and how much longer it takes than the antimony
salts to produce its effects.

A Surra rat on the fourth day of the disease was treated with 1 c.c. ot a
2 per cent. solution of arsenophenylglycin (practically the same dose as given
to the other rats).

1909.] Haxperimental Treatment of Trypanosomasis, 361

Blood was taken and showed the following :
4 hour after injection... Trypanosomes showed slight increase of motility.

1 » » .. Ditto.

2 hours és ... Ditto, but more marked,

3 » % ... Trypanosomes not quite so active and fewer in
number.

ee, : .... Trypanosomes now very few in number.

yy % ... Only one or two trypanosomes to be seen in a
preparation.

5 55 % ... No trypanosomes seen.

In these specimens no swollen, breaking up, or “ battledore” forms were
seen: the trypanosomes simply disappeared.

On the Effects of the Drugs used upon the Trypanosomes in the Living Body.

In studying the therapeutic effect of the various drugs tried, including
metallic antimony in a state of finest division, repeated observations of the
peripheral blood were made in order to observe the effect of the drug upon
the trypanosomes, and to ascertain when the trypanosomes entirely dis-
appeared from the blood. The first stage noticed of the effect of the drug
was a great increase in the motility of the trypanosomes, followed by a gradual
slowing down to movements slower than normal. At this stage there is a
tendency for the whole trypanosome to swell, and to become bloated in
appearance. The swelling of the trypanosome continues until it becomes
almost spherical in form, or oftener ‘“battledore” shaped; the protoplasm
becomes indistinct, and the flagellum appears to be attached to only one side
of the periphery ; the macro-nucleus is fairly distinct, but it eventually breaks
up, and then the swollen mass disintegrates. The spleen at this time is full
of these broken up masses of trypanosomes, and as the nuclei will still stain,
a plasmodial appearance is seen in films of bits of nuclei dotted about in a
granular ground. These stages can be observed after treatment with all the
salts of antimony used, and are well marked after the administration of the
metal, in which case, however, the stages are slower. The soluble salts,
lithium and sodium antimony] tartrates, effect the total disappearance of the
trypanosomes in about one hour. Metallic antimony, when given in the
various media tried (Lambkin’s medium, olive oil, cod liver oil, heavy paraftin
oil, egg-yolk), brings about this disappearance in from two-and-a-half to four
hours, according to the medium used: the first noticeable effects being
produced in about half an hour. In the case of egg-yolk and olive oil the
blood is free from trypanosomes in two-and-a-half hours. This would seem

362 Mr. Plimmerand Capt. Fry. Further Results of [June 28,

to show that some portion of the metal introduced must be changed into
some soluble form very rapidly; but apparently after the reaction of the
tissues occurs the antimony becomes more or less shut off, and absorption
must take place very slowly, as traces of the metal, apparently unaltered,
have been found as late as six to seven weeks after the injection.

Sodium antimony lactate and antimony sodium calcium lactate were
found to act rather more slowly than the above (see Table above), the time at
which the trypanosomes had completely disappeared varying from three to
four hours.

It was noticed in these experiments that trypanosomes, though obviously
drug-affected when the blood was taken, remained alive on the slide outside
the body for a long time after all forms had disappeared from the circulating
blood.

Further details of the time taken for the various drugs to act will be
found in the sections upon sodium antimony lactate, antimony oil, antimony
egg-yolk, and arsenophenylglycin.

On the Action of Trypanocidal Substances in vitro.

Experiments have been carried out with a view of throwing light on the
more exact nature of the changes which are produced in trypanosomes when
they are brought into contact with trypanocidal substances. The general
principles we have observed in these experiments have been: 1. To dissolve
the drug in some fluid so that when it is added to the infected blood it will not
cause osmosis to occur in the cellular elements of, or trypanosomes contained
in the blood. (The various substances were dissolved in a 0°89 per cent. salt
solution, isotonic with rat’s blood which was used in these experiments.)
2. To use always equal volumes of the solution and of the affected blood.
3. To use blood at the time when the trypanosomes are just becoming very
numerous, so as to avoid the presence of old, feebly moving forms, which are
always present in the later stages of an acute infection. The method of
observation has been to watch the behaviour of the trypanosomes when in
contact with the, various solutions of the drug under the microscope.
A measured drop of blood and of the solution are mixed on a slide with
care: the mixed drop is then covered with a sufficiently large cover glass,
and this is sealed with vaseline.

It has been found possible in this manner to exactly determine the
dilutions at which the various drugs used cease to have an instantaneously
trypanocidal action; further, in higher dilutions, by carefully watching the
changes taking place in the trypanosomes, it is possible to determine the
dilution at which no effect is produced, and between these two points

1909.| Haperumental Treatment of Trypanosomiasis. 363:

the periods of time necessary to ensure immobility and death of the
trypanosomes can be ascertained. By a comparison of the results obtained
a very good estimate of the probable action of any drug when given to an
affected animal can be arrived at.

For instance, sodium and lithium antimony] tartrates were found to act, in.
the same dilutions, in a manner fairly comparable to their antimony content,
and to their action on the trypanosomes in an affected animal. Again, with
atoxyl a much higher concentration of the drug was necessary—it had to be
about ten times stronger—in order to obtain the same destruction pictures,
results corresponding with the rapidity of the disappearance of trypanosomes
from the peripheral blood of affected animals when treated with the above
drugs.

In the case of the two new lactates mentioned above, their therapeutical
value was accurately foretold by a preliminary study of their action in vitro
in the manner described. In all these experiments controls have been
carried out: it has been found that trypanosomes will live and retain their
activity for hours when infected blood and the diluting fluid alone are mixed
together.

The various changes taking place in trypanosomes on coming into contact
with a dilute trypanocidal drug, commencing with their preliminary extra-
ordinary increase of activity, and their subsequent swelling up, immobility,
and disintegration, can be watched in all their different stages in this
manner. These effects resemble very closely the changes which take place in
the trypanosomes in the peripheral circulation of an animal treated with
antimony.

The following tables show the effects produced by the different substances
in their various dilutions.

Dilutions of sodium antimony] tartrate in 0°89 per cent. salt solution
mixed with Sarra rat’s blood, in equal parts. The control in all cases is
equal parts of blood and 0:89 per cent. salt solution.

Dilutions,
Time. Control.
1—500. 1—1000. 1—5000. 1—10,000.
: |
Motionless | Motionless | Few active forms | Trypanosomes active | 1 min. Very active. |
1) 5 Motionless Few active forms ; 10 mins. 5
rest sluggish
oy 9) 55 All sluggish 30 ,, a
» 51 - Motionless 1 hour Y :
}

364 Mr. Plimmer and Capt. Fry. Further Results of [June 28,

Dilutions of lithium antimonyl tartrate in 0°89 per cent. salt solution
mixed with Surra rat’s blood, in equal parts.

Dilutions.
Time. Control,
1—6500. | 1—1000. 1—5000. 1—10,000. 1—20,000.
Motion- | Motion- | Some active |. Active trypanosomes Very active 1 min, | Very active.
less less trypanosomes trypanosomes
rs 3 Motionless Some active Many active 10 mins. 5
trypanosomes trypanosomes
7 4 3 Practically no motile Few active 30 5
trypanosomes seen, trypanosomes
only 1 or 2 inaslide. | seen. Tendency
Tendency to clump to clump
5 * * Motionless 1 or 2 active 1 hour 3

forms seen. Rest
motionless

In a dilution experiment with lithium antimony] tartrate made with the
blood of a Surra rat after a second recurrence, after treatment with antimony
(metal) and on first recurrence with lithium antimony] tartrate, the
trypanosomes in vitro appeared to have a greater resistance to the dilute

drug than the stock strain.

A comparison of the following table with the previous one will demonstrate

hist ——

Motionless

A few active forms present

1—1000.

Dilutions.

1—5000.

A number of active forms present
A few active forms seen..............-0+»-0
Motionless .........

see eee

This bears out our experience that the recurrences become less and less
amenable to antimony as they increase in number.
The following table shows the action of atoxyl and lithium antimonyl
tartrate compared in the above manner :—

1909.]

Experimental Treatment of Trypanosonuasis.

365

Dilutions of atoxyl.

Dilutions of lithium antimony] tartrate.

Time.
1—500. 1—1000, 1—5000. 1—500. - | 15000. 1—10,000.
|
Trypanosomes, | Active Active 1 min. | Trypanosomes,| All markedly | All fairly active.
all active | all motionless | affected
Active but a ss 5 mins.| Motionless: Motionless Less active.
affected commencing
| | | disintegration |
Less active Sluggish, but = [pba oe Only débris | 5 Some still moving; |
| still many seen tendency to clump. |
active
Practically | Nearly all Many | 2 hrs 53 Disintegrated | Motionless; some
motionless | motionless; | moving disintegration.
| 1 or 2 active | still
| forms seen |

Concentrated decoction of quassia in 0°89 per cent. salt solution mixed
with Surra rat’s blood in equal parts.

Dilutions. |
Time. Control.
1—500. 1—1000. | 1—5000. | 1—10,000.
|
Very active Very active Very active | Very active 1min. | Very active.
2 ” LE | ” 10 mins | »
” 237 32 77 30 bh) ”
” 3? bP) | x” 1 hour
Less active Less active Less active Less active 2hours | Less Serine

The conditions of the dilutions and the control were precisely similar at

the end of two hours.

There was no swelling nor clumping.

Dilutions of arsenophenylglycin in 0°89 per cent. salt solution mixed with

Surra rat’s blood in equal parts.

Dilutions.
| Time. | Control
1—100. 1—500. | 1—1000. 1—5000. | 1—10,000.

Very active Very active Very active Very active Very active) 1lmin. Very active.

Irritated : move-' Activity 3 55 - | 10 mins. 3
mentsrapidand inereased |
convulsive

Nearly motion- Sluggish = ay “ BOs Pe

less

Motionless 3 a 1 hour

37

366 Mr. Plimmer and Capt. Fry. Further Results of [June 28,

Experiments in vitro performed with the Blood of a Normal Rat which had
been treated uith Antimony.

Experiments were made in order to ascertain whether the blood of a rat
which had been treated with antimony would show any active trypanocidal
powers im vitro. Although in the case of an infected animal all the
trypanosomes in the peripheral blood would have been destroyed in about
an hour, no noticeable trypanocidal effects were shown by the blood of a
treated rat in the following experiments.

A normal rat had 5 minims of a 1 per cent. solution of lithium antimony]
tartrate injected subcutaneously : its blood was taken at 15, 30, 60, and
70 minutes after the injection, and was mixed with an equal quantity of
blood from a Surra rat containing many trypanosomes; the mixed bloods,
taken at the times mentioned, were examined under the microscope at
various intervals from 5 to 30 minutes after the mixing, and the
trypanosomes were found to be entirely unaffected, so that the blood of the
treated normal rat did not have any trypanocidal effect added to it by the
dose of lithium antimony] tartrate. The Surra rat, whose blood was used
for this experiment, was then given 5 minims of a 1 per cent. solution of
lithium antimony] tartrate :—

Blood was taken and showed the following :
10 minutes after injection... Trypanosomes affected: movement very rapid.
20 - 2 ... Many “battledores.”
40 - s Trypanosomes greatly decreased in number

all “ battledores.”
60 3 Bs ... Blood quite free from trypanosomes.
A normal rat was given four doses subcutaneously, one every other day,
of 5 minims of a 1 per cent. solution of lithium antimony] tartrate:
24 hours after the last dose a drop of its blood was mixed with a drop of
blood from a Surra rat in which trypanosomes were plentiful. The mixture
was watched under the microscope for half an hour, but no effect was
produced: the blood of the treated animal behaving just as the blood of the
control, an untreated rat.
A normal rat was given subcutaneously 10 minims (a lethal dose) of a
1 per cent. solution of lithium antimony] tartrate, and its blood was mixed
at half an hour, one hour, and one-and-a-half hours after the injection with
an equal part of an emulsion of trypanosomes prepared from the lungs, liver,
and heart’s blood of a Surra rat just dead. Each of the mixtures was
examined up to 30 minutes, but no effect whatever was produced on the

1909.| Eaperivmental Treatment of Trypanosonuaasis. 367

trypanosomes. These experiments may be compared with those recorded
on p. 396.

Experiments with Antimony upon Dogs.

Since the date of the last paper a large number of experiments have been
made with antimony in various forms upon dogs suffering from Surra. Of
the five dogs mentioned there one remains alive and well at the present
date, more than a year after inoculation.

Our experiences with dogs show that they are extremely susceptible both
to the disease and also to antimony: they are therefore not quite suitable
animals for these experiments, although they have all lived many times the
length of the untreated disease, that is 14 days. Five of the dogs were
treated with small doses of sodium antimony] tartrate in their drinking
water, but the disease is so acute in dogs that this method of giving the
drug, although it appeared to have some effect in postponing the reappearance
of the trypanosomes in the blood, did not produce results sufficiently
encouraging to warrant further experiments.

With regard to the experiments made with metallic antimony suspended
in egg-yolk, the initial experiment was so encouraging as to make a further
trial necessary. In this case the dog at the first relapse was given
20 minims of a 24 per cent. suspension: there was no local reaction, which
in dogs is of frequent occurrence after the administration of antimony in
any form, and the trypanosomes, which were very numerous, were entirely
absent from the blood in 24 hours; the dog remained quite free from them
for 48 days, and gained 3 lbs. in weight, and appeared perfectly well. The
recurrence was very sudden, as the dog was perfectly well up to the moment
when he was seized with a series of fits which ushered in the recurrence,
from which he did not recover. A rat treated at the same time as this dog
with 5 minims of the same suspension is alive and well more than 100 days
after this one dose.

Many of the dogs mentioned in the table below have died with fits and
paralyses and other nervous symptoms, but we are not certain whether these
are due to the disease or to the antimony. Im certain of the dogs the
treatment has appeared to alter the acute disease into a chronic one, and in
one of these more chronic cases there was a considerable excess of cerebro-
spinal fluid and a cellular exudation around the vessels in the brain, very
similar in incidence and extent to that described and figured by one of us
in rats dead from infection with Trypanosoma gambiense.*

There is a curious uncertainty in the local effects produced in dogs by

* ©Roy. Soc. Proc.,’ B, vol. 79, p. 95.

368 Mr. Plimmer and Capt. Fry. Further Results of [June 28,

antimony, whether injected subcutaneously or intramuscularly, and they
vary from time to time in the same dog; sometimes little or no effect is
produced, and sometimes the suppuration and necrosis produced are sufficient
to kill the animal.

We have recently given 24 injections of lithium antimony] tartrate sub-
cutaneously to three dogs in the greatest possible dilution: of these three
places have suppurated slightly, although the conditions under which they
were given were similar to those under which the 21 other doses were given.
(These dogs are now living and well 53 days after inoculation, and they have
had no recurrences.)

The following table gives a synopsis of the treatment, etc., of Surra
dogs :—

369

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Trypanosom

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370 Mr. Plimmer and Capt. Fry.

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372

Observations on the Urine in Chronic Disease of the Pancreas.

By P. J. CamMIDGE (M.D. Lond.).

(Communicated by Sir Victor Horsley, F.R.S. Received April 5,—Read
May 20, 1909.)

Modern researches on the physiology of the pancreas have shown that it
plays a much more important part in digestion than had formerly been
supposed, and also indicated that it exerts a very considerable influence on
the internal metabolism of the body. The present investigations were
commenced early in 1901, at the suggestion of Mr. Mayo Robson, with the
object of throwing further light on the nature of these metabolic processes.
and discovering, if possible, some more reliable means of diagnosing diseases.
of the pancreas than is usually afforded by the clinical signs.

The condition of the blood in patients suffering from diseases of the
pancreas was first investigated, and subsequently attention was devoted to
the urine. The clinical bearing of the results of these observations has
been dealt with in my Arris and Gale Lecture, and Mr. Robson’s Hunterian
Lectures, delivered at the Royal College of Surgeons in 1904,* and also in
a paper I read before the Royal Medical and Chirurgical Society in 1906.+

In the course of a series of experiments designed to discover whether
glycerine, the soluble product of the disseminated fat necrosis met with in
pancreatitis, or some derivative of it, was excreted by the kidneys, I found
that if the urine of a person suffering from an inflammatory affection of the
pancreas were boiled with hydrochloric acid, the excess of acid neutralised
with lead carbonate, and the freed glycuronic acid precipitated out of the:
acid solution with tri-basic lead acetate, treatment of the filtrate with
phenylhydrazin yielded a crystalline product which appeared to vary in
amount with the stage and intensity of the disease, while normal urines and.
specimens from patients suffering from diseases in which there was no
reason to think that the pancreas was involved gave no reaction. In this
communication I propose to describe a series of experiments I have
conducted into the nature of this product, and also the results of animal
experiments designed to discover whether the reaction depended upon
changes in the pancreas itself or was due to alterations in the other tissues.
of the body brought about by a disturbance of its metabolic functions.

For the purpose of the former investigations 4 litres of urine from

* ‘Lancet,’ March 19, 26, April 2, 1904.
+ ‘Roy. Med. Chi. Soc. Trans.,’ 1906.

Urine in Chronic Disease of the Pancreas. Be

each of eight patients, under the care of Mr. Mayo Robson, were collected
and separately examined, but as the methods employed and the results
obtained were similar in all it will not be necessary to deal with them
individually. The diagnosis of “pancreatitis ” in these cases was based upon
the clinical symptoms, analyses of the feces, and the condition of the
pancreas found at operation, while in one instance it was confirmed by
histological examination of the gland after death. In five the pancreatitis
was associated. with, and was probably dependent upon, the presence of
gall-stones in the common bile duct; two had had symptoms of “ indigestion ”
for several years, which in one was believed to have originated in an attack
of typhoid fever, and in the other had come on insidiously, but was
associated with evidence pointing to excessive putrefactive changes in. the
contents of the intestine and an abnormal intestinal flora. In one there
was sub-acute pancreatitis with disseminated fat necrosis, and an impacted
gall-stone in the ampulla of Vater; post-mortem examination in this case
showed an abscess in the tail of the pancreas and staining of the walls of the
duct of Wirsung, with bile for a distance of about 3 inches from its junction
with the common bile duct. Sections of the gland on microscopical
examination showed a considerable over-growth of the interlobular connec-
tive tissue with some small round-celled inflammatory infiltration and
vacuolisation of the acinar cells, which stained feebly.

The urine from these patients, which had been preserved with chloroform,
was filtered to remove suspended matter, and to the 4 litres were added
400 cc. of hydrochloric acid (sp. gr. 1:16). The mixture was placed in
a flask with a funnel in the mouth to act as a condenser, and the flask
heated on a sand-bath until the contents boiled. After being kept gently
boiling for 10 minutes the flask was cooled in running water, the urine
made up to its original volume with distilled water, and the excess of acid
neutralised by slowly adding 1600 grammes of lead carbonate. On the
completion of the reaction it was filtered through a moist filter-paper, and
the acid filtrate shaken with 1600 grammes of tri-basic lead acetate. The
filtrate from this was made alkaline with ammonia, and the resulting
precipitate washed with distilled water until the washings no longer acted
upon red litmus paper. The precipitate was then suspended in 200 cc.
.of distilled water, made faintly acid with hydrochloric acid, treated with
a stream of sulphuretted hydrogen, and the precipitated lead sulphide
removed by filtration. The clear filtrate was gently warmed on a water-
bath and, when free from sulphuretted hydrogen, was again well shaken
with 30 grammes of tri-basic lead acetate, filtered, and the filtrate made
alkaline with ammonia. The precipitate that formed, after being washed

2E 2

374 Dr. P. J. Cammidge. Observations on the —_[ Apr. 5,

with distilled water until the washings were neutral to litmus, was
suspended in 100 c.c. of distilled water, treated with sulphuretted hydrogen,
filtered, and the filtrate gently warmed to expel the excess of gas. It was
then cooled and again filtered. The resulting clear solution was then
examined as follows :—

A. The fluid gave all the usual carbohydrate reactions: Molisch’s test
was positive ; heating with concentrated sulphuric acid caused it to quickly
blacken ; Moore’s test gave a brown coloration ; Tollen’s ammoniacal silver
nitrate solution showed a black precipitate at room temperature in a few
minutes ; alkaline solutions of copper sulphate and bismuth were readily
reduced, but a solution of copper acetate in acetic acid (Barford’s test) was
not reduced, showing that the carbohydrate was not dextrose. The presence
of free aldehyde was excluded by the absence of any reaction with a solution
of rosaniline decolorised by sulphur dioxide.

B. Examined with the polariscope the fluid was found to be optically
inactive.

C. Fifty cubic centimetres of the solution were mixed with 2 grammes of
phenylhydrazin hydrochlorate and 6 grammes of sodium acetate, and heated
in a water-bath at 100° C. for two hours. On cooling, a dense flocculent
light yellow precipitate appeared. Microscopically, this was found to
consist of long, flexible, hair-like crystals, which, on being irrigated with
33-per-cent. sulphuric acid, disappeared within a few seconds of the acid
reaching them, suggesting that they were either a pentosazone or
maltosazone.* The precipitate was filtered off, well washed with cold
distilled water, and recrystallised from hot 10-per-cent. alcohol three times.
It was then dried in a hot-water oven, cooled in a desiccator, and examined.

(1) The crystals were found to be soluble in water at 60° to 70° C,, like a
pentosazone.t

(2) A melting-point of 178° to 180° C. was obtained with seven of the
specimens ; one, that obtained from the patient with sub-acute pancreatitis,
softened at 160° C., but did not completely melt until the temperature
reached 178° to 180° C.

(3) Estimation of the nitrogen-content by Dumas’s method gave 17:02 per
cent., 17:01 per cent., 16°62 per cent., 17-00 per cent., 16°40 per cent.,
16°86 per cent., 17°11 per cent., 17:08 per cent. of nitrogen, readings which
fall within the limits of experimental error of the calculated 17-07 per cent.
of nitrogen for pentosazone.

* “Roy. Med. Chi. Soc. Trans.,’ vol. 88, p. 265.
+ Neubauer and Vogel, ‘Analyse des Harns,’ 1898, p. 88.

1909. ] Urine in Chronic Disease of the Pancreas. 375

D. The aniline acetate test, when applied to the original solution,* gave a.
uniform bright red colour. The phloroglucin test was also positive, showing
a bright red colour at the boiling-point and forming a dark purple precipitate
after boiling for about one minute. Spectroscopical examination of a
solution of this precipitate in 93-per-cent. alcohol revealed an absorption
band in the green, to the right of D. Tollen’s orcin test, and Bial’s reagentt
gave a dull red-brown colour and formed a precipitate, but no green~tint
was observed. An extract of this made with four parts of amyl alcohol and
one part of ethyl alcohol showed, however, a dark band between C and D
when it was examined with the spectroscope. The solution showed no gas
formation or diminution of its reducing powers after being incubated with
brewer’s yeast for 24 hours at 37° C.

These results indicated that the fiuid prepared in the manner described
from the urine of patients having the clinical signs and symptoms of
pancreatitis contained an unfermentable sugar giving the reactions of a
pentose. A careful examination of the urine in these cases, and also in
over 500 others that have given a positive “pancreatic” reaction, failed to
reveal any free sugar to which the reaction might be due, and it was
therefore evident that the pentose was not free as such in the urine, but
was derived from some antecedent substance in the course of the reaction.
Numerous attempts have been made to isolate this and determine its nature,
but so far without success. It does not appear to be precipitated by
alcohol, ether, ammonium sulphate, magnesium sulphate, lead acetate,
mercuric chloride, or calcium chloride. Treatment of the urine with
benzoyl chloride and sodium hydrate has also given negative results.

Beside the case already mentioned, in which a detailed examination of
the pancreas was made, it has been possible to investigate the condition
of the gland after death in 26 others in which the urine had been examined
during life. In 9, where no reaction had been obtained, it appeared to be
normal both macroscopically and microscopically. In 13 there was evidence
of chronic inflammation: these had all given a positive reaction. Four
proved to be cases of cancer of the pancreas, 2 of these had given a positive
reaction and 2 a negative result. Of the 13 that showed chronic pancreatitis,
12 were of the typical interlobular type, and 1 was in an early stage of
inflammation with no marked increase of fibrous tissue, but an interlobular
and intercellular infiltration of small round cells and granular changes in
the acinar cells.

The occurrence of this reaction in the urine of patients who presented the

* Mullikin, ‘Identification of Pure Organic Compounds,’ 1904, p. 33.
+ ‘Deutsch. Med. Woch.,’ 1902 and 1903.

376 Dr. P. J. Cammidge. Observations on the | Apr. 5,

clinical symptoms of pancreatitis, and its absence in those whose pancreas
was not apparently diseased, together with the post-mortem evidence I had
been able to obtain, suggested that it was due either to some degenerative
change in the tissues of the pancreas or to a disturbance of metabolism set
up by disease of the gland. To decide this question, and also to obtain
confirmation of the dependence of the reaction upon disease of the pancreas,
I arranged a series of experiments upon animals, the surgical part of which
was kindly carried out for me by Mr. H. C. G. Semon, in the Pathological
Department of the University of Freiburg. A detailed account of these
experiments will be given subsequently, and I shall now only deal with those
points that bear upon the questions at issue.

The urine from the dogs employed for the experiments was drawn by
catheter both before and after the operations. Each specimen was shaken
with a few drops of chloroform, sealed in a glass vessel, marked with a
distinguishing number and letter for subsequent reference, and despatched
to me in London.

The normal urine, taken before operation, gave no reaction in any of the
dogs examined.

(1) A sub-acute pancreatitis was set up in the first dog by injecting a very
small quantity of turpentine, less than 1 c¢.c., into the pancreatic duct.
A specimen of the urine withdrawn 16 hours after the operation gave an
exceedingly well-marked reaction, the whole bulk of the fluid being filled
with a light yellow flocculent precipitate, which on microscopical examination
was found to consist of long fine crystals that dissolved in 33-per-cent.
sulphuric acid in 5 to 10 seconds. Filtered off, and purified by re-crystallisa-
tion from 10-per-cent. alcohol, it was found to melt at 178° to 180° C.
A second specimen taken 24 hours after the operation gave a similar, but
less marked, reaction.

Three days later a much larger dose of turpentine (1°5 c.c.) was injected,
but by mistake this was introduced into the common bile duct instead of the
pancreatic duct. The urine withdrawn six hours after this operation
contained a large amount of urobilin, but gave no “pancreatic ” reaction.
Two other samples taken at the end of 18 and 24 hours respectively after
the operation also yielded no osazone crystals.

A small piece of the pancreas was excised at the time of the second
operation. Microscopical examination of this showed no small celled
infiltration or over-growth of connective tissue, but the nuclei of the acinar
cells were indistinct and the protoplasm was highly vacuolated. When the
animal was killed 48 hours later examination of the pancreas revealed no

pathological changes.

1909.] Urine in Chronic Disease of the Pancreas. 377

(II) Ina second dog chronic pancreatitis was induced by passing a silk
thread from the duodenum along the duct of Wirsung and leaving the loose
end hanging free in the intestine.* The urine withdrawn three days after
the operation yielded crowds of long fine yellow crystals, soluble in
33-per-cent. sulphuric acid in 5 to 10 seconds, and which melted at
178° to 180° C. A second specimen taken one week after the operation
showed many typical erystals. A third sample obtained two weeks after the
operation gave some crystals, but the reaction was not as well marked as
that given by the preceding specimens. A fourth sample obtained one week
later still gave a fairly well-marked reaction.

At the end of the next week the pancreas and part of the duodenum were
removed and the cut ends of the intestine united by a Murphy button.
Examination of the excised portions showed that the thread was still in
position in the pancreatic duct and hanging into the duodenum. The whole
pancreas, but more particularly the head of the gland, was thickened and
felt heavier than normal. Microscopical examination of sections cut from
various parts showed a marked over-growth of the interlobular connective
tissue, especially in the neighbourhood of the duct of Wirsung and its larger
tributaries. The epithelium of the duct was detached and in places lay loose
in the lumen. The periphery of the gland was not so markedly affected,
although here, too, there appeared to be some increase of fibrous tissue and
a few round cells were seen in and around the ducts.

Immediately after the second operation the bladder was emptied by
catheter. A specimen of urine withdrawn 15 hours later was found to
contain 1:8 per cent. of reducing sugar, as estimated by Bang’s method;
16 per cent. of fermentable sugar, estimated by Lohenstein’s saccharometer,
and the polariscope showed 1:2 per cent. of dextro-rotatory sugar. Treatment
with phenylhydrazin gave a dense precipitate of coarse greenish-yellow
erystals that were insoluble in 33-per-cent. sulphuric acid in five minutes,
and on re-crystallisation from 70-per-cent. alcohol melted at 204° to 205° C.,
thus corresponding to dextrosazone. Forty-five cubic centimetres of the
filtered urine were boiled with 3 cc. of hydrochloric acid for 10 minutes,
the excess of acid neutralised with lead carbonate, and the glycuronic acid
removed by shaking with tri-basic lead acetate. The lead in the filtrate was
then removed by treatment with a stream of sulphuretted hydrogen and
subsequent filtration. After being heated to drive off the excess of
sulphuretted hydrogen, the filtrate was cooled and mixed with half its bulk
of distilled water. Yeast was then added, and the mixture incubated at

* Of. Carnot, ‘Gilbert et Thoinot, Traite de Médicine et de Thérapeutique,’ 1908,
fase. 20, p. 238.

378 Dr. P. J. Cammidge. Observations on the — [Apr. 5,

37° C. for 18 hours, when, as it was found that a control specimen no longer
gave a reaction for sugar, it was cooled, filtered, and the filtrate treated with
phenylhydrazin. Examination 24 hours later showed no crystalline deposit,
either to the naked eye or microscopically. The animal was found dead
three days after the operation, and post mortem no. trace of pancreatic tissue
could be discovered.

(III) The pancreas of a third dog was extirpated on September 9. Two
days later it died from gangrene of the duodenum. A specimen of urine
was, however, obtained before death, and 18 hours after the operation. This
was found to contain 3-9 per cent. of reducing sugar (Bang), and 3°8 per
cent. on fermentation (Lohenstein). Examination with the polariscope gave
32 per cent. of dextro-rotatory sugar. The osazone crystals melted at
204°—205° C., and were insoluble in 33-per-cent. sulphuric acid in
five minutes. A specimen of the urine examined by the same method as
that just described gave no “pancreatic” reaction after the fermentable
sugar had been removed, there being no crystalline deposit on macro-
scopical or microscopical examination of a preparation left undisturbed for
24 hours.

The results of the examination of the samples from Dog I and of the
specimens obtained from Dog II after the first operation agree with and
confirm my experience in the human subject. They show that while normal
urine gives no “pancreatic” reaction, specimens from animals in which
pancreatitis has been set up, either by a chemical irritant, such as
turpentine, or bacterial infection and partial blocking of the pancreatic
duct, give a characteristic reaction. The accidental injection of turpentine
into the common bile duct of Dog I served to prove that the reaction first
obtained was not due to the turpentine itself or to the manipulation of the
organs in the course of the operation, for, although considerably larger than
the first dose which was injected into the pancreatic duct, it gave rise to no
urinary “ pancreatic ” reaction. The pathological changes found in the pancreas
of Dog II, when it was removed at operation, agree with those described by
Carnot as being present in similar experiments performed by him, and
correspond to the chronic interlobular pancreatitis met with in man as the
result of an infection of the pancreatic ducts.

The results of the examination of the urines of Dogs II and III, after
total extirpation of the pancreas, tend to show that the changes which give
rise to the “pancreatic” reaction in the urine are dependent upon the
presence of the pancreas, and are probably to be referred to changes in the
gland itself and not to disturbances of metabolism in other tissues brought
about by interference with or perversion of its functions, for Dog III gave

1909. | Urine in Chronic Disease of the Pancreas. 379

no reaction after the operation, and in Dog II the reaction which had been
obtained on four occasions during the preceding three weeks disappeared
after removal of the pancreas.

It may be objected that the modification of the procedure made necessary
by the presence of the fermentable sugar interfered with the reliability test,
but that this is not the case has been shown by the results obtained on
examining the urines from several patients suffering from glycosuria
associated with disease of the pancreas by the same method. One in
particular demonstrated this very clearly, and also showed how an exceed-’
ingly well-marked reaction may diminish in intensity as destruction of the
pancreas progresses, and finally disappears when advanced glycosuria has been
established. The patient was first seen in December, 1906; there was then
an abdominal tumour which was suspected to be pancreatic, but an exami-
nation of the urine gave no “ pancreatic” reaction, and there was also at that
time no sugar. An exploratory examination was performed by Mr. Mayo
Robson, and a growth was found in the first part of the duodenum, but quite
free from the pancreas. On January 18, a second specimen of urine was
examined and found to be free from sugar, but it gave a well-marked
“pancreatic” reaction, suggesting that the pancreas was then involved in
the disease. At the request of the patient’s friends the abdomen was
re-opened a few days later and it was then found that the growth had
invaded the pancreas as had been suspected. In the early part of May, 1906,
examination of the urine showed 5°25 per cent. of sugar and a modified
“ pancreatic ” reaction gave many fine crystals soluble in 33-per-cent.
sulphuric acid in 5 to 10 seconds. A month later the sugar had increased
to 7 per cent., and a much less marked “ pancreatic” reaction was obtained.
In July the urine contained 7°25 per cent. of sugar and the “ pancreatic”
reaction gave only a few crystals. In August, 75 per cent. of sugar was
present, and no crystals were found on carrying out the modified “ pancreatic ”
test. In October the urine contained 9°5 per cent. of sugar and the
“pancreatic ” reaction was negative. The patient died on November 5.

The indications afforded by the experimental, pathological, and clinical
evidence so far obtained all point to the so-called “pancreatic” reaction in
the urine being due to active degenerative changes in the pancreas, and, so
far as I have been able to determine, to these alone. The fact that the sugar
giving rise to the reaction is apparently a pentose suggests that it is probably
contained in a derivative of the pancreas nucleo-protein which passes into the
blood as a result of the degeneration of the gland cells. In view of the
constant presence of a pentose in the nuclei of the cells of other organs and
tissues it might be thought that if this were true, degeneration in these

380 Urine in Chronic Disease of the Pancreas.

would also furnish a pentose-yielding substance which might pass into the
urine, but this does not appear to be the case. The reasons why the pancreas
is probably more liable to yield a pentose complex that can be split up and
so recognised in the urine are two: first, according to Griind,* the percentage
of pentose in the dry weight of the pancreas is nearly five times as great as
in any other organ of the body (pancreas 2°48 per cent., liver 0°56 per cent.,
thymus 0°56 per cent., kidney 0°49 per cent., muscle 0°11 per cent.); second,
the pentose contained in the nucleo-protein of the pancreas and thymus
is said to be more loosely combined and more readily set free than the
corresponding sugar in other tissues.} With regard to the first point,
however, the relative bulks of the organs have to be taken into account, and
it is conceivable that if the whole of an organ, such as the liver, were
simultaneously involved in some degenerative change, it might yield as
much or more pentose-containing substance as the pancreas under similar
conditions.

Only a small part of the field of research opened up by these investigations
has as yet been touched upon. It is hoped that by further experiment it
may be possible to isolate the mother-substance giving rise to the pentose
obtained from the urine in cases of pancreatitis, and also that a fresh series
of animal experiments may furnish information as to the chemical changes
in the body associated with diseases of the pancreas that precede and lead
up to the disturbances of internal metabolism that give rise to diabetes.

* Hoppe-Seyler’s ‘ Zeit. f. hysiol. Chem.,’ vol. 35, p. 111.
+ Blumenthal, “Dis. of Metabolism,” v. Noorden’s ‘ Clin. Med.,’ 1906, p. 262.

(381

A Method of Estimating the Total Volume of Blood contained
in the Inving Body.
By J. O. WAKELIN Barratt, M.D., D.Se. Lond., and WARRINGTON
Yorke, M.D, Liverpool.

{Communicated by Prof. Sherrington, F.R.S. Received May 7,—Read May 20,
1909.)

(From the Liverpool School of Tropical Medicine.)

The determination during life of the total volume of blood contained in the
living body is usually effected by Haldane’s method.* This consists in first
estimating the percentage hemoglobin content of the blood and then
determining the total hemoglobin content of the circulating fluid, the latter
being effected by administering a known volume of carbon monoxide.and
observing the extent to which the hemoglobin of the red cells is in
combination with this gas. Copeman and Sherrington} determined the
volume of the blood in the living body by injecting a measured volume of
0°75-per-cent. solution of sodium chloride (sp. gr. 10046) and observing the
resulting fall in specific gravity of the blood.

Recently, while making an investigation upon hemoglobinemia, we found
it necessary to make estimations of the total amount of blood in the living
body. This we carried out by: (1) making a hemocrit estimation of the
relative proportions, by volume, of red cells and plasma; (2) injecting a
known quantity of dissolved hemoglobin into the blood stream and
determining the degree of the resulting hemoglobinemia, from which the
amount of plasma present can be calculated. The method employed thus
consists of two procedures, namely the estimation: (1) of the percentage (by
volume) of plasma contained in the blood (A); and (2) of the total volume of
plasma contained in the body (B). The total volume of the blood is
a x 100. :

1. To carry out the first procedure 0°20 c.c. of a 1-per-cent. solution of
potassium oxalate was placed in a glass capsule and about 1 c.c. of the blood
of the subject of examination, retnoved from a vein, added. The volume of
the mixture was then carefully measured, a hemocrit determination made,
and the percentage of plasma in the undiluted blood calculated therefrom.

* “The Mass and Oxygen Capacity of the Blood in Man,” ‘Journ. of Physiol.,’ 1899—
1900, vol. 25, p. 331.

+ “Method for Determining the Quantity of Blood in a Living Animal,” ‘Journ. of
Physiol.,’ 1890, vol. 11, p. 8 ; also “The Specific Gravity of the Blood,” zbid., 1893, p. 71.

382 Drs. Wakelin Barratt and Warrington Yorke. [May 7,

An example will make this clear. In an experiment upon a rabbit the
volume of the mixture was 1:12 c.c., and the proportion of plasma in the
mixture determined by the hemocrit 85:3 per cent. The proportion, by
volume, of plasma in the undiluted blood was therefore

(= x 112-020) = = 821 per cent,

2. To carry out the second procedure a solution of hemoglobin was
prepared, usually by laking the red blood cells of the animal whose blood
volume was to be determined, though in some experiments the red blood cells
of another animal of the same species were employed instead of the animal’s
own red cells. To this end the required amount of blood was taken from a
vein, usually by means of a cannula or hollow needle, and added to a sufficient
quantity of a 1-per-cent. solution of potassium oxalate to prevent clotting. The
mixture was then centrifugalised, the supernatant plasma completely
removed, and to the red cells distilled water added in amount just sufficient to
produce Jaking. When this had occurred, solid sodium chloride was added in
the amount required to produce a 0°85-per-cent. solution of this salt. The red
cell constituents which separated out on the addition of sodium chloride,
forming a reddish white precipitate, were then separated by centrifugalisation
and a dark red solution obtained. The strength of this solution was
determined by matching a portion of it, suitably diluted, with a solution
containing a known percentage by volume of red blood cells, the solution
being prepared by adding a measured amount of oxalated blood, previously
submitted to a hemocrit determination, to distilled water. The matching
was sometimes carried out in a Zeiss comparison spectroscope, but more
frequently we employed the simpler plan of estimating the percentage of
hemoglobin in the diluted solution by means of v. Fleischl’s hemoglobin-
ometer, the scale of which had been previously standardised by means of
solutions of hemoglobin representing known percentages by volume of red
blood cells. In this way the strength of the hemoglobin solution employed,
which usually represented the amount contained in 25 to 40 per cent. of its
volume of red cells, is determined. A measured volume of this solution,
corresponding to a known volume of red cells, was then injected into a vein
of the animal whose blood volume was to be ascertained. As soon as the
injection was completed a sample of blood was taken from a vein on the
opposite side of the body, added to a known quantity of a 1-per-cent. solution
of potassium oxalate, and the volume of the mixture measured. The mixture
was then centrifugalised and the percentage of dissolved hemoglobin
~ determined as above described.

1909.] Method of Estimating Total Volume of Blood, etc. 3838

If the plasma, obtained before injection, contained a small percentage of
dissolved hemoglobin, as usually happens in the human subject, this was
subtracted from that present after injection of hemoglobin solution.

When the total amount of hemoglobin injected (C) has been determined,
and also the percentage of hemoglobin contained in the blood plasma after
injection (D), the total amount of blood plasma after injection (E) is calculated
from the formula

From this the amount of fluid injected was subtracted, and to it should be
added the amount of plasma withdrawn, if the animal had been bled before
injection. By way of illustration the continuation of the experiment upon a
rabbit, already referred to in (1), is shown below, the amount of hemoglobin
(C) being expressed as before in terms of red blood cells in the moist
condition, the volume of red cells being given instead of their weight in order
to avoid calculation of the latter, and the percentage of hemoglobin (D) being
similarly expressed in equivalent volume of red cells.

2 Percentage of Total volume of blood
acct OF dissolved hemoglobin dissolved | Total volume of | [ hsemocrit determination
ae d sah in blood plasma blood plasma. gave 82°1 per cent. by
; after injection. Cc volume of plasma
: = x 100. : :
C. D. in undiluted blood].
0°74 c.c. dissolved in 0°85- 0°73 per cent. 101 c.c. after in-| 97x100:0 _ 118
per-cent. NaCl solution, jection 821 ete
the total volume of fluid .. 97 c.c. before
being 4:0 c.c. injection

In no case could any change in the general condition of the rabbits be
observed after the injection of dissolved hemoglobin. In man the injection
of dissolved hemoglobin, obtained from the subject’s own red cells, in amount
sufficient to cause the blood plasma to contain as much hemoglobin as was
present in 1 per cent. of its volume of red blood cells, also produced no
alteration of the general condition. Aseptic precautions were employed
throughout these procedures.

The amount of hemoglobin can be varied within wide limits. If, however,
the blood plasma contains less dissolved hemoglobin than would be
represented by 1 per cent. of its volume of red blood cells, the natural colour
of the normal plasma may interfere with the hemoglobinometer determination.
This is likely to occur if the blood plasma before injection is unusually dark,
as sometimes occurs in pathological conditions attended with red cell

384 A Method of Estimating the Total Volume of Blood, ete.

destruction. In such cases, and also when the degree of hemoglobinemia was
slight, we employed a Zeiss comparison spectroscope, provided with a cell of
variable height, the estimation being made by matching the oxyhemoglobin
bands of the plasma with those of a solution of hemoglobin of known
concentration.

The accuracy of this method of estimating the total volume of the blood
depends upon the precision with which : (1) the hemoglobinometer readings
(or the comparison spectroscope determinations) are made; (2) the condition
of the plasma immediately after injection is ascertained.

In our observations the variation of successive heemoglobinometer readings
of the same solution of hemoglobin did not exceed + 2 per cent. of the mean
reading.

The mode of ascertaining the degree of hemoglobinemia immediately after
injection is a matter of importance. If only a small percentage of
hemoglobin, representing less than 1 per cent. by volume of red cells, is
present, no correction is ordinarily required when the plasma is obtained
within three minutes of the injection, which generally occupies about one
minute. If, however, a sample of blood cannot be obtained so soon after
injection, or again if a much higher degree of hemoglobinemia has been
produced, then an amount of dissolved hemoglobin sufficiently large to affect
the determination of the blood volume may have disappeared before the
sample is obtained. In such cases, two or three determinations of the degree
of hemoglobinemia require to be made at short intervals as soon as possible
after injection. From these the percentage of dissolved hemoglobin present
in the plasma immediately after injection can be calculated. In a series of
observations upon the rate of disappearance of dissolved hemoglobin from
the blood plasma in the living body, which will shortly be published,* the
mode in which this calculation may be made will appear.

The advantage of the above method lies in the ease of its application, and
the circumstance that the injection of hemoglobin is not attended with any
recognisable alteration of the general condition.

* ‘ Annals of Tropical Medicine and Parasitology,’ 1909, vol. 3, p. 1.

385

Preliminary Note on Trypanosoma eberthi (Kent) (= Spirocheta
eberthi, Liihe) and some other Parasitic Forms from the
Intestine of the Fowl.

By C. H. Martin, B.A., Demonstrator of Zoology in Glasgow University, and
Mourtet Ropertson, M.A., Carnegie Research Fellow, Assistant to the
Professor of Protozoology in the University of London.

(Communicated by Prof. J. Graham Kerr, F.R.S. Received June 18,—Read
June 24, 1909.)

[PuaTE 8.]

In the eleventh volume of the ‘ Zeitschrift fiir Wissenschaftliche Zoologie,
published in 1862 (p. 98), Eberth described, in a remarkable paper, “ein
kleines Infusorium” which he had found abundantly in the ceca of various
birds (fowl, partridge, duck, goose); he described it asa flattened crescentic
form measuring 0:012 to 0:014 mm. long by 0:006 to 0-008 mm. wide, with
a wide and a narrow extremity, the latter of which is drawn out into a slight
point. He distinguished the true body of the animal, in which he sometimes
thought he saw a nucleus, from the conspicuous “hautige Saum,” the
movements and appearance of which he described and figured very clearly.
He considers that this form is related to the form seen by Leydig in the gut
of Piscicola, Pontobdella, and Ixodes, which Leydig had believed came from
the blood of fish, and those described in the blood of various fish and of the
frog. Eberth, however, remarked that he could not find his parasite in the
blood of the infected birds, though he found two other flagellates, which he
does not further describe, in the intestine.

Leuckart, in his first edition (1863) of ‘Die Parasiten des Menschen’
(vol. 1, p. 140), placed the animal described by Eberth in a new genus,
Senolophus; in his second edition, however, under Stein’s influence
(vide infra), he came to the conclusion that this animal “ Vermutlich
gleichfalls nichts Anderes als eine Zrichomonas ist, bei der die Anwesenheit
des Geisselapparates tibersehen wurde ” (2nd Edition, p. 312).

Stein (1878) was inclined to consider the parasite as a Trichomonas of
which the anterior flagella had been overlooked. (‘Der Organismus der
Infusionsthiere, Abtheilung, III, 1. Halfte, p. 79. “Sollte nun das vordere
End, wie ich vermuthe, noch mit zwei Geisseln versehen sein, so wiirde
dieser Parasit, aus dem Leuckart sogleich eine neue Sawnolophus gemacht hat,
unbedingt zur Gatt. Trichomonas gehoren.”)

The next reference to this form that we have found is to be met with in

386 Mr. C. H. Martin and Miss M. Robertson. [June 18,

Saville Kent’s ‘Monograph of the Infusoria’ (1881), where it is named
Trypanosoma ebertht. Saville Kent has apparently overlooked Leuckart and
Stein’s references to this form, and his theory as to its possible connection
with the trypanosoma of the frog need not be considered here.

Biitschli (1889), in his account of the Protozoa, gave Eberth’s figures of this
form and placed it in the genus Trypanosoma with the true blood parasites.

Doflein, in 1901, gave Eberth’s description and figures of this form, which
he seems inclined to connect with some of the flagellate parasites seen in
“ Gefliigel-Diphtherie.” The evidence on which he does this seems rather
scanty and he himself remarks (p. 60): “ Mir scheint aus dem Studium der
Literatur hervorzugehen, dass zwei oder mehr verschiedene Arten in den
Angaben mit einander vermengt worden sind. Es wire sehr verdienstlich
in dieser interessanten Frage Klarheit zu schaffen.”

Laveran and Mesnil (1904), in their ‘Trypanosomes et Trypanosomiases,’
state, on p. 354, “Il parait bien certain que le 7rypanosoma eberthi, Kent, vu
par Eberth dans le tube digestif de la poule, n’est pas un Trypanosome, c’est
sans doute un Trichomonas, comme Stein, puis Leuckart, en ont fait les
premiers la supposition.”

Liihe (1906), in a short note in his account of the blood parasites in
Mense’s ‘ Handbuch der Tropenkrankheiten,’ has changed the name of this
form from Zrypanosoma eberthi to Spirocheta eberthi, owing to the presumed
absence of a free flagellate end to the undulating membrane. We think,
however, that if Liihe had himself seen this form he would not have placed
it among the Spirochetes. It is rather interesting to note that in spite of
the numerous references to this common parasite in later literature, and its
triple change of name up to the present, Eberth appears to have been the
only observer who had actually seen it. :

Methods.

Most of the stained work was done on wet films fixed either with Flemming
or corrosive-acetic. The films were stained either with iron hematoxylin,
hemalaun, Delafield’s hematoxylen, Giemsa, or Twort. The three first all
gave good results.

General Conditions in the Ceeca and Rectum.

During the course of this preliminary work we have examined the cecal
and rectal contents of 14 fowls of various ages and at various stages of
digestion, and have met with the flagellate parasites of four types,
A, B, ©, D.

Before, however, proceeding to the description of these forms, it would be

1909. | On Trypanosoma eberthi (Kent), etc. - 387.

well to consider shortly the varying conditions which these parasites have'
to withstand in the course of digestion. Unfortunately, very little seems to
be known as to the part played by the czca in the metabolism of the bird,
and apparently much the same statement may be made as regards the
conversion of the fluid intestinal contents into the more solid feeces in any
animal. The body temperature of birds is given by Tigerstedt as lying
between 39°4 to 43°°9 C. The temperature of one of our fowls measured in
the axilla was 39°5 C., which is possibly rather low. eh

The condition of the cecal content can vary very greatly; in what we
have termed the normal cecal condition the ceca are full of a light brownish
fluid containing a large number of small gas bubbles. (In one Ivish hen
there was an enormous development of gas, and a strong smell of
sulphuretted hydrogen; the presence of the sulphuretted hydrogen was
possibly correlated with the presence of a rounded plump bacterium found
in very large numbers in this hen.)

The most characteristic feature of the bacterial flora in this stage of
digestion is a very active vibrio which has been met quite high up the
intestine. When the ceca are in this condition, the rectum is frequently:
empty, and if it is full the contents, though hard, are largely made up of
' fibres and husks of corn. In what we term the rectal condition of the cxca,
the czeca are filled with a dark firm mass; the rectum then may be filled with
the same substance, or iu may be empty, in which case the animal has.
probably recently defecated. In the transitional stages between the
normal and rectal conditions the bacterial flora undergoes a marked
change, the vibrio becomes much rarer, and large numbers of cocci and long
slender bacteria are met with. It is evident that in the course of this
change of the nature of the cecal contents, very complicated physical and:
chemical factors may be at work, not only owing to the action of the
walls of the alimentary canal itself but also to that of the bacteria. To
single out only one of these factors, the question of the changes in the
osmotic pressure of the surrounding medium of the parasite becomes a very
important one.

It is a general practice for workers on the intestinal protozoa to carry out
extended observations in a solution of sodium chloride, isotonic with the
blood, and as regards the fowl, Hamburger, in his recent work on
‘Osmotischer Druck und Ionenlehre, gives two determinations : on p. 176
the figures 0°45 to 0'417 per cent. of sodium chloride are mentioned as the
result of plasmolytic methods on blood corpuscles. On p. 458 he gives 0°591
to 0°605 per cent. sodium chloride as a figure arrivedat by the freezing-point
method. We used a tap water solution of 0°7 per cent. NaCl, but. we think,

VOL. LXXXI.—B, 2 F

388 Mr. C. H. Martin and Miss M. Robertson. [June 18,

that if one regards the events in the course of absorption by which the
liquid contents of the cca and intestine are converted into the practically
solid feces, 1t becomes difficult to believe that this solution can at best
represent more than one out of a series of many stages.

The Flagellate Parasites.

The flagellate parasites found in the cecum, as has been stated above, can
be divided into four groups, A, B, C, D, of which we only need here
particularly to consider A, B, and C. D is a sharply marked type, found in
small numbers on two occasions, with an anterior and a posterior trailing
flagellum, which can be coiled round the body. It is of very small size,
roughly half the size of the smallest A form seen, and of approximately
torpedo shape.

The chief difficulty in dealing with the remaining three groups, A, B, C,
is that although large numbers of individuals can be found in which the
characters of each group are sharply marked, yet numerous transitional,
forms are to be seem. Under these circumstances we do not feel inclined,
until we have obtained a great deal more evidence from artificially infected
chicks raised in an incubator, to decide definitely between the two alterna-
tives of considering A, B, C as various forms of a mixed infection or as
stages of a single life cycle. The small amount of evidence we have at
present points clearly to a transition from A to B, and we have observed
some cases of which the most natural explanation would be to regard
A, B, C as stages in one life cycle.

A. Trypanosoma eberthi, Kent. Plate 8, fig. 1—This is the form which
we are inclined to believe Eberth had before him when he wrote his
description of Z’rypanosoma eberthi. It is characterised by a rather elongated
body, a very well marked undulating membrane, along the edge of which a
flagellum runs from the anterior end of the animal to end freely at the
posterior end. The living animal, when seen on a cold stage in salt solution,
is easily picked up by the rippling movement of the undulating membrane.
In the living animal the nucleus can be readily seen as a rather cone-shaped
light area containing some small granules lying in the anterior region of the
animal. Along the base of the membrane a well marked line can be seen,
and in its neighbourhood there is always one and sometimes two rows of
blocks. In a suitably placed animal an axostyle, apparently resembling that
described in Trichomonas, can be seen. In preparations stained with
hemalaun and eosin, the nucleus is readily recognisable, and a fairly large
round body is seen at the anterior end at the origin of the flagellum ; it is

1909. | On Trypanosoma eberthi (Kent), etc. 389

probably the kinetonucleus. The line along the base of the membrane stains
blue, while the blocks and edge of the membrane take up the eosin very
vividly. In hemalaun preparations which have not been stained with eosin,
the blocks and the edge of the membrane may stain blue.

B. The B Form (Trichomonas Condition). Figs. 2 and 3—The B form
may be described as a typical Trichomonas of variable size, apparently
resembling the form described by Wenyon from the mouse. Usually the body
is more massive than in the A form, from which it is easily distinguished
by the presence of three long conspicuous flagella arising from the anterior
end.

In all other points, nucleus, axostyle-line, and blocks, B agrees with A.
In living forms, two types of movement have been observed: one closely
resembles that of A, the undulating membrane being in active motion, while
the anterior flagella trail passively. The second type of movement is
characterised by the activity of the anterior flagella, which strike out in front

of the animal. In the films stained with hemalaun and eosin; the anterior °:

flagella, which are longer than the body, are very conspicuous.

C. C Form (Monocercomonas Condition). Figs. 4 and 5.—This form is
roughly egg-shaped. On the blunt extremity there is a well marked
eytostome, in the neighbourhood of which four long flagella rise. The body .
is usually filled with food vacuoles containing bacteria, and the animal is
quite active even on the cold stage. The nucleus in the unstained forms can
sometimes be seen as a rather highly refractile spherical mass near the
anterior end, and there is no trace of an undulating membrane. In stained
forms there is no trace of the blocks or line; the nucleus is a spherical object
with the chromatin condensed in the membrane, though in some cases there
may be a large internal karyosome.

Possible Transitional Stages.

In addition to these forms, small, rounded, generally motionless forms were
met with in which the typical nucleus and blocks of the A form were seen,
Besides these rounded forms, far more elongated forms were found with a
similar nucleus and the blocks; these showed an indication of an undulating
membrane in the active motion during life. In these elongate forms,a well
marked axostyle was seen. If we now return to the forms A and.B, the
similarity between them in all other points except the absence of the three
anterior flagella in A is so great that it almost amounts to identity. Qn one
occasion we saw a living B torm with an undulating membrane and post riorly
directed flagella at 12.45 p.M.; at 1.25 P.M. a distinct«split | was seen along the
middle of one of the three flagella. It is tempting to suppose that the change

2 2

390 Mr. C. H. Martin and Miss M. Robertson. [June 18,

from A to B consists in a splitting off of a free flagellum from the
undulating membrane, this flagellum splitting again to form the three anterior
flagella of the later stage. This would appear to explain the difference
between the two types of B movement mentioned above, the forms with
posteriorly directed flagella representing the early stages of this transition
from the A to the B form. The differences between B and C are at first
sight very marked, but it is interesting to note that C forms, with the
rudimentary undulating membrane, a line, and a nucleus approximating to the
B type, were met with.

Infection.

Every fowl which we examined was infected by one or other of these
flagellate forms, usually in large numbers, as is shown in the following

table.
Fowl. | Date. | Locality. Condition. Ceecal contents. Rectal contents. Flagellates.
I | 15.4 | Great House, | 2 years old; | Light brown liquid, with gas| Darker; more solid | Spirochete
Abergavenny fat bubbles ; vibrio abundant in small quantity present,
ALB; C:
II | 17.4 |Holley’s Farm,| Old hen | Dark and of an almost earthy | Full of consistent] A, B, D.
Abergavenny consistency ; numerous bac-| matter
teria and cocci
TIT | 19.4 | Limavady, Old hen; | Light brown fluid; numerous | Same as cecum ...... A, B, C.
County Derry| very fat gas bubbles; HS; plump
bacteria numerous
IV | 20.4 |Holley’s Farm, Old hen; | Cecai contents dark and firm | Almost empty......... B, C
Abergavenny fat
Vi} 20.4 Llanfoist Chick; Czxcal contents dark and firm | Ready to defecate ... C.
60 hrs. after
hatching
VI | 20.4 Llanfoist Old hen |. Brown fluid contents ............ Rectum empty ...... A,B
VII | 21.4] Great House,| Old hen; | Firm and dark .................. Recently defecated B, C
Abergavenny | very fat Numerous
cysts
VIII | 24.4 Llanfoist Chick ; Firm and dark ...............+0+ Just about to defe- C.
10 days cate
IX | 24.4} Limavady, Old hen; | Brownish fluid .................. Dark solid mass,| A, B, C
County Derry| very fat largely composed
of husks
XK | 244] Llanfoist Chick ; Brownish fluid ................+ Eimip byzeseesseceeereeeee C.
10 days
XI | 25.4} Limavady, Old hen; | Brownish fluid ...............:.. Dark solid mass,| A, B, C
County Derry| very fat mostly husks

Martin x Robertson.

London Stereosco)

909%] On Trypanosoma eberthi (Kent), etc. 391

Infection is probably only effected by food contaminated by feces con-
taining cysts of the parasites. We propose in the course of the summer
further investigation in connection between these interesting forms upon
artificially infected chicks hatched out in an incubator.

DESCRIPTION OF FIGURES.
[PLate 8.]

The figures are drawn at a magnification of about 4500 diameters. Zeiss 2 mm.
250 mm. tube, and 12 oc.

Fre. 1.—A form (7. eberth2), showing nucleus, undulating membrane, blocks, and axostyle,
In typical A forms the free flagellum is longer.

Fies. 2 and 3.—B forms (Trichomonas), showing three flagella, undulating membrane,
staining line under membrane, axostyle and nucleus.

Fies. 4 and 5.—C form (Monocercomonas), showing four flagella, cytostome, nucleus,
and food vacuoles,

Fie. 6.—Small rounded non-motile A form.

Fic. 7.—A form, small, showing blocks and axostyle and nucleus, also indication of undu-
lating membrane.

Fic. 8.—Form possibly intermediate between B and C.

392

The Possible Ancestors of the Horses liwwing under
Domestication. .

By J. C. Ewart, M.D., F.R.S., University of Edinburgh.”
(Received May 15,—Read June 24, 1909.)
(Abstract.)

During. the later part of the Nineteenth Century it was generally taken
for granted—(1) that “ the seven or eight species of Equide now existing are
all descended from an ancestor of a dun colour more or less striped ”;* (2) that
the common ancestor of the living horses, asses, and zebras was connected
by a single line of descent with the four-toed “fossil” horses of the Eocene
period ; (3) that the domestic horses are descended from a Pleistocene species
characterised by large molars with a long anterior internal pillar, a large,
heavy head, and coarse limbs; (4) that in various parts of Europe and Asia
domestic races increased in size and were improved in make, speed, and
disposition as a result of artificial selection and favourable surroundings.

On the Continent it seems to be still generally assumed that the domestic
breeds have descended from a single species, but in England and America
many naturalists now believe—(1) that domestic horses have sprung from
several wild species, connected by several lines of descent with three-hoofed
species of the Miocene period ; and (2) that while some of the wild ancestors
were adapted for living in the vicinity of forests and upland valleys, others
were adapted for a steppe, plateau, or desert life.

Of possible ancestors of the domestic breeds, the following may be
mentioned :—Lquus sivalensis, EL. stenonis, E. gracilis (Owen’s Asinus fossils),
EL. namadicus, E. fossilis, and HL. robustus. These species mainly differ in the
teeth, size and deflection of the face, and in the bones of the limbs. In the
first three species, the grinding surface of the anterior internal pillar (a fold
of enamel on the inner surface of the cheek teeth) of the premolars and first
molar is short, in the last premolar, pm. 4, it may only be one-third the
length of the crown; in the second three species, the anterior internal
pillar is long—at least half the antero-posterior length of the crown.

One of the ancestral types (Z. robustus) was broad brewed and had a short
face, almost in a line with the cranium; another (Z. sivalensis), also broad

* Darwin, ‘ Animals and Plants,’ vol. 2, p. 17.

+ The latest suggestion is that domestic horses are the descendants of Equus fossilis,

Riitimeyer, a Pleistocene species closely allied to the wild horse of Mongolia—
E. przewalskit.

Possible Ancestors of the Horses ling under Domestication. 393

browed, had a long, tapering, strongly deflected face; a third (£. fossilis) had
a long, narrow face, not so strongly bent downwards as in &. sivalensis; and
a fourth (#. gracilis) had a fine, narrow, but only slightly deflected, face.

In £. gracilis the middle metacarpal (cannon bone) was so slender that the
length was seven and a-half times the width, while in F. robustus the length
of the metacarpal was sometimes only five and a-half times the width.

Of these possible ancestors, the first three occur in Pliocene deposits, the
second three have only hitherto been found in Pleistocene deposits.

Equus sivalensis, of the Siwalik deposits of Northern India, is the oldest
true horse known to science (i.e. the oldest one-hoofed horse with long
(hypsodont) molars), and, as it measured about 15 hands, it is the largest of
the Old World “ fossil” horses. This ancient Siwalik horse was characterised
by long, fairly slender limbs, and a long, tapering face, deflected to form an
angle of nearly 20° with the base of the cranium. In addition to having
a large head, a convex profile, and long limbs, /’. sivalensis seems to have been
characterised by a long neck, high withers, and a tail set on so high that the
root was well in front of the point of the buttock. Nothing is known of the
ancestors of the horse which suddenly made its appearance in Pliocene times
amongst the foot hills of the Himalayas, but it may be safely assumed that
E. sivalensis very decidedly differed from Pliohippus, the small “fossil” horse
of the late Miocene and the early Pliocene deposits of America, from which
some believe all the recent Equide are descended.

It used to be said that #. sivalensis could not be regarded as an ancestor
of domestic horses because of the shortness of the anterior pillar of the cheek
teeth. I find, however, that in some modern horses the anterior pillars are
decidedly shorter than in £#. sivalensis, and that in some of the short-pillared
domestic horses the face is nearly as strongly deflected on the cranium as in
EH. swalensis. There is hence no longer any reason for assuming that this
ancient Indian species had no share in the making of domestic breeds. But
in the absence of a large and representative collection of skulls of domestic
horses it is impossible to say which modern breeds are indebted to the large-
headed, long-limbed race which in Pliocene times frequented the area to the
east of the Jhelum River, now occupied by the Siwalik Hills.

Mr. Lydekker thinks JZ. sivalensis, or some closely allied race, “ may have
been the ancestral stock from which Barbs, Arabs, and Thoroughbreds are
derived.” When more skulls are available for study and when the phases
through which equine skulls pass during development and growth have been
worked out, it will probably be ascertained that broad-browed horses with
a prominent interorbital region—a forehead convex from side to side as well
as from above downwards—and a long, tapering, strongly deflected face have

394 - Dr. J.C. Ewart. Possible Ancestors of the [May 15,

in: great part descended from a species closely allied to ZL. sivalensis, but that
horses with a broad flat forehead, and the face short and nearly in a line
with the cranium, are at the most only remotely related to Z. sivalensis.
Further inquiries will probably also show that some Indian breeds as well
as some of the unimproved races of Central Asia (¢.g., certain long-faced
Kirghiz horses with a sloping forehead and long ears) in many of their points
agree with ZL. sivalensis of the Pliocene deposits of Northern India.

The second possible ancestor mentioned is Hqwus stenonis of the Pliocene
deposits of Europe and North Africa. In a typical specimen of this species,
with the teeth in an intermediate state of wear, all the anterior pillars of the
premolars and molars are shorter than in JZ. swvalensis, while in a specimen
with the teeth well worn the longest pillar may be only one-third the length
of the grinding surface of the crown. At no age are the pillars of the
molars more than half the length of the crown. Whether the face was long
and tapering and strongly deflected in #. stenonis has not yet been
determined, but from the limb bones collected it is evident that the horse
with short-pillared molars, which in Pliocene times frequented the valley of
the Arno, sometimes reached a height of nearly 15 hands.

It is generally supposed #. stenonis either became extinct towards the close
of the Pliocene age or was modified to form varieties with long-pillared
molars. It is conceivable that some of the descendants of £. stenonis
acquired long-pillared molars, but it by no means follows that all the
Pleistocene horses of Europe with the anterior pillars more than half the
length of the crown are related to or derived from . stenonis—some of them
may have been the descendants of H. namadicus. Be this as it may, horses
with teeth of the E. stenonis type existed in the south of Scotland during the
first and second centuries, and horses with short-pillared cheek teeth are
‘still in existence. In some of the skulls from the Roman fort at Newstead
the anterior pillar of the third and fourth premolars only measures 9 mm.,
which is only about half the length of the pillar in #. namadicus and other
“fossil.” Pleiostocene species. Further, in one of the first century Newstead
‘skulls the first premolar is as large as in JL. stenonis, and the face (as broad
and long as in #.-stvalensis) forms an angle of 18° 6’ with the cranium.

In all probability further inquiries will show that the short-pillared
species (with metacarpals as long but somewhat thicker than in Z. swvalensis)
widely distributed over Europe and North Africa in Pliocene times played
an important part in the making of Shires and other heavy modern breeds. -
‘ The only other possible ancestor dealt with in this contribution is the one to
‘which I have given the name Hqwus gracilis. Owen arrived at the conclusion
that Pleistocene horses “ had a larger head than the domesticated races,” and

1909. | Horses living under Domestication. 395

that even in small varieties the teeth were nearly as large as in a modern
cart horse.

Having come to these conclusions, it is not surprising that when it fell to
his lot to describe small equine molars from the drift overlying the London
clay and from a cavernous fissure at Oreston, near Plymouth, he decided that
they could not belong to a true horse and (on the assumption that they
belonged to an extinct ass or zebra) formed for them the species Asinus
Sossilis.

In addition to the small second and third molars described and figured by
Owen, there is in the British Museum a small first molar from Oreston. The
anterior pillars of the second and third Oreston molars are more than half the
length of the crown, as in horses of the “forest” type, but the pillar of the
first molar, m. 1, from Oreston is only about one-third the length of the
crown as in Pliohippus and LE. stenonis.

Except in size, the small teeth from Oreston and other Pleistocene deposits
bear little resemblance to the molars of asses or zebras, but they are
practically identical in enamel foldings as well as in size with the molars of
a smal] (12:2 hands) slender-limbed horse in the possession of the Auxiliaries
who garrisoned the Roman fort at Newstead in the south of Scotland about
the end of the first century.

In addition to small equine teeth, the Devonshire Pleistocene deposits have
yielded a small slender metacarpal. This metacarpal (from Kent’s Cave,
near Torquay), is 220 mm. long and 30°25 mm. wide—the length is hence
7-27 times the width, as in fine-boned Arabs.

As might have been anticipated from a study of the teeth, the Kent's
Cave metacarpal belongs to a very much finer-limbed race than the small
horse of the “elephant” bed at Brighton. On the other hand, the Kent’s
Cave metacarpal very closely agrees with the metacarpals of the small
Newstead horse.

This small first-century horse in teeth and limbs agrees with Exmoor,
Hebridean and other ponies of the Celtic type, ze. with ponies characterised
by a small fine head, large eyes, slender limbs, five lumbar vertebre, and by
the absence of the hind chestnuts and all four ergots.

It hence follows that the small equine of the English Pleistocene (Owen’s
Asinus fossilis), instead of being an ass or a zebra, is a true horse, which in
the metacarpals, as in the “ pillars” of the premolars and first molar, differs
but little from Pliohippus of the late Miocene and early Pliocene American
deposits.

Remains of a small horse with teeth and limbs like Lquus gracilis (Asinus
Jossilis, Owen) have been found in the Pliocene deposits of Italy and France

396 Dr. J. C. Ewart. Possible Ancestors of the {May 15,

and in the Pleistocene deposits of France and North Africa. The Italian
and Auvergne slender-limbed horse has generally been regarded as a small
variety of £. stenonis. By Pomel and other paleontologists the French
variety was known as .Z. ligeris, while the North African variety, named
Equus asinus atlanticus by Thomas, was regarded by M. Boule as closely
allied to, if not the ancestor of, zebras of the Burchell type.

The slender metacarpals from the valley of the Arno and Auvergne so
closely resemble the Kent’s Cave metacarpal, and the teeth from Perrier and
Puy de Dome in France and Lake Karar in Algiers so closely resemble the
small teeth from Oreston, that 2. ligeris and £. asinus atlanticus may be
regarded as varieties or races of #. gracilis.

There are good reasons for believing that #. gracilis varied to form a.
northern and a southern variety. Remains of a slender-limbed northern
race have been found in deposits belonging to the Neolithic, Bronze, and
still later ages in Britain and on the Continent. At the present day the
purest representative of this northern variety is the Celtic pony. Hence
this northern variety may be known as Lqwus gracilis celticus.

Remains of a slender-limbed southern variety have not yet been found
in recent deposits in North Africa, but fine-limbed ponies without ergots and
hind chestnuts are sometimes met with in the south of France, and slender-
limbed horses without hind chestnuts—horses almost certainly of North
African descent—are occasionally met with in the West Indies and Mexico.
In the French, and still more in the wartless ponies of Mexico, the limbs
are longer than in the Celtic ponies, the coat is finer, the mane less full,
and the “taillock,” so well developed in the northern variety, is very small.
As the southern variety in all essential points agrees with Prof. Ridgeway’s
fine bay horse of North Africa (#. caballus libycus), it may be known as
E. gracilis labycus.

Slender limbs and the absence of ergots and hind chestnuts are apparently
as distinctive of members of Z. gracilis as an upright mane and the absence
of hind chestnuts are distinctive of asses and zebras. Hence, when, as a
result of crossing varieties possessing four ergots and four chestnuts, slender-
limbed individuals without ergots and hind chestnuts appear in any area, it
may be assumed that the horses of that area include Z#. gracilis amongst their
ancestors.

From inquiries made and from crossing experiments it has been ascertained
that ponies of the Celtic type occur in the Faroe Islands and Iceland, in the
Western Islands and Highlands of Scotland, in the west of Ireland, in Wales,
Exmoor, and the New Forest, and in Norway and Finland.

Further crossing experiments have made it evident that the yellow-dun

1909. | Horses living under Domestication. 397

fjord horses of Norway are mainly a blend of the Celtic and “forest” types,
that the Shetland ponies, though usually having the conformation of the
“forest” or EH. robustus type, are in’ part of Celtic origin, and that some of
the mouse-dun Tarpans of the Russian steppes are a nearly equal blend of
the Celtic and E. przewalskii types. .

Prof. Ridgeway arrived at the conclusion that in the fine bay horse of
North Africa there is a frequent tendency to stripes on the back, legs,
shoulders, and face, to a blaze on the forehead and to white “ bracelets.”
Experiments made with four types of Arabs and with Russian, Mongolian,
Indian, and Borneo ponies, English, Irish, Iceland, and Norse ponies support
the view that the Pleistocene ancestor of the modern slender-limbed ponies
with short-pillared molars was of a yellow or bay-dun colour with a narrow
dorsal band and bars on the legs, but had neither “ bracelets” nor a blaze.
As stripes are most numerous on broad-browed horses, they have probably in
most cases been inherited from ancestors of the Z. robustus or EH. sivalensis
types.

As to the part played by #. gracilis lbycus in forming domestic breeds,
nothing very definite has been made out: Prof. Ridgeway says all the
improved breeds of the world are a blend in varying degrees of. the bay
horse of North Africa with thick-set, slow, dun and white horses of Europe
and Asia allied to #. przewalsku. A number of hybrids bred at Woburn by
the Duke of Bedford afford little, if any, evidence in support of the view
that Barbs, Arabs, or Thoroughbreds include amongst their ancestors horses
of the Prejvalsky or “ steppe ” type.

Slender-limbed horses with a wide flat forehead and a nearly straight
profile appear to be a blend of Z#. gracilis libycus (Ridgeway’s £. caballus
libycus) and horses of the #. robustus (“forest”) type, while slender-limbed
strains with a fine narrow face, a well set-on tail, and a mane that clings to
the neck, probably most accurately reproduce the variety of #. gracilis which
in prehistoric times inhabited North Africa.

398

Hillhousia mirabilis, a Giant Sulphur Bacterium.
By G. 8. West, M.A., D.Se., F.L.S., and B. M. Grirrirus, B.Sc.

(Communicated by J. Bretland Farmer, F.R.S. Received July 7, 1909.)
[PLATE 9.]

The organism which forms the subject of this paper has been under the
observation of one of us for a number of years, but its true nature was not
immediately recognised. It has been found in various parts of England and
Ireland since 1892, always occurring in very stagnant pools and marshy bogs.

Until quite recently it was only found very sparingly amongst Alge,
Infusoria, ete., generally in situations where there was much decomposing
organic matter. During the past winter, however, the organism has been
found in abundance in the mud of the shallower part of an old pool in
Worcestershire. This has enabled us to make cultures of it, and to make
a study of its general biology and structure. We have named it Hillhousia
mirabilis,*

The cells are solitary but gregarious, occurring freely among other
organisms and numerous small particles of decaying organic matter. They
secrete very little or no mucus, even in a culture, so that colonies are not
built up.

They are for the most part shortly cylindrical, with hemispherical ends,
and are usually straight. Some individuals exhibit a very slight curvature,
and others may be observed which are slightly attenuated towards each
extremity. The cells are from one and three-quarters to three and a-quarter
times as long as broad.

For a unicellular bacterium the dimensions of this organism are
phenomenally large. The diameter of the cell varies from 20 to 33 py, and
averages about 26. The length varies from 42 to 86 pw, and averages
about 60 pu.

In its normal healthy condition the cell is packed with globules of oily
amorphous sulphur of various sizes, the largest of which have a diameter of
about 10 (Plate 9, figs. 1 to 3). These globules are very crowded, and,
when isolated, are seen to be somewhat irregular, although rounded (fig. 17).
Their refractive nature gives the organism almost a black appearance under

* The generic name “ Hillhousia” has been given in honour of Prof. W. Hillhouse, the
retiring Professor of Botany in the University of Birmingham, to whom both authors owe
much in the way of kindly advice and criticism.

Hillhousia mirabilis, a Giant Sulphur Bacterium. 399

the microscope, but a pale yellow or yellow-green colour is quite obvious on
careful inspection by transmitted daylight.

Owing to the large quantity of sulphur they contain, the cells are very
heavy, sinking in the water almost like sand-grains.

The cells are motile, exhibiting slow oscillatory and rolling movements.
The organism is a peritrichous bacterium with several hundred short cilia
disposed all over the exterior of the cell-wall. The cilia can be seen imme-
diately on fixation either with a 5-per-cent. carbolic acid solution or with
a 40-per-cent. formalin solution. The action of these reagents results
in a cessation of the movements of the cilia in from 10 to 20 seconds, during
which period many of them are thrown off and become disintegrated. Some
of these cilia exhibit a contractile or wriggling movement after having been
thrown off, indicating that the contractility of the cilium is not dependent
upon attachment at the base. The cilia are plainly observed by the use of
the above reagents, but only within a few seconds of the moment of fixation
(figs. 15 and 16).

The cilia can also be clearly seen in active movement by examining the
living organism by means of the Zeiss or Leitz dark-ground illuminator. It
is likewise possible to demonstrate the presence of cilia by immersing the
living organisms in a drop of indian ink. The cilia appear as a halo around
the cell, and the minute particles of ink can be seen in rapid motion on the
edge of the halo. This movement is of a different character from the
Brownian movement of the minute particles in the water. The tiny particles
of ink can be seen to be lashed about and driven into small eddies.

Cell-division is relatively slow, and in no observed instance was a single
division completed in less than 24 hours, the time occupied in many cases
being as much as 48 hours.

The Sulphur Globules—The globules of sulphur are enormously larger and
much more densely packed than in any of the other known sulphur bacteria.
They are dissolved by glacial acetic acid,* by boiling in a solution of
magnesium sulphate; and also by prolonged boiling in potassium chlorate.
They turn almost black on boiling in ferrous sulphate ; and a brownish-black
precipitate is found in the cells after boiling in lead acetate. A 23-per-cent.
solution of commercial formalin entirely removes the globules. Carbon
bisulphide penetrates the cell-wall only with difficulty, but after penetration
quickly dissolves the sulphur.

When the living organisms in a pure condition are placed in distilled water

* This was pointed out by Corsini in the case of Beggiatoa ; cf. A. Corsini, “ Ueber die

sogenannter Schwefelkérnchen die man bei des Familie der ‘Beggiatoace’ antrifft,”
‘Centralbl. fiir Bakteriol.,’ II, 1905.

&

400 Dr. G. S..West and Mr. B. M. Griffiths. [July 7,

the sulphur globules disappear in about 48 hours, the sulphur being oxidised
in the process of respiration. The oxidation of the sulphur can best be
demonstrated by allowing a culture to remain undisturbed for several days
in asmall quantity of water containing minute traces of lime. The organisms
gradually lose all their sulphur, and a considerable deposit of granular crystals
of calcium sulphate is formed.

By allowing the bacteria to dry on a slide, and then irrigating with water,
the sulphur is obtained in small rhombic crystals (fig. 18). Crystals can also
be obtained by allowing the formalin solution in which the bacteria have
been fixed to slowly evaporate. These do not appear, however, to be crystals
of pure sulphur.*

From the general behaviour of the sulphur globules, there are many reasons
for supposing that the sulphur is not pure, but exists in some kind of loose
combination, possibly with proteid material. The crystals obtained by drying
the organisms and then irrigating with water are only formed owtside the
cells. After irrigation the colloidal sulphur, or sulphur-compound, passes
through the wall of the cell, without causing the latter any injury, and
crystallisation of the sulphur takes place in the surrounding medium.

Cultwres.—Like other sulphur bacteria, Hil/housia will not grow on gelatine
or agar.t We have obtained the best cultures in tap water containing minute
traces of sulphuretted hydrogen.

Straining the mud through coarse muslin enables one to obtain almost all
the bacteria, mixed only with various small living organisms, finely divided
flocculent organic matter, and small sand-grains. The great weight of the
bacteria can now be utilised to obtain a pure collection, as it is possible to
remove the other organisms by means of a fine pipette, while the large
sulphur bacteria and the small sand-grains remain as a sediment. If this
sediment be now transferred to a watch-glass it is possible to separate the
major portion of the sand-grains by a judicious tapping of the glass while held
in a slightly inclined position. In this way the bacterium can be obtained
mixed only with very minute sand-grains. We have not yet succeeded in
obtaining a culture of the bacterium entirely free from these tiny fragments
of silica, although some method of chemotaxis might possibly solve the

* After several hours’ treatment with 40-per-cent. formalin, the individual bacteria are
found to be encrusted with a thick deposit of radiating crystals. On the addition of
more water to the solution the crystals are dissolved. If weaker formalin is used, the
crystals are never formed, as the substance (some compound of sulphur) is dissolved as
fast as it is produced.

+ Winogradsky, in ‘Ann. de l'Institut Pasteur,’ 1889, pp. 49 and 50, has stated his

inability to obtain cultures of Beggiatoa, Thiothriz, Chromatium, etc., on solid culture,
media.

1909.] Hillhousia mirabilis, a Grant Sulphur Bacterium. 401

difficulty. The culture should now be shaken up at intervals, and very small
quantities of sulphuretted hydrogen water added every few days.

Such a culture will thrive for a time, although the multiplication of the
organism is relatively very slow.

The organism thrives best when the flocculent organic matter, after
straining, is allowed to remain in the water. Under these circumstances the
bacterium remains healthy, and can be kept for a very long time without any
addition of sulphuretted hydrogen water.

Experiments in obtaining pure cultures are still proceeding, and discussion
of this part of the investigation is for the present deferred.

Keeping the organism in the original mud in which it is collected, without
constant change of water, proves unhealthy and ultimately fatal. This is
due to the accumulated excess of sulphuretted hydrogen in the water, which
causes the bacteria to lose their sulphur. The addition of a strong solution
of sulphuretted hydrogen to a culture also causes a solution of the sulphur
and the death of the organisms.

Light is unnecessary for the perfect growth of this bacterium, cultures
thriving as well in complete darkness as in diffuse light.

In mass and by reflected light a culture presents a greyish-white
appearance.

Cytological Structure—Yormalin has been found the most useful fixing
reagent on account of the fact that the sulphur globules are removed at the
same time. A 23-per-cent. solution of commercial formalin will completely
remove the sulpbur in the course of a few hours.*

Individuals fixed in this way show a network of protoplasm which occupies
the interstices between the sulphur globules, the position of the latter being
indicated by the large clear spaces (figs. 5 and 19). Hmbedded in the
protoplasmic network are numerous minute granules of very variable size.
(These are well shown in fig. 19.)

The cell-wall is highly resistant to reagents, but becomes much more
permeable after the organism has been dried. It contains no cellulose, and
stains yellow on the addition of iodine. It dissolves only with difficulty in
sulphuric acid, does not dissolve in an ammoniacal solution of cupric
hydrate, and in many ways it is suggestive of fungus-cellulose. Its great,
resistance to reagents is probably due to the presence of a considerable
proportion of chitin.

On the addition of 5-per-cent. carbolic acid to the living organisms, the
cell-wall swells up and becomes lamellose, indicating that it is not of

* The removal of the sulphur is probably brought about by the small quantity of free
formic acid present in commercial] formalin,

402 Dr. G. 8. West and Mr. B. M. Griffiths. [July 7,

homogeneous structure (figs. 13 and 14). There appear to be several firm
layers, with intervening layers which become somewhat gelatinous on the
addition of carbolic acid. The innermost layer is a firm one, but the outer-
most layer, which can only be demonstrated by special methods, is apparently
gelatinous.*

Nothing of the nature of a definite nucleus is present in the cell, but as the
protoplasmic network includes many conspicuous granules, very careful tests
for nucleins have been made.

Staining has given very indefinite results. Erythrosin and methylene
blue were of little use. Safranin and carbol-fuchsin were found the most
useful stains, and double staining with safranin and gentian-violet gave good
results. Carbol-fuchsin stains the protoplasmic network very well, but in no
instances were the included granules distinctly brought out. Cover-glass
preparations gave much better results than any other method. The granules
of the network do not appear to have an affinity for any of the stains used,
and they cannot be regarded as chromatin granules.

Microchemical tests for nucleo-proteids have been carefully repeated many
times, using cultures of the organism fixed in 24-per-cent. commercial
formalin. As stated before, by this means the sulphur globules are removed
and the fixed protoplasmic network can be experimented upon. Owing
to the impermeability of the cell-wall it was found necessary to allow the
operations to extend over a considerable period.

Treatment with concentrated sodium carbonate removed fully nine-
tenths of the granules, while the network remained clear and refractive
(fig. 21).

A 10-per-cent. salt solution removed a large proportion of the granules,
probably about five-sixths of them, and the network was again left clear
except in the central part of the cell, where there appeared to be a concen-
tration or shrinking together of the protoplasmic strands (figs. 10 and 11).
For this reaction, and also the previous one, the cultures were exposed to the
reagent for rather more than 14 days.

Treatment with dilute potash (5 per cent.) gave a variety of results due
probably to the degree of penetration of the potash in different individuals.
In most cases, after about 10 days, the granules were for the most part
dissolved, and the network to a great extent disorganised (fig. 12).

A number of cultures were treated with acidulated pepsin-glycerin, and
in these cases the network was for the most part digested, whereas the
majority of the granules remained unchanged. Where the network had only

* There is evidence of this even in the active State of the organism, as the cells have
a tendency to stick to the bottom of the vessel’in which the culture is growing.

1909.] Hillhousia mirabilis, a Giant Sulphur Bacterium. 403

partly disappeared, the granules had the appearance of highly refractive beads
strung on fine threads (fig. 20).

The above tests, taken collectively, furnish evidence which goes far to
prove that a considerable proportion of the granules present in the general
protoplasmic network consist of nucleo-proteids.

Lastly, the bacterium was tested for phosphorus. A considerable quantity
of a culture (pure except for very minute grains of silica) was incinerated on
platinum foil, and kept at a red heat for several minutes. The ash was then
treated in small tubes with a reagent consisting of 10 cc. of nitric acid to
1 gramme of ammonium molybdate, and kept at a temperature of 50° C. for
a week, after which period numerous minute crystals of an intense yellow
colour were present in all the slides prepared. These crystals belong to the
eubic system, and there is every reason to regard them as crystals of
ammonium phospho-molybdate. Slides of the reagent only, kept for the same
period at 50° C., showed no trace of such yellow crystals.

From this test we conclude that phosphorus is present in the bacterium,
and therefore that some nucleo-proteid is present.* The previous tests
indicate that this nucleo-proteid is in the form of small granules in the
protoplasmic network, and the staining proves the granules are not particles
of chromatin.

Thus, Hillhousia is a very prumitive unrcell in which chromatin has not been
developed, and the particles of nucleo-proterd (possibly of the nature of linin) are
scattered evenly through the whole protoplasmic network of the cell.

Although the cytological structure of AHillhousia can be studied with
comparative ease, it must not be assumed that other and less easily investi-
gated bacterial cells have a similar structure. The sulphur bacteria may be
of a low type, and it is quite probable that among the various known
groups of the Schizomycetes there are bacteria in which the cytological
structure is of a somewhat higher order.

The present investigation is only of a preliminary character, as much work
yet remains to be done in obtaining pure cultures, and in further working out
the cytology of the organism.

Summary.

Hilihousia mirabilis is a sulphur bacterium of giant proportions, and is

much the largest solitary bacterium which has so far been discovered. Its

average length is about 60 » and breadth about 26 p.
The organism is a peritrichous bacterium with a large number of short

* Galeotti (in ‘ Zeitschr. fiir physiol. Chemie,’ vol. 25, 1898, p. 48) has definitely
demonstrated the occurrence of a nucleo-proteid in certain bacteria.

VOL. LXXXI.—B. DG

404 Hillhousia mirabilis, a Giant Sulphur Bacterium.

cilia. It occurs among decaying organic matter in the mud of shallow fresh-
water pools.

Each individual contains a protoplasmic network in the wide meshes of
- which large globules of sulphur (probably not pure, but in loose combination
with proteid material), are located. The network includes numerous small
granules, a considerable proportion of which consist of some nucleo-proteid.
None of them are chromatin granules.

The cell-wall is firm and has great powers of resistance to reagents. It is

not homogeneous, and 5-per-cent. carbolic acid demonstrates its lamellose
character.

The multiplication of the organism is relatively slow, one division
occupying upwards of 24 hours.

EXPLANATION OF PLATE.

Fics. 1—16, each xX 500; Fics. 17—21, each x 1000.

Fics. 1—3.—Drawings of living specimens of Hillhousia mirabilis to show the dark
refractive sulphur globules practically filling the whole cell.

Fic. 4.—Specimen kept in tap water for one week. The sulphur globules have been
almost entirely used up in respiration.

Fic. 5.—Individual after being in a 24-per-cent. solution of commercial formalin for
several days. The sulphur is completely removed and the protoplasmic net-
work becomes very obvious. Note the numerous granules in the network.

Fie. 6.—Outline of a curved cell. These are rarely observed, the great majority of the
cells being straight.

Fies. 7—9.—Outline of three distinct specimens showing three of the principal stages in
simple cell-fission. In fig. 9 the constriction is almost complete.

Fies. 10, 11.—Two individuals after treatment for 14 days with 10-per-cent. NaCl.
Many of the granules in the network have been removed, and there is a
decided contraction or shrinking of the central parts of the network.

Fie. 12.—Individual after prolonged treatment with 2-per-cent. KOH. The protoplasmic
network is largely disorganised, and a large number of the granules have been
dissolved.

Fies. 13, 14.—Two cells after treatment for about 15 minutes with 5-per-cent. carbolic
acid. The cell-wall exhibits a lamellation, and the sulphur globules have
coalesced into a central irregular mass. ;

Fies. 15, 16.—Two cells immediately on treatment with 40-per-cent. commercial
formalin. The numerous short cilia are very readily observed for a brief
period, and many of them can be seen to be thrown off into the surrounding
liquid.

Fic. 17.—Isolated sulphur globules, showing their irregular form, obtained by crushing
the living cell.

Fic. 18.—Small crystals (rhombic prisms and rhombohedra) of sulphur obtained by
allowing the living organisms to completely dry up. ‘These crystals are
formed outside the organisms, the colloidal sulphur passing through the cell-
wall to the outside after irrigating with water.

coy. Soc. Proc. B., vol. 81, Pi. 9.

West & Griffiths.

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HILLHOUSIA MIRABILIS. WEST & GRIFFITHS.

Development of Trypanosoma gambiense in Glossina palpalis. 405

Fic. 19.—Single specimen after treatment with 24-per-cent. commercial formalin, showing
both the parietal and the more internal portions of the protoplasmic network. —
The granules are shown only in the upper parietal portion of the network.

Fic. 20.—One extremity of an individual after treatment for 14 days with acidulated
pepsin-glycerin. Only the granules of the surface network are represented ;
these stand out very clearly, but the protoplasmic network itself has been for
the most part digested.

Fig. 21.—One extremity of an individual after treatment for 14 days with a concentrated
solution of Na,CO;. Only the surface network is represented. The proto-
plasmic network remains clear and distinct, but most of the granules have
been dissolved.

The Development of Trypanosoma gambiense i Glossina
palpalis.

By Colonel Sir Davin Brucs, C.B., F.R.S., Army Medical Service ; Captain
A. E. Hamerton, D.S.O., and H. R. Bateman, Royal Army Medical
Corps; and Captain F. P. Mackin, Indian Medical Service. (Sleeping
Sickness Commission of the Royal Society, 1908.)

(Received July 5, 1909.)
[Piares 10 anp 11.]

The following experiment is so complete in itself that no apology is offered
for publishing it by itself. In 1903 the Sleeping Sickness Commission of the
Royal Society came to the conclusion that the carrying of infection from
a sleeping sickness patient to a healthy person by the Glossina palpalis was
a mechanical act, and required no previous development of the parasite
within the fly. The Commission also held that the power of transferring
the disease was lost to the fly 48 hours after it had fed on an infected person.

Koch and Stuhlmann, in German East Africa, described developing forms
in Glossina, but did not succeed in infecting healthy animals by the injection
of these forms.

Kleine, in German East Africa, at the end of 1908, succeeded first in
showing that Glossina palpalis could convey Trypanosoma brucei some 50 days
after the fly had fed on an infected animal.

It seems, at first, strange that this fact should have escaped notice for
15 years, and can only be accounted for by assuming that it is an event of
the rarest for a fly to be found which fulfils the unknown conditicns
necessary for the development of the trypanosomes in its interior. If we
assume that it is only one fly in a hundred or in a thousand in which this

VOL, LXXXI—B. 2H

406 Col. Sir D. Bruce and others. Zhe Development of [July 5,

development takes place, then the difficulty of observing the phenomenon can
be understood.
Take the following experiments, for example :—

Table I—Fles caught in an Infected Area, kept for some days, and then
fed on Healthy Animals.

Trypanosoma brucei—Glossina morsitans.

: No. of days before
Expt. Place. Observer. ane a ae ee infection or under Result.
observation.
210 Zululand Bruce 5 _ 82 64 Negative.
242 x : 30 11 56 ‘
232A a 5) 50 15 384 “1

These experiments seemed to show that if flies caught in a highly infected
district, into which a horse could not be taken even for a few hours without
contracting nagana, are kept without food for a few days—say three to five—
they are then incapable of conveying infection. This appeared to be a strong
proof that the duration of infectivity in the fly was a short one, since, if this
were not the case, 1 of the 85 flies ought to have been in a condition capable
of infecting, having, of course, been infected at some previous date in the
“fly country.” It may be repeated, that these flies were caught in a most
highly infected district, so that if Glossina morsitans can remain infective for
50 or 60 days, 1 at least of the 85 ought to have been in the condition which
made it capable of conveying the disease.

This development of the trypanosomes in the fly is strikingly like what
occurs in the test-tube with Novy’s medium. A thousand tubes are inoculated
with Trypanosoma brucei: the trypanosomes all appear to die off, but 20 days
afterwards a peculiarly resistant individual is found in one tube of the
thousand, who has adapted himself to the new enviroment, and soon
multiplies into myriads. What it is which enables this particular individual
to adapt itself to such altered conditions is unknown. It is the merest
speculation to call it a sexual act and pick stout forms as females and slender
forms as males.

Again, because this late development of the trypanosomes enables
a particular fly to remain infective for 100 days, or even possibly for
the remainder of its life, it by no means follows that this is the usual
method of infection. The mechanical transference of the disease is proved
up to the hilt, and for every case which falls a victim to the rare late-infected
fly, a thousand must be infected by direct mechanical transference.

1909. | Trypanosoma gambiense 77 Glossina palpalis. 407

SUMMARY OF THE EXPERIMENT WHICH FORMS THE SUBJECT OF THIS
APER.

Before describing at length the experiment which forms the subject of this
paper, we may summarise it as follows :-—

1. On March 5, 1909, 60 Glossine palpalis caught on the lake shore were
placed in two cages, 30 in each. The flies were fed on two infected monkeys
for 2 days. They were then starved for 72 hours to get rid of mechanical
transference. The following 5 days they were placed on a healthy monkey,
and every successive period of 5 days, or thereabouts, on a fresh monkey, up
to 86 days, when the experiment came to anend. The result was, that the
first two monkeys remained healthy, but that all the following monkeys, up
to 75 days, became infected with 7rypanosoma gambiense.

2. If 7 days be deducted for the incubation period, then the flies first
became infected 18 days after their first feed on an infected animal.

3. There is some evidence that among the 60 flies only 1 was infective.
Fifty-four days after the beginning of the experiment each cage was placed
on a separate monkey. Up to that time both the cages of flies had been fed
on the same animal. Cage A contained, after 54 days, 11 flies. Cage B,
4 flies. Cage A continued to infect monkeys for 21 days more, making
a total of 75 days. Cage B did not infect. Again, as was natural, the flies
gradually died off during the experiment, and as each fly died it was carefully
dissected and examined for trypanosomes. Not a single trypanosome of any
kind whatever was seen in any dissected fly up to 75 days, when a fly died
in Cage A which was found to be swarming with trypanosomes similar to
Trypanosoma gambiense. After the death of this fly, Cage A ceased to be
infective, and when the experiment was stopped the remaining flies were
killed off and dissected, but among them not a sign of a trypanosome could
be seen. In the same way the flies remaining in the non-infective Cage B
were examined, with a similar negative result.

4, Here follows an interesting and unique observation. A tiny drop of
fluid taken from the gut of the 75-day fly injected under the skin of a
monkey gave rise to Sleeping Sickness after an incubation period of eight
days. This, so far as we are aware, is the first time this has been recorded.

5. It will be seen from the detailed experiment that the flies were starved
for three days between several of the experiments. This, of course, was to
get rid of the fallacy of mechanical transference.

6. It may be said that perhaps these monkeys became infected by some
other means than the flies in the cage—for example, by other biting flies, or
by contact. To this it may be answered that there are more than 200

7 isl

408 Col. Sir D. Bruce and others. The Development of [July 5,

monkeys under observation here, sick and healthy. They are all examined
twice a week, but during the last eight months not a single case of accidental
infection has taken place.

DETAILS OF THE EXPERIMENT.
Experiment 663.

To ascertain if development of Trypanosoma gambiense takes place in the
interior of Glossina palpalis, and if so, how long does the fly remain infective.

March 5, 1909.—Two batches of Glossina palpalis caught on the Lake
shore, consisting of 30 flies in each batch, were fed on monkeys,
Experiments 568 and 214, whose blood contained numbers of Trypanosoma
gambiense.

March 6.—The flies again fed as on the 5th, to ensure that as many as
possible should get a feed of the infected blood. Nearly all the flies fed on
one or other occasion. The flies are kept in a moist atmosphere at 22° C.

The following table gives the principal details of the experiment :—

Table II.
Day of Result.
Date. experi- Procedure. Remarks,
ment. Positive. | Negative.
1909.
Mar. 5 — Flies fed on infected monkey ;

6 1 ” ” » ye

7 2 Flies starved 72 hours

8 3 ” ”

9 4)
10 5
itil 6 Fed on Monkey 579 ............ —
12 7
13 8
14, 9
15 10
16 11 ‘ mod GET ee AT RERS =
17 12
18 13)
19 14)
20 15
21 16 l if ba INES D tare +
22 17
23 14)
24. 19
25 20 |
26 21 ¢ 5 Pe) Ue e
27 22 |

28 23 J

29 | 24

30 25

31 26 b ‘ Lion te GBM clea tetoagr a

vACOT el 27 |
2 28 J

1909.]

Trypanosoma gambiense in Glossina palpalis.

409

Procedure.

Result.

Positive. | Negative.

Remarks.

Fed on Monkey 655

722

” 3)

Starved for 72 hours

Fed on Monkey 727

Starved for 76 hours

(oe A fed on Monkey 735 ..

| ” B ”

Starved for 74 hours

(Cage A fed on Monkey 749...
|

|

eee ”

Starved for 72 hours

(Cage A fed on Monkey 765...

L ” B » ”
Starved for 72 hours

Cage A fed on Monkey 848...

Starved for 72 hours

Cage A fed on Monkey 911...

Experiment stopped.

ses eeensices

(elias

» 148...

764...

May 13.—Flies
remaining in
Cage B killed
and dissected,

| May 19.— Fly
| 866 found dead
in Cage A and
dissected. Did
not feed on
Monkey 848.

Expts. 848 and
911 healthy on
7th June, 1909.

Remaining flies
killed and dis-
sected.

= ee ed ee ee ee eee

410 Col. Sir D. Bruce and others. The Development of [July 5,

Remarks on the Experiment.

Everyone will agree that this is a most interesting experiment. It is
evident that a single infected fly did all the mischief, and by good luck this
fly was detected. Captain A. E. Hamerton, D.S.0., had charge of the
experiment at first, and on his leaving Mpumu about the beginning of May,
it fell to Sergeant A. Gibbons, Royal Army Medical Corps. Both are
to be congratulated on the results, which are the outcome of care and
thoroughness. Captain F. P. Mackie had the good fortune to dissect the fly
which did the injury, and which will be fully described later.

INCUBATION PERIOD.

From the experiment may be drawn the incubation period in monkeys
bitten by a late-infected fly.

It is remarkable how regular this is in those monkeys which gave a
positive result. This shows how very infective Fly 866 was. Apparently
each time it bit it infected.

The following table gives the period of incubation in each case :—

Table III.
| Trypanosomes Number of days
| Date. Experiment. | Flies first fed. appeared in before trypanosomes
blood. appeared in blood.
|
1909. 1909. 1909.
March 19 652 March 19 March 30 11
59 owes 653 4 eed April 2 9
5 (oe 654 oe 29 eG 8
April 3 655 April 3 x ls} 10
cS 672 aie 8s 7
ans 722 tes 20 7
18 7217 as » 24 6
28 735 2S May 5 ui
May 5 749 May 5 ee wilal 6
Re Ae 765 Rien ey 5

Leaving out the first experiment, 652, as it is doubtful as to the exact day
Fly 866 became infective, this gives an average incubation period of seven
days. It would therefore appear that Fly 866 probably infected each
animal on the first day it bit it, showing how dangerous such an infected
fly is.

1909. | Trypanosoma gambiense 1 Glossina palpalis. aes

DESCRIPTION OF THE Glossina palpalis, Fuy 866, WHICH WAS DISSECTED
75 DAYS AFTER HAVING FED ON A MONKEY WHOSE BLOOD CONTAINED
Trypanosoma ganbiense.

Experiment 866.

May 19, 1909.—Dissected a Glossina palpalis, which was found dead to-day
in Cage A of Experiment 663. On removing the viscera by the usual
method, the mid-gut was seen to be of a pale salmon-pink. A small
quantity of its contents, examined in the fresh condition, was found
to contain enormous numbers of trypanosomes. ‘The tube of this part of the
intestine was absolutely crammed with active, seething masses of these
flagellates. In regard to the other parts of the fly, nothing was seen in the
proboscis. In the proventriculus one trypanosome only was found. The
salivary glands contained large numbers of altered-looking trypanosomes,
the fore-gut many large stout forms, with bright granules. The crop was
empty and showed nothing. The Malpighian tubules, hind-gut, and
proctodzeum also were drawn blank.

In addition to examining these organs in the fresh condition, smears were
made and stained. The examination of these stained specimens gave the
following results :—

The salivary glands.—These had been carefully removed before the intestine
was opened, and therefore had no chance of being fouled. As will be seen from
the coloured drawing (Plate 10, fig. 1), the trypanosomes found in these glands
differed from those seen in the-intestine. The bodies are very irregular in
shape, and contain, besides a reddish-stained nucleus, dark deeply-stained coarse
chromatin granules. The other cell contents remain unstained. Free
chromatin granules and flagella are to be seen scattered over the field.
Sometimes the bodies are definitely pear-shaped, with a flagellum coming
from the narrow end, and rarely a more definite trypanosome shape can b
seen; but never a true trypanosome.

[It is a matter of deep regret that an inoculation experiment was not made
with an emulsion of part of the salivary glands. ]

The fore-gut.—The fore-gut contained many trypanosomes. The cytoplasm
stains a pale blue, and the nucleus a reddish-purple. The micronucleus is
not distinctly seen in some of the trypanosomes, but when it is, it is
always distinctly posterior to the nucleus. The protoplasm contains many
coarse darkly-stained chromatin granules. The undulating membrane is
less marked than in the normal blood trypanosome, and the flagellum, which
usually springs from a micronucleus-like body, is less deeply stained
(Plate 11, figs. 6—13).

412 Col. Sir D. Bruce and others. The Development of [July 5,

The mid-gut.—The mid-gut contained innumerable trypanosomes of the
gambiense type. Some are dividing, and all have a well-marked nucleus and
micronucleus, the latter at or near the posterior extremity. The protoplasm
contains many chromatin granules, and an undulating membrane and
flagellum are present (Plate 10, figs. 6—16). Many groups, or rosettes,
composed of 15 to 20 individuals, occur, the flagella pointing outwards
(Plate 11, fig. 1). .

The proboscis, proventriculus, thoracic gut, crop, hind-gut,and Malpighian tubes
contained no trypanosomes.

The most interesting thing in this description of the examination of
Fly 866 is the condition of the salivary glands. How these trypanosome-
like bodies, or derivatives of trypanosomes, got into them is a mystery, and
we will content ourselves at present with merely placing the bare fact on
record until the salivary glands of similarly infected flies are examined.

There is one fallacy which might be pointed out. It is assumed that
Fly 866 became infected on the first or second day of the experiment. It is
possible that it became infected when feeding on the fifth day on an animal
which showed trypanosomes in its blood a day or two later. This, however,
is unlikely, as no other fly showed trypanosomes on dissection.

In order to make the story more complete, on Plate 10, figs. 1—4, is repre-
sented the 7rypanosoma gambiense from the blood of one of the monkeys on
which the flies were fed at the beginning of the experiment, and on Plate 11,
figs. 2—5, are shown Trypanosoma gambiense from the monkey which became
infected from the contents of the mid-gut of Fly 866.

PrRoporTION OF INFECTED FLIES TO NON-INFECTED IN NATURE.

In the experiment under consideration it is seen that, in artificially-
infected flies, only 1 in 60 showed the phenomenon of late infectivity. In
nature the proportion must be less, as many of the flies, in many places at
least, can never have fed on an animal whose blood contained Zrypanosoma
gambiense.

That there can be but few under natural conditions Table IV shows. The
table is made by subtracting the flies fed on the animal during the last
seven days, before trypanosomes were found in the blood, this being the
incubation period, from the total number. The experiments consist in
catching tsetse flies in the infected area, bringing them to the laboratory and
placing them straightway on healthy animals.

The first two experiments were made with Zrypanosoma brucei and
Glossina morsitans, and it would appear from them that 104 and 108 flies

1909.

Trypanosoma gambiense 77 Glossina palpalis.

413

Table [V.—Table to show Probable Number of Naturally infected Flies

per thousand.

|
No. of flies Result.

Probable No.
fed before of naturall
BEDE Pikes. ‘DIDS infection infected flies
took place. | Positive. | Negative. | per thousand.
Trypanosoma brucec—Glossina morsitans.
225 Zululand Bruce 104 | + 9°6
236 i 35 108 + | 9-2
Trypanosoma gambiense—Glossina palpalis,
94 Uganda Bruce and Nabarro 89 + 11:2
130 pe Bruce, Nabarro, and 850 + 1:2
Greig
131 mY 506 + 19
136 3 Nabarro and Greig 723
228 ‘5 Greig and Gray 866 + 1:2
301 i Ps i 2299
45 Leopoldville | Dutton, Todd, and 457
: Hannington
46 » » ” 552
1284 River \ 3 25
139 3 9 ” 262
141 z ‘ 4 52
182 Kasongo in a 211
198 , ss si 2659 fe 04
203 be bby 2) 1789
213 ” ” ” 717
52 Uganda Bruce, Hamerton, 41
Bateman, and
Mackie
214 i ‘A 3284. + 0°3
568 ” ” ” 178 ab 5°6
571 ” ” 6 850 + 1-2
53% 2 ? ” 21
612 ” ” ” 615 a0 1°6
674 » er op 2315 + 04
7

were used respectively before an infective

* Animal died.

explains why Bruce’s 85 flies failed to infect.
In the experiments with Zrypanosoma gambiense and Glossina palpalis the

one was found. This perhaps

average is 2'0 per thousand. It is, of course, impossible to tell how many

of these positive experiments were infected by mechanical transference or by

a late-infective fly ; but, in any case, the proportion is small.

If this were

not so, all the native population of the Lake shore, and most of the Europeans
in Uganda, would long ago have been blotted out.

414 Col, Sir D. Bruce and others. On a [July 5,

DESCRIPTION OF PLATES.

Puate 10.

Smear preparation of salivary glands of Glossina palpalis, Experiment 866, stained
Giemsa, showing irregularly shaped trypanosomes, with unstained protoplasm, reddish-
coloured nuclei, and deeply stained chromatin granules. Note the chromatin granules
scattered singly about the field, each surrounded by a pale area, fig. 1. x 2000.

Normal 7rypanosoma gambiense from monkey, Experiment 568, on which the flies were
fed at the beginning of the experiment, figs. 2, 3, 4, and 5. x 2000.

Trypanosomes from the mid-gut of infected fly, Experiment 866, figs. 6—16. x 2000.

PLATE 11.

Rosette form from the mid-gut, fig. 1. x 2000.

Trypanosoma gambiense from the blood of monkey, Experiment 868, into which a tiny
drop of the contents of the mid-gut of Fly 866 had been injected, figs. 2—5. x 2000.

Trypanosomes from the fore-gut of Fly 866, stained Giemsa, figs. 6—13. x 2000.

A Note on the Occurrence of a Trypanosome in the African
Elephant.

By Colonel Sir Davin Brucz, C.B., F.R.S., Army Medical Service ; Captains
A. E. Hamerron, D.S.O., and H. R. Bareman, Royal Army Medical
Corps; and Captain F. P. Macxig, Indian Medical Service. (Sleeping
Sickness Commission of the Royal Society, 1908.)

(Received July 5, 1909.)

[Puate 12.]

As trypanosomes have never been reported as having been observed in
the blood of the African Elephant, the Commission thought it would be of
interest to note this observation.

In Laveran and Mesnil’s book on trypanosomes, translated by Nabarro,
on p. 261 it is stated that “the occurrence of Surra (Zrypanosoma evans) in
elephants in India and Burmah is practically proved. In this connection we
have only the statement of G. H. Evans that, in 1893, 14 out of
32 elephants died of the disease in Burmah.” The year 1893 is almost
prehistoric for trypanosomes. At that time observers had even failed
to distinguish between the common rat trypanosome—TZrypanosoma lewisi—
and that of Surra. It may well be, then, that Evans was mistaken in his
diagnosis of the species causing this large mortality in elephants.

The African elephant, in whose blood this trypanosome was found, was

4 Bruce, &c. — toy. Soc. Proc. B. Vol. 81, Plate 10.

) M.E. Bruce del. West,Newman chr.

| Bruce, &c.

M.E. Bruce del.

10

Roy.Soc. Proc. B. Vol. 61, Plate 11.

Fearn IT : cee % .
ca pea ay 8 gee 42 6 °@
22 © Gee: oe —o 5 ‘© ©

West, Newman chr.

/
i
| qi
||
|
i
|
) fi

1909. | Trypanosome in the African Elephant. 415

shot by Mr. L. C. Lea-Wilson, of the Uganda Company, Limited, at a spot
two miles from the eastern shore of Lake Albert, near Ngogole, about
31° 10’ E. lat. and 1° 30’ N. long. It is to be regretted that none of the
blood was injected into a dog, donkey, or ox, in order that a fuller study of
this trypanosome might have been made. As it is, all the material
available are a couple of smears made by Mr. Lea-Wilson and sent to the
Commission.

Morphology of the Trypanosome of the Elephant.

Method of Fixing and Staining.—The two slides received from Mr. Lea-
Wilson were fixed in osmiec acid vapour and alcohol, stained in Giemsa, and
decolorised in orange tannin.*

Length.—F¥or method of measurement see the same paper, p.16. As will be
seen from the coloured plate, which was drawn by Sergeant Gibbons,
R.A.M.C., this trypanosome is of medium size. The average length of
18 individuals is 18°5 microns: maximum 21, minimum 15.

Breadth—On an average the breadth at the thickest part is 3 microns.

Shape——tThis trypanosome is of the Trypanosoma brucet type, inasmuch as
it has a well-developed undulatory membrane and free flagellum. As will
be seen from the drawing (Plate 12), one noteworthy feature it has is the
uniformity in size and shape of the different individuals. The posterior end
is blunt, or conical, reminding one somewhat of the head of a seal, with the
bulging micronucleus for an eye. The body thickens as far as the middle,
when it gradually tapers away to the anterior end.

Contents of Cell—The protoplasm is clear and particularly free from
granules.

Nucleus—The nucleus is compact and sharply defined from the neigh-
bouring protoplasm. In shape it is round, or oval, and often lies nearer the
anterior extremity than the posterior. Its length averages 2 microns.

Micronucleus.—The micronucleus is small, round and distinct. It is
situated close to the posterior extremity, and often appears to bulge above
the surface.

Undulating Membrane.—The undulating membrane is well developed and
thrown into well-marked folds.

Flagellum.—tThe flagellum stains deeply. It runs from the micronucleus
along the edge of the undulating membrane, beyond which it projects
as a free flagellum for some 5 or 6 microns.

* Vide ‘ Roy. Soc. Proc.,’ Series B, vol. 81, p. 16.

416 On a Trypanosome mm the African Elephant.

Conclusions.

In our present state of knowledge it seems impossible to name trypano-
somes from their form alone. We were, however, much surprised, a short
time ago, by Sir John McFadyean separating with ease Trypanosoma brucer
from Trypanosoma evansi. If this can be done for such closely related
species, surely it should be possible to do it for all. To assist to this end it
would be well if observers would adopt one method of fixing, staining, and
measuring. In the ‘Third Report of the Wellcome Research Laboratories,
Khartoum, facing p. 30, there is a coloured plate of trypanosomes, stated to
have a magnification of 1000. On measuring one of them it is found to have
a magnification of between 2000 and 3000. Then, again, many of the
trypanosomes depicted are dividing forms, which is misleading.

The method of measuring must also make a difference. For example, in
Laveran and Mesnil’s book the length of Trypanosoma brucei in the rat is
given as 26 to 27 microns, whereas by our method of measuring the average
length of 20 individuals is 22°8 microns: maximum 25, minimum 20,

The trypanosome of the elephant has an average length of 18°5 microns:
maximum 21, minimum 15, a well-developed undulatory membrane and free
flagellum. The trypanosomes with free flagella are Trypanosoma brucei,
cazalbour, evansi, gambiense, pecaudi, and soudanense. It probably is neither
Trypanosoma cazalbowi nor pecaudi, on account of its well-developed
undulating membrane and uniform size. Under the circumstances it is
impossible to decide as to its identity with Zrypanosoma brucei, gambiense, or
soudanense, but if a guess were hazarded then it would be Trypanosoma
soudanense.

Until the nature of this species is better known we propose to name it
Trypanosoma elephants.

Foy. Soc. Proc. B.Vol. 81. Plate 12.

West,Newman chr.

417

The Ferments and Latent Infe of Resting Seeds.

By JEAN WHITE, M.Sc., Victorian Government Research Scholar.

(Communicated by D. H. Scott, F.R.S. Received March 3,—Read
March 18, 1909.)

This subject was suggested to me by Prof. Ewart as an outcome of his long
series of experiments on the longevity of seeds.* For the most part, I have
confined my attention solely to the seeds of cereals as being of the greatest
importance in agriculture.t

The scheme proposed for the carrying out of these investigations
necessitated the procuring of old grains; this was not an easy matter,
but after several months I was very fortunate in having seeds forwarded
from the Agricultural Department, Victoria; Dookie Agricultural College,
Victoria; the Chamber of Commerce, South Australia; and the Hawkesbury
Agricultural College, New South Wales. For these I am much indebted to
Messrs. J. Knight, H. Pye, and W. Potts, whom I now take the opportunity
of thanking for the trouble they have taken.

Many of the seeds obtained from the Agricultural Department of Victoria
were travelling samples, and had been in the possession of the department
for from 8 to 10 years, but whether they had been harvested the same
season or the previous one to that in which they were put up is not exactly
known. The greater number of the specimens sent from South Australia
and New South Wales were accompanied by information as to the exact
date of their harvesting.

The oldest grains available were samples of wheat received from South
Australia which had been stored for 21 years.

The old specimens of barley, oats, and rye were amongst those previously
referred to which were obtained from the Agricultural Department, Victoria,
and whose minimum age must be at least 8 to 10 years. In all the above
cases, certain of the different samples of grains had completely lost all their
power of germination, so that they were exactly what I required in order to
be enabled to carry out one section of the work. I was not quite so fortunate
as reyards the maize, for the oldest seeds in my possession were grown only
41 years ago, and had only partially lost their germinating power.

* ‘Proceedings of the Royal Society of Victoria,’ vol. 21 (New Series), August, 1908.

+ The whole of the work in the following paper has been carried out in the Botanical
Laboratory of Melbourne University under Prof. Ewart’s supervision, who has also
critically tested and verified certain experiments and written the summary at the end of

the paper. The expenses of the work were defrayed from the Research Scholarship and
Apparatus Fund of the Victorian Government.

418 Miss J. White. The Ferments and [Mar. 3,

The paper is divided into sections, the first of which relates to the relative
germinating powers of grains of various ages, and as far as possible obtained
from different sources.

The second section deals with the connection (if any exists) between the
age of the seeds and the persistence of their enzymes, with special reference
to any possible co-relation between the germinating power retained by the
stored grains and their enzymes.

The third section is a detailed account of experiments performed on the
seeds at temperature extremes, more especially concerning their germinative
capability and their enzyme reactions, as in the previous section.

The fourth section is a brief account of experiments concerning the
respiratory activity of certain seeds in a more or less dried condition and the
results obtained therefrom.

1. Germinating Power of Seeds.

The germination capacity of all the specimens received was tested, and
the data so obtained are shown in the columns below. From 50 to 100 seeds
from each packet (the number depending on the quantity of material at my
disposal) were sown on damp blotting paper placed in glass basins, which
were put under a glass frame in the conservatory to which air had free
access. The temperature of the conservatory was kept fairly constant at
about: 23° C.

The comparative germinating capabilities are given in the following

table (A) :—

Germination Table A. (Wheat.)

Age. RES Place of origin. Age. ae Place of origin.
6 months 100 S. Australia. 83 years 32 S. Australia.
Giese: 100 Victoria. Ssaey 3 Victoria.
} years 100 S. Australia. op 32 S. Australia.
aes 100 Victoria. See gn (0) Victoria.
2t 5; 100 8. Australia. 103 ,, 28 8. Australia.
ate 100 Victoria. WOe (6) Victoria.
Gyan an 100 S. Australia. Hy 12 S. Australia.
4h, 100 ee ine oy 0 Victoria.
a es 92 Victoria. We op 4 8. Australia.
ue 90 New South Wales. Use 35 0 oo
64 ,, 77 S. Australia. 15% ,, 0 5
Goes 42 Victoria. Ue oy 2 3
63s, 39 New South Wales. || 17, 18, 19,
Tex. oy 68 S. Australia. 20, and 21 i 0) .
(eas 16 Victoria. years

1909. | Latent Infe of Resting Seeds. 419

Germination Table B. (Barley, Oats, Maize, and Rye.)

: Percentage Bit
Kind of seed. | Age. | Beane Place of origin.
IIE? cooncotboescdcucoucec | 1% years 100 | Victoria.
giles ee 100 eee
Fe |. BeAOe SE CORERECCEOOCOn een 72 | a
pad Renpecaeatocar paererce 4b, 54 New South Wales.
ape OL (anseooey nee eeeeceee cae 18 | Victoria.
Lconneecnocdconeetere arr Or ans ) fe
OTE, saeco ete ee eae 96 init es
Bayi ccetesesccssdatce-lensee: ee 80 sy
rf} eon ced bouassebapsEBEoecG| 4y yy 68 -
eA) peodenccaseasoacuapodoce = 35 = ap
eg athna eral o 9 saialnce steele op 3
Aare ane 6 months 100 +
5, | “odeoHBeeeroassoDInOse | Al years 60 New South Wales.
TRI® ccocenecceesnvoneusecore 6 months 100 Victoria.
wp SnasagoEDneonscecanctiece 45 years 32 5
Fatih Sgoobodcaocaneedaacbcdoas | GE rg 0 | x
I

In those .cases in which different samples of Victorian seeds of the same
age were experimented with, the average number which germinated is given
in the tables. The majority of the seeds were sent in cotton bags, and the
method in which they had been stored was not stated. In the case of
wheat, however, taking for granted similar conditions of storage, the fact
that the South Australian specimens retained their germination capacity
for a longer period than the Victorian specimens, and the Victorian
specimens retained it longer than those from New South Wales, shows that
the drier the climate the longer is the life of the seed. On further reference
to the wheat table, it appears that there is a well-marked drop in the
germinating power of the grains after about the fourth year, and from
thence it descends more or less irregularly, reaching zero in 11 to 17 years,
according to the character of the sample and the conditions of storage.

2. The Relation between the Longevity of Seeds and their Contained Ferments.

The investigations were carried out with seeds freshly harvested and with
stored seeds which had lost the faculty of germination.

The object of these investigations was to determine, as has been previously
stated from time to time by different authorities, whether the loss of the
germinating power was concomitant with, and caused by, or in any way
related to, the disappearance of the capability of enzyme action in the seed.

The seeds used were wheat (Zriticwm vulgare), barley (Hordeum sativum),
oats (Avena sativa), maize (Zea mais), and rye (Secale cereale).

In every instance, except in the maize, seeds were employed in which the

420 Miss J. White. The Ferments and [ Mar. 3,

germination capacity was entirely lost, but of the oldest specimens of maize
obtainable 60 per cent. of the seeds sowed germinated.

Samples of the same seeds which had been tested for loss of germinating
power as described in the preceding section were first tested for the presence
of diastase ferments.

1. Diastase—The method adopted for the precipitation of the diastase was.
the same as that described by Darwin and Acton in the ‘ Physiology of
Plants, p. 305. Ten grammes of the seed were ground to a fine powder in
a hand coffee mill, and placed in a bottle containing 100 c.c. of slightly warm
distilled water, which was shaken for two hours with the aid of a water-
motor rocker.

The mixture was then filtered and the filtrate concentrated at 50° C. under
decreased pressure to about half its original volume. To the filtrate was.
added enough 90-per-cent. alcohol to produce a white flocculent precipitate.
This precipitate was separated by filtration, and the filter-paper containing
the precipitate was placed in an exhausted desiccator over sulphuric acid
during the night. When dry, the precipitate was scraped off the filter-paper
by means of a sterilised knife, and dissolved in a small volume of cold, boiled
water.

Equal quantities of this aqueous solution were put into each of three
test-tubes which had been previously sterilised, and the contents of one
tube C were thoroughly boiled. A very thin starch solution was prepared,
and, when cool, equal quantities of the starch solution were added to each of
the test-tubes B and C and also toa fourth test-tube D. Of the four test-tubes
A, B, C, D, A contained only the original aqueous solution of the precipitate.
B contained about equal quantities of the aqueous solution of the precipitate
and starch solution. C contained the same as B, with the exception that the
aqueous solution of the precipitate had been boiled and allowed to cool
before the addition of the starch solution. D contained only starch solution.
The four test-tubes were placed in a bath at a constant temperature of 50° C.
After about one or one and a-half hours the test-tubes were removed from
the bath, and the contents of each were tested for the presence of reducing
sugar by means of Fehling’s test. All through the experiments only 1 drop of
No. I Fehling was added to each test-tube and 2 drops of No. II. In every
instance, as may be observed from the detailed lists of experiments given
below, the presence of reducing sugar was detected in the contents of the
test-tube B, while no sign of reduction was noted in the contents of any.of
the other test-tubes.

The results of these experiments, which were performed with many varied:
specimens obtained from widely different sources, denote conclusively the

1909. | Latent Lrfe of Resting Seeds. 421

presence of a more or less considerable quantity of a diastase ferment in the
precipitate obtained from the extract of the crushed resting seeds. This
ferment was present as such, and not as a zymogen in the resting seed, and
was destroyed by boiling, as shown by the experiments performed with the
test-tubes C. ;

The experiments were performed as far as possible in pairs, in one of which
freshly harvested grain, and in the other old grain of the same kind, whose
germinating power was lost, were tested.

It was impossible to carry out any additional control experiments with the
aid of commercial diastase, for this was found in all cases to contain reducing
sugar, and it was not found possible with the means at hand to prepare
sufficient quantities of pure diastase from the material available.

It has been stated that the artificial addition of commercial diastase to
ungerminable or feebly germinable seeds may bring about or increase their
germination,* and the idea that ferments are connected with the vitality of
seeds is a fairly prevalent one.

In order to test this statement, samples of such seeds were sown on damp
blotting paper, and a little dissolved commercial diastase was added to them.
Similar numbers from the same packets of seeds were sown on damp blotting
paper and, instead of dissolved diastase, a little plain water was poured over
them. In addition, two similar sets of seeds were sown as above, but the
seed coat of each seed, both in those treated with dissolved diastase and with
water alone, was pierced once with a needle. In this way the entry of the
ferment was assured even in the presence of a more or less impermeable
integument.

Not all the seeds which were tested for the presence of diastase were thus
treated, chiefly owing to the lmited quantity of the available material, but
sufficient experiments were performed with each kind of seed to ensure
accuracy of results.

Effect of Addition of Diastase—The addition of the dissolved diastase to
intact seeds does not materially affect their germinating power, and in no
case does it bring about germination in otherwise non-germinable seeds. The
presence of external diastase aids the development of bacteria and interferes
with the aeration of the seeds, which may be sufficient in some cases to

* Thompson, ‘Garten-Flora, vol. 45, p. 344, 1896; Waugh, ‘Ann. Rept. Vermont
Agric. Exp. St.,’ 1896—7 ; ‘Science,’ N.S., vol. 6, p. 950, 1897; Sharpe, ‘Mass. Hatch
Exp. St.,’ 1901, p. 74.

+ Brnyning, Jr., F. F., “ Relation entre le pouvoir germinative et V’activité diastatique
de graines non-germées” ; Albo, G., ‘Bull. della Soc. Bot. Ital.’ 1908; ‘ Archiv des Sci.
phys. et nat.,’ vol. 25, p. 45.

VOL. LXXXI.—B. 2 7

422 Miss J. White. The Ferments and [ Mar. 3,

produce a slight lowering of the percentage germination by preventing the
germination of feebly germinable seeds.

The effect of pricking the seeds is apparently injurious, for, as a general
rule, less of the pricked seeds developed than of the unpricked ones. In
addition, samples of all these seeds were tested for the presence of diastase
while in the resting condition, 5 to 20 grammes being used for extraction
according to Darwin and Acton’s method. The precipitate was allowed to
act on starch solution for one hour, and then tested for reducing sugar.

Considering the case of the wheat first, as set out in the following tables, it
will be observed that while age materially affects the germinating power of
the seeds, it does not apparently materially influence the quantity of diastase
enzyme present in the seed, nor its activity, at any rate up to an age of
20 years.*

In every instance, strong reduction was produced by Fehline’s test, and the
variations in the degree of reduction noted in the tables were extremely
slight, and might be due to differences between the samples when originally
harvested.

The different samples of barley gave closely similar results, and the same
applies to the oats, rye, and maize.

The oats exhibited the greatest amount of variation, and though in all
cases a distinct reduction was obtained on testing for diastase, the amount
present in some of the resting seeds was small. Shght differences in the
amount of reduction are in part produced by extraneous factors, such as the
strength and quantity of the solutions used, the fineness to which the seeds
are ground, and the detailed treatment during extraction. These results do
not coincide with those obtained by Acton,+ who found that an extract from
wheat grains which had been stacked for 28 years exercised no diastatic¢
action on thin starch solution. He offers the suggestion that the diastase
present in the freshly stored grains had been destroyed by oxidation, or by
the influence of micro-organisms. Thus reference to the two first cases of
oats cited in the tables shows that 10 grammes of fresh oats, with .a
germination capacity of 100 per cent., produced exactly the same reduction
on testing as was produced by 10 grammes of oats from 8 to 10 years old,
with a germination capacity of ni/, under as precisely similar conditions as
possible.

The least reduction was produced by maize, the strength of reduction in

* Brocq-Rousseau and Gain (‘Compt. Rend. Acad. Sci. Paris,’ vol. 146, 1908, p. 545).
state that peroxidase enzymes appear in seeds up to 20 years of age and may persist in
some cases for 100 to 200 years.

+ ‘Annals of Botany,’ vol. 7, No. 27, September, 1893.

1909. | Latent Life of Resting Seeds. 423
Diastase Enzymes.
Reduction Percentage | Percentage of Pl
Kind of seed. Age. with Fehling’s| of seeds seeds germinated ¢ ace
test. germinated. | with diastase. eoHRe as
Wheat ......... 21 years Fairly strong | 0 — | §. Australia.
=) eoaaeno00 20; Strong 0) — ie
5) Lonaneoden Ne) 5 | Fairly strong | @) 0 (unpricked) Me
| 0 pricked
PMIE vociemsig in Sie, | Very strong | (@) — .
9. Boadadnge 17 » yy) 0 re | 2
peed BO shrabien 16 ,, | Strong (0) — “
ROD sistent 5 months | 3 100 — Victoria.
Diastase Enzymes.
* | |
Bednction Percentage Percentage of Pl f
Kind of seed. Age ¥ iE ont of seeds _ seeds germinated | Se
| Fehling’s : : sa | origin.
Eure tiet germinated. with diastase.
| |
\WWANGEIBS aeetasesopedacs 13 years Strong (@) — 8. Australia.
Re i hein Ss 1a is 2 ith el
Mee ee, nha ie 12 = is
er Nt cake ceeetc I@ = 5. Fs 28 24 (unpricked) sf
20 (pricked)
BMY rirenattae. nin: Ore. is 32 28 (unpricked) 5
co et ee ee Sut | 32 Bis) Pe | 4
a RS aN ie 53 62 as | a
Wheat (Steinwedel)} 8 to 10 years e @) == | Victoria.
» (Duleith) ...| 113 years of 0 = 55
Wheat (Indian a . 4 O (unpricked) | a
King)
Barley ieacssncsssiics 5 months ms: 100 — BS
» (Algerian) | 8 to 10 years u @) O (pricked) se
0 (unpricked)
5, (Chevalier) |8 to 10 years} Slight (0) = Re
Barley (Hallet; 4 years Very 54 51 (unpricked) | New South
Chevalier) strong Wales.
Oatsmaiy cect sncnaes aaa Slight 100 80 ie Victoria.
», (Golden) ...... 8 to 10 years a (0) 0 (pricked) a |
0 (unpricked)
» (Algerian) ...) 5 years 5 56 56 (unpricked) 3
Rate reine biciaceleraseerncte 3 4 Fairly 84 — u
strong
op NO ee ta aa An i 96 = fs
pails | ace seOes SB aceLIBEE ot 60 — RS
yy) (Calcutta) <3) 4a Slight 72 — 2
epee Ca phase mre ep ies Fairly 76 — ki
strong
IR” nobosdcctoooooned 6 months Strong 100 — Re
BRR aceon ink sae 43 years 35 32 32 5
% - poondagebsado60609 8 to 10 years rs 0 — 4
WIENS): agandaecpoaoe6e 6 months Slight 100 — ‘
Maize (Golden| 44 years 5 60 52 New South
King) Wales.

A24 Miss J. White. The Ferments and [Mar. 3,

the two specimens used being the same; but as material which had entirely
lost its power of germination was not obtainable, the results in this case are
not perhaps quite so convincing as in the case of the other cereals.

It might be urged that since the resting seeds were not stored-in an
absolutely dry condition, the persistence of the diastase might be due to its
being reproduced as fast as it decomposed, but a comparison of the South
Australian results with those from Victoria and New South Wales does not
give any evidence of this.

2. Proteolytic Enzymes.—The first method adopted for the demonstration
of these ferments was approximately the same as had been employed by
Prof. Vines in his paper on “The Proteases of Plants,’* the difference being
that in this case from none of the seeds had the integuments been removed
before grinding.

The grain, 10 grammes in each case, was crushed by the hand mill, and put
into 100 c.c. of distilled water and shaken for two hours. The material was
then filtered and the filtrate was used as the digestive solution without
precipitation of the enzymes.

Into bottles containing 50 c.c. of this solution were put 3 drops of hydro-
eyanic acid and 0:2 gramme of well-washed fibrin which had been carefully
preserved in spirit. Into a similar bottle were put 50 cc. of the same
solution which was thoroughly boiled, and when cold 0°2 gramme of washed
fibrin and 3 drops of HCN were added to this bottle also. These bottles
were put into the oven at a temperature of 36° C. As in the ease of
diastase, control experiments in which commercial pepsin was employed
were rendered impossible owing to the constant presence of traces of
peptone mixed up with the pepsin, which was obtained both in the form of
a powder, and as scales, but always containing the same impurities.
Attempts to separate the pepsin and peptone by fractional dialysis failed.

After about 20 hours in the oven, application of the biuret test showed
the presence of shght traces of peptone in the unboiled specimens, whilst
the boiled specimens when similarly tested only gave the violet colour
characteristic of undigested proteids.

On treating some of the aqueous solution from the seeds in exactly the
same way as the above, with the single exception that no fibrin was added
to the bottle, application of the biuret test showed the presence of minute
quantities of peptone, thus indicating the occurrence of autolysis. The
results obtained in this way are tabulated along with those obtained when
dealing with solutions prepared by another method to be now described.
In order to diminish autolysis and to obtain more satisfactory results, the

* “Annals of Botany,’ vol. 20, 1906, p- 115.

1909. ] Latent Life of Resting Seeds, 425

enzymes were precipitated from their solutions. The method followed was
almost identical with that described by Dean* when dealing with the
proteolytic enzymes of Cucurbita pepo. Twenty grammes of the grain were
ground and mixed with 100 cc. of cold, boiled water, and shaken for
two hours. The mixture was then filtered and the filtrate precipitated by
a volume of saturated ammonium sulphate equal to that of the original
filtrate. A white, more or less flocculent precipitate was thrown down,
which was filtered off and dried overnight in an exhausted desiccator over
strong H2SOx.

The precipitate was scraped off by means of a sterilised knife as before,
and dissolved in a small amount of cold, boiled water.

In testing for fibrin-digesting enzymes, 1 c.c. of the aqueous solution
produced above was put into each of three sterilised test-tubes A, B, and C.
The contents of the test-tube C were boiled well and allowed to cool. Into
B and C were put 0-2 gramme of well-washed fibrin, and 5 drops of
chloroform were added to each of the three test-tubes. The three test-tubes
provided with corks were placed in the oven at 35° C. and left for an
interval of time varying from two to four days. -

Examination of the contents of all these test-tubes for the detection of
peptone showed that B and C both gave faint biuret reaction, the boiled as
well as the unboiled specimens.

A little of the liquid from each test-tube was examined under the micro-
scope, with the result that it was found to be swarming with bacilli, by which
the conversion of proteids into peptones had been wholly or partially effected.

This difficulty regarding the bacteria was due to the prolonged time
necessary for the action of these ferments to become manifest; and it was
evident that the addition of 5 drops of chloroform, and the subsequent
plugging of the test-tubes, was inadequate to maintain antisepsis, so that
it was necessary to adopt more stringent measures to destroy the bacteria,
but still not to impede in any way the action of any digestive ferments
which might be extracted from the resting seeds.

After many unsuccessful attempts to find some means of effectively
fulfilling these conditions, the required end was attained by soaking the
grain in strong chloroform for about five minutes after weighing it, and
grinding it in the mill while still wet. Care was taken that all the bottles,
apparatus, and water employed throughout the experiments were thoroughly
sterilised beforehand. Prior to putting the test-tubes into the oven 5 drops
of chloroform were added, and the tubes were stopped with plugs of cotton
wool. In this way complete sterility was produced.

* © Botanical Gazette, vol. 39, May, 1905, “ Proteolytic Enzymes,” p. 331.

A426 Miss J. White. The Ferments and [ Mar. 3,
The-detailed results are enumerated in tabular form below, from which it
will be seen that a proteid digesting enzyme is present, which is- destroyed
by moist heat at 100° C. As in the case of diastase, germination tests were
performed in order to discover whether the germinating power was affected
by the addition of commercial pepsin solution, or bore any relation to the
persistence of the proteolytic enzymes in the resting seeds. Throughout the

experiments the temperature of the oven was between 34° OC. and 40° C.

Fibrin-digesting Proteolytic Ferments.

| |
: | Percentage
- Reaction Percentage | :
Kind of seed. Age. | Method. | ee oe with biuret fence Or ae SESE of
| digestion. Leet eos tion with origin.
| : 4 pepsin.
Wiheaitinnccxccsennsete 6 months Vv. 50 hours Faint 100 100 Victoria.
ceataceeheeitos 21 years V. COL a; 0 — S. Australia.
PE Ey Die Vice pil d6Os aces iy 0 = 2
99. | Teleialejereisevilaisie)sjes 6 ” D. 50 ” »” 0 We a
%) (Indian Gale D. ARs 6 8 4 | Victoria.
King)
AMA SCE DS eagaetaaatonc 2h ae D. 40, 5 68 68 8. Australia.
ISEWBIO\7! soe conbooeaDe 6 months Vv. (30) 5. x 100 100 Victoria.
» (Algerian) | 8 to 10 years We 9) 5, % 0 (0) 49
» (Golden 8 to 10 years We (RO 5 Very faint 14 8 op
Drop)
Barley (Hallet 43 years D. 48, Faint 54 25 New South
Chevalier) Wales.
Barley eaeacaseceees op 10), CORE. A 100 — Victoria.
(OSS) cddonasdonrasnde 2 gs We BO 55 Very faint 56 40 %
5, (Golden)......|8 to 10 years We 60 ,, 3 (0) 0 i
PHUEiascacdacen Gots ® years Wo G0) yy on 92 81 x)
Stee eh eee 2: ,, D. GORE Faint 96 92 Bs
Soh nee diek E 44, D. 60 ,, 3 62 58 .
JSHIO) bunsoanoeesoceD 6 months D. 64, Fairly good 100 96 %
CPN Sec Ree ct sti 43 years D. 64, : 32 29 35
0) dpapmeceenanbes |8 to 10 years| D. & V.| 64 ,, 3 0) 0 59
WIEMAP ssdosoncoaaee 6 months VE WO gp Slight traces 100 — %
5 (Golden 4h years |D.&V.| 70 ,, 5 | 60 60 New South
King) | Wales.

V. = Vine’s method of preparing the proteolytic ferments, ‘ Annals of Botany,’ No. 78, April, 1906, p. 115.
D. = Dean’s method, ‘ Botanical Gazette, vol. 39, May, 1905, p. 331.

Reference to the preceding tables shows that a fibrin-digesting enzyme is
present in minute quantity in the resting grains of the cereals investigated,
and that apparently its amount is not appreciably influenced by the age of
Judging the amount of the ferment by its activity the amount
present is small in all the seeds tested, the maximum activity being
possessed by the rye seeds, and the minimum by the maize. The addition of
commercial pepsin solution to the seeds does not in any case increase their
percentage germination, and where the percentage germination is low tends
to lower it still further.

the grains.

1909. | Latent Life of Resting Seeds. 427

Test for Hrepsin.—Further investigations were carried on in order to find
out whether erepsin ferments were present in the resting grains. The mode .
of precipitation of the enzyme has been previously described, the same
method being employed as was used in testing for pepsin. Before grinding,
the seeds were soaked for about five minutes in strong chloroform, as was
done when testing for the existence of fibrin-digesting ferments in the seeds.
One cubic centimetre of the aqueous solution of the precipitate produced by
the saturated ammonium sulphate was put into each of three sterilised test-
tubes A, B, and C. The same difficulties arose in the case of the samples of
prepared pancreatin as were met with in the diastase and pepsin, the
samples purchased giving the tryptophane reaction owing to the presence of
amide impurities. As in the case of the fibrin-digesting enzyme, the
contents of the test-tube C were boiled and allowed to cool. About 0:2 c.c.
of Witte peptone was added to each of the tubes B and C; 5 drops of
chloroform were dropped into each of the three test-tubes, and the mouths
of the tubes were plugged with cotton wool. The three test-tubes were
placed in the oven, which was kept at a temperature of 35° to 36° C,
throughout the series of experiments. The test-tubes were left in the oven
for about three days, during which time they were occasionally shaken at
intervals.

The test adopted for the detection of products of erepsin digestion such as
amides was the tryptophane test. About 4 or 5 drops of bromine water
were added to each of the contents of the test-tubes A, B, and C, and without
exception tryptophane was produced in the contents of the tube B, whilst
no trace was observed in A or C.

The results demonstrate the occurrence of an erepsin ferment in the
seeds, which is destroyed in water at 100° C. Experiments were also
performed to determine the percentage of seeds capable of germination under
the action of a solution of commercial pancreatin in water. The results of
these experiments are stated in a special column of the tables below.

As in every instance the tryptophane reaction was well marked, in order
to economise material the experiments for the detection of erepsin were limited
to one sample of fresh and one of old grains.

The quantity of erepsin present must be fairly considerable as judged by
the degree of activity of the extract.

Reference to the tables shows that no favourable effect on the germina-
tive capacity of the seeds is noticeable as the result of soaking in
weak pancreatin solution. The addition of a weak solution of pancreatin
did not favour germination, but rather the reverse, except in the case of
43-year-old rye. On repeating this latter test, however, the seeds only gave

428 Miss J. White. Zhe Ferments and [ Mar. 3,

Erepsin Ferments.

Time eet: Per- _| Percentage
Kind of seeds, Age. Method. | acting on} phane GORE aaa jane
peptone. | reaction. | 8°Pmmna- Ne OE OMe
tion. pancreatin.
| |
\WAEGENG soanoqcensenpon 2% years D. 65 hours Good 100 98 S. Australia.
», (Marshall’s | 8 to 10 years D. Gopamees Pn 14 iit Victoria.
Prolific)
Barley (English)...| 4% years D. 65 5; 3 44 44, s
, (Algerian) | 8 to 10 years D. Goa = (@) (0) 4
AtS\\seewaeccontonee | 13 years D. Gdi 5; 3 92 73 a
» (Golden) ...... | 8 to 10 years 1D), Ga . 0 0 Be
TiVA®) © nonanrinootinnsnaon | 6 months D. 65%"); 5 100 —_— es
Ey. a beetaece Raereeaes '8 to 10 years D. Goa ss () (0) sf
“cil nohahatetteceeaey | 4 years D. Gales ae 34 30 -
IWS) Asondacnononne 6 months D. 65 ,, | Fairly good; 100 — a
», (GoldenKing)| 43 years D. Gomme it 60 60 New South
Wales.

D. = Dean’s method of extracting erepsin, ‘ Botanical Gazette,’ vol. 39, May, 1905, p. 331.

30 per cent. germination after treatment with pancreatin, so that the
apparent rise was of accidental origin.

In concluding this section of the paper, the net results may be briefly
summarised as follows :—

1. Although the germinating power continually decreases with advancing
age, the enzymes persist comparatively unaffected.

2. Diastase is present in fairly large quantities in both fresh and old
resting seeds of wheat, barley, oats, rye, and maize, being least active in the
latter.

3. A fibrin-digesting ferment is present in traces in all the above-mentioned
seeds.

4, Erepsin is present in considerable amount in all the above-mentioned
seeds,

5. All these ferments are destroyed by being raised to the temperature of
boiling water in the presence of moisture, but they are not destroyed by the
immersion of the seeds for about five minutes in strong chloroform.

6. The maximum quantity of all the enzymes occurs in rye, and the
minimum quantity in the maize.

7. The addition of dissolved ferments does not increase the percentage
germination of old seeds, and where any effect at all is produced tends to
lower it.

1909. | Latent Life of Resting Seeds. 429

The Effect of Extremes of Temperature on the Germinating Power and
Enzyme Contents of Seeds.

High Temperatures—All the enzymes present in the resting grains are
completely destroyed by moist heat at 100° C., and the same is true of the
germinating power of the grains, no signs of germination being apparent
in the seeds of cereals which have been immersed in boiling water for a few
minutes. This appeared to suggest the possibility of some co-relation existing
between the germinating power and enzyme reaction of seeds regarding their
powers of resisting high temperatures, irrespective of the fact that no such
co-relation exists between these two phenomena as regards their capacity for
withstanding time.

To test whether any such connection really exists, many experiments were
carried out, using dry instead of moist heat.

It is important that the seeds should be as nearly completely dry as
possible, and for this reason before being used for an experiment they were
taken from the store room, which was the driest place obtainable, and placed
in sulphuric acid desiccators kept in an oven at about 35° C. for a week or
more. The first series of experiments was carried out at 100° C., and the
method adopted was as follows :—The dried grains were placed in perfectly
dry test-tubes, which were fitted into holes in a sheet of cardboard. The test-
tubes containing the seeds were placed in a vessel containing boiling salt
solution, and the bulb of a thermometer was put into one of the test-tubes
among the seeds. The sheet of cardboard prevented any steam reaching the
open ends of the test-tubes, so that the grains were kept perfectly dry. The
erains were kept at a temperature of 99° to 100° C., for different intervais
of time varying from 4 hour to 16 hours, and on removal from the test-tubes
their germination capacity and enzyme reactions were investigated. The
results of these investigations may be obtained in detail from the following
tables.

The germinating power became gradually weakened as the interval of time
during which the grains were subjected to this high temperature increased,
but the same did not apply to the enzymic activity, for after 16 hours’
exposure to 99° or 100° C. the actions of the enzymes were apparently in
no wise impaired.

"A different method had to be adopted in order to raise the temperature of
the grains above 100° C. This was done by spreading the seeds, which had
been previously dried in the desiccator, as before in a single layer, and placing
them in an oven heated to the required temperature. The temperature
of the oven was first raised to 120° C. and kept constant for an hour. The

430 Miss J. White. The Ferments and [ Mar. 3,

seeds thus treated were afterwards tested for their germinative capacity and
their enzyme reactions, the former of which was found to be entirely lost in
each kind of seed used, while the latter was still evident, though in certain
cases it was markedly diminished.

The highest temperature at which the slightest possible traces of enzyme
reactions remained visible was 130° C., when faint signs of saccharification
of starch were produced by the diastase extracted from resting grains of
barley subjected to this temperature. Throughout this series of experiments
only fresh seeds were employed, whose germinating capacity before exposure
to the high temperatures was approximately 100 per cent.

After the grains had been heated above 120° C. the nature of the
precipitates was apparently changed. While the bulk of the precipitate was
seemingly as copious or even more so than before the heating of the seeds,
it was much more soluble in water than that obtained from the same material
unheated, and the filtrate was thinner and less glutinous than before. This
was especially pronounced in the rye, for the filtering process in this case
lasted about two hours, while the same process with the fresh seeds which had
not been exposed to high temperatures occupied as many days.

The tables appended below show the results of these experiments in detail,
v.¢c. the effects produced in the seeds on exposure to abnormally high
temperatures; the effects of extremely low temperatures on the seeds will be
dealt with later.

The methods of precipitating both the diastase and the proteolytic
enzymes were the same as those employed in the preceding section of the
paper. |

Briefly summarising the results set down in the tables, it is found that the
most resistant of all the ferments to extremes of heat is the diastase of
barley, which is not absolutely destroyed till the grains have been heated to
131° C. for an hour. The least resistant of the enzymes is apparently the
fibrin-digesting enzyme, for it is destroyed entirely at 124° C. in every kind
of seed tried. This result may, however, possibly be connected with the fact
that the quantity of this ferment present even in the fresh grains is
extremely small.

Whether the slight variation in the resistant power of the diastatic and
proteolytic enzymes of different grains to dry heat indicates the existence of
specific varieties of the different enzymes must remain for the present an
open question, but in any case the most exact experiments merely indicate
that the ferments in question are no longer capable of extraction and do not
say whether they have been actually destroyed or merely coagulated and
rendered insoluble. The coagulation temperature in the different seeds might

1909. | Latent Life of Resting Seeds. 431

Temperature Extremes. High Temperatures.

: .
Time | Percentage Reduction | | . p
Kind of seed. ue seeds | germina- | with Fehling’s | Re | Heyetophane
exposed.| _ tion. test. |
Op hours.
Wiheat* sees .cca. 99—100 33 48 Strong Faint Good.
Barley sees 99—100 4 32 % 54 2
Oats yh ssuhcnies 99—100 ss 48 ” »” ”
Wiheasitic cost ccc §9—100 1 24 90 » ”
ed tea loglbiy 99—100 44. (6) 3 35 0
JeETAYy neaceoene 99—100 43 6 oi » ”
Oats ah eee 99—100 4h 24 x ny »
Barley Gag atmuce 9§9—100 63 0) ” rr ”
HRV renscmicine saad: 99—100 63 (0) a on ’
Maazel cian see 99—100 $ @) , 35 on
\KHIO@E i ooonanneane 99—100 16 0) ; % 5
iBarleyay basses: 99—100 16 0) 5 Fe oO
ORNS s5 5 otecementeee 99—100 16 (0) 9 » »
Wheaties... 122 1 0) ci Very faint Fairly good.
TRO Rpnecaes aeenate 122 1 0 Faint 5 Good.
IVEanIZey east cnc 122 1 0) Faintest trace as Very faint.
Wiheaty ic. 124. 1 0) Strong None Fairly good.
Rive ea estiaons de: 124 1 (0) Faintest trace 55 Faintest trace.
INIEHA®) sepoboseo ose 124 1 (0) None ui None.
Oatseet seek. ss: 126 1 (0) Faintest trace 9 0
Waheaticn. .coceaces 127 1 0 Faint s Faint.
Oats iis Sol} Lz 1 0) None A None.
WING Bitte gon bacon 128 1 O Faintest trace ‘A Faintest trace.
es he Mi A 130 1 (0) None x None.
Bamnley, | eases 130 1 @) Faintest trace 509 »
Oartstets sec. vin 130 1 @) None " »
led, Gasuduede 130 1 O ib 24) »

* [In my paper on the vitality of seeds, wheat and barley are given as withstanding a day’s
dry heat at 100°C. The error is due to the transcription of 1 h. into 1 d., and the records are for
one hour’s heating and not one day’s, the somewhat higher percentages being possibly due to more
perfect drying.—Alfred J. Ewart. ]

naturally vary somewhat, since their structure, composition, and power of
retaining moisture all vary to a certain extent.

The diastase and the erepsin of the resting seeds appear to be almost
equally resistant to dry heat, or at least there is more variation between the
diastases of different resting seeds than between the diastase and erepsin of
the same seed.

Above 100° C. no seeds of any kind were found to be capable of germina-
tion, and the germinating power was absolutely lost in those seeds which had
been subjected in a dry condition to a temperature of 99° to 100° C. for
55 hours. Just* showed that as seeds are dried their resistance to dry heat
increases, and von Hohnelt found that many fully dried seeds could with-
stand an hour’s exposure at 110° C.

* “Cobn’s Beitriige,’ vol. 2, 1877.
+ ‘ Haberlandt’s' Wiss.-prakt. Unters.,’ vol. 2, 1877.

432 Miss J. White. The Ferments and [Mar. 3,

Low Temperatures.—The exposure of the grains to low temperatures, both
in the dry and moist conditions, had different effects from their exposure to
high temperatures. The mode of carrying out these investigations was as
follows: Seeds of fresh wheat, barley, oats, rye, and maize were dried in the
same manner as when testing for the effects produced by abnormally high
temperatures, and placed in perfectly dry glass tubes, which were carefully
sealed off, but which previously to sealing had been weighted with shot.

Samples of the same kinds of seeds were put together with shot into
loosely woven muslin bags, and the tubes and bags were lowered into a flask
of liquid air. The weighting of the tubes and bags was necessary owing to
the specific gravity of the liquid air being about equal to that of water.

The liquid air remained in the flask for about three days and all the seeds
were completely immersed in it for fully two days.

The seeds were removed from the liquid air and some of each kind were
set for germination, while corresponding seeds from the same packets which
had not been subjected to the temperature of the liquid air were also set to
serve as controls. Also some of the seeds from the tubes and muslin bags
were ground up and their ferments precipitated as before. Neither the
germinating power nor the enzyme reactions appeared to be appreciably
affected in the case of any of the cereals by the exposure to the extreme
cold of the liquid air, the temperature of which is approximately —200° C.
No constant difference was noticeable between the effects of exposure in
sealed tubes and of exposure in muslin bags where the seeds were in direct
contact with the liquid air.

The slight drop in the percentage germination after exposure to liquid air
in sealed tubes in the case of barley and rye, and in the case of wheat, oats,
and rye where the seeds were in direct contact with the liquid air, is
probably the result of these samples containing a few seeds whose power of
germination was at a low ebb. In any case the differences are very small,
and would be almost within the limit of error, were they not all on the same
side.

As no means were available of obtaining lower temperatures, it was
impossible to arrive at the satistaction of destroying the ferments by
‘abnormally low temperatures, if this be possible. Somewhat similar sets of
experiments were performed by Brown and Escombe,* who kept various
kinds of seeds exposed to liquid air enclosed in vacuum-jacketed tubes for
110 hours, and then slowly thawed them. They proceeded to test the
germinating power of these seeds together with control specimens, but did
not discover any appreciable difference between that of the seeds which had

* ‘Science,’ N. Ser., vol. 8, 1898, p. 215.

1909. | Latent Life of Resting Seeds. 433

been exposed to liquid air and the control specimens which had not been so
exposed. The same conclusions were arrived at by Thiselton-Dyer,* who
subjected seeds to a temperature of —250° C. for a shorter period.
Becquerel+ also performed experiments dealing with this subject.

The results of the experiments performed are as follows :—

Temperature Extremes. Low Temperatures.

Pitentacst| Percentage _ Reduction Biuret Tryptophane é
Kind a ereee a germination | with reaction | reaction after| Contained
pespedy | Peo | after Fehling’s after fibrin) Witte-peptone in—
| (normal). | liquid air. | test. | digestion. digestion.
| | |
Whe! 100 | 100 | Strong Faint | Good | Sealed tube.
Barley ...... 100 | 96 | 33 | ” ” | ”
Ontsipe hoe: 92 92 ” | ” ” | ”
Rye mone soee0 | 96 SO ” | ” ” ”
Maize. ...:.. 90 90 | 2 | ” | ” ”
Wheat ...... 100 96 ¥ | 9 | 5 | Muslin bag.
Barley Beate 190 | 109 | 2 ” | ” ”?
Oatsi ices. 92 90 | ” | ” | ” | ”
Rye eee eunece | 96 92 ” | ” | ” | ”
Maizey). S225. 90 90 | ” | ” ” | ”

As regards the ferments, there was not the faintest perceptible difference
between those precipitated from the two sets of seeds, although it must be
remembered that a difference will only be perceptible when a relatively large
part of the original amount of ferment has been destroyed or rendered
inactive or insoluble.

It is of great interest to note that the enzymes present within the resting
grains of the five different genera of cereals employed throughout these
experiments are not destroyed when the thoroughly dried seeds are subjected
to the extraordinarily wide range of temperature of —200° C. to +120° C.,
de. arange of 320° C.

The enzymes of a few varieties of seeds such as the diastatic ferment of
barley retains a certain amount of its activity when the range of temperature
through which the seeds have been exposed is —200° C. to 130° C., wea
range of 330° C.

The range of temperature through which the capacity for germination is
retained is from —200° C. to 100° C., 7.2. a range of 300° C., above this the
power is apparently entirely lost.

The conclusions arrived at in this section serve to substantially verify that
drawn from the last section, that the capacity for germination is not

* © Roy. Soc. Proc., vol. 65, p. 362, 1899.
+ © Ann. Sci. Nat., Bot.,’ ser. 9, vol. 5, 1907.

434 Miss J. White, The Ferments and [ Mar. 3,

dependent upon the existence of enzymes in the resting seeds of the cereals
mentioned, although the question will not be absolutely closed until it is
found possible to germinate seeds which contain no enzymes in the resting
condition, or in which these enzymes have been destroyed. Since enzymes
appear to retain their activity within a wider range of conditions than does
the capacity for germination, this is likely to be a matter of the utmost
difficulty, or may be impossible.

Before concluding the series of experiments in connection with this section
of the paper the resistance to extreme cold of certain other varieties of
seeds was also tested. Some of the different kinds of seeds were tied up in
loosely woven muslin bags, together with shot to ensure their sinking when
immersed in liquid air. The bags were lowered into a flask of liquid air in
which they were left for one and a half days.

One hundred of each kind of seeds from the liquid air were set to
germinate on damp blotting paper in a special germinating box, whilst
100 of each kind which had not been exposed to the extreme temperature of
liquid air were set to germinate on damp blotting paper alongside them.
The varieties of seeds employed were chosen from sorts possessing widely
differing characters, including some which, being sensitive to desiccation,
might also be sensitive to extreme cold.

The names of the seeds used are enumerated in the tables, accompanied by
the relative numbers which germinated under normal conditions and after
exposure to liquid air respectively. The third column contains data supplied
by Prof. Ewart for comparison between the resistance to extreme cold and to
desiccation.

Reference to the table shows that in not a single instance were the seeds
entirely killed as the effect of their immersion in liquid air.

The influence of the low temperature is naturally most pronounced in
the case of samples with a comparatively low germination capacity in which
a number of the seeds are only just able to germinate under the most —
favourable conditions.

In the case of the carrot seed, freezing appeared to increase the percentage
germination, but on re-testing the original seeds a percentage germination
of 65 was obtained ; possibly the first test was discontinued too soon.

The liquid air apparently exerts a retarding influence on the germination
as, except in the isolated case of the cress seeds, in which signs of germination
were apparent in 100 per cent. of both sets of seeds one day after sowing,
germination was always noticeable in the seeds grown under ordinary
conditions before those which had been subjected to the intense cold of

liquid air.

1909. | Latent Life of Resting Seeds. 435

Germination Tables.

i]
Percentage
Kind of seed. germination. | Liquid air. Resistance to desiccation.
(Normal.)
Apple (Pyrus malus) ......... 12 4, Sensitive to severe desiccation.
Turnip (Brassica campestris) 91 88 43 per cent. after 42 days’ desicca- |
| tion at 37°C.
Cress (Lepidium sativum) ...| 100 100 30 per cent. after 4 weeks in abso-
lute alcohol.
Carrot (Daucus carota) ...... | 36 to 65 59 Lasts 10 years in dry air.
Haricot (Phaseolus multi- 100 90 2 per cent. after 45 days’ desicca-
florus) | tion at 37° C.
Hemp (Cannabis sativa)...... 24 | G Nil after 15 days’ desiccation at
| 37°C.
Mustard (white) (Brassica 85 72 Lasts 10 years in dry air.
alba)
MOOI CRUNUS) “rane. -2 sno -e «em | Si 13 Sensitive to prolonged extreme cold |
(De Candolie).
Parsnips (Peucedanum sati- | 45 18 Sensitive to extreme desiccation.
ovum)
Ras Leva erie toc cassies oeeroesnsees 28 | 1 os - 3
Pea (Pisum sativum) ......... 95 75 Nil after 42 days’ desiccation at
37° C. |
Radish (Raphanus sativus)... 97 88 Lasts 10 years in dry air.
Ricinus cambogiensis ......... 100 | 100 R. communis 40 per cent. after 28 |)
days’ desiccation.
Sunflower (Helianthus 70 | 65 51 per cent. after 42 days’ desicca-
annuus) ; tion at 37° C.

The fact that in every experiment except two there is a lower percentage
germination in the severely frozen seeds, and that in no instance is the:
reverse the case, signifies that to some extent freezing in the liquid air is:
deleterious to the germinative power of seed. Another noteworthy
observation is that there does not appear to be any particular class of seed
which is more injured by the extreme cold than any other class, ¢y., of three
kinds of oily seeds tested, viz., Hemp, Helianthus, and Ricinus, while the first
was strongly affected, the second was little, and the last-named seed not at.
all injured by —200° C. for two days.

The starchy seeds of cereals are, however, as resistant to the effects of
exposure to liquid air as are the oily seeds.

Lobelia erinus seeds were selected as good subjects for experiment on the:
strength of the statement of De Candolle* that dry seeds of Lobelia erinus:
lose their vitality sooner at very low temperatures than at ordinary ones.

The results tabulated in this paper show that the vitality of some is lost,,
but as 13 per cent. were found to germinate after exposure for one and
a half to two days to a temperature of approximately — 200° C., it is probable.
that if the time of their exposure were increased somewhat the vitality of all.
the seeds would be destroyed.

* Pfeffer, ‘ Physiology of Plants,’ Engl. translation, vol. 2, p. 234.

436 Miss J. White. The Ferments and | Mar. 8,

= From the third column on the list it can be seen that, on the whole,
though not without exception, the resistances to extreme cold and to extreme
desiccation are approximately parallel.

4. The Respiratory Activity of Resting Seeds.

In this series of experiments the respiration of certain other characteristic
kinds of seeds was tested in addition to the foregoing cereals.

Whether dried seeds respire at all, and if they do to what extent, is one
of the most discussed problems in plant physiology, especially in connection
with the views as to whether the life in resting seeds is merely at a low ebb
or is entirely suppressed.

The apparatus employed was Aubert’s improved form of that of Bonnier
and Mangin, and, as was stated in a previous paper dealing with the
respiration of gynecia,* the machine gave complete satisfaction, provided
that certain precautions were taken. Before each set of experiments the
mercury was removed from the apparatus and was thoroughly cleansed by
several washings in strong hydrochloric acid, followed by several washings
in distilled water, and then being passed through a filter to dry.

This precaution was found to be of extreme importance, for in the presence
of any impurities such as zine in the mercury, the inlet of a sample of air
into the tube produced oxidation of the zinc, and a consequent diminution
of the volume of the sample of air when allowed to stand in the apparatus
for a short time.

The NaOH used was a 40-per-cent. solution, and the pyrogallic acid was a
saturated solution diluted to one-fourth its original strength.

The seeds employed were the ordinary cereals, and also Lucalyptus globulus,
Acacia melanoxylon, Cytisus laburnum, Setaria italica, Ricinus cambogiensis,
Cannabis sativa, and Pinus insignis—and the experiments were performed in
four series.

1. The seeds used were tested as received from storage.

2. The seeds before being tested for their respiratory activity were dried
in the oven for eight days at a constant temperature of 45° C.

3. Samples of the seeds after drying for eight days at 45° C. were further
heated in the oven for three days. During the daytime the temperature
of the oven was 100° C., whilst at night the temperature fell to about 70° C.

4. Some of the above seeds were still further heated to about 130° C.,
when all were killed, and the gaseous exchanges were again tested.

For each respiratory test a weighed quantity of each seed was passed up

* “Annals of Botany,’ 1908.

1909. ] Latent Life of Resting Seeds. 437

into the upper part of a narrow, perfectly dry test-tube, containing a known
volume of air, over mercury. These test-tubes had been previously sterilised
by dry heat in all cases, although with the thoroughly desiccated seeds this
precaution is not really necessary, except as a means of drying the tube.

The tubes containing the seeds and mercury were set up vertically in a
shallow dish of mercury, where they were kept for from 5 to 15 days.
After this time had elapsed, samples of the contained air were drawn
into the Bonnier and Mangin apparatus, in which their composition was
ascertained.

Those seeds which were found to emit no carbon dioxide in their ordinary
stored condition were not further tested for signs of respiration in the more
completely desiccated state, but the quantity of moisture was ascertained in
every kind of seed used. The relative amounts of water present in the
seeds at different stages of desiccation are set down in a special table which
follows the respiration tables given below. Throughout these experiments
the seeds used were the freshest obtainable, and several analyses were
made of each sample of air, the results tabulated below being the mean of

these analyses.
Respiration of Seeds.

| | | Mgrms. | Percentage
Vol | CO, per | volume
Kind of seed. State. Weight. | ‘Ge eee |Time.| grm.of | of O,
| <ul | seeds per | absorbed |
| | day. per day. |
|
|
| grms c.c. | days | |
| Acacia melanoxylon ......... | Asstored | 2 Siemon .OLO05 0°15
| AMEN SATIOE .26cecseccecrecces a en Se |e | 0-0 0:07
| Cannabis sativa ............... es ae ZN og O50 0°3 |
| Cytisus laburnum ............ | = 5 4 6 |; 0°001 O15 |
| Eucalyptus globulus ......... | 3 2 Z| = Ge Ws 0°05
Hordeum sativum (barley) . ee Olam lea 1S | OW 0-014
| Setaria italica ..........0.0-- rs 2 5 6 | 0-001 0-03
Pinus insignis.....0.000.0.c000 ss fe te 5 6 | 0-000] O13 |
Ricinus cambogiensis ......... . 5 7 6} * 6-0 O10 |
Secale cereale .......0...1..000s sp 6 hols 6 | 0:°004 O15 |
Triticum vulgare ......0.02. . hee 18 Si Oats 2°5
LGB OT ET OICC GOR ECO ER ECOEEE “e |; 36 | 15 5 0 004. 0°02 «(|
|
Acacia melanozylon ......... Dried at 45° C.| 2-0 | 8 5 0-0 0-14
in dry heat
for 8 days
Setaria ttalica ..,....+-.002.. 3 1-2 6°5 4, 0-0 0-02
PUNUS UNSUGNIB.. 502; ..02000500s- i Noo AG i 5°5 | 4 0°0 0-06
Secale cereale ........0.ccc0000s op La 20 6 5 0-0 0:02 «(|
Triticum vulgare ............ re Wea ized 6 5 0-0 0-00
Geach a. a ie es | 2-0 Sle 5 0-001 0-12
| AN) se (it aie ai Sie oh ae | Further dried | 2-0 Dkoale go 0-001 0-05
| | for 3 days at
| ! 100° ©., dry heat
VOL. LXXXI.—B. 2K

[ Mar. 3,

The Ferments and

Miss J. White.

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1909. | Latent Life of Resting Seeds. e439

Comparing the preceding tables it is seen that a feeble respiratory activity
is shown by some of the seeds examined in the ordinary stored condition,
which varies according to the seed and to the amount of moisture it contains.
Oats, hemp, barley, Eucalyptus, and Ricinus showed no evolution of carbon
dioxide, and the trace of oxygen absorbed may be the result of chemical oxida-
tion or physical absorption.

Respiration was surprisingly active in the fresh wheat as obtained from the
seedsman, although not more than 1/300 part of what it is in an active
seedling. Further, the evolution of carbon dioxide ceased after a comparatively —
small degree of desiccation. Reference to the moisture tables shows that
the percentage of moisture contained by the wheat was not relatively large as
compared with the other seeds employed.

As might be expected from the fact that the seeds of Acacia melanoxylon are
completely covered by a cuticle, which in the fully dried seed can withstand
nearly 45 minutes’ immersion in strong sulphuric acid without becoming
permeable to water, the percentage of moisture eliminated by slow desiccation
in dry heat is less marked than in any of the other varieties of seeds. The
carbon dioxide evolved from the air dried seeds does not amount to 1/10000

of the amount evolved from an active seedling, and is, in fact, nearly within
- the limit of error, and may possibly be the result of oxidations taking place
in the arillar appendages of the seeds.

In any case, all respiratory activity as evidenced by the gaseous exchanges
completely ceased after the same seeds had been desiccated at 45° C. for
seven or eight days. For purposes of control various dead seeds, fragments
of dead wood, etc., were similarly tested in the air dry condition. In no
case could any sign of an evolution of carbon dioxide be detected, and the fact
that in some cases a trace of oxygen disappeared is not surprising, considering
the structure of the materials tested, and their large bulk relatively to the
amount of the enclosed air.

The above results indicate that respiration is not a function of completely
dry seeds, nor even of seeds after a mild degree of drying, for only in one
isolated instance, that of Zea mazs, was there the faintest trace of any apparent
respiratory activity present after remaining at 45° C. for one week. The
amount of carbon dioxide produced in this case was less than one-millionth
of that produced by an active seedling in the same time, and was evidently
the result of the slow outward diffusion from the bulky seeds of carbon
dioxide formed while in the air dry condition. The same applies to the traces
of oxygen absorbed, as the gaseous relationships inside and outside the seed
become equalised.

This is made even more evident by the fact that the greater number of, the

2K 2

440 Miss J. White. The Ferments and [ Mar. 3,

fresh seeds in the air dry condition as obtained from the seedsman exhibited
no respiratory activity whatever, although they contained quite appreciable
quantities of water. Among these non-respiring seeds Ricinus is included,
though in another species of Ricinus, Becquerel* states that an active inter-
change of gases does occur in the dried seeds, which he asserts to be a purely
non-vital chemical action.

The experiments of Kolkwitzt carried out on barley illustrate the important
effects produced on the respiration of seeds by the presence of moisture in the
seeds. He found that—

1 kilogramme barley grains at summer temperature gave off 3°59 m. mg.

of CO2 in 24 hours when 19 to 20 per cent. of water was present ;

14 m.mg. with 14 to 15 per cent. of water; and

0°35 m. mg. with 10 to 12 per cent. of water.

Becquerel? also discusses the effect of the presence of moisture in the
seeds on the respiratory activity of the seeds in his extensive researches on
the latent life of seeds, a short account of which also appears in the
‘Comptes Rendus,’ vol. 143, No. 26, December 24, 1906, p. 1177.

Summary.

The resting seeds of cereals such as wheat, maize, barley, oats, and rye all
contain diastatic, fibrin-digesting, and ereptic ferments in appreciable
amount. These ferments retain their activity without appreciable change in
stored dry seeds for 20 or more years, that is long after the power of
germination has been lost, which takes place in wheat after 11 to 16 years,
barley 8 to 10 years, oats 5 to 9 years, maize and rye over 5 years. The life
of the stored seeds is largely dependent upon the climatic conditions, a dry
climate favouring longevity. Thus South Australian wheat lasts longer than
that stored in Victoria, and still longer than that obtaimed from New South
Wales. The difference is, however, not shown strongly until after the
fourth or fifth year, South Australian wheat being still one-third germinable
after 9 to 10 years, whereas wheat stored in Victoria had entirely lost its
vitality by this time.

No relation was noted between the vitality of seeds and the persistence of
enzymes in them, but since the enzymes persisted longer than the power of
germination, the question as to whether germination could take place in the
absence of any pre-existent enzymes remains to be answered. In any case

* “Comptes Rendus,’ vol. 148, 1906, p. 974.
+ ‘Ber. d. Bot. Gesell.,’ vol. 19, p. 285, 1901.
} ‘Ann. Sci. Nat., Bot.,’ ser. 9, vol. 5, 1907.

1909. | Latent Life of Resting Seeds. 441

no otherwise non-germinable seeds could be excited to germination by the
addition of any kind of enzyme, and where the germination was feeble the
addition of enzymes usually lowered the percentage germination and often
delayed germination also to some extent.

The erepsin appears to be more abundant than the pepsin, but otherwise in
the cases of all three ferments greater differences are shown between
different samples of the same age than between different seeds, or between
the same seeds of varying ages. Pepsin appears, however, to be more
abundant in rye than in any other cereal, and is almost absent from maize.
Dry oats, barley, and wheat can in part resist a temperature of 99° to 100° C.
for 1 to 44 hours; after 6 hours’ exposure all are killed, but the ferments are
apparently unaffected. All the ferments are destroyed after an hour’s dry
heat at 130° to 131° C. The pepsin appeared to be least (1 hour at 124° C.),
the erepsin more (1 hour at 124° to 128° C.), and the diastase, especially of
barley, most resistant to dry heat (1 hour at 124° to 131° C.).

Two days’ exposure to liquid air, although it delays the subsequent
germination and may also decrease the percentage, does not absolutely destroy
any of the seeds tested and does not appreciably affect the ferments in any of
the cereals. The dry diastase of barley is therefore able to withstand a range
of temperature of —200° to +130°C. It is therefore thermally a highly
stable chemical compound.

Many seeds, including all cereals, give off appreciable quantities of carbon
dioxide when stored in the air dried condition, but others show no signs of
respiration whatever. The respiration of air dried wheat is especially pro-
nounced, but in practically all cases every sign of respiration ceases when the
seeds are moderately desiccated, although in the case of large seeds like
maize minute traces of carbon dioxide may continue to escape for a time.

SPECIAL BIBLIOGRAPHY.

Vines, “ Proteolytic Enzymes in Plants,” ‘ Annals of Botany’ :—
(i) Vol. 17, pp. 237—264.
(ii) Vol. 17, pp. 597—616.
“ The Proteases of Plants” :—
(i) Vol. 18, pp. 289—317.
(ii) Vol. 19, pp. 149—162.
Gii) Vol. 19, pp. 171—187.
(iv) Vol. 20, pp. 113—122.
Vol. 22, pp. 103—114.
J. W. T. Duvel, “The Vitality and Germination of Seeds,” U.S. Department of
Agriculture, Bureau of Plant Industry, Bull. No. 58.
Dr. G. Albo, “Les Enzymes et la Faculté Germinative des Graines,” ‘Archives des
Sciences Physiques et Naturelles, Cent-treiziéme année, vol. 25; “La Vita dei

442 Prof. E. A. Schiifer. [July 22,

semi allo stato di riposo,” ‘Estratto dal Bullettino della Societa Botanica
Italiana, Firenze, 10 Novembre, 1907.

M. Edouard Morren, “ Note sur le Role des Ferments dans la Nutrition des Plantes,”
‘Académie Royale de Belgique,’ 21 Octobre, 1876.

M. Ch. Des Moulins, “ Relatifs 4 la Faculté Germinative conservée par quelques Graines
Antiques,” ‘ Actes de la Société Linnéenne de Bordeaux,’ Juillet, 1846.

Adrian J. Brown, “Enzyme Action,” ‘Transactions of the Chemical Society,’ 1902,
vol. 81. j

Arthur Dean, “On Proteolytic Enzymes.—I,” ‘Botanical Gazette, May, 1905, vol. 39,
No. 5.

Acton, ‘ Annals of Botany,’ vol. 7, No. 27, September, 1893.

Just, ‘Cohn’s Beitriige,’ vol. 2, 1877.

Brown and Escombe, ‘ Science,’ N. Ser., vol. 8, 1898, p. 215.

Von Hohnel, ‘ Haberlandt’s Wiss.-prakt. Unters.,’ II, 1877.

Dyer, Thiselton, ‘ Roy. Soc. Proc.,’ vol. 65, p. 362, 1899.

Becquerel, Paul, ‘ Ann. Sci. Nat., Bot.,’ ser. 9, vol. 5, 1907 ; ‘Comptes Rendus,’ vol. 143,
1906, p. 974; ‘Comptes Rendus,’ vol. 143, 1906, p. 1177.

Kolkwitz, ‘ Ber. Deut. Bot. Gesell.,’ vol. 19, p. 285, 1901.

Croontan Lecture.—The Functions of the Pitwtary Body.
By E. A. ScHarer, F.R.S.

(Lecture delivered June 10,—MS. received July 22, 1909.)

The observation of P. Marie (1885) that the disease to which he has
given the name “acromegaly ” is associated with tumours of the pituitary
body has caused this organ to attract the recent attention of pathologists to a
greater degree than any other of the structures which were formerly classed
together under the generic name “ ductless glands.” Since Marie’s descrip-
tion of that disease, very many cases have been recorded, and in most of
these the same association has been noticed.

The most striking sign of acromegaly is the increased growth of certain
parts of the skeleton, especially the lower jaw and the extremities of the
limbs, with hypertrophy of the connective tissue; indeed, the enlargement
of the hands and feet is frequently the first change which calls attention to
the existence of the disease, the patient finding that his gloves and boots
are becoming too small for him. In the later stages there is dorsal kypho-
scoliosis. Headache is a prominent symptom, polyuria is often present, and
the eyesight is frequently affected. Acromegaly usually occurs in adults,
often about middle age, although it may begin during adolescence. An
allied affection—(pathological) gigantism—which occurs before normal

1909. | The Functions of the Pituitary Body. 443

growth is completed, is accompanied, in addition to many of the above
symptoms, by an increase in length both of the limb bones and of the trunk,
so that the patient affected attains an altogether unusual stature. This
also has been found in most cases that have been examined to be associated
with tumours of the pituitary body and a concomitant enlargement of the
‘sella turcica. It is probably the case that the changes in the skeleton in
acromegaly and gigantism are due to the same cause, operating, perhaps,
at different stages during the progress of growth, and that this cause is to
be found in an alteration in the functions of the pituitary body.* Assuming
from what is known of the above diseases that the functions of this organ,
or of a part of it, have to do with the growth and nutrition of the skeletal
tissues, 1t has still to be decided whether the increased and abnormal growth
which is met with in them is due to diminution or excess of the activity of
the gland. The former view was taken by Marie, who noticed—as others
have done since—that the nature of the tumour which is found after death
is often such as to have produced complete destruction of the organ,
a cancerous or sarcomatous formation having been frequently described.
Those who uphold this view look upon the gland as in some way—probably
by means of an internal secretion or hormone—regulating the growth of the
skeleton, which in the absence of such regulation proceeds abnormally.
But the opposite view (Tamburini, 1894, and Woods-Hutchinson, 1894) has
also been advocated, viz., that the symptoms of acromegaly and of gigantism
are due to a hypertrophic condition of the pituitary, or, according to the
very probable suggestion of Woods-Hutchinson, of its anterior lobe alone,
which may be considered to produce too great a quantity of a hormone
which stimulates bone-growth. The most important argument in favour of
this view is derived from the fact that the pituitary tumours which have
‘been found to be associated with the acromegalic condition have in many
instances, especially where the tumour has not been unusually large, been
described as a simple glandular hyperplasia of the anterior lobe. And ina
few cases of acromegaly which have been noted to be unaccompanied by any
distinct enlargement of the pituitary, the glandular cells have been described
as unusually full of the granules which are generally regarded as indicative
of the secretory activity of the cells.

On the other hand, the numerous instances in which after death the
glandular substance of the organ has been found entirely destroyed and
replaced by cells of the types met with in malignant growths seem at first
to offer difficulty to the acceptance of this view, and to favour the opinion

* Cf. Woods-Hutchinson (1898, 1900). According to Woods-Hutchinson, the connec-
tion between acromegaly and gigantism was first suggested by Cunningham (1891).

444 Prof. E. A. Schiifer. [July 22,

propounded by Marie that the symptoms have been produced by suppression
of the internal secretion. But to this it may be replied that in such cases—
as, in fact, is not unfrequent in tumours of glandular organs—it is possible
to assume that the tumour at its beginning was non-malignant and of the
nature of a simple hyperplasia, the malignancy of character being estab-
lished later, and only then proceeding to destruction of the glandular’
type of cell.

The chronic character of the affection favours on the whole this supposi-
tion. For it appears to be established experimentally that complete
extirpation or destruction of the gland is incompatible with continuance of
life for more than a few days at the utmost. If we assume—which we are
not entirely justified in doing—that destruction by disease will have a
similar result, then we should expect, if acromegaly be due to hypertrophy
and increase of activity of the gland, that as long as such a tumour is merely
glandular and benign, the series of symptoms which characterise the disease
would gradually develop, and it is a known fact that in most recorded cases
it has pursued a slow course with a gradual development of the characteristic
signs. It is unlikely that at this stage the tumour which is forming is
already malignant, and especially that it has assumed a sarcomatous
character, which is that which has perhaps been most often described
post mortem in this disease. It is more probable that the malignant character
has become developed shortly before death, and by no means improbable
that death has resulted from entire suppression of the function of the gland
owing to destruction of the normal glandular cells by those of a malignant
nature. One must at the same time bear in mind the existence of other
possible causes or contributory causes of death, such as the pressure of the
tumour upon the base of the brain and the mere existence of malignant
disease. But in many cases the tumour has not been of sufficient size
to justify death being attributed to these causes, and the assumption that it
has resulted from destruction of the normal glandular tissue is probably correct.

If the symptoms of acromegaly are due to an excess of secretion from the
gland, one would not expect amelioration as the result of pituitary feeding.
Campbell Geddes (1908) mentions a case in which the patient became
rapidly worse when pituitary and ovarian extracts were given. In this case
the pituitary was 30 times the normal weight, and showed simple hyper-
trophy of the anterior lobe tissue only. There was no polyuria, although
this condition is often found either with or without glycosuria in both
acromegaly and gigantism. Its occurrence is best explained, as will be
presently seen, by supposing the posterior lobe, or, at least, the pars inter-
media, to participate in the hyperfunctioning of the anterior lobe.

1909. | The Functions of the Pitwtary Body. 445

Before proceeding further it is necessary to refer to certain facts relative to
the structure, development, and functions of this organ.

Development.

Regarding its development, it is known to have a double source of
formation, a hollow extension from the buccal ectoderm towards the base
of the brain being met by a hollow extension of the neural ectoderm
occupying the situation of the future infundibulum. The two extensions
eventually grow together and constitute the pituitary body, the buccal
ectoderm, which loses its connection with the alimentary tube, forming the
anterior lobe and pars intermedia, 7.c. by far the larger portion of the organ ;
while the neural ectoderm becomes developed into the posterior or nervous
lobe. This retains in some animals its hollow connection through the
infundibulum with the third ventricle of the brain, although in man and
other Primates it becomes entirely solid.*

Structure.

The structure of the parts which are thus so differently formed in the
embryo is also, in the adult, entirely different. For while the pars
nervosa s. posterior shows no development of any tissue which can be
supposed to possess either nervous or glandular activity—consisting as it
does mainly of neuroglia elements with very few vessels—the pars anterior
is formed of a highly differentiated epithelium-like tissue, very richly
supplied with large and thin-walled capillary blood-vessels, many of its cells
being filled with granules such as are characteristic of glandular structures.
The appearance of the pars anterior is, in fact, precisely that of an organ
which has the property of forming a secretion which is passed from its cells
directly into the blood-vessels, and one would, without hesitation, class it
amongst the internally secreting glands.

The pars anterior in man and in most animals is separated from the
posterior lobe by a cleft-like cavity, which is the remains of the original
hollow of the outgrowth from the buccal ectoderm. But the epithelial
tissue immediately adjoining this cleft, and especially that which impinges
on the pars nervosa, is of a different character from that of the pars anterior.
The cells are less distinctly granular: they tend to be arranged in islets
separated by intervening tissue which is continued between them from the

* This description of the development and structure of the mammalian pituitary body
is based upon the investigations of Herring (1908), which is itself supplementary to
and in many particulars confirmatory of the results of former observers.

AAG Prof. E. A. Schifer. [July 22.

pars nervosa, and many of the islets are hollow, forming small vesicles
which are occupied by a “colloid” material. The inter-epithelial tissue is
far less vascular than that of the pars anterior. But this tissue also
exhibits “colloid,” which is contained in spaces prolonged into the pars
nervosa, and the same material can even be seen discharging into the pro-
longation of the infundibulum which enters the pars nervosa. Indeed, in
some animals (¢.g. cat) the infundibulum extends as a hollow canal as far as
the pars intermedia, and this canal receives the colloid secretion of that
part of the gland. The pars intermedia differs, therefore, from the pars
anterior not only in the structure of its cells but also in the fact that its
secretion—which is no doubt represented by the “colloid” material—is, in
all probability, not taken up directly into the blood but is passed into the
infundibulum and thus into the third ventricle.

The discovery of this difference, which must be regarded as a fact of
great importance in the physiology of the pituitary, is due to the investi-
gations of Herring (1908), who has further found that the amount of such
colloid which is discharged into the infundibulum is greatly increased after
removal of the thyroid in animals. It is true that an increase in the
amount of “colloid” in the pituitary body had previously been noted after
thyroidectomy ; but this “colloid” was located by previous observers in the
anterior lobe, and was supposed to pass into the blood-vessels, whereas it
has been shown by Herring to be a product of the cells of the pars intermedia
and to pass into the infundibulum and third ventricle.

Functions.

The first investigations of a strictly physiological character which were
instituted to determine whether the pituitary body possesses any active
function were those of Oliver and myself (1895). We found that aqueous.
or saline extracts—which may be boiled without losing their activity—
produce, when injected into the blood-vessels, a rise of blood-pressure which
is comparable to that produced by similar extracts of the suprarenal capsules.
We further showed that this effect is produced by an action upon the
peripheral arteries, which are caused strongly to contract, in this also
resembling the action of the active principle obtained from the suprarenals ;
but far more prolonged, and not due to the presence of that substance in
the extract. We did not in these experiments obtain any marked effect.
upon the rate of the heart’s beat, an acceleration of which is a characteristic
feature of the action of suprarenal extract, after the vagi have been cut or
paralysed (prior to which there may be some inhibition). Nor did we

1909.] The Functions of the Pitutary Body. 447

differentiate between the action of the different parts of the gland, having
used extracts of the whole pituitary body.

Howell (1898) carried the investigation further, and added considerably
to our knowledge of the action of extracts of the gland. He split it
into anterior and posterior parts, and determined that whilst the extract
of the former is without physiological activity when injected into a vein,
that of the latter produces the effects upon blood-pressure and _blood-
vessels which Oliver and I had obtained from extracts of the whole gland.
Howell further found the rise of blood-pressure to be accompanied by a
slowing in the action of the heart, and that both the raised blood-pressure
and slow cardiac rhythm might be maintained for a considerable time. And
that if a second dose be administered intravenously within a certain time—
which varies from half an hour to an hour or more—after the first dose,
these effects are not repeated—in other words, a certain immunity is
established which only slowly passes off.

Swale Vincent and I (1899) repeated Howell’s observations. We were
able to confirm them in almost every particular, but found that the cardiac
slowing described by Howell is not constant, and that when present it is
not abolished by section of the vagi or the action of atropine. It is,
therefore, of peripheral origin, and not due to the same cause as the
inhibition which often accompanies the action of adrenin, which is brought
about by an action upon the cardio-inhibitory mechanism in the bulb. We
also found that not only is there generally no rise of blood-pressure resulting
from a second or third dose of the extract of posterior lobe, but there is
invariably a fall, which, however, lasts only a short time. We showed
that this fall of blood-pressure is due to a depressor substance acting upon
the blood-vessels; that the substance is soluble in alcohol, in which the
pressor substance is insoluble; and that it is not identical with cholin, which
has a similar action, and might be supposed to have been extracted from the
nervous tissue of the lobe. These facts have now been corroborated by
many experimentalists, and it has recently been shown that they hold
good tor extracts of the human pituitary (Halliburton, Candler, and Sikes,
1909).

But the action upon the circulatory organs does not exhaust the effects of
such extracts. For in the course of certain experiments which Dr. R. Magnus
and I were conducting in the summer of 1901, we incidentally noticed, as one
of the results of intravenous injection, a marked increase in the flow of urine
from the ureters. Pursuing the subject further, in association with Herring
(1906), the fact became evident that the aqueous extract of the posterior lobe
—including the pars intermedia, which comes away with it when it is

448 Prof. E. A. Schiifer. [July 22,

separated from the anterior lobe—has a specific action both upon the renal
vessels and upon the kidney cells. For whereas this extract produces
contraction of most of the arteries in the body, it has the opposite effect upon
those of the kidney, causing them to dilate, although this dilatation, which
is very marked and lasting, may be preceded by a short period of contraction.
The increase in flow of urine, although no doubt greatly assisted by the
dilatation of the kidney vessels, which is coincident with a rise in general
blood-pressure caused by contraction of other arteries, is not entirely produced
by the vascular changes. For it may occur without them, as in the case
when a repeated dose of thetextract 1s administered intravenously within a
short interval. In such cases, as we have seen, the rise of blood-pressure may
fail altogether, or even be replaced by a temporary fall, and there may also
be no further dilatation of the kidney produced ; nevertheless, the diuretic
effect may still occur, and this can only be explained by supposing that there
is some substance in the extract which acts by directly stimulating the
secretory activity of the cells. Moreover, I have had occasion to observe
that the converse of this experiment may occasionally be obtained, and this
with a first dose; the normal effects of rise of blood-pressure and dilata-
tion of kidney being produced without any increase in flow of urine (see
fig. 1).

It is further noticeable that in a large proportion of experiments
a common phenomenon is a temporary diminution or cessation of urine
flow, even although the blood-pressure is raised to a considerable extent and
the kidney volume markedly increased ; conditions which—on the mechanical
or filtration theory of urine secretion—should inevitably produce diuresis.
There is, in fact, very often at first an inhibition of secretion (followed in the
majority of cases by the characteristic secretory activity), even although the
vascular conditions are throughout favourable to the occurrence of free
secretion.* The extract is therefore liable to cause two effects which are
antagonistic to one another. The most reasonable explanation of this is
afforded by the supposition that the gland contains not only a substance
which stimulates the kidney cells to activity but also another substance
which depresses their activity, and this to so great an extent in certain
cases that the kidney ceases to secrete, although all the vascular conditions
for urine secretion are of the most favourable character. Nevertheless, the
secretory substance usually ultimately proves the more potent: or it may be
that the kidney cells are more susceptible to its influence.

* These facts are illustrated by several of the tracings given in the paper in the

‘Phil. Trans.’ for 1906 by Herring and myself. This paper deals exclusively with the
effects upon the kidney and urine-flow of extracts of pituitary.

Yy. 449

tutary Bod

The Functions of the Pi

1909.]

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aunyoa haupyy

450 Prof. E. A. Schafer. [July 22,

These two substances are not the same as those which influence the blood-
vessels of the kidney, which may also be affected in opposite ways. For
although the most striking effect upon kidney volume is to produce augmen-
tation, this augmentation is in a large number of cases preceded by a
temporary diminution. But the temporary diminution of volume of the
kidney is not the cause of the temporary diminution or cessation of urine-
flow, which, as above noted, is frequently seen as the first effect of an intra-
venous injection ; for the diminution (or cessation) of flow may last long after
the diminution of volume has disappeared, and even after this has become
replaced by a large augmentation of volume.*

Finally, an important fact in the physiology of the pituitary body is that
which was first satisfactorily determined from the experiments of Paulesco
(1907), viz., that this organ, small though it is (it weighs about $ gramme in
man), is essential to life. Animals from which it is removed were found by
Paulesco inevitably to die, usually within 48 hours—indeed, sometimes
within 24 hours, although in others the fatal result was delayed to three or
four days. Previous observers had obtained contradictory results, some
denying that the removal or destruction of the gland produces any appreciable
result (Friedmann and Maas, 1900, Lo Monacho and Van Rynberg, 1901),
others averring that it is fatal but that life may be prolonged after removal for
several days or weeks (Marinesco, 1892, Vassale and Sacchi, 1892, Narbutt,
1903). But the methods which were adopted before Paulesco’s work was
published were not calculated to inspire confidence that the removal of the
gland was complete, since in them attempts were made to arrive at the
situation of the pituitary either through the base of the skull or through the
vertex. By both methods the difficulties of the operation are very great, as
are the liabilities to hemorrhage and to injury of adjacent parts of the brain.
Moreover, no clear view can be obtained of the gland by those methods, and
the operator works largely in the dark. Paulesco arrives at the gland from
the side through the temporal bone, which is removed freely on both sides,
an incision also being made in the dura mater. Through this on the one side
a retractor is introduced, and the side of the brain gently elevated until the
reddish-yellow pituitary body is seen lying in the sella turcica. It can, in the
dog, easily be shelled out of this with a small curette, and the brain may then

* These observations of Herring and myself upon the effects of pituitary extracts upon
the renal blood-vessels and on the secretory functions of the kidney have been confirmed
by J. Pal (1909), who states, however, that the dilatation effect is produced on the
peripheral branches only of the renal arteries, the main trunks participating in the
constriction which is produced in the vessels generally. Pal also finds that the coronary
vessels participate in the general constriction of blood-vessels caused by pituitary extract,
which in this respect also differs from suprarenal extract.

1909. | The Functions of the Pitwtary Body. 451

be allowed to resume its normal position and the wound closed. The animals
show, on recovery from the anesthetic, at first, as a rule, no adverse symptoms
and take their food readily ; but in the course of the second day they begin to
exhibit lassitude, and they die, without any very clear cause, within 48 hours.
Paulesco performed numerous experiments on various animals belonging to
different classes of vertebrata, always with a similar result. Control experi-
ments in which there was a rehearsal of the whole operation, but without
actual removal of the pituitary, produced no effect; death, therefore, must
have resulted from the removal of this body.

Similar experiments have been carried out in the Hunterian Laboratory
of the Johns Hopkins University, by Harvey Cushing, in conjunction with
Reford (1909). Their experiments have been entirely on dogs, altogether 16
in number.* The method employed is in the main that used by Paulesco,
but with one or two improvements of technique. The results of complete
extirpation were uniform and entirely confirm Paulesco’s conclusion. Livon
(1909) has also performed confirmatory experiments.f

These experiments exhibit the serious danger that lurks in any proposal for
entire removal of the pituitary for tumours. It is clearly necessary that some
portion be left in order that the functions of the gland, which are essential to
life, may be carried on. For it. has not been found possible either by
grafting pituitary or by feeding with pituitary substance to sustain life after
removal of this organ—as in the otherwise analogous case of complete
thyroidectomy.

It is not yet known which part of the pituitary is essential to life. It is
almost impossible to remove one part alone, so closely are they dovetailed
into one another; indeed, the pars nervosaand pars intermedia are in direct
continuity, and both are almost completely enclosed within the pars anterior.

Paulesco states that the mere separation of the pars nervosa from the
infundibulum has sometimes proved equally fatal with the actual removal of
the gland. In view of the discovery by Herring that the secretion of the
pars intermedia discharges through the pars nervosa into the infundibulum,
this statement of Paulesco is of great interest. But it still requires
confirmation.

* Cushing has since reported 100 cases of total or partial hypophysectomy, ‘ Journ
Amer. Med. Assoc.,’ July 24, 1909.—[Note added August 3, 1909.]

+ It has recently been denied by Fichera (1906) and by Gemelli (1908) that destruc-
tion of the hypophysis is followed by a fatal result. I am convinced, however, from my
own experience, that in such cases the destruction could not have been complete. My
experiments in this direction, although less numerous, confirm those of Paulesco and of
Cushing and Reford, but I have not thought it necessary in the present communication
to refer to them in detail.

A452 Prof. E. A. Schiifer. [July 22,

Masay (1908) has endeavoured to produce “ pituitary insufficience,”
i.e. a diminution or interference with the functions of the gland, by
preparing an antiserum or cytotoxine by intraperitoneal injection of
a guinea-pig with an emulsion of dog’s pituitary at intervals of two days,
and after five injections collecting the blood of the guinea-pig, centrifugalising
it, and injecting the serum (about 10 cc.) under the skin of a dog. After
two or three such injections the dogs, according to Masay, show symptoms
which he interprets as due to pituitary insufficience, viz. loss of flesh,
muscular weakness, especially in hind limbs, and modifications in the
skeleton, accompanied by histological changes in the pituitary ; the symptoms
constituting, according to Masay, a veritable cachexia hypophysipriva.
But such experiments require to be multiplied and carefully controlled before
the results can be accepted as produced by changes in the pituitary body.

Various views other than those here set forth have been taken regarding the functions
of the pituitary. Cyon, who was one of the first to study the effects of intravenous
injections of pituitary extracts, considers that the gland secretes several active substances,
one of which in particular acts upon the regulator nerves of the heart—especially the
vagus—increasing the force of the beats and slowing the action of that organ, this being
accompanied by a raising of blood-pressure ; an action somewhat like that of muscarine.
Cyon also states that direct excitation of the gland im situ, whether electrical or
mechanical, is capable of producing effects of a similar character to intravenous injection.
According to Pirrone (1903) and to Livon (1908) these effects are not due to excitation of
the hypophysis but of adjacent parts of the brain or its membranes. But Cyon states
that after extirpation of the pituitary the effects are not got ; nor according to him is
a rise of blood-pressure obtained in the carotids on compressing the aorta in a hypophy-
sectomised animal. Masay (1908) has repeated these experiments and obtained results
similar to those of Cyon, but was inclined to attribute them to operative shock, although
recognising that this explanation offers difficulties. Cyon believes that the pituitary is
an organ which is closely inter-related to the thyroid, being set in activity by differences
of pressure within the skull, and influencing the flow of blood to the brain through the
thyroid.

Rogowitz (1889) and others have regarded the pituitary as supplementary or vicarious
in its functions to the thyroid apparatus, this term including the parathyroids, but it is
difficult to reconcile this view with the results yielded by experiments on the effects of
extracts of the two glands and on the results of extirpation.

An antitoxic function has also been ascribed to the organ, certain observers looking
upon the gland as destined to neutralise poisons of bacterial origin or even poisonous
substances produced by the tissues. This view was suggested, but apparently afterwards
relinquished by Marie, and has been upheld by Guerrini (1904), Gemelli (1906), and
Thaon (1907), who describe structural appearances in the pituitary after poisoning with
bacterial products and with certain drugs which they regard as evidences of a functional
reaction or hyperactivity. Such a conclusion does not, however, appear to be justified
by the facts observed.

It seems, at any rate, clear that we must look upon the anterior lobe as
different in function from the posterior lobe (including the pars intermedia),
and it is advisable to study these parts as far as possible separately.

1909. | The Functions of the Pituitary Body. 453

PRESENT OBSERVATIONS.

During the last two years I have been engaged in attempting to elucidate
the question of the function of the several parts of the organ. Dr. W. Cramer
at first, and more lately Dr. H. Pringle, have materially assisted me in
chemical examinations connected with the research, and Dr. Pringle has
given me help in various other ways, including the histological examination
of the pituitaries. I am indebted to Messrs. Burroughs, Wellcome, and Co.
for a supply of material in the shape of pituitaries in both the fresh and
prepared condition.

Most of the pituitary material used has been obtained from fresh ox
pituitaries which had been kept for a few days (while being collected and
sent) in a bottle with chloroform. Each gland was then taken, its connec-
tive tissue capsule removed, and the larger anterior part separated from the
much smaller posterior part—the latter including also the pars intermedia.
The separated portions were spread thinly on glass and dried thoroughly on
a warm plate at a temperature of about 40° C., and in this state were
powdered and preserved.

ANTERIOR LOBE.

Feeding Experiments.

With this material a series of feeding experiments has been planned and
in part carried out on white rats, both young and adult, the animals being
usually kept three or four together in a cage, with a similar number,
generally from the same litter, in a second cage as a control. The cages are
contrived so that the urine and feces are separately collected, and are
contaminated as little as possible with the food, which has consisted of bread
and milk in a certain constant proportion made into the consistency of
a thick paste. This paste is placed within a cylindrical beaker. Upon the
top of the paste, but within the beaker, rests a heavy metal disk with
a central hole large enough for the snout of a rat to pass through. By this
means the animals are permitted to feed at any time, the metal ring always
falling as the food decreases, so that the rats cannot scatter the food over
the cage as otherwise they are apt to do. The amount of food consumed was
determined (in some of the experiments) each day by weighing the beaker and
its contents. The urine was allowed to accumulate in a vessel containing
abundance of thymol for a regular number of days—usually four or seven—
and was then examined for amount, reaction, specific gravity, percentage of
urea, and in some cases for phosphorus content. The feces were removed
daily and reserved when it appeared necessary; they showed no difference

VOL. LXXXI.—B. 2 1

454 Prof. E. A. Schiifer. [July 22,

obvious to the eye between those of the pituitary-fed and control animals.
The pituitary-fed rats received with their bread and milk a‘ certain small but
constant amount of the dried gland, either from the anterior or from the
posterior lobe, the proportion of pituitary substance to the bread and milk
food being extremely small. The control animals were fed and kept
in a completely similar fashion, except that in place of pituitary an equal
amount of the dry powdered substance of some other gland—usually testicle
or ovary—was added to the bread and milk. The weight of the animals was
regularly recorded.

Some of the details of one experiment performed in this manner may
here be given. A litter of eight rats was taken immediately after being
weaned and divided into two groups (A and B) of four each; the two groups
being, as it happened, of exactly the same weight. All were males with the
exception of one of the rats of Group B. Both groups were at first put
upon the bread and milk diet alone for four days; it was found that during
this period Group B tended to increase in weight slightly faster than
Group A. To the diet of Group A was then added a small constant amount
of the dry powdered material derived from the anterior lobe of ox
pituitaries. Group B, which were kept as controls, received in place of this
an approximately equal amount of dried powdered material prepared from
the testicle; for which was, later, substituted a similar material prepared
from ovaries.* At an advanced period of the experiment a small amount of
powdered material derived from the posterior lobe of the pituitary was
mixed with the powdered anterior lobe which was being given to Group A.
The experiment was started on March 4 of this year and terminated on
June 3.

At the commencement of the experiment the average weight of each rat
was 44:25 grammes in each group. A week before the termination of the
experiment (7.¢. on May 27) one rat of Group A and the female of Group B
were killed. At this time the average weight of each rat of Group A was
160 grammes; of each rat of Group B, 131 grammes. After eliminating
the female, which weighed only 113°5 grammes, and one of the (male) rats of
Group A, which weighed 170 grammes, the three remaining (male) animals
belonging to Group A were found to average 165 grammes; while the three
remaining (male) animals belonging to Group B averaged 142 grammes.

At the termination of the experiment the remaining animals, which were
all healthy, vigorous males, were killed and X-ray photographs were taken of
them. These have yet to be examined and measured.

* The amount of calcium in the substances given to the two groups was nearly the
same : if anything the balance was against that given to Group A.

The Functions of the Pitwtary Body. 455

1909.]

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456 Prof. E. A. Schifer. [July 22,

The experiment is here set out in tabular form and some of the results
are given in the form of charts (figs. 2 and 3). In the table the total weight
of the animals in each group, the total amount of urine collected in the
given time, its reaction and specific gravity, and the percentage of urea are
shown.

The chart (fig. 2) shows the relative rate of increment of weight of
Groups A and B, the dates being marked upon the abscissa at proportionate

Grammes

Dates4 8 I2 16 20 24 28 1 4 8 15 22 29 6 B 20 27
March April May

Fic. 2.—Chart showing Rate of Growth of two groups of Rats from same litter. Group A
with addition of anterior lobe of pituitary to food ; Group B, controls. The addition
was commenced on March 8.

intervals, whilst the weights in grammes are given as ordinates. The
drop in weight from May 6 to May 13 was due to the fact that the
feeding of the animals during part of that time was restricted to only
two hours a day. This chart shows that whereas before the pituitary
feeding began there was a tendency for the rats belonging to Group A
to increase in weight less rapidly than those of Group B, after eight
days of the addition of pituitary to the food of A these show a steady
increase on Group B, the increase being continued up to the end of the
experiment.

The chart (fig. 3) shows the relative amount of urine per diem in the two
groups, calculated per kilogramme weight of animal. The ordinates

1909. ] The Functions of the Pituitary Body. 457

represent cubic centimetres: the dates as before are marked proportionately
on the abscissa. The correspondence between the two curves is almost
complete: the fluctuations, which are considerable, and are probably caused
by varying meteorological conditions—especially, perhaps, temperature—

r diem.
Leal
o
le)

H
Nv
°

me and pe
lee!
H
o

80

7oO

Urine in c.c. per kilogram
wo
fo)

Dates+ 8 IZ 16 20 24 28 I 8 15 22 29 6 B 20 27
March April May

Fie. 3.—Chart of Urine Secretion of the Groups A and B.

synchronise in a singular manner :* only towards the end of the experiment
and after the animals of Group A had received a certain small proportion of
posterior lobe, intermingled with the dried anterior lobe powder which they
had previously been taking, is there any distinct difference in the relative
amount of urine per gramme weight of animal, and even this does not show
itself immediately.

The fluctuations in the urea excretion correspond on the whole with those
of the amount of urine, but there was less urea excreted in the pituitary-fed
animals than in the controls.

This experiment is given to show the manner in which we are
endeavouring to approach the problem as to the effects of pituitary feeding
upon growth rather than with the intention of drawing positive conclusions
from it. We must await the result of other experiments still in progress

* The necessity of working with a duplicate set of control animals is well illustrated
by the curves in this experiment. Without such control the very considerable fluctua-

tions, which are probably due to meteorological conditions, might easily be misinterpreted
as effects contingent on the experiment.

458 Prof. E. A. Schifer. [July 22,

and projected before attempting to decide whether pituitary feeding has the
distinct influence upon growth which might be inferred from this
experiment.

Here the observations of Cerletti (1907) and of Sandri (1907) may be briefly
referred to.

Cerletti injected young animals (intraperitoneally). As the result he found that the
bones of the avimals receiving the pituitary emulsion were after some time, as compared
with controls, somewhat shorter as regards the diaphyses but larger as regards the
epiphyses. If Cerletti’s results are to be accepted, they appear to indicate if anything
a retardation in growth of the bones, at any rate in the direction of length. Sandri fed
young mice with pituitary—apparently with the pure crude gland to the exclusion of
other food. He states that this caused an arrest of growth, but there seem to have been
no controls made with food of a similar percentage composition. Sandri also injected
young guinea-pigs with an emulsion of the gland, and found that in these also growth
was diminished.

Our experiments have certainly not shown any arrest of growth as the result of
pituitary feeding.

Grafting Experiments.

Another method by which we are endeavouring to investigate the effects
of pituitary secretion upon growth is that of implanting pituitaries of other
individuals of the same species in various parts of the body, such as the
brain, the subcutaneous tissue, the muscular tissue, the peritoneal cavity,
and the kidneys. But so far these experiments have failed to throw any
clear light on the question by reason of the fact that we have not in
any case obtained a permanent graft of the implanted organ. The chief
result which has been noted is a temporary increase in the amount of urine
secreted, a result due either to absorption of the diuretic substance which
the transplanted pituitary contained, or, perhaps, to a temporary functioning
of the implanted organ preceding its degeneration. All the animals in
which this attempt to implant the pituitary has been made—including dogs,
cats, monkeys, and rats—have remained healthy and have been killed after
a certain lapse of time. In no case have we been able on post-mortem
examination to substantiate the presence of the characteristic epithelial
structure of the pituitary at the site of implantation. In one experiment,
which is still in progress and a chart of which is appended (fig. 4), the
animals (rats) with pituitary grafts grew at first at exactly the same rate
as the controls of the same litter and sex. But after three weeks the
controls for some unexplained reason lost weight for a few days, and have
hardly as yet managed to catch the others up.

The chart of urine-secretion (fig. 5) follows almost exactly the same course
in the two groups: the effect of the implantation in these animals was
therefore to all appearance nil.

The Functions of the Pituitary Body.

459

Ir 18 25 3 8 15 22
June

6 3
July

Fie. 4.—Chart showing Rate of Growth of two groups of Rats, A? and B?, of the same
litter, A? with attempted pituitary implantations ; B? controls.
were made on May 4.

per week
H H
hm d

oa ©
QS

The implantations

mal &
8
}

Urine in c.c. per kilog. weight of ani

boa 18 25 I 8 15 22
June

Fia, 5.—Chart of Urine Secretion of the Groups A? and B?.

6
July

460 Prof. E. A. Schiifer. [July 22,

PostTERIOR LOBE.

The experiments hitherto mentioned have been concerned mainly with an
attempt to elucidate the functions of the anterior lobe of the pituitary ;
those that we have next to consider had mainly in view the diuretic
properties of the secretion of the gland, which former investigations have
shown to be most probably connected with the posterior lobe, at least with
the pars intermedia. The experiments on this subject may be referred to
under three heads, viz., feeding, grafting, and stimulation by injury.

Feeding Experiments.

There seems to be little doubt that the exhibition by the mouth of an
active water-extract of the posterior lobe may greatly increase the amount
of urine secreted. We have frequently, although not invariably, obtained
such increase in animals; where it has failed to oceur—as, indeed, may
happen also with intravenous injection—the failure is probably due to some
special condition in the individual which renders him irresponsive to the
excess of pituitary substance which is passing into the blood. What this
condition may be we need not stop to inquire, but it is noteworthy that the
activity of the gland in promoting diuresis often appears greatest in cases
in which the amount of urine which was previously being passed is less
than usual.

In connection with this part of the investigation, Mr. Harold Stiles was
good enough to allow an active extract of the posterior lobe of the ox
pituitary to be tested upon two children under his care, both convalescent
after operations and otherwise in good health; these may be given as
instances of the effect of the extract.

In the first of these cases, a boy, aged 10 years, during a period prior to the
exhibition of the extract, was secreting an average of 28 fl. oz. of urine per
diem, while during and immediately after the period that the extract was
being administered the average secretion was 38 fl. oz.

In the second case, that of a girl, aged 94 years, the average amount of
urine in the period prior to the administration was unusually low, viz., only
9 fl. oz. per diem, whereas during and immediately after the period of
administration it rose to an average of 29 fl. oz., and was one day as much as
35 fl. oz.

Instances of marked increase of urine-secretion as the result of the
clinical administration of pituitary extract exist in the literature of the
gland. Marinesco (1895) gives the results of the treatment of three cases
of acromegaly with tablets of pituitary. In the first the average amount of

1909. | The Functions of the Pituitary Body. 461

urine was increased from about 1 litre to 14 to 2 litres per diem; in the
second from 1100 to 1300 c.c.; and in the third case, which was already
diabetic, the increase was from 16 to 21 litres. J. Azam (1908) observed a
marked diuresis as one of the effects of administration of 0°3 to 0-4 gramme
ox pituitary in cases of infectious fevers.

_ The accompanying chart (fig. 6) of a feeding experiment upon rats may
also be here given in illustration of the diuretic effect of pituitary feeding.

©
°

@
fe)

Ww
°

Dates 21 28 1 8 15° 22 29 6
May June July

Fie. 6.—Chart of Urine Secretion of two full-grown Male Rats, A‘ and Bi‘, fed on bread
and milk, to which in the case of A‘a small addition of dry sheep’s pituitary was
made on and after June 1, while B* was given as a control a similar addition of dry
testicular substance.

The chart shows the amount of urine in cubic centimetres per kilogramme
and per diem secreted prior to and during the administration (along with
the ordinary bread and milk food) of a small amount of sheep’s pituitary.
In this case the whole gland was used, a little of the dry powder, which had
been kept for several years, being added to the food. Two large male rats
were chosen of about the same weight (250 grammes), one for use as a
control. To the food of this one an equal amount of dry testicular substance
was added during the time that the other one was receiving pituitary sub-
stance. A preliminary observation was first made, extending over rather more
than two weeks, no addition being made to the ordinary food. As a result
of this it was found that one of the two rats secreted rather less urine per
kilogramme body-weight than the other. This one (A*) was selected for the
pituitary addition, and the other (B+) for the control. During the first week
of pituitary feeding the curve of the urine secretion of A* is approaching
that of B+; during the second week it crosses it,and in the subsequent weeks
it maintains a higher position, so that in place of secreting about 25 per
cent. less urine than B*, as was the case before the feeding with pituitary

462 Prof. E. A. Schiifer. [July 22,

began, it now secretes about 25 per cent. more. The same fact is illustrated
towards the termination of the experiment which is illustrated by the chart
given in fig. 2.

Grafting Experiments.

The effects upon the secretion of urine of grafting pituitary have already
been referred to in connection with growth, but it may be of interest to
record some of the results on urine-secretion which have been yielded in our
attempts to effect the implantation.

A cat, which was passing, prior to the operation, 207 cc. of urine per
diem (average of 15 days), was found to pass during the 15 days succeeding
the implantation of the pituitary of another cat into its peritoneal cavity
an average of 276-c.c., the greatest increase being during the first week after
the operation. In another cat the average amounts were 180 c.c. (before)
and 233 c.c. (after). In a monkey the amounts recorded were 202 c.c. and
255 cc. respectively—for a daily average during 16 days before and after
the implantation. Of two rabbits operated upon in this way, the effect pro-
duced in one was hardly noticeable, but in the other the amount of urine rose

170

150

100

Urine in c.c. per kilog. and per diem.

50
Dates 5 ? IF LT ZZ 5 Zo
Marc! April
Fie. 7.—Chart of Urine Secretion of two groups of full-grown Rats (three in each group) :
Group A’ with pituitary implantation into muscles of back; Group B, controls.
The implantations were made on March 9.

1909. | The Functions of the Pituitary Body. 463

from an average of 50 c.c. before the implantation to an average of over
100 e.c., and on one day to 150 e.c. during the week following.

The result was in all these cases temporary, and the effect of implantation
upon the urine gradually disappeared, no doubt coincidently with the
destruction and absorption of the implanted tissue, which, as I have already
mentioned, we failed to find on post-mortem examination.

Similar results have been obtained with rats. Thus in one experiment a
graft of pituitary was made into the muscles of the back in three full-grown
rats, three others of similar dimensions being employed as a control. The
amount of urine passed before the operation was rather more in the control
animals than in those selected for the implantation. After the grafting the
amount of urine per gramme weight of animal showed a decided increase,
and this increase persisted for some weeks. This is shown in the accom-
panying chart (fig. 7),in which the curve A! shows ‘the amount of urine in
cubic centimetres per kilogramme weight of the pituitary-grafted animals,
and curve B! the amount in the controls. The urine was collected at first
every four days, subsequently with a seven days’ interval.

Stimulation of Piturtary by Injury.

With the view of determining what effect injury to the pituitary might
have upon the performance of its functions, we have in some animals exposed
the gland by the method of Paulesco and subjected it either to mechanical
injury or to partial destruction by means of a feeble thermo-cautery. Before
the operation the animals were all approximately in nutritive equilibrium.
The results are striking, and may be illustrated by giving the main results
of three experiments, all on dogs.

(a) A dog weighing 5°5 kilogrammes and taking each day 180 grammes of
dog biscuit was passing, immediately before the operation, from 30 to 40 ce.
of urine per diem. The pituitary was subjected to partial injury by means
of a warm, but by no means hot, electro-cautery ; the animal recovered
without a bad symptom, except that during one or two days it was thought
to turn towards the operated side in walking. The day after the operation
40 cc. of urine was collected; the next day 180 c.c.; the next 230 cc;
the next 102 c.c., the amount gradually coming down, although an average
amount of 114 ¢.c. was maintained during the whole period of 19 days that
the animal was kept alive, whereas the average of the 19 days prior to the
operation was 54 c.c. It may be noted that, contrary to what occurred in
the other cases, the amount of water consumed was in this case greater during
the after period than during the period prior to the operation.

464 Prof. E. A. Schiifer. [July 22,

(0) A dog weighing 9°5 kilogrammes was passing, during 11 days prior to
the operation (which was similar to the last), 110 cc. urine per diem.
During the 11 succeeding days the average was 182 c.c., and the increase was
maintained until the animal was killed 10 days subsequently. The greatest.
amounts passed were on the third, fourth, and fifth days after the operation,
the amounts on these days averaging 266 c.c. The average daily amount
of water taken for the 11 days before operation was 400 c.c., for the 11 days.
after operation 310 c.c.

(c) A dog weighing about 6 kilogrammes had its pituitary exposed by the
method of Paulesco and Cushing, and with a blunt instrument the gland
was mechanically injured and partially broken. During the four days.
preceding the operation the average daily amount of urine secreted was
119 ec. During the four days subsequent to the operation the average
amount was 192 c¢.c. After the fourth day in this case the average fell to a.
normal amount. The average daily amount of water taken during the
four-day periods was 322 e.c. before and 235 c.c. after the operation.

(d) Another dog weighing about 9 kilogrammes was subjected to exactly
the same operative procedure, the brain being exposed from the side and
raised so as to bring the pituitary body freely into view. But the gland
was not touched nor intentionally injured in this case. The wound was
closed in the same manner as in the dogs in which the pituitary had been
mechanically injured. The polyuria which was displayed in the other three
cases was only exhibited on the second and to a less extent on the third day
in this dog, and this is probably to be accounted for by the fact that the
animal refused its ordinary diet of dog biscuit and water on the day
following the operation and was given milk instead: of this it consumed
450 c.c., whereas the amount of water which it was in the habit of taking
with the biscuit rarely exceeded 200 cc.

The microscopical examination of the pituitary in the first two dogs
(a and 6) reveals no serious injury, but in both blood is extravasated into
the central cavity, and there is marked increase of the colloid secretion of
the pars intermedia—which previous experiments have shown to be probably
associated with the diuretic function of this gland. The pituitary of the
third dog has yet to be examined, but there is no doubt about its having
been injured.

The bearing of these results upon the polyuria which occurs in injuries.
and tumours affecting the base of the brain is of considerable clinical
interest. Such cases are well known to surgeons and physicians, and are
frequently recorded. The polyuria and glycosuria have generally been set:
down to injury of a hypothetical centre at the base of the brain. Even

1909. | The Functions of the Pituitary Body. 465

when associated, as these symptoms often are in acromegaly, with tumours
of the pituitary, they have not been usually ascribed to an increased activity
of that gland.* Indeed, in many cases of acromegaly, polyuria does not
occur. Doubtless this is due to the hypertrophy and increased activity
being confined to the anterior lobe, which is the part usually involved in
this disease. Often it does not occur until the disease is more advanced,
and may then be due either to the hypertrophy involving the pars
intermedia or to this part being stimulated mechanically by the adjacent
growth.

Conclusions.

1. The pituitary body consists of three parts: (1) the pars anterior,
formed of vascular glandular epithelium ; (2) the pars intermedia, formed of
a less vascular epithelium secreting “colloid”; (3) the pars nervosa,
consisting mainly of neuroglia, but invaded by the colloid of the pars
intermedia, which passes through it into the infundibulum of the third
ventricle. These parts differ from one another in function.

2. The function of the pars anterior is probably related to growth of
the skeletal tissues, including cartilage, bone, and connective tissue in
general. The chief evidence in favour of this is derived from the fact that
hypertrophy of the pars anterior is associated with overgrowth of the skeleton
and of the connective tissue in growing individuals, and of the connective
tissues especially in individuals in whom growth has ceased. These effects
are probably produced by hormones.

3. The function of the pars intermedia is to produce a “ colloid” material
which contains active principles or hormones acting upon the heart, blood-
vessels, and kidneys. Probably there are several such hormones acting upon
blood-vessels and kidneys independently, and also acting antagonistically ; so
that according to circumstances either a rise or fall of blood-pressure, an
increased or diminished secretion of urine, may be produced, and the effects
on the kidney may be independent of those on the blood-vessels. The
hormones. which appear to be most active are those which produce contraction
of the blood-vessels in general, with dilatation of the renal vessels and

* Rosenbaupt (1903), who describes a tumour of the pituitary associated with poly-
uria, states that he is “loth to assume that this is due to the pituitary tumour, since
there are no physiological grounds to support such a view.” Arid Steinberg (1897)
remarks that in the “die alte Casuistik” it was not uncommon to associate pituitary
tumours with polyuria and glycosuria, but that it is more probable that the latter
symptoms are due to an overlooked condition of acromegaly. More recently, Borchardt
(1908) has suggested that the glycosuria which is so often recorded in acromegaly may be

associated with the hypertrophy of the pituitary, but does not especially connect it with
the pars intermedia.

466 Prof. E. A. Schifer. [July 22,

increased activity of renal cells, but there appear to be others which cause
constriction of renal vessels and diminished activity of renal cells; the effects
of these latter are generally less lasting. There is also usually an inhibitory
effect produced on the heart.

4. Extirpation of the pituitary body is incompatible with survival during
more than two or three days. Injury of the organ when not extensive
causes no pronounced symptoms other than increased secretion of urine,
which is accompanied by increased production of colloid by the pars
intermedia. Complete removal of a pituitary tumour in man should not be
attempted, since entire removal of the gland would in all probability be
speedily fatal. ;

5. Acromegaly and gigantism appear to be due to an increase of function
of the anterior lobe alone. It is this lobe which is always in the first
instance hypertrophied in those affections. If the posterior lobe is involved
polyuria is likely to result. The fatal termination which ultimately occurs
in acromegaly—but which may be long deferred—is probably associated with
a change in the nature of the tumour, which from being a mere glandular
hyperplasia becomes of a sarcomatous nature, while the normal tissue becomes
destroyed.

6. The addition of a small but regular amount of pituitary substance to
the food produces an increase in the amount of urine secreted. This effect is
obtained from the pars intermedia and posterior lobe, not from the anterior.
Implantation of the pituitary of another individual of the same species may
produce a similar effect on the urine, causing an increase of secretion which
may last a short time but soon disappears.

7. The addition of a small amount of pituitary substance to the food
appears to favour the growth of young animals: it does not impede or
restrict their growth. The attempts at implantation of pituitary in young
animals have not in these experiments been followed by any deterioration in
growth as compared with controls; if anything, there are signs of improved
nutrition. But we have not succeeded in establishing permanent grafts,
and any result which might be looked for could only be of a temporary
character.

LITERATURE.

A very full biblidgraphy of the subject of the pituitary body and of acromegaly in
relation to it is given by Masay in his thesis (see below), published in 1908. The follow-
ing papers are more particularly referred to in the preceding pages—some of these are
subsequent to Masay’s thesis :—

Azam, J. Thése, Paris, 1908.
Ballet and Laignal-Lavestine. ‘Nouv. icon. d. 1. Salpétriére,’ 1905. (Case of acro-
megaly associated with enlargement of anterior lobe only.)

1909. | The Functions of the Pituitary Body. 467

Borchardt. ‘ Zeitschr. f. klin. Med.,’ vol. 66, 1908. (Glycosuria in acromegaly is asso-
ciated with, and is due to, the hypertrophy of the pituitary.)

Boyce and Beadles. ‘Journ. of Pathology,’ 1892—1893.

Cagnetto, ‘ Virch. Archiv,’ vol. 176,1904. (Two cases of pituitary tumour, one without,
the other with, acromegaly : the former a destructive sarcoma.) Also ‘Arch.
p. L. sci. med.,’ 1907.

Cerletti. ‘R. Accad. d. Lincei,’ 1906 and 1908 ; alsoin ‘ Arch. ital. de biol.,’ vol. 47, 1907.

Cunningham, D. J. ‘Journ. Anat. and Physiol.’ vol. 13, 1879; ‘Trans. Roy. Irish
Academy,’ vol. 29, 1891.

Cyon. ‘ Pfliiger’s Arch.,’ vols. 71—87, 1898-1901. Also ‘Compt. rend.,’ 1907.

Fichera. ‘Lo Sperimentale,’ 1906. (Destruction of Pituitary in Fowls.)

Friedmann and Maas. ‘ Berliner klin. Wochenschr.,’ 1900.

Geddes, A. Campbell. Thesis for M.D. Degree, University of Edinburgh, 1908 (as yet
unpublished).

Gemelli. ‘Arch. p.1. sci. med.,’ vol. 30, 1906; ‘ Arch. ital. de biol.,’ vol. 50, 1908 ; ‘ Folia
Neurologica,’ 1908.

Guerrini. ‘ Riv. di patol. nerv.,’ vol. 9, 1904; ‘ Arch. d. Fisiol.,’ 1905.

Herring, P. T. ‘Quarterly Journal of Exper. Physiology,’ 1908 (several papers).

Howell. ‘Journ. Exper. Med., vol. 3, 1898.

Launis and Roy. ‘C. r. soc. biol.’ 1903. (Diabetes in tumour of pituitary associated
with acromegaly.)

Livon. ‘C. r. soc. biol.,’ 1898; ‘Journ. de Physiologie,’ 1909.

Lo Monacho and Van Rynberg. ‘R. d. r. Acc. d. Lincei,’ 1901.

Magnus and Schafer. ‘ Proc. Physiol. Soc., Journ. Physiol.,’ 1901.

Malcolm, J. ‘Journ, Physiol.,’ vol. 30,1904. (Effect of pituitary feeding on output of
N, P, Ca, and Mg.)

Marie, P. ‘ Revue de méd.,’ 1885—86 ; see also ‘ Brain,’ 1889.

Marie and Marinesco. ‘ Arch. d. méd. expér.,’ 1891.

Marinesco. ‘C. r, soc. biol.” 1892 and 1895; ‘Bull. d. 1. soc. méd. d. hépitaux de
Paris,’ 1895.

Masay, F. ‘Ann. d. 1. soc. roy. des sciences méd. et nat. de Bruxelles, 1903 ; “ L’hypo-
physe,” Thése, Bruxelles, 1908.

Narbutt. ‘Die Hypophysis Cerebri, etc.” Diss. St. Petersburg, 1903. (Abstr. in
‘Physiologiste Russe,’ vol. 5, 1907.)

Oliver and Schafer. ‘Journ. Physiol.,’ vol. 18, 1895.

Osborne and Vincent. ‘Journ. Physiol.,’ vol. 25, 1899.

Pal, J. ‘ Wiener med. Wochenschr.,’ 1909.

Paulesco. ‘Journ. de Physiol.,’ vol. 9, 1907 ; and “‘ L’hypophyse du cerveau,” 1907.

Pirrone. ‘ Riv. med.,’ 1903. (Abstr. in ‘ Review of Neurol.,’ J.)

Redslob. ‘Klin. Monatschr. f. Augenh., 1905. (Case of fracture of base of skull with
diabetes insipidus. )

Reford and Harvey Cushing. ‘Johns Hopkins Bulletin, vol. 20, 1909.

Rogowitsch. ‘ Ziegler’s Beitrige,’ vol. 4, 1889.

Rosenhaupt. ‘ Berliner klin. Wochenschr.,’ 1903.

Salvioli and Carraro. ‘Arch. p. 1. sci. med.,’ vol. 31, 1907. (Action of extracts injected
intravenously.)

Sandri. ‘Riv. d. patol. nerv. e ment.,’ vol. 12, 1907.

Schafer and Herring. ‘ Phil. Trans.,’ B, 1906.

Schafer and Vincent. ‘Journ. Physiol.,’ vol. 25, 1899.

Souza-Leite. “ DelAcromégalie,” These, Paris, 1890. (Translated for the New Sydenham
Society by P. 8. Hutchinson, 1891.)

468 Mr. J. H. Orton. Occurrence of Protandric [June 8,

Sternberg. “ Die Akromegalie,” 1897. (New Sydenham Society’s Translation, 1899.)
Tamburini. ‘Centralbl. f. Nervenh.,’ 1894.
Vassale and Sacchi. ‘ Riv. sper. d. fren.,’ 1892.

Widal, Roy, and Froip. ‘Rev. de méd.,’ 1906. (Case of Acromegaly without enlarge-
ment of pituitary, but with great increase of cyanophil cells and eysts.)
Woods-Hutchinson. ‘Trans. Pan-American Medical Congress,’ 1894; ‘New York

Medical Journal,’ vol. 67, 1898, and vol. 72, 1900.

On the Occurrence of Protandric Hermaphroditism in the Molluse
Crepidula fornicata.

By J. H. Orton, A.R.C.S., Marshall Scholar in the Royal College of Science,
London (Imperial College of Science and Technology).

(Communicated by Prof. Arthur Dendy, F.R.S. Received June 8,—Read
June 24, 1909.)

Introduction.

Crepidula fornicata is a streptoneurous Gastropod belonging to the
Calyptreide, a family of the Tenioglossa. It was first introduced into
England from America about 1880 (1), when it was imported with American
oysters. In America it is found on the east coast from Labrador to Florida,
but in England so far as is known, it is confined to the Essex and
Lincolnshire coasts, occurring, however, in abundance in shallow water in
the neighbourhood of the mouths of the Crouch and Blackwater rivers. The
conditions on the Essex coast seem to be highly favourable for its growth
and propagation ; indeed, so favourable, that within five or six years it has
over-run the oyster beds at West Mersea. By attaching themselves very
strongly to oyster-shells they cause the oyster fishermen much trouble, and
it may be remarked, by competing for food and oxygen with the oysters may
become a cause of much more serious trouble in the future. To obtain food
the animals raise the anterior part of their shell, and extending the head to
the front edge of the shell, move it slowly from side to side: at times the
whole shell may be similarly turned slowly round to the one side or the
other.

Crepidula fornicata is sedentary for the greater part of its life. It forms
“chains,” as Prof. Conklin calls them, by the curious habit the individuals
have of fixing themselves in linear series one on the top of another as in
fig. 1. Chains of as many as 12 individuals have been found. Viewed as a

1909.] Hermaphroditism mm Mollusc Crepidula fornicata. 469

Fie. 1.—On the left ; Dorsal View of a single Shell: a, anterior ; p, posterior. On the right;
a Postero-dextral View of a Chain attached to an Oyster-shell (SH). (# nat. size.)

A, shell of proximal individual ; B, shell of second individual, and so on. The symbols
adjacent to the shells denote the secondary sexual characters of the inhabitants (see text).

whole, a chain is seen to form a spiral of about half a turn, bending over to
the right.

On close examination of the chains it is found that when an individual settles down
upon another it places the right antero-lateral border of its shell close to or touching that
of the individual upon which it settles ; since, however, the individual shells are roughly
semi-ellipsoidal in shape, with the longitudinal axis a little concave to the right, and have
the left side a little more convex dorso-ventrally than the right, it follows that the chain
must form a right-handed spiral.

In a typical chain the twisting is accentuated by the gradual decrease observable in
the size of the shells: the shell of the bottom or “ proximal” individual being the largest,
and that of the top or “distal” individual the smallest. The “proximal” individual
always fixes the chain to some surface, as the shell of a dead or living animal, a pebble, or
a piece of broken pot or glass. In America, chains of two or three individuals are found
on the ventral surface of the King-crab (2, p. 10).

The following facts indicate that when a chain is once formed, none of the
individuals separate :— :

(1) The accurate fitting of each shell into the crevices and irregularities of the surface
or shell upon which it occurs; hence, only short periods of separation of the
individuals could be possible ; nosuch periods have, however, been observed.

(2) In soft rock the proximal individuals wear a deep impression of the edge of the
shell by the lateral movement executed in the search for food. In these cases the
surface to which the middle of the foot is attached is not worn down, so that the
animal becomes fixed on a boss of rock, which thus fits loosely into the aperture
of the shell. ;

(3) Animals detached from a surface are apparently incapable of refixing themselves
after a certain age, which I have not determined ; for, immediately an animal is
detached the sucker-like foot becomes “cupped” by a strong contraction of its
muscle fibres ; subsequent relaxation of the fibres does not seem to be possible.

(4) Prof. Conklin states that old individuals sometimes become permanently fixed by a
calcareous secretion of the foot (2, p. 11).

VOL. LXXXI.—B. 2M

470 Mr. J. H. Orton. Occurrence of Protandric [June 8,

There would seem to be no doubt, therefore, of the permanence of the chains.
All the young ones, however, are motile, moving about by alternate exten-
sions and contractions of the flat muscular foot.

Crepidula fornicata has hitherto been described as dicecious, with a
“marked sexual dimorphism” (2, p. 16), the males having been estimated
by Prof. Conklin as being on the average three-quarters the size of the
females. Those individuals were apparently called males, which had a
muscular, cylindrical, and tapering outgrowth, the penis, on the right side
of the head just behind the tentacle, as in fig. 2, ¢. Individuals having no

3g 3 9

Fig. 2.—Illustrations of the Five Categories of Individuals in C. fornicata.

The animals were taken out of their shells and drawn from life (dorsal view), the
mantle being turned back over the visceral sac. The branchial filaments are drawn only
of the male. p., penis; w., uterus; p.7., rudimentary penis ; w.7., rudimentary uterus 3.
a., anus : ant. f,, anterior part of foot ; ¢.¢., branchial filaments ; ep., epipodium ; ew. ap. u.,
external aperture of uterus; f, foot; ~, intestine; m.v., mantle vessel; 7.@., external
renal aperture ; sp.gr., sperm groove ; v.s., visceral sac. (Nat. size.)

1909.| Hermaphroditism in Mollusc Crepidula fornicata. 471.

such outgrowth on the head, but possessing a slightly spirally constricted
tube, the uterus or external part of the oviduct, projecting on the right side
of the mantle, were apparently called females (fig. 2, 2). In dissecting a
number of these animals I came across an hermaphrodite form, that is a
form possessing both penis and uterus as in fig. 2 ¢.

Before the commencement of this work, Prof. Dendy had kindly given me a large
collection of the radule of Crepidula fornicata obtained by him at West Mersea,
suggesting that I might investigate and compare the variability of the English and
American stocks with respect to radular characters. I took the matter up, but after reading
Prof. Conklin’s work (2, pp. 19 to 25), on the genus Crepidula, decided to extend the
investigation. Fresh material was obtained from West Mersea, and all of this was
preserved in order to permit of correlation of all characters.

It was thought that the chain relations of individuals, and the sex relations
in the chain would be interesting. A few entire chains were therefore
preserved, and the relative position of the individuals recorded: thus the
sex relations of the individuals in the chains were brought out as displayed
in Table I.

Table L.
Ref. No. Ac B. C. 1D), K. EF. G. H. 1

@hainyloi eee @ .., @ ©) Se ONY 0s cy ae Ont ar lar RR Ae

Pema OF si EL Fe: Were Orta Chaves Aa Olen Me ht ore RL

RS pie ORE OL Ere OWL ANON EOL aS iS". 3 3

Soe VW St ey Ee 2 fe) OO, CN Ce TE iy ene

eee Pecan Oe ae iunane sy

Sy MiGr annette” 2 Opnlostier sae eae Soe Se

In Table I the individuals ina chain are denoted by the letters, A, B,C,...
etc., the proximal individuals being denoted by A, the next to the proximal
by B, and so on. The chains therefore, read horizontally :—

Forms with a well-developed penis are represented by the symbol ¢

” ” uterus ” 3) 2
of 5 penis and uterus 3 -¢

If the chains were found to be naturally complete,* two dots are placed after the last
symbol, thus— ¢:

If the chain were doubtfully complete, one dot was placed similarly, thus— 3°

If the chain were known to be incomplete, only the symbol was used— ¢

* Usually, in a naturally complete chain, the “distal” individual is a very young one.
If, however, the distal individual is large, one can generally tell if it has had another
individual on its back by the absence of the periostracum in an elliptical area on the right
side of the shell.

bo

M 2

472 Mr. J. H. Orton. Occurrence of Protandric [June 8,

A glance at the Table shows that all the individuals at the proximal ends
of the chains are females, all the more distal individuals males, and those
in the middle of the chain hermaphrodites. Jn discussing the records with
Prof. Dendy, it occurred to us that the hermaphrodite condition appeared to
be a stage in the life-history of all the individuals, and that as the males
were the youngest, the hermaphroditism, if successive, must be protandric.
Prof. Dendy suggested that an examination of young specimens would settle
the matter. I immediately examined a collection of 48 young ones, and
found them to be all males. Subsequently, 1000 young ones, namely, motile
forms of the average size of about 1 cm., have been examined and found to be
all males.* In a few cases the penis is a mere protuberance behind the
tentacle, but as these are the smallest individuals—even larger ones requiring
microscopical examination—they are doubtless the youngest males.

Sex Relations in the Chains.

A systematic examination of the sex relations of the individuals in the
chains was begun at this stage. Observations of about 350 chains were
recorded. A sample of the records made is shown in Table V (p. 476).

At the outset of the examination, however, it was found necessary to adopt
additional categories to the g, ¢,and @ ; for all stages were found on the
one hand between ¢ forms and ¢’s with a rudimentary uterus, and on the
other hand between ¢ forms and ?’s with a rudimentary penis. The
number of stages found, however, intermediate between ¢ and ¢ was
small compared with the number of stages found intermediate between

2 and ¢.
The following arbitrary categories were adopted :—

Males with a rudimentary uterus are represented by
(WOKS Shy7S0H) 80) bs ccoeen so acoosrhoagsonoodonGsBnDE0sngequDG0D0DsGREGGR $ ur. (= uterus rudimentary).
See fig. 2, 3 u.r.
Forms intermediate between ¢ ur. and ¢ are repre-

sented by the symbol ..............csersecsceseneceeneceeees ¢ u.s. (= uterus small).
Females with a rudimentary penis are @ represented by
THOS) 2)7200) OO sngoponooooocoDSaboncnoEsNSouoIDIGD SON DBENOIBAgAq00. 2 pr. (penis rudimentary).

See fig. 2, 2 p.r.
Forms intermediate between ¢ and ? p.r. are repre-
sented by the symbol ...........c.seseceeeeeneesenceseeers ¢ p.s. (= penis small).

Occasionally individuals were found with both a small 5 p.s. (= penis small and uterus

uterus and asmall penis ; such are represented thus ia small),

In the records will be noticed here and there symbols which are bracketed,
and another symbol or series of symbols placed alongside, and enclosed with

* As determined by the possession of a penis.

1909.]| Hermaphroditism in Mollusc Crepidula fornicata. 473

these in a square bracket, thus:—[(¢)(¢)¢]. This indicates that younger
and smaller individuals settled down on members of the main or primary
chain, and formed secondary or side-chains; occasionally even tertiary chains
were found.

A comparison of the chains, even in Table V, brings out the regularity of
the positions in which the different categories of individuals occur. As
before, the ?’s are found at the bottom of the chains, the ¢’s at the top, and
the ¢’s in the middle. Between the more distal ?’s and the ?’s are found
the ? p.r.s; between the more proximal g’s and the ¢’s or distal females
are found the ¢ w.7.’s.

Hence, in a typical chain a series may be constructed to read from the top
to the bottom, thus:—¢, gur., gus, ¢, ops, 2 pr, 2.

It is interesting to note this series is just what one would expect to find if
3’s passed successively through the different stages indicated, becoming finally
?’s. It is certain, as will be shown later, that such a change does occur.

All lengths of chains occur from as many as 12 individuals in a chain down
to one. A single individual was regarded as settled if its shell fitted
accurately the irregularities of the surface upon which it was found, and if
this surface were found to be clean. It is to be remembered that the records
do not bring out the facts that in a// chains there is typically a decrease in
size from proximal to distal individuals.

In reviewing the records it is seen that a chain of a given length may be
formed by various combinations of the five sex-forms (see chains 91, 338, 340,
in Table V, p. 476), but the relative positions in the chains of individuals of
the different categories are, with rare exceptions, constant. Occasionally it
was found that an ¢ form otcurred between two @’s.

Chain Formation.

From a study of the records therefore, it would seem that chains are formed
as follows :—

A male settles down on some surface, but before another male creeps on
to its back it may pass through the series of changes from ¢ to ? as shown
in Table II. Chains of one individual may be said to be at Stage I, chains
of two individuals at Stage II, and so on.

At any time while the single male is changing into a female, another male
might creep on to its back, settle down permanently, and form a Stage II
chain. The new comer may then change to a female, and thus a Stage IT
chain might be found in any of the conditions represented in Table ITI.
Similarly, another male might settle down upon a Stage II chain at any
condition of the latter, and, changing in turn into a female, would form a

474 Mr. J. H. Orton. Occurrence of Protandric [June 8,

Stage ITI chain in one of the conditions represented in Table IV. Similarly,
Stages IV and V, and so on, would be formed, and tables could be drawn
up to indicate their different possible conditions.

Table II.*

Table III.*

| No of times
B. | found among |
records made.

No. of times

. | found among

records made.

Stage I (a) ... : 51 Stage II (a2) .../3 3b: 4
ees (DQ) cool) GS Hs 8 | 10) op (6) ..j|d um bd: 1
Le desosmurdeni(C) ears Clue | 4 Be uae eat) ae 3

DMG) a2 on | 11 » @)- sal fas Bs 12
5. (@) cal 2 | 40 Be USe)\inecal® Bo 30
GA)” tacall 3 ur 0
” (9) oon 2 g . 10
ECA) APS 2 pr 5
” (%) .../9 OF: 4
Table 1V.*
IN B. C D ee ine eas:
g records made.
Stage III (a) é é: (0)
rF (6) ... sh 3: 0)
” (c) ... 3 6: 0
“ (d) ... Pate é: 5
” (e) .. 3 3: 11
” (f) our. b : i
” (9) co} és 8
” (2) Y pr. é 8 16
” (2) 9 és 5
ee) g our 0
” (”) ° g : 3
03 (2) 2 2 pr 1
” (m) ? 2 : 1 e

* 1. In these tables the chains read horizontally and the life-histories of the individuals may
be read vertically.
2. Some of these chains occur as side-chains.

On examining the whole of the records made of the natural chains, I find
that all the conditions of chains shown in Tables II, III, and IV occur except
Stage I (0), Stage II (/f), and Stage III (a), (0), (c), and (1).
of times each condition in these stages is found in the whole of the records
is put in the right-hand column in Tables II, III, and IV. Probably the
earlier conditions of Stage III chains do not oceur because the ¢ period of life
will already have been passed by “A” individuals before more than one or
two males have been able to settle down on them. Since nearly all conditions

The number

1909.] Hermaphroditism in Mollusc Crepidula fornicata. 475

of Stages I, II, and III are found, I have no doubt that chains are formed as is
indicated above. The relative frequencies of different lengths of chains in all
the chains examined—including those in Table V—may be gathered from a
glance at the curve in fig. 3.

From fig. 3 it is seen that the longer the chains are the less frequently do

68

Frequencies.
is’)
N

—-
-———-— --—

9 10 II Ue,

I 2 3 5 6 Zl

No. of individuals.

Fic. 3.—Curve showing frequencies of different lengths of chains among 336 chains.
Ordinates = frequencies ; abscissee = individuals in chain.

they occur. Chains of 9, 10, and 11 individuals were not recorded once, and
only one chain of 12 individuals was seen.

The age of change from ¢ to 9, and the length of time required for the
formation of a chain of given length are being investigated.

Some points in the records of the chains are noteworthy. The scarcity of
3g ur.’s is noticeable. Probably their rarity may be accounted for by the
following facts.

(1) The uterus develops in the wall of the mantle, and is not visible
externally until partly developed:* only those individuals were
recorded as ¢u.7.’s in which the end of the uterus projected from the
mantle.

(2) The chains have only been examined between February and May this
year: it is possible the change may occur rapidly at some other
period of the year.

Single chains often afford evidence of the change from ¢ to ?; such
a series as 2p.7. 2p.r. iS common; moreover, in these cases a
gradual decrease in size of the penis from the ¢ to the proximal
?p.r. is also often observable; a point not brought out in the
records.

* The development of the uterus is being investigated.

[June 8,

Occurrence of Protandric

Mr. J. H. Orton.

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478 Mr. J. H. Orton. Occurrence of Protandric [June 8,

Relation of Primary to Secondary Sexual Characters.

Having established the fact that a completely continuous series in the
reduction of the penis, and correlated increase in size of the uterus occurs, it
became clear that continuity of the primary sexual characters should also be
found, Accordingly, an investigation was made with this object in view, by
means of serial sections of the gonad.

The gonad in all forms consists of two or three main tubes extending the whole
length of the visceral mass, giving off tubular diverticula, which anteriorly divide and
subdivide to form a loose compound tubular gland, but which posteriorly divide at
most a few times, or are mere blind ceca. The cells lining the tubes proliferate, and
some are shed into the lumina as germ cells—eggs or sperms.

In the male the main tubes of the gonad open into a vas deferens, from a dilatation of
which a narrow tube leads to a groove on the dorsal surface of the body. This groove runs
towards the head from the anterior end of the visceral sac to the base of the penis, and
is continuous with a groove in the latter. In the female the main tubes of the gonad
open into the oviduct. A receptaculum seminis opening into the oviduct has been
described (3). The relation of the gonad to the gonaducts in the ¢ forms is being
investigated. The colour of the gonad in the ¢ is brownish red, that of the ¢ brownish
red or orange, occasionally yellow near the uterus, that of the ? yellow.

Sections of the ¢ forms were naturally cut first, but an examination of
the gonad in all those investigated revealed nothing but ova. Sections of the
“proximal” males were then prepared, with the result that both ova and
sperms were found in the gonad. An examination of the gonad of all the
males in a chain indicates, as far as observations go, that there is a gradual
increase in ova in the gonad the nearer a male is to the most distal ? ; but
even the smallest ¢’s examined have a few ovain their gonad.* It therefore
seems doubtful whether pure males, 7c. males with only sperm in the gonad,
are ever found in Crepidula fornicata. Hence the necessity of defining the
term “male.” The term “male” is usually applied to an organism which
produces only sperm in its gonad. As, however, in most of the higher
animals a special part of the body is modified into an intromittent organ, the
presence of such an organ is adopted asa criterion of maleness. Usually this
is a fair inference. In species presenting no sexual dimorphism, such as
Amphioxus and Echinoderms, one resorts to the true criterion of maleness or
femaleness, namely the production of ova or sperms in the gonad. In
Crepidula fornicata, therefore, to be strictly accurate one should examine the
gonad of every individual possessing even a well developed penis before
committing oneself as to its sexual character. As a matter of convenience,

* Since G. Smith (4) has found ova in the gonad of the ¢’s of many species of
Crustacea, it would seem that a careful microscopic examination of the gonads of all
males, made in the light of these observations, might bring out important facts bearing
on the nature of maleness.

1909.] Hermaphroditism in Mollusc Crepidula fornicata. 479

however, I have adopted the simpler plan of calling all forms with a penis,
males; it being understood that nothing more than the presence of the
external male character is implied.

The youngest forms, however, are doubtless exclusively males as regards
function, and the oldest forms probably exclusively females.

Examination of the gonad, then, at different periods in the life-history of
Crepidula fornicata, makes clear that at first it produces ripe sperms only,
but becoming with advancing age more and more egg-producing, until finally’
it is probably entirely egg-producing. There is, therefore, no doubt that all
the individuals of this species are born as males, and change in the course of

- their life-history into females.

It is interesting to note that all stages of the gonad may occur among the
individuals of a single chain. A comparison of the primary with the
secondary sexual characters made at any phase in the life-history of an
individual shows that the development of the former is always in advance of
the latter ; indeed, the primary sexual characters forecast the secondary sexual
characters.*

It has been shown that the right antero-lateral borders of the shells of ad/
the individuals in a chain are very close together; since the penis of the
male and the external aperture of the uterus of the female are also on the
right side anteriorly, it follows that any male inachain could transfer sperms
to any female; but no such transference has yet been observed.

Crepidula fornicata appears to be the only one of the many species of the
genus which has taken to the habit of forming chains of more than two
individuals ;+ in several other species, however, namely, C. adunca, C. plana,
C. conveza, a male is often found mounted on a female(5). Crepidula
Jornicata is also the only species of the genus yet described as hermaphro-
dite, but probably other species are hermaphrodite, as will be shown below.

In Crepidula fornicata it would seem, therefore, that chain-formation and
hermaphroditism are in some way causally connected. Knowing, as we do,
that most of the genus Crepidula are sedentary in habit, and that
sedentariness is associated throughout the whole animal kingdom with
hermaphroditism ; knowing, further, that a closely allied species, Crepidula
plana, shows at least signs of, ifnot complete hermaphroditism (see below), it
would seem that chain-formation is an adaptive phenomenon, which has
arisen along with, and favoured the acquisition of, protandric hermaphro-
ditism.

* In view of the conception of a sexual formative substance (4, p. 85), this phenomenon

is not without significance.
+ See footnote, page 482.

480 Mr. J. H. Orton. Occurrence of Protandric [June 8,

Chain-formation, along with protandric hermaphroditism, effects a strict
economy of the sperm of the males, since the sperm is probably all transferred
to the females; moreover, this arrangement probably ensures cross-
fertilisation of all the females. In this respect chain-formation with
protandric hermaphroditism is an advantage over permanent hermaphroditism
with self-fertilisation, and no doubt leads to as great productiveness as would
obtain in a motile unisexual condition. Crepidula fornicata, therefore, has
become adapted to a sedentary life without losing any of the procreative
advantages of a free-living habit.

The individuals forming side-chains are almost always on the left side
of the primary chain, and therefore cut off from all sexual relations with
any ot the individuals of the latter. Here would appear at first sight to be
a mal-adaptation, but individuals settling on a primary chain are quite
comparable with those settling down on foreign objects ; if the phenomenon
is not due merely to chance, however, the former would appear to be the more
gregarious,

Occurrence of Dwarf Females.

The variation in size in this species is indeed remarkable; it has heen well
described by Prof. Conklin (5, pp. 4388 to 440). Size, besides being dependent
on the usual conditions, is also determined by the extent of the surface
of attachment. If individuals settle down on a small pebble or other surface
where expansion is impossible, they remain permanently dwarfed. Mature
dwarfed females have been found in such situations as small as 1:8 cm. long by
1 cm. wide, their shells being generally somewhat thickened, especially
around the edge. On a flat surface of unlimited extent individuals may
grow to a size of 5°5 cm. long by 3°5 em.wide. Cvrepidula fornicata is thus
able to regulate its shell-forming metabolic processes to the individual
requirements. A similar readiness to adapt itself to its situation is exhibited
by C. plana; but an even greater difference in the size of the extremes has
been observed in this species by Prof. Conklin (2, p. 12), a race of dwarfs
having been described by him as being one-thirteenth the size of the larger
forms !

The smallest females found in Crepidula fornicata are the dwarts
mentioned above: they often occur in the middle of an oyster-shell surrounded
by chains, the posterior ends of the shells of which converge on the middle of
the oyster-shell, thus preventing expansion of the enclosed individual.
Dwarf females, however, often have ¢ forms or ¢ p.r.’s fixed upon them, and
since single settled males are found quite as small, there is no doubt that
these small females have once been males and are really dwarfed. These
dwarfs, as in the case of those of C. plana, appear to be dwarfs merely by

1909.| Hermaphroditism im Mollusc Crepidula fornicata. 481

reason of their environment, being only, as Prof. Conklin provisionally calls
them, “ physiological varieties.”

In some cases chains were observed in which the proximal individuals were more or
less dwarfed, but as the posterior ends of the shells of dwarfs tend to overgrow the
surface to which they are attached, especially if such be a small pebble, the dorsal surface
of the shell of the proximal individual offers a larger surface than did the pebble, hence
the “B” individuals in such chains are able to and do grow bigger than the “A”
individuals. In such chains the largest individual is found about the middle, and is often
an ¢ orevena ¢.

Chains dredged up just off the shore at West Mersea were found to consist of larger
and more numerous individuals than those dredged up from “the Main,” about 20 miles
farther down the coast; these, however, may be phenomena arising from the usual
conditions which determine size, as food supply, temperature of medium, chemical com-
position of medium, and so on.

Sex Phenomena in Allied Species.

Prof. Conklin has made estimations of the relative average volume of the
6 ’sand 2 ’s of several species of Crepidula, obtaining the following relations :—

The males of C. plana are +4, the size of the females.

t tn
3 Cadunca >, = o .
9 C. convera. 4, + m5 “s
‘A C. fornecata ,, 4 F ss

He therefore naturally concludes that “There is then a marked sexual
dimorphism in these molluscs, the mature females being generally much
larger than the males; the females are sedentary, the males locomotive
(2, p. 16).

In another place (5, p. 441) he further states : “ In all species of Crepidula
the males are smaller than the females....” And again (5, p. 442) he
states “That in the case of the other species named (conveza, adunca,
navicelloides, plana) the males are never immovably fixed to one spot ... ,
their shells also are not distorted so as to fit irregular surfaces as is the case
with the females. In all cases locomotion is limited to small individuals. The
young of all species and both sexes crawl about freely and rapidly. In
C. convexa individuals of both sexes retain this power to a limited extent,
but the large females of adunea, navicelloides, and plana become firmly fized,
whereas the males of these species remain small and retain, to a certain extent,
their power of locomotion* .... In C. plana the shell of the male is more
nearly round than that of the female, and is usually more sharp-pointed at
the apex ... . [In a] number of individuals the older part of the shell has
the male characters, while the newer part has those of the femalet+ “In

* The italics are mine.
+ 2, p. 16.

482 Mr. J. H. Orton. Occurrence of Protandric [June 8,

such animals the penis is usually very small, and in some cases has almost
entirely disappeared. Quite a complete series of stages in the degeneration
of this organ was observed from the fully formed organ on the one hand toa
minute papilla on the other. Sections of such animals show that neither
male nor female sexual cells are produced at this time(!) The evidence seems
to favour the view that we have in these cases an example of protandric
hermaphroditism, but I am net able to assert that this is really the case,
although I have spent much time in attempting to decide it.”
From these quotations the following facts are brought to hght :—

(1) The males in all species of Crepidula are smaller on the average than
the females.

(2) The females of the species of adunea, navicelloides, and plana are fixed,
but the males are motile.

(3) The adult females and males of C. convexa are motile to a limited
extent.

In the light of the present observations on Crepidula fornicata, I have no
hesitation in concluding that C. plana is also a protandric hermaphrodite, as
Prof. Conklin suspected. It is highly probable also that the species, adunca
and navicelloides are protandric hermaphrodites, but there is not sufficient
evidence available for a judgment on C. convexa.*

A careful research on the proportions of the young males and females, and
on the sexual character of the young of the various species of Crepidula, may
bring out an interesting series of stages in the evolution of protandric

hermaphroditism.
Sex Phenomena in the Streptoneura.

It is significant that Pelseneer should remark (6, p. 124) that “sedentary
species (of Gastropods) often possess a rudimentary penis.” Stimulated by
this statement, I examined a collection of 160 Calyptrea chinensis, and found
that all the small ones, about half the number examined, were ¢’s, while
the larger ones were either ¢, ?p.7., or ¢, but were nearly all ?p.7.’s.
Since all the small ones are males, however, it would seem that this species
is also a protandric hermaphrodite. An investigation is being made of the
primary sexual characters to decide the question. It is probable, therefore,

* July 20.—Since the above was written, Mr. E. Smith has drawn my attention to a
chain of C. navicelloides (probably =dilatata), exhibited in the cases of the British
Natural History Museum. I was kindly allowed to examine the Museum collection of
Calyptreeidee, and in a collection of C. dilatata from Ancud I found the following chain—
A, 2 p.7.; B, é. In another collection of the same species from Patagonia, out of seven
individuals the three smallest were ¢’s, the others being either 2 pr. or 2. Thus
stronger evidence is adduced for the above statements.

te

1909.]| Hermaphroditism in Mollusc Crepidula fornicata. 483

that protandric hermaphroditism may be found to be much more common in
the Streptoneura than is thought at present. Pelseneer mentions 10 other
Streptoneurous hermaphrodites, one of which, Entoconcha, is known to
be protandric(6, p. 159); three others, ntocolax, LEntosiphon (7), and
Exteroxenos (8), are probably protandric. Six others occur, Valvata,
Bathysciadium, Odostomia, Cocculina, Oneidiopsis, Marsenina, of which I have
not found descriptions. C. fornicata may now be added to this list.
Hence, it would appear that one of the chief distinctions between the
Streptoneura and the Euthyneura is beginning to break down.

The sex phenomena observed in Crepidula fornicata support in a striking
‘manner G. Smith’s view (4, pp. 88, 89) of sedentarily-induced hermaphroditism,
that is, suppression of females; moreover, the genus may be reasonably
expected to offer stages in the evolution of this hermaphroditism, and so
afford a means of testing the above-mentioned view. In the early stages of
its evolution we should expect to find :—

1. A small percentage of young females among the spat.

2. Adult females of two kinds—

(a) Those born as females.
(6) Those born as males.

Recent Researches on Gametogenesis—besides the known fact that some
Tznioglossa have two kinds of spermatozoa (6, p. 125)—give some hope that
the two latter categories might be distinguishable by the cytological
characters of their gametes.

Summary.

Crepidula fornicata is a Streptoneur of the family Calyptreide.

Individuals of this species associate permanently in linear series to form
“chains.” All lengths of chain composed of upwards to as many as 12
individuals have been found.

All the young are able to creep about, but the adults are sedentary.

The individuals in a chain offer a transitional series from maleness to
femaleness both in primary and secondary sexual characters. Since all the
young ones are males, the species is a protandric hermaphrodite.

Dwarf females occur as “ physiological varieties.”

Allied species and a species of an allied genus will very likely be shown to
be protandric hermaphrodites.

There is good reason for thinking that this sex phenomenon may be even
more widely spread in the Streptoneura.

Since the males in this species change into females, it would seem in this

484

Protandric Hermaphroditism in the Mollusc, ete.

case that it 1s the male which possesses the potentialities of both sexes.
A solution to this problem is offered, if, as seems likely, allied species present
an evolutionary series in the acquisition of protandric hermaphroditism.

I wish here to express my thanks to the College authorities for the
facilities afforded me during the research. JI am also deeply indebted to
Prof. Dendy and Mr. Darbishire for important suggestions and valuable

eriticisms.

(1)
(2)
(3)
(4)

(5)
(6)
(7)
(8)

REFERENCES.

Dodd, ‘ Malacological Soc. Proc.,’ vol. 1, 1893, pp. 31, 32, and p. 19.

Conklin, ‘Journ. of Morphology,’ vol. 13, 1897, pp. 10—25.

Haller, ‘ Morphologisches Jahrbuch,’ vol. 18, 1892, p. 514.

Smith, ‘Fauna and Flora of the Gulf of Naples, Monograph XXIX, 1906,
“ Rhizocephala,” pp. 91, 92.

Conklin, ‘ Acad. Nat. Sci. Philadelphia Proc.,’ 1898, pp. 438, 441, 442.

Pelseneer, Lankester’s ‘ Treatise of Zoology,’ vol. 5, 1906, ‘Gastropoda,’ pp. 124, 125.

Koehler and Vaney, ‘ Revue Suisse de Zoologie,’ vol. 11, 1903, p. 36.

Bonnevie, ‘Zool. Jahrbiich. Anat. und Ontog.,’ vol. 15, 1902, p. 735.

485

The Elasticity of Rubber Balloons and Hollow Viscera.*
By Prof. W. A. OSBORNE, with a Note by W. SUTHERLAND.

(Communicated by Prof. J. N. Langley, F.R.S. Received July 5, 1909.)

(From the Physiological Laboratory, University of Melbourne.)

Introductory Theory.

In an elastic balloon the relation between the internal excess pressure and
the tension of the wall can be readily caleulated if we assume that the
balloon is spherical and that the material is homogeneous and of negligible
weight. If we suppose the balloon divided into two hemispheres by a plane
horizontal partition, the area of this partition will be wr? and the downward
force on the upper surface due to the excess pressure p will be wr2p. The
balloon wall meets the partition at right angles along a length 277. Hence
if T is the tension in the wall, the upward force exerted by this tension on
the partition is 27rT. But as these two forces must be equal we have

Trp — Amie iso that p= 2 0/r (1)

When such a balloon is filled without stretching the wall the pressure
inside is equal to the prevailing atmospheric, and the radius 7) may be
termed the initial radius. If we assume that the balloon is perfectly
obedient to Hooke’s law, then

T; = K (m—7)/70;
but from (1) we learn that
oe T;/ ‘1;
hence, by substitution, pi = 2K/[m—2K/1,

i)
or 4 (-=—n1) Ke (2)
To

That is to say, the pressure will increase with radius asymptotically to
2 K/7, and if we plot radius against pressure we shall obtain a rectangular
hyperbola.

The original object of the following research was to investigate the elastic
behaviour of various hollow viscera. Before doing so I decided, however, to
carry out a number of experiments with rubber balloons, using the pressures
found with varying radii as fundamental data.

* This research was completed before I became aware, from a reference in Boruttau’s
‘Lehrbuch der Medizinischen Physik,’ that a similar investigation had been carried out
by R. du Bois-Reymond, and the results published in the ‘ Festschrift fiir Rosenthal.’ On

obtaining the latter, I found sufficient difference in the treatment of the subject to
warrant publication of this. [See also A. Mallock, ‘ Roy. Soc. Proc.,’ vol. 49, p. 458.]

Olt, 109,0-0;4 18), 2N

486 Prof. W. A. Osborne and Mr. W. Sutherland. [July 5,

Methods.

The balloons were of the common variety sold as children’s toys, and were
of varying sizes. A balloon was firmly tied to a glass capillary cannula and
held vertically in a glass flask which was immersed up to the neck in an
Ostwald thermostat (fig. 1). The temperature of the latter was kept (with
a maximum variation of 0°1 C.) at 35°5 C. The glass cannula was
connected by means of fine-bore pressure-tubing to one limb of a capillary
T-piece, a second limb of which led to a water manometer, whilst the third
limb was connected with a burette for
admitting measured volumes of air. The
connecting tubes were made as short and
of as narrow bore as possible so that the
contained volume of air could be neglected
in calculation. The burette at its lower
end was connected with a levelling tube
containing mercury, and at its upper end
had a three-way tap. In one position of
the tap a sample of air of definite volume
and at atmospheric pressure could be taken
from the outside air; on the tap being
turned this air could be driven through the
connecting tubes into the balloon, the
mercury being accurately brought to the
beginning of the bore of the tap. Con-
versely, the balloon could be deflated in
measured decrements by the same burette.
The water manometer consisted of a straight
glass tube of 3 mm. bore firmly tied toa
vertical scale, and connected at its base
with a shorter vertical tube on which was

ihe. il. a mark. By means of a three-way tap,

capable of connecting the manometer either

with the outside air or with an elevated reservoir of water, the level of the

water in the shorter limb could be brought to its mark, allowing direct

readings to be made from the scale as well as preventing change in the
volume of the tube system.

The radius of the balloon was calculated from the volume of the air

admitted by the usual formula. This involved two assumptions, first that

the balloon was spherical, and secondly, that the volume of the enclosed air

1909.| Elasticity of Rubber Balloons and Hollow Viscera. 487

was the same as that of the air admitted at atmospheric pressure., With the
exception of the early stages of inflation and last stages of deflation, when
the radius approached the initial value, the balloon could be regarded
asa true sphere. As, further, the greatest pressure within the balloon was
always a negligible fraction of the prevailing atmospheric, I have not
thought it necessary to make any calculated correction as to the volume of
the contained air.

When experiments were performed on a hollow viscus, some water was
placed in the partially immersed flask, and a few drops placed in the interior
of the viscus itself so that the air within and without should be saturated
with water vapour.

Experiments on Balloons.

When air was admitted in measured increments to a fresh balloon, and
the reading taken a definite time (three minutes) after entrance of each
increment, it was found that the pressure rose quickly to a maximum and
then on continued inflation fell slowly. This is typically exemplified in the
experiment illustrated graphically in fig. 2.

24.0

Pressure in m.m. H,0.

40

I Zz 3

4 5
Radius in centimetres
Hire. 2:

It will be seen from this experiment that, over a considerable range, two
values of radius can be given for each value of pressure. This can be
demonstrated as a class experiment in the following way: Two balloons of

2N 2

488 Prof. W. A. Osborne and Mr. W. Sutherland. [July 5,

equal dimensions are tied to two limbs of a T-tube and inflated by the third
limb, which can be closed by a tap. Asa rule one of the balloons inflates
well, the other remaining small. On closing the tap in the inflating tube
the contents of one balloon can be discharged into the other by squeezing
with the hand. If the air be worked backwards and forwards a few times
to equalise the “history ” of each, it will be found that if the balloons are
approximately equal in volume they will remain so for a few seconds, in
a state of unstable equilibrium, and then one of the balloons will partially
deflate itself into the other. The balloon which is now the larger, if
squeezed until its volume is slightiy less than that of the other and then
let go, will continue to deflate until equilibrium is reached.

These experimental results appeared to be utterly at variance with what
was deducible from the theory of a perfectly elastic balloon. Amongst
the many articles dealing with the elasticity of rubber to which I had
access, I found one which promised to throw some light on my results.
O. Frank* assumes a somewhat modified Hooke’s law. According to him
the pressure dP in a sample of section g and length # associated with a
shortening dz is given by the formula

adP/q = Edz/z,
in which unit initial length and unit initial cross sectional area are not con-
sidered, but length and areasuch as they are when the change dz is produced.
If xz is the original length and a the final, he calls A = (#,—2)/z the
specific extension. For the total tension in a strip of unit width and of
initial thickness 2 his final result on p. 608 can be written

20
(1+A)?"

Substituting this value of T in equation (1), we get
_ 4EHA%
m= + AP
But in the case of the balloon the specific extension A = (71—7)/70,

therefore

IE 2) Wye

pe ene LO,

py = 4 Hzro as
According to this equation the pressure in an inflating balloon will rise
to a maximum when 7; = 27, and will approach asymptotically to zero when
7, increases indefinitely. But I may say at once that this approach to zero
pressure is never given in balloon experiments, so that Frank’s analysis
fails to explain the results obtained. One may indeed state a priori that as

* © Annalen der Physik,’ vol. 21, p. 602, 1906.

1909.|] Elasticity of Rubber Balloons and Hollow Viscera. 489

investigations on elasticity are generally confined to substances where the
maximum extension is always a small fraction of the initial length, and as
Frank’s experiments did not follow rubber further than linear extensions
to double the initial, it would be almost idle to expect that laws deduced
from these experiments could be applicable to the large and two dimensional
stretchings of an inflated balloon.

The difficulty in explaining the rise of pressure and the subsequent partial
fall on inflation is, I believe, more apparent than real. This crest is due,
I take it, to a disturbing factor which, for lack of a better name, may be
called initial rigidity. This view is supported by the following facts :—

1. If s fresh balloon is inflated, so that the pressure is anywhere on the
rise or fall of the crest, it will be found that the pressure does not remain
at a constant value, but tends to fall. In fact, to obtain a graph such as
fig. 2, the convention had to be adopted of reading the pressure after a given
interval of time—3 minutes. But the fall had by no means stopped when
the reading was taken, and could be detected even some hours after infla-
tion. An attempt to register the pressure after a long interval of time
when no further fall might be expected, failed owing to the fact that some
of the air diffused out, as was proved by deflating the balloon in measured
decrements.

2. If a balloon is inflated a second time (care being taken that the elastic
limit has not been reached in the first inflation) the crest is always less
pointed than in the first inflation. A third inflation gives a more obtuse
convexity than the second, and so on. The longer a balloon remains
collapsed the steeper is the rise and fall of pressure on inflation. This is
particularly marked if the collapsed balloon is exposed to light.

3. When an inflated balloon is deflated in measured decrements and the
corresponding pressures recorded, in the vast majority of cases the pressure
falls to zero without any rise being manifested. I obtained this pronounced
hysteresis constantly in my earlier experiments, and was inclined to look
upon it as the invariable behaviour of a balloon during deflation. Fig. 3
gives graphs for two typical instances.

But a rise of pressure may be obtained on deflation if certain conditions
are fulfilled. The rubber must be in good condition, the inflation should not
be taken far past the maximum pressure, and the return by deflation should
be carried out at once. The rise of pressure, however, is never more than a
few millimetres of water. The better condition the rubber is in the blunter
is the inflation crest and the less abrupt is the deflation fall of pressure.
Conversely, the more the rubber has been exposed in a deflated state to light
the sharper is the crest and the more abrupt is the deflation fall.

490 Prof. W. A. Osborne and Mr. W. Sutherland. [July 5,

500

400

Pressure in m.m.H20.

100

Radius in centimetres.
Fte. 3.

I may mention in this connection that if inflation be carried out
immediately after deflation the rise of pressure does not follow the same
gradient as the deflation fall. It is much steeper, and a crest may be
obtained. An illustrative specimen is the following (fig. 4) :—

It is easy to demonstrate, however, that the more a balloon is inflated and

200

2U.

0)
rs)
a
fe}
tl

H
le)
is)

Pressure in m.m.H

On
fo)

;

°
H
On

2 3
Radius in centimetres.
Fia. 4.

1909.|] Elasticity of Rubber Balloons and Hollow Viscera, 491

deflated, provided that the elastic limit is not approached too closely, the
nearer does the inflation pressure gradient approach the deflation. We
may regard this as due to the partial removal of the disturbing initial
rigidity. :

4, If a balloon be inflated until the pressure, after the usual crest, falls
and tends to remain constant, and be kept inflated for some time, say
24 hours, and then be rapidly deflated and once more inflated in measured
increments, the graph displays no crest and may be a true hyperbolic curve.
The following experiment illustrates this important fact :—

A balloon was inflated until the pressure ceased falling, and was kept
inflated in the thermostat for 24 hours. It was then rapidly deflated and
- the usual inflation by the burette commenced. On plotting pressure against
radius (fig. 5), I was struck by the regularity of the graph, and recollecting
that a balloon of perfect elasticity would give a rectangular hyperbola,

250
ro)
nu
xx
¢ 150
£
£
fe
=)
9)
ip)
p
a

ee

(o)

I 2 3 A 5 6
Radius in centimeters.
HIGas Ds

proceeded to ascertain if such were the case here. If this were a rectangula
hyperbola, the asymptotes being parallel to the co-ordinate axes, it ought to
_ satisfy the equation (7—a) (p—0) = ¢.

To calculate « and 0 I used the ordinary three-point method. The value
for a was found to be 2°8, that of 6 287°8.

From radius = 3:29 to radius =437 the product (r—a)(b—y) is a
constant. To illustrate this graphically we can plot b—y against the
reciprocal of r—a, and should obtain a straight line passing through the
origin. This is shown in fig. 6.

492 - Prof. W. A. Osborne and Mr. W. Sutherland. [July 5,

250

200)

0
: P

100

°
50
ie) Zz 4 6 8 I 12 1-4 16 18 Z 22
Reciprocal of 7-a.

Fig. 6.

As another instance of the applicability of this equation to a balloon, the
deflation values already given in fig. 3 may be cited. Calculation by the
three-point method gives here a = 2°03, b = 263.

Conclusive as these values are that the rubber balloon, when initial
rigidity is removed, follows the equation (7—«a) (p—b) =e, it will be at
once obvious, from the values of a and 6 found here, that this is certainly
not the behaviour of a perfectly elastic substance giving equation (2). For
one thing, the value for a is far removed from zero and is suggestively close
to that of the initial radius in the two cases inyestigated. I abandoned the
theoretical analysis of my results at this stage, and handed over my data on
balloons and on bladders to Mr. William Sutherland, who has kindly
complied with my request to comment upon them (see p. 497 below).

Rubber Balloons at the Elastic Limit.

In the course of this research a curious result was obtained with every
balloon which I inflated beyond the elastic limit. I invariably found that, .
before the balloon burst, the pressure, over a considerable range, was a linear
function of the volume. Of the many instances obtained I will pick out two,
one giving a close approximation to a straight line on plotting volume against
pressure (fig. 7).

One of the more divergent types is that given in fig. 8, which is a
continuation of the same experinient as fig. 2.

As a rule, the straight line rises abruptly, producing discontinuity in the
graph.

1909.| Llasticity of Rubber Balloons and Hollow Viscera. 493

500 peel —
| Burst
400 |
S
a
Be
¢ 300
£
<=
2 200
“a
rH)
o
=
a
100
S00 800 900 1000 1,100 1,200 1,300 1,4.00 1500 1600 1,100 1800
Volume in cubic centimetres.
Fie. 7.
250

bd
°
{eo}

H
on
fe)

HH
le}
°

Pressure in m.m.H, 0.

L

fo) 1,000 2,000 3,000 4,000 5,000 6000 7,000
Volume in cubic centimetres.
Fie. 8.

Experiments with Hollow Viscera

In these experiments attention was confined chiefly to the bladder, as its
shape approaches more closely to the spherical than other viscera, Experi-
ments on lungs proved impossible, owing to the remarkably low bursting
pressure of the superficial air cells. A number of observations were made
with bladders taken from the recently killed animal, but the erratic behaviour
of the living muscular tissue did not allow of a definite pressure being
assigned to any stage of inflation. Consistent results could only be obtained

by experimenting with bladders some time (24 hours) after the death of
the animal.

494 . Prof. W. A. Osborne and Mr. W. Sutherland. [July 5,

Fig. 9 shows the results of an experiment with the bladder of a large
Newfoundland dog 24 hours after death. As with the balloon, I anticipated
that here a hyperbolic curve was present, and calculated by the three-point
method the value for a to be 0:071, b to be 179. Here it will be seen that
from radius 1:93 to radius 2°88 a distinct approximation to a rectaneular

350

300

250

bd
le}
{e)

Pressure in m.m.H,0. |
GQ
re)

100

50

° I 2 3 4 5
Radius in centimetres.

Fic. 9.

hyperbola is manifest. But even here, though @ can be made zero without
appreciably altering the constancy of c, the value for 0 likewise does not
allow us to apply to this bladder the formula for a perfectly elastic
substance.

A number of bladders of various animals were investigated. I give here
the results obtained with the bladders of two monkeys and a cat (fig. 10).

It must be remembered that the elastic tissue of a viscus is not a homo-
geneous membrane, but a web of elastic fibres with a variable amount of
inextensible white fibres intermixed. This fact must always complicate
physical investigations on the elasticity of animal membranes, even if the
isolated elastic fibres present obeyed some simple physical law.* When we

* A research on the elastic constants of the ligamentum nuche is at present being
conducted in my laboratory.

1909.| Elasticity of Rubber Balloons and Hollow Viscera. 495

consider the complex xolotropism of a visceral wall, it is indeed surprising
that approximations to uniform behaviour, such as are illustrated in fig. 10,
should be shown at all.

140

100
ro)
is)
x=
= 60
£
aS
2g
ri
my 60
ry
‘=
oo

40

20

Co) 0-5 I 15 Zz 25 3 35 4
Radius in centimetres.
Fic. 10.

R. du Bois-Reymond has conjectured that in hollow viscera the pressure
may fall with increasing volume. I may state at once that I have never
found this. What sometimes does happen (and to this Du Bois-Reymond’s
statement is possibly due) is that, on extensive inflation, one of the coats of
the organ may give way and lead to a marked drop in pressure. The sudden-
ness of the drop will always indicate the true nature of the fall, and if the
organ be now deflated and then inflated again, a consistent rise of pressure
will be obtained. Moreover, as I have endeavoured to show, a fall of pressure
on continued inflation is only found in balloons manifesting initial rigidity,
and such initial rigidity is altogether absent from animal membranes kept
moist.

A bladder always displays some hysteresis on deflation, but I have found
that this hysteresis can be made negligibly small—(1) if the elastic limit is not
approached too closely ; (2) if the inflation and deflation are carried out by
very small increments and decrements respectively; and (3) if on deflation

496 Prof. W. A. Osborne and Mr. W. Sutherland. [July 5,

some time be allowed to elapse at each stage before reading the pressure, as
this always tends to rise somewhat. When the elastic limit of a bladder is
reached, the gradient of the pressure rise is very steep and the rise is not
a linear function of the volume.

There is always a danger that in investigations on elasticity one may forget
that the viscus in question in the living animal is supplied with reactive
muscle, and that only when this muscle is fully inhibited can the pure
physical elasticity of the walls play a predominating part. It is a mistake to
describe the flow of blood in the systemic arteries as a flow of liquid in elastic
tubes. Such is certainly the case in the aorta, and possibly in the larger
arteries, but in the arterioles and smaller arteries only when the muscle is fully
inhibited or killed. To describe the circulation as occurring through a system
of muscular tubes, with some elastic tissue aiding the muscles, would be more
accurate. Similarly with the bladder antl other hollow viscera (except the
lung), the elastic tissue acts merely as an adjuvant to the muscle, economising
the work of the latter; but it is the muscle which plays the preponderating
part in determining the tension of the visceral wall.

Conclusions.

1. When initial rigidity is present in a rubber balloon, the pressure on
inflation rises rapidly at first, then falls, and tends to remain at a constant
value until the elastic limit is reached.

2. Such a balloon on deflation displays a marked hysteresis. Only rarely
will the pressure rise on deflation.

3. If initial rigidity be abolished by keeping a balloon inflated some time
and then rapidly deflating, the pressure on a new inflation rises consistently.
On plotting pressure against radius in such eases a rectangular hyperbola may
be obtained, satisfying the equation

(r—a) (p—b) =e,
where @ is close in order of magnitude to the initial radius, and 6 is a
constant greater than p. The behaviour of such a balloon is, however,
far removed from that of a sphere of perfectly elastic and isotropic
material.

4. When the elastic limit is reached in a rubber balloon the pressure is a
linear function of the volume.

5. Hollow viscera approximately spherical, such as the bladder, do not
display initial rigidity, and never give a fall of pressure with increasing
volume. When the elastic limit is reached, the pressure is not a linear
function of the volume.

1909.] Elasticity of Rubber Balloons and Hollow Viscera. 497

6. In the bladder of a large dog, giving sufficient range between the
assumption of globular form and the elastic limit to allow analysis of the
graph of pressure against radius, it was found that the equation

(r—a)(p—b) =e
was followed. In this case «@ was practically zero; but like the rubber
’ balloon the behaviour was not that of a perfectly elastic and isotropic
substance.
Note on the foregoing Paper by W. SUTHERLAND,

From the purely physical point of view the simplest way to prepare for a
theoretical interpretation of experiments such as these is to fix attention in the
first instance on tension per unit area.

Let the tension per cm.” be ¢ in the balloon or bladder which has radius 7
and thickness z. Let initial values of these, when ¢=0, be 7» and ~%.
Consider the equilibrium of a hemisphere. It experiences a pull 2arzt
from the other hemisphere. But on account of the excess p of the pressure
inside the sphere over that outside the hemisphere is subject to a thrust
mrp ; thus

TAG) = ae CE — Pi (1)

If, as in studying the surface tension of bubbles, we fix attention on 2¢, the

total tension across unit width of cross-section of the bounding wall, and call

it T, we have

(5) = elt) Ge. (2)
According to Hooke’s law, we write
ae; (r—70)/70, (3)

where E is a modulus of elasticity appropriate to the conditions of the
experiment, which in the present case are equal tensions in two dimensions
and no external stress in the third dimension. For substances such as
rubber and most organic tissues which have a compressibility, small in
comparison with their deformability, E for small strains is twice the ordinary
Young’s modulus for small strains. But when large strains are used, as in
these experiments, E can no longer be treated as a constant. It is a function
of the strain. This appears when we compare Prof. Osborne’s formula
(p—b) (r—a) =c with (1) and (3), after elimination of z by the relation

rz = Ty'% expressing incompressibility, for we get

_ b(r—a)+e _ 2 Kz (r7—70) 0 4
eS fig as

This makes E a complicated function of r.

498 Prof. W. A. Osborne and Mr. W. Sutherland. [July 5,

In the experiments with the dog’s bladder, @ is nearly 0, so that this
takes the simpler form E = de
interpretation. But to connect the results for the tissue of dog’s bladder
with those for other tissues the modulus of elasticity E can be regarded from
a different point of view. In experiments on dead muscle, for instance, the
muscle is stretched by different weights, the amount of stretching produced

, which is still too awkward for

by each being recorded. As the muscle is lengthened its cross-section is
diminished, but, as a rule, no account is taken of this fact. This is because
more interest is taken in the behaviour of the muscle as a whole, or of a
single representative muscle fibre, than in the intensity of the tension or
the tension per cm.” of cross-section of the muscle. For the gastrocnemius of
the frog stretched by amounts /—/), by weights w up to95 grammes, C. Henry
has shown* that the following formula holds:

1—1) = 6°55 log (1+w/6'10), (5)
J—l) being expressed in mm. and w in grammes weight. For other tissues
with a wide range of elastic properties, A. Goyt finds the same formula to
apply with appropriate values in place of 6°55 and 6:10. But the physical
explanation given for (5) by Henry is not sound, as he interprets 1+w/610
in the form (610+ w)/6:10 to mean that there is at the beginning a tonus of
the muscle equivalent to a weight 61 grammes. If there is stress in the
muscle at the beginning it must be self-equilibrating, and it is not correct
mechanics to fix upon one part of this internal stress, called the tonus, and
treat it as a sign of a not otherwise demonstrable external force denoted
above by 6:1. But, guided by the success of (5), we can arrive at a simpler
formula which is capable of legitimate and easy physical explanation. Let
us suppose that the elongation /—/) caused by wis related to w by the following
equation :

(Ib) Jw = a—b (I—h), (6)

where @ and 0 are constants for a given tissue.

This means that the average elongation caused by unit weight, that is to
say (l—J))/w, diminishes with increasing w in such a way that the diminution
is linear in the total elongation produced by w. When 6 = 0, we have the
usual Hooke’s law for small strains. It is possible to give a theoretical
molecular explanation of (6), though it would not be appropriate here. In
the case of the frog’s gastrocnemius, for values of w from 30 to 95 grammes,
it gives the elongation 7—/, with a maximum error of 1°6 per cent., and
from 0 to 30 grammes with a maximum error of 16 per cent., the

* “Compt. Rend.,’ vol. 162, 1906, p. 729.
+ Ibid., p. 1158.

1909.| Elasticity of Rubber Balloons and Hollow Viscera, 499

corresponding error for (5) being 17 per cent. But it is probable that in
neither case are these really errors of formula, because with the smaller
weights there is liability to considerable experimental uncertainty while
“taking up the slack” of the specimen.

It is interesting to see how the type of formula (6) applies to
Prof. Osborne’s experiments on the bladder of a dog. We must treat the
experimental facts so that they are as similar as possible to those of muscle.
If we return to (1) we see that 7 pr? corresponds with w the weight used to
stretch muscle, although it stretches the bladder wall in two directions at
right angles to one another. The chief effect of this stretching in two
directions is to replace E as measured on a strip cut from the bladder wall
and stretched only in one direction by 2E. From the experiments we get

ae = 0:00121—0:000318 (r—7), (7)
with the following comparison :—

Gna sets oass 1336 1-680 ~ 1927 2421 2:285 2429 2672 2:878
exper. ... 0 14 40 55 63 68 77 87

PR CONC! socio 0 BE 50 58 64 68 76 82
Taietocedé ss +k 3220 3637 3959 4252 4493 4655 4856

pore pera OO kOe yy 2T7 410% 560) | 620

jo Calon renee 95 116 142 182 243 Sylls} = esya¥53

At the two lowest pressures after 0 the discrepancy between calculation
and experiment is large, but can plainly be ascribed to the taking up of
slack in the experiments. The formula fits the facts satisfactorily over the
very great elongation from r = 2121 to r= 3-220. Beyond that the
formula ceases to give the connection between p and 7 in a useful manner,
but on that account it by no means loses its physical significance. If we
write (7) in the form

p = (r—71)/ ar? {000121 —0:000318 (7—7)}, (8)
we see that for values of +—7) greater than 3 the difference 0:00121—
0:000318 (r—7) becomes small compared with either 0:00121 or
0:000318 (7—7). Hence a small error in 7 produces a much larger relative
error inp. With this fact in view it appears that (7) gives a good account
of the physical happenings in the wall of the bladder during the large
elongations up to 7 = 4°856.

The form (7) can be applied to the experiments on a deflated rubber
balloon, but not to those on an inflated.

500

The Modes of Division of Spirocheeta recurrentis and §. duttoni
as observed in the Living Organisms.
By H. B. Fantuam, D.Se. Lond., Christ’s College, Cambridge, Assistant to
the Quick Professor of Biology in the University, and ANNIE PORTER,
B.Se. Lond., University College, London.

(Communicated by Prof. G. H. F. Nuttall, F.R.S. Received July 26, 1909.)
(From the Quick Laboratory, Cambridge.)

The exact mode of division of Spirochetes is still a matter of controversy.
Some workers, as Prowazek, and most of the German school of protozoologists,
consider that Spirochetes divide longitudinally, while Novy, Swellengrebel,
Laveran and Mesnil, and most French workers consider that they divide
transversely. Unfortunately, the direction of division of Spirochetes has
been made a criterion of their protozoal or bacterial nature. Too much stress
appears, from this point of view, to have been laid on the mode of division in
Spirochetes. Fantham (June, 1907, and January, 1908), in the case of
Spirocheta balbianii and S. anodonte, and Dutton and Todd (November,
1907), in the case of S. duttont, state that both longitudinal and transverse
divisions occur, while Breinl (November, 1907) figured (but did not describe
in detail) both modes of division in S. duttont. Fantham worked on both
living and stained material. Miss Mackinnon (1909), working in the Quick
Laboratory, has recently shown that both modes of division occur in
S. recurrentis. We hope to show in this paper, from observations on the
living organisms, that both methods of division certainly do occur in
Spirocheetes ; also how these processes are brought about, and to put forward
suggestions explaining the phenomena.

The subject is one of great difficulty, and the intrinsic difficulty has
not been lightened by the methods adopted by various investigators. Too
much reliance has been placed on the examination of fixed and stained
preparations of Spirocheetes, to the exclusion of evidence which might have
been derived from observation of the living organisms. It is only recently
that the importance of observations of living material in protistology has been
recognised, and even now it is not sufficiently appreciated.

During the past year we have observed living S. recwrrentis and S. duttone
at various times. The investigations were conducted at first independently
(those on S. duttont by A. P., and those on S. recurrentis by H. B. F.);
afterwards we worked in collaboration and carefully checked and correlated

The Modes of Division of Spirocheeta recurrentis, etc. 501

each other’s observations. As a result, we believe that we have arrived at a
solution of the difficult problem of the mode of division of Spirochetes.

Material and Methods.

The blood of tame mice infected with Spirocheta duttoni and S. recurrentis
(Russian variety) was used as the source of the Spirochetes. The prepara-
tions of freshly drawn peripheral blood were transferred at once to the stage
of a microscope enclosed in a thermostat kept at blood temperature (37° C.).
In this way the Spirochetes could be observed for many hours.

At other times, for purposes of comparison, no thermostat was used,
and the preparations were examined at room temperature. Freshly drawn
blood from the heart, liver, spleen, and kidneys of infected mice was also
examined. The objectives used were Zeiss’ 2 mm. apochromatic homogeneous
immersion, or Zeiss’ ;4,’’ achromatic, with compensating ocular 8.

Careful observations were also made on living Spirocheta anodontw, in
order to determine whether there is a uniformity in the methods of division
in the Spirochetes of mammalian blood, and in the Spirochetes occurring in
the crystalline styles of Lamellibranchs.. Thin portions of infected styles
from Anodonta cygnea were examined, the preparations being kept at the
temperature of the room. Other portions of infected styles were allowed to
dissolve in water, and preparations were made. Living specimens of
S. anodonte were under observation for as long as 50 hours.

Longitudinal Division.

Longitudinal division of \S. duttuni and S. recurrentis is best seen when the
blood under examination contains relatively few Spirochetes. We have
found such to be the case at the onset of the infection. At this time there
are but few Spirocheetes in the blood, and their movements can be followed,
though it is at all times a difficult task. As the number of Spirochetes
during the early stages of infection is few, the danger of confusing longi-
tudinal division with entanglement figures is at a minimum. This possible
source of error has been carefully considered and eliminated.

The Spirochetes about to divide longitudinally are slightly thicker than
the other forms, and are somewhat slower in their movements. At the
onset of division, waves resembling the peristaltic waves seen travelling along
the intestine of an insect begin to pass down the body of the Spirochete.
These waves were carefully differentiated from the spirals of the body of the
parasite. A split appears at one end of the organism, and two distinct free
ends are seen. The undulations continue to pass down the body, each free
part having its waves vibrating in unison with the other. As the length of

VOL. LXXXI.—B. 20

502 Dr. H. B. Fantham and Miss Annie Porter, [July 26,

the free portions gradually increases, they diverge from one another, and
their appearance resembles that of the arms of a Y. The divergence
continues until, at the actual moment of separation, the two daughter
organisms are practically in a straight line, that is, there is an angle
of 180° between them. When the two individuals are quite free of one
another, they usually remain quiescent for a few seconds, and then move off
in different directions.

Both 8S. duttoni and S. recwrrentis divide longitudinally after the manner
outlined above. We have repeatedly seen this occur in life in both these
Spirochetes, and also in S. balbiani and S. anodonte. The time required for
complete division is variable. The complete longitudinal division of
S. recurrentis has taken ten minutes, that of S. duttoni thirteen minutes, but
much variation was shown by different specimens.

Transverse Division.

Transverse division of Spirocheetes of the blood has been definitely stated
to occur by various workers, but few of them state precisely what use was
made of living and of stained material respectively. Naturally, therefore,
details of the exact processes of transverse division are wanting. We have
made a careful study of the behaviour of living S. duttoni, S. recwrrentis, and
S. anodonte during transverse division, and under conditions resembling, as
far as possible, those that obtain in the host.

Transverse division occurs in very long, thin individuals, whose move-
ments are somewhat slower than those of other Spirocheetes in their vicinity.
The division is initiated by the appearance of waves passing from each end
of the organism towards its centre. When the waves reach some particular
spot at or near the centre that serves as a node, they meet and die out,
return waves passing rapidly in the opposite directions towards either end.
A second set of waves then passes from the ends in the direction of the
centre; these meet and die out, and return waves, travelling in opposite
directions proceed to each end of the organism. These processes are
repeated many times, the velocity of the waves increasing meanwhile. As
time goes on, the nodal region of the organism becomes thinner, and finally,
after several extremely rapid sets of waves in succession, the tenuous centre
parts, and two complete short organisms are produced. The parent organism
appears to increase slightly in length during transverse division. The
individuals resulting from the division usually swim away in the ordinary
manner,

The time needed for complete transverse division varies with the
individual Spirochete, but we have observed specimens of both S. duttoni

1909.] Modes of Division of Spirocheeta recurrentis, etc. 503

and 8, recurrentis that took from fourteen to sixteen minutes for complete
transverse division.

The processes of transverse division, as seen in the larger S. anodonte,
exhibit yet further details. One of us (H. B. F.) has already shown that

Spirochetes move by («) an undulatory flexion of the body for forward
progression, and (8) a corkscrew motion of the body as a whole. It may be
of interest to note that in the transverse division of S. anodonte, as observed
in the living organism, reversal of the direction of the corkscrew or helicoid
motion of the parasite may occur. In a specimen about to divide trans-
versely, each of the halves vibrating about a node may reverse its direction
of torsional movement; for example, a right-handed spiral may suddenly
become a left-handed one and vice versd. The resultant strain at the node
probably aids in the actual transverse division.

General Remarks.

The fact that both longitudinal and transverse division take place explains
the occurrence of thick and thin and of long and short forms of the same
species of Spirochete. Everyone who has worked on Spirochetes is
cognisant of the occurrence of such polymorphism.

Longitudinal division of S. recwrrentis and S. duttoni, as before mentioned,
is best seen when the blood contains relatively few Spirochetes—at the
beginning of infection and also at the end. Breinl (1907) noted the
occurrence of longitudinal division “especially at the time of the dis-
appearance of the parasites from the peripheral circulation.” Numerous
long tenuous Spirochetes are present in the blood during the height of the
infection, and these divide transversely. While allowing for the reactions of
the host upon the parasites, which reactions might induce the different forms
of division at different periods, it is also possible that the nwmber of
Spirocheetes present in the blood at any given period may have some effect
on the direction of their division. The greater space necessary for
longitudinal division occurs at the onset of infection, when the Spirochetes
are few. As the parasites grow in length and increase in numbers, it would
appear easier for transverse division to take place.

We have, then, clearly shown that there is a distinct periodicity in the
direction of division exhibited by S. recwrrentis and S. duttoni. Naturally
there is a time when both forms of division go on side by side. The
conflicting statements regarding the direction of division of Spirochetes are
thus explained and reconciled.

From the foregoing it is clear that the direction of division of Spirocheetes
cannot be used alone as a criterion of their protozoal or bacterial nature.

2) O) 7A

504 The Modes of Division of Spirocheeta recurrentis, etc.

The Spirochetes exhibit characteristics of both Protozoa and Bacteria. The
reasons for considering them as Protozoa have been well set forth by
Nuttall (1908), while Swellengrebel (1907) considers them to be Bacteria,
belonging to the family Spirillacew, On the whole, we consider that the
protozoal characteristics of Spirochetes preponderate over their bacterial
characteristics.

We have much pleasure in thanking Prof. Nuttall for the material used in
these researches.

Summary.

1. The observations recorded in this paper were made on living
Spirochetes. We have previously examined much fixed and stained
material. It is very necessary to examine living material, as results based
only on stained preparations are not always reliable.

2. Both longitudinal and transverse division occur in Spirochetes as seen
in S. recurrentis, S. duttoni, S. anodonte, and S. balbianii.

3. Longitudinal division of S. reewrrentis and S. duttoni is best seen when
there are but few Spirocheetes in the blood. ‘This is the case at the onset of
infection and at its close. In longitudinal division, rapid waves pass down
the body of the Spirochete. At one end a split occurs, which gradually
widens. Waves travel down each of the diverging daughter forms, which
ultimately lie at an angle of 180° with one another. The daughter
Spirochetes then separate. Organisms about to divide longitudinally are
slightly stouter than the others.

4, Transverse division of S. recurrentis and S. duttoni also oceurs. It is
initiated by the appearance of waves passing from both ends towards the
centre of the organism (which centre acts as a node). These waves meet and
die out, and return waves pass rapidly from the centre towards each end.
These processes are repeated many times, the frequency of the waves
increasing and the nodal region becoming thinner. Finally, after a succession
of very rapid waves, division occurs at the node and two complete daughter
organisms result.

5. There is a periodicity in the direction of the division of S. recwrrentis
and S. duttoni. At the onset of infection, longitudinal division occurs. This
is followed by transverse division of the Spirochetes when the infection is at
its height, while, with the diminution in numbers of the parasite as the
infection draws to an end, there is a reappearance of longitudinal, division.
Naturally, there are times when both forms of division occur together. Our
observations relating to periodicity were made on peripheral blood of the
host.

Origin and Destiny of Cholesterol in the Animal Organism. 505

REFERENCES.

Breinl, A, (XI, 1907), “On the Morphology and Life History of Spirocheta duttoni,’
‘Ann. Trop. Med. and Parasitol.,’ vol. 1, pp. 4835—488, 1 pl.

Dutton, J. E., and Todd, J. L. (XI, 1907), “ A Note on the Morphology of Spirocheta
duttoni,” ‘ Lancet,’ 1907 (ii), pp. 1523—1525.

Fantham, H. B. (VI, 1907), “Spirocheta (Trypanosoma) balbianii (Certes), its Movements,
Structure, and Affinities ; and on the Occurrence of Spirocheta anodonte (Keysselitz)
in the British Mussel, Anodonta -cygnea,” ‘Ann. Mag. Nat. Hist.,’ ser. 7, vol. 19,
pp. 493—501 (prelim. communic.).

Fantham, H. B. (1, 1908), “Spirocheta (Trypanosoma) balbianii (Cevtes) and Spirochetu
anodonte (Keysselitz): their Movements, Structure, and Affinities,” ‘Quart. Journ.
Microse. Sci.,’ vol. 52, pp. 1—73, 3 pls.

Mackinnon, D. L. (1909), ““Observations on the Division of Spirochetes,” ‘ Parasitology,’
vol. 2, pp. 267—280.

Nuttall, G. H. F. (VIII, 1908), “ Spirochzetosis in Man and Animals” (Harben Lecture I1),
‘Roy. Inst. Publ. Health Journ., pp. 449—464.

Swellengrebel, N. H. (VIL, 1907), “Sur la Cytologie comparée des Spirochétes et des
Spirilles,” “Ann. Inst. Pasteur,’ vol. 21, pp. 448—465, 562—586, 2 pls.

The Origin and Destiny of Cholesterol in the Animal Organism.
Part VL—The Excretion of Cholesterol by the Cat.

By G. W. Exits and J. A. GarpNer, Lecturer in Physiological Chemistry,
University of London.

(Communicated by Dr. A. D. Waller, F.R.S. Received August 14, 1909.)
(From the Physiological Laboratory, South Kensington, University of London.)

In an earlier paper®™ of this series the results of a number of estimations
of the cholesterol content of the feces of a dog fed on a variety of diets—
animal and vegetable—were described. It was shown that the cholesterol
found in the case of meat diets could be entirely accounted for by that
present in the food, and from a general survey of the whole of the results,
the opinion was expressed that the whole of the cholesterol of the bile is not
excreted in the feeces, and must therefore have been either totally destroyed
or reabsorbed in the gut along with the bile salts. In the case of a diet of
raw brain, it was found that the cholesterol was not excreted as such, but
entirely in the form of coprosterol. This was subsequently} found to be the
case when cats were fed on either raw or cooked brain.

* “Roy. Soc. Proc.,’ B, vol. 80, 1908.
+t ‘Roy. Soc. Proe.,’ B, vol. 81, 1909.

506 Messrs. Ellis and Gardner. Origin and [Aug. 14;

A few months after the appearance of our paper, Chosaburd Kusumoto*
published a series of estimations of the cholesterol content of the feces of
dogs fed on horseflesh, and horseflesh with the addition of measured
quantities of fat bacon or carbohydrates. His results showed that the
cholesterol content of the food was considerably greater than that of the
feces. He also found that on meat diets variable quantities of coprosterol
were always excreted with the cholesterol, and that the proportion of
coprosterol increased with the fat in the food. This explains our observations
on brain diets, the putrefactive changes which cause the formation of
coprosterol being favoured by the presence of fats. The daily outputs of
cholesterol observed by Kusumoto are somewhat greater than in the case of
the dog we used, but his daily rations were much larger. He gives no data
to indicate the purity of the specimens of cholesterol weighed. As dogs are
omnivorous feeders, it seemed desirable to examine the feces of more truly
carnivorous beasts, and for this purpose the cat was selected. The feces
collected during each diet period were dried in the water oven, roughly
powdered, or, if too greasy, ground with plaster of Paris, and extracted
thoroughly for many days in a Soxhlet’s apparatus with ether. The ethereal
extracts were treated in the manner fully described in our papert on “ The
Cholesterol Contents of Eggs and Chicks.”

Experiments in which cats were fed on meat diet :—

I. A cat, which had previously been fed on raw brain diet, for the purpose
of another experiment, for 14 days, during which it went down in weight
from 2°8 to 2°3 kilogrammes, was fed for 14 days on a diet of lean cooked
horseflesh. During this period it devoured 3125 grammes of the meat, and
increased in weight steadily, its weights taken every third day being 2:3, 2°5,
2°5, 2°7, and 2°9 kilogrammes. The total dried feces weighed 98 grammes,
and yielded after the treatment described 2554 grammes of unsaponifiable
matter in the form of an oily mass. This was crystallised from alcohol
repeatedly, but only 0°2052 gramme of pure crystalline matter was obtained.
This melted at 938°—99° C. and had the characteristic crystalline form of
coprosterol. The mother lquors were evaporated to dryness and treated in
pyridine solution with excess of benzoyl chloride. On pouring into water
the benzoate of coprosterol was thrown out of solution, and on washing
with a small quantity of alcohol was sufficiently pure for weighing. The total
weight of coprosterol obtained was 1:°397 grammes, which corresponds to an

* “Uber den Cholesterolgehalt der Hundfaces bei gewohnlicher Ernihrung und nach
Fiitterung von Cholesterin,” ‘Biochemische Zeitschrift,’ vol. 14, 1908, pp. 411 and 416.
+ ‘Roy. Soc. Proce.,’ B, vol. 81, 1909, p. 129.

1909.| Desteny of Cholesterol in the Animal Organsm. 507

output of 0-098 gramme per day. If we take Dormeyer’s* figure, 0°23, as
the percentage of cholesterol in dry muscle, and assume that the cooked meat
contained 62 per cent. of moisture,f the animal should have received in its
food some 2°7 grammes of cholesterol. A considerable quantity of cholesterol,
therefore, must have been absorbed by the animal, which was putting on
flesh during the whole experiment.

II. A cat, which had previously been fed on a diet of cooked brain for
14 days, during which it lost weight from 2°9 to 2°5 kilogrammes, was fed
for 14 days on lean cooked horseflesh.. During this period it devoured
2940 grammes of the meat, and its weights taken every other day were 2:5,
2°5, 2°7, 28, 2'9, and 2°8 kilogrammes. The total weight of dried feces was
92 grammes, which gave 3:02 grammes of unsaponifiable matter. On
repeated crystallisation from alcohol, 0°588 gramme of pure coprosterol,
melting at 93°-—98° C., was obtained. The mother liquor on benzoylation
yielded a further quantity of coprosterol benzoate. The total coprosterol thus
obtained weighed 1:077 grammes, corresponding to an output of 0-077 gramme
per day. Calculating as before, the animal received in its food 2°56 grammes
of cholesterol.

II. In this experiment a healthy cat was fed for 14 days on 1550 grammes
of lean cooked meat—beef and mutton. Its weight remained practically
constant during the experiment. 44 grammes of dry feces were obtained,
which yielded 1°331 grammes unsaponifiable matter. On treatment this
gave 0:27 gramme of white crystalline matter, melting at 129°—132°, which
appeared to be a mixture of cholesterol and coprosterol. A further quantity
as benzoate was isolated from the mother liquors. Total weight of cholesterol
and coprosterol, 0:5486, corresponding to an output of 0:032 gramme per
day. Calculating as before, the animal received in its food 1:35 grammes of
cholesterol.

IV. As the experiments described are possibly open to the criticism
that the sameness of the diet over a long period may have affected the
metabolism, in this experiment we took four cats and fed them for seven
days on a diet of raw lean bullock’s heart. The animals appreciated this
food and took it greedily. They consumed in seven days 5166 grammes, the
daily ration of each animal being of the same weight. On the eighth day
each animal had a meal of cooked wheat germ, which had previously been
freed from fat and phytosterol by extraction with ether, in order to sweep
out the gut. The feces were very oily in character and difficult to dry at

* ©Pfliig. Archiv,’ 1896, vol. 65, p. 99.

+ A sample of this cooked horseflesh was dried at 100° C. and found to contain 62 per
cent. of moisture.

508 Messrs. Ellis and Gardner. Origin and [| Aug. 14,

80° C: The weight of partially dry stuff was 362 grammes. The weights of
the animals were taken at the beginning of the experiment and periodically
afterwards with the following result :—

| At beginning. On third day. On last day. |
i |
|
|

lbs. ozs. lbs. ozs. lbs. ozs.

Catry TEs 3 Stee 8 2 7 14 7 12

[7 Veena eegces He” ise 6 14 7 oD

lp CPR 28322 6 2 6 1 5 a
LV eee Sa al ee ake! 5 1G ee

The feeces were extracted in a Soxhlet’s apparatus with ether for 19 days
and yielded 43985 grammes of unsaponifiable matter. This was a dark stiff
vaseline-like substance. The unsaponifiable matter was repeatedly crystal-
lised from alcohol, but it proved exceedingly difficult to purify. Eventually
0-05 gramme pure coprosterol, melting 99°—102°, was obtained. The impure
crystalline crops and all the mother liquors, after evaporating to dryness, were
separately treated in pyridine solution with excess of benzoyl chloride. On
pouring into water the crude benzoates which separated were treated in a
similar manner to that described in a former paper.* 1°7272 grammes of
benzoate was obtained. This melted at 125°—128° without showing any
play of colours. It appeared likely that we were dealing with a mixture of
cholesterol: and coprosterol benzoates, and the substance was, therefore,
fractionally crystallised from ethyl acetate. The first crop of crystals on
heating began to soften at 130° C. and melted to a turbid liquid at 141°C.,
which became clear at 165° C. On cooling, the play of colours characteristic
of cholesterol benzoate was shown in a well-marked manner. A microscopic
examination showed that it consisted of the characteristic plates of cholesterol
benzoate with a comparatively small quantity of coprosterol benzoate. A
later crop, which a microscopic examination showed to consist mainly of
coprosterol benzoate with only a very few crystals of cholesterol benzoate,
melted at 117°—119° to a clear liquid. Pure coprosterol benzoate melts at
120°—121° C. Evidently, therefore, a mixture of cholesterol and coprosterol
was excreted, the total weight beimg 1'4004 grammes, corresponding to an
output per day per cat of 0:05 gramme.

Heart muscle contains between 0-066 and 0-071 per cent. of cholesterol,t
so that the animals received in their food about 3°5 grammes of cholesterol.

* “Origin and Destiny of Cholesterol in the Animal Organism,” Part IV, ‘ Roy. Soc.
Proc.,’ B, vol. 81, p. 129.
+ “Cholesterol Content of Heart Muscle,” ‘Journ. Physiol.,’ vol. 38, 1908, ‘ Proc.,’ p. 1.

1909.| Destiny of Cholesterol in the Animal Organism. 509

- The results of these experiments are summarised in the following table :—

Total weight

aoe Duration | El Wali of cholesterol
: cholesterol = ofun- | : Output per |
Experiment. ‘ken i of diet | _ onifiable | and copro- ___ Deficit. pfaot ae ea
* age | period. sana sterol passed Beene. |
Pcs | Bee in feces.
grammes. | grammes. grammes. grammes.
I ory 14 2-554 1-397 1-303 0-098 |
| II 2-56 14 | 3:02 1°‘077 1-483 0-077 |
iil 1°35 14 1°331 0 5486 0°8 0:032 |
EV 3:5 28 4.°3985 1 -4004 2 “099 0°05 |
|
}

It is clear from these results that cats behave similarly to dogs when fed
on meat diets, but the tendency for the change of cholesterol into coprosterol
-appears to be greater in the case of cats.

The total cholesterol of the food should of course be increased by that
poured into the gut in the bile during digestion. No data are, however,
-available for forming any estimate of these quantities.

‘In the hope of ascertaining whether the whole or any of the cholesterol of
‘the bile was excreted in the feces in the case of artifical diets as free as
possible from cholesterol or phytosterol, and if so, whether under such
conditions any reduction to coprosterol took place, the experiments detailed
‘below were undertaken. We had some difficulty in finding suitable food, as
cats are dainty animals and will not eat freely of substances that are in the
least distasteful to them, and we thought that any attempt to starve an
animal into eating any particular food would be likely to vitiate the results.
Further, it was necessary that the diet should contain all the constituents
‘required to keep the animal in good condition.

Ultimately the following diets were selected :—

(1) 90 grammes of white bread mixed with the white of one egg were
moistened with a dilute solution of Liebig’s extract of meat and lightly fried.
About 4 grammes of cream were then added.

(2) About 200 grammes of germ of wheat, which had previously been
thoroughly extracted with ether, were mixed with a little Liebig’s extract
dissolved in hot water to a stiff paste. This was incorporated with about
-30 grammes of suet, and the paste baked for two or three hours in a dish in
a hot oven. The suet used was purified as far as possible from cholesterol by
dissolving in ether and precipitating with alcohol several times. An analysis
Showed that this purified fat still contained 0118 per cent. of cholesterol
either free or in the form of esters.

The animals experimented on partook of these foods readily and appeared
to thrive on them.

510 Messrs. Ellis and Gardner. Origin and [Aug. 14,

V. A cat weighing 3°5 kilogrammes was fed for 17 days on the above-
mentioned bread and egg diet, and the feces were collected during the last.
15 days. During the period in which feces were collected, the animal ate
1390 grammes of bread, the whites of 18 eggs, and about 65 grammes of
cream. The weight of the cat remained quite constant until the 10th day of
the experiment, after which it gradually decreased to 3:2 kilogrammes ;
300 grammes of dry feces were collected, which after extraction yielded
1'735 grammes of unsaponifiable matter asa dark oil. On recrystallisation
from alcohol three times, 0°6075 gramme of white crystalline matter, melting
at 125°—138° C. was obtained. From the residues 0°2604 gramme of
benzoate was prepared. A microscopic examination of the crystalline matter
showed that it was a mixture of cholesterol with probably some phytosterol-
like substance from the bread. Reckoning the whole as cholesterol, the total
weight obtained was 0°8126 gramme, corresponding to an output of
0:05 gramme per day.

VI. This cat was fed for 17 days on the bread-egg-cream diet, with the
addition during part of the time of 2 grammes of cholesterol, given in

():25-gramme portions. Altogether the animal ate 1710 grammes of bread,

the white of 17 eggs, and about 60 grammes of cream. It liked the food, and
during the experiment increased in weight from 3°2 to 3°3 kilogrammes. The

total weight of dry feces was 488 grammes and yielded 2-48 grammes of

unsaponifiable matter of a crystalline nature. After twice crystallising from

alcohol, 1:935 grammes of white crystalline matter were obtained. This was.

again recrystallised from alcohol, and the main crop consisted of almost pure
cholesterol, melting at 143°—144° C. The final mother liquors yielded a
minute amount of matter, crystallising in star-shaped aggregates of needles,
not unlike coprosterol in appearance. The residues, on benzoylation in
pyridine, yielded 0°1723 gramme of benzoates. Assuming that the whole
crystalline matter consisted of cholesterol, the total amount was 2°07 grammes,

a quantity only a little greater than the weight of pure cholesterol given to:

the animal.

VII. Four cats were fed for 10 days on the above-mentioned diet of
extracted germ of wheat and purified fat, the feces being collected during the:
last nine days. The animals took the food readily and ate during the period
1980 grammes of wheat germ and 308 grammes of fat, the total weight of
which, when cooked as described, was 3916 grammes. The weights of the
cats during the experiments were as follows.

490 grammes of dry feces were collected and yielded on extraction.
3°3246 grammes of unsaponifiable matter of an oily semi-solid consistency.

After several crystallisations from alcohol, 11495 grammes of white:

1909.| Destiny of Cholesterol in the Aimmal Organism. my

First day. Third day. Sixth day. Ninth day.
lbs. ozs. lbs. ozs. lbs. zs. Ibs. ozs.
7 14 a ud 8 (6) 8 2
q 6 7 6 7 4 7 2
6 14 6 10 6 4 6 2
5 14 by 5 6 5 4

crystalline matter were obtained, which melted at 135°—137° C. This
consisted mainly of cholesterol, for a portion, after recrystallisation again from
alcohol, melted at 142° C., and another portion, on treatment in ether acetic
acid solution with bromine, according to Windaus’ method, gave cholesterol
dibromide, melting at 120°—122° C., in fair yield. The mother liquors, after
recrystallisation from alcohol, yielded a small amount of matter, which
under the microscope had the appearance of a mixture of cholesterol and
phytosterol. The residues, after separating the above-mentioned 1:1495
grammes of cholesterol, were benzoylated in pyridine solution and 0°5049
gramme of fairly clean benzoate was obtained. This melted, after recrystalli-
sation from ethyl acetate at 146°—147° C., to a turbid liquid, which cleared
at 170° and on cooling gave a brilliant display of colours: Reckoning the
whole of the crystalline matter as cholesterol, 15472 grammes were obtained,
corresponding to a daily output of about 0:04 gramme; or, if we subtract the
quantity of cholesterol contained in the fat given with the wheat germ, which
amounted in all to 0°36 gramme, the daily output, independent of food, was
0:033 gramme. No trace of coprosterol was discovered.

VIII. A cat, weighing 1°7 kilogrammes, was fed on a diet prepared similarly
to the last, but without any fat, for 17 days. It consumed altogether
630 grammes of extracted germ of wheat, and produced 93 grammes of dried
feces. The weight of the unsaponifiable matter was 0°6930 gramme and
fairly crystalline. From this 0°5495 gramme of cholesterol was obtained,
corresponding to an output per day of about 0:03 gramme.

IX. A cat was fed as in Experiment VIII for eight days, but during the
first five days it received, mixed with its food, small quantities of pure
phytosterol. It consumed altogether 250 grammes of the extracted germ
and 1:41 grammes of phytosterol. The weight of dry feces was 68 grammes
and this yielded 1°7415 grammes of unsaponifiable “matter as a greasy
crystalline solid. On crystallisation from alcohol, 1°3545 grammes of white
crystalline matter, which appeared to consist of almost pure phytosterol. An
attempt was made to separate any cholesterol from this by conversion into
the dibromides by Windaus’ method, but without success. A small quantity
of dibromide separated out on standing after the addition of the acetic acid

512 Messrs. Ellis and Gardner. Origin and | Aug. 14,

solution of bromine to the solution of the substance in ether; this was filtered
off and reduced in glacial acetic acid solution with zinc dust. The product,
which should have been cholesterol, had it been present in any quantity, on
heating began to soften at 137° and was not completely melted until 142° C.
An examination of the crystals under the microscope showed that they con-
tained phytosterol. The soluble dibromide treated in a similar way gave a
substance melting at 138°—140° C. A microscopic examination showed that
this was largely phytosterol.

The residues, after the separation of the 1°3545 grammes of phytosterol,
were treated in pyridine solution with benzoyl chloride; 01196 gramme of
benzoate was obtained. This, after recrystallisation from alcohol, melted at
145°—146° C.'to a clear liquid and on cooling showed colours, though not
very brilliantly. On carefully examining the crystals under the microscope,
they were found to consist of phytosterol benzoate and none of the typical
square plates of cholesterol benzoate could be seen. The conversion of
phytosterol into the benzoate by the pyridine method is by no means quanti-
tative, so that we do not know whether all was recovered from the residues.
Altogether 14487 grammes of phytosterol were recovered, including, of
course, cholesterol if present. All the phytosterol given in the food was,
therefore, excreted unchanged, but whether accompanied by cholesterol we
cannot say. There could not, however, have been much.

X. Immediately after the conclusion of the last experiment the diet was
continued, but with the substitution of cholesterol for phytosterol. The
experiment lasted 12 days, cholesterol being given with the food on the first
eight days only. During the period the animal ate 620 grammes of germ
and 2 grammes of cholesterol; 138 grammes of dried feces were collected
and the yield of unsaponifiable matter was 2°1775 grammes in the form of a
brown solid. After recrystallising twice from alcohol, 1°5455 grammes of
cholesterol, melting at 145°—147° C., were obtained. This figure is rather
low owing to an accident, but the amount lost was under one-tenth of a
eramme. The residues on benzoylation yielded 0-4386 gramme of cholesterol
benzoate. The total cholesterol obtained was, therefore, 1°89 grammes, so
that the total amount excreted could not have been greater than the weight
of the cholesterol administered.

Discussion of the Results.

The conversion of cholesterol into coprosterol in the gut of the cat appears
to take place only in the case of meat diets, and then the change is not
necessarily complete. The two cats in Experiments I and II which yielded
only coprosterol had been previously fed for some time on sheep’s brain. The

1909.| Destiny of Cholesterol in the Animal Organism. 513

others, which had previously been fed in an ordinary way and led the
ordinary domestic life, yielded a mixture of cholesterol and coprosterol. The
animals fed on the artificial and vegetable diets gave no coprosterol. This
recalls the experiences of Miiller,* who found that in man a prolonged milk
diet resulted in the excretion of cholesterol and not coprosterol. In all our
experiments on meat diets the total cholesterol and coprosterol excreted was
considerably less than that taken in with the food. Without considering the
cholesterol poured into the gut with the bile, the percentage loss in
Experiments 1 and II together was 53 per cent.; in Experiment III, 40 per
cent.; and in Experiment IV, between 59 and 60 per cent., an average loss of
0:08 gramme per day. Two alternative explanations of these results suggest
themselves.

(1) The hypothesis put forward in an earlier paperf that cholesterol is a
substance which is strictly conserved in the animal economy: that when the
destruction of the red blood corpuscles and possibly other cells takes place in
the liver, their cholesterol is excreted in the bile, and that the cholesterol of
the bile is reabsorbed in the intestine along with the bile salts, and finds its
way into the blood stream to be used in cell-anabolism ; and further, that any
waste of cholesterol might be made up from that taken in with the food.
This latter process would of course be limited in man and carnivorous animals
by the change of cholesterol into coprosterol, and in herbivorous animals by
the fact that their normal food does not contain cholesterol, but isomeric
substances such as phytosterol, which would have to be converted into
cholesterol before utilisation. Further evidence in support of this hypothesis
in the case of herbivorous animals was brought forward by Miss Fraser and
one of us in a paper? on the “ Inhibitory Action of the Sera of Rabbits fed on
Diets containing varying Amounts of Cholesterol on the Hemolysis of Blood
by Saponin.”

(2) The change of cholesterol into coprosterol is generally supposed to be
one of simple reduction brought about in the intestine by the bacteria of
putrefaction. We have, however, no experimental evidence that coprosterol
is a simple reduction product of cholesterol, as it is quite different from any
of the bihydrocholesterol derivatives hitherto produced in the laboratory ; and
further, attempts to bring about the change im vitro by means of bacteria
have so far been unsuccessful. Whatever the exact nature of the change

* “ Reduction of Cholesterol to Coprosterol in the Human Intestine,” ‘Zeit. physiol.
Chem.,’ 1900, vol. 29, pp. 129—135.

+ “Origin and Destiny of Cholesterol,” Part III, ‘Roy. Soc. Proc., B, vol. 81, 1909,
p. 109.

{ “Origin and Destiny of Cholesterol,” Part V, ‘Roy. Soc. Proc.,’ B, vol. 81, 1909,
pp. 280—247.

514 Origin and Destiny of Cholesterol in the Animal Organism.

may be, however, it may be accompanied either by a total destruction of a
portion of the cholesterol, which in view of the great chemical stability of
the molecule of this substance is unlikely, or a change of a portion into some
non-crystalline oily product.

We do not think, however, that a comparison of the total weights of the
unsaponifiable matter of the feces given in Table I with the weights of
cholesterol in the food bears out the second explanation, more especially when
we remember that the latter weights should be increased by the quantities of
cholesterol poured into the gut with the bile during digestion. The weights of
unsaponifiable matter are, moreover, generally higher than the truth, as they
are often rather difficult to dry, without drastic means, and often contain
traces of soap.

If the first-mentioned explanation were strictly true, we should not have
expected to find any cholesterol in the feces of the cats fed on the artificial
cholesterol-free diets—the feces should have been cholesterol-free, just as are
those of herbivorous animals. Small quantities of cholesterol were, how-
ever, found. In Experiment V, on bread, egg, and cream diet, the cat
excreted 0:05 gramme per day, a minute fraction of which, however, may
have been due to cream; and further, in this weight is included the
phytosterol of the bread. In the case of the cats on extracted germ
of wheat, in Experiments VII and VIII, the quantities excreted were
0:033 and 0:03 gramme per day respectively. These values may also have
contained traces of phytosterol left in the germ after extraction. Whether
these quantities are large enough to represent the whole of the cholesterol of
the bile daily poured into the intestine, no data are available to determine.
If, however, we adopt the data given for dogs, the values are undoubtedly too
low. Further, the quantities of feces produced per day on the vegetable
diets were very much larger for a given weight of food than in the case of
meat diets, and possibly this may have caused some of the cholesterol to
escape absorption.

In the case of Experiments VI, IX, and X, however, in which known
quantities of cholesterol or phytosterol were added to the daily rations of the
artificial foods, no excess of cholesterol above that administered was recovered
from the feces.

From the point of view of deciding whether in the case of carnivorous
animals the cholesterol of the bile is normally reabsorbed along with the bile
salts in the intestine, these results are inconclusive. Experiments are, how-
ever, in progress to compare the effect on the blood of the addition of
cholesterol to artifical diets such as those used in the experiments detailed
in this paper. The results of these experiments we expect to give more

Supposed Presence of Carbon Monoxide in Normal Blood, ete. 515

definite information on this point, and 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 the opportunity of expressing our thanks,

On the Supposed Presence of Carbon Monoxide in Normal Blood
and in the Blood of Anmals anesthetised with Chloroform.

By G. A. BucKMASTER and J. A. GARDNER.

(Communicated by A. D. Waller, M.D., F.R.S. Received August 12, 1909.)
(From the Physiological Laboratory of the University of London.)

While engaged in the study of the gases of the blood during the various
stages of anesthesia by chloroform, we found after absorption of the carbon
dioxide and oxygen extracted by the blood pump an amount of residual gas
far in excess of any amount that could be regarded as nitrogen remainder plus
leak of apparatus.* We have found as the result of many experiments,
carried out to determine this particular point, that practically all the chloro-
form present in the blood of anesthetised animals comes off with the gases
of the blood when these are extracted at 40°C., so that the excessive residual
gas is in large part chloroform vapour, or its decomposition products. The
exact method of procedure of analysis of these gases and the effects of the
presence of this chloroform on the methods of analysis will form the subject
of a forthcoming paper, but we quote the following experiments to show what
percentages of chloroform may be present:

Cat, weight 3 kilos.; chloroformed for one hour with an air-chloroform
mixture 2—3 per cent., 54 c.c. of dark blood withdrawn from carotid artery.

The gases extracted with the pump at 40° C. were mixed with excess of
pure moist oxygen and passed through red-hot spiral platinum tubes. The
products of combustion were collected in ammonia. This was exactly
neutralised with nitric acid and titrated with silver nitrate (1 ¢.c. = 0-001 Cl) ;
17 c.c. of silver nitrate were required = 0:01986 gramme CHCl; = 3°7 cc. of
chloroform vapour at 0° and 760 mm.

* The blood pump employed was the Tcepler as modified by Barcroft (‘Journal of

Physiology,’ vol. 25, p. 265), with certain modifications for this particular work. These
wwill be described in our paper on the blood gases in chloroform anesthesia,

516 Messrs. Buckmaster and Gardner. Supposed [Aug. 12,

The blood therefore contained 0:0348 gramme of chloroform per
100 grammes of blood, so that the blood gases evolved contain about.
10 per cent. by volume of chloroform vapour.

To quote another experiment :—

Cat, weight 3°5 kilos.; chloroformed with an air-chloroform mixture of
2 per cent., 54 c.c. of dark blood withdrawn from carotid artery for analysis.
of gases, Immediately before and after withdrawing this, two extra samples
were taken of 10 c.c. each for analysis by the method which we call the
method of Nicloux.*

Volume of gases extracted = 30°53 at 0° and 760 mm. This gas was
analysed as in former experiment; 19-9 c.c. of silver nitrate were required
(1 e.c. = 0:001 Cl), which corresponds to 0:0223 gramme of chloroform =
4:15 cc. of chloroform vapour at 0° and 760 mm. = 00419 gramme of
CHCl, in 100 grammes of blood.

Sample I (Nicloux method) gave 0°0396 gramme of chloroform per
100 grammes of blood.

Sample II (Nicloux method) gave 0:0403 gramme of chloroform per
100 grammes of blood.

The blood gases were always evacuated from blood at 40° C., and at this.
temperature and under the conditions we employed for anzesthetisation it is.
clear that practically all the chloroform which is calculated to be in blood
can be recovered as chloroform vapour.

In 1894 Gréhant+ recognised the presence of a combustible gas in the
blood. This he considered to be carbon monoxide. The amount of this in.
blood he determined with his grisoumeétre, an instrument by means of which
he could measure the quantity of carbon monoxide fixed by hemoglobin in:
an atmosphere which contained one part in 60,000.

As the result of experiments carried out on dogs in Paris, Desgrez and
Niclouxt stated that carbon monoxide is not only a normal constituent.
of the blood gases, but that the blood of these animals, when aneesthetised by
chloroform, contained an augmented quantity of this gas: “Les animaux
soumis 4 Vanesthésie par le chloroforme nous ayant fourni un sang notable-
ment plus riche en oxyde de carbone que leur sang normal.” This
conclusion is drawn from their experiments, which we summarise in the
following table.

Their method of estimating carbon monoxide consisted in passing the

* “Tosage de Petites Quantités de Chloroforme,” ‘ Extraits du Bulletin de la Société
Chimique de Paris,’ 3rd series, vol. 33, p. 321, 1906.

+ Gréhant, ‘Comptes rendus,’ November 8, 1897, and ‘ Les Gaz du Sang,’ p. 109, 1894.

t ‘ Archives de Physiologie,’ No. 2, April, 1898, p. 377.

17

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Presence of Carbon Monoxide in Normal Blood, ete.

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518 Messrs. Buckmaster and Gardner. Supposed [Aug. 12,

blood gases over iodine pentoxide at 150° C., and estimating the iodine
liberated by the method of Rabourdin. Beyond the fact that carbon
monoxide decomposes iodic anhydride under these conditions, whereas neither
hydrogen nor methane will do this, Desgrez and Nicloux bring forward no
evidence whatever that the liberation of iodine in their experiments is due
to carbon monoxide from the blood.

They explain the production of carbon monoxide in chloroform anesthesia,
in the light of observations of Desgrez,* that chloroform is decomposed by
a solution of potash 1 : 8 with production of carbon monoxide according to
one of the following equations :—

CHCl3+ 2KOH = 2KCl+H,0+ HC1+CO ;

CHCl3+ KOH = KCl+2HC1+ C0.
But the blood is certainly not an alkaline fluid in the sense that even an
extremely dilute solution of potash is: the blood is both alkaline and acid
according to the indicator chosen. This reaction cannot therefore be
regarded as lending support to their conclusions.

The statement that carbon monoxide is contained in blood was apparently
confirmed by Saint-Martin.| This observer worked with large quantities of
blood, 500 ¢.c. being taken for an experiment. The blood was mixed with
oxalate of potassium solution in the proportion of 1 gramme of the salt to
500 grammes of blood. The gases evacuated from this mixture at 45° C.
by the blood pump were rejected, and on the subsequent addition of 250 c.c.
of saturated tartaric acid solution a further liberation of 30 to 40 c.c. of gas
occurred. This proved to be a complex mixture of gases among which was
carbon monoxide, which he absorbed with ammoniacal cuprous chloride.

In two papers, Lepine and Bouludt state that in 25 c.c. of dog’s blood they
could find no carbon monoxide, though working with the apparatus devised
by Desgrez and Nicloux. In blood taken post mortem of severe cases of
anemia, carbon monoxide is present, whereas in control cases which were
non-anemic the presence of this gas could not be detected. After injections
of oxalate of calcium or tartaric acid into the circulation as much as 0°4 c.c.
of carbon monoxide per 100 c.c. of blood was found. Practically no experi-
mental details however are given in their papers.

Finding all these results difficult to understand, we incidentally, during the
course of some other experiments, made an examination of a sample of the
blood of a cat which had undergone a prolonged anesthesia, both spectro-
scopically and by Haldane’s method, without being able to detect the

* ‘Comptes Rendus,’ November 15, 1897.
+ ‘Comptes Rendus,’ February 14, 1898.
{ Lepine et Boulud, ‘Comptes Rendus,’ p. 56, 1905, and p. 302, 1906.

1909.] Presence of Carbon Monoxide in Normal Blood, etc. 519

presence of any carbon monoxide; and without denying the conclusions of
Desgrez and Nicloux, stated in a paper published in 1906,* that “ we were
unable to accept the view that the combustible gas appearing during
chloroform-narcosis is carbon monoxide, a product of the decomposition
of chloroform within the organism.” This led Prof. Nicloux, in his work
‘Les Anesthesiques Generaux,{ to make an attack on us and represent that
we had denied the accuracy of his results on the strength of a single experi-
ment by a spectroscopic method on a cat, whereas his experiments, he remarks,
were performed on dogs. In reality our observations were made by the
admittedly delicate method of Haldane, in which weak solutions of blood are
examined in long glass tubes. We were therefore led to examine the
conclusions of Desgrez and Nicloux in more detail.

Is there any carbon monoxide in the gases of the blood which can be detected
by passing the gases through solutions of oxy-hemoglobin ?

It will be seen from the table we have given that Desgrez and Nicloux state
that normal blood contains 1°6 ¢.c. of carbon monoxide per litre of blood. In
that of anzsthetised animals they find amounts of 2°6 cc, 24 ce, and
6-9 cc. On the assumption that a litre of blood yields 600 c.c. of mixed gases
at 0° and 760 mm., then the CO-content of normal blood is 0:27 per cent.,
which in chloroform anesthesia may reach 1:15 per cent. by volume. The
actual quantities of gases these observers examined were about 15 c.c.

We have minutely followed the directions contained in Haldane’s papers,t
using mixtures of air and carbon monoxide. In the first experiments 500 c.c.
of air with 4 cc. of carbon monoxide = 0°8 per cent. CO were used.
Measured volumes of this mixture were slowly bubbled through 1 : 100
solutions of freshly defibrinated cat’s blood. The tubes so treated were
compared with control solutions, the two solutions being compared either
undiluted or equally diluted. Long columns of these solutions were
examined in long glass tubes, with the following results :—

Volume of mixture
Series. of air+CoO in cc.

ih 50 Hb+CO detected without dilution.
25 Ditto.
15 Ditto.
II 50 Ditto.
25 Ditto.
. 10 Ditto.
5 Detected on dilution in long tubes.
* “Roy. Soc. Proc.,’ B, vol. 78, 1906, p. 414. + Paris, 1908.

t ‘Journal of Physiology,’ 1898, vol. 18, and zbedem, 1899, vol. 19.
2P 2

520 Messrs. Buckmaster and Gardner. Supposed [Aug. 12,
In another series of experiments mixtures of 500 cc. of air and 1 ce. of
carbon monoxide = 02 per cent. by volume were used.

Volume of mixture
employed in c.c.

50 Hb+CO recognised readily: 0-4 ¢.c. of the blood in water
compared with control similarly prepared.

25 Equally well marked : 0:4 ¢.c. of blood in water compared with
control similarly prepared.

10 Difference from control quite noticeable.

From these results it is clear that the quantities of carbon monoxide stated
by the French observers to be present in blood gases should be readily
recognisable by this method, for half the amounts stated to be present in
normal blood are recognisable.

In order to ascertain whether carbon monoxide was present in the blood of
aneesthetised animals the gases of cat’s blood, never less and generally more
than 60 c.c. in volume, were evacuated from 108 ec. or more of blood at
40° C., without the addition of any acid, until no further trace of gas could
be obtained. The chloroform used for anesthetisation was chloroform puriss.
B.P. (made from acetone) washed with water, shaken with excess of anhydrous
potassium carbonate, filtered and then distilled.

To quote some of the experiments :—

Duration of Volume of very
anvesthesia. dark blood.
Ny, iy) ey anh, C.C.

i 12 52—2 19 54
FiO 27 54

The total gases were slowly bubbled through 1 Foo fresh defibrinated cat’s
blood and this examined against control solutions both diluted and undiluted.
The solutions were also examined with Michael’s tintometer. No differences
whatever could be detected either by ourselves or by four separate
independent workers in the laboratory, all of whom were unacquainted with
the purpose for which the observations were being made.

IL. 191 c.c. of blood, from two cats deeply anesthetised for 1 hour 6 mins.
and 1 hour 40 mins. respectively, were evacuated. The gas so obtained was
slowly bubbled through 1: 100 blood solution and compared with control
blood solution through which an artificial blood gas containing chloroform
vapour was bubbled. No difference whatever in tint could be recognised
with the naked eye either in diluted or undiluted solutions by ourselves or by
five independent workers in the laboratory. This result was confirmed by

1909.] Presence of Carbon Monoxide in Normal Blood, ete. 521

comparisons made with Michael’s tintometer. Differences could, however, be
readily detected when 0:2 ¢.c. of blood of similar dilution 1: 100 saturated
with CO gas was added to either of the original solutions through which the
blood gases or artificial mixture had been bubbled.

Duration of Volume of
anzesthesia. blood.
hrs. ¢.c.
Il. 2 DA
20 min. later. Dik

Gases bubbled through as before, examination as above. No difference
whatever could be detected.

Duration of Volume of
anesthesia. blood.
Ing” ail, c.c.
IV. i= 30 a4
30 min. later. 54

The total gases as before bubbled threugh 1 : 100 human defibrinated
blood. On examination as before, no differences whatever could be detected.

These experiments, we subinit, conclusively show that when the blood of
anesthetised animals is evacuated at 40° C. until no further gas is evolved,
the gas obtained contains no recognisable trace of carbon monoxide, and,
therefore, if this is present it is present in far smaller quantities than Desegrez
and Nicloux state to be the case in normal blood gases. Therefore, chloroform
is not decomposed in the organism or in the blood with production of carbon
monoxide. If there was any carbon monoxide really present in their experi-
ments, this must have been due either to the acid which they added before
evacuation of the gases, a view from which they explicitly dissent, or have
been produced in their experiments.

-It now became necessary to arrive at some explanation for the iodine
evolved in the experiments of Desgrez and Nicloux, and the most obvious
line of research was to study the effect of chloroform vapour on iodic
anhydride at various temperatures, and also the effect of heat alone on this
substance. For this purpose the following apparatus was employed. The
gases of the blood, mixtures of air and chloroform or other gases under
investigation were swept over the iodine pentoxide by a current of oxygen
aspirated at a rate varying from 1 to 2 litres an hour. This plan was
adopted to obviate the entrance of air of the laboratory, which might
contain traces of carbon monoxide; and as a further precaution the oxygen
was passed through a long combustion tube filled with red hot copper oxide.

522 Messrs. Buckmaster and Gardner. Supposed [Aug. 12,

The general arrangement of the apparatus is shown in the accompanying
figure. The oxygen current passes from left to right through (1) combustion
tube, (2) bulbs containing 40 per cent. potash, (3) bulb containing the gas
under examination, (4) a U-tube containing powdered potash, the proximal
limb of which was slightly moistened, (5) a calcium chloride drying tube,
(6) tube containing iodic anhydride, (7) absorption vessel containing 10 cc.
of a 10 per cent. potassium iodide solution to absorb any liberated iodine
(this was kept cool during the experiment), and (8) aspirator.

The bulb (8) was either one of Waller’s densimeter bulbs of 235 c.c.
capacity for mixtures of chloroform and air, or, in the case of bloed gases,
a 200 cc. tube with stopcocks at either end.

Lffect of Heat on Iodine Pentoxrde.

Dr. Wade, of Guy’s Hospital, who has for some time past been engaged in
studying the methods of estimation of small quantities of carbon monoxide,
kindly informed us, when we commenced these experiments, of the fact which
he had observed that iodine pentoxide invariably gave off iodine when first
heated, and that this evolution of iodine never actually ceased, although the
rate eventually became steady if the temperature was maintained constant.
This we can entirely confirm. We used iodine pentoxide tubes of a special
kind devised by Dr. Wade, a full description of which, along with his results,
he is about to publish. For this reason we give no further account of our
experiments in this direction.

Effect of Chloroform Vapour on Iodine Pentoxide at various Temperatures.

The above-mentioned tubes were thoroughly cleaned and filled with 28 to
30 grammes of powdered iodine pentoxide and heated for a short time in
aniline vapour, but in no case was any considerable quantity of water vapour
evolved. The oxide was then heated in a current of purified oxygen for 24 to
48 hours or longer, the tube being placed in the vapour of a suitable liquid,
boiling at the temperature we desired to use in an actual experiment, until the

1909.] Presence of Carbon Monoxide in Normal Blood, etc. 523

rate of evolution of iodine per hour became approximately constant. Different
specimens of iodine pentoxide, and even different tubes made from the same
sample, were found to differ considerably in this respect. These differences
may in part have been due to traces of impurity in the iodine pentoxide,
or more probably to different states of physical aggregation. A specimen
obtained from a French source kindly recommended to us by Prof. Nicloux,
which qualitative tests showed to be pure, after prolonged heating at
157° C. evolved iodine equivalent to 1 c.c. of N/1000 sodium thiosulphate in
two hours, but we never reduced the amount below this value. We hoped at
first to be able to determine for each tube the constant loss of iodine per
gramme per hour at the temperature employed in our particular experiments,
but experience soon showed that, in addition to the difficulty of keeping all
conditions sufficiently constant, when chloroform vapour had been passed
through a tube the “constant” underwent change. It was therefore found
more satisfactory to make a blank determination of the iodine liberated in a
given time by heat alone before each experiment and sometimes also after.
The temperatures we employed were 100°C., the boiling point of xylene
(137° C.), the boiling point of bromobenzene (157° C.).

The chloroform used in our experiments was made from acetone and
carefully purified for us by Dr. Wade. It boiled at 61°14 to 61°15 C. at
760 mm. and had a specific gravity 15008 15/15.

Experiment I—0:0588 gramme of chloroform, weighed in a thin bulb, was
placed in the vessel (3) and the bulb broken. The chloroform vapour was
swept over the iodic anhydride in a current of oxygen at the rate of 1 to
1} litres per hour. The current was maintained for 81 minutes. Iodine
equivalent to 1029 cc. N/1000 sodium thiosulphate was liberated. In a
control experiment which had previously been made under similar con-
ditions in 104 minutes, iodine was liberated equivalent to 1:5 c.c. N/1000
sodium thiosulphate. Thus each gramme-molecule of chloroform liberated
2°6 grammes of iodine, 2.¢. about 1/100 of a gramme-molecule of iodine.

In the following experiments, mixtures of air and chloroform vapour
(amounting to 255 c.c. in each case), the concentration of which was deter-
mined by Waller’s densimetric method, were passed through the apparatus
at different temperatures. A few of the results are given in the following
table (Table I1).

A very large number of experiments were made, and it was found that
the quantities of iodine liberated varied markedly with different tubes and
with alteration of the conditions of the experiment. This was particularly
the case in experiments conducted at 100° C.

524 Messrs. Buckmaster and Gardner. Supposed [Aug. 12,
Table II.
| Iodine liberated | Iodine
eas Duration | in N/1000 sodium | liberated
CHO in | eeten| of oxygen | thiosulphate in | by CHCl,
Temp. ae Care a 2 in | Current in only in Remarks.
Fistral Cae _ | terms of
ment. lGontrol: Experi- | thio-
| ment. | sulphate.
: per cent. h. m. | ho oimby ilu hove: cic: C.c.
157 GQ i BS yy. By | Gt 57°45 51°55 Gas in aspirator
| | | smelt of CHCI;.
137 ZNO) |) VA =O | 1 20 4°5 8:5 | 4:0 )\|In these experi-
| | ments N/1000 was
| | | actually used, so
| thatthe figures are
| | t| only approximate.
| | | The gas in aspi-
| | | rator smelt dis-
| | tinetly of chloro-
137 LO 740) | ak 740) | 3°0 40 ORS) eetorm:
| |
100 9°2 | ik do | 1 30 15 | 24:0 22°65

[ October 12, 1909.—« It was further noticed that when the U-tube (4) con-
taining solid potash was eliminated from the circuit, the quantities of iodine
evolved underwent a marked diminution.

When iodine pentoxide is boiled under a reflux condenser with pure chloro-
form the action is very slow. One gramme of pentoxide boiled with 5 c.c.
of pure chloroform for three hours only gave iodine equivalent to 18°7 c.c.
of N/1000 sodium thiosulphate.
appear to proceed much more rapidly, as the following results of experiments
in which iodine pentoxide and chloroform were heated in sealed tubes at.

At higher temperatures the action does not

temperatures of 100° C. and 137° C. respectively indicate :—

| (1) Weight Volume of | T ature, | Duration of | Iodine liberated in terms of
HW ose TO |p) Chhilomotorsny fp eS | heating. | N/1000 thiosulphate.
| | |
| |
gramme. c.c. hour. C.c.
1 i | 100 | 1 13 ‘9
| 1 1 | 137 | 1 21-1
i i

In control experiments in which the same weights of iodine pentoxide
were heated without chloroform, the quantities of iodine set free were equiva-
lent to 1:2 and 1°4 c.c. of thiosulphate respectively.

It was clear from these results that although iodine pentoxide reacts with
pure chloroform with the liberation of iodine, the amounts so produced are

1909.| Presence of Carbon Monoxide mm Normal Blood, ete. 525

insufficient to account for some of the quantities found in the experiments
quoted in Table IT.

It is known from the experiments of Desgrez*, and of Thiele and Dentt,
that chloroform is decomposable by aqueous potash in the cold with the
production of carbon monoxide, and it seemed not unlikely that in this
reaction an explanation was to be found for the comparatively large amounts
of iodine evolved when U-tube (4) was in the circuit.

That this was the case is proved by the following experiments :—

Effect of Solid Potash on Chloroforne.

Experiment I—Two stout test-tubes filled with mereury were inverted in
a bath of mercury. Into one a piece of dry solid potash was introduced, and
into the other a piece of potash, the surface of which was moistened with
water. A few drops of chloroform were then passed into each tube. In
both cases a slow evolution of gas commenced at once, and after standing
overnight 3—4 cc. of gas had collected above the mercury. This gradually
increased in quantity until at the end of three days the tubes were almost
full of carbon monoxide.

Experiment IT —In this experiment a comparison was made between the
quantities of iodine liberated when a 5 to 6-per-cent. mixture of chloroform
and air was passed over iodine pentoxide (A) with the U-tubes 4 and 5 in
the apparatus figured above filled with calcium chloride, (B) with the

-tubes 4 and 5 filled with roughly-powdered potash, that in the first limb
of 4 being slightly moistened. The experiments were conducted otherwise
under rigidly similar conditions. The following results were obtained :—

| | | | | Todine liberated in | Difference
| Composition Duration | Duration | terms of N/1000 between
| Stiegl of CHCl; | of oxygen | of oxygen thiosulphate in | expt and
| Hage | Hicmp: and air | current | current | Sey control
| mixture. | in control.| in expt. | in e.c. of |
| | | Control. | Expt. | thiosulphate. |
| | | |
a i | | i
2C5 | | hours. hours) ||) (cre) vere:
A 1S fies MA OOD wih |\Raet 2 DB | ae | nes | Bes
using CaCl, | |
tubes |
| | | | |
Eells? Boi a ay A ten aes 181 °5
using KOH | | |
tubes | | |
i | |

* “Comptes rendus,’ 1897, vol. 125, p. 782.
+ Thiele and Dent, ‘ Annalen,’ 1898, vol. 302, pp. 223—274.

526 Messrs. Buckmaster and Gardner. Swpposed | Aug. 12,

Hxperiment II[.—1'd litres of a 2-per-cent. chloroform-air mixture were
driven by means of a current of purified oxygen through a Y-tube, one limb
of which was connected with a U-tube containing calcium chloride A, the other
with a U-tube containing roughly-powdered potash B. After passing
through these tubes the gas was allowed to bubble slowly through two small
wash bottles, each containing 30 c.c. of a freshly prepared dilute solution of
human blood. The apparatus was so arranged that the gas passed through
the parallel U-tubes and thence through the wash bottles at the same rate.
At the end of the experiment it was found that the blood through which the
gas from tube A (calcium chloride) had passed contained no trace of carboxy-
hemoglobin, but that in connection with tube B (potash) was almost wholly
converted from oxyhzmoglobin to carboxyhemoglobin.” |

Lffect of Blood Gases in Ancesthesia on Iodine Pentoxide.

From a large number of experiments we give a few results in the following
table. In all cases there was a minimal amount of experimental interference
with the animal. The blood was always taken from the carotid artery.
The gases were evacuated at 40° C. Percentage of chloroform inhaled
= 2:3 per cent. CO, absorbed before experiment, and U-tube 4 always in
cirewit :—

Table III.
Duration of ice ee:
Vol Temp. oe Brat uEreay N/1000 sodium Todine
Weight of | Duration of of ‘lo od of thiosulphate in | liberated
cat, anesthesia. | jo.on | experi- owing to
aac’ | ment. CHO.
Experi-| , Experi-
| Control. eit Control. | “ent.
kilos. mins. c.c. ° min. min. cic; c.c. secics
2°8 48 54 157 120 120 2°68 | 18°81 16 -13*
2°8 55 54: 157 112 222 1°8 41°45 37 88 +
3:5 78 54 100 120 120 15 9-0 75
* Blood very dark. + Blood very dark. Animal near asphyxia.

It seems clear, therefore, from all these experiments, that the iodine
liberated by the blood gases of an animal anesthetised by chloroform is due
partly to the vapour of chloroform and partly to the carbon monoxide
produced by the action of chloroform vapour on the potash in U-tube 4, but
not to the carbon monoxide that might be liberated in the organism.

In order to obtain further confirmation of this, experiments were made in

1909.|] Presence of Carbon Monoade in Normal Blood, etc. 527

which the blood gases before analysis were freed from chloroform by shaking
with a few cubic centimetres of alcohol in the laboratory tube of the gas
analysis apparatus, and, finally, from traces of alcohol vapour by shaking
with ice-water. We quote one experiment :—

Todine liberated in
Duration of oxygen terms of N/1000
Daration wcvelume current in | sodium thiosulphate
oe of of Temp. oF
sao a anesthesia.| blood. | eet Seare oa
Control. BEGDOR Control. gpa
| ment. ment.
|
kilos. hour. C.c. ° min. min. C.c. c.c.
3 1 54 151 135 135 iL o7/ 2

The gas, therefore, liberated iodine equivalent only to 0:3 cc. N/1000
thiosulphate, a quantity which may very well be ascribed either to the
incomplete elimination of the chloroform or to the variation of the control
value of the tube.

[October 12, 1909.—“ Finally, in order to settle the question quite
conclusively, we made the following experiment :—

A cat weighing 3°6 kilos. was anesthetised by means of carefully purified
chloroform for 1 hour 15 minutes; 108 cc. of blood were withdrawn
in two lots of 54 e.c. from the carotid artery. The gases were completely
pumped out of this blood at 40° C., and divided into two portions. The one
half was slowly bubbled through 1:100 blood solution, and examined
‘by Haldane’s method in the manner described in the earlier part of this paper.
No trace of carboxyhemoglobin could be detected.

The other half of the blood gas was allowed to stand for 45 minutes in
ithe laboratory tubes of the gas analysis apparatus in the presence of a few
Jumps of moistened potash. It was then transferred to the measuring tube,
and thence bubbled through 1:100 blood solution as before. The presence
of carboxyhemoglobin was obvious in this without dilution.

The results were confirmed spectroscopically in both samples before and
after the addition of reducing agents.”

Conclusions.

1. Our experiments lend no support to the view that carbon monoxide is a
normal constituent of the blood gases. We think that the small quantities of
iodine found in Desgrez and Nicloux’ experiments are due to the decomposition
-of iodine pentoxide at the temperature (150° C.) of their experiments.

528 Mr. W. J. Young. Hexosephosphate formed by [July 30,

2, Chloroform is not decomposed in the blood with the formation of earbon
monoxide.

3. [October 12, 1909.—“ The iodine liberated in the experiments of Desgrez
and Nicloux on aneesthetised animals was due to some extent to the direct
decomposition of the iodine pentoxide by the chloroform vapour contained in
their blood gases, but mainly to the carbon monoxide produced by the action
of this chloroform on the potash over which they passed the blood gases in
order to free them from carbon dioxide. This explanation is quite in accord-
ance with their observations that the amount of iodine liberated increases.
with the duration of the anesthesia.” ]

We express our thanks to the Government Grant Committee of the Royal.
Society for the funds which they have placed at our disposal for our work.

The Hexosephosphate formed by Yeast-juice from Hexose and
Phosphate.

By W. J. Youn (Biochemical Laboratory of the Lister Institute of
Preventive Medicine).

(Communicated by A. Harden, F.R.S. Received July 30, 1909.)

It has been shown by Harden and Young* that—(1) the rate of fermenta-
tion of glucose by yeast-juice is greatly increased by the addition of a soluble
phosphate ; (2) this rate soon diminishes until a constant rate is attained,
which is only slightly greater than that of the original yeast-juice and
glucose; (3) during this period of increased fermentative activity, the
phosphate undergoes some alteration, and at the end of the period is no
longer present in a form precipitable by magnesium citrate mixture. The
authors suggested in the first paper that a combination of the phosphate with
the sugar, to form a phosphoric acid ester, had taken place, and more recently
embodied this suggestion in the form of an equation—

2CgHi20¢ + 2R’oH PO, = 2COz + 2C2H,O + CgHw On POsR’2)2 + 2H.0.
It has also been shown that the same phenomena occur when fructose or
mannose is used in place of glucose.t

* “Chem. Soc. Proc.,’ 1905, vol. 21, p. 189 ; ‘ Roy. Soc. Proc.,’ B, 1906, vol. 77, p. 405_
+ ‘Roy. Soe. Proc.,’ B, 1908, vol. 80, p. 299.
t ‘Chem. Soc. Proc.,’ 1908, vol. 24, p. 115; ‘ Roy. Soc. Proe.,’ B, vol. 81, 1909.

1909. | Yeast-juice from Hexose and Phosphate. 529

In a paper on the changes that take place in the phosphorus compounds of
plants, etc.,* Iwanoff stated that, during the fermentation of sugar by pressed
yeast, the inorganic phosphates of the yeast are converted into organic
compounds. This observation, however, remained unknown to the author of
the present paper until the publication of a later paper by Lwanoff,t in which
he described the formation of a phosphoric acid compound during the fermen-
tation of glucose or fructose in the presence of sodium phosphate by yeast
which had been previously treated with acetone (“zymin”). By precipitating
with copper acetate, decomposing with sulphuretted hydrogen and evaporating,
he obtained the free acids, corresponding to these compounds, in the form of
dark brown syrups, which when prepared from glucose contained 19°6 to
20 per cent., and from fructose 13-4 to 14 per cent., of phosphorus. No other
analyses were given in the paper. These syrups gave the characteristic
reactions of aldo- and keto-groups, and yielded osazones when heated with
phenylhydrazine, that from the glucose compound melting at 142°, and that
frem the fructose compound at 125°. Iwanoff concluded that these bodies
were compounds of phosphoric acid either with a triose or with methylglyoxal,
and that they were different according to the sugar from which they were
derived.

At the time when Iwanoff’s paper appeared, a considerable amount of work
had been done by the author on this reaction, and a preliminary note was
published describing the isolation of the compound by means of its lead salt.t
It was shown that this salt had the empirical formula C3H;0.(PO,Pb), and
when decomposed with sulphuretted hydrogen yielded an acid which was
slightly dextro-rotatory, and which, when the solution was boiled, underwent
hydrolysis with formation of phosphoric acid and a levorotatory reducing
‘substance.

The present paper embodies the results of further work on this substance,
and also on the similar compounds obtained by fermenting fructose and
mannose in place of glucose. No difference has been detected between the
compounds obtained from these three sugars.

Preparation of the Lead Compound.—A solution of 0°6 molar di-sodium or
potassium hydrogen phosphate (R’2HPO,) was added to a mixture of yeast-
juice and excess of glucose at 25°, in such an amount that rapid fermentation
was set up. The quantity of phosphate which may be used varies with
different juices,§ and the necessary amount was determined in each case in

* “Travaux de la Société des Naturalistes de St. Pétersbourg,’ vol. 34, 1905.
+ ‘Zeit. physiol. Chem.,’ 1907, vol. 50, p. 281.

{ Young, ‘Chem. Soc. Proc.,’ 1907, vol. 23, p. 65.

§ Harden and Young, ‘ Roy. Soc. Proc.,’ B, 1908, vol. 80, p. 299.

530 Mr. W. J. Young. Hexosephosphate formed by [July 30,

a small sample by measuring the rate of evolution of carbon dioxide under
similar conditions. When the rate of fermentation had fallen, more
phosphate was added, and the additions continued so long as this acceleration:
could be produced. The mixture was then boiled, and filtered from the
coagulate formed.

In a typical experiment, a mixture of 440 cc. of yeast-juice and
55 grammes of glucose was employed, and 440 c.c. of the potassium:
phosphate solution (37 grammes, K2HPO,) were added 80 cc. at a time..
When fructose is used in place of glucose, much more phosphate may be
added ; thus in one case a mixture of 495 ¢.c. of yeast-juice and 200 grammes.
of fructose was employed, and 1000 ec. of the phosphate solution
(104:4 grammes K2HPO,) were added, 100 cc. at a time.

The liquid, after boiling and filtering, was always found to contain a little
free phosphate, and this was removed by adding a solution of magnesium
nitrate, making alkaline with potash, stirring well, and allowing to stand for
afew hours. The filtered liquid was then neutralised with acetic acid, lead
hexosephosphate precipitated by the addition of lead acetate, and washed
repeatedly with water by means of a centrifugal machine, until the washings
no longer reduced Fehling’s solution. It was found that this precipitate
invariably contained traces of some nitrogenous material, and it was there-
fore suspended in water, decomposed with sulphuretted hydrogen, and the
excess of this gas removed after filterimg by passing a current of air through.
the solution. The liquid was then neutralised with potash and the lead salt
reprecipitated with lead acetate, the process being repeated until the preci-
pitate, after washing, was free from nitrogen. Usually two or three such
precipitations were found sufficient. The salt was then filtered off and dried,
first on a porous plate and then in a vacuum over sulphuric acid. It was
thus obtained as a white, amorphous powder, free from nitrogen, which, on
decomposing with sulphuretted hydrogen, yielded an acid solution containing”
no free phosphate precipitable by magnesium citrate mixture.

The preparation of yeast sold under the name of “zymin,” which is
obtained by treating pressed brewers’ yeast with acetone, may be used in
place of yeast-juice for the preparation of hexosephosphate. In one
experiment 10 grammes of zymin, 10 grammes of glucose, and 100 c.c. of
water were incubated at 25°, and 75 c.c. of a 0°3 molar solution of potassium
phosphate added, 15 c.c. at a time. After rapid fermentation had ceased the:
mixture was filtered without boiling, and the lead compound obtained from
the filtrate as before.

Lead compounds have been prepared from glucose, fructose, and mannose,
and the analyses of several different preparations of these compounds are

1909. | Yeast-jwice from Hexose and Phosphate. . 638i

given in Table I. The mannose employed was obtained by the hydrolysis of
ivory-nut, and was purified by being converted into the phenylhydrazone,
which was recrystallised from water and decomposed with benzaldehyde.

Table I.

ie ; Percentages.
Noe Origin of | Weight CO.

H,O. | Mg,P,0,.| PbSO,.

compound. | taken.

C H 12, Pb

1 | Glucose ...| 0°5261 | 0°1870 | 0:0777 —_— — 9°69 | 1°65 | — —
0 -6269 — —_— 0:°1775 | 0°5080 | — — | 7°88 | 55-34

2 FA ...| 0'4745 | 0°1696 | 0 :0732 — — 9°75 |} 1°73 | — —
0 5993 — _— 0:1704 | 0:4774 | — | — | 7°92 | 54-40
3 | Fructose ...| 0°3040 — —- 0:0890 | 0°2484 | — — | 8°15 | 55°81
4 ee O4503) = a 0-1887 | 0:3649| — | — | 8:27 | 55-34
5 | Mannose ...| 0 °3614 — -—— 0:1021 | 0:2931-| — — | 7-87 | 55-39
CeHypO4(RO{ Pb) s requixes) cece. secession ceciee-ieo> 9-60 | 1°33 | 8:27 | 55-20

Hexosephosphoric acid was obtained from the lead salt by suspending it in
water, decomposing with sulphuretted hydrogen, and removing the excess of
this gas from the filtered lquid by means of a current of air. An acid
liquid was thus obtained which could be titrated with standard alkali,
phenolphthalein being used as indicator, in a similar manner to phosphoric
acid. In Table II several examples are given in which the amount of
hexosephosphoric acid was estimated both by titrating the solution with
decinormal alkali, and by decomposing with nitric and sulphuric acids,
precipitating with magnesium citrate mixture, and weighing as magnesium
pyrophosphate. The numbers all refer to 10 c.c. of the solutions.

Table II.
Hexosephospborie acid.

bo Titration, 7

Origin. ec, N/10: Mg,P30;.
From titration. From Mg.P,0-.

GrlUCOSe? = erciscisrseeiet biacioe aalsiecians 198 0 1086 0 1683 01664
Reb SG ersh ne e, 63-4 0 °3458 0 5390 0 5296
MMi Why) Seemncnpasiaseonmanasiceen 99-0 0 5434 0 8415 0 °8323
STD Re a aadeatamonacemeamentas 9°6 00517 0 0816 00792
ip || Japa bovooabdaedpouDgbuEDo 29 °2 0 °1559 0 -2482 0 :2388
JOTI HOSE: con sponnovedicnooncenanen 38 °7 0 °2124 0 3290 0 *3253
Naw Weammose) aiiysct. dtetiing ios seinoss 32 °6 0 "1802 02771 0-2761

When the solution was evaporated on the water-bath a charred mass was
left which contained free phosphoric acid and had a strong odour of caramel,
whilst even on evaporation under reduced pressure, or at ordinary tempera-

532 Mr. W. J. Young. Hexosephosphate formed by {July 30,

tures in a vacuum over sulphuric acid, decomposition occurred and free
phosphoric acid was formed. The solution was found to give Mohlisch’s
a-naphthol reaction. It reduced Fehling’s solution only after some hours in
the cold, rapidly on boiling: this reduction may be due to the hydrolysis of
the compound with formation of a reducing hexose. This is known to take
place, as an alkaline solution of the sodium salt that had been left standing
for two or three days at room temperature was found to contain free
phosphate, and a levorotatory substance which reduced Fehling’s solution
in the cold in a few minutes. The same change was brought about more
rapidly when the solution was boiled.

The acid was found to have a less reducing power when boiled with Pavy’s
amimoniacal copper solution than corresponded to an equivalent quantity of
glucose. Thus five preparations from glucose gave reductions of 0°754,
0-788, 0°767, 0°732, and 0°800, an equivalent quantity of glucose being taken
as 1. No osazone could be obtained, as on heating with phenylhydrazine
acetate decomposition took place, and phenylhydrazine phosphate was
formed. Attempts were also made to prepare insoluble hydrazones or
osazones with phenylhydrazine, methylphenylhydrazine, benzylphenyl-
hydrazine, parabromophenylhydrazine, and nitrophenylhydrazine, but in
no Gase was any success attained.

No differences could be detected between the acids obtained from glucose,
fructose, or mannose. They all yielded the same products on hydrolysis,
formed salts having the same properties, and had approximately the same
dextro-rotatory power. Table III gives the rotations of a number of
preparations of these acids.

Table III.
| Gtaeneneebnee | Observed rotation |
No. Origin. | Wee Per! in4-dm. tube. | Temp. Pas
| at | + | aF
eo}

1 Gillucoselpeere steer 0-079 0-097 20 3-07

2 35 | 0-539 0-671 18 3-1l

3 5 | 0-246 0 364 19 3°70

A iS sog0db09 3408000 0-114 0-161 16 3°53

5 Beli, | Rarie: Meee | 0-088 0°131 13 3°72

6 Silent iaR a tantaas | 0-178 0°278 16°5 3°90

7 PWN AL ale oe | 0-148 0-179 16 3-02

8 Pr iieescndoccogbosss 0-247 0 °333 15 3°38

9 1 i 2 NR LAN 0-529 0 695 16°5 3-28
10 Se A en Ee A 0-425 0 560 25 3:29
11 Fructose ............... 0°247 0°315 16°5 319
12 r teen el MOLT 0-416 18 3°31
13 OT Aes Nic Ota 0143 0-181 17 3°15
14 Mannose ..............- 0-152 0-189 19 3°12
15 A le REN RS 0°555 0-926 17 417
16 Yeast-juice ............ 0-053 0-074 13 3 50 |

1909. | Yeast-jwice from Hexose and Phosphate. 533

All these acids were prepared from the lead salts with sulphuretted
hydrogen, excepting No. 13 which was obtained from the barium salt by
means of dilute sulphuric acid. No. 16 was obtained from yeast-juice to
which neither sugar nor phosphate had been added, whilst zymin was
employed for the preparation of Nos. 6 and 11.

It will be observed that the differences between the values for the acids
derived from fructose and mannose are of the same order as the differences
among the glucose preparations themselves. With so low a rotatory power
a small experimental error or a trace of optically active impurity would
cause an appreciable difference in the ep, and the differences seen in the
table are probably due to these causes. The rotations of the barium salts of
these acids, which are slightly soluble in cold water, were also determined,
and were found to be approximately equal. These are given in Table IV.

Table IV.
Cee ner Rotation in *
Origin. P 4 dm. ay 17°5.
10 c.c.
+ +
Glucose .....c.ceeee: 0 -0665 0-085 3 20
IDI EORO={2) podnebadse- 3008 0 0692 0-088 3°18
Mannosetenss.scasecece 0 0656 0 :082 3°13

Hydrolysis of the Acid.

(a) Production of Phosphoric Acid and a Levorotatory Reducing Substance.—
When the solutions of the acid were boiled, phosphoric acid was gradually
set free, the reducing power to Pavy’s solution increased, and the solution
became levorotatory. Table V shows the rate at which the first two
of these changes took place when a solution of hexosephosphoric acid
prepared from glucose was boiled in a flask fitted with a reflux condenser.

The phosphoric acid is expressed as grammes of magnesium pyrophosphate

Table V.
Time, in hours. | Free phosphate. Reduction.
0 0 0 °258
2 0-167 0 :297
4 0 .:238 0 °342
Gi 0 304 0:371
11 | 0 °365 ee
15 0-401 0°373
27 | 0-467 0°371

i)
©

VOL. LXXXI.—B.

534 Mr. W. J. Young. Heaxosephosphate formed by [July 30,

and the reduction as the number of grammes of glucose to which it
corresponded. The original solution contained 0°7167 gramme of the acid
in 200 cc. (approximately 0:01 molar), corresponding when completely
hydrolysed to 0°511 gramme of magnesium pyrophosphate, and equivalent
to 0'414 gramme of glucose. Samples were taken out after the intervals
stated, and the free phosphate estimated with magnesium citrate mixture
and the reducing power with Pavy’s solution.

The solution after about seven hours’ boiling became very dark coloured,
some of the reducing substance formed evidently being decomposed.

The acids obtained from fructose and mannose behaved in a similar
manner on boiling, in every case a levorotatory substance and phosphoric
acid were formed. The following Table VI gives a number of examples of
this change in rotation on the hydrolysis, at 100°, of the compounds prepared
from all three sugars and, in one case, No. 6, of that obtained from yeast-
juice to which nothing had been added.

Table VI.
|
Rotation in 4-dm. tube.
No. Origin. {ane of boiling,
ee ce Before. After boiling.
+ _—
1 Glucose ......... 6 0°161 0 °299
2 Jul “ 0 558
14 55 0°658
2 ae OR 10 0 634 0-756
3 alte Ie 6 0-543 0-924
A Fructose ......... 10 0-416 1°514
5 Mannose ......... 6 0 +230 O 204
6 Yeast-juice ...... 16 0074 0 500

As glucose is converted by alkalis into a levorotatory mixture of
glucose, mannose, and fructose, it was thought that the treatment with
alkali in the separation of the free phosphate, during the preparation of the
lead salt, might have altered the hexosephosphate in such a manner as to
account for the levorotatory sugar being obtained on hydrolysis. Prepara-
tions of the lead salt were therefore made without removing the free
phosphate, the solution being kept always slightly acid, but the method
being otherwise the same as before. The hexosephosphoric acids obtained
from these acid preparations were found to have approximately the same
specific rotatory power as the others. The strength of the solution used was
estimated by determining the total phosphorus, and making allowance for
the small quantity of free phosphoric acid, which was precipitated in

1909. | Yeast-jwce from Hexose and. Phosphate. 535

a separate quantity with magnesium citrate mixture. In Table III, already
given, Nos. 5, 6, 12, and 14 refer to acids prepared in this way, and it will be
seen that they cannot be distinguished from those which had undergone the
alkaline treatment. On hydrolysis, also, the same levorotatory solution
was obtained as is shown in Table VII.

Table VII.
|
Rotation in 4-dm. tube.
No. Origin #5)/ Dane ot Roving,
: Before. After boiling,
+ —

il Glucose ......... iil 0 056 0 °363
2 ee 8 0-131 0°370
OU ene ea aaa cee 6 0-637 1 +184
4, Fructose Eanes 2 0°317 0 :276
Fr iat SOcLnG 4 0 °574

MALT Stee 6 0-956

Pi eae e. 8 : 1-212

(b) Nature of the Reducing Substance—To determine the nature of this
reducing substance, the mixture from the hydrolysis of a preparation from
glucose was exactly neutralised to litmus with barium hydrate, and the
precipitation of the barium salts of phosphoric and unchanged hexose-
phosphoric acids completed by the addition of two volumes of absolute
aleohol. After filtration, the solution was evaporated at 35° to 40° under
reduced pressure to a thick syrup. This was found to reduce Fehling’s
solution in the cold, and to be levorotatory. It gave the red coloration
characteristic of fructose when heated with resorcinol and hydrochloric acid
(Seliwanoff). When this syrup was treated at 0° with milk of lime, an,
insoluble calcium compound similar to calcium fructosate was formed. This:
was filtered off, washed with water, suspended in water, and decomposed
with carbon dioxide, and the filtered liquid treated with animal charcoal and
evaporated under reduced pressure.

The colourless syrup thus obtained reduced an alles solution of copper
elycocolate after 12 hours in the cold, a reaction which, according to Pieraerts,*
is only given by fructose. It readily formed an osazone when heated with
phenylhydrazine in the usual manner, and this, after recrystallisation, first
from alcohol and then from a mixture of pyridine and water, melted at the
same temperature as glucosazone, viz., 206°, whilst a mixture of this compound

* “Chem. Zentral., 1908, vol. 1, p. 1854. ;
7. B)

536 Mr. W. J. Young. Hexosephosphate formed by [July 30,

with glucosazone melted at 206° to 207°. The analysis also corresponded to
that of glucosazone—
0°1222 gramme gave 16:2 c.c. nitrogen at 16°4 and 7743 mm.
N = 15°76 per cent.
Glucosazone, CigH2204N4, requires N = 15°64 per cent.

The fructose and mannose acids, when treated as above, yielded syrups
having the same properties as that from the glucose acid; in both cases
glucosazone was obtained from them.

All these syrups were found to induce rapid fermentation in a mixture of
yeast-juice and glucose with such an excess of phosphate that fermentation
was only proceeding at a very slow rate, and this induction has been shown
to be brought about by the addition of fructose, but not of glucose or
mannose.*

Finally, the ratio of the rotation to the reducing power was obtained and
was found to be approximately the same as that found for pure fructose.
The reducing power was determined by means of Pavy’s ammoniacal copper
solution and, as this was standardised, each time it was used, with a solution
of pure glucose it has been found convenient to express the reduction per
100 c.c. of solution by the number of grammes of glucose which would reduce

the same amount of copper, and to compare the rotation of the solution in
Rotation

a 4-dm. tube with this number. In Table VIII, this ratio, Reduction’ =

eduction
given for the hexoses obtained by means of milk of lime from the products of
hydrolysis of the acids derived from all three sugars, and it will be seen that
it agrees in every case fairly well with that found for pure fructose.

Table VIII.
— Rotation in Reduction as glucose :
Sugar from— | A aerate per 100 cc. Ratio.
°
Gilacose/acid ins. .s:sc--2--cne 1-806 0512
Fructose acid (1) ........ are 0 ‘962 0 284
= (QB) onascaceson 0°516 0°151
~Mannose acid............--.... 1 ‘376 0-441
Pure fructose (1) ............ 0-607 R 0-161
> (@)) stcronacabss 0-829 © 0°219

The solutions, after removal of the phosphate and hexosephosphate and
before the treatment with milk of lime, always possessed a greater reducing

* Harden and Young, ‘Chem. Soc. Proc., 1908, vol. 24, p. 115; ‘Roy. Soc. Proc.,
B, 1909.

1909. | Yeast-juice from Hexose and Phosphate. 537

power than a solution of fructose having the same optical activity. The
ratio of the rotation to the reduction was always lower than that for pure
fructose, and varied from 1°36 to 2°78. This might be accounted for by the
presence of glucose or mannose, and experiments were made to ascertain if
these sugars were present; in no case, however, could either be detected.
The insoluble hydrazone of mannose was not obtained with phenylhydrazine,
nor was a hydrazone formed when the syrup was treated with methyl-
‘phenylhydrazine in the manner described by Neuberg* for the separation of
glucose or mannose from fructose. They were also examined for glucose by
the method by which Lobry de Bruyn and van Ekenstein+ detected glucose
in the mixtures obtained by the action of alkalis on fructose or mannose.
This consisted in removing most of the fructose by precipitating as calcium
fructosate or by extracting successively with alcohol, ethyl acetate, ether, and
acetone, and identifying glucose in the residue either by converting into
a-methyl glucoside by means of methyl alcoholic hydrochloric acid, or by
oxidising with dilute nitric acid and precipitating the acid potassium salt of
the saccharic acid formed. Neither of these compounds could be obtained
from the syrups under examination.

In order to ascertain whether these low ratios were due to the action of
the phosphoric acid on the fructose after it was set free, a mixture of these
compounds in the same relative proportions as would be contained in
hexosephosphoric acid was boiled for some hours, and the phosphoric acid
then removed by means of barium hydrate and alcohol in a similar manner
to that described for the hydrolysed acid. The results of two experiments
are given in Table IX.

Table IX.
}

Time, in hours. — Rotation in 4 dm. tube. | Reduction per 100 c.c. Ratio.
@yi0 7-424 1-927 3°87

. 10 3 °832 1-082 354
15 2 580 0-791 3°26

(2) O 0-711 0-188 3°78
10 4, -250 1-276 3°33

In both these experiments the solutions became very dark coloured and
a considerable quantity of fructose had been destroyed. It will be observed
that in both cases the ratio became less on boiling, but the lowest value
obtained was much higher than the numbers found for the product from
hexosephosphoric acid. It is possible that the fructose as it is being formed

* ‘ Ber.,’ 1902, vol. 35, p. 959.
+ ‘Rec. Trav. Chim.,’ 1895, vol. 14, p. 156.

538 Mr. W. J. Young. Hexosephosphate formed by [July 30,

is much more susceptible to the action of phosphoric acid. On the other hand,
these low ratios may be due to the presence of some other reducing substances
having a less leevorotatory power than fructose.

Salts of Hexosephosphorie Acid.

In addition to the lead salt already described, several other salts have been
prepared. Attempts to obtain the potassium and sodium salts were made by
adding the hydrates to the acid solution until neutral to phenolphthalein, and
evaporating the solution under reduced pressure. A sticky mass was left
which would not crystallise, and which decomposed on keeping.

Solutions of the alkali salts slowly decomposed on keeping, free phosphate
being formed and the solution becoming levorotatory. This decomposition
causes a small error in the estimation of the free phosphate present in
a mixture of this salt and hexosephosphate by means of magnesium citrate
mixture. Many of the salts are more soluble in cold than in hot water ;
thus, when magnesium citrate is added to a solution of sodium hexose-
phosphate, no precipitate is formed until the mixture is heated; a white
precipitate then comes down, which redissolves on cooling. The manganese,
barium, and calcium salts behave in a similar manner.

Silver Heaxosephosphate, CgHi904(POsAg2)2—This salt was obtaimed by
neutralising the free acid with caustic soda, adding the calculated quantity of
silver nitrate and then half the total volume of alcohol. The precipitate was
filtered off, well washed with a mixture of water and alcohol, dehydrated
with alcohol and ether, and dried over sulphuric acid in a vacuum, and it was
thus obtained as a white amorphous powder. It was exceedingly unstable,
and the whole preparation was carried out by red light. Even when dry the
salt darkened on exposure to light, whilst when heated with water it was at
once reduced to metallic silver.

The following analyses were carried out with different preparations of this
salt :—

Table X.
i
Percentages.
oe Weight :
Origin. reer AgCl. Mg.P:0;.
Ag. 184
Giuicose) (1) Perea 0 -2340 01740 0 -0644, 55 94 7-67
59 2) ees ...| 0°3976 0 -2989 0 °1057 56 54 7 40
Fructose (1) ..........0.-.. 038293 0 -2439 0 0893 55°75 7°55
43 (PA i opesbedeldatiogs 0 3214 0 :2378 0 -0909 55 "70 7 87
CGH i O4(POsAge)o VEQUIVES....seseereereerneaneees 56°24 8 08

1909. | Yeast-juice from Hexose and Phosphate. £589

Barium hexosephosphate, CsHi90«4POsBa)2, was obtained by adding a
solution of barium chloride to a solution of potassium or sodium hexose-
phosphate, and warming the mixture on the water bath. A granular white
precipitate was thus formed, which was found to be more soluble in cold
than in hot water. This was purified by dissolving in cold water and
reprecipitating by warming the solution. It was then filtered off, washed
with warm water, and dried over sulphuric acid in a vacuum. At 17%5
100 c.c. of solution were found to contain 0°665, 0°692, and 0°656 gramme of
the barium salts derived from glucose, fructose, and mannose respectively
The following analyses were obtained for this salt :—

Table XI.
ee |
Percentages.
Origin. Wetshe BaSO, | Mg,P,0..
Ba. Ie
Gilarcose (wii seesck<acecee 0 :2578 0 +1994 0 -0960 45°50 10 36
Co | Wee Nias ooo g aatie age | 0 °4317 = 0 °1538 = 9 -92
Fructose eX cebasiocarsecnoC 0 4009 0 -3081 0 °1485 45°23 10 °32
sbaéocRoocebeeo 0 °5221 0 -3933 | 0 -1902 44°34 10 ‘14
| Ree tea SJEROCIONECODEOASPere 0 -4186 0 °3179 | 01538 44°70 10°22
| 6Hp0,(PO,Ba)2 requires....,........2.0.-s000+ 44°92 10°16

Calcium Hexosephosphate.—A calcium salt of the acid derived from glucose
was obtained by adding calcium chloride to a solution of the sodium salt,
and completing the precipitation by adding alcohol. When dried over
sulphuric acid in a vacuum it gave the following analysis :—

0:3488 gramme gave 0°1958 gramme COz, 00917 gramme H,0,
0:0890 gramme CaO, and 0°1778 gramme Mg»P20;.
C = 16°81, H = 2:92, Ca = 18-24, P = 14-20 per cent.
CgH04(PO.Ca)2+ H20 requires C = 16°59, H = 2:76, Ca = 18°41,
P = 14-28 per cent.

Source of the Hexosephosphate-—In order to show that this compound was
formed from the added phosphate, two quantities of 50 c.c. of yeast-juice
were incubated at 25°, one with 30 c.c. of a 30-per-cent. solution of glucose
and the other with 30 c.c. of a 0°3 molar solution of potassium phosphate
containing 30 per cent. glucose. After two hours both were boiled and
filtered, 30 cc. of each filtrate were treated with the same quantity of
magnesium nitrate and potash to remove any free phosphate and, after

540 Mr. W.J. Young. Hexosephosphate formed by [July 30,

standing for a few hours, the solutions were filtered, neutralised with acetic
acid and lead hexosephosphate precipitated with lead acetate, washed and
dried over sulphuric acid in a vacuum. The mixture to which the phosphate
had been added gave 5°03 grammes of lead salt, whilst the other only yielded
1:35 grammes. This experiment also shows that yeast-juice itself contains
a small quantity of hexosephosphate, and experiments have already been
quoted to show that this compound has similar properties to that formed
when phosphate and sugar are added. The same hexosephosphate was
obtained by means of zymin in place of yeast-juice as already mentioned,
and as in this case the mixture was not boiled before the precipitation of the
lead salt, it follows that the salts of this acid were present in the mixture as
such, and were not formed from a more complex body by the boiling, which
was always necessary when yeast-juice was employed. A hexosephosphate
has also been detected in the extract obtained by boiling pressed brewers’
yeast with water.

Molecular Weight of Hexosephosphoric Acid.—The analyses of the salts of
this acid gave no information as to its molecular weight, smce the same
percentage composition would be obtained if the acid had the formula
C3H;02(PO.H2) and not CgHi 90. POsH2)2. The first compound might be
expected on hydrolysis to yield glyceraldehyde or dioxyacetone, and these
might polymerise to form hexoses; so that the fact that fructose was formed
when the acid was hydrolysed does not do away with the possibility of the
smaller formula.

As no derivatives of the acid could be obtained, such as a hydrazone or an
osazone, analysis of which would have settled the question, recourse was
made to physical methods. The molecular weight of the acid was calculated
from the difference between the freezing point of its solution and that of
water, and was compared with that calculated by taking into consideration
the extent to which it was dissociated in solution as determined by the rate
at which it hydrolysed cane sugar. The freezing points of three solutions of
different concentrations of the acid prepared from glucose was found by the
ordinary method, the amount of acid in solution was estimated by titration
and checked by the total phosphorus content, and the weight of water in the
solution was obtained from the specific gravity. By way of comparison with
a compound of known constitution and of a similar nature, corresponding
determinations were carried out with a sample of synthetical glycero-
phosphoric acid, which was purified by means of its lead salt in exactly the
same manner as the hexosephosphoric acid. The results are given in
Table XII, Nos. 1, 2, and 3 being those obtained from the hexosephosphoric
acid, and Nos. 4 and 5 from two concentrations of glycerophosphoric acid.

1909. ] Yeast-jwice from Hexose and,Phosphate. . 5AL

Table XII.
uae Grammes per | Specific Weight of : Molecular
210; | Oana ly 100 c.c. gravity. water. Wepzessions. weight.
1

1 1°75 15°13 1:075 92°37 1 °747 177

2 0:99 8-42 1:042 95 -78 0 :932 178 |

3 0°55 4°68 1-022 97 52 0 °585 169 *4 |

4 1°65 14 ‘21 1 :063 92 09 21538 135 °5 |

5 0:97 8°38 1 037 95 °32. 1 °800 127 ‘7

The formula CgH1)0.(PO,H2)2 corresponds to a molecular weight of 340,
-and the smaller formula to 170, whilst glycerophosphoric acid, C;H;O2(PO.H2),
has a molecular weight of 172. Thus the value obtained, although low, was
much greater than that found for elycerophosphorie acid, which only differs
in composition from the smaller formula by two hydrogen atoms.

The cane-sugar hydrolysis was carried out at 25° with semi-normal acid,
and the rate was compared with that found for semi-normal hydrochloric
and glycerophosphorie acids under similar conditions.

Table XIII gives the figures found for the hexosephosphoric acid and
Table XIV those for the glycerophosphoric acid. Allowance has been made
in the tables for the rotation due to the acids themselves. Solutions of the
hexosephosphoric and glycerophosphoric acids of the same normality were
also kept by themselves under similar conditions for the same time, and no
alteration took place in their rotations.

Table XIII.—Hexosephosphoric Acid.

K x 10#
Time, in minutes. Rotation. Bie A—za. (K tell log a ).
A-—zx
0 + 26 83 (0) 84 ‘88 —
30 25 *95 0°88 34°00 3 °700
60 24 98 1°85 33 °03 3 “945
90 24 ‘O01 2 82 32 ‘06 4069
120 23 °12 83 27/l 31°17 4-070
160 22 ‘05 4°78 30 ‘10 4-001
180 21 44. 5°39 29 47 4051
210 20 *66 6:17 28°71 4-026
240 19 :95 6°88 28 :00 3 976
310 18 17 8°66 26 °22 3 999
330 U7 372 9-11 25°77 3 984.
360 17 -06 9°77 25-11 3 ‘965
390 16 °32 10°51 24°37 3 9938
420 15-70 11°13 23°75 3974
o) —8 05 34°88
Mean. ceveeses 3 981

542 Mr. W. J. Young. Hexosephosphate formed by [July 30,

Table XIV.—Glycerophosphoric Acid.

Time, in minutes. Rotation. a, | A—z. | K x 10%
0) + 26°36 0 34°41 —
31 25°51 0°85 33 °56 3 503
60 24°79 1°57 32 84 3 °380
90 24 °06 2°30 32°11 3 °338
120 23 °30 3°06 31°35 3 °370:
150 22 64 3°72 30°69 3 °312
182 21°86 4°50 29 ‘91 3344
210 21°28 5°13 29 -28 3 °339
240 20 *54 5°82 28 °59 3 °353
270 19 -89 6°47 27 94: 3 °350
300 19 °26 7°10 27°31 3 °345
330 18 64 ehe, 26 ‘69 3°3438
360 18 ‘01 8°36 26 05 3 °358

a —8 05 34°41
Mean......... 3 °3538

Semi-normal hydrochloric acid under the same conditions gave the constant
K x 10* = 22°466,

It follows from these rates that the ratio of hydrogen ions in the solution
of hexosephosphoric acid to those in the hydrochloric acid was 3-981 to
22:466 or 17:7 to 100, whilst in the case of glycerophosphoric acid the ratio.
was 3°355 to 22-466 = 14:9 to 100. As hydrochloric acid is almost completely
dissociated, these numbers may be taken as a measure of the concentration of
hydrogen ions in the solutions.. Glycerophosphoric acid is dibasic and a
solution which is semi-normal by titration will contain only half as many
molecules as a semi-normal solution of hydrochloric acid, or 50 molecules for
every 100 of hydrochloric acid. If the glycerophosphoric acid be assumed to
dissociate into two ions, then out of every 50 molecules 14:9 are dissociated
into 14:9 x 2 = 29°8 ions, and 50 —14°9 = 35:1 molecules remain undissociated.
The solution will thus contain 29°8+ 35:1 = 64°9 units, and the freezing point
depression will correspond to a molecular weight of a x 172 = 132°5 ; those
actually observed gave 135°5 and 127-7. Similarly with the hexosephosphoric
acid, if the larger formula obtain, the acid is tetrabasic and a semi-normal
solution will only contain one-quarter as many molecules as a semi-normal
solution of hydrochloric acid, or 25 molecules to every 100 of hydrochloric
acid. If the dissociation take place into two ions, of these 25 molecules
17°7 are dissociated into 17-7 x 2 = 35:4 ions, 25—17°7 = 7°3 molecules remain
undissociated, and the total number of units will be 734+35°4 = 42°7. The
molecular weight of the acid from the freezing point of this solution will be

oe = 199; the observed values were 177, 178, and 169-4.

1909. | Yeast-juice from Hexose and Phosphate. 543:

If, on the other hand, the compound have the smaller formula, with a
molecular weight of 170, it will be a dibasic acid and the solution will
contain 50 molecules to every 100 contained in a solution of hydrochloric
acid of similar normality. The number of dissociated molecules corresponding
to the hydrogen ions present will be 17:7, yielding 17°7 x 2 = 35:4 ions,
50—17'7 = 32°3 will be undissociated, and the total number of units will be
35°44 32:3 = 67°7. The molecular weight, as deduced from the freezing point,
170 x 50

67°7

The observed depression thus corresponds more closely to that which would
be expected from the larger formula, although the agreement is not so good
as that in the case of glycerophosphoric acid. This low result may be due to
some hydrolysis in the solution, which would make the number of units still
greater. If the hexosephosphoric acid had the smaller formula, it would be
expected to depress the freezing point at least to the same extent as glycero-
phosphoric acid, since it only differs from this by two hydrogen atoms, and
is slightly more dissociated in solution than this acid. These results thus
point to the acid having the formula CgHi)04( POsH2)o.

Further evidence against the acid being a derivative of glyceraldehyde is
also afforded by the following facts:—When the acid was reduced with
sodium amalgam, no glycerol was obtained, as might have been expected if
it were a compound of glyceraldehyde. When glycerophosphoric acid was
heated with hydriodic acid in a current of carbon dioxide, it was reduced to

would therefore be — 55),

isopropyl! iodide, which was detected by passing into alcoholic silver nitrate in
the manner in which glycerol is estimated by the method of Zeisel and
Fanto. On the other hand, hexosephosphoric acid gave no volatile iodide
when treated in this manner.

Constitution of Hexosephosphoric Acid.—lt has not yet been found possible
to obtain any evidence with regard to the position of the phosphoric acid
groups in the molecule. The facts that the compound does not reduce
Fehling’s solution in the cold until after a long time, and that no hydrazones
or osazones could be obtained, render it possible that the acid does not contain
an active carbonyl group, and it may be that this group is involved in the
reaction with the phosphoric acid.

Glucose, fructose, and mannose all appear to yield the same hexose-
phosphoric acid when fermented by veast-juice in the presence of phosphate.
One explanation of this is that these sugars are changed, before conversion
into hexosephosphate, into some form common to all three. The only
differences between glucose, fructose, and mannose are in the groups or
arrangement of the groups attached to the first two carbon atoms:

544 Hexosephosphate formed by Yeast-jwce from Hexose, ete.

CHO CHO CH,0H
H-C-0H OOH Go
OH-C-H OCH OHCH
HC-OH H-C-OH HC-OH
HCOH H-C-OH 1-C-0H
CH.OH CHLOH (HOH
Glucose. Mannose. Fructose

and all these sugars have the same enolic form:
CHO CHOH CH,0H

| Ul

CHOH COH CO
| == | —> |
CHOH. CHOH. CEO:
| —> | mead

CHOH CHOH CHOH
| |

CHOH CHOH CHOH

I | |
CH,OH CH,OH. CH,OH
Glucose and mannose. Enolic form. Fructose.

and are converted into one another in alkaline solution,* and it is conceivable
that the hexosephosphate is a derivative of this enolic form.
If the reaction take place according to the equation:

2CgHi20¢6+ 2R’2H POs. = 2CO2+ 2C2H60 + CgHi90u( PO4R’s)2 4+ 2020,

two molecules of sugar are concerned, and another possibility is that the
phosphoric acid groups are attached to a new molecule, which is formed by
the combination of portions of each of these two sugar molecules. In this
case, also, the hexosephosphoric acid might be expected to have the same
composition when formed from different sugars.

Summary.—t|. The compound formed during the accelerated fermenta-
tion of glucose, fructose, and mannose by yeast-juice, in the presence of
a soluble phosphate, is a salt of an acid which probably has the formula
C6Hi004(PO1H2)2, and may be isolated by precipitation of its lead salt.

2. The free acid may be obtained in solution by decomposing this lead
salt with sulphuretted hydrogen.

.3. The acid is very unstable and readily decomposes on keeping, or on
evaporating even at ordinary temperatures in a vacuum over sulphuric acid,
with formation of a reducing substance and phosphoric acid.

4, It reduces Fehling’s solution only after some hours in the cold, rapidly
on boiling, whilst no osazones or hydrazones have been obtained from it.

* Lobry de Bruyn, ‘ Rec. Trav. Chim.,’ 1895, vol. 14, p. 201.

Comparative Power of Alcohol, Ether, and Chloroform, etc. 545

5. No differences have been detected between the hexosephosphoric acids or
their salts, whether derived from glucose, fructose, or mannose.

6. On hydrolysis of the acid by boiling, phosphoric acid is set free and
fructose formed. No other hexose could be identified, but the solution, after
hydrolysis, was always less levorotatory than a solution of pure fructose of
the same reducing power.

7. The salts of lead, barium, silver, and calcium have been prepared.

[The compound containing phosphorus, which was considered to be phenyl-
hydrazine phosphate, has since been examined by von Lebedew (‘ Biochem.
Zeits.,’ 1909, vol. 20, p. 113), who regards it as a phenyl hydrazido-phosphoric
acid compound of hexosazone. A re-examination of this substance by the
author leads to the conclusion that it is in reality a derivative of hexose-
phosphoric acid, but decisive results as to its constitution have not yet been
obtained.— November 15, 1909.]

The Comparative Power of Alcohol, Ether, and Chloroform as
measured by ther Action upon Isolated Muscle.
By Aucustus D. WALLER, M.D., F.R.S.

(Received and Read June 24, 1909.)

The object of the following communication is twofold: (1) to present the
results of a careful comparison of the physiological effectiveness of certain
narcotics, and (2) to illustrate the degree of accuracy of which such com-
parisons are susceptible by the systematic use of the sartorius muscle of the
frog as an indicator.

Method.—The two sartorius muscles of a frog are dissected out and the
portions of bone to which they are attached are ligatured with fine copper
wires serving as conductors. The muscles are set up in the two vessels
V, V and connected with two myographic levers that record their movements
on two smoked plates L, R. The connections with the secondary coil of an
inductorium (Berne model) are as given in the diagram, so that both muscles
are traversed in series by the same current in the same direction. The
muscles are directly excited once every 10 seconds by maximal break
induction shocks. Each observation consists of three parts: a first part
consisting of the normal responses of the muscle immersed in normal saline
(0°6 per 100 NaCl in tap water); a second part consisting of the responses
while the muscle is immersed in an experimental solution; a third part

546 Dr. A. D. Waller. Comparative Power of [June 24,

consisting of the responses while the muscle is replaced in normal saline.
The solutions are changed by being run off through a tap and run in from
a pipette, care being taken that the volume of fluid is always the same.
The induction currents are kept going automatically throughout an experi-
ment, excepting during the short periods required for changing the solution.
The apparatus used for this purpose consists of: (1) a Berne coil fed by a
2-volt accumulator ; (2) a Brodie clock with interruptions set at six per
minute; and (3) a relay key, ze. that shown by G. R. Mines at the July,
1908, meeting of the Physiological Society.

As a general rule of procedure in any comparison between the effects upon
two muscles L aud R of two solutions A and B, a first comparison is made
between the effects of A on L and of B on R, and a second comparison of
the effects of B on L and of A on R. Each complete experiment thus
comprises two pairs of simultaneous trials of two solutions in reversed order of
action, and constitutes an experimentwm crucis in the strict sense of the term.

Electrical excitation of the muscles while immersed in the experimental
solutions—in spite of the fact that the induction currents are in large
measure short-circuited by the solution—-was systematically adopted in
preference to excitation of the muscle after the solutions had been run off,
because it affords a more complete picture of the gradual effects of such
solutions. Currents of sufficient strength are taken to give maximal excita-
tion in spite of the derivation,

Double Myograph to test Action of Substances in Solution.

>

The diameter of the muscle vessel was slightly less than 3 em., so that
30 c.c. of fluid gave in it a column about 5 cm. long, more than sufficient to
‘keep the muscle wholly immersed.

1909. | Alcohol, Ether, and Chloroform, ete. 547

The exciting currents passed through the muscle and solution—principally
through the former by reason of the copper wires by which it is attached—
are taken of such strength as to give assuredly maximal effects. Their
direction is not a matter of indifference, the contractions being always
unequal to the two directions of excitation; as a rule, but not always, the
more effective direction was from tibial to pelvic end, and this was therefore
taken as the ordinary direction of exciting currents. But this is not a very
essential point, all that is really necessary is to keep to one direction during
experiment. Unpolarisable electrodes are also unnecessary, as, indeed, may
be readily seen from the records obtained. The magnification of contraction
by the lever was x 2.

(From October 9 onwards I used narrower muscle tubes, in order to use
up less fluid for each bath, and to have a greater density of current passing
through the immersed muscle.)

Alterations of current distribution caused by alterations of resistance of
the experimental fluids; the oligodynamic action attributable to the use of
copper wire ; small differences of room temperature ; the possible excitation
of intramuscular nerve as well as of the muscle itself, are the principal
circumstances that have been considered and recognised to be negligible in
the present connection. On the other hand, every care has been taken to
secure constant strength of stimulation and constant pressure of the
myographie levers against the recording surfaces, which are moved past
the levers in tandem by the same clockwork. The influence of considerable
differences of temperature was specially examined (vide infra).

By preliminary experiments it was found that conveniently graded effects
upon muscular excitability were produced by a 5 per 100 solution of alcohol,
by a 1 per 100 solution of ether, and by a 1 per 1000 solution of chloroform
(by volume in each ease). These strengths are of the order of molecular
(58 c.c. per 100) in the case of alcohol, decimolecular (1:05 per 100) in that
of ether, and centimolecular (0°8 per 1000) in that of chloroform.

Thereafter solutions were made up on a molecular scale, taking as the
standard of reference a molecular solution of absolute alcohol, and as the
first terms of comparison a decimolecular solution of ether and a centi-
molecular solution of chloroform as tabulated below.

. 5 c.c. per 100 c.c. saline
Sp. gr. MUD to give molecular solutions,
1
IAT CoWOl gre Gases ake sties | 0°79 46 5:8
OTs Ree ee ee | 0-72 74. 10°3
Chloroform ..............- 1 ‘50 119 °5 7°95

548 Dr. A. D. Waller. Comparative Power of [June 24,

Comparisons were systematically made (1) between alcohol and chloro-
form ; (2) between alcohol and ether ; and (3) between ether and chloroform:
Such comparisons were, whenever possible, made upon the same muscle,

preliminary experiments having shown that two or more successive intoxica-

tions, if not too profound, by the same strength of solution, are of equal
gravity.

The principal indication of the comparative effects of reagents consists in
the rate at which the contractility is abolished in solutions (in 0°6 per cent.
NaCl in tap water) of variotis strengths. The rate and amount of return of
contractility in 0°6 per cent. NaCl affords confirmatory evidence, of which,
however, we have not made systematic use, having done no more than note
the facts: (1) that at equal times of immersion the time required for
recovery augments with augmented strength of solution, and (2) that at equal
strengths of solution the time required for recovery augments with
augmented time of immersion.

Comparisons may be established between : the effects of two solutions upon
the same muscle successively ; or between the effects of two solutions upon
two muscles simultaneously; and each kind of comparison has its own
obvious advantage and disadvantage. By the method we have adopted of
simultaneously recording the contractions of two muscles in series, we secure
the advantage of both plans, and minimise the disadvantage of successive
comparison by reversing the solutions on the two muscles. Other obvious
advantages of the double method are that we get double the number of
observations, and that we can readily tell whether an accidental irregularity
is due to the stimulus or to the muscle or to the solution.

We may also compare the effects of different solutions upon different
muscles, but in such comparisons from muscle to muscle we must take care
that the conditions of observation are, as far as possible, identical. We may
not, e.g., compare fresh with stale muscle, nor muscles of greatly unequal bulk,
nor muscles taken from healthy and unhealthy frogs, nor results obtained at
different temperatures. Nevertheless, comparisons of this order are practi-
cally available, for under similar conditions the results of experiment with a
given solution are closely similar upon different muscles; the “idiosyncrasies ”
of different muscles are not a very disturbing factor, although, as might be
expected, effects are more rapidly produced with very small than with very
large muscles.

The two chief fallacies in their order of importance are: (1) a variation in
length of the column of fluid, and (2) a considerable variation of temperature.
_ As regards the column of fluid, it is evident that this must be kept of
constant length during an observation, since the fluid forms a derivation cireuit

7909) Alcohol, Ether, and Chloroform, ete. 549

surrounding the muscle, which is traversed by only a small fraction of current.
The effect of varying the length of column is easily shown by adding or
taking away fluid while a series of contractions is in progress. I have, there-
fore, always been careful to replace fluids by pipette as exactly as possible. If,
as has sometimes happened, the difference of excitability and contraction in
two muscles has been grossly unequal—even more so than in the case of the
pair of muscles used for the record of fig. 1—I have thought it permissible to
adjust the tubes in their holders upwards or downwards so as to alter the
current lines in suitable degree. But once fixed in position, the tubes must
not be shifted again; the level of fluid must be kept unaltered throughout
experiment.

Differences of resistance between different fluids are in most cases of little
moment, ¢.g. a cubic centimetre of chloroform does not increase the resistance
of a litre of saline enough to influence the exciting current traversing the
muscle. In some cases, however, differences of resistance may be such as to
affect the current distribution and the response of the muscle, e.g.a 10-per-
cent. solution of alcohol in saline has an appreciably higher resistance than
saline alone.

It may be objected to the method that excitation is not restricted to the
muscular substance, but includes intramuscular nerve tissues. To meet this
objection I compared the effects on fully curarised and on uncurarised muscle,
and found that they were indistinguishable. This fact, however, is of little
weight, inasmuch as immersion in normal saline is of itself sufficient to
remove the effect of curarisation. But, on reflection, the objection itself is
of little weight. As is well known, the direct excitability of muscle outlasts
its indirect excitability wid nerve; loss of all contractility is of necessity
loss of direct excitability, and whatever might be said as to the beginning of
an observation, there can be no doubt that at its end we are dealing with
muscle and muscle only; even at the beginning of an observation, since we
are using a strength of current more than sufficient to excite muscle as well
as nerve, the contraction must be by direct muscular excitation; and even if
ib were not, if it were by indirect excitation at the beginning, the com-
parative results of, eg. the action of ether and chloroform, would remain
acquired. To use as a test object excitation of the nerve of a nerve muscle
preparation would, of course, be a different method, by which the tissue
specially under investigation would be the end-plate. To use as an index the
minimal strength of stimulation giving contraction would again be a different
method, by which at first indirect and later direct excitability would be
investigated. I have avoided both these proceedings, and have preferred to
follow the method described because it is more practicable and less ambiguous

VOL. LXXXI.—B. 2R

Comparative Power of [June 24,

Dr. A. D. Waller.

550

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1909. ] Alcohol, Ether, and Chloroform, ete. bok

in its results. I felt justified in making this choice by the results of experi-
ments made many years ago on direct and indirect excitability, and on the
junction between nerve and muscle.*

Gam...

LTT

AMA
Co al - Il O15 m,
ti oooncill 0:10 m.

SS SSS SS SS SS Ee ee ee eee

Fic, 2.—August 21, 1908. Effect of Ether Solution at Strengths of 071, 0°15 and 0:2 m.
(= 1, l'5 and 2 per cent. by volume).

ET A
| tut |
MIN,

AhhorwSforrnne ide by vt

: pe es fe ee pee ee

Fie. 3.— August 20, 1908. Comparison between the Effects of Chloroform in 1/1000 and
1/2000 dilution in normal saline (= m/80 and m/160) and of Ether in 1/100 dilution
(= m/10).
In chloroform 1/1000 contractility is abolished in 5 minutes.
1/2000 ” ” 12 ”

”

* “Experiments and Observations relating to the Process of Fatigue and Recovery,”
First Report, ‘ British Medical Journal,’ July, 1885 ; Second Report, ‘British Medical
Journal, July, 1886. In the present connection the principal conclusion of the investi-
gation (carried out during my tenure of a research scholarship to the British Medical
Association) was to the effect that the junction between nerve and muscle is functionally
a weak link in the neuro-muscular chain, being the first to suffer in its transmitting
function in fatigue (by indirect excitation) in intoxications (“curarisation ”) and in
pathological degeneration.

2 1D

552 Dr. A. D. Waller.

Minutes.

0 to}5

5 to 10

10 to 19

19 to 24

24 to 34

35 to 40

40 to 50
50 to 55

55 to 70

70 to 73!

73 to 83
83 to 88

Fie. 4.—August 22, 1908.

Comparative Power of [June 24,

I. Alcohol v. Chloroform.

Left.

In normal saline ...
=

tility lost in 4
minutes.

In normal saline ...

x
In alcohol, 1 mol.
solution, contrac- = 8
c
|
4
I

In chloroform, —= aX
m/100 solution, =
contractility lost —
in 3 minutes.

fi 2

In normal saline ... 4 == e
iS ieee mae
Ie as \

|
J
y tion, contractility lost
g in 3 minutes.
—-s |
c

Right.

In normal saline.

solution, contractility
lost in 2} minutes.

: 7 chloroform, m/100
|
fie

‘In normal saline.

Tn alcohol, 1 mol. solu-

In normal saline.

(Continuation of record not reproduced.)

In alcohol, 1 mol. solution, contractility lost
in 5 minutes.

men ormallesa line pe eerceeerenreeeerescieseindras

In chloroform, m/100 solution, contractility «
lost in 4 minutes.

nen orm als aline yer seessrkeraeerteec reese

In ether, 0:2 mol. solution, contractility
lost in 1 minute.

iinimormalesaline mene reeeseeeter eect er reres

In alcohol, 1 mol. solution, contractility lost
in 5 minutes.

In chloroform, m/100 solution, contractility

lost in 5 minutes.

In normal saline. fag
In chloroform, m/100 solution, contractility

lost im 34 minutes.

In normal saline. RE
In chloroform, 0-02 mol. solution contractility

lost in 13 minutes.

In normal saline. ; ;
In alcohol, 1 mol. solution, contractility lost in

5 minutes.

Simultaneous Record of Left and Right Sartorius Muscles.

Comparative Effects of Ethyl Alcohol (molecular sclution in normal saline, 5°8 ¢.c.
per LOO) and of Chloroform (centimolecular solution in normal saline, 0°08 c.c. per
100). Temperature = 20°. In the continuation of this experiment, comparisons
were made with 0°2 m. ether and 0:02 m. chloroform (not reproduced).

It appears from this experiment that chloroform of centimolecular strength

is slightly more effective than alcohol of molecular strength.

From a further experiment made with chloroform at 0:009 m. as compared
with alcohol of standard strength, it is found that at this strength chloroform

1909) Alcohol, Ether, and Chloroform, ete. 553

is considerably less effective. The physiological equality of chloroform with
our alcohol standard is between 0°010 and 0:009 m., nearer to the former
than to the latter value; we have therefore taken as a sufficiently close

approximation that
sep C=1A or 100A=1C.

‘ ( | | i : | | | i ie i

Minutes.

35 to 40 In normal saline...... In normal saline.

36 to 41 Im alcohol, 1 mol.
solution, contrac-
tility lost in 38
minutes.

mol. solution, con-
tractility lost. in
} minutes.

| In chloroform, 0:009
|

41 to 49 In saline............... In saline.

49 to 56 In chloroform, 0:009
mol. solution, con-
tractility lost in
6 minutes.

y

| In alcohol, 1 mol.

t solution, contrac-
tility lost in 33

| minutes,

56 to 63 In saline............... > In saline.
J
(Continuation of record not reproduced.)
HORCOM OMe UNIESA LIM Gece acyrencnencioaeiwee.heaseteices In saline.
75 to 82 In chloroform, 0:009 mol. solution, In alcohol, 1 mol. solution, contractility
contractility lost in 5 minutes. lost in 33 minutes.
CY We ffs} Isai GeulNYe: 5. ecboosopn0ccsanquancooovgB09nbO NK In saline.
88 to 94 In alcohol, 1 mol. solution, contrac- In chloroform, 0:009 mol. solution, con-
tility lost in 3 minutes. tractility lost in 5 minutes.

Fie. 6.—August 25, 1908. Simultaneous Record of Left and Right Sartorius Muscles.
Effects of Ethyl Alcohol, 1 m. solution, and of Chloroform, 0:009 m. solution.
Temperature = 20°.

554 Dr, A. D. Waller. Comparative Power of [June 24,

This relation is not confined to this particular strength. In an experiment
with 2 m. alcohol and 0:02 m. chloroform, the two reagents produced
substantially equal effects (October 6).

II. Alcohol v. aiien

Similar considerations apply to the estimation of the relative physiological
efficiency of alcohol and ether.
As compared with a molecular solution of ethyl alcohol it was found—

That a 01 mol. solution of ether was too weak,
» 0:2 ” bP]
oy PILE A 4 shehtly too strong,

too strong,

and that the closest approximation to equality of effects was obtained
with 0:13 and 012 mol. solutions, from which it is concluded that
molecule for molecule ether is between seven and eight times as powerful
as alcohol.

III. Chloroform v. Ether:

Similar considerations apply to the estimation of the relative physiological
efficiency of chloroform and ether.

We commenced by comparing the effects of a 1-per-cent. (by volume)
solution of ether and a 1-per-1000 solution of chloroform, these two strengths
being respectively equal to 0'1 m. and 0°012 m. The ether solution proved to
be considerably the weaker of the two.

For the next trial we took double the strength of ether, viz., 2 per 100 or
0'2 m., and found that at this strength the ether solution was considerably
stronger than a chloroform solution of 0°8 per 1000 (=0:01 m). Ina
further comparison between ether, 0°15 m., and chloroform, 0°01 m., the
former was still considerably the stronger, and from further trial of 0-13 m.
and of 0°12 m. ether we finally determined as nearest to physiological
equivalence :

0:01 chloroform = 0:12 ether.

1909.]

Minutes.

Otod In saline

5to12 In ether

Olam, E.

12 to 20 Im saline......... 4

L
20 to 26 In chloroform... |

26 to 84 In saline.........

Alcohol, Ether, and Chloroform, ete.

955

Right.

In saline.

In chloroform.

ee ee,

+ In saline.

fm ether.

fare | j
1

In saline.

Sa nee dccereeeeeel
Cat eae ere

(Continuation of record not reproduced.)

AGtOIADS, CInvsaline 2), cccc6ccc ees as /aeote os sieves
45 t0 52 In chloroform, contractility lost in
43 minutes.

In saline.
In ether, contractility lost in 6 minutes.

ESP) HO GIL hha, EEN hts \eyg5 con andootaodasnkcenedecacdceon In saline.
61 to 68 In ether, contractility lost in 4 In chloroform, contractility lost in 7
minutes. minutes.

_ Fie. 7.—August 26, 1908. Simultaneous Record of the Effects of Chloroform, 0-01 m.,
and of Ether, 0:12 m., on Two Sartorius Muscles.

The general conclusion from the foregoing experiments is given in the

following tabular summary :—

Physiological Equivalence.

By molecules. By weight. By volume.

| SCT Cle cnecoeccccseccc: 100 100 100

ibibo. 27. on ene 12 19°3 21°3
Chloroform ............... 1 26 1°4

I,e.1 molecule chloroform = 12 molecules ether = 100 molecules alcohol. T.e. a chloroform
molecule is 12 times as powerful as a molecule of ether and 100 times as powerful as a molecule
of alcohol.

[By weight approximately, 1 gramme of chloroform = 8 grammes of ether = 40 grammes of

alcohol.
By volume approximately, 1 c.c. of chloroform = 15 c.c. of ether = 75 c.c. of alcohol. ]

556 Dr. A. D. Waller. Comparative Power of [June 24,

Influence of Temperature upon the Rate of Intoxication.

In my earlier experiments upon the rate of intoxication of muscle by
alcohol, ether, and chloroform, I paid no particular attention to the tempera-
ture beyond noting that the ordinary room temperature during those
experiments was comparatively steady at 19° to 21°. But as the degree of
precision of which the method was susceptible became apparent, I undertook
to examine the quantitative effect of the temperature factor.

The first experiment in this direction (August 18) was made with a
5-per-cent. solution of ethyl alcohol for the purpose of testing the influence
of temperature upon the velocity of the reaction between alcohol and muscle
upon which the abolition of contractility depends. At 19° muscular con-
tractility was abolished in 7 minutes; at 30° muscular contractility was
abolished in 24 minutes; the velocity of reaction in this case was augmented
in very similar degree to the augmentations with raised temperature observed
in the saponification of ethyl acetate and in cases of vegetable activity.*
In these cases it has been observed that the velocity is increased between
twice and thrice with a rise of 10°; in the case of alcohol and muscle the
reaction was accelerated nearly threefold by a rise of temperature of 11°.

Similar results were obtained as regards the effect of raised temperature
upon velocity of reaction in the case of chloroform and in that of ether.

In the experiment of August 25, the times of abolition of contractility by
a 0:02 mol. solution of chloroform (1°6 c.c. per 1000) were—

TAT BIL Sica eter 2 min. and 24 min.

IOS ose Oumins4D secs eae

In the experiment of August 27 (fig. 8) the times of abolition of con-
tractility by a 0°15 mol. solution of ether (1'5 cc. per 100) were—

Avi 20F) exeriee 4 min. and 44 min.

1+ als
wecone Q » ” 2 ”

cin
|

Fic. 8.—August 27, 1908. Effect of Ether Solution, 0°15 m., on a Sartorius Muscle at
20° and at 28°.

* Cohen, ‘ Beitrage iiber Physikalische Chemie,’ 1901, pp. 37, 48, and 45.

1909. | Alcohol, Ether, and Chloroform, ete. OD

In an experiment of August 28, in which the times of abolition by a
0:01 mol. solution of chloroform (0°8 cc. per 1000 cc. saline) were
taken by stop-watch, the numbers were noted as 4 minutes at 20°, 2 minutes
at 30°, 14 minutes at 37°.

[ Wote (added September 1)—In a report recently presented to Section I of
the British Association at Winnipeg, I have brought forward evidence to
show that effects of the two anesthetics, chloroform and ether, are simply
additive, 7.c. the sum of their individual effects.

Taking, ¢.g., a mixture composed of (1 gramme chloroform+8 grammes
ether) per 1000 c.c. saline, I find that the solution is twice as powerful
as a solution of 1 gramme CHCl; per 1000, or 8 grammes Et20 per 1000.

Taking, as a point of departure, that 1 ec. CHCl; is physiologically
equivalent to 15 cc. Et,0, I find that the saline solution of a mixture
composed of equal volumes of chloroform and ether is approximately half
as powerful (actually rather more than half) as the saline solution of a
volume of chloroform equal to that of the volume of mixture in solution.

Assuming, as before, that 1 c.c. CHCl; = 15 ce. Et20, I calculate that the
physiological power of a mixture used in clinical medicine composed of two
volumes CHCl; and three volumes Et,O is 0°27 as compared with the power
of chloroform taken = 1:00.

Similarly, that the theoretical value of the well-known A.C.E mixture
(one volume alcohol+two volumes chloroform-+ three volumes ether) referred
to the same standard is 0:23.

To put these estimates to the test of experiment, a careful comparison was
made of three freshly-prepared solutions, containing respectively —

(1) 2°5 ec. per 1000 of the mixture (2C + 3E).
(2) lee. per 1000 of chloroform alone.
(3) 2:5 ec. per 1000 of the mixture (LA+ 2C+3E).

In correspondence with the fact that the theoretical equivalent amount,
in the case of the first solution = 2°3 c.c., and in that of the second solution
= 2'7 c.c. (as compared with 1 c.c. in the second or standard solution), it was
found that the effect of the first solution came out slightly above that of the
standard solution, while that of the third solution came out slightly below
that of the standard. ]

Note——Dr. Veley has been kind enough to give me the following calcula-
tion, from which it appears that we are really dealing with an alteration of
reaction velocity :—

558 Comparative Power of Alcohol, Ether, and Chloroform, ete.

At a temperature of 20° atime 4 min. was required for the abolition of

contractility.
”» 30° ” 2 ” 2» ”» bP)
” Bo » 1:25 ” ”? o> ”
Hence at the end of each minute,
ia) (Gey) (CD) canes 0-25 unit change took place> Referring to unity,
5 NCER) Beas 0:5 3 s } ratio of numbers
Pear GELB) ete 0°6 é. ll oe
By Esson’s formula,* (x1 —«) = m log (t1—T).
log 2 = 3010 log 3-4 = 5051
log 1
3010 5051
log 273+ 30 = 4814 log 273-++37 = 4914
log 273+ 20 = 4669 log 2734+ 20 = 4669
0145 0245
BOLO oa. HOW an, ;
Diag = 28: 545 7 20'S. Mean, 20:7.
Hence 0:0145 x 20'7 = 0:3002 = 1:99 cale, 2°00 found.
OOS sx 20 SS O02 = QQ 3°20

The graph of log «’/« in terms of log t/t is a straight line and is the most
convenient form of representation.

* ©Phil. Trans.,’ A, 1895, vol. 186, p. 861.

559

Studies on the Structure and Affinities of Cretaceous Plants.

By Marie C. Stopes, Ph.D. D.Sc. F.LS., Lecturer in Paleobotany,
Manchester University, and K. Fuut, Ph.D., Assistant Professor of
Botany, Imperial University, Tokio.

(Communicated by Dr. D. H. Scott, F.R.S. Received May 13,—Read
May 27, 1909.)

(Abstract.)

The authors comment on the importance of the work done on the flora of

the Paleozoic period, and the botanical interest that would attach to similar
petrifactions of plants from all ages of the Mesozoic period. They have had
the good fortune to find excellently preserved material from the Cretaceous
of Northern Japan.
In the present paper they describe 18 plants from this material, which is
extraordinarily rich. As hitherto there has been very little known from
anatomical material of plants of this age, the present paper is by no means
final, but is in the nature of a pioneer chart of the ground.

The petrifaction of the cells of the plants is often extremely good, though
the fragments are not so complete as could be desired. The plant structures
include stems, roots, leaves, cones, fern sporangia, and even an Angiospermic
flower, the first petrifaction of a flower to be described. The débris le
together in the nodules in much the same way that the débris lie in the
Coal-balls of the Paleeozoic, though they are mixed with fragments of shells.
The latter are largely Ammonites and serve to determine the age of the
petrifactions.

The flora as a whole represents an interesting mixed flora such as has not
hitherto come to light among petrifactions.

Roughly speaking, the flora seems to have consisted of about one-third
Angiosperms, slightly more than one-third Gymnosperms, and the rest of
ferns and lower plants. The anatomy of the early Angiosperms being such
a desideratum in botany, their presence in the petrifactions renders them
doubly interesting, and particularly when they are found in so evenly balanced
a mixed flora.

All the specimens described in this paper were cut in Tokio in the botanical
department by the authors.

The plants described are as follows :—

Petrospheria japonica, gen. et spec. nov. A fungus which has numerous
microsclerotia,in the periderm of one of the Angiosperms, Sawrwropsis.

560 Drs. M. C. Stopes and K. Fuji. Studies on the [May 18,

Schizwopteris Tanslevi, gen. et spec. nov. The sorus and sporangia of a
Schizeeaceous fern.

Fasciostelopteris mesozoica, gen. et spec. nov. The stem and petiole of a
fern with a dictyostelic anatomy. Probably allied to the Dicksoniacee.

Fern rootlets, in excellent state of preservation, showing the diarch stele of
the leptosporangiate ferns.

Zamiophyllum cordaitiforme, gen. et spec. nov. The leaf of what appears
to be some plant of Cycadean affinity, the anatomy bearing con-
siderable resemblance to that of Cordaites.

Yezonia vulgaris, gen. et spec. nov. A Gymmosperm, of which stems,
unthickened twigs, leafy axes, are all very plentiful. It is the
commonest plant in the material, and at the same time the most
unique. In the anatomy of both main axis and foliage it is not like
any known type.

Yezostrobus Oliverii, gen. et spec. nov. The fructification of a Gymnosperm,
the cone bearing simple scales with seeds, one on each, which are
like those of Cycads in some respects, but have a nucellus standing up
entirely free from the integument with a well marked epidermis
between.

Though continuity is lacking between these two plants, there seems
considerable ground for suspecting them of belonging to the same plant from
anatomical points of likeness.

Araucarioxylon tankoensis, spec. nov. Secondary wood, showing remarkably
clear pittings in the transverse sections.

Cedroxylon Matsumuru, spec. nov. Well preserved secondary wood.

Cedroxylon Yendow, spec. nov. Secondary wood, with traumatic resin
canals.

Cunninghamistrobus yubariensis, gen. et spec. nov. A cone, as its name
implies, belonging to the family of the Cunninghamias, with its
external appearance partly preserved and the cone scales and axis
fairly well petrified. The seeds have apparently been scattered.

Cryptomertopsis antiqua, gen. et spec. nov. Stem with leaves attached, the
foliage very like that of a Cryptomeria.

Saururopsis niponensis, gen. et spec. nov. The stem and attached roots of
an Angiosperm, probably to be included in the Saururacee.

Sugloxylon Hamaoanum, gen. et spec. nov. The secondary wood of an
Angiosperm.

Populocaulis yezoensis, gen. et spec. nov. The stems of an Angiosperm,
with cortical tissue.

1909.] Structure and Affinities of Cretaceous Plants. 561

Fagoxylon hokkaidense, gen. et spec. nov. The secondary wood of an
Angiosperm.

Sabiccaulis Sakurvi, gen. et spec. nov. Minute stems, and older twigs of
an Angiosperm, with cortex, and well preserved and characteristic
anatomy.

Cretovarium japonicum, gen. et spec. nov. The flower of an Angiosperm, of
which there are several specimens.

Of this list of plants, the commonest, z.c. those which have yielded the
greatest number of specimens in the course of the work, are Yezonia,
Sabiocaulis, and Cretovarium. It is noteworthy that these are among the
most unusual and the most interesting of the plants.

The authors acknowledge much assistance in the work from the Royal
Society Government Grant Committee, which made it possible for one of
them (M. C. 8S.) to attempt the work; and from the various departments of
the Imperial Government of Japan in the course of collecting and preparing
the material.

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DAVID JAMES HAMILTON, 1849—1909.

AFTER protracted illness, the death of Prof. David James Hamilton, M.B.,
LL.D., F.R.S., F.R.S.E., F.R.C.S.E., took place at his residence in Aberdeen on
February 19, 1909.

The subject of this memoir was born sixty years ago at Falkirk. He
received his medical education at Edinburgh University, from which he
graduated as M.B.in 1878. He subsequently held several clinical appoint-
ments: two in Edinburgh, where he acted as House Surgeon in the Royal
Infirmary, then as Resident in the Chalmers Hospital ; a third in Liverpool,
where he held a Resident Surgeoncy in the Northern Hospital. Even at this
early period of his career he was strongly attracted towards pathological
problems, for the study of which his thorough acquaintance with the
physiology of that period formed an all-important basis. This predilection
for a branch of medical science at that time much neglected, was happily
confirmed when the Triennial Astley Cooper Prize was awarded him for
a thesis dealing with “The Diseases and Injuries of the Spinal Cord,” in
which the result of much careful observation and research was embodied.

This mark of appreciation effectually determined Hamilton’s path in life,
and in order to acquaint himself more thoroughly with the thought and
technique of other schools, he proceeded to the Continent, visiting Strassburg,
Munich, Vienna, and Paris in succession. After thus spending two years in
close contact with the leading European pathologists of the time, he returned
home in 1875 on his appointment as Assistant to the late Prof. Sanders in
Edinburgh University. Somewhat later he became pathologist to the Royal
Infirmary, a post which furnished him with ample facilities for the prosecution
of observation and research. At this time he organised a course on morbid
histology, which was largely attended, mainly by young graduates, who were
not slow in recognising the vigour, resource, and thoroughness of their teacher.

An even wider opportunity for studying the most effective methods of class
instruction presented itself in the winter of 1880-1, when, owing to the
ill-health of Prof. Sanders, necessitating the appoitment of a temporary
substitute, Hamilton found himself entrusted with the entire duties—
educational and administrative—of the University Pathological Department.

Before the endowment of the Erasmus Wilson Chair in 1882, the
University of Aberdeen had no teacher in pathology apart from the
professors of Practice of Medicine and of Surgery, who did what the dis-
charge of their more obvious duties permitted to remedy the deficiency.
On his election as the first occupant of the Chair of Pathology, Hamilton
naturally found that his resources were limited; it may be said that they
were non-existent, for he inherited no department and but the scantiest
equipment. But his enthusiasm and determination of purpose, coupled with
exceptional physical yigonr, fully qualified him to deal with the difficult

i@iy, WOOUi—a a

i Obituary Notices of Fellows deceased.

position, so that in a surprisingly short space of time he was presiding over
classes of systematic and practical study in which his able and inspiring
teaching was illustrated by a wealth of material, amongst which his models
of pathological conditions, moulded and coloured to perfection, were marvellous
counterfeits of reality. Practically all these accessories were prepared
either by his own hands or under his direct supervision. Though his most
pressing needs were satisfied for the moment, others speedily presented
themselves. With the active development in bacteriology then proceeding,
the requirements of pathological departments demanded an accelerated
expansion ; more experimental and teaching accommodation had to be found,
apparatus to satisfy the needs of an elaborate technique to be procured, but
by unfaltering effort Hamilton seldom failed in obtaining that which he
deemed essential in order to keep his laboratories fully abreast of the
requirements of the time. It would, in fact, have been almost impossible
for those who presided over the University’s coffer to have rejected the
vigorous and conclusive appeals which he made to their liberality.

Hamilton’s exceptional qualifications as a teacher were speedily recognised.
They were endowments rather than acquirements. Some scientists with
high versatility in observation and investigation possess a less conspicuous
qualification for imparting their enthusiasm and information to others, but
he was a born teacher whose clear incisive style, carefully ordered facts, and
closely argued theories compelled the attention and conviction of his hearers.
By some he has been styled dogmatic, but if he sometimes pronounced a
decided judgement on a contested point, it was always preceded by a fair
statement of the observations and theories of others. That his own views
were decidedly and vigorously instilled, was no doubt a factor in the success
of his method of instruction, in so far that the student was left with a
perfectly clear conception of the standpoint adopted by his master.

Whilst his duties at Marischal College and at the Royal Aberdeen
Infirmary (in which institution in his capacity as pathologist he had entirely
remodelled the post-mortem department) occupied his time very fully,
Hamilton was devoting much of his evenings to preparing his ‘Text Book
of Pathology’ for publication. On its appearance in 1889, the book was
received in most quarters with unqualified approval. It was recognised as
being a thorough and comprehensive work of reference in the various aspects
of pathological study, based on the teachings of physiology to ensure a due
comprehension of morbid function, minute, clear, and practical in its details
of conditions and methods, illustrated with an exactitude and _ skill
unattainable except by those possessing not merely the requisite scientific
knowledge, but an artistic temperament to perfect the delineation.

That all critics should be entirely satisfied with the work was naturally
impossible: too little attention paid to the vital manifestations of morbidity,
too much to structural abnormality, the experimental aspect of study unduly
subordinated, these were amongst the strictures; but when the critic had
sufficiently vindicated his own standpoint, he usually showed himself

David James Hamilton. ill

constrained to praise much of the text and all of its illustrations. It
was no small assistance to Hamilton in this literary work that his earlier
investigations had brought him into the closest contact with very various
questions of a pathological character; he had not limited himself to a
narrow field of observation, but had been truly catholic in his selection of
objects of study. A recital of some of his chief contributions to pathology
may illustrate this point.

In 1879 he commenced a series of very capable articles dealing with
various morbid conditions of the lung. The papers appeared at brief
intervals in the issues of ‘The Practitioner’ throughout a period of two
years, so that the name of the author became familiar to medical man and
specialist alike. (It may be mentioned that Hamilton particularly desired
to accentuate the value of pathological study in its proper relationship to
diagnosis and treatment.) Topographical knowledge of the central nervous
system was enriched by his contributions on the conducting paths in the
brain, and especially by his work on the corpus callosum in adult and
embryonic conditions. (In prosecuting these researches he was assisted by
a grant from the Royal Society.) In 1882 a communication was made
by him to the Royal Society of Edinburgh, in which he dealt with a physical
explanation of diapedesis, and illustrated his theories by novel and ingenious
mechanism.

Much originality of idea, expressed with his wonted lucidity, is dis-
played in his studies relating to sponge grafting, and to embolic infarction,
as well as in articles bearing upon such matters as the influence of heredity
in disease, the pathology of gastric dyspepsia, and the alimentary canal as a
source of contagion.

Hamilton showed a keen interest in the pathological conditions occurring
amongst domestic animals, especially those which contribute to the food
supply of man, and his later work in this direction is important,
not merely from its bearing upon agricultural economics, but also as
a practical addition to current knowledge of invasion and_ resistance.
His inquiry into the relationship of human to bovine tuberculosis
strengthened by its results his opposition to the views adopted by Koch,
whilst his laborious investigation into the etiology, symptomatology, and
prophylaxis of certain disorders occurring amongst sheep led to very inter-
esting and valuable conclusions.

This—his last, and probably his most important work—must be referred
to in some detail. The commencement of this inquiry is now remote, for it
was in 1881 that Hamilton was sent to the Island of Skye by the Highland
and Agricultural Society as the expert member of a committee charged with
investigation of the disease known as “ Braxy ” (morbus subitarius ovis). The
time of year chosen for the expedition proved unfavourable for this purpose. It
was not until 1897 that Hamilton found himself able to resume the research :
in that year he visited the Fort William district, with other localities where
disease was rampant, observing the symptoms and topography not only of

a 2

iv Obituary Notices of Fellows deceased.

braxy but of other members of the group of disorders with which it is
frequently confounded by stock-masters and shepherds. From year to year
these excursions to various infected areas were repeated with a similar object,
and as their result, augmented by official information mainly derived from
Ireland, he was able to settle and map out the topography of braxy. But
this was merely a preliminary to the much more important work which was
instituted by the Departmental Committee appointed by the Board of
Agriculture in 1901, upon which Hamilton acted as chairman and expert.
Plans of action were agreed upon and observation stations organised in
localities where disease occurred endemically amongst sheep, the chief being
located at Kielder, in Northumberland. The report of this Committee
appeared three years ago in the form of a Blue book. It deals chiefly with
braxy and louping-ill, though touching upon several other disorders which are
held to be individually distinct. Hamilton fully confirmed the description
of the braxy micro-organism, first recognised by Ivar Nielsen in 1888, and
further, added several important observations relating to its seasonal activity
and manner of invasion, together with a suggested method of prophylaxis.
The louping-ill disease (chorea paralyticu ovis) which had been a veritable
mystery, was finally unravelled, the bacillus found, its characteristics under
cultivation studied, its manner of development and invasion traced, its
seasonal activity explained, a plan for protection against it elaborated and
tested with most encouraging results. The pages of the report testify to the
prolonged and arduous nature of a research which, step by step, led the way
to an entirely satisfactory and practical issue. Apart from the information
they contain bearing upon seasonal, receptivity towards infection, the nature
of intestinal infection, the duality of symptoms produced by the toxine
originated by the same micro-organism, together with other matters which
may have their signification for man as well as for the animals which
Hamilton observed, the work has a direct bearing upon agriculture of a
far reaching character. The need for similar investigation into other
disorders of the sheep, which are touched upon incidentally in the report,
is indicated; it was, indeed, Hamilton’s intention to make a thorough
study of these individually, but their elucidation was not to be at his hands.

The attachment of his former pupils was suitably shown in 1906, when,
on the completion of his twenty-fifth year of service in the University, a
volume of ‘ Studies in Pathology ’ was prepared in his honour, to which many
old students, now professors and lecturers in other schools, were contributors,
His jubilee coincided in point of time with the celebration of the quater-
centenary of Aberdeen University. He appeared to be at that time in the
enjoyment of his accustomed vigour, but not long afterwards symptoms of a
disquieting character became apparent: exertion fatigued him, vigour of step
and speech were less manifest, and although he only relinquished his work at
intervals under pressing medical advice, it was evident that he was losing
ground, By the end of 1908 he was confined to his room and unable to be
present at the funeral of his wife when her decease occurred somewhat later.

ee Ra ee ne

3

Dawid James Hamilton. Vv

Under the influence of this bereavement and the progress of his malady,
Hamilton’s condition became steadily worse ; he recognised the impossibility
of recovery and resigned his Chair; five months later, in the spring of 1909,
death terminated his suffering.

As we have endeavoured to summarise the work of a lifetime, it is fitting
that we should glance for a moment at the outstanding characteristics
of the man who accomplished it. Hamilton possessed a strongly detined
individuality, intense and ardent, firm in upholding a conviction, direct and
enthusiastic in supporting it. Finesse, compromise, and ambiguity were
alike foreign to his nature; if he was frank and outspoken he was incapable
of harbouring feelings of bitterness or resentment towards others who held
different views. To his intimate friends there was a perennial freshness
and geniality in the relationship: his keen sense of humour, interpreted by
the expressive grey eyes and the musical infectious laugh, added to the
charm of his company. Beyond his work he had wide interests and deep
sources of pleasure; he loved nature and keenly appreciated the artistic,
whether in form or colour. On several occasions he lectured on artistic
themes, architecture included, always exhibiting a fine enthusiasm for work
which he recognised as harmonious and genuine. In music he was a
connoisseur and was himself possessed of a melodious voice.

Hamilton was a member of many learned societies and bodies ; his
election to the Fellowship of the Royal Society in 1908 and _ his laureation
as LL.D. by his Alma Mater soon after, were tokens of appreciation which
caused him peculiar gratification. Had he lived to the spring graduation of
the current year he would have received a similar recognition from the
University in which he had served with much distinction throughout a period
of twenty-six years.

Hamilton was twice married: his first wife died seventeen years ago ;
his second, a daughter of Mr. John Wilson, of Falkirk, predeceased him by
a few months.

Jo dh, C.

val

WILFRID HUDLESTON HUDLESTON, 1828—1909.

In the history of Geological Science two classes of individuals have, at
various times, contributed to its advancement, namely, the amateur
investigator and the professional worker. Of these the amateurs were
certainly amongst the earliest in the field, and indeed it may be truly said
that, but for their labours,
the initiation of the Geo-
logical Society and the
Survey itself as a pub-
lic department would
scarcely have met with
so early a reception in
this country.

Thanks to our univer-
sities and public schools,
well-trained professional
workers have now become
so numerous that there
seems little room left for
the amateur; neverthe-
less, so fascinating is the
science that geology is
still pursued with marked
success by many private
persons, purely con amore
and often as a leisure
hour pursuit or an
agreeable concomitant of
travel. Foremost amongst
those non - professional
geologists, who devoted
his life for many years to
this science, must be placed the name of Wilfrid Hudleston Hudleston
(formerly Simpson).

Born at York, June 2, 1828, Wilfrid Hudleston Simpson was the eldest
son of Dr. John Simpson, of Knaresborough, and is, on his father’s side, a
descendant of three generations of Yorkshire “ medicine-men.” His mother,
née Klizabeth Ward, was heiress of the Hudlestons of Cumberland, and in
1867, on succeeding to the family estates, Wilfrid, by letters patent, assumed
the name of Hudleston, by which he is best known among geologists.

From 1831 to 1834 he resided with his parents in Harrogate, where his
first playfellow was Henry Clifton Sorby—who afterwards became so
distinguished a geologist and a President of the Geological Society of London.

‘

Wilfrid Hudleston Hudleston. vil

Young Simpson received his early education at St. Peter’s School, York,
from which he was transferred to Uppingham School, and subsequently
entered St. John’s College, Cambridge, where he graduated B.A. in 1850. Up
to this time, as a school-boy and an undergraduate, Wilfrid had evinced no
special predilection for geology. In his last term he attended Sedewick’s
lectures and was much impressed with the manner and appearance of that
distinguished geologist. His earliest scientific pursuit was ornithology,
commenced at fifteen years of age whilst still at Uppingham School. At
Cambridge he was associated with Alfred Newton from 1848 and with
J. E. Law, making many expeditions in Northumberland, Cumberland, and
N.W. Ireland. Afterwards he visited the Norwegian coast and here he became
acquainted with John Wolley (author of ‘Ootheca Wolleyana’), and with
him he collected in Finland and Lapland, also with Alfred Newton and John
Wolley in 1855 and in 1856 in the Island of Oland and Sweden.

In 1857 he joined Canon Tristram and Osbert Salvin in an ornithological
expedition to Algeria. Together they explored the Eastern Atlas, visiting
Tunis, Constantine, Kef, ete. The years 1859—60 were chiefly spent by
Wilfrid Simpson in Greece and part of Turkey (the Dobrudscha, now
Roumania). His last ornithological trip was made in 1861 to Switzerland.

On leaving Cambridge he devoted some time to the study of the Law and
was called to the Bar in 1853, but never practised.

From 1862 to 1867 Mr. Wilfrid Simpson held a commission in the Kent
Artillery (Militia), and performed yearly garrison duties at Dover Castle.
About this time he also began a special course of scientific studies, selecting
more particularly Natural History and Chemistry. He studied in Edinburgh
under Playfair and Stephenson Macadam, and subsequently, for three sessions,
at the Royal College of Chemistry in London under Hoffmann, Frankland,
and Valentine. At that time he was uncertain whether to take chemistry or
geology as his main subject of pursuit, when an accident decided in favour of
the latter. In 1866 he met Marshall Hall at Chamounix and on their return
to England he was speedily introduced to many’ persons interested in
geological science, of whom Prof. Morris may be regarded as the chief.

Prof. Morris had a wonderful influence over his pupils and associates, and
this was just the attraction which Mr. Simpson, who had now (1867)
assumed the patronymic of Hudleston, required to enlist him as a geological
recruit, and in due course to make him a “knight of the hammer” for the
rest of his life. A close friendship was thus formed which was only
terminated by the death of Morris in 1886.

In 1867 he was elected a Fellow of the Geological Society, and in 1871
a Member of the Geologists’ Association. Of this Association he became
Secretary in 1874, and during the three years of office he spent much time
in preparing reports on the various districts visited, some of which are of
considerable extent and importance. In 1872 he published his first original
paper (with Mr. F. G. H. Price) “On Excavations at the New Law Courts.”

His papers on “The Yorkshire Oolites” (1873—78) and “The Corallian

vill Obituary Notices of Fellows deceased.

Rocks of England” (with Prof. J. F. Blake (1877) ) soon established his
reputation as one of our leading geologists.

Mr. Hudleston was elected President of the Geologists’ Association in
1881, and his great services rendered to that body during so many years
were marked, in March, 1892, by the presentation of an illuminated address.

From 1886 to 1890 he filled the post of Secretary to the Geological
Society of London and, in succession to Sir A. Geikie, he was elected President
(1892—94). In these years, in addition to his official duties both in
connection with the Geological Society and the Geologists’ Association,
Mr. Hudleston proved himself an able and prolific contributor to the
literature of geology. Some twenty papers were written by him on the
field-geology of various districts, eight on Chemical Geology, for which he had
always a strong predilection, dealt with the Lizard Rocks ; the Gneiss Rocks
of the N.W. Highlands; Sterry Hunt’s Chemical Essays; King and
Rowney’s views on Hozoon Canadense; the Diamond-rock of S. Africa;
and Sterry Hunt’s views on Serpentines. In Paleontology he published
Monographs on the Corallian Gasteropoda of Yorkshire (1880—81). The
Gasteropoda of the Portland Rocks (1881) and of the Oxfordian and Lower
Oolites of Yorkshire (1882—85), the Gasteropoda of the Inferior Oolite
(1887—96), this latter comprising 514 quarto pages of letterpress and
44 quarto plates of fossils; a Catalogue of British Jurassic Gasteropoda
(with Mr. E. Wilson), and papers on the Fossils of Western Australia and
South Australia.

To this period must also be added Presidential Addresses to the Geologists’
Association “On Deep-Sea Investigation” (1881), on the Geology of
Palestine (1882, with additional notes in 1885), to the Malton Field
Naturalists’ Society (1884), and the Devonshire Association (1889); lastly,
two Presidential Addresses to the Geological Society (1895—94), and one,
later, as President of Section (C) Geology, British Association, Bristol
(1898).

On the death of his old friend, Prof. Morris, in 1886, Mr. Hudleston
succeeded him as one of the editors of the ‘Geological Magazine,’ to which
journal, since 1879, he had been a frequent contributor, and continued so
until his death in the present year. He was a keen student of recent and
fossil mollusca and one of the founders of the Malacological Society.

In 1886, accompanied by Dr. Henry Woodward, F.BR.S., and Mr. C. E.
Robinson, M.Inst.C.E., he carried out some experimental dredgings,
with a Brixham trawler and her crew, along the English Channel, and in
and near Torbay, in order to study marine mollusca and observe their living
habitats. In the following year he engaged a Grimsby steam trawler and
her crew, and accompanied by Mr. C. E. Robinson and the late Martin F.
Woodward, of the Royal College of Science, he spent three weeks in a
dredging cruise in the English Channel and along the French coast.

Mr. Hudleston resided for many years in Cheyne Walk, Chelsea, but
removed in 1883 to Oatlands Park, Surrey. This suburban residence,

ry

Wilfrid Hudleston Hudleston. 1x

however, interfered with his scientific engagements, and he again took up
a residence in town at 8, Stanhope Gardens, South Kensington.

In 1890, he married Miss Rose Benson, second daughter of the late
William Heywood Benson, Esq., of Littlethorpe, near Ripon.

Early in 1895, Mr. Hudleston, accompanied by his wife and his friend,
Prof. J. F. Blake, F.G.S., left London for Bombay. After leaving Prof. Blake
duly installed as Organising Curator of the Museum at Baroda, to which he
had just been appointed, Mr. Hudleston journeyed onwards towards the
north-west frontier of India. The geological results of this expedition are
~embodied in “ Notes on Indian Geology,’ read before the Geologists’ Associa-
tion in December, 1895 (see ‘ Proc. Geol. Assoc., vol. 14, p. 226, 1896).

He presided over or took part in the Councils of numerous scientific
societies. He was elected, in 1889, President of the Devonshire Association
for the Advancement of Science, the Yorkshire Naturalists’ Union, and the
Malton Field Naturalists’ Society; and had been for years a Vice-President
of the Dorset Natural History Field Club. He also served as a Member of
Council of the Royal Geographical Society, and as President of the Geological
Section of the British Association at Bristol in 1898.

In 1897, Mr. Hudleston was awarded the highest honour which the Council
of the Geological Society could bestow, namely, the “ Wollaston Gold Medal,”
in recognition of his valuable contributions to our knowledge, including
chemical, mineralogical, paleeontological, and stratigraphical geology. Special
reference was made by the President, Dr. Henry Hicks, F.R.S., to his mono-
graph on “The Inferior Oolite Gasteropoda,” contained in the volumes of
the Paleontographical Society, which, with the services of four collectors in
the field and in cleaning, developing, etc., occupied a period of over twenty
years, the descriptions filling 514 quarto pages of letterpress and 44 quarto
plates of figures. This fine collection of types has, since the death of
Mr. Hudleston, been transferred, as a gift, to the Sedgwick Memorial
Museum, Cambridge.

Two later papers deserve special mention, namely, the investigation of the
structure of “Creechbarrow in Purbeck” (‘ Geol. Mag.,” 1902—3), and that
“On the Origin of the Marine (Halolimnic) Fauna of Lake Tanganyika ”
(‘Geol. Mag.,’ 1904).

In his earlier years, before he became known as a geologist, he took a keen
interest in ornithology, and was instrumental in founding, in 1858, in con-
junction with the late Prof. Alfred Newton, of Cambridge, Mr. John
Wolley, and others, the British Ornithologists’ Union; and so lately as
December 9, 1908, they commemorated the Society’s fiftieth anniversary.
To mark the occasion, the Society presented a gold medal to each of the four
surviving original members, of whom Mr. Hudleston was one.

In connection with the Armstrong College, Newcastle (in the University of
Durham), Mr. Hudleston provided the site and advanced capital for erecting
a Marine Biological Laboratory at Cullercoats, Northumberland, to be named
the “ Dove Laboratory” (after a great ancestor of his family, Eleanor Dove):

x Obituary Notices of Fellows deceased.

It was erected and equipped at a cost of £4000, and was opened in July,
1908, by the Duke of Northumberland. The building is suitably provided
with a fine aquarium with numerous tanks, and a large room on the ground
floor where experiments on pisciculture may be conducted under improved
conditions. It has, in addition, a library, a lecture room, workrooms, etc.
The Director is Prof. Meek, M.Sc., under whose direction the entire equip-
ment of the building was carried out.

Mr. Hudleston was a Justice of the Peace for the West Riding of York-
shire and for East Dorset. He purchased the East Stoke Estate in 1897, for
the sake of the shooting, having all his life been a keen sportsman. His latter
years were divided between West Holme, Wareham, and his town residence,
8, Stanhope Gardens. He died at West Holme, January 29, 1909, in his
eivhty-first year.

Mr. Hudleston’s life was marked by untiring energy, directed with
a steady purpose throughout. As a man of science, may be mentioned the
numerous oftices he held in connection with the Geological Society, the
Geologists’ Association, and many other bodies. No fewer than 58 memoirs
and papers, extending over a period of 32 years, attest to his energy and
ability.

As an ornithologist and a traveller he accomplished much. During his
sojourn in the Kast he acquired a fluent knowledge of modern Greek as well
as Arabic. As a magistrate and a landed proprietor he was always earnestly
desirous to fulfil his duties; while as a sportsman, both with gun and rod, he
exhibited the same keenness as with his geological hammer or in his chemical
laboratory and museum.

For many years Wilfrid Eiadlestion lived much alone, having but a small
number of intimate friends: Prof. Morris, F. G. Hilton Price Henry
Woodward, H. W. Monckton, and some few others. Hence the social side
of his life was never fully developed. But his earliest ornithological
friendships for Prof. Alfred Newton, John Wolley, O. Salvin, Canon Tristram,
and J. EK. Law remained the strongest and warmest throughout his life, and
were only separated by death and as he drifted apart from them in his later
geological pursuits.

H.We

X1

Xi SIR GEORGE KING, 1840—1909.

GrorcEe Kina, the only son of Robert King and Cecilia Anderson, was born
at Peterhead, where his father was a bookseller and his maternal grandfather
was Collector of Inland Revenue, on April 12, 1840. While King was still
a child his father moved to Aberdeen, and joined in partnership his elder
brother George, a bookseller in that city, where another brother, Arthur,
founder of the Aberdeen University Press, was a printer. Partly in con-
junction with this press, the firm of G. and R. King developed a publishing
business which rendered good service to the north-east of Scotland at a time
when difficulties of communication delayed supplies of literature from
London, or even from Edinburgh. The partners were members of a family of
Independents, a denomination never numerous in Scotland. Both were men
of strong character and much ability, and took an active part in promoting
the interests of the Congregational community. The senior partner, George,
belonged to the Scottish Society of Antiquaries, and was one of the three
founders of a still flourishing local Liberal newspaper.* He was for forty
years closely connected with the administration in Aberdeen of the Scottish
poor-law, and published an essay ou “Modern Pauperism ” which attracted
the attention of social statesmen fifty years ago. He also wrote an historical
review of the origin and condition of the Congregational churches in
Aberdeenshire and Banffshire, in which, while holding the views of an Inde-
pendent as regards the organisation of the Anglican Church, he found the
Presbyterian form of church government equally unjustifiable on historical
erounds. The junior partner, father of the subject of this notice, was the
author of a meritorious historical work, ‘The Covenanters in the North,’
published in 1846, shortly after his death from phthisis, at the early age of
thirty-six, in November, 1845. As an Independent, the author was able to
handle his subject with sympathy, and at the same time without the bias so
often apparent in Presbyterian writings. King’s mother died, also of phthisis,
at the age of forty, in June, 1850.

Left an orphan at ten, King became the ward of his uncle George, who in
the autumn of 1850 transferred him from the preparatory academy he had
hitherto attended, to the Aberdeen Grammar School, then under the rectorship
of Dr. Melvin, one of the foremost classical schools in Scotland. Two pupils
named King, were already there, one in the fifth, another in the second class ;
the subject of this notice on entering the first class, taught by Mr. (after-
wards Sir William) Geddes, was therefore, to the masters, King “ tertius,” to
his schoolmates “Tertius” for short; this agnomen stuck to him till his

* Arthur King, of the University Press, also engaged in newspaper enterprise, and
became proprietor of the first penny paper published in the north of Scotland.

xu Obituary Notices of Fellows deceased.

undergraduate days. At school “Tertius,” in spite of constitutional delicacy,
was an apt pupil; his guardian, partly on this account, partly because of the
boy’s poor health, decided to train him to be his successor in the firm, and
effect was given to this design when King left school in 1854. The
experiment did not succeed as his guardian had hoped, though it cannot be
said that this effort to determine King’s career was altogether thrown away.

Contemporaries of King who survive recall the proceedings of a literary
society connected with the church to which his uncle belonged. Young
King, while in business, was a leading member; his contributions to the
discussions, we are told, showed erudition and insight beyond his years. The
society subscribed for papers then rarely seen in Scotland; among them the
‘Saturday Review, for which King developed an especial liking that, except
when the paper was edited by F. Harris, he retained to the last. It may be
noted that when, later in life, King left the Independent community, he
transferred his allegiance to the Anglican, not the Calvinist, Church. Not
improbably King’s sympathies with lterature and art, and -his practical out-
lock on life, inay have been paternal inheritances. But it is clear that this
association with a bookselling and publishing business aided in developing
his keen and sure taste, so appreciated by intimate friends, so unsuspected by
others. Nor can it be doubted that his instinet for the essential, his mastery
of complex detail, and his genius for organisation, which combined to render
his public services so valuable, benefited by their exercise durimg the
apparently fruitless and certainly irksome years of King’s business life.

With the utmost affection and regard for his uncle, King never threw his
heart into the business of the firm. As asmall boy he had shown, in country
walks with his father, all the interest of a child in birds and flowers with
more than the usual power of remembering their names and peculiarities.
But in the absence of parental stimulation and in spite of delicate health,
this interest in natural objects, in place of being inlubited by the literary
drill of a classical school, gradually developed into an overpowering taste for
scientific study in general, and for zoological and botanical pursuits im
particular. His innate ambition was to be a naturalist; his accidental
attachment to a business failed to suppress his devotion to natural study.
His spare time was given to field excursions ; his enthusiasm gained him the
“freedom” of the arcana of local nurserymen. His pursuits led King to
introduce into the business premises specimens in which he was interested.
These his uncle contemptuously termed “scroggs”; King’s addiction to
their study caused the worthy man genuine grief and much indignation.
After his eighteenth year King’s general health greatly improved, and it
was Clear that the situation could not persist. King continued in business
until he reached his majority, but immediately thereafter he announced to
his guardian his decision to devote what remained of his patrimony to
acquiring a medical education, then the only avenue to a scientific career.
His uncle, so far from expressing surprise, gave his approval to this decision,
and thereafter did all in his power to further his nephew's designs. The

en ee

Sir George King. Xl

relationship between the two men was perhaps more sympathetic after King
entered the University of Aberdeen in 1861 than it had been before.

King’s gift of lucid expression and his aptitude for business may, like his
instinct for literature and art, have been paternal inheritances. The origin
of his faculty for observation seems more obscure. It may have been derived
from the maternal side, for among his undergraduate contemporaries was
a relative,* in whom, as a boy, the love of natural history was also strongly
developed, though in this instance the capacity for close and accurate
observation was ultimately applied in a somewhat different field.

How distinguished as a student King proved, may be gathered from the
fact that his contemporaries, after his first medical session, changed the old
agnomen “ Tertius” to “Optimus.” The improvement in health which had
begun in his eighteenth year continued ; he had still to exercise unusual care,
but the close of his university curriculum found him in more robust health
than at its commencement. His capacity attracted the attention of all his
teachers; those who exercised most influence upon him were the Professors
of Botany (Dickie), Materia Medica (Harvey), and Anatomy (Struthers).
King attended Dickie’s class in 1861, and in 1862, when Dickie was
incapacitated by illness, King was assistant to the deputy-professor,
Dr. Dickson.f In 1863, when Dickie resumed work, and again in 1864, King
continued to be assistant in the botanical department, and the attachment of
the two men only ended with the death of the gentle and distinguished
Dickie in 1882. The question as to the career King should adopt already
exercised his chief and himself. King’s predilection was towards erypto-
gamic botany; this was no doubt encouraged by so able an algologist as
Dickie, who applied for counsel to Sir W. J. Hooker. Hooker’s suggestion
was that King should follow the example of his son, Dr. (now Sir J. D.)
Hooker, and join the Naval Medical Service. But, while studying Materia
Medica, Royle’s ‘Manual’ led King to that author’s other works ; these, with
Thomson’s ‘ Narrative’ and Hooker’s ‘ Journal,’ induced a desire to serve in
India. There seemed little hope of this, recruitment of medical men for India
having been suspended since 1860. But in April, 1865, the Indian Medical

* Dr. James Rodger, whose father was an able mathematician and a successful man of
business, and whose mother was a sister of King’s mother, was one of King’s class-fellows
as a medical student. He graduated in 1865 along with King, and, like King, with
highest academical honours. An accurate anatomist and a sound pathologist, Rodger
was Senior Demonstrator of Anatomy under Prof. (afterwards Sir John) Struthers at
Aberdeen from 1866 till 1871, and was Pathologist to the Royal Infirmary there from
1869 till 1886, when he was appointed a physician to the institution, and continued a
member of its staff until his death in 1900. But the evidence is not conclusive ; we must
assume that King’s father was at least capable of appreciating his child’s interest in
natural history, and we know that Rodger’s father was an intimate friend of Prof.
Dickie, F.R.S., enjoyed a considerable reputation as a local botanist, and took a keen
interest in scientific matters generally.

+ Alexander Dickson, afterwards Professor of Botany at Trinity College, Dublin
(1866), Glasgow (1868), and Edinburgh (1879),

X1V Obituary Notices of Fellows deceased.

Service was reopened and a son of Prof. Harvey, already in a sister service,*
threw up his commission and entered the Indian one. King decided to
follow young Harvey’s example; after attaining his M.B. in 1865, he joined
the Indian Medical Service on October 2 of that year, and having spent
the winter at Netley, left for India in the following March. Before
embarking King paid a visit to Kew, and on the voyage was able to render
the earliest of his many services to that establishment and to India.
Dr. Hooker entrusted to his care the first medicinal Ipecacuanha plant sent
to India; this King delivered in safety at the Royal Botanic Garden,
Calcutta, when he reached that port on April 11, 1866.

On landing, King was attached to the General Hospital, but on May 9 he
was transferred to the Medical College Hospital, Calcutta, and was appointed
house surgeon there on May 18. This post the principal advised him to
resign on August 20; in the performance of his duty he had contracted
fever, followed by an attack of pneumonia, which endangered his life and
threatened to hght up the phthisical tendency of his boyhood. Already his
work had commended him to his seniors, who used their influence to procure
his transfer to the drier and healthier climate of Upper India. He was
posted to military duty on August 29, and reached Agra on September 4,
when he was attached to the 41st Bengal Infantry. On December 13, 1866,
he again fell ill, and on returning to duty was given medical charge, on
January 9, 1867, of the civil station of Muttra; but a month later his
administrative chief, again with the object of promoting his recovery,
procured his transfer to the still drier climate of Central India, where, on
February 17, le took over medical charge of the 1st Central India Horse at
Goona. This post he held till December 4, when he was transferred to the
Political Department in Rajputana, taking up his duties at Deoli, in Marwar,
on December 24, 1867. During the following year he served there and at
Mount Abu, and afterwards at Jodhpur, but early in September he was
selected to officiate as Superintendent of the Botanic Garden at Saharanpur,
a post which he filled from December 9, 1868, till November 22, 1869. As
his temporary appointment at Saharanpur drew to an end, King found
himself at the parting of the ways. His energy and ability impressed all
those with whom he came in contact, and some glimpse of his organising
faculty had been given in connection with famine work in Rajputana. The
medical authorities were anxious to secure his services as a Deputy Sanitary
Commissioner, and orders to take up civil medical duty on his relief at
Saharanpur were issued. But the chief political officer in Rajputana had
been equally impressed, and, having learned that the Forest Department was
in need of competent officers, strongly advised the Conservator in the
North-west Provinces to ask for King’s services. King was accordingly
invited in September, 1869, to accept an Assistant Conservatorship. The
forest appointment offered greater scope for the utilisation of his botanical

* Dr. R. Harvey, C.B., D.S.O., subsequently Director-General, Indian Medical Service,
in which capacity he died at Simla, Panjab, December 1, 1901.

al

Sir George King. XV

knowledge, and was accepted by King, who was thereupon placed in charge
of the Dehra Dun forests, taking up his new duties on December 3, 1869.

At Agra, Muttra, and Saharanpur King laid the foundation of that
knowledge of the plants of the Upper Gangetic Plain, shown in a contri-
bution by him to the ‘N.W. Provinces Gazetteer. At Goona the flora of
Central India did not suffice to occupy all his leisure; the balance he
devoted to an ornithological survey of the district and to the preparation of
a series of skeletons for the anatomical museum of his university. This
work he continued in Rajputana, where he also made large botanical collec-
tions, investigated the plants used as food in times of scarcity, and studied
the vegetation from what is now termed the cecological standpoint. At
Saharanpur he found time, amid his administrative duties, for morphological
and economic botanical studies.

The forest work at Dehra Din gave King freer scope for the display of
his powers. Zeal, energy, and candour were in him combined with a charm
of manner which attached to him those with whom he had dealings. He
could direct without damping the enthusiasm of those who served under him,
while commanding the confidence of those under whom he served. Honest
difference of opinion he seemed to find attractive; his anger never was
provoked save by attempts at intrigue or subterfuge. These qualities were
sorely needed in the Assistant Conservatorship with which he was entrusted.
The situation he had to face is disclosed “in a ‘Memorandum on the Dehra
Dhoon Government Forests, presented to Government in April, 1871. To
help him in his task he had been invested with the powers of a subordinate
magistrate of the first class within forest limits on March 18, 1870; in
performing it he virtually saved these forests from extinction. The nature
of his achievement during the fifteen preceding months may be gathered from
a judgment delivered on May 21, 1871, of which the following passage forms
part :—“I do not think it would be right of me to close this case without
putting on record some expression of opinion regarding the zeal displayed by
Dr. King, and the debt which the Forest Department owes him. For years
a most complete system of bribery and corruption had been going on in the
Forest office ; Government property to the value of thousands of rupees had
been yearly stolen with the connivance of the Forest officials. Dr. King
had hardly been in office a month when he saw how affairs stood, and before
the end of six months he had obtained convictions against the principal
offenders. The amount of labour Dr. King had to go through to obtain this
satisfactory result is beyond belief. Day and night he never rested;
through the most impenetrable jungles in the most unhealthy seagon he
forced his way. No man, woman, or child who could throw any light on the
subject was left unseen and unquestioned ; once he got a clue he never let
it drop; account-books and papers sent into the heart of foreign territory to
be beyond grasp he ferreted out and laid before the Court. The result
is that he has thrown light on all the most secret transactions. The
character of all the officials in the Forest Department has been clearly

XV1 Obituary Notices of Fellows deceased.

portrayed; the various ways Government had hitherto been defrauded and
robbed have been found out and exposed; if Dr. King’s successors do not
take advantage of this it is their fault. The benefit which should accrue to
the Forest Department by Dr. King’s labour, if followed up in a proper
spirit and with ordinary energy, is incalculable.”

Little more than a year after joining the department King was appointed,
on January 28, 1871, to officiate as Additional Deputy Conservator in charge
of the Kumaon Forest Division, and on March 2 he was recommended for
permanent promotion to this higher grade. While acting as Deputy
Conservator King was ordered to prepare a “Report on Forest Con-
servancy, etc., for Raneekhet,” one of the N,W. Himalayan _hill-stations.
This report he submitted in June, 1871, and its nature may be gathered
from the ‘Gazette’ of India for September 9, which officially states: “That
the Governor-General in Council has read Dr. King’s very interesting report
with great satisfaction, and cordially endorses the praise bestowed upon it by
His Honour the Lieutenant-Governor. The recommendations contained in
it are excellent, and His Excellency in Council trusts they will be borne in
mind and carefully carried out.” King further made a careful study of tea-
pruning under the conditions that prevail in the Kangra Valley, and
although it had reference primarily to the N.W. Himalaya, a successful
tea-garden manager in N.KE. India once remarked of King’s paper that
“before reading it he had pruned with his hands, after its perusal he could
prune with his head.” During his forest service King formed extensive
botanical collections, and was acquiring the knowledge displayed afterwards
in his ‘Gazetteer’ “List of the Plants of Garhwal, Jaunsdr Bawar, and
Dehra Dun.”

The recommendation that King should be made a permanent Deputy
Conservator was accepted by Government. Owing, however, to there being
no vacancy in the N.W. Provinces, this promotion, it was decided, must
involve his transfer to Burma. But before this arrangement could be
carried out King’s forest service came to an end. Dr. Thomas Anderson,
Superintendent of the Royal Botanic Garden, Calcutta, and of Cinchona
Cultivation in Bengal, fell a victim, in 1869, to the energy and zeal with
which he had for years been labouring to establish plantations of cinchona
in Sikkim. He had to be invalided to Europe, where he died in October,
1870. On March 10, 1871, the Secretary of State for India selected King
as suecessor to Anderson (India Office despatch, March 23; Government
of India Order, May 22). On being relieved of his forest duties King left
for Calcutta and entered on his new charge, which included the Professorship
of Botany in the Medical College of Bengal, and where the tasks before him
were heavy ones, on July 10, 1871.

Two cyclones of extreme severity which swept over Calcutta in 1864 and
in 1867 had ruined every park and garden in the neighbourhood. The
Botanic Gardens, formerly famed for possessing one of the finest collections
of trees in the East, had been reduced to a comparatively naked plain,

ht.

Sir George King. xvi

thoroughly exposed to sun and wind, and therefore favourable to the growth
of rank grasses which smothered the young trees and shrubs planted to
replace the uprooted veterans. Roads and paths were insufficient in
number and unsatisfactory in condition and alignment. The site of the
garden, being part of the rice-swamp which forms the Gangetic delta, was
far from suitable to the successful cultivation of many desirable indigenous
and exotic species. The residences of the garden employés, native and
European alike, were inadequate and insanitary. The accommodation for
the herbarium and library was cramped and inconvenient; the herbarium
collection, though extensive, was very unequal. The labours of Roxburgh,
Buchanan, and Wallich from 1793 to 1828 had brought together the richest
botanical collection hitherto made in Asia. But in 1828 this collection was
taken to Europe, and Wallich, then on leave in England, dispersed it on
behalf of the East India Company with a generosity so lavish that nothing
was left for the institution at whose cost and on whose behalf it had been
formed. Something was done to repair this injury by Wallich himself on his
return (1832—46), by Falconer (1846—54), by Thomson (1854—59), and by
King’s predecessor Anderson (1859—69), while the generous aid of Kew had
provided Calcutta with a substantial share of the contents of the East India
House cellars. But great leeway had still to be made up in order to render
the Caleutta Herbarium commensurate with the needs of so important
a botanical centre.

The problems connected with cinchona were of equal importance and of
even greater difficulty. The Sikkim plantations, begun by Anderson ten
years before, and pushed on with a zeal which cost that indefatigable officer
his life, were an established fact when King assumed control. The policy of
Government had been to act, as in the case of tea, only as a pioneer. So
soon as it could be shown that private growers were in a position to under-
take the enterprise, Government was prepared to dispose of these experimental
plantations and retire from the field. In the case of cinchona this policy
could not be carried out. The experience of these ten years had proved that
in spite of every encouragement and assistance, the cultivation of cinchona in
Northern India is, owing to natural causes, unprofitable to private enterprise.
Government itself had therefore to attempt the economic separation, from the
bark produced on its own plantations, of the alkaloids this bark contained,
and to utilise these alkaloids in combating the ravages of malaria. Attempts
in this direction had been made before King assumed charge ; these attempts
had not been attended with success. But there was another equally important
problem to be dealt with. The bark of those cinchonas that had so far been
most successfully grown in Sikkim and that were therefore chiefly represented
in the Government plantations, is bark that, while rich in total alkaloids, is
relatively poor in quinine, the most important of these alkaloids. It was
therefore King’s ambition to replace the kinds then most largely cultivated
by others whose bark is rich in quinine, and eventually to separate this
quinine in such a manner as to obviate financial loss to Government. With

VOL, LXXXIL—B. ° b

XvVill Obituary Notices of Fellows deceased.

characteristic energy King attacked these problems: as regards the first,
urging the employment of a competent quinologist; as regards the second,
himself attempting its solution on the spot. His zeal almost involved him in
the fate that had befallen Anderson. Exposure and fatigue during his work
on the plantation induced severe illness, which became ageravated while on
an official visit to the Madras plantations in July, 1872. He developed
symptoms of phthisis, and from July 16 was placed on the sick list at
Coonoor. A month later his condition was so serious that he was invalided
to Europe, and his friends hardly ventured to anticipate his return. After
-a year, spent mostly on the Riviera, his health, however, became so improved
‘that he was able to resume his duties on November 5, 1873.

In 1874, King obtained the approval of Government for the improvements
required in the Botanic Gardens as a scientific centre and a place of public
resort. His designs involved prolonged work and considerable outlay. The
funds required could only be gradually allotted; that they were granted at
all gives ample proof of the enlightened liberality of the Bengal Government
and the confidence which King’s administrative gifts inspired. During the
next nine years the Gardens were practically reconstructed. By excavating
a series of lakes and ponds, so designed as to produce a variety of pleasing
effects, sufficient soil was obtained to raise the level of the whole of the
grounds. These sheets of water were connected by underground pipes, and
the whole system was so arranged as to be kept at a uniform level by
pumping water from the contiguous river. Many footpaths and carriage
roads were laid out so that visitors can drive to any part of the garden.
Elegant conservatories and a noble palm-house were built. New potting
sheds, tool stores, and propagating pits were supplied and good dwelling-
houses were provided for the members of the garden establishment both
native and European. A handsome fireproof herbarium, on the lines of that
at Kew, was erected to accommodate the rapidly-growing collection of dried
plants and the valuable library. Minor improvements were added in
subsequent years, but by 1883 the heaviest of King’s garden work was over.

While effecting these improvements, King steadily added desirable species,
indigenous and exotic, to the collections of living plants. Whenever this
was compatible with the health of the plants and the production of pleasing
effects King arranged his species with regard to their affinities, so that one
part of the great garden can boast its fine palmetum, pandanetum, .
bambusetum; elsewhere other natural groups are similarly treated. But
King had all the horror of the true lover of plants for a pedantic arrange-
ment of species in.rectilinear blocks according to what are conventionally
termed natural families and regardless of the conditions suited to particular
species. The first duty of a gardener he held to be the proper culture of his
plants; the needs of the species grown, not the pragmatical requirements of the
methodist, were his chief consideration. Here and there he aggregated with
the happiest results groups of species from some particular geographical area,
thus reproducing plant associations which, though unmeaning except from

Sir George King. X1X

the artistic standpoint to the average European, are readily appreciated by
intelligent native visitors. His operations were controlled with a singular
prescience for ultimate effects and his years of unremitting toil were amply
rewarded. The whole place bears the impress of King’s influence and care,
and the charm and beauty of its lakes and groves, its avenues and lawns for
which the Royal Garden at Calcutta is now so justly famed, serve as an
adequate memorial to his energy, patience and skill as a landscape gardener.

King’s work in the Botanic Garden by no means exhausts his achievements
in this direction. Shortly after his return to India in 1873 the amenities of
Calcutta were enhanced by the addition of a zoological garden. King was
appointed an original member of its Committee of Management, the
Lieutenant-Governor in person serving as President. The site selected was
occupied by a collection of miserable native huts; in a few years, under
King’s skilful guidance, it became one of the most attractive public resorts
in India. Very soon afterwards the site of a summer residence for the
Lieutenant-Governor was acquired at Darjeeling. The demesne in which the
mansion stands was laid out under King’s eye and the consummate art with
which he employed the constituents of a natural forest and blended the
effects produced within the area treated with those to be obtained from
adjacent hill slopes and distant views has rendered these grounds at once the
ideal of what such a place should be and the despair of those who would
repeat the results. Again, in 1879, when, with the help of private
munificence, Government was able to provide at Darjeeling a temperate
annexe to the Botanic Garden at Calcutta, King was given administrative
charge of this new garden. By a combination of the methods employed for
the “Shrubbery” grounds at Darjeeling with those applied to the Zoological
Garden at Calcutta, King in a few years created another place of public resort
at once beautiful and instructive.

When King resumed charge of the Cinchona Department in 1873, he
found that a quinologist had been appointed; he was, therefore, for the
moment relieved of his anxiety with regard to factory operations. The officer
appointed, Mr. C. H. Wood, devised a satisfactory method of extracting the
mixed alkaloids from cinchona bark, and to King fell the delicate duty of
creating a market for the resulting product, known as Cinchona Febrifuge.
He brought to this task all the qualifications of an expert business man,
surmounting every difficulty, and firmly establishing the distribution of the
article on commercial, as opposed to eleemosynary, lines. But on October 13,
1877, Wood was transferred to Calcutta and King was placed in administra-
tive charge of the factory, and in August, 1879, Wood, for domestic reasons,
resigned Government service. When Wood retired he had not yet devised
an economic method of separating quinine; he had, however, left his process
for extracting febrifuge in excellent working order. In Mr. J. A. Gammie,
the resident manager of the plantation, King had an able and resourceful
lieutenant, who had worked in conjunction with Wood and was thoroughly
competent to conduct Wood’s febrifuge process.

b 2

2G Obituary Notices of Fellows deceased.

Difficulties due to natural causes at first impeded King’s substitution of
the cultivation of yellow, or quinine-yielding, cinchonas for that of red
cinchonas, in which the proportion of quinine is low. But for the skilful
co-operation of Gammie these difficulties must have proved insurmountable,
though, great as they were, they proved trifling as compared with other
difficulties which only the confidence that he inspired in his immediate
superiors enabled King to defeat. From the same source came difficulties
connected with manufacture. When Wood resigned the quinologist’s post
in 1879, King had been deputed to Java to study the working of the Dutch
cinchona department in that island. When he returned to India, on
December 5, 1879, King found himself appointed to act as his own
quinologist. The situation, for one whose passion was for thorough work
and yet who was not himself an expert chemist, was full of difficulty; but
the situation had to be faced, and he faced it with courage. Wood, after his
return to England, took the keenest interest in the work, striving in his own
laboratory to master some economical mode of obtaining quinine, while
Gammie made trial of his suggestions on a commercial scale in the factory
in Sikkim. Eventually King himself conduced to the ultimate success.
He spent the summer of 1884 on furlough in Europe. Botanical studies on
which he was engaged necessitated a visit to the Dutch herbaria. While in
Holland he acquired some valuable information as to a process for separating
quinine, which he at once made known to Wood, who was thereby led to
devise a process more hopeful than any previous one. Gammie was able to
visit England on furlough in 1885; he studied the details of the new process
in Wood’s laboratory, and on his return to India found that it was practicable
on a commercial scale. The separation of pure quinine on the spot without
involving financial loss to Government was at last possible, and by the
end of 1887 a factory had been established and the manufacture of quinine
on commercial lines was in full operation. In reporting this event, King
was content to recount “the generous way in which Mr. Wood, without any
pecuniary reward, initiated and invented it [the process] in his private
laboratory, while Mr. Gammie perfected it in the Government factory.
Without Mr. Wood the process would not have been invented, while without
Mr. Gammie it would not have been successfully applied to manufacture.”

The achievement was, after all, only a step towards the realisation of the
original design of Government to supply the people of India, on a self-
supporting basis, with quinine at a nominal cost. The attempt to supply the
article on an eleemosynary basis had, indeed, already been made, police
outposts being utilised as the distributing agency. But this humane effort
was promptly defeated by small capitalists, who bought up the whole supplies
as soon as these reached the various outposts, in order to resell the drug at a
handsome profit and yet at rates which undersold the regular vendor. A
firm of European merchants, inspired partly by genuine philanthropy, partly
by a legitimate desire to extend their business, had also essayed the task, but
had been compelled to abandon it, owing to the impossibility of organising a

Sir George King. XXl

reliable distributing agency. Clearly, therefore, no special agency could be
economically created: some already existing one must be utilised. Clearly,
too, the drug must be sold at a rate which, while securing Government
against loss, should at the same time eliminate temptation on the part of
outsiders to exploit the humanity of the authorities. King saw at a glance
how the desired result might be attained, but the confidence he inspired in
Government was not in itself sufficient to ensure the success of his design,
which demanded not only the consent but the enthusiastic support of other
heads of departments. In gaining this support the charm of his personality
was as effective as it had been in securing the co-operation of his colleagues.
The scheme involved the sale at every post-office of Government quinine,
made up in doses of five grains each. Hach dose was to be enclosed in a
neat sealed packet. Each packet was to be sold for one farthing, and,
together with brief instructions in the various vernaculars, was to bear the
Royal Arms as a guarantee of genuineness. To encourage postal officials to
push sales, a small commission was to be allowed, and facilities for
replenishing stocks were to be offered. To eliminate the risk of adulteration
and pilfering, the making-up of the packets was to be entrusted to the
Prisons Department, who would receive the quinine in bulk direct from the
Cinchona stores. In perfecting the scheme, King worked in co-operation
with the Financial Secretary to Government, Mr. (now Sir Herbert) Risley.
But the Postmaster-General for Bengal, Mr. Kisch, devised the procedure
regulating the actual sales; the ingenuity of the Superintendent of the
Alipur Jail, Mr. Larrymore, hit upon a method of cheaply, rapidly, and
accurately dividing the quinine into doses, and the expert advice of the
Government Printer, Mr. Lewis, guided the details connected with the
preparation of the envelopes. The success of the scheme depended on the
precision with which each department did its share of the work, and on the
accuracy of the calculations with regard to the cost of each operation. These
calculations had of necessity to be so close as to leave no margin ; an error
at any point might easily have involved financial loss. The scheme, fully
matured, was put into operation in 1893, and worked from the outset
without a hitch. To the officers of these co-operating departments King
attributed the success, after thirty years of effort, of the design enunciated
by Government when it introduced cinchona to India :—*To put the only
medicine that is of any use in the cure of the commonest and most fatal of
Indian diseases within the reach of the poorest.”

In 1874 King also commenced on a definite plan, the details of which he
wisely subordinated to current exigencies, a survey of the vegetation of the
countries within the sphere of influence of the Calcutta garden. These
include the Eastern Himalaya, Bengal, Assam, Burma, the Andaman and
Nicobar Islands, and the Malayan Peninsula. His first object was to fill up
gaps in the Calcutta Collection rather than to investigate afresh areas
already examined; to this end he sent independent collectors to unvisited
districts or attached them to military expeditions or survey parties. But

-o-4 Obituary Notices of Fellows deceased.

his efforts were largely aided by personal friends, and in no branch of his
work was his magnetic influence more potent than in this. He imparted to
officers of Government, both civil and military, to missionaries, planters and
travellers some share of his own enthusiasm, and many of the most valuable
additions to the Calcutta herbarium were the result of his endeavours in
this direction. His multifarious duties left him few opportunities for
personal travel, but he never allowed them to impede his constant super-
vision of the work of his botanical artists. He was thus enabled to bring
together a collection of specimens and drawings far surpassing in extent and
value that dispersed in 1828, and to take a considerable share in the task of
supplying material for the use of Sir Joseph Hooker, while that botanist was
engaged from 1872 to 1897 in preparing the ‘Flora of British India.’ It
was therefore fitting that when, in 1891, the botanical officers serving in the
different Presidencies were linked together in one department, King was
appointed the first Director of the Botanical Survey of India. In this
capacity he urged the necessity for the preparation of a series of local or
regional floras to supplement Hooker’s great work. His proposals, after
being approved alike by the local governments concerned and by the
Supreme Government, encountered difficulties akin to those he had
experienced in connection with cinchona, so that nothing beyond what he
himself could accomplish had been done in this direction when he left India
in 1898. In the end these difficulties were overcome, and the work he had
shown to be necessary has already been partly accomplished.

As Professor of Botany at the Medical College of Bengal, King was a lucid
and effective teacher, and in the course of study to which he subjected his
pupils he, with the approval of Government, effected at the outset alterations
which to his practical mind seemed improvements. The course, as he found
it, was modelled on those adopted in medical schools in Britain, where the
teacher was either content to coach his students to the point required to enable
‘them to pass an examination on some prescribed standard, or was prone, if
enthusiastic, to endeavour to bring his pupils to some approximation to his own
standard of botanical knowledge. The first method King held to be a waste
of the time both of teacher and taught; the second, even if the laws of
supply and demand had rendered it desirable, he found to be impossible. His
students, with hardly an exception, were young men who had suffered from
what he held to be the injurious incubus, a western literary education; with
minds often originally bright, their natural powers of observation had been
inhibited and sometimes atrophied by close attention to the written word.
In consideration of the fact that the real purpose of their presence in college
was to acquire a practical knowledge of surgery and medicine, he deemed it
his duty to treat the subject he taught as purely ancillary to this laudable
end, His teaching therefore resolved itself, not into a course of Botany in
the ordinary acceptation of the term, but into a steady application of
botanical facts and truths to the training of the various senses of his
students. If in the end they did come to know a good deal about the subject,

Sir George King. XX

this King considered a purely incidental result. _ The object he strove, and
strove successfully, to attain was to habituate his pupils to the art of observing
natural facts, and to accustom them to the ordeal of giving reasons for the
faith that was in them when confronted with objects that, though similar in
externals, were essentially different.

The exercise of King’s business capacity was not limited to the departments
which he administered. His local Government appointed him a member of
the visiting Board of the Bengal Engineering College, an institution with
whose objects as a technical school he was in entire sympathy, and in whose
progress he took a warm and effective interest. He was appointed by its
Chancellor a Fellow of the University of Calcutta, and was long a trusted
member of the Senate, for a time also representing the medical faculty on the
Syndicate. He represented the Supreme Government as a Trustee of the
Indian Museum, an institution for which he did much important work,
especially during a number of years when he was Chairman of the Trustees ;
from his resignation of that office till his retirement from Indian service he
was Vice-chairman. When in 1894 the Government of India organised an
enquiry into the indigenous drugs of the country, King was appointed
Chairman of the Central Committee, and served in this capacity till he left
for Europe. He tooka deep interest in the welfare of the Asiatic Society of
Bengal, and although he did not often accept a seat on its Council, he was
always the trusted adviser of the Society’s officers in matters relating to its
natural history side. He was an active member of Council, and at one time
President of the Agricultural and Horticultural Society of India.

It is somewhat interesting to observe that, in spite of his early distaste
for business, King’s public services should have derived their chief value
from his remarkable business aptitude, and that although the extent and
gravity of his official duties did not prevent the simultaneous prosecution of
purely scientific studies, the possession of this business aptitude deprived him
for many years of any opportunity of presenting ordered statements of his
results. It is equally interesting to find that, as regards his scientific work,
the line which he took was not that towards which his tastes naturally led.
When King reached India in 1866 his botanical interests were centred on
physiological and morphological problems, and especially on those connected
with eryptogams. Here, again, circumstance proved stronger than pre-
dilection. The comparative poverty of the floras of Central India and
Rajputana led him to expend his surplus energy in important zoological
studies ; during the rest of his career these were given to systematic work |
connected with flowering plants. His practical mind realised, from the time
he took charge of the Saharanpur garden, that however enticing his favourite
studies might be, the path of duty for him led elsewhere ; that the immediate
needs of people and Government alike demanded that every official Indian
botanist should devote himself to aiding Hooker in the prosecution of his
fundamental undertaking of providing recognisable descriptions of Indian’
phanerogams; and that until this floristic study was completed, the time for

XXIV Obituary Notices of Fellows deceased.

indulging in the work he personally preferred had not yet come. His duties
as a forest officer taught him how diffieuls and yet how essential the -
recognition of species that are of economic importance may be; his experience
then and afterwards, when engaged in the formation of a first-rate herbarium
collection, led him to realise how frequently competent field workers, whose
results in obtaining material for the study of herbaceous plants or shrubs may
leave nothing to be desired, are deterred by what are doubtless serious
physical difficulties from supplying specimens that adequately illustrate
arboreous types. His sense of the extreme importance, from the industrial
standpoint, of full accounts of the constituents of the Indian forests, led him
by precept and example constantly to strive to remedy this well-known
defect. With all this he fully realised the desirability of advancing our
knowledge of Indian cryptogams, more especially in regard to their
connection with pathological problems, but he failed, for once, to convince
Government how desirable it was to add a competent eryptogamist to the
garden staff. He did what he could to remedy the defect by referring
material to experts in Europe, and here again his personal influence was
of incalculable benefit to India. A friend, Dr. Cunningham,* devoted
much of his scanty leisure to questions connected with vegetable pathology ;
another friend, Dr. Barclay,+ was an ardent student of the life-histories of the
Uredinee. For many years these two workers dealt on King’s behalf with
critical references relating to the field of study which King was precluded
from entering and their generous co-operation with him in the public interest
only ended with the departure from India of the one and the untimely
death of the other. Throughout his active career King kept himself abreast
of what was done in most branches of botanical activity, but intimate friends
alone were aware of the pain it gave him to observe the gradual drifting
apart of workers in different lines of research. What grieved him most was
the hostility, especially when this was veiled, sometimes displayed by men
whose work connected with what they termed “scientific” botany he held in
high regard, towards “systematic” botany. This attitude on the part of
students of problems which naturally attracted himself, towards conscientious
workers in the field to which circumstance and a sense of duty confined him,
caused King deep distress.

The: fact that King’s scientific attainments were on a level with his
administrative gifts, though unknown to the world at large, could not be
concealed from those with whom he corresponded on botanical subjects, and in
1884 his university conferred on him the degree of LL.D., while in 1887 he
was elected into the Royal Society. He had since 1874 in reality done much
critical work, but it was not until 1887, when the progress made with his
garden improvements and especially in connection with the manufacture of

* D. D. Cunningham, C.1.E., F.R.S., Professor of Physiology, Calcutta, and Secretary
to the Sanitary Commissioner with the Government of India.

+ Arthur Barclay (1852—1891), Secretary to the Director-General, Indian Medical
Service.

Sir George King. XXV

quinine seemed to justify the step, that King commenced the publication of
important contributions to botanical literature. In that year the enlightened
liberality of the Government of Bengal enabled King to found the ‘ Annals
of the Royal Botanic Garden, Calcutta, a series of sumptuous volumes in
which he proceeded to publish amply illustrated monographs of difficult
and unwieldy genera and families. The first of these deals with “the
species of Ficus of the Indo- Malayan and Chinese countries.” On
this work he had bestowed the labour of much of his scanty leisure
for eleven years, during which time he had examined the material
preserved in every important European collection. The objects he
had in view were altogether practical ones; the genus was _ selected
because of its being largely composed of trees, many of them being of
economic importance, and the monograph was primarily intended to break
ground for Sir Joseph Hooker and to assist that author in subsequently
dealing with its species. The work, however, is marked by such accuracy,
lucidity, and completeness that it at once placed King among the foremost
systematic botanists of his time, and its appearance was rapidly followed by
that of equally finished works on Quercus, Castanopsis, Artocarpus, and
Myristica, all prepared with the same object and selected for the same
reasons. When King visited Java in 1879 he had an opportunity of seeing
something of the rich vegetation of Malaya, which made on his mind an
ineffaceable impression. From Singapore he paid a botanical visit to Johor,
in company with his friend Archdeacon (now Bishop) Hose. He collected
personally in Penang and Province Wellesley, and was subsequently able to
arrange for the systematic botanical exploration of Perak. In 1886 facilities
were afforded, at the request of King’s friend, Sir Hugh Low, to Father
Seortechini, who had also made extensive collections in Perak, to commence
the preparation in the Calcutta herbarium of a flora of that State. Scortechini,
unfortunately, soon afterwards died, bequeathing to the Calcutta Garden all
his specimens, drawings, and notes. Sir Joseph Hooker and Sir Hugh Low
now begged King himself to undertake this very urgent task, and in 1889 he
commenced single-handed a floristic study of the whole Malayan Peninsula.
As preliminary to the preparation of a local flora of the region—the first of
the series of such floras, whose publication for the various provinces of India
he was two years later officially entitled to urge—King began to issue, in the
‘Journal of the Asiatie Society of Bengal, a series of contributions intended
to serve as “ Materials for a Flora of the Malayan Peninsula,” but prepared
with such care that they form a satisfactory substitute for a final work.

Five instalments of this great undertaking, completing the Thalamiflore,
were issued up to 1893, but in the case of two important families, Magnoliacez
and Anonacez, the study of the Malayan forms involved a careful examina-
tion of extra-Malayan material, the results of which were embodied in two
great monographs simultaneously published in the garden ‘ Annals.’ In
1895, King, having attained the age of fifty-five, was, under Indian rules, due

b 38

XXV1 Obituary Notices of Fellows deceased.

to retire on April 11 without being able to qualify for the pension payable
after thirty years’ service. In consideration of the importance of the work
he had in hand, his service was extended for two years, and on July 1, 1895,
he was further permitted to resign his chair at the Medical College so as to
leave more time for the floristic work on which he was engaged. In 1896
with the eighth part of the ‘Materials, King completed the Disciflore, and
in 1897 he was granted a further extension to permit him to carry still
further his Malayan work and to complete a sumptuous monograph of the
‘Orchids of the Sikkim Himalaya, of great importance to horticulture, for
which he provided the text, while one of his Cinchona officers, Mr. R.
Pantling, prepared the illustrations. Towards the end of 1897 his health,
which since 1873 had been uniformly good, was’ completely undermined by a
severe attack of fever, and his medical advisers peremptorily ordered the
termination of his service. But before he left India on February 28, 1898,
after more than thirty-two years of devoted service to the people and the
Government, he had the satisfaction of seeing the issue of the orchid mono-
eraph, and had carried his Malayan work to the middle of the Calyciflore, at
the end of the tenth fasciculus. On reaching England, King, resumed at
Kew, his work on the Malayan flora. The state of his health, however,
prevented his making great progress during 1898, and in 1899, owing to his
consenting to serve as President of the botanical section of the British
Association at its meeting at Dover, he was able to accomplish less than he
had hoped. He had, moreover, under medical advice, to spend each winter
and spring on the Riviera, and soon realised that he might never finish the
task he had allotted himself. - He faced the contingency with characteristic
practicality. By arrangement with his friend, Mr. H. N. Ridley, Director of
the Singapore Botanic Garden, that botanist undertook the elaboration of the
Monocotyledonous families, while King worked out the remaining Dicoty-
ledons, and when, in 1902, with the issue of the thirteenth part, King had
finished the Calycifloree, he was joined by his friend, Mr. J. 8S. Gamble, in the
elaboration of the Corolliflore. For three more years King took his full
share in the joint work, which now made rapid progress; after 1905 partial
loss of sight and progressive infirmity led to his enforced abandonment
of active participation in the task, and the only share he could take in the
preparation of the twenty-first part, whose issue coincided almost to a day
with his death and completed the Corolliflore, was the examination of the
sheets as they passed through the Press. He had, however, the satisfaction
of seeing the issue in 1907 of the first portion, and the completion in 1908
of what remained of Mr. Ridley’s contribution to the great undertaking
begun in 1889.

King’s reputation as a landscape gardener was well known; it brought
him honorary association with various horticultural societies and was
recognised by the award of the Royal Horticultural Society’s Victoria Medal
in 1901. The value of his services to humanity in connection with the

Sir George King. XXVil

separation and especially the distribution of quinine brought him honorary
membership of the Pharmaceutical Society, the grade of Officier d’Instruction
publique, the gift of a ring of honour by the Czar Alexander III, and the
Companionship, in 1890, of the Order of the Indian Empire. His work as
a systematic botanist was also widely appreciated; he was a corresponding
member of the Bavarian Academy, an honorary member of the Royal
Botanic Society of Belgium and of the Deutsch Botanische Gesellschaft, one
of the six honorary British Fellows of the Botanic Society of Edinburgh, and,
a distinction that gratified him more than any other, an honorary member,
after he left India, of the Asiatic Society of Bengal, with which he had been
connected since 1867. The University of Upsala presented him with
a medal in recognition of his botanical studies, and the Linnean Society, to
which he had been elected in 1870, awarded him its Linnean medal in 1901.
On January 1, 1898, he was, in recognition of his long and distinguished
service, created a K.C.LE., and immediately after his retirement a number of
his personal friends united in obtaining a medallion portrait in bronze, which
was placed, with a similar portrait of his friend, Dr. Cunningham, who for
many years was Secretary to the Committee, in the Zoological Gardens
which King had designed. A replica of King’s portrait was placed in the
Royal Botanic Garden whose beauty he had restored. At San Remo, where
he wintered yearly from 1898 till his death, another memorial, connected with
a public institution whose welfare he had much at heart, will bear lasting
witness to his quiet but effective devotion to the cause of practical
philanthropy.

King married, in 1868, Jane Anne, daughter of Dr. G. J. Nicol; during
his illness in 1897 she was with him in India. As he was slowly recovering,
Lady King’s health gave way. On the homeward voyage she gradually
sank ; she died in London the day following their arrival in England. From
this blow King never fully recovered; its effect became more and more
apparent as the solace of strenuous work was denied him. The hemorrhagic
tendency of early life reasserted itself, and led to the rupture of a retinal
vessel which deprived him of the use of an eye. The tendency steadily
increased, and the melancholy induced by the feeling that his days of
usefulness had ended was mercifully relieved by an apoplectic seizure to
which King succumbed at San Remo on February 12, 1909. His remains
were interred, as he had desired, where he died.

Kine’s wide knowledge, which extended to most branches of science and
embraced many aspects of art and literature, was accompanied by a natural
modesty and a personal charm that rendered intercourse with him extremely
pleasing, though literary or artistic friends rarely came to know of his
scientific tastes, and scientific acquaintances had still fewer opportunities of
appreciating his critical acumen. But these, and other friends outside either
category, fully understood his innate goodness and courtesy, his transparent
candour, his shrewd sense, and his keen but kindly wit. A wise counsellor

XXVlil Obituary Notices of Fellows deceased.

and an unfailing friend, he was loved by all who were privileged to know
him. King’s life was spent in doing with his might what his hand found to
do, and if others have made more striking contributions to natural knowledge,
none have rendered more self-sacrificing and devoted service to the nations

of India and to the science of Botany.
Dare

INDEX. 10 VOL. LXXXI. (B)

Acidalia virgularia, cross-breeding of two races of (Prout and Bacot), 133.

Alcohol, ether, and chloroform, comparative power of, as measured by action on isolated
muscle (Waller), 545.

Ancestral gametic correlations of a Mendelian population mating at random (Pearson),
225.

Armstrong (H. E.) The Origin of Osmotic Effects. II.—Differential Septa, 94.

Bacilli, influence of glucosides on growth of acid-fast, and new method of isolating
tubercle bacilli (Twort), 248.

Bacot (A.) See Prout and Bacot.

Bacteria, etc., differentiated by rate of diffusion into living cells (Ross), 97 ; effects of
nitrogen-fixing, on non-leguminous plants (Bottomley), 287 ; sedate reactions
of certain, and application in detection of tubercle bacilli (Ree), 314.

Bacterial endotoxins, effect on opsonising action of rabbit serum (Hewlett), 325.

Bacterium, a giant sulphur (West and Griffiths), 398.

Balloons, rubber, and hollow viscera, elasticity of (Osborne and Sutherland), 485.

Barratt (J. O. W.) and Yorke (W.) A Method of Estimating the Total Volume of
Blood contained in the Living Body, 381.

Bashford (E. F.) and Murray (J. A.) The Incidence of Cancer in Mice of Known Age,
310.

Bateman (Capt. H. R.) See Bruce (Sir D.) and others.

Bayliss, (W. M.) The Properties of Colloidal Systems. I.—The Osmotic Pressure of
Congo-red and of some other Dyes, 269.

Blood from diseases in man, presence of hem-agelutinins, etc., in (Dudgeon), 207 ;
in cholera, variations in pressure and composition of (Rogers), 291 ; in living
body, method of estimating total volume of (Barratt and Yorke), 381 ; —— supposed
presence of carbon monoxide in (Buckmaster and Gardner), 515.

Blood-platelets, vacuolation of (Ross), 351.

‘Bottomley (W. B.) Some Effects of Nitrogen-fixing Bacteria on the Growth of Non-
leguminous Plants, 287.

Brown (A. J.) The Selective Permeability of the Covering of the Seeds of Hordeum
vulgare, 82.

Bruce (Sir D.) and others. A Trypanosome from Zanzibar, 14 ; Trypanosoma ingens,
n. sp., 323; —— The Development of Trypanosoma gambiense in Glossina palpalis,
405 ; —-— A Note on the Occurrence of a Trypanosome in the African Elephant, 414.

Buckmaster (G. A.) and Gardner (J. A.) On the Supposed Presence of Carbon
Monoxide in Normal Blood and in the Blood of Animals anesthetised with
Chloroform, 515.

Cammidge (P. J.) Observations on the Urine in Chronic Disease of the Pancreas, 372.

Cancer, incidence of, in mice of known age (Bashford and Murray), 310.

Carbon monoxide in normal blood and in blood of chloroformed animals, supposed
presence of (Buckmaster and Gardner), 515.

Cholera, variations in pressure and composition of blood in (Rogers), 291.

xxx

Cholesterol, origin and destiny of the organism, Part III (Dorée and Gardner), 109 ;
— Part IV (Hllisand Gardner), 129 ; Part V (Fraser and Gardner), 230 ; ——
Part VI (Ellis and Gardner), 505.

Colloidal systems, properties of (Bayliss), 269.

Colloids, electrolytes and (Wood and Hardy), 38.

Crepidula fornicata, protandric hermaphroditism in (Orton), 468.

Croonian Lecture (Schiifer), 442.

Darbishire (A. D.) An Experimental Estimation of the Theory of Ancestral
Contributions in Heredity, 61.

Dendy (A.) The Intracranial Vascular System of Sphenodon, 290.

Desmids, distribution of British (West and West), 165.

Diffusion of stain, etc., into living cells, determination of coefficient of rate of (Ross), 97.

Dorée (C.) and Gardner (J. A.) The Origin and Destiny of Cholesterol in the Animal
Organism. Part II].—The Absorption of Cholesterol from the Food and its
Appearance in the Blood, 109.

Dudgeon (L. 8.) On the Presence of Hem-agglutinins, Hzem-opsonins, and Hzmolysins

in the Blood obtained from Infectious and Non-infectious Diseases in Man.
(Second Report), 207.

Elasticity of rubber balloons and hollow viscera (Osborne and Sutherland), 485.

Electrical state of living tissues, effect of heat upon (Waller), 303.

Ellis (G. W.) and Gardner (J. A.) The Origin and Destiny of Cholesterol in the
Animal Organism. Part 1V.—The Cholesterol Contents of Eggs and Chicks, 129 ;

Part VI.—The Excretion of Cholesterol by the Cat, 505.

Ewart (J. C.) The Possible Ancestors of the Horses living under Domestication, 392.

Fantham (H. B.) and Porter (A.) The Modes of Division of Spirocheta recurrentis and
S. duttont as observed in the Living Organisms, 500.

Fermentation of glucose, etc., by yeast-juice (Harden and Young), 336.

Flowers, colours and pigments of, with reference to genetics (Wheldale), 44.

Fraser (M. T.) and Gardner (J. A.) The Origin and Destiny of Cholesterol in the
Animal Organism. Part. V.—On the Inhibitory Action of the Sera of Rabbits fed
on Diets containing Varying Amounts of Cholesterol on the Hemolysis of Blood by
Saponin, 230.

Fraser (Sir T. R.) and Gunn (J. A.) The Action of the Venom of Sepedon hemachates of
South Africa, 80.

Fry (W. B.) See Plimmer and Fry.

Gardner (J. A.) See Buckmaster and Gardner, Dorée and Gardner, Ellis and Gardner,
and Fraser and Gardner.

Genetics, colours and pigments of flowers, with reference to (Wheldale), 44.

Gluten, physical state of (Wood and Hardy), 38.

Goitre, summary of researches on etiology of (McCarrison), 31.

Griffiths (B. M.) See West and Griffiths.

Gunn (J. A.) See Fraser and Gunn.

Hadwen (S8.) See Nuttall and Hadwen.

Hem-agglutinins, hem-opsonins, etc., in blood from diseases in man (Dudgeon), 207.
Hamerton (Captain A. E.) See Bruce (Sir D.) and others.

Hamilton (D. J.) Obituary notice of, i.

XXXl

Harden (A.) and Young (W. J.) The Alcoholic Ferment of Yeast-juice. Part 1V.—The
Fermentation of Glucose, Mannose, and Fructose by Yeast-juice, 336.

Hardy (W. B.) See Wood and Hardy.

Heape (W.) The Proportion of the Sexes produced by Whites and Coloured Peoples in
Cuba, 32.

Heredity, experimental estimation of ancestral contributions in (Darbishire), 61 ;

Theory of ancestral contributions in (Pearson), 219.

Hermaphroditism in molluse Crepidula fornicata (Orton), 468.

Hewlett (R. T.) The Effect of the Injection of Intracellular Constituents of Bacteria

(Bacterial Endotoxins) on the Opsonising Action of the Serum of Healthy

Rabbits, 325.

Hexosephosphate formed by yeast-juice from hexose and phosphate (Young), 528.

Hillhousia mirabilis, a giant sulphur bacterium (West and Griffiths), 398.

Hordeum vulgare, selective permeability of seed coverings of (Brown), 82.

Horses living under domestication, possible ancestors of (Ewart), 392.

Hudleston (W. H.) Obituary Notice of, vi.

Innervation, reciprocal, of antagonistic muscles (Sherrington), 249.

Jaundice in dog, malignant, discovery of a remedy for (Nuttall and Hadwen), 348.
King (Sir George) Obituary Notice of, xi.

McCarrison (R.) A Summary of further Researches on the Etiology of Endemic Goitre,
3.

Mackie (Capt. F. P.) See Bruce (Sir D.) and others.

Martin (C. H.) and Robertson (M.) Preliminary Note on Trypanosoma eberthi (Kent)
(= Spirocheta eberthi, Liihe), and some other Parasitic Forms from the Intestine of
the Fow], 385.

Medals, award of, 8.

Murray (J. A.) See Bashford and Murray.

Muscle, action of alcohol, ether, and chloroform upon (Waller), 545.

Nerves of atrio-ventricular bundle (Wilson), 151.

Nitrogen-fixing bacteria, effects on growth of non-leguminous plants (Bottomley), 287.

Nuttall (G. F.) and Hadwen (S.) The Discovery of a Remedy for Malignant Jaundice
in the Dog, and for Redwater in Cattle, 348.

Obituary notices :—
Hamilton (D. J.), i.
Hudleston, (W. H.), vi.
King (Sir G.), xi.
Opsonising action of rabbit serum, effect of bacterial endotoxias on (Hewlett), 325.
Orton (J. H.) On the Occurrence of Protandric Hermaphroditism in the Mollusc
Crepidula fornicata, 468.
Osborne (W. A.) and Sutherland (W.) The Elasticity of Rubber Balloons and Hollow
Viscera, 485.
Osmotic effects, origin of (Armstrong), 94.
Osmotic pressure of Congo-red and some other dyes (Bayliss), 269.

Pancreas, the urine in chronic disease of (Cammidge), 372.
Pearson (K.) The Theory of Ancestral Contributions in Heredity, 219 ; On the
Ancestral Gametic Correlations of a Mendelian Population mating at Random, 225.

XXX11

Phytoplankton, British freshwater, with special reference to Desmids (West and West),
165.

Pituitary body, functions of the (Schafer), 442.

Plimmer (H. G.) and Fry (W. B.) Further Results of the Experimental Treatment of
Trypanosomiasis : being a Progress Report to a Committee of the Royal Society,
354.

Porter (A.) See Fantham and Porter.

President’s anniversary address, 1.

Prout (L. B.) and Bacot (A.) On the Cross-breeding of Two Races of the Moth Acidalia
virgularia, 138.

Rayleigh (Lord) Anniversary Address, 1908, 1.

Redwater in cattle, discovery of a remedy for (Nuttall and Hadwen), 348.

Robertson (M.) See Martin and Robertson.

Rogers (L.) The Variations of Pressure and Composition of the Blood in Cholera ; and
their Bearing on the Success of Hypertonic Saline Transfusion in its Treatment, 291.

Ross (H. C.) On the Determination of a Coefficient by which the Rate of Diffusion of
Stain and other Substances into Living Cells can be measured, and by which Bacteria
and other Cells may be differentiated, 97; ——— The Vacuolation of the Blood-
platelets: an Experimental Proof of their Cellular Nature, 351.

Russ (C.) The Electrical Reactions of certain Bacteria, and an Application in the
Detection of Tubercle Bacilli in Urine by means of an Electric Current, 314.

Schafer (E. A.) The Functions of the Pituitary Body (Croonian Lecture), 442.

Seeds, resting, ferments and latent life of (White), 417.

Sepedon hemachates, action of venom of (Fraser and Gunn), 80.

Sexes, proportion of, produced by whites and coloured peoples in Cuba (Heape), 32.

Sherrington (C. 8.) Reciprocal Innervation of Antagonistic Muscles. Fourteenth
Note.—On Double Reciprocal Innervation, 249.

Sphenodon, intracranial vascular system of (Dendy), 290.

Spirocheta eberthi (Liihe), preliminary note on (Martin and Robertson), 385.

Spirocheta recurrentis and S. duttoni, modes of division of (Fantham and Porter), 500.

Sutherland (W.) See Osborne and Sutherland.

Trypanosoma eberthi (Kent), Preliminary Note on (Martin and Robertson), 385 ;
gambiense, development in Glossina palpalis (Bruce and others), 405 ; ingens,
n. sp. (Bruce and others), 323.

Trypanosome from Zanzibar (Bruce and others), 14;
African elephant (Bruce and others), 414.

Trypanosomiasis, further results of experimental treatment. of (Plimmer and Fry), 354.

Twort (F. W.) The Influence of Glucosides on the Growth of Acid-fast Bacilli, with a
New Method of Isolating Human Tubercle Bacilli, 248.

note on occurrence of, in

Urine in chronic disease of pancreas (Cammidge), 372.
Venom of Sepedon, action of (Fraser and Gunn), 80.

Waller (A. D.) The Effect of Heat upon the Electrical State of Living Tissues, 303 ;

; The Comparative Power of Alcohol, Kther, and Chloroform as measured by
their Action upon Isolated Muscle, 545.

West (G. 8.) and Griffiths (B. M.) Hélihousia mirabilis, a Giant Sulphur Bacterium, 398.

West (W. and G.S.) The British Freshwater Phytoplankton, with Special Reference to
the Desmid-plankton and the Distribution of British Desmids, 165.

XXXlll

Wheldale (M.) The Colours and Pigments of Flowers with Special Reference to
Genetics, 44.

White (Jean) The Ferments and Latent Life of Resting Seeds, 417.

Wilson (J. G.) The Nerves of the Atrio-ventricular Bundle, 151.

Wood (T. B.) and Hardy (W. 8B.) Electrolytes and Colloids.—The Physical State of
Gluten, 38.

Yeast-juice, alcoholic ferment of, Part IV (Harden and Young), 336; —— hexose-
phosphate formed by (Young), 528.

Yorke (W.) See Barratt and Yorke.

Young (W. J.) The Hexosephosphate formed by Yeast-juice from Hexose and
Phosphate, 528 ; —— See Harden and Young.

END OF THE EIGHTY-FIRST VOLUME (SERIES B).

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Series B. Vol. 81. | No. B 545.

BIOLOGICAL SCIENCES.

CONTENTS.

; Page

Address of the President, LORD RAYLEIGH, O.M., D.C.L., at the
Anniversary Meeting on November 30, 1908 : ‘ : : ; I

A Trypanosome from Zanzibar. By Colonel Sir DAVID BRUCE, C.B.,
M.B., F.R.S. D.Sc, LL.D., Army Medical Service, and Captains
A. E. HAMERTON, D.S.O., and H. R. BATEMAN, Royal Army

Medical Corps. (Plates 1 and 2) : heres : ; : : 14
A Summary of further Researches on the Etiology of Endemic Goitre. By
ROBERT McCARRISON, M.B., B.Ch., Captain, Indian Medical Service . 3

The Proportion of the Sexes produced by Whites and Coloured Peoples in
Cuba. By WALTER HEAPE, M.A., F.R.S., Trinity College, Cambyder:

(Abstract) . : 3 : 32
Electrolytes and Colloids.—The Physical State of Gluten. By oe
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The Action of the Venom of Sepedon heemachates of South Africa. By Sir
THOMAS R. FRASER, M.D., LL.D., Sc.D., F.R.S., Professor of Materia
Medica, University of Edinburgh; and JAMES A. GUNN, M.A., B.Sc.,

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Series B. Vol. 81. No. B 546.

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The Selective Permeability of the Coverings of the Seeds of Hordeum
vulgare. By ADRIAN J. BROWN, Professor of Brewing in the
University of Birmingham. : : : 2 3 4 : : 82
The Origin of Osmotic Effects. II.—Differential Septa. By HENRY E.
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On the Determination of a Coefficient by which the Rate of Diffusion of
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which Bacteria and other Cells may be Differentiated. By HUGH C.
ROSS, late Surgeon R.N., Pathologist to the Royal Southern Hospital,
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The Origin and Destiny of Cholesterol in the Animal Organism. Part III.—
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London, and J. A. GARDNER, Lecturer in Physiological chemistry
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The Origin and Destiny of Cholesterol in te Loe ee Part [V.—
The Cholesterol Contents of Eggs and Chicks. By G. W. ELLIS and
J. A. GARDNER, Lecturer on Physiological Chemistry, University of

London. : . 29
On the Cross-breeding of Two Recs of te Moth ai es By
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The Nerves of the Atrio-ventricular Bundle. By J. GORDON WILSON,
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The British Freshwater Phytoplankton, with Special Reference to the
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pb andsG., Ss WEST WAS D Seb IS.- oe ee OR

On the Presence of Hzem-agglutinins, Hzem-opsonins, and Heemolysins in
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The Theory of Ancestral Contributions in Heredity. By KARL PEARSON,
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Reciprocal Innervation of Antagonistic Muscles.- Fourteenth Note——On
Double Reciprocal Innervation. By C. $. SHERRINGTON, D.Sc., F.R.S. 249
The Properties of Colloidal Systems. I.—The Osmotic Pressure of Congo-
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College, London . z : 4 : : : : A : Oe
The Intracranial Vascular System of Sphenodon. By ARTHUR DENDY
F.R.S. (Abstract) . . 3 : gh : : : . 290
The Variations in the Pressure and Composition of the Blood in Cholera ;
4 and their Bearing on the Success of Hypertonic Saline Transfusion in its
: Treatment. By LEONARD ROGERS, MLD., F.R.C.P., F.R.CS., LMS.,
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The Effect of Heat upon the Electrical State of Tae Tissues. Bs
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The Incidence of Cancer in Mice of Known Age. By E. F. BASHFORD,
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BIOLOGICAL SCIENCES.

No. B 549.

CONTENTS.

‘Trypanosoma ingens, n. sp. By Colonel Sir DAVID BRUCE, C.B., F.RS.,
Army Medical Service; Captains A. E. HAMERTON, D.S.O., and
H. R. BATEMAN, Royal Army Medical Corps; and tase
F. P. MACKIE, Indian Medical Service. (Plate 7) ;

The Effect of the Injection of Intracellular Constituents of Bacon (Bartel
Endotoxins) on the Opsonising Action of the Serum of Healthy Rabbits.
By R. TANNER HEWLETT, M.D. 5s

The Alcoholic Ferment of Yeast-juice. Part 1V.—The Fermentation of
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HARDEN, F.R.S., and W. J. YOUNG

The Discovery of a Remedy for Malignant ede in ihe Bez ac ioe
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ee HADWEN, D.V.Sci. eee) of the Department of Agriculture,

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The Elasticity of Rubber Balloons and Hollow Viscera. By Prof.
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Hudleston Hudleston ; Sir George King te . i— xxviii

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igi eoampereters

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