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PROCEEDINGS

5 OF THE
ROYAL SOCIETY OF LONDON
SERIES B
CONTAINING PAPERS OF A BIOLOGICAL CHARACTER
ViOu ening

a ~~
Bex yons. de La

* i PRINTED ror THE ROYAL SOCIETY anp SOLD BY >
aan HARRISON AND SONS, ST. MARTIN’S LANE,
es A 3 on : PRINTERS IN ORDINARY TO HIS MAJESTY,

NOVEMPER, 1907,

KA

LONDON:
HARRISON AND SONS, PRINTERS IN ORDINARY TO HIS MAJESTY,
ST. MARTIN’S LANE.

CONTENTS.

SERIES B. VOL, LXXIX.

No. B 528.—February 22, 1907.
PAGE

On Opsonins in Relation to Red Blood-cells. By J. O. Wakelin Barratt, M.D.,
esc: Loud. Communicated by Dr. C. J. Martin, F.R.S. ........0.0sescoeccevssee i 1

The Structure of Nerve-fibres. By J. S. Macdonald, the University of Sheffield.
Communicated by Professor Sherrington, F.B.S. ..........cccccccescacscscescecsesssace 12

The Influence of Increased Barometric Pressure on Man.—II. By Leonard
Hill, F.R.S., and M. Greenwood, Jr., M.R.C.S., British Medical Association
Hse cree MSC TOMO A Ves tht a aa 5 rin faint e Baic/t¥t ales Aocelan Ss vse de cae edie de saivadsoiy she PON salem blaald egrets 21

On the Inheritance of Certain Invisible Characters in Peas. By R. H. Lock, M.A.,
Fellow of Gonville and Caius College, Cambridge. Communicated by W.
Ls PPO SOMME by Sed sae eater et ahi sate ceiedais daca dies dda o bene tedadeaoasecnada salubuiee dade demueaels 28

On the Occurrence of Encystation in 7rypanosoma grayi Novy, with Remarks on
the Method of Infection in Trypanosomes generally. By E. A. Minchin, M.A.
Communicated by Professor E. Ray Lankester, F.R.S. .........cccscssceseesoevssecees 35

On the Transpiration Current in Plants. By Henry H. Dixon, Sc.D., Professor
of Botany in Dublin University, and Director of Trinity College Botanic
Gardens. Communicated by Professor J. Joly, FRG. .......ccccceceseececeeeeeenes 4]

Nitrification of Sewage. By George Reid, M.D., D.P.H. Communicated by
LONE OT NG OLE ME tyr ecu lhe Col ocst edt ccatc grab saoeake claeebes anaes ceva dt eledavcake drepeun pais 58

The Relation of the Kidneys to Metabolism.—Preliminary Communication. By
F. A. Bainbridge, M.A., M.D., and A. P..-Beddard, M.A., M.D. Communicated
Prmteroressor ler Er, Starling, HORS: + ficcccisstsdsses seeds sons vve cesses seve wba voeseadennst 75

No. B 529.—April 4, 1907.

Address of the President, Lord Rayleigh, O.M., D.C.L., at the Anniversary Meeting
Gm MTEL RONG) Hc s Jassie die acedan slean de eeu ba eds dncweeladduweacheGevectead cutpepadseiese. 83

Further Observations on the Effects produced on Rats by the Trypanosomata of
Gambia Fever and of Sleeping Sickness. By H. G. Plimmer, F.L.S8. Com-
Tmuetted) by Dr. C.J. Martin, F.R.S, , (Plate 1) .....ccccescceetegdeienneeden seein at 95

1V
No. B 530.—April 18, 1907.

On Intravascular Coagulation in Albinoes and Pigmented Animals, aad on the
Behaviour of the Nucleo-proteids of Testes in Solution in the Production of
Intravascular Coagulation. By George P. Mudge, A.R.C.Sc. Lond., F.Z.S.,
Lecturer on Biology at London Hospital Medical College (University of
London), and at the London School of Medicine for Women (University of
iondon). Communicated by Dr. A. D. Waller, FIRIS] ie sssees eee eeeeseeeee

The Chemistry of Globulin. By William Sutherland, M.A. Melbourne. Com-
municated by Dr. C. J. Martin, FLRS. .:...2:.) 00+ onsseeeberebenee cee stcen© saee ae eeeeeee

Observations on the Life-history of Adelea Ovata, Aimé Schneider, with a Note on
a New Gregarine from the Gut of Lithobius forficatus. By C. C. Dobell, B.A.,
Scholar of Trinity College, Cambridge. Communicated by J. J. Lister, F.R.S.
GRIAGESIOFATIO:S)' 03.000 <achece oacanoectvesn see oeuedesmouee bees pee eee mean Cees too Ut tee ERE ER EE aEee

The Natural and Induced Resistance of Mice to the Growth of Cancer. By
E. F. Bashford, M.D., J. A. Murray, M.B., and W. Cramer, Ph.D. Com-

municated by Dr. J. Rose Bradford, WR.S. (..:.0ckcccc.sccsssoccscne oes 1:2 sche eee

No. B 531.— May 9, 1907.

On the Combining Properties of the Opsonin of an immune Serum. By Robert
Muir, M.D., Professor of Pathology, University of Glasgow, and W. B. M.
Martin, M.B., Carnegie Research Scholar. Communicated by Dr. C. J. Martin,
10] ENS OAc Go BeR OR OER CEU Rene Se Once Mon on Abr made cinngc yAddondd AshandsaccuSdauaesccUsen cooco0s

A General Consideration of the Subaérial and Fresh-water Algal Flora of Ceylon.
A Contribution to the Study of Tropical Algal Ecology. Part I.—Subaérial
Alge and Algee of the Inland Fresh-waters. By F. E. Fritsch, D.Sc., Ph.D.,
F.L.S., Assistant Professor of Botany, University of London, University College.
Communicated by Professor F. W. Oliver, FURIS.. ....c.nccene.2ss sseecoeesepeeeeeeneeeee

Experiments made to determine the Conditions under which “Specific” Bacteria
derived from Sewage may be present in the Air of Ventilating Pipes, Drains,
Inspection Chambers and Sewers. By Major W. H. Horrocks, R.A.M.C.
Communicated by Colonel David Bruce, C.B., F.R.S., R.A.M.C. ............ceceeeeee

No. B 582.—June 5, 1907.

On the Pathology of the Dropsy produced by Obstruction of the Superior and
Inferior Venz Cave and the Portal Vein.—Preliminary Communication. By
Charles Bolton, M.D., Assistant Physician to University College Hospital.
Communicated by Professor Sidney Martin, FR:S. (.:..s:tcece--sesccessse ase seeeneee ne

The Influence of Increased Barometric Pressure on Man. No. 3.—The Possibility
of Oxygen Bubbles being set free in the Body. By Leonard Hill, F.R.S., and
M. Greenwood, Jun., M.R.C.S., Research Scholar of the British Medical
PASS OCIALIOME Wee aisles siceele suis enotielstetsaeelets cae hes 5 EPA ESAT Roo topnraboriag Sad 20052027

The Inheritance of Flower Colour in Antirrhinum majus. By Miss M. Wheldale,
Assistant Lecturer in Botany and late Bathurst Student, Newnham College,
Cambridge. Communicated by W. Bateson, F.R.S. (Plate 4) .........ceeeeceeee es

PAGE

103

130

155

164

187

284

7

Longitudinal Symmetry in Phanerogamia. By Percy Groom, M.A., D.Sc. F.LS.

Communicated by Dr. D. H. Scott, F.R.S. (Abstract).......... cc cecee ee eeeneeeeees

The Estimation of Chloroform in the Blood of Anesthetised Animals. By
G. A. Buckmaster, Assistant Professor of Physiology, University College,
University of London, and J. A. Gardner, Lecturer on Physiological Chemistry,
University of London. Communicated by Dr. A. D. Waller, F.R.S................

Cyanogenesis in Plants. Part VI.—On Phaseolunatin and the Associated Enzymes
in Flax, Cassava, and the “ Lima Bean.” By Professor Wyndham R. Dunstan,
M.A., LL.D., F.R.S., T. A. Henry, D.Sc. (Lond.), and 8. J. M. Auld, Ph.D.
(Wiirzburg) PE EIA: MOAT ge akc cides Hiachoy ses teceoeehraccnes Leases eee ee

Capillary Electrometer Records of the Electrical Changes during the Natural Beat
of the Frog’s Heart. (Preliminary Communication.) By Francis Gotch, M.A.,
MRCS oo oa os fre drones ae nations o Baw aeie Vav'scttewaane save th adaesrvle rosin ed guciacsdc ent duiedigus

The Fermentation of Glucosides by Bacteria of the Typhoid-coli Group and the
Acquisition of New Fermenting Powers by Bacillus dysenterve and other
Micro-organisms. Preliminary Communication. By F. W. Twort, M.R.C.S.,
L.R.C.P. Communicated by Leonard Hill, M.B., F.RS. ........cccccceceeeseeeseeees

On Reciprocal Innervation of Antagonistic Muscles.—Tenth Note. By C. 8.
RAEI BONO NSC REISS Lat hands. eWiclbaics cs hy mags acideaa¥a evisun begets cabvesa ondgeunesaeuees

No. B 533.—July 8, 1907.

Studies on Enzyme Action. IX.—The Enzymes of Yeast: Amygdalase. By
R. J. Caldwell, B.Sc. (Lond.), Leathersellers’ Company’s Research Fellow,
Chemical Department, City and Guilds of London Institute, and S. L.
Courtauld, B.A. Communicated by Professor Armstrong, F.R.S. ..............008

Studies on Enzyme Action. X.—The Nature of Enzymes. By Henry E.
mrmctrong, K.R.S., and EK. Frankland Armstrong .......2..:sccec0cccscecscsoevesesenes

The Solubility of Air in Fats, and its Relation to Caisson Disease. By H. M.
Vernon, M.D., Fellow of Magdalen College, Oxford. Communicated by Dr. J.8.
feemeti td est MMOD CES ee ee SPM le Sines cciee with ofc MESS Noa slec anc uh east cemececanl eh etenude bette

On Mitosis in Proliferating Epithelium. By J. O. Wakelin Barratt, M.D.,
D.Sc. Lond. Communicated by Professor C. S. Sherrington, F.R.S. ...........2.64

Increase in the Complement-content of Fresh Blood-serum. By J. Henderson-
Smith, M.B., Philip Walker Student of Pathology in the University of .Oxford.
Communicated by Professor Gotch, FURS. ..........cccc0sesccascssscccsccsescoscscceaseas

The Correlation of the Ovarian and Uterine Functions. By E. 8. Carmichael, M.B.,

F.R.C.S.E., and F. H. A. Marshall, M.A., D.Sc. Communicated by Professor
rere SE Let MME Ur ean A cricln noe cos chckec ange oks scieudvaotedepece eed cubive noewaduabeveceusenes

The Ascent of Water in Trees: (Second Paper). By A. J. Ewart, D.Sc. Lond.,
Professor of Botany, University of Melbourne. Communicated by Francis
Darwin, For. Sec. R.S. (Abstract) ........ccccscescsccseceeseeeeeecseeees gan be at eeaten ee

PAGE

309

315

350

360

378

The Réle of the Various Elements in the Development and Regeneration of Bone.
By Sir William Macewen, F.R.S: (Abstract).......:.........0+-ceceessee et ea

An Experimental Enquiry into the Nature of the Substance in Serum which
influences Phagocytosis. (Second Communication.) By George Dean, M.A.,
C.M., M.B., Chief Bacteriologist, Lister Institute. Communicated by Dr.
Cod Martin, WARIS. ..t.c.cecescoicnsessesodeaccensesceoccessenoe seco tet teeta aaa

Croontan LecturE:—On Globulins. By W. B. Hardy, M.A., F.RS. ...............08

No. B 534.—September 6, 1907.

Fatty Degeneration of the Blood. By 8. G. Shattock and L. 8. Dudgeon. Com-
municated by Professor J. Rose Bradford, F.R.S. | .2...\.00...2.4.2-02-0ceee ee eee eee

Some Points in the Development of Ophiothrix fragilis. By E. W. MacBride, D.Sc.,
F.R.S., Strathcona Professor of Zoology in McGill University, Montreal .........

Croontan Lecture :—On the Structural Constituents of the Nucleus, and their
Relation to the Organisation of the Individual. By J. Bretland Farmer, D.Sc.,
Sec sent atacd sued «ped eschoiteecs coasesuens oces ous onQievehiedye Utes inet ents Uae en eae ea

On Certain Phenomena of Inactivation and of Inhibition exhibited by Precipitin
Antisera. By D. A. Welsh, M.A., B.Sc., M.D., Professor of Pathology, and
H. G. Chapman, M.D., B.S., Demonstrator of Physiology in the University of
Sydney. Communicated by Dr. C.J. Martin, PARIS.) ..2....:...0-:s0 see ene ene

Miadesmia membranacea, Bertrand ; a New Paleozoic Lycopod with a Seed-like
Structure. By M. Benson, D.Sc. F.L.S. Communicated by Dr. D. H. Scott,
IRS: CA DSUTACL) . o.cccc.ssesckeocehensse veieseoscsesecinu uatuleeteicals Sone etee ee eE tae

On the Identification of Chitin by its Physical Constants. By Igerna B. J. Sollas,
Fellow of Newnham College, Cambridge. Communicated by Professor W. J.
No(O) | EVCHS | M4] athe lek WR Ran Nl Ser ee RUE NR I 8 a anagbacconon a:

The Inhibitory Action upon Subsequent Phagocytosis, exerted on Active Normal
Serum by Inactive Normal Serum through which Bacilli have been passed.
By J. C. G. Ledingham, M.A., M.B. (Aberdeen), Assistant Bacteriologist,
Lister Institute. Communicated by Dr. C. J. Martin, F.R.S. ..........c:sccceeues

Contributions to our Knowledge of the Poison Plants of Western Australia. By
K. A. Mann, Government Analyst, and W. H. Ince, Ph.D., F.C. Communi-
cated by Professor J. Norman Collie, F.R.S. (Abstract)......0.2...:.ccc.scscuseeceee

Observations on the Life-history of Leucocytes. Part IJ.—On the Origin of the
Granules. By C. E. Walker, Assistant-Director of the Cancer Research
Laboratories, University of Liverpool. Communicated by Professor C. S.
Shermmeton, Ris. WOR late 5) co. ls cccedeesShs 0s seus loceess od seag ee ee

Observations on the Life-history of Leucocytes.—Part III. By C. E. Walker,
Assistant-Director, Cancer Research Laboratories, University of Liverpool.
Communicated by Professor J. B. Farmer, F.R.S. (Plate 6) ..............cecceseees

399

413

440

446

465

473

474

482

485

491

vil

The Time taken in passing the Synapse in the Spinal Cord of the Frog. By
Florence Buchanan, D.Sc., Fellow of University College, London. Communi-
cated by Professor Sherrington, LL.D., F.R.S. (Abstract)...........ccccsceseesenees

A Preliminary Summary of the Results of the Experimental Treatment of
Trypanosomiasis in Rats. By H. G. Plimmer, F.L.S., and J. D. Thomson,
M.B., C.M. Communicated by Sir Ray Lankester, K.C.B., FLR.S. ...............

No. B 535.—November 18, 1907.

The Pressure of Bile Secretion and the Mechanism of Bile Absorption in Obstruction
of the Bile Duct. By Percy T. Herring, M.D., and Sutherland Simpson, M.D.,
D.Sc. Communicated by Professor Schafer, F.R.S. ........cccescccceveecccncsaceacs sae

Further Studies of Gastrotoxic Serum. (Progress Report.) By Charles Bolton,
M.D., Research Scholar of the Grocers’ Company. Communicated by Professor
MeMRIPaLAIALSIAPME LMA Ac CE*ULOU G4): .2e Leases wvedaiesnislccet's eles acca teste see Coens delnden vdareea ener

On the Relation between the Output of Uric Acid and the Rate of Heat Production
in the Body. By E. P. Cathcart, M.D., and J. B. Leathes, M.B. Com-
me eC MON MLC; Se WiartiNs WBS. .iiccicccccsserus conesesecssecnsdsacecavcesseedeacues

Implantation of Actively Proliferating Epithelium. By J. O. Wakelin Barratt,
M.D., D.Sc. Communicated by Professor Sherrington, F.R.S. ...........cceeceeees

The Rate of the Assumption of Chloroform by the Blood during Anesthesia. By
G. A. Buckmaster, Assistant Professor of Physiology, University College,
University of London, and Jj. A. Gardner, Lecturer on Physiological Chemistry,
University of London. Communicated by Dr. A. D. Waller, F.R.S................

Function of the Red Corpuscles in Chloroform Anesthesia. By George A.
Buckmaster, Assistant Professor of Physiology, University College, University
of London, and J. A. Gardner, Lecturer on Physiological Chemistry, University
of London. Communicated by Dr. A. D. Waller, F.R.S. .............cccceeecseeseeees

On the Rate of Elimination of, Chloroform from the Blood after Anesthesia. By
George A. Buckmaster, Assistant Professor of Physiology, University College,
University of London, and J. A. Gardner, Lecturer on Physiological Chemistry,
University of London. Communicated by Dr. A. D. Waller, F.R.S................

OBITUARY NOTICES OF FELLOWS DECEASED.

PAGE

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505

517

533

541

546

555

566

579

ill

SIR JOHN SCOTT BURDON-SANDERSON, Bart. 1828—1905.

SIR JOHN SCOTT BURDON-SANDERSON was connected on both his father’s
and his mother’s side with men of great distinction. His father, Richard
Burdon-Sanderson, of West Jesmond and Otterburn Dene, was the son of
Sir Thomas Burdon, several times Mayor of Newcastle. His grandmother
was a sister of John Scott, famous afterwards as Lord Eldon, and of William
Scott, who became Lord Stowell.

Richard Burdon went to Oxford for his University education, where he
took first-class honours, obtained the Newdigate, and became a Fellow of
Oriel College. He married the only daughter of Sir James Sanderson,
Bart., M.P., who had been Lord Mayor of London; on his marriage he
took the name of Sanderson. There were five children, two sons and three
daughters ; the eldest son, Richard Burdon-Sanderson, died from the effect
of injuries received in the Abbott’s Ripton railway accident of 1876; one of
the daughters married Robert Haldane, of Cloanden, and two of her sons,
the Right Hon. Richard Burdon Haldane, M.P., and Dr. John Scott Haldane,
F.R.S., are well known, the former for his great public services, the latter
for his contributions to science. John Scott Burdon-Sanderson, the fourth
child, was born at North Jesmond, Northumberland, on December 21, 1828;
the old Manor House had become unsafe owing to the undermining of the
adjacent collieries, and the new house at West Jesmond was being erected
for the future residence of the family.

In common with many other distinguished men of that period he was
not sent to school, but was educated at home by tutors under the immediate
supervision of his father. This home at West Jesmond, in the North
Country, with its surrounding of swelling moors and rocky streams, fostered
in the boy that love of nature and appreciation of natural objects which
was to lead him into the biological side of natural science. He retained
to the end of his life the impression of these early years, and always showed
a special delight in wide space, the swell of hills stretching out to meet
the sky, and all that airy freedom which is so indelibly bound up with
moorland scenery. Shooting, fishing, and riding were the natural recreations
of such a boyhood, but probably the greatest joy was experienced in scouring
the stretches of wild country on horseback or on foot, often in the company
of his father’s huntsman, a most intelligent man, who knew every bird that
flew. In later years there were few things which he found so exhilarating
as a tramp over the heather, the enjoyment of which became much enhanced
when he had developed his scientific knowledge. As a boy he delighted
in the observation of wild animals and in the discovery of wild flowers ;
later he became a keen field botanist, experiencing all the satisfaction of
the systematist, who, by naming a wild flower, places it into its proper

b 2

iv Obituary Notices of Fellows deceased.

place in the scale of plant life, experiencing also that peculiar pleasure
which the field botanist knows when he comes upon a rare wild flower
or one growing in unsuspected localities. In such surroundings were slowly
forged the links which were to bind him to the study of living phenomena,
and make him regard both pathological and physiological phenomena from
a biological standpoint. There is little doubt that, with his inclinations,
temperament and powers of observation, he possessed gifts which might have
made him a great naturalist. .

His father may have recognised the significance of these features of his
son’s character, as he undoubtedly recognised his ability, but the fame of the
boy’s great-uncles, Lord Eldon and Lord Stowell, inclined him to believe that
a lecal training would give the lad the best opportunity for their display.

An event, however, occurred which fixed the boy’s own ideas as to his
future. Dr. Mortimer Glover, of Newcastle, came to Otterburn to attend
the elder brother, who was dangerously ill. Dr. Glover was an exceedingly
able man, with a vivid sense of the great possibilities of his profession ;
he was at the time occupied in the experimental investigation of chloroform,
and continued this work in his spare time at Otterburn. There is little
doubt that he recognised young Burdon-Sanderson’s intelligent interest in
biological subjects, and that this friendship suggested to the boy the career
of his life.

When Mr. Burdon-Sanderson discovered that his son’s desire was to enter
the medical profession, he wisely gave up his own views as to his future, and
sent him in 1847 to Edinburgh, where he remained for about four years,
_ taking the degree of M.D. in 1851, and obtaining the Gold Medal. Edinburgh
was at this time the chief medical school in the United Kingdom, and the
teaching staff comprised men of very great distinction; two of these seem
to have exercised a particular influence upon young Burdon-Sanderson,
Goodsir, then Professor of Anatomy, and Hughes Bennett, the Professor
of Physiology; this was especially the case in regard to Bennett, who
turned his thoughts and efforts towards the scientific and experimental
side of medical science. It is somewhat remarkable that his earliest scientific
investigations should have been related to subjects which were to employ
for many years his mature powers.

In the Journal of the Royal Medical Society (a gathering of Edinburgh
students for the discussion of scientific subjects) an entry occurs which states
that, in 1850, a dissertation was read by John Scott Sanderson, of Newcastle,
on “ Vegetable Irritability.” In the Edinburgh Monthly Journal of Medicine
of 1851 there is an extensive criticism by John Scott Sanderson of the
views held as to the metamorphosis of the red blood corpuscles; the
criticism is founded on numerous experimental observations made by
the author upon the blocd of the spleen and other organs; from the title
page of this paper, it seems that he was at this time Vice-President of the
Physiological Society of Edinburgh.

It thus appears that, at the age of twenty-three or twenty-four, he had

Sir John Scott Burdon-Sanderson, Bart. v

grasped some of those fundamental principles which were to be displayed in
all his later scientific work. He recognised the importance of studying
living processes, and the fundamental nature of such characteristics of these
processes as are general in their distribution; for instance, excitability or
irritability ; he recognised also the paramount necessity of experimental
investigation for the determination of a physiological problem. A further
notable characteristic of his mental attitude towards biological and medical
science is displayed by the step which he took when the Edinburgh
curriculum had been completed. He appears to have realised early in
his medical career that, since the study of living processes, whether normal
or abnormal, was a study of their chemical and physical aspects, 1t was
essential to possess an adequate knowledge of the methods used in chemistry.

The completion of medical education by a visit to a Continental medical
school was in vogue at this time, and so Burdon-Sanderson went in the
autumn of 1851 to Paris, with the definite intention of spending a con-
siderable part of his time in working practically at organic chemistry under
Gerhardt. In a little daily journal he dotted down brief indications as to
the main occupations of each day; the diary is somewhat irregular, but
is of much interest, since it affords a glimpse of the nature of his work.

In Paris he placed himself under Gerhardt, but soon began systematic

work of a practical kind in the laboratory of Wurtz. He devoted himself
especially to the study of the organic compounds which are found in animal
tissues, and commenced a research into the constitution of such substances as
could be extracted from muscle, particularly kreatine.
_ In the diary there are brief references to chemical work on a large variety
of compounds, including kreatine, uric acid, urea, hippuric acid, benzoic acid,
leucine, glycin, bile compounds, choleate of soda, stearic acid, elaic acid, oleic
acid, lactic acid, and pulmonic acid, this last having a query annexed to it.

On January 16, 1852, there is a note to the effect that he read a paper at
the Paris Medical Society “which was not understood.” It was a busy
winter; time was found for attending the embryological lectures given by
Coste, for visiting once or twice a week some of the Paris hospitals, and for
witnessing the experimental demonstrations given by Claude Bernard.
Several of those who had been his fellow students at Edinburgh were in
Paris at this time; among these the names of Pavy and Marcet frequently
occur in the little diary. It appears that he joined these two friends in
attending the experimental demonstrations and lectures given by Claude
Bernard, and seems to have immediately fallen under the maguetic spell
cast by the intellectual personality of this great physiologist. His veneration
for Bernard lasted throughout his life, and in later years he used to say,
pointing to the bust which stood upon the shelf above his study table, that
Bernard was the most inspiring teacher, the most profound scientific thinker,
and the most remarkable experimental physiologist that he had ever known.
On March 1, 1852, there is the following interesting entry in the diary:
“Went with Marcet to Bernard’s, who showed us much attention ;” followed

vl Obituary Notices of Fellows deceased.

the next day by, “ Went with Harley and Wason to Bernard, who introduced
us to Majendie!!”; on March 13, 1851, occurs the entry “ Was with Bernard
performing several experiments.” Among other entries scattered along the
diary are the following : “ Performed operation for obtaining pancreatic juice on
a rabbit; did not succeed in introducing the cannula”; “injected a pigeon ” ;
“ operated unsuccessfully on a dog for pancreatic juice ;” “ operated success-
fully on pancreatic duct,” etc. A more elaborate note-book of this date in
his handwriting gives a synopsis of Bernard’s experimental lectures, and
treats some subjects in much detail, notably, the rdle and nature of glycogen
and the phenomena of diabetes. He was in Paris during the exciting period
of the Coup d’ Etat, December 2, 1851; in 1852 he came back to England for
Easter, but returned to Paris for May and June, mainly in order to finish
some of his chemical work. He had now decided to settle in London, this
resolve being largely determined by his approaching marriage with Ghetal
Herschell, the daughter of the Rev. R. H. Herschell, and sister of Farrer
Herschell, who subsequently became Lord Chancellor. In this union, which
took place in 1853, he found, in the highest sense of the word, the helpmeet
for him; all who knew Sir John and Lady Burdon-Sanderson are well aware
of the peculiar strength of the ties which bound them, whilst those who
have enjoyed the privilege of more intimate friendship realise that the great
powers of the husband were fostered by the congenial atmosphere which the
devoted care and loving sympathy of the wife ensured. In the serenity of
true domestic felicity, a happiness which was to last without a check for over
fifty years, he started in London as a practising physician. His scientific
bent inclined him to take some scientific post if it should be offered him,
and, in recognition of his extensive knowledge and undoubted ability, such
offers soon occurred. He was in 1854 appointed Medical Registrar to
St. Mary’s Hospital, whilst a little later he was made Lecturer in Botany,
and finally Lecturer in Medical Jurisprudence at the medical school attached
to this institution. In 1854 he contributed, among other things, a masterly
article on “Vegetable Reproduction” to Todd’s Cyclopedia of Anatomy
and Physiology, thus showing that his botanical interests remained as strong
as ever.

In 1856 there occurred an event which may be considered as the first and
greatest landmark in his scientific career, for it was the event which deter-
mined the specific character of his life’s work ; in this year he was appointed
Medical Officer of Health for the extensive district of Paddington. That
a post of such responsibility should be offered to a man under thirty years of
age, and within three years of his arrival in London, is striking evidence
of the high opinion which had been formed of his powers; these were now
to be successively focussed on those branches of medical science in which he
was to achieve acknowledged eminence. From practical sanitation he rapidly
became an authority upon the scientific aspects of hygiene, and this study
led by a natural extension to wider aspects of pathology, and ultimately back
to physiology.

Sir John Scott Burdon-Sanderson, Bart. vil

He retained his Paddington appointment for eleven years, one notable
incident, the cholera epidemic of 1865-66, occurring towards the end of
his period of office. His rare abilities and scientific acumen were soon
recognised by the late Sir John Simon (at that time Mr. Simon), the Medical
Officer of the Privy Council, and in 1860 he was appointed an Inspector in
the Medical Department of the Council. In the discharge of these duties he
~ sent in many sanitary reports, which were so valued that, although he ceased
to be an acting inspector in 1866, he was frequently asked to undertake
scientific Investigations for the Government authorities, and continued to
contribute to the annual reports of the Privy Council and Local Government
Board for a further period of ten years. His contributions contain the
results of experimental investigations on a number of scientific problems,
which have subsequently become of extreme importance in connection with
the prevention and etiology of infectious and other diseases. Sir John
Simon, in his “ Review of English Sanitary Institutions,” gives some details
which display the scope of Burdon-Sanderson’s early work in practical
sanitation, but a true appreciation of the debt which civilisation owes to
Burdon-Sanderson can only be derived from a perusal of the reports
themselves. It appears that from 1860 to 1864 he co-operated with
others in collecting reliable information as to the utility of vaccination,
and it was largely on the strength of this mass of information that a Bill
to amend the Vaccination Law was laid before the Legislature, and that
the important sanitary statute relating to this matter was passed in 1866.
But the mental attitude of Burdon-Sanderson towards the problems presented
by hygiene was one which impelled him to undertake a more profound
experimental study of morbid processes, the subject being treated along
chemical and physical lines without losing sight of the essential biological
setting. In 1865 he commenced for the Medical Department of the Privy
Council a laborious investigation of the chemical aspects of infective processes,
and in particular of those which were associated with the presence of
contagious diseases in animals. The special portion of this work with which
his name will be always identified, is that devoted to the study of cattle
plague, undertaken towards the close of 1865 for the Royal Commission
appointed to investigate this very important matter. He ascertained that
the true contagium could be separated by physico-chemical means from the
animal tissues which contained it, and that the extracts of these tissues
lost, in consequence of such separation, their infective power. This work
brought the experimental study of contagious diseases in this country into
line with the work of Pasteur upon fermentation and putrefaction. Another
suggestive feature of his work at this time is furnished in the reports which
he produced dealing with the subject of tuberculosis. The researches
described in these reports are models of. exactitude, and the descriptions
of the way in which chronic tuberculous infection invades the organism
is in all essentials that which is still put forth. At this period there was
grave doubt as to the causative factor of tuberculosis, and even the

5

Vill Obituary Notices of Fellows deceased.

possibility that the origin of the infection was bacterial in its nature was
scarcely recognised by pathologists. In 1869 Sanderson’s mental attitude
as to the causative factors of contagious diseases was one which distinctly
foreshadowed the views held at the present day. Alluding to the difficulties
which were brought forward, he states explicitly that, as regards the most
important of them, they disappear “if we suppose that the contagious
process is connected with the unfolding of organic forms.” At that time
stress was laid upon chemical aspects of these pathological processes irre-
spective of their biological significance ; Burdon-Sanderson, however, recog-
nised the profound importance of these hypothetical micro-organisms. Thus,
he states, with regard to such processes, that “it is difficult to see how
the phenomena could be accounted for by their chemical properties, whereas
there is good and scientific ground for anticipating that the solution of the
problem will some day be attained by the investigation of the morphological
phenomena which attend the infective process.” A characteristic expression
of his belief in the experimental method, as the instrument for furthering
our knowledge of the subject, occurs in the sentence, “I am strongly
impressed with the conviction that, if the investigation is to be successful,
it must be experimental in every stage of its progress.” In this, as in
general pathology and physiology, he took up the attitude that the experi-
mental study of the processes, normal and abnormal, indicated the sole
course which could be steered, if the navigator would avoid shipwreck, upon
the numerous rocks and shoals which beset this difficult and little known
region.

The extent to which the value of this pathological work was recognised by
his contemporaries is shown by the appreciative language of eminent
scientific men. Thus, in 1870, Huxley drew attention to it in the remark-
able address entitled “ Biogenesis and Abiogenesis,’ which he delivered as
President of the British Association. In this he pointed out that the report
of Mr. Simon, the Medical Officer to the Privy Council, contained in an
appendix a most valuable essay “On the Intimate Pathology of Contagion,”
by Dr. Burdon-Sanderson, “ which,’ he went on to say, “is one of the
clearest, most comprehensive, and well-reasoned discussions of a great
question which has come under my notice for a long time.”

In 1873 Burdon-Sanderson appears to have been firmly convinced that
living forms were in reality the causal infective agents, and, as regards these
forms, he stoutly opposed those who advocated the theory of spontaneous
generation. At this time he was Professor Superintendent of the Brown
Institute, a post which he held for seven years, from 1871 to 1878. One of
the most suggestive of the many fruitful ideas which abound in this
prolonged inquiry into infectious diseases occurs in this period, when, in
concluding a report upon the nature of anthrax, he brought forward
experimental evidence showing that the passage of anthrax virus derived
from the ox through the bodies of guinea-pigs diminished its virulence for
the bovine species. In concluding a report on this and allied subjects,

Sir John Scott Burdon-Sanderson, Bart. ix

he said: “The question whether this fact, like the analogous one of the
mitigation of human small-pox by transmission, can be directly applied to
a practical purpose, I leave to be determined by future inquiry.” Three
_ years afterwards Pasteur elaborated his system of protective inoculation
against anthrax.

All this time Burdon-Sanderson still retained his hold upon that wide
field of human experience which is offered to every practising physician.
He was Assistant Physician to the Brompton Hospital for Consumption
from 1859 to 1867, and full physician to the same hospital from 1867 to
1871. At the Middlesex Hospital he was Assistant Physician from 1863
to 1870, and he had, in addition, a certain amount of private practice,
although his time for this was necessarily much encroached upon by his
scientific occupations. In 1870, however, he definitely gave up clinical
practice and all his hospital appointments in order to devote more energy
and time to scientific pathology and physiology. The summer of this year
is the second great landmark in his career, for, in this, the forty-second
year of his life, he succeeded Michael Foster as Professor of Practical
Physiology and Histology at University College, London, and, when
Professor Sharpey retired in April, 1874, he was appointed Jodrell
Professor of Human Physiology, the two chairs being for this purpose
merged into one. In order to strengthen the teaching staff, Schafer, who
for several years had been the actual teacher of practical histology, was
made Assistant Professor, and entrusted with the histological part of the
work, to which was subsequently added elementary physiology.

Burdon-Sanderson held the Jodrell Professorship until 1883, when he went
to Oxford as the first Waynflete Professor of Physiology, under a scheme
which had been recommended by the University Commission.

The twelve years of his professorial life in London, from 1870 to 1883,
‘were of great fertility from many points of view. As already indicated,
sis long-continued investigations in subjects connected with public health,
led him to forecast that protective treatment, by means of modified sera,
~which has now become of such great importance; but the duties of a purely
physiological chair naturally concentrated his attention more and more
upon the phenomena exhibited by normal tissues. He regarded these
phenomena as being capable of exact presentation, only in so far as they
could be described in the terminology of physics and chemistry; for this
purpose it was necessary to utilise the precise methods employed in these
‘sciences, and he spent much thought as to the best way of modifying
chemical and physical procedures so as to adapt them for the special
purpose of physiological investigation. Even in connection with his previous
‘clinical work, this point of view had come to the front, as is evidenced by
his careful investigation of the pulse, for which he made certain modifica-
tions in Marey’s sphygmograph, and obtained an extraordinary number of
.sphygmographic records taken in health and in disease. It is also shown by
ithe exact methods he used for determining the amplitude of the respiratory

x Obituary Notices of Fellows deceased.

movements, among these being that of a recording stethometer for measuring
the movements of the chest wall, the instrument being designed by himself
for this purpose.

Probably his earliest strictly physiological inquiry of an experimental
kind was that carried out for the Royal Medico-Chirurgical Society in 1862
and 1863 as to the best methods for the resuscitation of the apparently
drowned ; these investigations led to the adoption in 1864 of the Sylvester
method by the Royal Humane Society. Having started on this purely
physiological investigation, he was led on to others, and worked upon .the
respiratory and circulatory mechanisms of mammals for several years prior
to his being appointed to the Physiological Professorship at University
College. |

In 1867 he was elected a Fellow of the Royal Society, mainly for the
importance of his researches into subjects connected with public health, but.
the value of his physiological investigations was also recognised, for im this.
year he was selected by the Council of the Society to give the Croonian
Lecture, the subject being “The influence exercised by the Movements of
Respiration on the Circulation of the Blood.” In 1872, in conjunction with
Foster, Lauder Brunton and Klein, he published the ‘Handbook of the
Physiological Laboratory, the special parts for which he was responsible
being those devoted to experimental investigations on the circulation and
on respiration.

His conception of physiology, as one prominent aspect of general biology,,
led him gradually into more recondite paths, and he began to devote his
energies to the investigation of certain fundamental or elementary aspects
of living processes. The nature of these aspects is set forth in the
remarkable address which he delivered in 1893 at Nottingham, as President.
of the British Association. The rule of action of all such processes is there
conceived to be “in each instance the interest of the organism as a whole
of which it forms part, and the exciting cause some influence outside of the
excited structure, technically called a stimulus.”

In 1872 he apppears to have commenced a series of experiments into the
conditions which determine that universally distributed phase of living
tissues termed excitability, and, with his characteristic craving for exact
knowledge, he associated with this series the experimental determination of
the precise history of an active process from the point of view of its time
relations. This involved the accurate measurement of the time occupied
by the development, maintenance and subsidence of that excitatory change
or state which a living tissue displays in response to an effectual stimulus.
The time relations of such excitatory responses are undoubtedly largely
dependent upon the character of the active change, and, when this is an
alteration in mechanical form or a development of heat, all methods of
recording are more or less vitiated by the circumstance that the instrument.
employed for recording the change has its own personal time; moreover,
even as regards the actual tissue, all observed changes of this character are

Sir John Scott Burdon-Sanderson, Bart. X1

gross ones, so compounded with incidental effects that it is difficult or
impossible to discriminate the finer details of the fundamental active
alteration.

One type of physico-chemical change, however, appeared to him to
approach more closely than others to that which is assumed to take place in
the actively responding excitable tissue; this type is that alteration of
electrical state which du Bois-Reymond had shown to be recognisable in
nerve and which had long been known to occur in muscles and notably in
the electrical organs of fishes. Acting on this view, Burdon-Sanderson
commenced his well-known researches into the characters of the electro-
motive phenomena displayed by excitable tissues, and the investigations thus
started were destined to occupy him for the remainder of his life, that is for a
period of thirty-five years. They form Burdon-Sanderson’s most noteworthy
contribution to physiological knowledge and illustrate in a striking way the
pertinacity with which he investigated a difficult physiological problem and
the necessity which he felt for utilising in such investigation recording
instruments of the utmost precision. Examples of the latter characteristic
are the time and labour which he devoted to devising suitable modifications
of the repeating rheotome for use with the galvanometer, and to perfecting
the employment for physiological purposes of Lippman’s capillary electro-
meter.

For some time the attention of Darwin had been directed to the
phenomena of insectivorous plants, especially his “beloved Drosera: a
wonderful plant or, rather, a most sagacious animal.” Darwin’s work on the
digestive powers of Drosera brought him into communication with Burdon-
Sanderson, and a letter written in July, 1873, shows that Darwin was struck
with the notion that the excitable cells of the plant might exhibit phenomena
in all essentials similar to those present in excitable animal tissues. Drosera
led to Dionza, which is characterised by the rapid character of the move-
ment of its leaf lobes when certain hairs upon their inner surface are
touched. The exquisite sensibility of the hairs to mechanical displacement
and the extraordinarily rapid character of the mechanical alteration in the
leaf attracted the attention of Burdon-Sanderson, who saw in this plant an
organ admirably fitted for the investigation of fundamental phenomena.

He therefore, about 1875, commenced an investigation as to the changes
which were associated with this active process and the conditions which
influenced their production and character. In the course of this inyuiry he
discovered that there was a pronounced electromotive change in the leaf
every time it passed into the active state. He then devised an ingenious
method for keeping the leaves forcibly open by fixation in plaster of Paris,
and demonstrated that although no mechanical movement could now take
place when an excitable hair was touched, yet each local stimulus of this
kind still evoked an excitatory response, since the same active electromotive:
change was cbserved. This active “electrical response ” was of considerable
magnitude, although of comparatively short duration, and its peculiar interest

X11 Obituary Notices of Fellows deceased.

lay in the circumstance that it was successively developed in all parts of the
leaf lobes, indicating the propagation of some active cellular change from the
seat of the stimulus to more remote regions.

In 1877 an account of this discovery, together with the experimental
determination of the time relations of this electrical response, formed the
subject of the Croonian Lecture of that year, the title of the lecture being
“The Mechanical Effects and the Electrical Disturbance Consequent on
Excitation of the Leaf of Dionwa muscipula.” Five years later, in 1882, a
more extended and detailed account of the phenomena was published in the
‘Philosophical Transactions, entitled “The Electromotive Properties of the
Leaf of Dionza in the Excited and Unexcited States.” From the careful
analysis which he had made of his experimental observations, he concluded
that the excitatory disturbance, “by the mode of its origin, the suddenness of
its incidence and the rapidity of its propagation, is distinguished from every
other phenomenon except the corresponding process in the excitable tissues
of animals.” “In the one case as in the other,” he continues, “we must
regard the electrical change as a visible sign of an unknown molecular
process.” The actual mechanical displacement he agreed with Pfeffer in
considering as probably related to “the diminution of the turgor or water-
charge of the protoplasm of the excitable cells.” The experimental evidence
which he now brought forward appeared to indicate two possible sources of
electromotive change; there was shown to be an initial or primary change,
sudden in its development, brief in its duration, and always of the same
general type; this he regarded as the electrolytic indication of the peculiar
molecular alteration in the plant cells which constitutes the excitatory state ;
it was succeeded by a second change of longer duration and often of different
sign, which was attended by a prolonged residuum or after effect; this he
regarded as associated with diminished turgor and consequent displacement
of water. It was only by accurate methods of recording that he was enabled
to discriminate between the primary and secondary effects and to show that
as regards the primary or initial change the active excitatory state is
fundamentally the same, whether it occurs in these vegetable cells or in
excitable animal tissues.

A third paper upon the electromotive properties of the leaf of Dionza
appeared in the ‘ Philosophical Transactions’ of 1888. This paper included
a number of experimental observations as to the behaviour of what he
termed “modified leaves”; the observations were made in Oxford, and
involved the use of a special double rheotome; the capillary electrometer
was also employed, and photographic records of the excitatory electrical
changes are given in the paper.

The observations show several interesting facts, but the chief points
brought out refer to the so-called “modification,” which is produced in
a leaf when it is subjected to the flow of even a weak voltaic current. The
modification reveals itself as a permanent alteration in the amount, and even
the sign of the electrical state of the inactive leaf-surface. It is localised to

Sir John Scott Burdon-Sanderson, Bart. Xl

the part which the modifying current has traversed, and is associated with a
remarkable diminution in the high electrical resistance of the tissue, this
diminution being strictly confined to the modified region. Finally, the
records give demonstrative proof that, when any such modified area is thrown
into the active state, either by direct stimulation, or by the arrival of a
propagated excitatory wave aroused elsewhere, then the electrical response
in this area, whilst it has the same time relations, may be entirely changed
as regards its sign. This extraordinary reversal is related to the sign of the
inactive tissue, and, when through a modifying agency this is altered, then
the sign of the active change may be similarly reversed.

From the constancy of this relationship, Burdon-Sanderson inferred “ that
the constantly operative electromotive forces, which find their expression ‘in
the persistent difference of potential between the opposite (leaf) surfaces,
and those more transitory ones, which are called into existence by stimula-
tion, have the same seat, the opposition between them being in accordance
with the general principle that, whereas the property which renders a.
structure capable of undergoing the excitatory change is expressed by
relative positwity, the condition of discharge is expressed by relative
negatiwity.”

Between the first and second papers on Dionea he had, whilst at University
College, London, pursued the same method of inquiry in the cardiac muscular
tissue of the frog. Huis investigations upon this subject are set forth ina
paper published in 1880 in the ‘Journal of Physiology.’ These researches
have been taken as a model for most subsequent work of this character,
whatever the tissue which has been utilised for such electro-physiological
investigations. The paper is entitled “The Time-relations of the Excitatory
Process in the Ventricle of the Heart of the Frog,’ and was followed three
years later by one “On the Electrical Phenomena of the Excitatory Process
in the Heart of the Frog and of the Tortoise, as Investigated Photographically.”
In both these investigations he had the assistance of Mr. F. J. M. Page. The
whole research has been long regarded as classical, through the exactitude of
the methods of observation, the rigorous determination of the influence of
various surroundings or accessory factors, and the lucid interpretation of the
phenomena. It conclusively demonstrated that the diphasic effect (or change
of electrical sign) which is observed when two contacts are placed on the
uninjured surface of the frog’s heart, is the algebraic sum of two monophasic
electromotive changes each of similar sign but developed successively one
under each of these two contacts. Hence it is not an indication of the
occurrence of opposite types of tissue change, but is simply the expression of
a similar type of monophasic change occurring at different times in different
localities. Finally, he demonstrated that the reason why the change begins
under one contact before it occurs under another one, is solely because
the excitatory state, like the muscular contraction, is propagated in the
differentiated cells which compose the cardiac tissue. For these researches he
was, in 1883, awarded a Royal Medal.

XIV Obituary Notices of Fellows deceased.

After his appointment to the Oxford Chair in 1882, Burdon-Sanderson
had to brave the turmoil of an opposition in which an antivivisectionist
outcry was utilised. This opposition was directed successively against a
proposed expenditure of £1,500 for apparatus in 1882, and of £10,000 in
1883 for erecting an appropriate physiological laboratory. In 1884 it
culminated in an attempt to throw out the decree authorising a maintenance
grant of £500 a year for the incidental expenses of the newly erected
Physiological Department. The details of the scenes which were associated
with the debates upon the subject in Convocation are now almost forgotten,
and it is unprofitable to revive them. It will be sufficient in this memoir to
say that, in spite of the virulent character of the feeling displayed by his
opponents, it was generally felt that Burdon-Sanderson came through the
storm with, if anything, an added dignity, both opponents and supporters
being impressed by his self-control under circumstances which might have
justly provoked his resentment. One who knew him well at this time,
Sir William Church, wrote that he would “ never forget his striking face and
figure as he stood quietly leaning against the wall of the Sheldonian Theatre
during the momentous debate in Convocation on February 5, 1884.”

The establishment of Burdon-Sanderson in Oxford, with the erection of the
Physiological Laboratory, and thus the foundation of that scientific study
which used to be called the Institutes of Medicine, may be regarded as the
third landmark in his career. It is true that it was to Sir Henry Acland that
Oxford owes the idea of resuming its part in medical education, but with
Sanderson’s arrival the creation of the Oxford Medical School passed from a
fruitful idea into reality, and it is now generally recognised that he achieved
the end for which he left London by establishing on an enduring, because a
scientific, foundation, the Oxford Medical School. He thus enjoyed the
rare felicity of living to see the purpose which he had so much at heart, in
all essentials, attained. An excellent Department of Human Anatomy was
erected during his Physiological Professorship, and when, in 1895, he resigned
the Physiological Chair, on his being nominated Regius Professor of Medicine,
he devoted his energies towards the efficient equipment of pathology. The
scientific progress of this subject had always been one of his great aims, and
his satisfaction was unbounded when, through the stimulus of a generous
donor, the University acknowledged its importance and erected an adequate
laboratory. He gave not only time and thought but material help towards
the task of making this new University department an efficient one, and left
in his will the sum of £2,000 in the hands of trustees “ for the support of the
Laboratory of the Pathological Department of the University of Oxford and
specially to provide for the expenses of research in pathology conducted in
the said laboratory or elsewhere.”

The most extensive of the researches which he carried out at Oxford were
upon muscle. In order to obtain exact records of the electromotive changes
during muscular activity, he devoted great attention to the working out of an
adequate method. Inspired by his enthusiasm, Burch took in hand the

Sir John Scott Burdon-Sanderson, Bart. XV

capillary electrometer, greatly improving its construction and developing
a method by means of which the graphic records of its well-known displace-
ment could be interpreted with accuracy. It thus became possible to
construct, from the actual photographic records, other derived curves which
should faithfully display the variations in the potential difference of a
tissue even when these variations were of very brief duration.

The whole method was elaborated by Burdon-Sanderson with scrupulous
attention to accuracy of detail; he left it the most exact method of its kind
employed in this or any other country, and the utility of the technique has
now received ample acknowledgment from all those who are engaged in
investigations upon the electromotive phenomena displayed by excitable
tissues. Others, inspired by his example and encouraged by his help, have
applied the method with success for the elucidation of the electromotive
changes occurring in nervous and other tissues, including those which are
found in the electrical organs of fishes. As regards the last-named struc-
tures, Burdon-Sanderson himself visited Arcachon in 1886, St. Andrew’s in
1887 and Plymouth in 1888, in order to investigate the electrical organs of
fishes, and published, in conjunction with F. Gotch, two papers upon the
electrical organ of the Skate in the ‘Journal of Physiology.’

The results of the investigations upon muscle are given in the papers
which he published in the ‘Journal of Physiology, and a comprehensive
account of the work and of his conceptions as to the excitatory process
in this structure, is set forth in the third of his Croonian lectures.

This lecture, delivered in 1899, was entitled “ The Relation of Motion in
Animals and Plants to the Electrical Phenomena associated with it.” It
deals first with the technique by means of which he endeavoured to obtain
precise information and then sets forth the main results obtained by its
employment in Dionea and in striped muscle. The electrical response is
described in various forms of muscular contraction such as twitch, tetanus
and reflex activity, also in the peculiar condition produced by veratria. In
summarising the indications which the phenomena seemed to him to afford,
he stated that “in striated muscle the primary effect of every excitation is
a process of oxidation having its seat at the excited part. It may be
surmised that this consists of two stages, namely, liberation of previously
intramolecular oxygen, and actual oxidation.” He further concludes that
“the monophasic variation may be taken as the type from which all other
forms of response to stimulation may be derived, either by repetition, pro-
longation or interference.”

An interesting point for electro-physiologists is associated with the con-
cluding sentence, which affirms that he believes the “muscle current” to be
“the manifestation of processes which have their seat at the surface of
contact between electrode and living muscle”; he thus definitely adopted
the view which is termed “ pre-existent” as opposed to the “alteration ”
conceptions of Hermann. The work upon reflex muscular activity was
continued almost up to the time of his death; a brief note relating to

XV1 Obituary Notices of Fellows deceased.

the phenomena of strychnia tetanus in the ‘ Proceedings of the Physiological
Society’ of 1902, is, however, the only publication on the subject, although
a large mass of information had been obtained.

It is only possible within the limits of this notice to allude quite briefly to
the many suggestive articles and addresses which are scattered along the
years of his scientific activity. They are chiefly related to the consideration
of pathological and physiological subjects; notable ones are the Addresses
delivered as Sectional President at the British Association in 1872, 1881, and
1889, the Address on the Cellular Pathology of To-day, delivered to the
Thirteenth International Medical Congress at Paris in 1900, and the masterly
Address, delivered in 1893 at Nottingham, as President of the British
Association. Among his pathological articles, probably the most conspicuous
is that on ‘“‘ Inflammation,” in Holmes’ ‘System of Surgery, which, although
written early in the seventies, is still accepted as the classical utterance upon
this subject. An extensive article of a physiological character was written in
1900 for the ‘Textbook of Physiology, on “The Mechanical, Thermal and
Electrical Properties of Striped Muscle.” }

Viewing his scientific work as a whole, the most remarkable feature which
it presents is the breadth of its scope: sanitary science, practical hygiene,
medicine, pathology, bacteriology, botany and physiology have all been
enriched by his activity. If one of these branches can be selected as having
been more particularly illuminated by his genius, the choice must fall on
pathology. In this his influence was unique; he was ahead of all his contem-
poraries in this country, both in conception and in method, and by leading
his successors into the true path-—that of experimental investigation—he
endowed English pathology with purpose, and gave this subject, as regards
this country, its proper scientific status. His influence upon physiology,
although profound, was not of such an epoch-making character; in one
respect, however, it has been most marked, and that is in connection with
the methods now adopted by all physiological teachers. It has been
suggested that Burdon-Sanderson was great as an original investigator rather
than as a teacher, but this suggestion is far from being borne out by fact.
A great teacher is to be judged by the ability and subsequent eminence of
his students and by the radical alterations which he has been able to initiate
in teaching methods; from both these points of view Burdon-Sanderson
achieved greatness. His lectures were extraordinarily suggestive, whilst his
personality and grace of expression held his audience. But a more solid
tribute to his teaching capacity is the change in teaching which he was the
first to effect, and which consisted in the introduction into the students’
course of practical work and thus of the experimental method.

As Jodrell Professor in University College, London, he organised classes for
the practical study of physiological chemistry and of the phenomena of muscle
and nerve. The printed syllabus of the work to be done formed an appendix
to the published synopsis of his physiological lectures; it was the first of its
kind, and inaugurated a system of practical instruction which now forms
part of the routine work in every University existing in this country. For

Sir John Scott Burdon-Sanderson, Bart. -Xvil

many years the methods employed by him for the teaching of students were,
in this respect, in advance of those which existed upon the Continent.

The stress which he laid upon this part of physiological training was the
logical outcome of his firm belief in the application of experimental methods
to medical science. Quite apart from the obvious advantages which such
practical teaching offers through its facilitating the acquisition of certain
facts, work of this character by students appeared to him of the utmost value
in engendering a mental attitude of primary importance. He believed that
it cultivated those powers of observation, experimental inquiry and reflection,
in regard to living phenomena, which have subsequently to be brought to bear
on the human subject by all those who undertake the responsible duties of
clinical practice. Throughout the whole of his life he held that, since medical
science remained inexact and emipirical unless it rested upon the sure
foundation of physiological and pathological knowledge, and since this
knowledge could itself be advanced only by experimental methods, the highest
interests of society were bound up with the practice of such methods.
Whether or not infectious diseases are a product of civilisation, they are
certainly fostered by man’s social habits and communicated by him to animals,
and the responsibility lies with man to utilise to the utmost his mental
powers for their removal or delimitation, This responsibility was appre-
ciated in its fullest extent by Burdon-Sanderson, and all his work and
teaching was tinged with his endeavour to faithfully discharge the trust
which his position and mental powers involved. He had in consequence an
earnestness of purpose which was felt by all those who came under his
influence. The words in which he set forth, in 1896, the greatness of
Ludwig as a teacher, are applicable to himself: men, many of whom were
already nearly of his own age, rallied round him, “attracted in the first
instance by his discoveries, they were held by the force of his character, and
became permanently associated with him in his work as his loyal friends and
followers, in the highest sense his scholars.” The large number of his
students who now hold official appointments in connection with medicine,
surgery, hygiene, pathology, and physiology, will acknowledge the truth which
the foregoing sentence expresses.

The present writer is one of those who, as a student, came, in 1874, under
the sway of his inspiring personality, and on reflecting as to the various
factors which gave this sway its extraordinarily potent character, it would
appear that the following features may be specially distinguished. His
commanding appearance and striking individuality ; the intellectual beauty
of his face, which is so well displayed in the print copied from a photograph taken
by Miss Acland, at Oxford; the peculiar charm of his manner and diction;
the sympathetic interest with which he followed the work and thought of
others, young or old; above all, that earnestness of purpose to which allusion
has been already made. Like Ludwig, he strove “to unite his life-work as
an investigator with the highest kind of teaching.” This life-work, extending
for over fifty years, coincided with what is generally regarded as the most

VOL, LXXIX.—B. ¢

XVill Obituary Notices of Fellows deceased.

eventful period in science; it is undoubted that as regards biology,
physiology, and pathology, the latter half of the nineteenth century has been
creative. The propitious moment in these provinces of science arrived about
the year 1850, and with the birth of the new movement came the men fitted
by their endowments to accelerate its progress. The part which Burdon-
Sanderson took in directing the course of this movement as regards pathology
and physiology, stamps him as one of those masters who mould the thought
of a generation.

Various distinctions, unsought by him but valued as indicating public
appreciation of work done for science, were bestowed upon him. He was
selected on five different occasions to serve on the Council of the Society, was
awarded a Royal Medal, was appointed Croonian Lecturer on three separate
occasions, was the recipient of many honorary degrees, was made a member
of the chief Foreign Scientific Academies, and in 1899 was created a baronet.
His appreciation of these distinctions caused no change in those traits which
endeared him to the large circle of his friends, for he always retained that
charming simplicity of character and of life which, when associated with
great intellectual gifts, increases the affectionate and reverent regard of others.

He was a man of wide sympathies, intellectual and artistic, with an
appreciative taste in art and music which was only realised by his more
intimate friends.

The breadth of his views and the commanding position which he held in
the scientific world augmented the regard in which he was held by the
University of Oxford and by the particular society, Magdalen College, with
which he was associated for twenty-three years, first as Professorial and later
as Honorary Fellow. The peculiar charm of his individuality and character
secured the devoted attachment of a wider circle. There are many to whom
he was that “true friend to whom you may impart griefs, joys, fears, hopes,
suspicions, counsels, and whatsoever lieth upon the heart to oppress it,”
many to whom the memory of his intimate friendship will remain as one of
the abiding joys of life.

During his tenure of the Regius Professorship of Medicine at Oxford, the
condition of his health was several times such as to cause his friends much
uneasiness, and particularly when, at the close of the year 1903, he felt
compelled to resign the Chair. It was a source of unbounded satisfaction to
him to realise that, by the appointment of Dr. Osler as his successor, the
continued development of medical study in Oxford was assured, and that the
task set before him when he left London in 1882 was practically accom-
plished. For him, as for many others who have spent their lives in the
service of science, the words of Bacon form an appropriate epitaph—“ But,
believe it, the sweetest Canticle is Nunc dimittis, when a man hath obtained
worthy ends and expectations.” EG:

(The photograph which is reproduced in the frontispiece to this account
was taken by Miss Acland, at Oxford, in the year 1894, who kindly lent the
negative for the use of the collotypers.)

XIX

HENRY BENEDICT MEDLICOTT, 1829—1905.

HALF a century ago, our knowledge of the Geology of India was very small
Some traverses of the Himalayas had been made, and notes had been
published of portions of the Peninsula of India; fossils had been recorded
and described from Spiti, the Salt Range, Sind, Cutch, Trichinopoly, and a
few other localities; coal had been found and to some extent worked, but
very little was really known of the different formations or of their relations
to each other. A comparison of Dr. Carter’s ‘Summary of the Geology
of India between the Ganges, the Indus, and Cape Comorin, published
in 1853, or of Greenough’s geological map (1854), with the Manual of the
Geology of India issued in 1879, and with the map accompanying it, will
show the great difference between the knowledge of Indian Geology at the
two periods.

Amongst the men who originated the Geological Survey of India and by
whom the great improvement in our acquaintance with the geology of the
country was secured, a leading position was taken by Henry Medlicott.
More of the numerous errors which had become almost stereotyped amongst
Indian geologists were corrected by him than by any other surveyor. To
take one example only. In the Peninsula of India there are, in the
classification adopted in the Manual, the following groups of formations below
the great Deccan basaltic series (Upper Cretaceous and Lower Tertiary) :—
(1) Metamorphic or Gneissic; (2) Transition; (3) Vindhyan; (4) Gond-
wana; the (5) Marine Jurassic and (6) Cretaceous beds being confined with
one unimportant exception to coastal fringes. The Vindhyan and all below
it are now believed to be pre-Cambrian ; the Gondwana ranges from Upper
Paleozoic to Lower Cretaceous. Now in the 1854 ‘Summary of the
Geology, all the Vindhyan and Gondwana, various portions of the Transi-
tion Rocks and most of the known limestones from Metamorphic to
Cretaceous were assigned to the “ Oolitic Series.” In his first season’s work
with his brother, J. G. Medlicott, Henry Medlicott distinguished the pre-
Cambrian Vindhyan from the Gondwana, thus at once clearing the way for
further progress. In his first published Memoir he arranged in sequence
the Vindhyan, Sub-Kymore or Lower Vindhyan and Bijawar or Transition
of Bundelkhand, and distinguished all from the Gneissic Series. In_ his
next Memoir he classified the rocks of the Himalayas, both the Siwaliks
and Nahuns, resting on the Nummulitic Subathu group, and the ancient
hill rocks, Krol and Blaini, associated with the older formations of the
mountains.

Henry Benedict Medlicott was born in Loughrea, County Galway, Ireland,
on August 3, 1829. He was the second of three sons of the Rev. Samuel

c 2

XX Obituary Notices of Fellows deceased.

Medlicott, Rector of Loughrea, and of Charlotte, daughter of Colonel
H. B. Dolphin, C.B.; his elder brother, Joseph G. Med fens was also for
several years on the Staff of the Geological Survey of India.

Henry Medlicott was educated in France, Guernsey, and Dublin, where
he graduated in Trinity College and took the degree of B.A. in 1850, with
ialenan and honours in the School of Civil Engineering. He took the M.A.
degree in 1870. He was thus from his early education an excellent French
Scholar, and his acquaintance with the classical works of the great French
geologists may be often traced in his writings.

In 1851 he joined the Geological Survey in Ireland, and in 1853 he was
transferred to the English Survey where, for a time, he worked with
My. Aveline in Wiltshire. He was next engaged by Dr. Oldham and joined
the Indian Geological Survey, then occupied in the Rajmahal Hills at
or near Bhagalpur on March 24,1854. His brother, J. G. Medlicott, had
joined the survey a year or two earlier. Before, however, Henry Medlicott
had commenced field work, he was appointed, in August, 1854, by the Court
of Directors of the East India Company, Professor of Geology in Rurki
College, and he remained in this post till October, 1862, when on some
additions being made to the staff of the Geological Survey, he rejoined it as
Deputy Superintendent for Bengal.

During his tenure of the Rurki Professorship, however, he did most
important work in field geology. By an arrangement with Dr. Oldham he
was allowed to occupy the field season in surveying, and thus he was enabled
to examine parts of the Nerbudda Valley and Bundelkhand in 1854—55 and
in 1856—57, whilst in the other years he was engaged in working out
the geology of the Lower Himalayas between the Ganges and the Ravi
(Hardwar to Dalhousie), and of the Siwalik Beds and their associates at the
base of the mountains, In 1857 he served as a Volunteer with the garrison
of Rurki against the mutineers of the Bengal Army, and on the close of the
Campaign was awarded the Indian Mutiny Medal for Special Service. An
instance of the service rendered may be added here.

Rurki during the outburst of the Sepoy mutiny was isolated, like most of
the small garrisons in the Upper Provinces, where the natives of the villages
were associated with the rebels. No European could possibly traverse the
country in safety. News was received at Rurki one day of a Christian
family who were held by the villagers as prisoners at a few miles distance,
and who were inimminent danger of being killed. It was arranged that Henry
Medlicott and one European companion should go out with an escort of
Sowars to endeavour to rescue these unfortunate people, but on the day before
the attempt the Sowars showed signs of mutiny and were disarmed.
Medlicott and his companion, whose name appears now impossible to trace,
left in the morning, and by sheer pluck, at the risk of their lives, succeeded
in bringing back the imprisoned people in safety, the villagers apparently
being so astonished at two Europeans coming among them that they
surrendered their prisoners. Only the outline of the story can be now

Henry Benedict Medlicott. XX

recovered ; it was told to the present writer in 1866 by Colonel Baird Smith,
who commanded the Rurki Garrison in 1857. |

On October 27, also in 1857, Mr. Medlicott was married at Landour to
- Louisa, daughter of the Rev. D. H. Mannsell. He has had three sons and
three daughters; only one son and one daughter, together with his wife,
survive him. The surviving son is a Civil Engineer in the Pubic Works
Department of the Indian Government; the two who have been lost were
one in the Civil Service of India and one in the Royal Engineers.

After rejoining the staff of the Geological Survey in 1862, Mr. Medlicott
was engaged for many years, first in one part of the country then in another,
generally by himself, though occasionally in charge of a party, in enquiring
into economical problems or in investigating geological questions, and not
unfrequently in clearing up difficulties that had proved too serious for his
predecessors. In 1862—63 he was in South Rewah, in 1863—64 in Behar, in
1864—65 he traversed Assam, from the Khasi Hills to the farthest eastern
extremity of the province, in 1865—66 he was engaged in Central India and
Rajputana. In other years he examined various tracts of Northern India
from the Jamu Hills of Kashmir, the Satpura ranges of the Central Provinces
and Chattisgarh to the Garo Hills in Assam. Twice he officiated as Super-
intendent of the Survey during Dr. Oldham’s absence on leave, once in 1870
for a short period, and in 1873—-74 for 17 months.

On April 1, 1876, Mr. Medlicott sueceeded Dr. Oldham as head of the
Department, and from that time the duties of his office kept him mainly in
Calcutta. The title of Superintendent was altered to that of Director in 1885,
but the change was confined to the title.

Dr. Oldham had been in weak health for some time before he retired, and he
naturally left many arrears for his successor to carry out. Foremost amongst
these was the preparation of a general account of Indian geology. This had long
been required ; a mass of scattered papers and reports, not a few of which were
in MS., represented about a quarter of a century’s progress with the systematic
examination of Indian geology, and before those who had helped from the
beginning had left India for good, it was essential that they should prepare
for their successors a record of work in which they had taken part. Moreover,
until a general description of the country, as a whole, was compiled, it was
impossible for any one either in India or in Europe, without an enormous
amount of labour, to gain a correct idea of its geological features. Henry
Medlicott at once set to work at this general account, and dividing the
chapters between Mr. Blanford and himself, brought it to a conclusion in
1879 and published the ‘Manual of the Geology of India,’ with a map, in
that year. It is unnecessary to poirt out the great importance of this book,
nor the assistance it has afforded, not merely to Indian yeologists but to those
throughout the world.

This, however, is only one of the subjects to which the new Superintendent of
the Survey devoted his attention. Of the effect of his superintendence on the
work of the staff a fair example may be furnished by the survey publications.

pean Obituary Notices of Fellows deceased.

From 1877 the records which, as describing recent work, are the best illustra-
tions of survey progress, doubled in size, and they certainly did not fall off in
the importance of the contributions printed, either economical or scientific.
Besides these Mr. Medlicott edited, during his tenure of office, about
10 volumes of the Memoirs and numerous parts of the Paleontologia Indica,
comprising Dr. Feistmantel’s descriptions of Gondwana plant fossils, most of
Mr. Lydekker’s accounts of the Siwalik and other vertebrata, and the largest
portion of Dr. Waagen’s great work on the Salt Range fossils, and of the
descriptions of Tertiary Corals and Echinoderms from Western India by
Dr. Duncan and Mr. Percy Sladen. There was the same thorough progress
in other directions: for instance, in the arrangement of the Museum and of the
Survey Office.

His work absorbed him entirely; living almost the life of a hermit, and
confining himself to his office, he devoted all his time to the Survey. He
was a man of a retiring disposition originally, not in the least from want of
energy or courage, but because the social pursuits of men in general did not
attract him; he had, in fact, something of the ascetic temperament. During
his life at the Museum, he not merely lived alone on the poorest of fare, but
he scarcely quitted the building. In the evening he took exercise on the
roof. He had retained some early habits, such as walking bare foot, and long
after he left Calcutta, a path along the flat Museum roof, worn by his naked
feet, could still be traced. Asa young man he had been strong and active,
and amongst other traits had acquired the power, not often possessed by
Europeans, of sitting like a native of the East on his heels. His method of
living he rarely cared to explain, though as a fact, whilst his own living
expenses were reduced to a minimum, far below the usual minimum of Anglo-
Indians, he was providing for several relatives and not merely for his own
family. The death of his brother, too, had added to a considerable extent to
his responsibilities.

After the completion of ti doth year, in 1884, Mr. Medlicott was retained
by the Government of India in his office until 1887. He retired on April 27
in that year, having served almost continuously for rather more than 33 years.
He was only on leave for six months in 1865, less than six months in 1871, and
again six months in 1884, or 18 months during the whole period of service.

Since his retirement he lived very quietly at Clifton, near Bristol, and
had taken but little part in scientific discussion, though always keenly
interested in the progress of the Indian Survey. His time, as long as he
remained in good health, was mainly devoted to philosophical problems,
to which he had always been devotedly attached. He published two short
pamphlets on “ Agnosticism and Faith,” in 1888, and on “The Evolution of
Mind in Man,” in 1892, and was engaged ona lareer work which has not been
finished. A few years since, a strain caused by bicycling, when, with character-
istic energy and determination, he had resolved that he would not be beaten by
a particular hill, led to serious heart symptoms, and, although for a time a
partial recovery was made, a relapse early in 1904 reduced him greatly.

Henry Benedict Medlicott. XX

The end came quietly on April 6, 1905, when he was seated in his
study. :

Henry Medlicott joined the Geological Society of London in 1856, and
received the Wollaston Medal in 1888, on his retirement from the Indian
Service. He was elected a Fellow of the Royal Society in 1877. He was a
Fellow of Calcutta University, and from 1879 to 1881 he was President of
the Asiatic Society of Bengal. He was also an Honorary Fellow of several
Foreign Scientific Societies, but he never made use of his titles, which he
regarded as simply “ official.” It may be remarked that he carefully omits the
letters F.R.S. after his name in all his survey publications, and even in the
“ Manual” he struck out the initials after his own name, whilst leaving them
after his colleague’s. Throughout his career, in fact, he appears almost
unnecessarily to have disregarded the honours to which he was entitled for
brilliant original work as well as good official service.

His policy as head of the Survey was to assist his colleagues in every way
to publish the account of their observations in their own words. Occasionally
the result was that their views drew upcn the authors replies froin other
officers who took different views, or from geologists who had another opinion
on the subject. This was especially the case in paleontological questions, on
which he never pretended to decide difficult points. The policy adopted is
perhaps not quite in accordance with official usage, but there can be no doubt
that the result of Mr. Medlicott’s principle was to bring out the facts in
a discussion, and not infrequently in a difficulty of high scientific and
economical importance, as in the various disputes over the Gondwana flora.
And not only did he obtain the help of the staff of the Survey in forwarding
the progress of Indian geology, but he succeeded in securing the valuable
assistance of the late General McMahon and others, who contributed obser-
vations on subjects of high interest in the geological history of the Indian
Empire. That he contributed no lengthy memoirs of his own, was simply due
to the fact that he regarded with horror any attempts to gain credit by others’
work. Short notes and an annual report were for many years his principal
contributions to the Survey publications. He was not a fluent writer, though
he could write strongly and earnestly, never so strong as when he was dealing
with attempts at injustice or plausible but misleading statements. He was
absolutely fearless, and cared but little of whose pet theory he was disposing,
if the theory was in his opinion worthless.

It is difficult to appreciate Henrv Medlicott’s work in India without some
acquaintance with the knowledge of the geology available when he entered
the service. So great a change has taken place in the half century that. has
elapsed since 1854, that it is scarcely possible to reconstruct the conditions
under which geological surveying was carried out 50 years ago. There were
no railroads and very few roads. Travelling was difficult and very slow, the
early surveyors had often to make their own maps in the wilder parts of the
country. Some idea of the Geology known in the days before the Survey may
be gained from Dr. H. J. Carter’s ‘Summary,’ which appeared in the same

XXIV Obituary. Notices of Fellows deceased.

year that Mr. Medlicott landed in India. In this remarkable work lime-
stones from every formation in India, from metamorphic to the Bagh and
Intertrappean beds, are included in the “Oolitic Series.” An equally
remarkable confusion is shown upon Greenough’s Geological Map of India.

Nothing had been done to clear up the nebulous condition of Indian
Geology before 1854. Henry Medlicott’s first season in India was signalized
by the earliest and most important step in the classification of the
Peninsular rocks, when he and his brother separated the Vindhyans north of
the Nerbudda Valley from the Gondwanas tothe south. The name Vindhyan
was given by Dr. Oldham, who, however, when announcing the discovery,*
stated that the separation had been made by the Medlicotts before he visited
the country. In the first memoir he wrote, in Vol. 2 of the Memoirs, Henry
Medlicott reduced the whole mass of Vindhyan, Infra-Vindhyan, and Bijawar
rocks that extend throughout Bundelkhand to a sequence which has received
no great subsequent alteration, the principal change, perhaps, being in the
substitution of the term Lower Vindhyans for Sub-Khymores. This masterly
paper, though published in a most imperfect condition, ill-arranged, and not
very clearly written, was not only the beginning of our accurate knowledge of
the Vindhyan and Infra-Vindhyan rocks, but laid a firm foundation on which
much has since been built. From his earliest work to his latest, these
wonderful azoic rocks of India were Mr. Medlicott’s especial favourites,
perhaps because all the knowledge of them was derived from purely physical
observations, and not interfered with by organic remains, which in the later
Gondwanas have not always proved an accurate indication of the age of the
beds or their relations to each other. Throughout his writings in India, and
throughout the Annual Reports issued during his Directorship, point after
point is brought up tending to the correlation of these formations in various
parts of India, and showing the relations between Karnul and Cuddapah,
Kaladghi, Gwalior, and many other similar rocks on the one hand, and the
Vindhyan and Bijawar on the other.

His next great work was the arranging of the rocks of the Lower
Himalayas in the Simla and neighbouring area and of the Siwalik rocks and
their associates at the base of the mountains, and the sketching of the
geological history of the Himalaya range on a definite geological basis. The
first has been one of the most difficult questions in Indian geology, and
although the work was commenced in 1855, it cannot be regarded even
now as nearly solved. The Tertiary Himalayan Beds have, however, been
regarded as fairly classified since Mr. Medlicott’s Memoir was published
in 1864. The important observations made in this Memoir are essentially
physical. The demonstration that -Himalayan elevation is shown by the
relations of the Lower Nummulitics and the older hill rocks not to have
begun before Tertiary times, and the beautiful illustrations proving the
permanence of the great river valleys by their coincidence with the

* Journ. Asiat. Soc. Bengal,’ 1856, vol. 25, p. 250., and more clearly in ‘Mem. Geol.
Surv. Ind.,’ vol. 2, p. 304.

Henry Benedict Medlicott. SESE

maximum of conglomerates in the Siwaliks, are amongst the observations
that connect our first clear ideas of Himalayan elevation with Henry
Medlicott’ s work.

It has been already noticed that not unfrequently Henry Medlicott was
occupied in clearing up difficulties that had been too great for his pre-
decessors in the Survey. An illustration may be taken en the geology of
the Khasi Hills. A comparison of his observations as recorded in the
seventh volume of the Memoirs should be made with the earlier account of
the same area by Dr. Oldham in the first volume. In the first account,
amongst other differences, the bedded Sylhet traps (Mesozoic) are not
distinguished from the ancient greenstone rocks of the inner hills, and
the occurrence of fossiliferous Cretaceous rocks was not recognized ; their
fossils, then supposed to have been Nummulitic, having been lost. The
alterations made in the geology by Henry Medlicott may be seen by
comparing the map and section, Plate 8, in Vol. 1, with the map and
section at p. 154 in Vol. 7. The earlier observations, it is true, were made
in the monsoon, when of course the ground was not as well exposed. Again,
the sketch map of the Satpura or Nerbudda coal area as corrected by
him in Vol. 10 of the Memoirs may be compared with the earlier
map published in Vol. 2. It is quite true that the principal discrepancy
in this case is easily explained. J. G. Medlicott, who not merely geo-
logically examined the country but who also to a considerable extent
surveyed the map, had practically completed the western portion containing
the typical Mahadevas without separating them from the underlying
Damudas. In the early part of 1856, Dr. T. Oldham, the head of the
Survey, went over the field, and almost at the end of the season discovered
that the Mahadeva was a separate series. He desired that this separation
should be recorded on the map. The result is, what may be supposed under
the circumstances, complete chaos, from which Henry Medlicott first rescued
the mapping of the Gondwanas. It is scarcely necessary to say that portions
of his Report were omitted by the then Superintendent of the Survey
from the printed copy, as he has mentioned in the Annual Report for
1880.*

In fact, throughout his life Medlicott had been the champion of whatever
he believed to be founded on scientific truth. Almost his only important
geological paper, outside of the Survey Publications, was that on “The Alps and
the Himalayas, a Geological Comparison,” issued in the ‘ Quarterly Journal of
the Geological Society’ for 1868, Vol. 24. This Paper narrowly escaped
rejection ; it was postponed for a time, but was finally published. Nor was
this hesitation wonderful, for the paper attacks, and in no doubtful way,
the conclusions of all the great Alpine geologists, v. Hauer, Gumbel, Studer,
Desor, and others, and there is no question that in the main Medlicott was
right. Some of the views expressed by him required and have since received
revision, but as an original description of mountain-building from a uniformi-

* ‘Rec. Geol. Surv. Ind.,’ 1881, p- 1.

xkvaly Obituary Notices of Fellows deceased,

tarian as opposed to a catastrophic point of view, it deserves far more
attention than it has received.

There can be no question of his strong uniformitarianism. One of his
objections to some of his colleagues’ work was against the use of faults to
explain abrupt boundaries. It was, however, characteristic of his love of truth
that when Mr. R. Oldham found that the typical Nahun-Siwalik section, to
which Henry Medlicott had so long referred as an example of deposition
against a pre-existing cliff, might after all be a simple fault, he revisited the
section and in the simplest manner admitted that he had misunderstood it.
Only those who remember the whole controversy about faults can have an idea
of how great a blow to his theories this must have been, and from some
experience of Directors of Surveys, there are few of whom it could be said,
that an error of so much importance was so frankly acknowledged. On
the larger question, whether, for instance, the great lines terminating the
Gondwana basins of Bengal, Orissa, and the Central Provinces are simple
faults, or whether they represent the boundaries of the old river valleys in
which the Gondwanas were formed, and to which they were limited, these
boundaries having subsequently been slightly crushed and dislocated, but
not greatly faulted, no complete answer has yet been given. In some cases
at least, for instance, in the Mopani field of the Satpuras, strong evidence
was brought forward by Henry Medlicott in favour of his view of original
limit, and even in the case of Raniganj, where unquestionably consider-
able faulting exists, the absence of outliers of the coal-bearing rocks south
of the field is a difficulty in supposing that a simple great upthrow alone
terminates the Gondwana area. Between the two authors of the Manual
of Indian Geology there was a difference of opinion on this point, and it
cannot be said that the question is completely settled.

The above may serve to recall a few of the services of Henry Medlicott to
the Geological Survey and to India, and some of the discoveries which he
made in science. It cannot do more than suggest the amount of labour that
he devoted to his work. His memory should remain as a striking example
of the thoroughly honest and capable geologist and as a worthy head of a
scientific branch of the Indian Government.

W Take

* [A melancholy interest attaches to this Notice by Dr. W. T. Blanford. It was written
by him at the request of the Council, and the proofs were duly sent to him, but they
arrived when he was already too ill to look at them,and he died before they could be
printed off and published. His genial sketch of his old friend and colleague was thus the
last task he accomplished in his own life.—Sec. R.S.]

XXVII

WILLIAM THOMAS BLANFORD, 1832—1905.

WiiuiaAm THomas BLANFoRD, though much of his working life was spent
out of his native land, began and ended his days in London. He was born
on October 7, 1832, at 27, Bouverie Street, in a house which, with the
adjoining manufactory, had been built by his father, William Blanford, the
site being now occupied by part of the offices of the Daily News.

From private schools at Brighton he went, at the age of 14, to one in Paris,
and there had a serious illness, probably diphtheria, just at the time of the
revolution of 1848. On leaving school he spent two years at Civita Vecchia
in a mercantile house, and on his return to England, in 1851, joined’ his
father’s business. But his younger brother, Henry F. Blanford, afterwards
the well-known meteorologist, and a Fellow of this Society, had already
become a student at the Royal School of Mines, and the elder one before long
yielded to the same attraction, and commenced his studies there in 1852.
During the vacations they worked at field geology with Ramsay (afterwards
Sir Andrew) in Wales, and mining, with Smyth (afterwards Sir Warington),
in Cornwall. Each passed out head of the list for his year, obtaining the
Duke of Cornwall and the Council Scholarships.

W. T. Blanford, on leaving the school in 1854, continued his studies at
Freiberg in Saxony, but next year both brothers were offered and accepted
posts on the staff of the Geological Survey of India, then in charge of
Dr. Thomas Oldham, arriving in Calcutta at the end of September.

Early in 1856 they, with an older officer, were sent to examine a coalfield
near Talchir, in the province of Orissa, south-west of Calcutta. The results
were important, though the coal was of little value, for they were able to
distinguish the measures containing it—afterwards called the Damuda series
—from an upper group which they named the Mahadeva and a lower one, the
Talchir, all forming part of the Gondwana system now familiar to geologists.
They also found that the Talchir group contained boulders sometimes 4 or
5 yards in diameter, embedded in a fine silt with smaller stones, facetted and
striated, which, they suggested, must have been transported by ice. For
some time the idea of a glacial epoch in late Carboniferous or early Permian
times was not received with favour, but it has since received ample con-
firmation.*

Afterwards Blanford visited Darjiling, and helped in surveying the
Rajmahal Hills, having a narrow escape from a band of mutineers on his
return to Calcutta, where he joined the Volunteer Cavalry Guards. From
the later part of 1857 to 1860 he was engaged in resurveying the Raniganj

* The subject was fully discussed in W. T. Blanford’s address to the Geological
Section of the British Association at Montreal in 1884.

XXVIll Obituary Notices of Fellows deceased.

Coalfield, and in visiting the Trichinopoly district and the Nilgiri Hills,
but in the cold season of the latter year he was sent to commence a
geological survey in Burma, where he visited Mandelay, not then in British
territory, and made zoological collections which added much to the knowledge
of the Burmese fauna.

Promoted after a furlough in England in 1862 to be Deputy Superintendent,
Blanford had charge of the survey of the Bombay Presidency, where he was
engaged for the next four years, also visiting Sind and Cutch. The position
of the Deccan traps in regard to the underlying Cretaceous and the over-
lying Nummulitic series was determined, as is described in the sixth volume
of the ‘ Memoirs of the Geological Survey of India.’

In September, 1867, Blanford was attached to the Abyssinian Expedition
to study the geology and zoology of the country. Landing at Zeila shortly
before Christmas, he accompanied the army to Magdala, afterwards remaining
for some time in the Bogos province. On his return to India in the autumn of
1868 he was released from field duties in order to work out his collections,
and then spent six months in England to complete his ‘ Observations on the
Geology and Zoology of Abyssinia.’ After returning to India in the late
autumn of 1869, he visited Sikkim next year with Captain Elwes, getting as
far as the Tibetan frontier, and was then engaged in the Godavari Valley and
the border of the Madras Presidency, chiefly in the coalfields, returning to
Calcutta when the hot weather began. By this time he had traversed the
whole peninsula on foot or on horseback, with the exception of some
20 or 30 miles, from the Arabian Sea near Surat to the Bay of Bengal at
Coconada.

In the autumn of 1871 he was ordered to join the Persian Boundary
Commission, and after a preliminary voyage to Basra and back started from
Gwadar, about 300 miles west of Karachi, marched to Shiraz with Major
St. John’s party, and then travelled alone through Ispahan to Teran to join
the other one under Sir Richard Pollock. After a short visit to the Elburz
Mountains, he returned to England from Enzeli on the Caspian, by
Astrakan, Moscow, St. Petersburg and Berlin to England, arriving at home
in September, 1872. Here he was obliged to remain on a two years’ leave,
for his health had suffered from the hardships of his work in Persia, but
he was fully occupied in preparing the volume on the ‘Zoology and Geology
of Persia’ for the Reports of the Boundary Commission.

After his return to India at the end of 1874, he was engaged during
the next three years on the geology of Sind, except for a three months’
exploration of the desert between the Indus and Jodhpur, which he crossed
in two directions; in the former region a continuous series was found to
extend from the Cretaceous (covered by representatives of the Deccan traps)
to the Pliocene. From 1877 to 1879, Blanford was chiefly engaged on
office work in Calcutta, one part of it being co-operating with Mr.
H. B. Medlicott, the Superintendent, in preparing the ‘ Manual of the Geology
of India.’ Then he returned to England on furlough, which was extended

William Thomas Blanford. Xxix

to two years to enable him to represent the Indian Government at the
meeting of the Geological Congress in Bologna. Returning to India in the
autumn of 1881, he undertook the survey of the country between the Punjab
and Quetta, till he was prostrated by a severe attack of fever. This
ultimately obliged him to return to England, where he arrived in April,
1882. Under medical advice he retired on pension after 27 years’ service
on the Indian Geological Survey, and though after a time he recovered his
health, his constitution was less robust than in former days.

Early in 1883 he married Miss Ida Gertrude Bellhouse, and settled at
Bedford Gardens, Campden Hill, converting a studio at the back into a
library, which was adorned by trophies of the chase collected in India.
Here three children—two sons and a daughter—were born, and the rest of
his life was spent. He enjoyed good health on the whole, though occasionally
liable to slight attacks of gout or bronchitis, but a year or so before his
death these attacks became more severe, and signs of failing physical powers,
happily unattended by mental weakening, began to make his friends anxious ;
but the end came more rapidly than they anticipated, for he passed away
after a brief illness on June 23, 1905, his last literary work being a notice
of his old friend and companion, H. B. Medlicott, contributed to these
obituaries.

Release from official duties simply gave Blanford more time for the work
which he loved, but Indian zoology now received more attention than geology,
although the latter was anything but neglected. He was also frequently
consulted upon survey matters by the authorities in England, and rendered
them many services. He became Secretary to the Geological Society in
1884, was elected President in 1888, serving for the usual period of two
years, and in 1895 was appointed Treasurer, holding the office till his death.
In 1884 he presided over the Geological Section, when the British Associa-
tion met at Montreal, and attended the Toronto meeting in 1897, afterwards
joining the party to Vancouver Island. He took part in the meetings of
the International Geological Congress at Bologna, Berlin, London, and Paris,
served on the Councils of the Royal, the Geographical, and the Zoological
Societies, and was a Vice-President of all. Before leaving India he had
been a President of the Asiatic Society of Bengal, and when visiting Canada
he received the degree of LL.D. from the McGill University, Montreal.
The Geological Society awarded him their Wollaston Gold Medal, and the
Royal Society marked its appreciation of the value of his Indian researches
by conferring on him one of the Royal Medals in 1901. He received the
Military Medal for the Abyssinian Expedition, the Cross of St. Maurice and
St. Lazare in 1881, and was made a Companion of the Indian Empire as
late as 1904.

Not many men have made more numerous contributions to scientific
literature or bestowed more pains on what they wrote. His first book,
‘Observations on the Geology and Zoology of Abyssinia,’ was published in
1870. It begins with a written account of the expedition, and nothing could ~

78.5.0 Obituary Notices of Fellows deceased.

be better, considering the inevitable difficulties, than the scientific portions.
He had succeeded in determining the true succession of the principal rock
systems in Abyssinia and in defining more accurately their character, and he
had collected zoological specimens, representing about 350 species of Vertebrata
and 500 others, mostly Mollusca. His second book was the volume in the
Report of the Persian Boundary Commission on the ‘ Zoology and Geology of
Persia, which maintains the high level of its predecessor and gives a very
suggestive account of the physical geology of a region characterised by
climatic extremes and a very low rainfall. The third was his contribution
to the ‘Manual of the Geology of India,’ equal in bulk to rather more
than one of the two descriptive volumes, a work the value of which is
universally acknowledged, and the fourth in order were the volume on
Mammals and two of those on Birds, contributed to the Fauna of British
India, the whole of which series, 17 volumes, he edited for the Government
of India.

His shorter papers, according to a list printed in the ‘Geological Magazine, *
exceed 175. The first, published in 1854, was geological, a short notice of a
section exposed in excavating for the West India Docks. The first zoological
paper (the fourth of the series), written in conjunction with his brother, and
published in 1860-1, was entitled “Contributions to Indian Malacology.”
The papers on the former subject mostly appeared in the publications of the
Indian Geological Survey, the ‘Quarterly Journal of the Geological Society,’
and the ‘Geological Magazine.” They cover a wide field, physical geology,
stratigraphy and paleontology, but it will suffice to mention three of a more
general character, characterised no less by breadth of view than by accurate
knowledge of the subjects. The first, his Address to the Geological Section
at Montreal, discussed the occasional conflict between the evidence for
geological age derived from marine formations on the one hand and terrestrial
(land and freshwater) on the other. The second, the Presidential Address to
the Geological Society in 1889, contained a clear account and some valuable
criticisms on the work done at the first and second meetings of the Inter-
national Geological Congress; the third, the corresponding address for the
next year, was ““On the Permanence or otherwise of Ocean Basins.” In this
- valuable contribution to a very difficult subject, Blanford exhibits the judicial
character of his mind and adopts neither extreme, holding that while the
general evidence in favour of the permanence of land areas and of ocean
basins is very strong, there are certainly exceptions, where large tracts of the
former have been submerged, and this for more than a thousand fathoms, and
cases where portions of the latter, now at least that depth, have been raised
above the sea to connect distant lands. His other writings show the same
mental qualities, for, in the words of an anonymous writer, “ His work in

* Decade 5, vol. 2 (1905), p. 1. It goes down to 1901. I have made great use of
this excellent article in drawing up the above notice, because I have been informed on
good authority that a near relation of Dr. Blanford supplied many of the facts, and he
himself revised the list of publications.

Thomas George Bond Howes. XXX1

every department, whether in zoology, geography or geology, has been marked
by thoroughness and accuracy.”

But if Blanford’s death has been a loss to science, it has been even greater
to his home circle and his many friends. Sound in judgment, free from
prejudices or any angularities, with a gentle courtesy of manner and a desire
to conciliate, nothing could make him depart from his own high standard of
honour or the path which his conscience approved. He was, in short, a man
emphatically to be trusted, for he exhibited in the daily work of life the calm
courage which had carried him through the difficulties, and sometimes the
dangers, of his earlier career, and while the last to seek his own advantage or
magnify his own work, he was the first to recognise merit in that of others
and ever ready to do them a service.

E..rGey eh

THOMAS GEORGE BOND HOWHES, 1853—1905.

THoMAS GEORGE Bonp HowEs, more commonly known as George Bond Howes,
was brought up in his boyhood at a private school. His education proper
he would no doubt himself have attributed to Huxley, with whom he was
for many arduous years associated at the Normal School of Science and Royal
School of Mines as successively pupil, assistant demonstrator, and deputy
professor. To Huxley’s person and memory he was ever devotedly attached,
and in 1895 he succeeded his celebrated master in the professorship of
zoology, the duties of which he had been in fact performing for the preceding
ten years.

While still Demonstrator of Biology at the Normal School, and at the same
time Lecturer on Comparative Anatomy at the Medical School of St. George’s
Hospital, Howes published, in 1885, ‘An Atlas of Practical Elementary
Biology, with a Preface by Professor Huxley, P.R.S.’ In this introductory
notice Huxley explains that in 1872 one of his first cares had been the
creation of a teaching collection for the use of his students, to contain, “in the
first place, a series of preparations and dissections illustrative of every
important fact in the structure of the animals and plants selected for study ;
and, in the second place, a corresponding series of drawings of the dissections,
of large size and executed in such a manner as to facilitate the comprehension
of the structures represented.” In carrying out this important scheme, he
candidly adds, “the whole burden of the work has fallen upon my former
demonstrator, Mr. T. J. Parker (now Professor of Biology at Otago) and
Mr. George Howes, who succeeded Mr. Parker, and now holds the office.” He
points out that,‘ A Course of Practical Instruction in Elementary Biology,’

rOO.011 Obituary Notices of Fellows deceased.

published by himself and Professor Martin in 1875, needed supplementing by
just such an atlas as Howes had supplied, “ which, while starting from part of
the work with which he has been occupied in our laboratory, contains so
many accurate and well-devised additional illustrations that it will be hardly
less useful to students who are engaged in the laboratory than to those who
work independently of it.”

In 1889 the course of elementary biology above mentioned was _ itself
revised and extended by Howes in collaboration with his friend Dr.
Dukinfield Scott, and in 1895 he edited and annotated the translation of
Wiedersheim’s ‘Bau der Menschen.’ Besides these services to scientific
literature, his pen yielded some forty original contributions, of which a
catalogue is given in the ‘ Proceedings of the Linnean Society’ for the present
year (1905). The longest and most important of the series is that which he
wrote, in conjunction with Mr. H. H. Swinnerton, ‘On the Development of the
Skeleton of the Tuatara, Sphenodon punctatus, with Remarks on the Egg, on
the Hatching, and on the Hatched Young. Material for this treatise was
supplied him with generous zeal by Professor Dendy, who used with great
success his point of vantage for procuring it, while holding the chair of biology
in the Canterbury College of the New Zealand University. Eggs of this
interesting animal, which is supposed to be “the sole surviving representative
of an early and widely ancestral amniote type,” were brought over, with tender
care, from New Zealand by Mrs. Dendy, and were incubated in the laboratory
of the Royal College of Science. Three out of the six precious specimens ran
the full developmental period. The young Sphenodons made good their ~
own escape by help of the horny shell-breaker with which Nature has
provided them, and led an observant and apparently contented life for three
or four months. They then succumbed, it is thought, as occasionally happens
with animals far higher in the scale of existence, to the effects of over-feeding
or of feeding at a time when they ought to have been asleep. Hospitality was
unthinkinely prolonged by their friends into the period which these lizards
or lizard-like creatures should normally have spent in peaceful hibernation.
Among the results arrived at by their elaborate treatise, Howes and
Swinnerton consider that they have added “fresh testimony to the belief in
the batrachian affinities of the living Sphenodon,” while strengthening the
demonstration of its chelonian resemblances and extending them to the
Plesiosauria. ‘“ Sphenodon,” they say, “judged from our standpoint, must be
regarded as the surviving representative of that group of animals ancestral to
all the living Sauropsida, and to at least the Dinosauria, Pterodactyla, and
Ichthyosauria of the past, if not of the Mosasauria and Dolichosauria also,
and unquestionably intimately related to the Anomodontia.” They recall the
view, much earlier advanced by Howes,* “that a sharp distinction may be
drawn between the coraco-sternum of the Batrachia (an archisternum), and the
costal-sternum of the Amniota (a neosternwm), the existence of which in any
Batrachian or Stegocephalian has not been proved.” In the succeeding para-

* ‘Nature,’ vol. 18, p. 269, 1891.

Thomas George Bond Howes. XXXill |

graph, they say, “ And. as to the skull, the now well-known fact that, whereas
in the Amniota the hypoglossal nerve-bearing region, truncal in origin, is
incorporated in the occiput, in the Batrachia these nerves are postoccipital,”
once again opens the gap between the Batrachia and Amniota; indeed, so
markedly that “our ideas may be systematised by applying to the former
condition the term Archicraniate, and to the latter that of the Syncraniate.”
Finally, they lead up to, but leave open, the question, whether terrestrial
vertebrates are descended from batrachian reptiles or reptilian batrachians ;
that is, from a group which possessed a coraco-sternum and an archicranium,
or from one which possessed a costal-sternum and a syncranium, “ structural
combinations which go hand in hand.” Every page of this memoir carries
evidence of the extremely careful investigation conducted by the joint authors.
But also it is marked throughout by the distinguishing characters which
Howes impressed on his own work. Acute and fearless criticism is frequent ;
equally or more frequent are expressions of admiring appreciation. At every
point astonishing familiarity is displayed with the literature of the subject in
all its bearings. The contributions of Germany and Japan, of Belgium and
France, and Russia, of England and New Zealand and the United States of
America, are all weighed in the balance. A paper is not neglected because it
is of ancient date, nor yet because it has only been published “ during the
passage of this memoir through the press,’ or even shortly after the revisal of
the proofs.

As may be gathered from his writings in general, Howes found his favourite
study in the anatomy of vertebrates. Nevertheless, when delivering addresses
as President of the Malacological Society, in 1896 and 1897, he showed a
commanding familiarity with all that had been written on the subject of
Mollusca. His addresses as President of the South-eastern Union of Scientific
Societies in 1900,and of Section D of the British Association in 1902, were
equally impressive as the fruit of wonderfully capacious reading combined
with high intellectual capacity. He was, however, by no means a voluminous
author, although it might well have been expected that he should be by those
accustomed to his voluminous fluency in expounding any topics which
appealed to his easily kindled enthusiasm. i

Howes was born on September 7, 1853, and may be said to have spent a
quiet, unostentatious, and comparatively uneventful life in accumulating and
dispensing vast stores of biological knowledge. He was elected a Fellow of
the Royal Society in 1897. He was long the treasurer of the Anatomical
Society, which he helped to found in 1887. From 1895 to 1903 he was the
highly efficient zoological secretary of the Linnean Society. He served on the
Council of the Zoological Society, and on its Reorganisation Committee, and
was more than once a vice-president. In everything he undertook it was with
him evidently only a question of doing the best work which the situation
demanded or permitted; but it was seemingly without question that he under-
took everything which he was asked to undertake. It is a sort of axiom in
the intellectual world that a man may be killed by worry, but not by work.

VOL. LXXIX.—B. ad

-

XXXIV Obituary Notices of Fellows deceased.

In the case of Howes, his ungrudging readiness to serve was mated with an
ambition to serve supremely well. It may seem unworthy to describe this
noble eagerness as containing in itself the element of worry, and yet one can
scarcely help believing that, as a result of too continuous effort, the brain
at last gave way beneath the strain of its own restless activity. Early in
1903 his health broke down, and without any substantial recovery in the
interval, he died on the 4th of February, 1905, in the fifty-second year of his
age. He leaves a widow and daughter, colleagues, distinguished pupils, and
many other warm friends, to deplore his loss. He was a man of religious
temper, cheerful, affectionate, generous. As already explained, the energy of
his life was given to science, but not to a lucrative branch of it. Had his
life been prolonged, he might have secured fame by authorship; as it is, his
reputation must largely rest on those whom he successfully trained in his
lecture-room and laboratory, and who gratefully acknowledge the debt they
owe to his wide learning, his skill as a teacher, his inspiring energy. There
can be little doubt that he long rejoiced in the prodigal expenditure of his
great powers, but in the concluding words of a stirring address, which he
delivered in 1900, we may fancy a touch of prophetic pathos and self-allusion
when he says, “ Higher ambition than that of adding to the sum of knowledge
no man can have; wealth, influence, position, all fade before it; but we must
die for it if our work is to live after us.”

T. R. ROS.

XEXV

FERDINAND BARON VON RICHTHOFEN, 1833—1905.

AMONG the active promoters of geology and geography during the last half
century, no one has filled a more distinguished place or has had a wider circle
of friends and admirers all over the world, than the illustrious Professor in
Berlin whose unexpected death on October 6 last sent a thrill of deep regret
through every civilised country. Up to the last he retained so much of his
youthful enthusiasm and power of work, that even those who knew him most
intimately and could watch him from day to day, saw little sign of old
age, and looked forward to a further continuance of his influence and assist-
ance in the scientific progress of future years. But his end came in the midst
of his labours. As he was working in his wonted place at his writing-table,
a sudden seizure deprived him of speech. It left him, however, so fully con-
scious that an early recovery was hoped for. But a little later another stroke
came while he was in a profound sleep, and after two days and a half he
passed peacefully away without illness or suffering of any kind.

The family to which Baron von Richthofen belonged is one of some position
in Silesia, where it owns extensive estates. Born at Carlsruhe in that country
on May 5, 1833, he received his early education at the Roman Catholic
gymnasium of Breslau, under ecclesiastics of whom he used in later life to
speak with respect for their learning and their skill in imparting knowledge;
yet he would refer regretfully to the monotonous restraints imposed by them
which, inasmuch as they curbed free and strenuous intercourse among boys,
had in his case, as he thought, induced a certain reserve and diffidence which
he had in vain endeavoured to overcome. He would sometimes contrast his
school experience with the very different training furnished by the public
schools of England. From school he went to the University of Breslau, and
then to that of Berlin, where he took his degree of Dr. Phil. in 1856.

Before completing his University career, he had already shown his strong
bent towards the studies to which his future life was to be devoted. Well
grounded in all the main branches of the natural sciences, it was in geological
and geographical studies that he took specially absorbing interest. The writings
of Leopold von Buch and Alexander von Humboldt appear to have had a
decisive share in directing his course, and he spoke in later years with
sratetul enthusiasm of his indebtedness to these two great masters. Von
Buch opened out for him the ample fields of geological research. The glowing
pages of Humboldt kindled in him that eagerness for geographical exploration
which carried him far and wide in the Old World and in the New. Perhaps
it may have been the influence of the example of these two heroes which drew
him at first into the study of ancient volcanic rocks; for Humboldt had begun
his scientific career by writing a little treatise on the basalts of the Rhine,
while Von Buch, fresh from the influence of Werner, had found in Landeck
and in his early journeys into Italy proof of the aqueous origin which his
master had so long claimed for all the basalts of the globe. Be this as it may,

dz

KKK VE Obituary Notices of Fellows deceased.

there can be no doubt that Richthofen, too, early felt the fascination of the
problems presented by volcanic phenomena and ancient igneous rocks. He
began, however, with a somewhat more adventurous flight into that difficult
department of Nature than had been attempted by his two geological masters.
He chose as the subject of his thesis for the doctorate a group of igneous
rocks, on which he wrote a Latin essay, “ De Melaphyro,” and as soon as his
University time came to an end, he started off on his first extended scientific
investigation, for which he selected the region of Predazzo, St. Cassian, and the
Seisser Alp. His observations on that area were begun in the summer of
1856, and were continued in subsequent years, but their publication did not
take place until 1860, when they appeared in his remarkable monograph,
wherein he arranged the sequence of the formations in that complicated
region, discussed the igneous rocks and their successive epochs of eruption,
worked out the tectonic arrangement of the several rocks, and propounded
the view that the dolomite masses of the Tyrol represent ancient coral-reefs.

During the next three years he had ample opportunities of extending his
knowledge and experience of field-work by journeys in the Nerthern Tyrol.
Vorarlberg, North-Eastern Upper Hungary and Transylvania. His published
papers of that period on the intrusive melaphyres of Moravia and on the
trachytes of Hungary show how keen was his early interest in the tectonics
and petrography of the igneous rocks, and at what a youthful period of his
career he had thoughtfully considered the family relationships of these rocks,
and, in particular, the important and independent character of the rhyolites.
At Vienna he came into contact with the distinguished group of geologists
and mineralogists who at that time flourished in the Austrian capital. Among
these men he was more especially drawn to Franz Ritter von Hauer, after-
wards for so many years the Director and guiding spirit of the Austrian
Geological Survey. It seems to have been as a consequence of this cordial
friendship that Richthofen was induced to become a volunteer on this Survey,
and to spend four of the pleasantest and most profitable years of his life in
hard systematic field-work among the Eastern Alps.

But his passion for geographical exploration in a wider and less-trodden
field than an European mountain-range came at last to be gratified in a
wholly unexpected way, whereby the current of his hfe was at once and
permanently changed. In 1860 he had the good fortune to be chosen
scientific member, with the rank of Secretary of Legation, in a Prussian
Embassy, sent out to negotiate commercial arrangements with the native
States of Eastern Asia. During this expedition he had an opportunity
of spending some months in Japan, and of visiting Formosa, the Philippines,
Java, and Siam. He did not, however, return with his comrades to Europe,
but, fired with the desire of making extensive explorations into the heart of
the Asiatic continent, he remained behind in the expectation of being able
to carry out what had been a long-cherished dream. JBut, after some
journeys in India and Cashmere, difficulties arising from the disturbed
state of China arrested his further progress, so that at last, postponing

Ferdinand Baron von Richthofen. XXXVI

his quest for some more favourable occasion, he crossed the ocean to
California, with the view of studying the metalliferous and volcanic .
regions of the western border of North America. The full development of
petrographical types of eruptive rocks in that remarkable territory soon
kindled anew his enthusiasm for this department of geology, and furnished
him with rich material for study. As the fruit of this research there
appeared, in 1868, his now classic paper on the “ Principles of the Natural
System of Volcanic Rocks,’ which was published in English, in the first
volume of the ‘Memoirs’ of the California Academy of Sciences. In this
essay the relations, classification and order of sequence of the rhyolites,
trachytes, propylites, andesites, and basalts were clearly expounded. A
philosophical discussion was likewise given of the relations of modern to
ancient eruptive rocks, of the origin of volcanic rocks, and of the connection
between the distribution of these rocks and the configuration of the surface
of the globe. One notable result of this residence in Western America was
to enable the geologist to obtain such a mastery of the English language as
is not often reached by a foreign man of science.

The design of Asiatic exploration, though necessarily postponed for a time,
had never been laid aside. Richthofen discussed the subject with some of
the American geologists, especially with J. D. Whitney, then in the height
of his activity and usefulness as a geological pioneer in the Far West. At
last the bold scheme was planned of making a prolonged geological survey of
the interior of China. On August 3, 1868, Richthofen left San Francisco
and recrossed the Pacific. He then began the series of journeys which,
during a period of four years, extended over most of the provinces of the
Celestial Empire. He made voluminous notes on the physical geography and
geology of that vast region, and on the distribution and condition of its
teeming human population. At the same time he formed an extensive
collection of the minerals, rocks, and fossils of the various districts in which
he sojourned. He was the first to reveal to the outer world the wide extent
of workable coal in China. The commercial importance of his observations
came to the notice of the European merchants in the East, and with the
view of inducing him to continue and complete them, the expenses of his
later journeys were partly defrayed by a contribution from the Shanghai
General Chamber of Commerce, to which, in 1870, he forwarded a series
of Reports on the economic and commercial aspects of the regions through
which he had travelled. But the large amount of scientific material which
he had amassed demanded years of patient elaboration.

He returned to Europe at the end of 1872, after twelve years of travel,
and, settling in Berlin, set to work on the preparation of his monumental
work upon China. The first; volume did not appear until 1877; the
second was published in 1882, the fourth in 1883, and the ‘Atlas’ in
1885. The third volume has never been issued, but it is to be hoped
that the large bulk of material collected by the author for its completion
may yet be given to the world. Instead of confining himself to the

XXXVII1 Obituary Notices of Fellows deceased.

region actually traversed by himself, Baron von Richthofen discussed the
general physical geography of Central Asia as a whole, and with great
skill explained the broad configuration and grouping of the mountain
chains of the region. Among the problems of which he presented a
new solution, perhaps the most interesting and important was that of
the Loess, which attains such colossal dimensions in the Celestial Empire.
He brought forward a large body of evidence to show that this wide-
spread and thick deposit is of olian origin, being due to the transport
of dust by the winds. The fourth volume bears ample witness to his
diligence and ability as a field-geologist and collector: it is devoted to the
Palzontology of the scene of his labours, and though he entrusted the
determination and description of his fossils to others, it 1s to his skill and
enthusiasm that we owe the establishment of the existence of the Cambrian,
Silurian, Devonian, and Carboniferous systems in China. He it was also
who brought to light the Chinese development of the floras of Carboniferous,
Jurassic, and Tertiary times. |
Richthofen’s geographical prowess was so well recognised, that in 1879,
after the publication of the first volume of his ‘China,’ he became Professor
of Geography in the University of Bonn. There he spent four happy years,
during which he issued the successive volumes of that wor. It was during
this interval that he married Irmgard, daughter of Karl Freihern v. Richthofen,
the gracious and accomplished lady to whom he had long been devotedly
attached, and who became thenceforward his helpful companion in his work
and in his journeys. As a teacher he soon showed that he possessed pre-
eminent educational gifts, whereby he attracted students around him, kindled
in them some of his own enthusiasm, and gave to geography a more defined
and a higher place as a mental discipline in the studies of a University. His
success in this sphere led to his being called, in 1883, to the Professorship of
Geography at Leipzig. But all who had watched his career realised that before
long he must inevitably be withdrawn to Berlin as the national centre where
most scope would be found for the development of his schemes as a teacher
and organiser of educational methods. The expected transference took place in
1886, when he became Professor of Geography at the metropolitan University
of Germany. There he threw himself with all his characteristic originality
and ardour into the task of devising and arranging such illustrations of
geography as would bring vividly before the eyes and minds of his students,
and of the general public, the characteristic features of land and sea, and the
appliances for scientific exploration and travel. In connection with the
University a Geographical Institute was organised by him, in which these
various collections were arranged, and where ample space was found for large
and small lecture rooms, map-drawing rooms, survey-practice rooms, a reference
library, and large reading-room, well furnished with current geographical
literature in all languages. In his later years he gave special attention to
the illustration of the sea, and succeeded in prevailing on the authorities to
build a Museum for Oceanography (“ Meereskunde”), in which he sought to

Ferdinand Baron von Rachthofen. XXX1x

display, by help of charts, maps, models, paintings, diagrams, and apparatus,
every feature of the sea which is capable of such illustration. Nor did he
confine his efforts to a strictly scientific field: all that could help to convey
.a sense of the practical work of navigation and exploration, or quicken the
popular interest in the navy, came within the scope of his aims. He journeyed
through the maritime countries of Europe in search of the best examples of
illustration for his purpose—models of ships of all ages, methods of ship-
building, and types of intruments employed in navigation. For spaciousness
and even luxuriousness of accommodation, for breadth of conception and for
the completeness with which the designs have been carried out, there is
probably no geographical establishment in the world that can equal the
Geographical Institute of Berlin, planned and carried out by the genius and
unwearied enthusiasm of its Director.

For many years he had taken keen interest in the welfare of the
Geographical Society of Berlin, and when he returned to the capital as his
permanent home, he had an opportunity of devoting his practical energies
to its improvement. He introduced several reforms in its organisation, and
communicated to it so much of his own personal activity and enthusiasm as
eventually to place it in the distinguished position which it now holds
among the geographical institutions of our time.

Beginning his career with geological studies in the field, Baron von
Richthofen passed into geographical investigation with the inestimable
advantage of a training in detailed observation and induction. He thus
became a geographer of the highest type. To him the mere addition of so
many hundred square miles of territory to what was already known of the
earth’s surface, and the opportunity of affixing the names of friends and
benefactors to peaks, promontories, rivers, and inlets were matters of no
moment; it was the grand features of land and sea that interested him, their
origin, their history, their relations to each other, their influence on the
progress and destiny of mankind. His geological apprenticeship with the
Austrian surveyors among the Eastern Alps, and his early researches in
mountain-structure and the behaviour of igneous rocks, could hardly fail to
give him that grasp of physical features, combined with that knowledge of
detail which are so often lacking in travellers and explorers. But besides his
scientific accomplishments he possessed in rare measure the personal qualities
which go so far to ensure the success of an explorer—health and strength
alike of body.and mind, a wide range of natural knowledge, courage, patience,
endurance, taet, and kindliness. It may have been the consciousness of the
possession of these qualities, combined with a recollection of the pleasure
which their exercise had given him in his varied wanderings in Europe,
America, and Asia, that led him, in response to many requests, to give a course
of popular lectures on scientific travel. These lectures he subsequently
extended, and in 1886, the year of his transference to Berlin, amidst all his
University and other work, they appeared as his admirable ‘Fiihrer fiir
Forschungsreisende.’ No one but a born and trained explorer, who had enjoyed

xl Obituary Notices of Fetlows deceased.

ample experience by flood and fell all over the globe, could have written such
a volume, so full of the ripest practical knowledge, so broad in its conception
of what exploration should be, and so clear and emphatic in its statement of
the accomplishments which are needed for the making of a successful traveller.
Every department of research is luminously presented in his chapters, which
may be regarded as a contribution of the first importance to geology and
physical geography. Among the shrewd pieces of advice in the book the
author recommends the explorer to travel alone, with only the attendants
necessary for his purpose, but without any friend or companion who would do
more than look after the material well-being of the expedition. Various
treatises with similar aims had been given to the world before the publication
of Richthofen’s volume, from the time of Ami Boué’s ‘Guide du Géologue
Voyageur’ onwards, but none of them so thoroughly deserves to be put into
the hands of every man who proposes to undertake the examination of new
or little-known regions, and who is willing to learn beforehand what is
expected of him by those competent to judge.

Baron von Richthofen, as a familiar and welcome participator in various
International Congresses, and an honorary member of many of the learned
societies of Europe, was one of the most widely known and most respected men
of science of his day. His tall figure and courtly presence made him conspicuous
wherever he came, while his quiet, earnest and kindly manner irresistibly
attracted those with whom he came in contact. Huis home at Berlin, where
he was greatly aided by the full intellectual sympathy of his devoted wite,
became a centre to which geographical pilgrims from all lands made their way.
With his students he was not merely the learned professor, but the personal
friend who guided and helped them while they were with him, and watched with
keen interest their subsequent progress when they left. The ‘Colloquium’ which
he established among them afforded ample opportunity for that friendly social
intercourse between teacher and taught which is so valuable an aid in the
higher education. This personal influence he cultivated so ardently that
perhaps few modern teachers have equalled him in the enthusiastic devotion of
his pupils. On his sixtieth birthday his old students at Bonn, Leipzig, and
Berlin offered him as their most welcome gift a ‘ Festschrift’ of geographical
essays, In evidence that, of the seed which he had sown so widely, some had
taken good root. While his original contributions to science will always
keep his name fresh as one to whom geology and geography stand greatly
indebted, it is probably his educational work in the organising and developing
of geography as a mental discipline in university training that has had the
most influence in his own lifetime. But this influence could never have been
so far reaching and profound had it not been based on the recognition of his
place as one of the great scientific travellers of the nineteenth century. He
leaves a blank in the ranks of science which none of his survivors can
wholly fill, and his memory will be warmly cherished by all who knew him
as one of the ablest, worthiest, and most lovable of men. A. G.

xli

FREDERICK WOLLASTON HUTTON, 1836—1905.

Captain F. W. Hutton, whose name has been so long and so intimately
connected with the Natural History of New Zealand, died at sea on
October 27, 1905, while on his way back to the Colony after a short stay in
England. He was born in 1836, at Gate Burton, Lincolnshire, of which
village his father was Rector. At the age of fourteen he was a midship-
man on a ship trading to Calcutta, but he soon left the sea, and became
a student at King’s College, London. Before the age of twenty he received
a commission in the 23rd Royal Welsh Fusiliers, and soon saw active
service in the Crimea, and afterwards in India during the Indian Mutiny,
where he was present at the Relief of Lucknow and in other engage-
ments. His bent for science was already showing itself in various ways, and
in 1860, on his return to England, he was elected a Fellow of the Geological
Society, and gained considerable practical acquaintance in that science with
the officers of the Geological Survey in Hampshire and the Isle of Wight.
Fortunately for New Zealand, Captain Hutton, in search of fresh fields of
work, turned his attention to that Colony, and, retiring from the army,
arrived there in 1866. Soon he became attached to the Geological Survey of
New Zealand, and commeuced that series of researches into the geology of
the Colony which lasted, with some breaches of continuity, throughout his
life. In 1871 he was appointed Assistant Geologist, and removed from
Auckland to Wellington; in 1873 he became Provincial Geologist of Otago
and came to Dunedin, and before long, in conjunction with the late Professor
Ulrich, he published an important work on the geology of Otago.

But Captain Hutton was not a man of one science only. During this time
he had already been working also on the zoology of New Zealand, and when
the position of Provincial Geologist lapsed with the abolition of the provinces
in 1876, he was appointed Professor of Natural Science at the Otago Univer-
sity, and had charge of the Otago Museum, which had been designed and
built under his device and direction, and which still contains so much
evidence of the work that he did at that time. About four years afterwards
he removed to Christchurch as Professor of Biology at Canterbury College,
and soon began to instil into his students some of his own enthusiasm for
natural science, as he had already done in Dunedin. Here, while continuing
his researches on geological and zoological subjects, he found it necessary, for
the sake of his students, to take up the study of botany, and with his natural
clearness of insight and ability to grasp readily the essential details of a fresh
science, he soon made himself familiar with his subject, and a little pamphlet
which he published on the structure of the common weed “shepherd’s purse’
received favourable notice at the time. | |
- He continued to occupy the chair of Biology at Canterbury College till the

xl Obituary Notices of Fellows deceased.

death of Sir Julius Von Haast, when he became, in addition, Lecturer on
Geology, and had temporary charge of the Canterbury Museum. Soon after,
on the resignation of Mr. Forbes, he was permanently appointed Curator of
the Museum. Thereupon he resigned his professorship of Biology, but con-
tinued to lecture on geology till about three years ago, when he found the
strain of lecturing in addition to his museum work pressing too heavily
upon him.

For nearly forty years he had been constantly engaged with his various
official duties, and had never revisited England, but early in the year 1905 he
was granted six months’ leave of absence, and he left New Zealand in March,
full of anticipation of the pleasure he would have in renewing old
acquaintances and of meeting personally the many scientific friends whom
he already knew so well by correspondence; but owing to a breakdown in
his health soon after his arrival in England, these pleasurable anticipations
were only in a very slight degree realised.

Such, then, is a brief outline of what may be called Captain Hutton’s
official life; but this only covers a small part of his activity: during these
busy years he found time to prepare and publish a long list of papers and
works embodying the results of original research, which he contributed
to the scientific journals of New Zealand, Australia, and Europe. Many of
these, as already stated, deal with geology, but a far larger number are
devoted to zoology, and they cover practically every class of animals from
mammals to protozoa, and it can safely be said that no other New Zealand
zoologist will ever be able to cover so wide a field. Indeed, Captain Hutton’s
work has made this unnecessary, and at the same time impossible. When he
arrived in New Zealand he found its zoology practically untouched; some of
the birds and more conspicuous animals had, indeed, been collected and
described by various scientific expeditions that had visited New Zealand ;
but even of these little was known, and all the others were unnamed and
unknown. ‘To the task of describing these, Captain Hutton set himself with
determination and enthusiasm, and not only did he possess the, necessary
knowledge and skill required for the commencement of the work, but he had
also the rare faculty of self-development with his work, and of acquiring new
methods of research and investigation as they were needed.

From his pen there came in rapid succession descriptions and catalogues
of fishes, mollusca, crustacea, worms, echinoderms, sea-anemones, and insects
of all kinds. Many of these appeared in the ‘Transactions of the New
Zealand Institute, to which he has been a constant contributor from the
commencement, and many of his catalogues were issued as separate publica-
tions. As the result of his labours, aided to some extent by that of his
students and others, whom he started and encouraged in the same task, the
preliminary work of naming and classifying the animals of New Zealand has
been to a great extent completed, and it is a great satisfaction to all zoologists
that he was able, with the assistance of several co-workers, to publish in the
early part of 1904 an ‘Index Faune Nove-Zealandix ’—.e., a complete list,

Frederick Wollaston Hutton. xl

with references of all animals hitherto recorded from New Zealand. A
survey of the pages of this work will give some idea of the number of the
animals that were described by himself, while the numerous congratulatory
letters on the completion of this work that were publicly presented to him
at a meeting of the Philosophical Institute of Canterbury in May, 1904, bear
eloquent testimony to the value placed upon his work by those best qualitied
to judge. More recently, under the title ‘Animals of New Zealand,’ he
has published a beautifully illustrated account of the air-breathing vertebrates
of New Zealand.

But Captain Hutton was not content with merely naming and describing
new species: no one knew better than he that this work, valuable though it
was, formed only the preliminary statement of the problems presented by
the phenomena of animal life, and these problems he never lost sight of.
To the particular problems of the origin of the New Zealand fauna and flora
he specially devoted himself, and brought to bear upon it all his wide
knowledge as a zoologist and his experience as a geologist. His contribu-
tions to the solution of this complex problem have made his name known
throughout the world as a recognised authority on all matters connected with
the geographical distribution of animals and plants. It was from their
bearing on this question that he took such a keen interest in the results of
the various Antarctic expeditions that have recently returned from their
fields of observations, and even after his arrival in England, he contributed
an important article to ‘Nature’ on the Antarctic continent.

But he attacked even wider questions still. One of his very first contribu-
tions to scientific literature was a review of Darwin’s ‘ Origin of Species’ in
the ‘Geologist’ in 1861—a review which gained from Darwin an appre-
ciative letter, of which Captain Hutton was justifiably proud—and throughout
his career Captain Hutton returned from time to time to the more philo-
sophical questions underlying the whole of natural science. Some of his
articles on these subjects were issued a few years ago in book form under the
title of ‘ Darwinism and Lamarckism.’ A later work, ‘The Lesson of Evolu-
tion,’ which contains his presidential address to the Australasian Association
at Hobart in 1902, and kindred essays, attracted much attention not only
from naturalists but from thinking men in general.

Captain Hutton naturally took a keen interest in the welfare of the
various scientific societies in New Zealand. As honorary secretary and
president of the Otago Institute in years past, he did much to establish it on
a firm and permanent. basis. During the whole of his stay in Christchurch
he was a most active member of the Philosophical Institute of Canterbury,
acting as secretary, treasurer, and on several occasions as president. He was
secretary of the Australasian Association for the meeting in Christchurch in
1891. Later on he was president of Section D (biology), and in 1902 he was
president of the Association itself, on the occasion of its meeting in Hobart.
On the retirement of Sir James Hector as manager of the New Zealand
Institute, about three years ago, Captain Hutton was prominent amongst

xliv: Obituary Notices of Fellows deceased.

those who promoted a Bill for the reconstitution of the Institute, providing
for the better representation of the various district Institutes on the Board of
Governors, and for an elected president, and when the new constitution came
into force in January, 1903, he was by common consent elected as first
president under the new constitution—a position he still held at the time of
his death.

As a teacher he was singularly clear and original in his methods of exposi-
tion, and possessed the valuable faculty of arousing enthusiasm in his pupils,
several of whom have become regular contributors to various departments of
natural science. Though he was not a public man in the ordinary sense, he
took his full share of public duties in all matters where he could contribute
special knowledge without becoming involved in questions of local politics.
Being thoroughly sincere, open, and straightforward in all his actions, he was
a keen and unsparing critic of anything that bordered upon pretence and
humbug, but to all genuine students and workers he was a warm friend and a
ready helper, and the loss of his valued advice and ripe judgment will be felt
by many.*

* For this Obituary Notice the Royal Society is indebted to Professor Charles Chilton,
M.A., D.Sc., .L.8., Canterbury College, Christchurch, New Zealand.

_—

xlv

GEORGE BOWDLER BUCKTON, 1818—1905.

_ In his address to the Royal Society in November, 1905, the then President,
- Sir William Huggins, reported among the year’s losses the death of George

Bowdler Buckton, who “to the deep grief of a large circle of friends, passed

away at the ripe age of 88, leaving behind him a noble example of unwearied

devotion to the successful prosecution of scientific work, notwithstanding
great physical infirmity.”

Most men who attain to scientific eminence find the study of a single
branch of science, or of one or two allied branches, sufficient to occupy a
lifetime. It is comparatively seldom that the same man is able (like
Buckton) to pursue with success two such dissimilar studies as Chemistry
and Entomology. There was also another unusual feature in his scientific
career. Most men who occupy themselves with Entomology and kindred
pursuits do so chiefly in their younger days, and more or less abandon the
study in later life, whereas Buckton, though always interested in Natural
History, devoted his earlier years to Chemistry and Physics, and the latter
half of his long life to the preparation of his valuable works on Entomology.

Born in or near London, on May 24, 1818, and brought up at Oakfield,
Hornsey, Buckton was the eldest son of a large family. His father, George
Buckton, was Proctor of the Prerogative Court of Canterbury, and his
mother, Eliza, was the daughter of Richard Merricks, of Chichester, Deputy
Lieutenant of the County.

At about the age of five years, Buckton sustained an accident which
permanently crippled him, a misfortune which he bore throughout: life with
courage and silence, but which must have been peculiarly painful to a man
of his large build and energetic temperament. Nevertheless, it is probable
that if he had been able to lead a more active life, he would not have had
the same opportunities of carrying on his scientific pursuits so steadily
and successfully.

As Buekton’s infirmity unfitted him for public school and University hfe,
he received tuition from the Rev. Oliver Lodge, Rector of Barking, and the
Rev. Dr. Meuse, formerly Headmaster of the Cholmondeley School. He was
popular with his class-mates, shared their escapades, and was often carried
pick-a-back by some stronger comrade on daring excursions. Necessarily
left much to his own resources, he became a good classical scholar and
an extensive reader. He also devoted himself successfully to music and
painting, which remained his favourite recreations throughout life. He
showed much ingenuity in the construction of appliances to illustrate his

youthful lectures on scientific subjects.

While still scarcely more than a boy, Buckton made the acquaintance of
Thomas Bell, who proved his close friend for more than 40 years, and by
whose encouragement he became an earnest student of Natural History,
beginning to form collections and make observations on bees, butterflies,

xlvi Obituary Notices of Fellows deceased.

shells, bats, birds, ete., round London and elsewhere. Most of the birds he
collected were shot and stuffed by his own hand; he also became a good
fisherman.

Astronomy was another of Buckton’s favourite studies, and, being always
alive to the interest of collateral issues in science, he started quite early
grinding telescopic specula. Frankland, with whom he afterwards became
intimate, vied with him in being the first English physicist to produce
specula on glass, which method had been lately inaugurated by the French
philosopher Foucault. Subsequently, Buckton worked specula of 12 inches
diameter, and, as an amateur, mounted them equatorially. }

On the death of his father, he moved into London, residing at Queen’s
Gardens, W., where he built himself a circular observatory on the leads
of his house, and made more than one telescope, using his own brass
turnings, etc. About this time he became the pupil, friend and assistant of
Professor A. W. von Hofmann (of whom, it will be remembered, Sir William
Perkin and other distinguished men were also pupils), at the Royal College
of Chemistry. His first recorded scientific paper was “ Observations on the
Deportment of Diplatosomine with Cyanogen,” published in the ‘Journal of
the Chemical Society’ (vol. iv, pp. 26 to 33), and subsequently republished
in translation, in several French and German periodicals in 1851 and 1852.
A succession of other papers followed, among the most important being those
relating to his discovery and isolation of the radical, mercuric methyl, ete.
(Proc. Roy. Soc.,’ vol. ix, 1857 to 1859, pp. 91, 92, 309 to 316; *Piul. ‘Trane;
1858, pp. 163 to 169; 1859, pp. 417 to 435). The last of the series, worked
out with the collaboration of William Odling, was upon aluminium
compounds, and was published in ‘Proc. Roy. Soce., vol. xiv, 1865, pp. 19
to 21. A full list will be found in the Royal Society’s Catalogue of Scientific
Papers.

Meantime Buckton had been unobtrusively carrying on his Natural
History studies, his earliest recorded papers on the subject being “On the
Application of Cyanide of Potassium to Killing Insects ” (‘ Zoologist, 1854,
vol. xiv, pp. 436 to 438) and “ Notice of several species of Bats captured in
England during the present autumn ” (‘ Proc. Linn. Soc., vol. ii, 1854, pp. 259
to 261).

In 1845, Buckton became a Fellow of the Linnean Society, serving in 1855-6
upon its Council, and coming into contact with Yarrell, Westwood, Saunders,
Huxley, Hooker, and other naturalists. In 1852 he joined the Chemical
Society, and in 1857 the Royal Society, when he became a member of its
Philosophic Club, the meetings of which he attended with great interest, till
extreme old age compelled him to relinquish them. In 1883 he became
a Fellow of the Entomological Society. He served on the Councils of all
these Societies from time to time. He was also elected a Member of the
Entomological Society of France; the Academy of Natural Sciences at
Philadelphia, ete. :

In 1865, Buckton married Mary Ann, the only sister of his friend
Professor Odling, of Oxford. He purchased the estate of Weycombe, at

George Bowdler Buckton. xlvu

Haslemere, Surrey, where he built himself a stone-gabled house, according to
his own designs, taking with him his observatory and transit instruments.
Here he lived a peaceful, happy life for the remainder of his days; and out
of eight children, five daughters and a son survive him ; likewise his widow.

After settling at Haslemere, although Buckton kept his private laboratory
and lathe room, and gave private lectures on Chemistry and Physics to his
children and friends, he devoted himself chiefly to Natural History, com-
mencing by studying Parthenogenesis in Aphides, which led up to the
first of his valuable series of Entomological works, his ‘Monograph of
British Aphides, published by the Ray Society in four volumes, from 1876
to 1883. The numerous illustrations to this work were made under the
camera lucida and lithographed and coloured by his own hand.

Continuing the study of the somewhat neglected sub-order Homoptera, he
published his ‘Monograph of British Cicade or Tettigide’ (2 vols,
Macmillan) in 1890-1891, in which he was assisted by his children, who
collected specimens and coloured many of the plates under his direction.
This was followed by the ‘ Natural History of Eristalis Tenax; or, the
Drone Fly’ (1 vol., Macmillan, 1895); and finally by a large and important
work on the Membracide of the World, issued by Lovell Reeve & Co. in
Ato. parts from 1901 to 1903, with many illustrations. A supplement to
this work was published in the ‘Transactions of the Linnean Society’ in
July, 1905, only two months before the author’s death. The original plates
of the Monograph of Membracidz have been presented to the Hope
Museum, Oxford.

Buckton’s deftness of hand in arranging his hundreds of microscopic
slides was remarkable; often in laying out the delicate nervous organisa-
tion of an insect, he would make use of the sting of a wasp, as the finest
available instrument. He was in constant communication with various
scientific societies at home and abroad, and carried on a large correspondence
with the unfailing energy and courtesy that reminds one of an earlier
period, when letter-writing was a fine art; this was distinctly perceptible
even in his business letters.

Buckton instructed his children himself till they were 9 or 10 years of
age in a variety of subjects, from Grammar and Languages to Euclid, Drawing
and Physiology. He had an unusual gift of exposition, and was always
ready to share his knowledge with others. He often gave tools or specimens
to small boys who showed interest in Natural History.

In spite of his infirmity, the charm of his personality deeply impressed
even strangers casually meeting him for the first time; and a friend writes,
“ None who ever met him could fail to be struck with his kindly courtesy,
his intense vitality, his wide range of knowledge, and his unflagging interest
in every topic affecting mankind.”

In his earlier days, Buckton travelled in Italy, France, and elsewhere,
saw the Commune, and even climbed the barricades, his great will-power
enabling him to surmount obstacles that might have daunted stronger
men.

xlvii Obituary Notices of Fellows deceased.

The Observatory which Buckton took with him when he removed from
London to Haslemere was erected in the grounds at some little distance
from the house; but his visits to it were discontinued after a serious accident
that befell him in 1882, when he overbalanced himself in trying to reach
the long focus of a Newtonian. He lay with a leg fractured in two places for
some hours before he was found; and his recovery, though complete, was slow.

Buckton interested himself sympathetically in all sorts of local matters,
acting as Treasurer and Chairman on various local bodies. In politics he
was a Conservative; in religion a moderate Churchman, with broad
sympathies, and he gave liberally to Church and Schools. Among his
large circle of friends he numbered many of eminence, among whom were
Tyndall and Tennyson, of whom, as they resided not far from Haslemere, he
saw a good deal.

Though sometimes swift and uncompromising in his judgments, and
naturally of a quick temper, he was nevertheless gifted with remarkable
self-control, especially as regarded his physical infirmity. Those most
intimately associated with him can recall no single instance of moodiness or
murmuring ; but when some unexpected hindrance presented itself, he would
allude, half-sadly, and half-humorously to his disability; a reference not
easily forgotten by those who heard it. It has been remarked that “the most
striking thing about him was his magnificent calm.”

Buckton kept his remarkable powers of work and energy to the last. The
finely-executed bust by R. Hope-Pinker, exhibited in the Academy of 1904,
plainly indicated how little old age had impaired his clear intellect and
vigour. He was finishing some water-colour sketches of Norway up to
within a few weeks of his death.

Buckton succumbed at last rather from the natural exhaustion of old age
than from any physical disease. He sustained a chill, followed by three
weeks of illness and suffering; but he was conscious to the last, and his
spirit passed peacefully away on the night of September 25, 1905, in the
presence of his wife and children.

After cremation, the ashes were buried, according to his wish,in Haslemere
Churchyard, in the presence of many relatives, friends, and members of the
various Societies to which he had belonged. At the time the village was
celebrating the subdued festivities of its Harvest Home, adding an appro-
priate calm and beauty to a somewhat unusual ceremony.

The present Lord Tennyson has written, concerning Mr. Buckton, “ Truly
a devoted, spiritual, knightly nature, with a faith as clear as the height
of the pure blue heaven. His views and my father’s upon Life, Death and
Immortality were very much alike. My father used to say, ‘My most
passionate and most earnest desire is to have a fuller and clearer knowledge
of God’” There is scarcely a preface to any of his larger works in which
Buckton does not incidentally reveal his deep-lying interest and trust in
things unseen. W...t., Kine,

xlix

CHARLES BARON CLARKE, 1832—1906.

CHARLES BARON CLARKE was born at Andover, Hampshire, on June 17, 1832.
He was the eldest son of Turner Poulter Clarke, J.P., and Elizabeth Parker.
His father was the son of Turner Poulter Clarke and Elizabeth Baron; his
mother was the daughter of James Parker and Elizabeth Ward. One of his
grand-uncles, Charles Baron, an enthusiastic gardener, was the founder of the
Agricultural Society of Saffron Walden. Baron’s sister, Clarke’s paternal
grandmother, shared her brother’s love of plants; three of her sons owed
largely to her influence a taste for scientific enquiry. These were Clarke’s
uncles, Joseph, a careful antiquarian, who founded the Saffron Walden
Museum ; Joshua, an able field botanist and a recognised authority on the
flora of Essex; and Benjamin, a systematic botanist of decided originality,
spoken of by Bentham as “one of our most careful observers.” Clarke
shared the tastes of two of his uncles; he took, in addition, a deep interest
in social science. The latter taste he seems to have inherited from his
maternal grandfather; his mathematical powers appear to have been derived
from his father and his paternal grandfather.

As a boy, Clarke was sent,to the preparatory school of Mr. Tomlinson, at
Salisbury. From this he went to King’s College School, London, where he
passed through the higher forms and then attended classes in the College
before proceeding to Cambridge. He entered at Trinity in 1852; graduated
B.A. and was bracketed Third Wrangler in 1856; was elected a Fellow of
@ueen’s in 1857; was called to the Bar at Lincoln’s Inn in 1858, and
appointed Mathematical Lecturer of his College in the same year; and took
the degree of M.A. in 1859. He continued a resident Fellow of Queen’s till
December, 1865, when he was appointed to the Bengal Education Depart-
ment.

On reaching India in 1866, Clarke was at first attached to the Presidency
College, Calcutta, but was soon appointed Inspector of Schools in the Eastern
Division of Bengal, with his headquarters at Dacca. Early in 1869, Dr. T.
Anderson, Superintendent of the Royal Botanical Garden at Calcutta and of
Cinchona Cuitivation in Bengal, was invalided to Europe; on Anderson’s recom-
mendation Clarke was appointed to officiate in both posts. Towards the end
of 1870, Anderson died in Scotland, but Clarke was not relieved of his duties
in the Botanical Department till July, 1871, when, on the arrival of Dr. (now
Sir) G. King, who had been appointed Anderson’s successor, he was able to
revert to his substantive appointment at Dacca. In 1874, Clarke was
transferred to the Presidency Division of Bengal, with his headquarters at
Calcutta, and in 1875 he was again transferred, as Inspector of Schools, to
the Northern Division, with his headquarters at Darjeeling.

In 1877 Clarke came to England on two years’ furlough; when this

VOL. LXX1X.—B. ¢

] Obituary Notices of Fellows deceased.

expired he did not return to India, but was placed on special duty at Kew, to
assist Sir J. D. Hooker in preparing the ‘Flora of British India.’ This
deputation continued till 1883, when Clarke reverted to duty in Bengal as
Inspector of Schools in the Presidency Division, and temporarily as a
Professor in the Calcutta Presidency College. In December, 1884, Clarke
was appointed to officiate as Director of Public Instruction in Bengal, and in
March, 1885, his services were placed at the disposal of the Government
of Assam, where he served as Inspector of Schools, with his headquarters at
Shillong, till his retirement, at the age of 55, in June, 1887.

On. his return to England, Clarke settled at Kew, to be near the Herbarium
at the Royal Gardens, where he worked without a break as a volunteer till
within a few days of his death, which took place, after a short illness, on
August 25, 1906.

The influence of Mr. Tomlinson on Clarke’s intellectual development
seems to have been considerable, and the foundations of the interest which
he took in geographical and historical studies appear to have been laid at
his preparatory school. At King’s College School he was much impressed
by the teaching of Mr. Hann, a man of marked individuality, who was
mathematical master.

As an undergraduate at Cambridge, Clarke, from his King’s College
connection, was one of a circle of close friends which included John (after-
wards Sir J.) Rigby, a Member of his College and subsequently Second
Wrangler in Clarke’s year, and Henry Fawcett, also of the same year but
a Member of Peterhouse. Clarke, who, both as an undergraduate and as
a resident Fellow, was Faweett’s especial intimate, had not known the latter,
or only knew him by sight, in his school days. Though a native of
Salisbury, Fawcett did not attend the same preparatory school ; Fawcett was,
besides, a year Clarke’s junior, and though they were contemporaries at
King’s College School, they were not class fellows. But from Easter, 1851,
till they went to Cambridge, both attended the same mathematical lectures
at King's College, and during this period began their lifelong friendship.
The migration of Fawcett to Trinity Hall brought him and, as a consequence,
his friend Clarke, into contact with Leslie Stephen, who had also been at
King’s College. As a result, Stephen was drawn into their circle and
became a lifelong friend of both. Characteristic of the members of this
brilliant coterie was an interest in discussions bearing on political economy
and a foible for unconventionality in matters of dress. In the case of
Clarke the interest continued to the last; the foible became second nature.

That a group of thoughtful undergraduates should take pleasure in the
discussion of intricate social problems is, fortunately, not unusual; nor is it
surprising that the feelings of youth should often induce the adoption of
advanced views. The outlook, however, not infrequently alters when the
realities of life are faced and the discussion of such problems are too frequently
left to the professional philanthropist and the party politician, whose inter-
pretation of economic truths is occasionally unconsciously affected by a natural

Charles Baron Clarke. li

enthusiasm or subconsciously influenced by a thirst for place. Clarke was
neither, and his views on political economy were therefore particularly
refreshing. He treated the subject as one of the exact sciences, and if the
sanity of his outlook seemed at times almost cynical, those who knew him
knew also that this was to be attributed to the subject and not to the man.
For the factors in a social problem never presented themselves to his mind as
mere abstractions, and any approach to cynicism was foreign to his kindly
nature.

While in residence at Queen’s, the penchant for what was then his favourite
study incidentally led to his taking an active part in the Cambridge Election
of 1863 which Fawcett unsuccessfully fought. He was still more active in
his friend’s behalf a year later, when Fawcett first stood for Brighton.
Between these two contests occurred the more memorable one, so vividly
described by Leslie Stephen, into which Clarke and Stephen flung themselves
with almost unacademic ardour, that resulted in Fawcett’s appointment to
the Chair of Political Economy at Cambridge. Clarke’s intervention in these
struggles was due to his personal affection for Fawcett, whose views on social
problems were substantially his own. The relative position of the two was,
however, far from being that of disciple and teacher. Though not a good
public speaker, as Fawcett was, Clarke’s conversation was easy and, from his
store of facts, he was remarkably ready in discussion. We learn from Leslie
Stephen that Fawcett made frequent use of Clarke’s often pithy expressions
on economic subjects. Where his interests were shared, whether his views
were accepted or not, political economy was a frequent topic in Clarke’s letters
and talk. Besides various pamphlets and letters on special problems, some
of these written at the request of Indian officials with reference to legislative
measures that were being contemplated or enforced, in which his views are
stated with lucidity and directness, Clarke has recorded his attitude towards
the subject as a whole in his ‘Speculations from Political Economy,’ published
in 1886.

The circle at Cambridge to which Clarke belonged was mainly a
mathematical one, but most of its members seem to have held the view that
the value of a mathematical training lay chiefly in its excellence as a branch
of intellectual gymnastics. It is not clear that this was precisely Clarke’s
view; the fact that he published no purely mathematical paper suggests
that he may have shared it. He was, though not technically a musician,
fond of good music, and while in India was interested in native, particularly
Bengali, music. The interest in this case was, however, mathematical rather
than esthetic, and that he was attracted to this side of the art as a whole
may be deduced from his note on the “Equal Temperament of the Scale ”
which appeared in ‘ Nature’ in 1883.

The members of this Cambridge circle had a house in the Lake Country
where they could stay during vacations. Here Clarke spent most, if not all,
of the Easters of his time at Queen’s; Leslie Stephen has recorded how,
during Clarke’s last visit, Easter 1865, he and Clarke made an ascent of the

e 2

li Obituary Notices of Fellows deceased.

Pillar Rock in Wastdale. Clarke, always a traveller, during long vacations
went further afield. In the earlier years he visited Scotland, once going as
far as Skye; in 1862 he visited Madeira; several visits, usually as one of
a climbing party, were paid to the Alps, the last of these being made in
1865. Mountain adventure was not, however, the only object of his
journeys ; his private herbarium shows that during all of them he was an ©
assiduous botanical collector. While engaged in his favourite occupation in
Madeira, his inexplicable presence on a lonely hillside and the aberrant
nature of his attire led to his being arrested as an escaped prisoner.

Clarke’s most sustained botanical work while he was a resident Fellow of
Queen’s was done, however, in England and chiefly in his own country,
Northern Hampshire. The results were embodied in a list of the flowering
plants of Andover, his birthplace ; this list, completed before he left for
India, was printed at Calcutta in 1866, shortly after his arrival there. The
modest title and remote origin of this first contribution to what was now to
be his favourite science failed to conceal the independence with which the
subject had been treated, and the trenchant criticism which it evoked led to
a reply which showed that in Clarke the reviewer had to deal with no mean
antagonist.

The work of an Indian Inspector of Schools involves much touring during
a considerable part of the year, and affords opportunities for a study of the
flora of the country traversed hardly to be surpassed. Of these opportunities
Clarke took full advantage during his service in Bengal and Assam,
supplementing them as occasion offered by vacation visits to districts and
provinces other than those within which his duties lay. During the first
two years and a half of his Eastern service Clarke travelled extensively in
East Bengal, where the physical conditions are such that the only practical
mode of conveyance is by boat. The considerable collections he had
amassed, and the bulk of his other belongings, were lost, early in 1868, by
the wreck of the country boat which for the time being he had practically
made his home; his own safety was due to his being a powerful swimmer.
Undiscouraged, he began afresh, and besides collecting largely in his own
division, was able to visit the Khasia Hills, Tipperah and Chittagong. He
occasionally complained that, owing to the pressure of official and adminis-
trative duties at the Botanical Garden, he was able to do “less real botanical
work during a year of his superintendentship than he could accomplish in
a month of his travelling appointment.” Still his temporary connection
with the Cinchona Department gave him an opportunity of studying the
flora of temperate Sikkim, enabled him to pay his first visit to the high
Himalayan passes, and led incidentally to his making a journey in Madras
and the Nilgiris. His transfer to Calcutta in 1874 permitted him to
partially explore the Sundribun forests of the Gangetic Delta, to visit Chutia
Nagpur and to make a holiday tour in the Punjab Himalaya. When
Darjeeling became his headquarters he was enabled to make three more
tours in Alpine Sikkim, to investigate temperate British Bhutan, to study

:

Charles Baron Clarke. li

the flora of the North Bengal plain, and to make a holiday journey which
lasted three months and a half, his most sustained purely botanical
excursion, in Kashmir and on the Karakoram Range. The collections
accumulated during these various journeys amount to 25,000 numbers,
representing about 5000 species. The whole he made over to Kew when he
reached England in 1877.

When Clarke returned to India in 1883 he made a second and more
extended tour in Chutia Nagpur, and in the protected native States to the
south-west, paid botanical visits to various portions of Central Bengal and
explored subtropical Sikkim and the submontane forests of the Terai and the
Duars. His transfer to Assam in 1885 enabled him to renew and extend his
acquaintance with the flora of the Khasia Hills, to explore the valleys of the
Brahmaputra from Dhubri to Sadiya, and of the Surma from Silhet to Silchar,

-as well as to visit the Naga Hills and Manipur. The collections amassed

during Clarke’s second period of service in India amount to some 10,000
numbers; a complete set of the specimens was presented to Kew after his
retirement in 1887. }

In the course of his Indian career Clarke was able to acquire, at first hand,
a knowledge of the Indian flora comparable in extent with that of Hamilton,
Wallich and Hooker, and only surpassed by that of Griffith. Clarke was an
admirable field botanist; his commissariat and equipment when travelling

- were Spartan in their simplicity; his physique was such that he seemed to

regard the ascent of a Himalayan slope or the passage of a tropical rice-
swamp much as he regarded an English geological excursion. His
indifference to climate was such that his clothing did not vary from what he
wore in England, whether he was on a Himalayan pass in early spring or in
the Gangetic plain in the hot season. His specimens were always carefully
selected and prepared. The precise and often full field notes, frequently
accompanied by serviceable analyses, were always made in the field; every

gathering received a separate serial number, so that misquotation of a plant

collected by Clarke is impossible; energetic and tireless, he always dealt with
the specimens of a particular date before he retired to rest, no matter how
long or trying the day’s march might have been. A feature of Clarke's work
as a collector was the especial attention given by him at particular times to
individual natural families. His notes show that from 1866—69 his interest
was great in Commelynacee, from 1869—71 in Cyrtandracee, from 1871—73
in Composite, in 1874 in Gentianacee, from 1874 onwards in Glumacece
generally, but after 1876 more particularly in Cyperacee, the family as to
which in later years he was to become the recognised authority. Another
characteristic of Clarke’s work was the comparative indifference with which
he regarded trees: as a collector of herbaceous species he has, perhaps, never
been surpassed. These two peculiarities were more marked during the first
phase of his Indian career than they were during the second, which began
after he had spent six years continuously at work at the Herbarium at Kew.
Clarke’s contributions to botanical literature during his first ten years of

liv Obituary Notices of Fellows deceased,

service in India include monographs of the Commelynacew and Cyrtandracee of
Bengal, published at Calcutta in 1874, and of the Indian Composite, also
published there in 1876; with six minor papers in the Linnean Society’s
‘Journal’ between 1870 and 1876. In 1874 he further earned the gratitude of
Indian botanists by reprinting, with a preface at his own cost, Roxburgh’s ‘Flora
Indica, which had become scarce and hardly procurable. While on furlough
during 1877—79, and on deputation at Kew during 1879—83, his additions
to botanical literature were more extensive. They include accounts of over
fifty of the natural families described in the second, third and fourth volumes
of Sir J. D. Hooker’s ‘ Flora of British India’; three minor papers in the
‘Journal of Botany’; ten in the Linnean Society’s ‘Journal’; and one, an
important review of the ferns of Northern India, in that Society’s
‘Transactions. During this period he also wrote two of the volumes,
that on Commelinacee, issued in 1881, and that on the Cyrtandree, issued in
1883, of De Candolle’s ‘ Monographiz Phanerogamarum.’

The family Cyperacew was, as we have seen, the last of a series of families
which had proved especially attractive to Clarke. This particular interest did
not become dominant till 1876, near the close of his first period of Indian
service; it continued so, however, during the rest of his career. While
helping forward at Kew, between 1877—83, the preparation of the ‘ Flora
of British India,’ he had little time to study his favourite family till shortly
before he left England; his first papers on the subject are two of the
minor contributions in the Linnean Society’s ‘Journal,’ already alluded to,
which appeared in 1883. But during his two last months in England
advantage was taken of his special knowledge in having the Indian species
of Cyperus rearranged. When Clarke was leaving for India, Sir J. D. Hooker
asked him to publish the results of his work, so as to aid later on in dealing
with the genus in the‘ Flora.’ On reaching India, Clarke similarly rearranged
the rich Indian material of the genus in the Calcutta Herbarium ; he
thereupon published, in the Linnean ‘Journal’ for 1884, an important
review of the Indian species of Cyperus in both herbaria.

Clarke’s only botanical contributions during his second residence in
India were notes on three of his botanical journeys, published in the
Linnean ‘Journal. During this period, however, he studied and rearranged
the Calcutta material of the order Cyperacew, and when he reached Kew
in 1887, after having critically examined and distributed the contents of
his second Indian Herbarium, he devoted the greater part of his time to a
detailed study of the Cyperacee of the globe, his object being to complete
a general monograph of this difficult family. As his work progressed he
gradually became’the recognised authority on the subject, to whom botanists
of every nationality sent their collections from all parts of the world for
identification. The results of his work in this direction are to be found
in numerous papers published in many periodicals at home and abroad.
The Cyperacee in most of the principal collections in Europe were also
submitted to him for critical examination and determination. In addition

Charles Baron Clarke. | ae

he wrote, at the request of Sir J. D. Hooker, the account of the Cyperacece
for the ‘Flora of British India, published in 1893—94, and at the request
of Sir W. T. Thiselton-Dyer the accounts of the same family for the ‘ Flora
Capensis, published in 1897—98; for the ‘Flora of Tropical Africa,’
published in 1901—02 ; and for the ‘ Index Flora Sinensis,’ issued in 1903—04.
His exhaustive acquaintance with the family was turned to further account
in defining, in a paper which appeared in the ‘ Philosophical Transactions ’ in
1893, the relationship that natural biologic regions, so far at least as
botany is concerned, bear to the tabulation areas which are most useful in
dealing with the distribution in space of living organisms. He utilised his
familiarity with Cyperacee a second time in 1898 in defining the limits of the
sub-subareas that comprise the tabulation area of British India. But his
monograph of the family as a whole, though completed at the time of his
death, remains still unpublished.

Clarke did not, however, confine himself exclusively to his favourite family
during the period from 1887 onwards. Besides publishing the results of his
Manipur journey, in the Linnean ‘ Journal,’ he prepared for Sir W. T. Thiselton-
Dyer accounts of three natural families for the ‘Flora Capensis,’ and
of five for the‘ Flora of Tropical Africa’; of two for Sir G. King’s ‘ Flora
of the Malayan Peninsula’; and of seven or eight for Schmidt’s ‘ Flora of
Koh Chang.’ One of the last pieces of work he did was to finish Lowe’s
‘Flora of Madeira.’ This is still unpublished ; the text has been completed,
but a memoir of Lowe on which Clarke was engaged when overtaken by his
last illness remains unfinished.

Clarke possessed a considerable general knowledge of Natural History, but
was not minutely acquainted with any special branch, and he rarely discussed
zoological subjects. His acquaintance with Geology was more extensive, and
though he seldom spoke at geological meetings, he frequently attended the
excursions of the Geologists’ Association, not only the shorter day or half-
-day meetings, but the longer excursions made by the members at Easter and
Whitsuntide, and in August, and was always ready to enter into the discussion
of such geological questions as arose during the excursions. He was hardly
an active fossil collector, and his interest inclined rather to those wide
problems in Geology where the science approaches the domain of Physio-
eraphy. He was frequently able to intervene in discussions on the
origin of river-systems, the formation of valleys, and similar topics, and to
describe, from his Indian experience, cases very much in point. The extent
and accuracy of his knowledge of Geography are incidentally shown in
those papers which deal with the distribution of plants and are more
directly seen in the various geographical text-books written by him for
Macmillan between 1878 and 1894. Equally extensive was his knowledge of
History generally and of English History in particular, though, if a
conclusion might be drawn from his conversation, his interest was greatest
and his knowledge most detailed with regard to the periods of the Tudors
and the Stuarts. Unfortunately, none of the results of his historical studies

lvi Obituary Notices of Fellows deceased.

have been published; for some time before his death he was engaged in
writing a History of England and had completed the manuscript to the reign
of James I. But the subject which, after Political Economy and Botany,
most interested him was, perhaps, Ethnology. His knowledge of this science
was extensive and his views were independent. He, however, published only
one ethnological paper, on the Stone Monuments of the Khasi Hills, in the
* Journal of the Anthropological Institute’ in 1874.

In Clarke were united all the qualities that gain esteem and hold affection:
Open and sincere, he was neither prone to praise nor ready to blame. The
peculiar charm of his manner seemed only to increase with years and longer
intercourse. Mental powers which excite admiration were in him combined
with singular modesty. Unselfish to a degree and unfailingly kind, he was
always ready to place his time and knowledge at the service of others.
Hospitable to a fault, the warmth of his welcome was only equalled by the
difficulty with which he could be persuaded to accept the hospitality of
others. |

Clarke became a Fellow of the Linnean Society in 1867 and of the
Geological Society in 1868. He joined the Council of the Linnean Society
for the first time in 1880 and so served during sixteen years ; he was a Vice-
President seven times and was its President during two years, 1894-96. In
1882 he was elected a Fellow of the Royal Society and served on its Council
during 1888—90. In 1897 he joined the Geologists’ Association ; in 1903, in
conjunction with Professor Judd, he acted as Director of one of its excursions.

| Die

lvi

LIONEL SMITH BEALE, 1828—1906.

LIONEL BEALE was born in the year 1828, in the parish of St. Paul’s, Covent
Garden. His father was a surgeon, and was one of the earliest Officers of
Health in London. While a medical student at King’s College, Lionel
Beale showed talent as well as industry, particularly in the study of
chemistry and anatomy; and before he had taken his degree he was invited
by Professor Acland to take the part of anatomical assistant in the Natural
Science School at Oxford. He remained there for two years, when he
returned to King’s College, and was succeeded by Victor Carus, of Leipzig.
In 1852 Beale established a laboratory where he taught “the use of the
microscope,” and gave courses of instruction in physiological chemistry, and
minute anatomy, normal and morbid. In 1853 he was appointed Professor
of Physiology in King’s College, Huxley being one of the candidates.

During these early years of his scientific life, Beale received guidance
and help from Dr. Todd, Mr. Bowman, and Mr. Sambrooke. Between 1851
and 1858 he seems to have published nearly one hundred papers, most of
them illustrated by his own skilful hand.

In 1859 he married Frances, only daughter of the Rev. Peyton
Blakiston, M.D., F.R.S., and took the lease of 61, Grosvenor Street, which
he retained until 1904. In a small greenhouse, and on a flat roof at the
back of this house, he, for many years, grew orchids, ferns, and bulbs with
skill and success. In the year 1882 he found it necessary to live in the
country on account of his wife’s health, and took a small cottage in a pine
wood at Weybridge, travelling to and from London daily. His garden
became well known amongst those interested in this pursuit, for he grew
successfully palms, Japanese plants, and bulbs. One of his friends there
was the late Mr. G. F. Wilson, whose wild garden at Wisley has now come
into possession of the Royal Horticultural Society.

In 1892 his wife died at Weybridge, and this loss and the constant
fatigue of railway-travelling began to affect his health. In 1896 he had
an attack of apoplexy, which laid him up for several weeks—his first serious
illness. In the year 1904 he returned to London to live with his son and
daughter-in-law in Bentinck Street, where his general health markedly
improved, although senile changes became more evident. In March, 1906,
he had another attack of cerebral hemorrhage. He became unconscious
on the next day, and passed peacefully away on the 28th. He was then
78 years old. He was buried in Weybridge Cemetery, as was his wish,
and in the presence of his near relations and a few of his oldest friends.
One of his sisters, Miss Sophia Beale, survives him, well known for her
sketches at home and abroad; and he leaves one son, Mr. Peyton Beale,
F.R.C.S.

lvii Olituary Notices of Fellows deceased.

Dr. Beale was a self-contained man, at least in his later years, of
somewhat less than the middle height, with good features and a pleasant
smile. He was remarkable for scarcely showing signs of his age until his
last illness, and always gave the impression of a busy aud happy man,
enjoying his scientific work and his garden with almost equal constancy.
His old friend, Mr. H. T. Wells, R.A., painted an admirable portrait which
will faithfully transmit his features to a distant day.

Dr. Beale’s reputation was early established as a histologist, an original
observer, and a skilful investigator, while his beautiful drawings added
greatly to the value of his anatomical papers and of both his completed
volumes on the same subject. He was admitted to the Royal Society in
1857,* and gave the Croonian Lectures in 1865 on the termination and
destruction of the ultimate nerve-fibres.

In 1871 the College of Physicians awarded him the Baly medal, a
distinction shared by him with Sharpey, Claude Bernard, Carl Ludwig,
Brown-Sequard, Heidenhain, Burdon-Sanderson, Michael Foster, Maurice
Schiff, Gaskell, Schafer, Pavy, Langley, and Pavloff. The College also
appointed him Lumleian Lecturer in 1875, when he chose for his subject
“ Life and Vital Action in Health and Disease.”

As a lecturer, he was clear, accurate, and instructive; aS an examiner,
patient, and sound in judgment. Strongly as he held his own views, he
never judged a candidate by his opinions, but by an impartial estimate of his
knowledge and ability. As a physician, he was courteous, shrewd, and |
practical ; as a colleague, honourable, and friendly.

Apart from his distinction as a practising physician, a demonstrator, and
lecturer, Beale deserves lasting reputation as a histologist. His first
investigations were on points of animal chemistry: the absence of chlorides
in pneumonia, ‘Medico-Chirurgical Trans.’ (1852), and the methods of
identifying glycose in the urine (1853). Then came observations on the
minute anatomy of the liver (‘ Phil. Trans.,’ 1856). His methods of soaking
tissues in dilute acid solutions led to valuable results in tracing the course of
ultimate nerve-fibres, and his staining with carmine was a fruitful method of
investigation. When he began his investigations, the admirable work of
Bowman on the histology of renal structures and of muscular fibres was the
most striking advance in this country, and Beale’s close relations with Dr. Todd
and Mr. Bowman were of great importance. He also had the merit of
distinguishing between the structure Protoplasm or, as he called it, Bioplasm,

* Copy of extract from Certificate of Dr. Lionel Smith Beale, February 4, 1856: —

The author of (1) a paper on the “ Minute Structure of the Liver,” ordered for
publication in the ‘Phil. Trans.’ for the present year ; (2) a paper on the
‘“‘ Absence of the Chlorides in the Urine in cases of Pneumonia,” published in
the ‘Medico-Chirurgical Transactions’; (3) a work on the ‘ Microscope, and
its Application to Clinical Medicine,’ 1854.

Distinguished for his acquaintance with the sciences of Physiology and Pathological
Chemistry.

Inonel Smuth Beale. lix |

which forms the nucleus and body of a cell, from the formed material
or intercellular structure without power of movement or growth or
reproduction. The one he called “germinal matter,” the other, “formed
material.”

The title of a very early paper of his was “ An Attempt to show that every
Living Structure consists of Matter which is the seat of Vital Actions, and
Matter in which Physical and Chemical Changes alone take place.”

The discovery and description by Schleiden and Schwann of the structure
and essentially identical properties of vegetable and animal cells-led directly
on to the observations of Goodsir and Redfern in this country, and to the
cellular pathology of Virchow. Beale’s researches were in the same
direction, and established the distinction between cellular and intercellular
or connective tissues.

It was, perhaps, unfortunate that the difference of view held by Beale
from that maintained by Huxley and many others in this country and
abroad was formulated as the question between “vitalism” and “materialism.”
The truth does not always lie between two extremes. If we invoke the
“vital principle” to explain every difficulty, we so far put a bar to the
advancement of science. But when we have traced all vital functions
to the primordial living ovum, and “ Nature’s self untwisted lies into its first
consistencies,” the untwisted knot is as hard to be “explained” as ever.
As Du Bois-Reymond said, Jgnoramus et Ignorabimus.

In estimating Dr. Beale’s work, we must remember the great importance of
a new method. The differential staining of tissues was the method he
introduced, and ultimately, his views on the nucleus and the cell were based
on the reaction of animal and vegetable nuclei and protoplasm to carmine
and other discriminating agents. He left no school behind him, but the
influence he had on his contemporaries was important. His two works
on histology, “ The Microscope in Clinical Medicine,” published in 1854, and
“How to Work with the Microscope,” published in 1857, covered to a large
extent the same ground. The former is a careful description not of the
instrument itself, but of needles, scalpels, scissors, methods of staining and
mounting sections, chemical reagents, and of drawing, measuring, injecting,
and staining. The second part is not a laboratory handbook, but a histo-
logical account of tissues and fluids of the body, both normal and morbid,
including the histology of tumours and of the vegetable and animal parasites
then known. It is well illustrated by drawings of the author’s selection
and execution. |

Simultaneously with the successive editions of ‘The Microscope in
Medicine’ appeared not less numerous issues of ‘How to Work with the
Microscope.’ It was dedicated to the writer’s old friend, Mr.Sambrooke. The
first part is devoted to a description of the instrument and its accessories, and
is followed by a systematic account of the structures—animal, vegetable, and
mineral-—revealed by the compound microscope. As in the earlier volume, not
only are the construction of the microscope and its accessories fully illustrated

bs Obituary Notices of Fellows deceased.

by the author’s drawings, but numerous woodcuts are also collected at the end
of the volume figuring the structure of human tissues.*

A third volume, of very different character, was published many years later,
and reached a second edition in 1882. It is a reprint of lectures on medicine
at King’s College, and is specially occupied with “Slight Ailments,” with no
reference to histology or carmine. It is of purely practical value, and by
its many shrewd observations shows a power of observation at the bedside
that no one would have expected who was only acquainted with his
histological works.

In his later life Dr. Beale wrote chiefly in defence of vitality as a cause of
living phenomena. He held strong convictions on its application to meta-
physics and theology, and on these subjects was much influenced by the late
Dr. Thring, of Uppingham, and Dr. Wace, the present Dean of Canterbury.

Professor Osler writes as follows of Beale’s reputation in the United States
and Canada (‘ Lancet, April 7, 1906):—* The influence of Dr. Beale as a
scientific investigator and a clinical physician was much more widespread than
perhaps was recognised in London or Great Britain at large. In part, at
least, the unfortunate terminology was responsible for the unpopularity
of his views in some quarters. Both in Canada and the United States there
are scores of men of my day who, like myself, knew Dr. Beale only by his
writings, who will hear of the death of their old teacher with sincere regret,
and who will recall with gratitude labour which often helped to lighten
their own.”

Titles of Some of the most Important Scientific Papers of Dr. Beale.

His first book was on “ The Microscope and its Application in Practical
Medicine: a Course of Lectures, 1854.”
1853. “On the Absence of Chlorides from the Urine in Cases of Pneu-
monia.” ‘ Med. Chir. Trans.’ :
1854. “The Microscope in Clinical Medicine.”
1856. “On the Anatomy of the Biliary Ducts.” ‘ Phil. Trans.,’ published
separately, with sixty photographs of the author’s drawings.
1857. “ How to Work with the Microscope.”
The two books were slightly varied in title in successive
editions and overlapped.
The fourth and last edition of “The Microscope in Medicine ”
was published in 1878.
The fifth and last edition of “ How to Work with the Micro-
scope” was published in 1880.
The Croonian Lecture delivered before the Royal Society of
Physicians, “On Life and Vital Action in Health and Disease,”
show that every structure consists of matter which is the seat of

* Both books were modified in title in successive editions.

Lionel Smith Beale. bxi

vital actions and matter in which physical and chemical changes
alone take place.
1862-3. “ Nerve-endings in Voluntary Muscles.” ‘Proc. Roy. Soc.,’ 1862-3.
“Minute Anatomy of Sarcolemma and Striped Muscle,” 1864.
‘Quart. J. Mier. Se.’
1863. Paper on “The Branching of Nerves in the Frog’s Bladder.”
‘Phil. Trans., 1863.
1864. “Termination of Nerve-Fibres.” bid.
1864. “Minute Anatomy of Sarcolemma and Striped Muscle.” ‘ Proce.
Roy. Soe.’
1865. He delivered the Croonian Lecture before this Society on “The:
' Ultimate Nerve-Fibres Distributed to Muscles and some other
Tissues.”
1865. “The Minute Structure of Dentine.” ‘Quart. J. Micr. Se.’
1869. “Minute Structure of Papille of Frog’s Tongue.” ‘Quart. J.
Mier. Se.’
1870. “Bioplasm.” bid.
1871-2. “ Nerves of Capillary Vessels.” bid.
1880. Presidential Address to the Royal Microscopical Society.

Dr. Gustavus Mann has favoured the writer with the following appreciation
of Dr. Beale’s histological work :—

“Looking back from our present knowledge, Beale’s greatest achievement
is to have recognised that a distinction must be drawn between the ‘ germinal’
and the ‘ formed’ matter of tissues. This view, first published in 1861, in “ The
Structure of the Simple Tissues,” was adversely criticised by Max Schultze
in “Das Protoplasma,” because ‘it lay outside the cell theory.’ How little
justified this dictum was has since been established, for Beale’s germinal
matter includes what we now call the nucleus and also cytoplasm in an active
state of metabolism. Beale was led to his conception by the different
behaviour of tissues towards his ammoniacal carmine solution. He wrote
‘all elements of fresh tissue which stain readily with carmine, alone possess
the power of growth and of producing matter like itself out of material ©
differing from it entirely in properties and powers. I therefore called it
germinal or living matter to distinguish it from the formed material. The
latter may possess remarkable properties and may undergo various physical
and chemical changes under the influence of heat, moisture, oxygen, etc. It
may permit some fluids to permeate it, and may interfere with the passage of
others. It may contribute to the stability of the organism, and perform
a variety of important functions, but it cannot take the place of the germinal
or living matter, nor in many cases does it exhibit its characteristic properties
after the death of the germinal matter belonging to it. As examples of
‘formed’ material, he mentions the white fibrous tissue of tendon, the matrix
of cartilage, and the body of red blood corpuscles. These far-reaching
conclusions were not mere speculation, but were the legitimate outcome of

lxu Obituary Notices of Fellows deceased.

a very careful study of the changes which the body, and particularly the
connective tissues, undergoes during its development from an embryonic to
the adult state; of the changes met with in fully-grown individuals wherever
there is a replacement of old tissues by new, as in the skin; and, finally, of
the alterations produced in normal tissues as the result of infection, both local
and general. In his “ Disease Germs,” many experiments are quoted in which
the marked proliferation account of the white blood corpuscles is carefully
depicted, and although we now know definitely that white blood corpuscles
are not the agents producing infectious diseases, the idea underlying Beale’s
theory is correct, for in the light of Ehrlich’s recent researches there is no
doubt that a cancer cell, z.¢., Beale’s germinal matter, may have its virulence
greatly increased under new conditions of environment.

“Beale did not fall into the common error of considering histology from
a purely anatomical point of view. He saw that in physiological research,
physics, anatomy, and chemistry must go hand in hand. By the first,
the physical phenomena may be elucidated. By the second, the minute
mechanism concerned in these phenomena is ascertained. By the third, the
nature of the chemical analyses and syntheses taking place in the living
organisms are to be determined. How much Beale valued the chemical
aspect of minute anatomy is shown by the care he devoted in his text-book
to micro-chemical methods, and by the labour he expended on urinary
deposits, the numerous figures being drawn with the greatest possible care,
and, above all, drawn to scale. He further recognised the acid reaction of
the ‘ germinal’ matter and the basic properties of ‘ formed ’ matter.

“To him also belongs the credit of having for the first time practised the -
method of fixing tissues by injection, for experience had taught him how
rapid are the alterations ensuing after death if due precautions be not taken
to prevent post-mortem changes. Among Beale’s discoveries the distribution
of the peripheral nerves and the minute structure of the cells of both the
central and peripheral systems are especially worthy of mention. Working
with the simplest methods, but gifted with the greatest patience and a natural
_ aptitude for microscopical research, he discovered that the sympathetic
ganglion cells of the frog were connected with two distinct processes, one of
which, the straight one, was continuous with the central part of the cell,
while the other or others were arranged spirally round the straight fibre and
were joined to the circumference of the ganglion cell. We now know that
Beale’s spiral fibre is in reality the termination of a nerve-fibre springing from
another cell and carrying stimuli to the ganglion cell. His methods of
examining tissues in dilute acetic acid allowed him to see delicate nerve-
fibres scarcely less numerous than those revealed by Ehrlich’s zntra-vitam
staining with methylene blue. It is only natural that Beale, having shown
by his simple method that the distribution of nerve-fibres in striped muscle
is so abundant, should have treated Cohnheim’s gold method for nerve-endings
and the results which Kiihne obtained by this method and Max Schultze’s
tetroxide method, sceptically. His conception of the distribution of the

Lionel Smith Beale. Lxiii

nervous system was that there are no free nerve terminations; that ‘in all
cases the terminal distribution of nerves is a plexus, network, or loop, and
hence that in connection with every terminal nervous apparatus there must
be at least two fibres, and that in all cases there exist complete circuits, into
the formation of which central nerve-cells, peripheral nerve-cells, and nerve-
fibres enter. This view of Beale has been revived to a great extent by
Apathy, and Pfitiger has recently expressed his doubts as to the correctness
of Waldeyer’s neuron-theory; but if we may place reliance on Ehrlich’s
intra-vitam staining with methylene blue, and on the various modifications
of Golzi’s method, there cannot be any doubt that nerve-fibres have definite
terminations, and that Beale’s conception of ‘circuits’ is not tenable. But
though there be no direct continuity between efferent and afferent nerves
of the cord, there is an indirect continuity by way of the tissues which are
supplied with ‘motor’ and ‘sensory’ nerves. An equilibrium can only be
established by both motor and sensory reflexes.

“ Beale was partly led to his view by the important discovery that the nerve
cells in the central nervous system of man, the dog, and cat, show a distinct
fibrillation on being treated with dilute acetic acid. Not only did he trace
these fibrils from the dendritic processes to the axis cylinder process, but
also from one dendron to other dendra. As far as the arrangement of nerve-
fibrils in nerve cells of the spinal cord is concerned, no new facts have been
added to what Beale published 43 years ago, although by recent methods
nerve-fibrils can be made plain to those who have no special gifts for
microscopic work. .

“ Microscopical researches were to Beale a labour of love, and all who study
his directions, how to become proficient in this line of investigation, cannot
fail to be drawn unconsciously to follow the paths Beale travelled, and to
endeavour to achieve as much as he did.”

P. H. P-8:

ThE ik
eC eA uc cae tae

LK

PROCEEDINGS OF

THE ROYAL SOCIETY.

Szecrion B.—BrIoitogicAaL SCIENCES.

On Opsonins in Relation to Red Blood-cells.
By J. O. WAKELIN Barratt, M.D., D.Se. Lond.

(Communicated by Dr. C. J. Martin, F.R.S. Received August 11,—Read
November 22, 1906.)

(From the Lister Institute of Preventive Medicine. )

In a previous communication* it was pointed out that opsonins, whose
existence in relation to bacteria 1s generally recognised, are also produced by
injecting foreign red blood-cells into the peritoneal cavity. The latter
variety of opsonin, which is thermostable, not being destroyed by exposure
to 08° C. for 30 minutes, is taken up by red blood-cells of the kind used for
injection. Such red blood-cells, after being thus “ sensibilised ” by opsonin,
are ingested by leucocytes.

Since it appeared obvious that the occurrence of phagocytosis might,
under suitable conditions of experiment, be made use of in order to discover
if a serum were capable of sensibilising red blood-cells, that is, if it contained
opsonin corresponding to the red blood-cells employed, it was determined to
ascertain if, proceeding along the line of investigation thus suggested,
a method for the estimation of red blood-cell opsonins could be devised. It
was further decided to study the nature of the action of such opsonins upon
red blood-cells. |

Estimation of Opsonin.

In attempting to make use of phagocytosis for detecting the presence

of opsonin in serum a serious difficulty is at the outset encountered owing to

* J.O. W. Barratt, “The Phagocytosis of Red Blood-cells,” ‘ Roy. Soc. Proc.,’ B, 1905,
vol. 76, p. 524.

VOL. LXXIX.—B. B

Dr. J. O. W. Barratt. [ Aug. 11,

the occurrence of spontaneous phagocytosis. When the red blood-cells of the
rabbit, for example, are injected into the guinea-pig, opsonin for such red
blood-cells develops in the latter’s serum, which (after destruction of comple-
ment (alexin) by heating for 20 to 30 minutes at 58° C.*) may now be used
to sensibilise the red blood-cells of the rabbit. If the sensibilised red blood-
cells are added to the leucocytes of the guninea-pig, contained in the (heated)
normal serum of the guinea-pig, extremely active phagocytosis of the red
blood-cells at once takes place. If, however, sensibilisation is omitted,
phagocytosis is still observed, though in considerably diminished degree.
Attempts to overcome the disturbing effect of this residual or “ spontaneous ”
phagocytosis have not as yet met with a sufficient degree of success to enable
estimations of opsonin to be carried out in such cases.

There is, however, another class of cases in which spontaneous phagocytosis
does not occur. Leucocytes contained in the serum natural to them do not
ingest the red blood-cells with which they are normally associated.+ If, for
instance, the red blood-cells of the guinea-pig are injected into the abdominal
cavity of the rabbit and the serum of the latter is employed for sensibilisa-
tion, the leucocytes of the guinea-pig, contained in (inactivated) guinea-pig
serum are now seen to ingest such sensibilised red cells, though if sensibilisa-
tion is omitted no phagocytosis whatever occurs.

A serum of this kind was prepared from a rabbit, which will henceforth
be referred to as Rabbit A. The serum was used for the series of experiments
recorded in the tables.

Rabbit A—Weicght, 2300 grammes. Intraperitoneal injections were made
as follows :—

On the 1st day the red blood-cells of 7:5 c.c. of guinea-pig’s blood.

On the 11th day the red blood-cells of 4°5 c.c. of guinea-pig’s blood.

On the 21st day the red blood-cells of 4°5 cc. of guinea-pig’s blood. '

On the 118th day the rabbit was bled to death. The serum obtained was
inactivated by heating to 58° C. for 30 minutes.

An attempt was first made to ascertain if opsonin could be removed from
the serum of Rabbit A by the washed red blood-cells of the guinea-pig,
added in varying amounts and acting upon the serum (diluted and undiluted)
for periods of time ranging from five minutes to five hours (Table I). During
this time the mixture of serum and red cells was continually shaken, either

* Otherwise hemolysis of the red blood-cells occurs.

+ All the experiments with sensibilised red blood-cells, given in the tables, were
accompanied with control experiments on normal red cells. In these, as in numerous
other control experiments not referred to here, the results as regards phagocytosis were
invariably negative.

1906. | On Opsonns in Relation to Red Blood-cells. _ 3

by hand or in a motor-driven shaker. The mixture was then centrifugalised
and the supernatant liquid pipetted off (great care being taken to ensure the
complete removal of the red blood-cells).

The washed red blood-cells of 0:005 c.c. of guinea-pig’s blood were next
added to 1 c.c. of the deopsinated serum, and the whole agitated for two hours
at 37° C. The sensibilised red cells, after being washed in 0°85 per cent.
sodium chloride solution, were added in excess to the leucocytes of the
guinea-pig in inactivated guinea-pig serum and kept on a warm stage (37° C.)
for two hours. The leucocytes were then examined under the microscope
and the percentage containing red blood-cells noted, 2000 to 4000 leucocytes
being enumerated. The percentages thus obtained are only approximately
correct,* as will be readily understood by reference to Table III, but
by making a number of tests under conditions as nearly as possible identical
in respect of every detail, fairly concordant results are obtained.

The results, given in Table I, showed that it was possible to remove
opsonin from the serum employed, provided that a sufficient number of red
cells were used. After the red blood-cells of one or more parts of guinea-
pig’s blood had acted upon two parts of the serum of Rabbit A, the serum
had completely lost its power of sensibilising red cells; in other words, it no
longer contained opsonin in recognisable amount. If the red blood-cells of
one part of guinea-pig’s blood were shaken with four parts of serum, sufficient
opsonin was usually left in the serum to sensibilise red cells; this was
especially the case when the period for deopsination was short. In
Table I no indication of the degree to which opsonin is removed is afforded
beyond the fact that a period of five minutes is too short to effect complete
removal at 37° C.

The enquiry now presents itself, how much serum is required to sensibilise
the red blood-cells ofa given volume of guinea-pig’s blood? To investigate
this point the series of observations recorded in Table II was undertaken.

* The degree of phagocytosis obtained, apart from the degree to which sensibilisation
of the red cells has been carried, depends chiefly upon the uniformity and the closeness
of distribution of leucocytes and red cells. To secure the maximum degree of
phagocytosis the cells must be evenly distributed and the leucocytes must not be
separated by more than a few cell breadths from the erythrocytes, for leucocytes do not
appear to recognise the presence of the latter until their processes come in contact with
them. Difficulty was occasionally experienced owing to the occurrence of some degree of
agglutination of the red cells after treatment with the rabbit’s serum ; separation could
usually be effected by shaking, provided the proportion of serum employed was not too

great (ce, = not above 4, compare Table III). The condition of the leucocytes also

affects the degree to which the red cells are taken up. If a sufficiently extended series

of tests are made the condition of the leucocytes tends to become the same in each
experiment.

B 2

4 , Dr. J. O. W. Barratt. [Aug. 11,

Table I—Deopsination of Serum of Rabbit A, by means of the Red Blood-cells of
the Guinea-pig. Temperature of Experiment, 37° to 38° C.

No. of Dilution Bs Phagocytosis.f [The duration of the deopsinating action of the red blood-cells

expt. . S on the serum is given in brackets. |
serum.

[5 min. }.|(1Omin. ].}[20 min. ].| [40 min. ]. inr J. (1ahe. [2hr. |.|[3hr. ]. adel 0 het

i lin 8 $

2 | Undiluted| 4 Lis

3 Lins 2 = |

4 | Undiluted| + = |

5 - ie. = ox os p=

6 TS, 4 ids, |

7 1 in 2 5 = = 24) =

8 lin 8 al os

9 | Undiluted | _,, af |
10 43 Ri iuabece + + ~_ a |
ipl lin 8 » | ttt +++ = a
12 | Undiluted| ,, |.+++ +++ a Srasr + |
13 lin 8 Spe | cee oe ay ++ + |
14 |Undiluted| , | +++ _ = = |
15 lin 8 » | tat +++ +++ +++ |
16 | Undiluted | ,, + + ++ + Fs |
17 1in8 » | tet _ +++ +++ |
18 Undiluted ” _ +4 ay te |
19 1 in 8 ao _ me at |
20 By » | tet] test | ttt +++
21 22 »9 + + ++

* The washed red blood-cells of guinea-pig’s blood (B) were added to the serum of rabbit A (S) in the
proportions given. Thus, a= $ means that eight parts of guinea-pig’s blood and one part of the serum of the

rabbit were taken.

+ In order to test phagocytosis, the red blood-cells of 0°005 c.c. of guinea-pig’s blood were sensibilised
with 1 c.c. of the deopsinated serum, diluted or undiluted, and then added to leucocytes obtained from
the guinea-pig. The sign — means that no phagocytosis occurred ; + that 0°2 per cent. to 0°7 per cent. of the
leucocytes contained red blood-cells; + + that 1 per cent. to 1°5 per cent. contained red cells; + + + that
the maximum degree of phagocytosis was obtained (2 per cent. to 5 per cent., in one instance 10 per cent.,in
another 20 per cent.).

In these experiments the red blood-cells of gradually diminishing amounts of
guinea pig’s blood were added to a given volume of the serum of Rabbit A,
and the whole shaken for two hours at 37° C., the red cells being then
washed and tested as to their power of exciting phagocytosis. It will
be seen that so long as the ratio of the former to the latter exceeds 3: 1 the
red blood-cells are quite insufficiently sensibilised and fail to excite phago-
cytosis when added to the leucocytes of the guinea-pig. When the propor-
tion is 1:1, phagocytosis becomes marked,* and no further increase in the

* The highest degree of phagocytosis obtainable was found to be that in which 2 to
6 per ceut. of the leucocytes had taken up red cells at the end of 2 hours at 37° C. (after
2 hours little or no increase occurred). This range represents the variation to be expected

under the most favourable circumstances. Percentages of 20, 12 and 10 were occasionally
obtained, but were quite exceptional. )

(1906. | On Opsonins in Relation to Red Blood-cells. 5

amount of serum employed causes any recognisable augmentation of the
degree of phagocytosis obtainable. From this it may be concluded that, as
far as phagocytosis is concerned, this ratio represents complete sensibilisa-
tion. This does not, however, represent the maximum amount of opsonin
which can be taken up, for it appears, from Table I, that the red blood-cells
of one part of guinea-pig’s blood in some of the experiments removed opsonin
to such an extent from four parts of serum that the serum was no longer
capable of sensibilising red blood-cells, though in other experiments sufficient
opsonin for this purpose was still left. An explanation of the difference in
the amount of opsonin taken up in the two cases is probably to be found in
the circumstances that phagocytosis is essentially a surface phenomenon
depending upon the degree to which the outer part of the red blood-cell is
sensibilised, and no advantage arises from the opsonin acting upon the cell
in its whole thickness.

A striking fact becomes evident from Table II, namely that dilution of
the serum does not arrest the process of sensibilisation of red blood-cells.
When the red cells of one part of guinea-pig blood are mixed with one part
(or more) of serum, sensibilisation occurs and phagocytosis results equally
whether the serum is undiluted or is mixed with as much as 500 parts of
0:85-per-cent. sodium chloride solution.

(Bsa tae ;
The ratio noted above (3 = | as representing the minimum relative

amount of serum necessary for the production of marked phagocytosis
enables an idea to be formed of the delicacy of the test of deopsination
employed in Table I. If the red blood-cells of 0°005 c.c. of the blood of the
guinea-pig are mixed with 1 c.c. of deopsinated serum, and are found to
become so far sensibilised that marked phagocytosis is obtained, it follows
that the serum contains, after deopsination, at least 0°5 per cent. of the amount
of opsonin originally present. If, however, phagocytosis is found, on repeated
testing, to be always feeble or to be absent, it follows that less than this
percentage is present. By adding the red blood-cells of varying amounts
of guinea-pig’s blood to 1 cc. of the serum, and noting the point at which
sensibilisation becomes incomplete and phagocytosis feeble or absent—and, as
is illustrated in Table II, this point is indicated with a fair amount of
clearness if a sufficient number of experiments are made, the same technique
being closely followed in each—it appeared to be feasible to ascertain
approximately the degree to which opsonin had been removed from the serum
in question.

Influenced by this line of thought, the series of determinations in
Table III, which forms a continuation of Experiments 8 to 21 in Table I,

Table I].—Experiments to Determine the Degree to which Red Blood-cells must be
Rabbit A, and the Red Blood-cells

No. of
experi-
ment.

OU

oO oa ns DB DB BD OO

* Phagocytosis was tested by sensibilising the washed red blood-cells of guinea-pig’s blood (B) with the serum

the rabbit’s serum were taken.

6 Dr. J. O. W. Barratt. [Aug. 11,

Duration Phagocytosis* (the dilution
of action of
red blood- fee Peet) i |

cells on e B — 32 16 8 4 | 3 2
serum. S) a ae z x S :
j
2 hours. 37° C. |
99 3)
3) 9 |
99 9 |
39 92 |
|
” ” i
” ”
” ” ae Pe oo ++
(1in 14) | (1 in 7) | (1 in 7) (1 in 7)
29 ay Sar a2
é (1in 10)| (1 in 10) (1 in 7)
| ta
” 9 é a a
| (1 in 100) (1 in 100)

(a0) (1 in 10)
|

| 23 33

instance 9 per cent., in another 10 per cent.).

Fe
was carried out. Portions of the serum of Rabbit A, diluted with an equal
volume of 0°85-per-cent. sodium chloride solution, were mixed with the
washed red cells of guinea-pig blood in the proportion of 4:1, for periods of
time varying from 5 minutes to 3 hours and 20 minutes, and subsequently
tested for the presence of opsonin, as in previous experiments (A to D,
Table III). A second (E to H), and third (I to M) series of the same character
were also carried out, the serum being diluted to 1 in 8 and 1 in 7
respectively. The tests of phagocytosis were made in groups, sometimes
with diminishing amounts of the same serum (arranged horizontally in
Table III), as in Experiments 1 to 18, sometimes with serum deopsinated
for different periods of time (these experiments are marked with an asterisk

(1 in 256)

+
(1 in 512)

After sensibilisation the red cells were washed in 85-per-cent. sodium
0'2 to 0°6 per cent. of the leucocytes ingested red blood-cells; + + that 0°8 to 1°3 per cent. contained

sensi
‘were

1906. | On Opsonins in Relation to Red Blood-cells. 7

bilised in order to excite Phagocytosis. The Serum employed was that of

those of the Guinea-pig.

of the serum is given in brackets).

Nl
a 1 - i 2 1 a =e she
a TOE 2° 3 4 6 8 12 a°
+++ +++ | +++ +++ t+
(1 in 128) (1 in 64) (1 in 32) (1 in 16) (1 in 8)
+ + | + . + ++
(1 in 512) (1 in 256) (1 in 128) (1in 64) | (1in 32)
+++ +
(1 in 16) (1 in 16)
++ +++ | +++
(1 in 128) (1 in 64) (1 in 32)
+++ +++ +++
(1 in 256) : (1 in 128) (1 in 64)
+++
(1 in 8) |
+++ |
(1 in 16) |
+++
(1 in 32)
+++
(1 in 512)
+++
(1 in 5)
+ + + +
(1 in 10) (1 in 10) (1 in 10) (1 in 10)
+++ fs ++ fut oh
(1 in 100) (1 in 100) (1 in 100) (1 in 100)
| +++
(1 in 100)

of Rabbit A in the proportions given.

chloride solution and added to the leucocytes.

Ss

Thus, ee 32 means that 32 parts of guinea-pig’s blood and 1 part of

The sign — means that no phagocytosis was observed ; + that

red cells; and +++ that the maximum degree of phagocytosis was reached (2 to 6 per cent., in one

—

and are arranged vertically in Table III) as in Experiments 19 to 30.
In spite of the variations exhibited by individual experiments fairly definite
limiting values are obtainable for most of the sera. Thus in the experiments
with Serum A a sharp diminution of sensibilising power is seen when the
ratio B/S is less than 1/25, whence it is concluded that 25 parts of serum
after deopsination contain only as much opsonin as one part originally did,
that is to say, the opsonin has been reduced to 4 per cent. of the amount
present at the beginning of the experiment; with B and C a distinct but less
marked change in the sensibilising power of the sera is seen, indicating
reduction of opsonin to 2 per cent. and 1 per cent. respectively of the
amount originally present ; the opsonin in D is obviously reduced to less than

[ Aug. 11,

Dr. J. O. W. Barratt.

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1906. | On Opsonins in Relation to Red Blood-cells. 9

1 per cent. of its original value, but beyond this its titre cannot be further
defined from the experiments made; and similarly with the remaining sera.

The experiments in Table III show that a very rapid removal of opsonin
takes place during the first five minutes, and that subsequently the removal
of opsonin becomes considerably slower, some of the opsonin originally present
still remaining in Sera D and H even at the end of several hours. As would be
expected, the removal rate is seen to be more rapid with the more concentrated
serum (A to D) than in the more dilute serum (E to H, and I to M).

The experiments in Table III show that, when spontaneous phagocytosis
is avoided, the estimation of red blood-cell opsonin may be carried out.
Sera from different sources may be compared as to their opsonic content by
determining the minimum amount of serum required to sensibilise a given
bulk of red cells so as to produce the maximum degree of phagocytosis.
An alternative method is to determine the minimal bulk of red blood-cells
required to deopsinate unit of volume of the sera.

The Interaction of Red blood-cell and Opsonin.

‘It is not possible from the data above given to arrive at a definite
conclusion as to the nature of the process occurring when red blood-cells
are sensibilised by opsonin.

It has been already pointed out that even considerable dilution of the
serum of Rabbit A did not abolish sensibilisation so long as the ratio

iB
s = 1 was preserved. This circumstance is readily explicable on the

assumption that a chemical reaction takes place, but it is also equally well
explicable on the assumption that a physical process of adsorption or
selective solubility occurs. The relation between concentration and
adsorption has been determined by Ostwald* to be
OC, = aC",

where C, is the concentration of the adsorbed substance, Cs that of the
substance in solution, and a@ and 7 are constants. By suitably choosing
the values of a and x, the value of C; could be left practically unchanged
when Cz was reduced to 1/500 of its initial value.t So far as the behaviour
of opsonins in respect of dilution has up to the present been investigated,

* ‘Lehrbuch d. allg. Chemie,’ 1891, vol. 1, 2te Auflage, p. 1096.

t For example, if a = 1,000,000 and n = 1, in which case (since c = 1000000) aj] but

1x 10°¢ of the opsonin originally present in the liquid would pass to the red blood-cells.
If m were less than 1, the proportion taken up by the red cells would be still greater.
Unfortunately the estimation of very small percentages of opsonin requires much larger
amounts of serum than can be obtained from rabbits.

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1906. | On Opsonins in Relation to Red Blood-cells. 11.

the results obtained do not permit a conception of the nature of the
interaction of red cell and opsonin to be formed.

In a stoichiometric reaction which proceeds slowly, not only is it possible
to establish the molecular character of the reaction by means of determina-
tions of the reaction rate, but the degree of the reaction may also be
ascertained. The general formule for stoichiometrical reactions are

t—t.= klog C’ fC”
for monomolecular reactions, and
7 = RYO’ -1/(C’Y]

for multimolecular reactions, where ©’ is the concentration at time z’, C’’ that
at time ¢’’, is unity for bimolecular reactions, and & is a constant. The
truth of these formule has been established for homogeneous systems.
Whether they are applicable to heterogeneous systems as in the interaction
of toxin and antitoxin has been called in question. In the case of the
heterogeneous system formed by red blood-cell + opsonin, the data above
given enable the applicability of the formule in question to be tested
empirically. In Table IV, from the data furnished by Table III which are
repeated, the values of & are calculated: (1) as for a monomolecular
reaction ; (2) for = 1; and (3) for » = 2.* The values of & for the first
and third formule exhibit wide variations; the values for n = 1 show a fair
correspondence, the average of all the experiments being 00014, while the
highest and lowest values are 0:0008 and 0:0025 respectively. Thus the
striking result is obtained that in the heterogeneous system under considera-
tion a relation is exhibited similar to that observed in a bimolecular

stoichiometric reaction occurring in a homogeneous system.

Summary.

It has been shown that—

1. By employing phagocytosis as a test of the presence of red blood-cell
opsonin, and avoiding spontaneous phagocytosis by suitable conditions of
experiment, quantitative determinations of the opsonic content of serum may
be made.

2. In the experiments made, the interaction of opsonin and red blood-cell
proceeded at a rate corresponding to that exhibited in a bimolecular
stoichiometric reaction.

In conclusion, I must express my indebtedness to the assistance I have
received from Dr. Allan Macfadyen, in whose laboratory this research was
conducted, and from Mr. J. A. Craw and Professor Donnan.

* The concentrations of red blood-cell and of opsonin are regarded as equivalent, since
the opsonin is present in the minimum proportion necessary to lead to full sensibilisation
of the red blood-cells.

-

12

The Structure of Nerve-fibres.
By J. S. MacponaLp, the University of Sheffield.

(Communicated by Professor Sherrington, F.R.S. Received July 31,—
Read November 22, 1906.)

Frog’s nerve-fibres present very different appearances in different portions
of their length, when teased and examined in Ringer’s solution. The main
distinctions affect the contents of the myelin sheath. To avoid mis-
understanding I propose to speak of this portion of the nerve-fibre as the
“intramyelin material,” since the term more generally used (“axis-cylinder ”)
has now become so completely identified with new appearances produced in
this material by the action of reagents.

Briefly summarised the main differences observed are as follows :—

(a) In places the intramyelin material exhibits the perfect transparency
and homogeneity of a clear solution.

(b) In places this limpidity is clouded by a fine punctate granulation.

(c) At others the granular appearance is much more marked. The
individual granules are of different sizes, and vary from obvious spherules
to just perceptible points.

(d) In other places again large hemispherical vacuoles make their appear-
ance, their bases upon the myelin sheath and their convex surfaces pointing
inwards towards the central axis of the fibre. These marginally placed
vacuoles are situated at irregular distances from one another, and are of
irregular size. They lie now on this side, now on the other side of the fibre.
The remainder of the intramyelin material, the more central portion, is here
less translucent and more granular.

(¢) The vacuoles here have extended, and by their more numerous
formation and communication with one another have joined to form an
irregularly spiral channel. The central and less translucent portion of the
intramyelin material also presents the appearance of a spiral, as it bends
now to this point, now to that point upon the myelin sheath at places where
a separation from the myelin sheath has not yet occurred. The curves of
this irregular spiral are now short, now long. The material of which it is
formed also varies in diameter, more swollen here, more condensed there.
The central mass is much longer than the straight tubular myelin sheath
within which it lies.

(7) In other places this central coagulum is of more uniform diameter and
forms a straighter line. It has undergone a diminution in length and in
thickness.

The Structure of Nerve-fibres. 18

The evidence of this change of length is sometimes very complete.
Imagine a stretched elastic string placed in the long axis of a tube with
flexible walls. Release the string and so let its elasticity come into play.
As the string contracts, the flexible tube will become arranged as a spiral

- curled around the axial string. Such a figure would be the reverse of the

appearance described above in which a straight tube enclosed a spiral string.
I do not say that I have seen evidence of tension so complete as this, but I
have frequently seen something similar, if something less.

_ Thus when the process of teasing. has left nerve-fibres arranged upon the
slide in undulating curves, the clot which forms within the intramyelin
material sometimes shortens to a length less than that of the fibre. In this
case the clot does not follow the curvature of the fibre, but passes straight
across from one point of maximal curvature to another.

Sometimes this central coagulum may be seen to show the usual signs of
longitudinal strain in a viscous cord. It is faintly marked by fine lines in
the main direction of the strain, and has a finely fibrillated appearance
which has led to a belief that this central mass is formed by the agglutina-
tion of a number of originally separate “ neuro-fibrils.” There is, however,
nothing in the appearances seen in teased nerve-fibres to warrant such a
view. The most severe test to which this view can be put is to seek, in
terms of it, for an explanation of the alterations in length which this
central mass undergoes. Such an explanation would require not only an
imagination of separate fibrils, but also their new endowment with the
property of elasticity.

In nerve-fibres teased in Ringer’s solution these appearances are usually
distributed in a definite order. The immediate vicinity of the cut end is
granular, this granulation is succeeded by a tract of homogeneous intra-
myelin material, and then all the successive appearances due to a gradually
increasing degree of coagulation occur in the order given above. The order
in which the appearances are arranged is sometimes complicated by less
extensive and subsidiary sets of appearances of the same kind arranged
around interpolated nodes of. Ranvier.

This balancing of appearances around injured points and nodes of
Ranvier introduces a complexity into the description of the normal character
of teased nerve-fibres. If there is any hurry to identify some one of these
appearances as the “ normal” one, it must be remembered that teased nerve-
fibres are traversed by the “current of injury” and must be expected to
exhibit evidence of consequent polarisation.

14 Mr. J. 8S. Macdonald. [July 31,

Nerve-fibres Teased in “ Normal Saline” Tinged with Tolwidine Blue.

I have, in a previous note, described the manner in which these nerve-
fibres are affected by the addition of neutral red to the “normal saline” in
which they were teased. This dye characteristically stains all injured points
and some of the nodes of Ranvier. As time elapses it also stains granules
which form at gradually increasing distances from these points. The most
notable fact about neutral red staining is the slow invasion of the nerve-
fibre by this process of granule formation. When I first replaced this basic
dye by another one of an apparently not very different character—toluidine
blue—I was surprised by the complete difference in result obtained. It is
true that here and there some stained plugs of material were visible at:
injured points, but I failed to find any trace of the progressive granule
formation so evident in the other case. The characteristic effects of
toluidine blue staining were obtained in quite another region of the teased
nerve-fibres, and had a very different character. This dye stains all those
regions of the nerve-fibre in which the coagulative changes occur, whereas
neutral red yields a picture of a slowly progressing change gradually
affecting that first portion of the nerve-fibre in which the intramyelin
material is at first seen homogeneous and transparent.

In these regions of coagulative change toluidine blue gives rise to
appearances which vary pari passu with the extension, and with the
intensity, of the coagulation present. Regions such as in unstained fibres
would be definitely granular, are loaded with blue granules distributed with
much precision across and along the intramyelin material. Such granules
leap suddenly into sight over long stretches of the fibre, and are not
originated therefore by any process which can be described as an invasion.
The tract of developed granules is bounded on the side nearer to the injury
by a clear, unstained and homogeneous region, the line of demarcation is
sometimes remarkably sharp. On the other side the granular tract passes
into a region in which coagulative changes have led to the development of
a central rope now stained an intense blue colour.

This curiously complementary behaviour of these two basic dyes is there-
fore of obvious interest in mapping out two different tracts of the teased
nerve-fibre. It certainly does not diminish this interest that in case of either
dye an appropriate adjustment in the saline solution in which the dye
is introduced to the nerve-fibre may be made to overcome this dissimilarity.
If Ringer’s solution is substituted for “normal saline,” or, better still, if a small
quantity of sodium carbonate, 0°02 per cent., is added to the Ringer, or even
when the sodium carbonate is simply added to “normal saline,” or even when

1906. | The Structure of Nerve-fibres. 15

the basic dye is itself added in increased quantity, then toluidine blue may
be made to reveal the complete set of appearances. In these solutions the
invasion of the homogeneous region may be watched in a nerve-fibre in which
the sudden appearance of granule formation in more distal regions and the
intensely stained coagulum is also marked. If, on the contrary, Ringer's
solution is substituted for “normal saline” in the case of neutral red,
nothing whatever, or at the most only faint traces of granule formation are
obtained even in the region near to the cut end. The neutral red stain
is enabled to penetrate the whole nerve-fibre best when present in small
quantity, 0°01 per cent., and when presented in unboiled “normal saline”
rendered faintly acid by the presence of carbonic acid. Toluidine blue
solution must be rendered faintly alkaline, or, at least, the calcic phosphate
of the Ringer’s solution must be present to prevent a development of acidity.
Neutral red is, on the other hand, ineffectual in the presence of a reagent,
preventing the development of acidity, and can be made to stain the distal
coagulated stretch of the fibre only when presented in association with a
slight trace of acid. There is an obvious conclusion to be drawn from this
series of facts.

There is one region of the nerve which neutral red stains when unaided by
any addition of acid. This is the point of actual injury, and a limited region
in its neighbourhood which gradually extends with lapse of time. The
inference is clear, that this is a tract of the nerve in which a certain amount
of acidity develops, or, at least, that it is the least alkaline portion of the
nerve. The behaviour of toluidine blue in the same region is also seen to
support this inference. Here it refuses to stain until alkalinity is obtained.
This might lead to the additional inference, that the region of coagulation
which toluidine blue stains unaided is itself more alkaline than any other
tract of the fibre.

It would seem therefore that at present we had done no more than map
out the teased nerve-fibre into regions of homogeneity and of coagulation, into
regions of less and greater alkalinity, and also that we have associated these
two separate attributes. There is a homogeneous region close to each injured
point which is gradually invaded and rendered granular by an acid tide
sweeping in from the point of injury. There is a more alkaline region, in
which different grades of coagulation occur.

The Distribution of Potassium Salts in Teased Nerve-fibres.

I have already pointed out that Macallum’s reagent may be used to prove
the existence of potassium in every portion of the length of a nerve-fibre

16 Mr. J. S. Macdonald. [July 31,

provided only that the portion tested be first injured previous to the applica-
tion of the test. The circumstance of injury reveals the presence of a highly
concentrated solution of potassium salt within the intramyelin material,
which makes an immediate appearance at every injured point. The bizarre
appearances produced by this method of mapping out injured points had been
observed by Macallum, but was left completely unexplained by him. Their
relation to injury is, however, unmistakable when sought for. Since these
points of injury can be multiplied indefinitely so as to include the whole
length of the nerve-fibre, it follows that potassium really exists everywhere
within the intramyelin material in some manner hidden from the action of
the reagent, and that the processes attendant upon injury are capable of
presenting it in a new condition ready to take part in the reaction. I have
discussed the probable meaning of this fact in a previous communication, and
the important conclusions which can be drawn from it as to the nature of
nerve-function. |

A slight modification in the method of use of this reagent brings out further
points of considerable interest. For if nerve-fibres are teased in “normal
saline ” and allowed to lie in this solution for some time before being tested
with Macallum’s solution, the potassium solution is found much more widely
distributed. It is now found in all those regions of the fibre, in which
toluidine blue can be used to reveal its own characteristic effects. Under
these conditions potassium is discoverable at each. site of injury, and in a
neighbouring tract to which it has diffused from this site. Potassium is also
now detected in all those regions in which I have described the occurrence .
of coagulative change. We are, therefore, introduced to a new and very
important conclusion. Potassium salts are also discoverable in regions
of the fibre distant from points of injury, provided that time is given for
their appearance. 7

Although not situated at injured points this more distal region of the
fibre has a secondary relation to injury of a most interesting kind. This
relation is at once made evident on reference to Bethe’s observations upon
the staining capacity of polarised nerve-fibres.

Bethe has made use of an ingenious experimental method, whereby the’
“fixation” of a nerve is secured during the traverse of a polarising current.
The nerve is then prepared for examination under the microscope, and is
stained in toluidine blue or in some other similar basic dye. Prepared in
this way the fibres are found to exhibit a remarkable distribution of staining
capacity. The anodal region is pallid, the indifference point is studded with
granules, and the kathodal region is intensely stained. It will be seen that
this set of appearances is exactly similar to that which I have observed in

1906. | The Structure of Nerve-fibres. 17

teased nerve-fibres stained with the same dye. Now I have already produced
evidence in favour of the view that teased nerve-fibres are traversed by an
electrical current—their own injury current. Bethe’s observations enable me
therefore to identify this distribution of staining capacity in teased nerve-
fibres as due to polarisation caused by this current. The pallid stretch of
homogeneous intramyelin material is the anodal region of the self-polarised
nerve. The deeply-stained region, in which coagulative changes are observ-
able, is the kathodal region where the injury current leaves the nerve-fibre »
to traverse the external circuit of the salt solution. We can now return to
the evidence obtained as to the distribution of potassium.

Injury suddenly elicits the appearance of strong solution of potassium at
the injured point. Given time, this solution diffuses into the neighbouring
stretch of the nerve-fibre. This phenomenon is accompanied by an electrical
phenomenon, the injury current. The traverse of the injury current polarises
the fibre and gives rise to the appearance of a new mass of potassium
solution, accompanied by appearances characteristic of coagulation at the
kathodal region.

Macallum has more recently published an account of the distribution of
the inorganic chlorides present in nerve-fibres. With the assistance of
Miss Menten he has penetrated the false character, which the vagaries of the
slowly-diffusing silver nitrate confer upon their apparent distribution, and
has discovered the fact that the inorganic chlorides are everywhere present
in great quantity in the intramyelin material. The drawings accompanying
this paper also reveal a very remarkable addition to this statement of
chloride distribution. In most of these illustrations it is easy to observe
indications of effects produced by the nature of the reagents made use of.
The proportion existing between the diameters of the myelin sheath, the
intramyelin material, and the whole fibre, are alone sufficient basis upon
which to make this statement. The intramyelin material does not occupy
one-half of its usual relative cross-section. Where this is not the case, and
the right proportions are maintained, the authors describe the fibres as
swollen. Wherever, in the illustrations, this swollen, but normal, appearance
of the fibre is present, the distribution of the chloride coincides exactly with
the appearance of teased nerve-fibres stained with basic dyes, and also with
appearances obtained by the use of Macallum’s reagent for potassium.
I have no hesitation whatever in urging, upon this basis of evidence, the
necessity of conjugating these facts. The salt present is potassium chloride.
It is present in extraordinary quantity. It is present in some masked
position in the structure of the intramyelin material. From this position
there is direct evidence that it is enticed by injury, and by the kathodal state.

VOL. LXXIX.—B, Cc

18 , Mr. J. S. Macdonald. [July 31,

Staining Due to Salt Concentration.

We may now turn to another striking fact which has made its appearance
in the course of these investigations. Basic dyes, toluidine blue, and neutral
red have been made use of to map out the distribution of a material,
which, upon further examination, turns out to be potassium chloride. Set
forth in these direct terms, there is nothing surprising in this statement,
since both dyes can be “salted out” from their solutions by the addition of
potassium chloride. Neutral red is a most insoluble body when tested in
this manner, and it was for this reason that I selected its staining effects as
a guide to the distribution of the potassium chloride solutions in nerve-fibres.
Toluidine blue is more soluble, nevertheless it is easily capable of being
“salted out” by solutions of the concentration that my electrical experiments
have led me to anticipate. I have also since found that it is more easily
“salted out” by potassium chloride in the presence of small traces of alkali,
0-03 sodium carbonate. This latter point has an obvious bearing upon the
salting out of toluidine blue in alkaline cell-structures, and more especially
at kathodal points.

This definite coincidence between toluidine blue staining and results
obtained by the use of reagents precipitating potassium and “ chloride”
renders it possible at once to advance a general proposition of some interest:
All the appearances made evident in the nerve-fibre by the use of toluidine blue
are due to the manner in which this dye is salted out from rts solutions by
potassium chloride.

There is also a further point. The appearances which Bethe developed in
nerves fixed in alcohol, or in alcohol and ether, I have obtained in nerves
freshly teased in “normal saline.” Bethe’s results coincide with mine, and
must be equally accepted as providing a picture of solutions of different con-
centrations of potassium chloride. Nor is this to be wondered at. Potassium
chloride is only slightly soluble in alcohol, although extremely soluble in
water. The addition of alcohol, the rapid dehydration which ensues, and the
replacement of the water by alcohol, must be definitely expected to leave the
salts present as precipitates. Wherever it exists the potassium chloride is
precipitated practically in the same quantity as it was originally present in
solution. Alcohcl fixation, therefore, is precisely calculated to leave in situ
precipitates of salt for subsequent micro-chemical investigation with toluidine
blue and similar dyes.

It is impossible to leave this point without remarking upon the general
bearing of this conclusion. The nerve-fibre cannot be the only case in which
considerations of this kind are worthy of examination. Granules in the

1906.| The Structure of Nerve-fibres. | 19

~ cell-bodies of secretory cells, or in the bodies of nerve-cells, are no doubt
elicitable by similar means, which also have the same significance. It is
possible that Nissl granules are of this order of importance. Failing definite
evidence upon which to make any assertion, it must at least be said that
: the well-known appearances accompanying “chromatolysis” are extremely
: suggestive in character. They would seem to be the consequences of such
changes in osmotic pressure as would result from the sudden liberation of
salts into solution.

Neurofibrils.

It is now possible to deal with the phenomena observed in that tract of
the teased nerve-fibres which corresponds with Bethe’s indifference point
and its granules. In teased nerve-tibres this region is naturally much more
extended, since the polarising “injury current” has a comparatively small
value and the changes in the intrapolar region are more gradual. Throughout
the whole of this region dense granule formations occur, and these are of
such a striking appearance when examined with high powers of the
microscope as to absorb a major share of attention. In my own case their

fascination left me at first with scant respect for the polar changes described
above. To a certain extent this interest is justified when it is considered
as the region least involved in polar changes, and altered, therefore, only
from the “normal” resting condition in so far as it is involved in the
transmission of an electrical current. For the present, however, it is
sufficient to deal with the light which these granule formations throw upon
the artificial structures known as neurofibrils.

In a previous communication I reported the fact that in nerve-fibres
teased in saline solution [ had never seen structures bearing any very close
resemblance to the neurofibrils described as making their appearance in
nerve-fibres “fixed” and stained in special reagents. Since making this
statement I have, however, seen structures which cannot be described as
eranules, since their length is considerable and their thickness much too
minute, and which yet cannot be called “neurofibrils,’ since they are too
short, too irregularly arranged, and since they anastomose. The resemblance
which they bear to the “neurofibrils” described as found in fixed nerve-
fibres is very much that borne by the anastomosing networks of fibrils
described as structural components of some nerve-cells. Fibrils such as
this I have only seen in some few out of the many nerves that I have teased.
Much more frequent, but still only of comparatively occasional occurrence,
are structures conveniently classed as bacillar granules, shorter but more

c 2

20 The Structure of Nerve-fibres.

characteristically much thicker than the fibrils. The latter are always in
much smaller number in each unit length of the fibre. Other forms seen
far more frequently are short ovoid and spherical granules and flakes of —
the precipitated dye. When all these forms are present they occur in a
definite order. The fibrils are found in the first portion of this stained
region abutting upon the unstained anodal tract, then follow bacillar granules,
then ovoid granules always associated with tracts of the fibre in which
definite kathodal staining and coagulative changes are found. In this latter
region spherical granules may also occur, but they are placed not in the
central coagulum but in the clearer fluid surrounding it. In one and the
same portion of the kathodal region ovoid granules may be seen enclosed in
the central mass, flakes may be seen upon its surface, and spherical granules
may be seen in the clear fluid surrounding it.

To understand the meaning of these granule formations and this difference
in their character, it is essential to remember the manner in which they
are thus associated with different degrees of coagulation. At the distal
extremity of the homogeneous anodal tract is placed the region in which
the first approach to visible coagulation takes place. In this place there
is no separation between a core and a surrounding fluid. Each unit of
volume still contains almost its normal amount of uncoagulated material.
The threads of coagulum are still surrounded by an almost normal intra-
myelin solution. It is in this region, where “ solid phase ” is still surrounded
by a great mass of “fluid phase,’ that the fibrils are sometimes seen.
Before I had puzzled out the meaning of the adjacent unstained anodal
tract, in which no granule formation takes place except as the result of
diffusion from the source of the injury current, I was greatly exercised by
this constant position of such fibrils as I had found. The bacillar granules
probably represent a further stage of degradation, in which, however, the
main mass of material is almost normally fluid in character. In both these
cases the dye is precipitated upon the outer surface of threads of coagulum.

As we proceed into districts nearer and nearer to the kathode an opposite
set of conditions arises. The greater portion of the proteid material is
precipitated or coagulated as a “solid phase.” Within the interstices of
this mass there are, however, entrapped collections of solution. It is
probable that here the granules represent such collections within which
the dye is precipitated.

At one end of the scale of changes solid threads are stained upon their
surfaces. At the other there is a similar staining upon the surface of a
coagulated mass, and within this again are intensely stained vacuoles.

In no place are neurofibrils of indefinite length existent, simply by reason

The Influence of Increased Barometric Pressure on Man. 21

of the gradually varied conditions present in the district where coagulation
is taking place.

REFERENCES.

_ A. B. Macallum, “On the Distribution of Potassium Salts in Animal and Vegetable

Cells,” ‘ Journal of Physiology,’ vol. 32, pp. 96-128, 1905.

A. B. Macallum and Miss Menten, “On the Distribution of Chlorides in Nerve Cells and
Fibres,” ‘Roy. Soc. Proc.,’ B, vol. 77, pp. 165-192, 1906.

Albrecht Bethe, ‘‘ Allgemeine Anatomie und Physiologie des Nervensystems,” pp. 272-298,
Leipzig, 1903.

J. S. Macdonald, “The Structure and Function of Nerve Fibres,” ‘Roy. Soc. Proc.,’ B,
vol. 76, 1905.

.

AMD. Cnn,

The Influence of Increased Barometric Pressure on Man.—ll.

By Lronarp Hitt, F.R.S., and M. GrEENwoop, Jr., M.R.C.S., British
Medical Association Research Scholar.

(Received October 6,—Read November 8, 1906.)

In a previous communication, we gave reasons for thinking that decom-
pression symptoms may be avoided by maintaining a steady rate of
20 minutes per atmosphere during this process.

The actual time selected was purely empirical, being based on the statistics
of Caisson Works, Diving Operations, and Laboratory Experiments. It is
clear that a more scientific foundation would be obtained if we could determine
(1) the rate at which the tissue fluids are saturated with nitrogen; (2) the
rapidity with which dissolved gas escapes during decompression. The most
direct method would be to analyse samples of arterial blood under various
pressure conditions; but this is not, unfortunately, practicable in the case of
man. Another way is the examination of venous samples under similar
circumstances. This plan can be followed, and we hope to communicate
some results in another paper, but the technic is difficult and still in need
of improvement. A third line of research is the indirect determination of
the tissue gases, and this will be discussed in the present communication.

If a condition of diuresis be produced by drinking considerable amounts
of water, the profuse secretion of urine which results will afford some
measure of the dissolved tissue fluid gases. We have proceeded in the
following way :—The subject of the experiment drinks at least a quart of
a warm water, and, after an interval of 10 to 15 minutes, enters the pressure
chamber, the pressure being then raised a definite amount. Directly the
desired pressure has been attained, he empties his bladder. Ten minutes
later the bladder is emptied again. Samples of the urine passed are then run

22 | Messrs. L. Hill and M. Greenwood. Influence of [Oct. 6,

into glass bulbs with rubber connections, care being taken to drive out any
air bubbles that may be present. The rubber junctions are then clamped,
and the dissolved gas subsequently pumped out by means of Hill’s blood-
pump, the volume determined, and the mixture analysed by the potash and
pyrogallic acid method.

In this manner, a sample of urine, passed since the pressure limit had
been reached, was available for analysis, and a series of specimens could be
obtained. It was first, of course, necessary to determine the amount of gas
dissolved by the urine under normal pressure conditions. Six analyses
were made, and the mean volume of dissolved nitrogen per 100 c.c., reduced
to 760 mm. and 0° C., was found to be 1:14 c.c. The individual observations,
made at different times and under different circumstances, exhibit only
a small. range of variation, they are 1:21, 1:19, 0°97, 1:10, 1:31, 1:07, 1:14.
The coefficient of variability determined by one of us (M. G.) is only
20992138799: |

We are thus entitled to assume that, in our own cases, the coefficient
of solubility for atmospheric nitrogen under normal barometric pressure, and
at body temperature, is between 0:11 and 0°12.

We next determined the changes in the amount of dissolved nitrogen with
varying pressures. The following are the results of a typical experiment :-—

Expervment 31, 31.5.06.

M. G.—compressed to +30 lbs. in 10 minutes (2.50 p.m.).

The bladder was then emptied, and at 2.55 the bladder was emptied again,
135 c.c. of urine being passed, and Sample 1 was taken.

2.56—decompression commenced.

3.20 (+15 lbs.)—the bladder was emptied again (320 c.c.).

3.45—decompression completed, bladder emptied again (310 c.c.).

4.1—another sample taken (135 c.c. passed).

At 4.55 and 6.10 other samples were taken.

Analyses.
| Percentage | Calculated N per cent.,
| Sample. Time and pressure. of supposing urine
| nitrogen. | followed the pressure.
1 Passed after 5 minutes under 30 lbs. ............ 2 522 1l-1x3 =3°'3
2 and 3 | Were spoiled
a At normal pressure, 16 minutes after decom- 2 373 cig E
pression
5 70 minutes after decompression theunaus Os cis geet LOT, Re
6 Gdio mimiUtes ater aekescc cies seme ees fnccene cma 1 023 Lat

‘ya aoe
4

1906. | Increased Barometric Pressure on Man. 23
In another experiment the following were the results :—

Percentage | Calculated N per ‘eat,
Sample. Time and pressure. of supposing urine
nitrogen. | followed the pressure.

1 AS Ubs., immediately .......s.cescsssesseessesseseeees 2 “64 L‘lx4= 4:4 |
2 3 S mim Bbess ALES auld. s.nnctceseeecceveere 4,57 1l1x4=4°4
; 3 ven oD Pd a cht 4°69 l1x4=44 |
4 trig EE Te PRS tas POE Salt 4°35 11x4=4°4 |
5 iiilieg ie TAN ree eee ee 4517 l1lx4=44 |
; 6 At 30 Ibs. during decompression .............0000 4, *252 11x3=3°3 |
i

These results, amply contirmed by our other experiments (wide tables,
pp. 26 and 27), merit some consideration.

First of all, we note that an exposure of more than five minutes to
a positive pressure of 30 Ibs. is necessary in order to produce saturation ;
the mean of five samples taken under these conditions is 1:99, as compared
with 3°3, the theoretical value.

Saturation appears to be attained in 10 minutes,* however. Thus, the
mean of seven samples, taken not less than 10 minutes after attaining the
above-mentioned pressure, was 3°32. For a pressure of +45 lbs. the same
statement appears to hold, but the time limit is probably slightly lower, as,
on two occasions, full saturation was obtained in eight minutes. The mean
of eight observations at this pressure, for exposures of less than 10 minutes,
is 4:23, the theoretical value being 4°4.

The next point to notice is that the escape of dissolved gas is a much
slower matter than could be expected. Thus, in experiments at +3
atmospheres, samples were taken when the pressure had fallen during slow
decompression to +2 and +1 atmospheres. The means of 7 and 8
observations at these pressures were, respectively, 3°63 and 2°75, we, the
samples were super-saturated.

These results, however, are not surprising, since some of the urine taken
in the specimen must have been separated from the blood while under the
higher pressure, hence we could not assert, on the strength of these

‘ observations alone, that any definite retardation occurs in the equilibrating
process. But, if we consider the analyses made after returning to the
normal pressure, we find very strong evidence in support of such a view.
In the experiment recorded above, a sample passed sateen minutes after
decompression contained 2°373 per cent. of nitrogen, in another experiment
the amount was 1°66, the mean of 5 being 1°64.

r\

&,

ab
3
@

* Saturation of the kidney under the conditions of diuresis is probably far quicker than
that of other and less vascular organs.

24 Messrs. L. Hill and M. Greenwood. Influence of [Oct. 6,

Of samples passed immediately after decompression not one yielded less
than 1:64 per cent., the mean of 6 being 1:99.

We are thus led to conclude that, even with a decompression rate of
20 minutes per atmosphere, the excess of dissolved nitrogen has not escaped
entirely from the tissue fluids, even 15 minutes after decompression. As the
matter is of importance, we must examine the validity of our method a little
more closely. The accuracy of the analytical process itself was tested by sub-
mitting a sample of boiled water to a vacuum in the Hill pump and
proceeding exactly as in the urine analyses. No measurable gas was
obtained, so that the error due to leakage may be regarded as negligeable.
In any case, we disregard analyses in which, owing to the presence of
unusually large quantities of oxygen, we can reasonably suspect con-
tamination with air. In general, only very small quantities of oxygen
were found.

The admixture of urine that had collected in the bladder while the subject
was exposed to a higher pressure would lead to erroneous results, but the
complete emptying of the bladder at each observation avoids this source of
fallacy. All the urine of the samples passed 15 minutes after decompression
must have been separated from the blood after the pressure had fallen to
+ 0 lb. with the exception of the amount present in the tubules and
renal pelves, this latter quantity being so small in comparison with the total
volume (135 e.c.) passed, that the resultant error can hardly be appreciable.

That our experiments are not sufficiently numerous to admit of definite
statement as to the exact time relations of the process may be granted ;
but the qualitative accuracy of the conclusion seems unassailable.

We have obtained a striking confirmation of our view in the following
manner. At the caisson works now being carried out by the London
County Council in connection with the new Rotherhithe Tunnel, a positive
pressure of 14—17 lbs. is employed and only three minutes allowed for
decompression.* We have analysed samples obtained from six caisson
workers obtained directly after leaving the air lock, with the following result :—

Nitrogen.
LIS 1:595
2 1:658
3 1:506
4 1414
5 1°747
6 1°744
* With such low pressures there is little risk in so short a decompression period.

+ The men had some beer to drink and most of them passed water just before
decompression.

1906. ] Increased Barometric Pressure on Man. 25

It must be admitted that these observations indicate in a manner not
to be disregarded the extreme danger of rapid decompression from high
: pressures. It might even be doubted whether 20 minutes per atmosphere
: is an absolutely safe allowance; indeed, a few fatal cases, recorded in the
literature of caisson works, in which this time appears to have been allowed,
support such a contention. It may well be, however, that a certain steepness
of pressure-slope between dissolved and free gas is necessary for the formation

;

of bubbles sufficiently large to produce injury.

So far as our observations go, with a decompression rate of 20 minutes an
atmosphere the dissolved nitrogen falls in a given time to the same level,
whatever be the height of pressure attained during the experiment. That
is to say, the excess of dissolved nitrogen on returning to zero is no greater
after exposure to three atmospheres than after raising the pressure to two
atmospheres only. It would seem that the velocity of escape diminishes
from some critical point. If this be so, it would be an argument for retarding
the later stages of decompression in order that an equilibrium may be
re-established on returning to normal conditions.

The causes underlying these results remain for subsequent consideration ;
in the light of other experiments we have carried out, we think it possible
that a physical explanation will be found adequate to account for the facts,
but our investigations are not yet sufficiently complete to permit its
immediate statement.

A complete table of our analytical results is subjoined; the figures queried
are possibly vitiated by experimental errors, we have no reason to doubt
the accuracy of the others.

Our general conclusions from the present investigation are :—

(1) The body fluids of man exposed to compressed air absorb nitrogen in
accordance with Dalton’s law.

(2) Saturation of the body fluids is attained after exposure to pressures
of +30—45 lbs. for from 10 to 15 minutes.*

(3) Even with a decompression rate of 20 minutes an atmosphere,
equilibrium between dissolved and atmospheric nitrogen is not
completed 15 minutes after decompression.

* Our results agree in principle with those deduced by V. Schrétter from theoretical
considerations, wide ‘Sauerstoff in der Prophylaxie und Therapie der Luftdruckerkrank-
ungen,’ Berlin, 1904, pp. 35—39.

26 Messrs. L. Hill and M. Greenwood. Influence of [Oct. 6,

Table I—Urine Analyses from Hill and Greenwood’s Experiments.
Individual Observations.

N, per cent. calcu-
lated, supposing

Pressure. N, per cent. found. Means. urine fsllomen
the pressure.
STO OEE werner neni eases 1:21, 1519; 0:97, TAO CSI Ofeeeeeecn eee 1°14 1
+30) libs. for less | 2°52, 1:67, 1:83, 2:41, U5. oss. e. ccc. connects 1-99 3°3
than 10 mins.
+ 30 lbs. for more | 3°32, 3°49, 3°29, 3°22, 3°42, 3°94, 2°57 ...... 3°32 3°3
than 10 mins.
+45 Ibs. for less | 2°64, 4°57 (8 mins.), 5°68 (?) (8 mins.), 4-29 4, *4,
than 10 mins. 4°28
+45 lbs. for more | 4°54, 3°78, 4°52 (?), 4°69, 4°35, 4°57, 3°46, 4 +23 4, °4,
than 10 mins. 3°94:
+ 30 lbs. during de-| 4°01, 3°47, 3°52, 4°25, 3°68, 3°13, 3°33 ...... 3°63 3°3
compression
+15 lbs. during de-| 2°40, 2°78, 2°96, 3°16, 2°60, 3°04, 2°35, 2°73 2°75 2-2 -
compression
+0 lb. on decom- | 1°84 (4 mins.), 2°21, 1°70, 2°30, 1°64, 2°24 1-99 1:1
pression
+0 Ib. 15 mins. | 1°34, 1:37, 2°37, 1°66, 1°45 (10 mins.) ...... 1 64 1
after decom-
pression
+ O®lb- more ; than ||, 1°28, 1:07, 102 yew ae net nese aenese eee: 1°13 1‘1
15 mins. after
decompression
Table ILI.
A. Rotherhithe Tunnel Workers.
N, per cent. calcu-
lated, supposing
Source of sample. N, per cent. found. unine fallaned
the pressure.
Pra cummnel (LADS) cp icnt ewe! 2 BOs Li) Seencdcnsdetuaseceecaeteace se Neceen ame 2°35
After decompression (decom- , 1°60, 1°66, 1°51, 1°41, 1°75, 1°74, 1°31, 1°63 1h
pression time, 33 mins.)

Increased Barometric Pressure on Man.

B.* From Admiralty Diver C.

: N, per cent. calcu-
Depth lated, supposing
es Time below and N, per cent. found. urine followed
the pressure.

SO ECCE oo. .cs yearns Below 46 mins. 20 secs. Ascended in 6 mins. 5 secs.—
: Sample taken 20 mins. 30 secs. after ascent:
ING LC Savage nce. Saatchi tod ude cacao eld Mdeule ovaa riod veens « le D
| SEM eas sa <ne 0s Below 46 mins. Ascended in 5 mins.—
" Sample taken 11 mins. after ascent : Nj = 1°60 ......... ded.
ig OME Leavsiien sins Below 44 mins. Ascended in 6 mins.—
bs Sample taken 5 mins. after ascent: N, = 2°11 ......... 1
bs a 20 . ¥ ING == OR) enc. 08 Et
4 SU) yt bh ae Below 46 mins. Ascended in 43 mins.—
‘i Sample taken 4 mins. after ascent: N, = 2°50 ......... 1-1
di Ms 203 3 2 ING = SBR eee nds: Lt

* For these samples we are indebted to Dr. Haldane, Lieutenant Damant, and Mr. Cato.

i The experiments were carried out with the help of a Government Royal
~~ «Society Grant, and with the aid of apparatus placed at our use by Messrs.
Siebe, Gorman and Co.

28

On the Inheritance of Certain Invisible Characters in Peas.
By R. H. Lock, M.A., Fellow of Gonville and Caius College, Cambridge.

(Communicated by W. Bateson, F.R.S. Received October 1,—Read November 22,
1906.)

In a paper which appeared recently in the ‘Proceedings of the Royal
Society,’ Messrs. Bateson and Punnett and Miss Saunders showed that the
visible appearance of certain characters in Sweet Peas and in Stocks was
due to the association together in the same zygote of factors belonging to
independent but complementary allelomorphic pairs. If both the com-
plementary factors were not simultaneously present, no hint of the presence
of the characters in question was to be detected without breeding tests ;
nevertheless, the presence and absence respectively of such a factor constitute
a pair of allelomorphs, and segregate in complete accordance with Mendel’s.
law.

The present communication deals with a similar but somewhat simpler
phenomenon exhibited by certain colour characters of the testa of the
ordinary field or garden pea—the species to different cultivated varieties of
which the names Pisum sativum and P. arvense have been given. The case
is simplified by the fact that here only one of the two complementary factors.
is by itself invisible. The presence of the second factor is invariably
accompanied by the appearance of a definite visible character, whether the
invisible factor is present or not. The present examples of “latency” are of
special interest on account of the classical nature of the material, as well as.
because it has been suggested that Mendel was in error in his own account.
of the characters of the testa in peas. It may be stated at once that this is
not the case. Mendel’s account is perfectly correct so far as it goes. The
grey colour of the testa, constantly associated with coloured flowers and
coloured axils, is a simple Mendelian dominant to colourless testas coupled.
with white flowers and green axils.

The testa may, however, show other colours in addition to the well-known
grey. In Ceylon, in 1902, the writer found in cultivation a pea, the appear-
ance of which strongly suggested that it came very near to the primitive
ancestor of cultivated peas. We are concerned here only with the characters
of the testa. This showed an essentially grey ground colour (greenish or
yellowish), and, in addition, two distinct sorts of coloured markings :—
(1) small specks of a very deep purple colour, the presence of which may be
briefly denoted by the letter p.; and (2) a mottling or marbling of brown,

On the Inheritance of Certain Invisible Characters in Peas, 29

very distinct, and closely resembling the pigmentation which is characteristic
of the cultivated variety known as “maple”; the presence of this character
will hereafter be referred to asm. The grey ground colour may further be

~ denoted as g. In the absence of these three characters the testa of a pea

is commonly nearly colourless, and this absence of colour in the testa is
constantly associated with white flowers and unpigmented axils. The latter
group of characters we shall call w. The native pea with which the following
experiments were made bears, then, the characters mpg. Its flowers were
purple and its axils coloured.

At a recent meeting of the Royal Horticultural Society, Messrs. Sutton
exhibited a cross between a white seeded variety and what was described as
a possible wild type of P. satiwwum, seeds of which had been obtained from a
plant found in Palestine by Mr. Arthur Sutton. On examining the seeds of
this cross, I found, as I had expected, that they showed both the purple
spots and the maple marking on a grey ground colour.

Of the colour characters referred to, the grey is determined by a greenish
insoluble pigment contained in all, or almost all, the cells of the testa. The
purple pigment exists in the form of a coloured cell sap, confined to certain
large cells of the subepidermal layer. The brown pigment seems to exist
chiefly in the cell walls of the outermost layer of I-shaped cells.

Early in 1903. the Ceylon variety, already described, was crossed with an
English pea known as “Sutton’s Satisfaction,” having colourless testas. The
testas of the seeds borne by the resulting plants were all precisely like those
of the native parent—m., p., and g. are all dominant.

In F, only 25 plants were raised, which showed the following characters :—

M..J. M.g. DG. g- Ww.
11 2 6 2 +

In the case of one of the four plants with white flowers, the nearly
colourless testas exhibited a very faint brown marbling, like the “ghost” of
a maple character.

These few plants were clearly insufficient to allow of any deduction being
made as to the characteristic proportion of Fz Three of the above m.p.g.
plants, however, yielded once more all the different types in Fs, being, there-
fore, clearly of the same heterozygous constitution as their parents;
169 plants of this description included the following :—

M.p.J. M.Je pg. g- W.
82 20 21 9 37

And the offspring of the remaining plants showed nothing to contradict the

30 Mr. R. H. Lock. On the Inheritance of [Oct. 1,

following theoretical explanation, which was published as a provisional
hypothesis in 1904.*

[ regarded m., p., and g. as each depending on the presence of the dominant
member of a separate pair of allelomorphs A—a, B—b, and C—e respec-
tively, C representing the grey character. And I further supposed that
A and B, though present, were unable to lead to the appearance of m. and p.
in the absence of C. B (p.) was totally inhibited in this way, whilst A might
just make its presence known by the appearance which we have described as
that of “ ghost” maple.+ !

In the accompanying diagram the combinations of the three pairs of
allelomorphs are set down at length, and it will be seen that, on the above
hypothesis, they lead to the proportion—

M.9.g. m.g. ViXCE g: W.

27 9 9 3 16 |

with which the result actually obtained and given above is in very fair

agreement. The expectation is 71.3, 23.8, 23.8, 7.9, 42.2, and the observed
numbers were 82, 20, 21, 9, 37.

AbC aBC | abC | ABc | Abe | aBe

| ABC abe

ABC{ ABC | ABC | ABC |} ABC ABC | ABC |} ABC | ABC
ABC | AbC | aBC | abC | ABe Abe | aBe | abe

ARO ARCO Ace ne lee! Ebon a nCalmacen inet
ABC | AbC | aBC | abC ABe _ Abe aBe abe

aBC | aBC | aBC | aBC | aBC | aBC | aBC | aBC | aBC
ABC | AbC | aBC | abC | ABe | Abe | aBe | abe

os

ab | abO Uab@allabO a] sbOullap@ulcabemleanG lane
ABC | AbC | aBC | abC ABe | Abe aBe abe

ABc | ABe | ABe | ABe | ABe | ABe | ABc | ABce | ABe
ABC | AbC | aBC | abC | ABc | Abc | aBe | abc
aBe } aBe | aBe aBe aBe aBe | aBe aBe aBe
ABC |} AbC | aBC | abC ABec | Abc | aBe abe

Abe | Abe | Abe | Abe | Abe | Abe | Abc | Abe | Abe
ABC | AbC | aBC | abC ABe | Abe | aBe | abe

On this supposition some of the whites must contain m. in a latent (or
partly latent) condition—Abc; others contain p. completely latent—aBc ;

* © Annals of the Royal Botanic Gardens, Peradeniya,’ vol. 2, p. 333.

+ Subsequent experiments showed that the non-appearance of the “ ghost” maple did
not necessarily imply the absence. of A. The degree of development of this faint brown
pigmentation on the testas of plants with white flowers seemed to depend to some extent
on the manner of ripening, and to vary from complete invisibility up to a fair degree of
brightness, and this sometimes in different seeds of the same plant.

‘
~
%
”
i

1906. |] Certain Inwsible Characters in Peas. 31

others again contain both m. and py—ABc. Finally, one white out of 16
contains neither—abe. ;

It seemed so certain that this was the true explanation that, in spite of the
fact that no case of the kind had then been worked out, I ventured to predict,
in the summer of 1904, that, when crossed with the simple grey type, some
of these whites would yield plants bearing purple spotted seeds—p.g.; others
seeds with the full brown marbling—m.g.; others again with both—m.p. ;
whilst some I expected to give only grey, like the grey parent used to
render visible the hidden factor, if present.

The actual crosses were carried out in 1905 upon plants of the fourth
generation (derived from heterozygotes in F2) with the result set down in
Table I.

One or more plants were raised successfully from each of 62 crosses, one of
the parents having in each case exhibited grey testas and the other white
(with or without the “ghost” maple). Hach cross is entered twice, once
according to the presence or absence of the purple character among the off-
spring, and once according to the presence or absence of the full maple
character.

Considering first the purple character, nine of the white-flowered plants
examined are found to have been heterozygotes in respect of the hidden
factor, whilst 51 plants gave rise to offspring showing only the dominant or
only the recessive character. The offspring were too few in number to allow
of exactly determining how many of the parents were truly homozygous, but
it is clear that a considerable majority must have been of this nature. The

Mendelian expectation in the second generation of descendants from a

heterozygote is a ratio of 3 homozygotes to 1 heterozygote.

In the case of the presence and absence of the maple character the result
was closely similar. Thus the latency of m. and of p. in white flowered
plants is demonstrated on a considerable scale, and the proportions in which
they appear is not inconsistent with precise Mendelian segregation.

A similar result was obtained in the case of the offspring of a cross made
by Mr. Bateson between Maple (m.g.) and Victoria Marrow (w.). On learning
that I was engaged upon the experiments already described, Mr. Bateson very
kindly handed over to me the crop obtained from this cross in F».

In F, the maple character was described as being simply dominant. In F2
there appeared, m.g. 38, g. 12, w. 19, which is very close to the expected ratio
of 9:3:4. Some of the seeds produced by two of the white flowered plants -
showed just the faintest possible trace of “ghost ” maple.

In this particular instance the “ ghost ” maple made its appearance, even in
later generations, in only a small number of those plants which could be

32 Mr. R. H. Lock. On the Inheritance of [Oct. 1,

shown to bear the latent factor for maple marking; m. is in fact almost, but
not quite invariably, completely latent.

Offspring of all the 19 white plants were crossed with members of various
pure strains of grey and purple seeded peas, in all of which maple marks
were wanting. Table II shows that seven of the F; plants examined were
heterozygous in respect of the hidden factor, whilst 22 yielded only dominant
offspring, and seven only recessive.

In the light of these experiments 1t becomes extremely probable that the
hidden factor—the cryptomere—comes always from the white flowered parent
and not from the coloured, as Tschermak supposed* in the case of several pea
crosses where reversion occurred in Fj, followed by the appearance of the ratio
Fo) ving.

To the behaviour of the maple character as thus described I know of no
exception. The purple character, on the other hand, does not, as it would
appear, Invariably follow the simple rule obeyed by this character as exhibited
by the coloured native pea of Ceylon. Hitherto among extracted whites
derived from the cross between the Purple Sugar Pea and different white
strains I have been able to find no example which does not yield exclusively
purple offspring when crossed with grey.

Table I.

Note.—57.1, 57.2, etc., indicate different plants of F, derived from seeds of the same plant
(No. 57) of F;. It will be seen that some such families include all dominant members, others
all recessive, and others again a mixture.

1g F;. F,. Jt
White (SS White eae A—__—
parent Coloured p. p. parent Coloured mM. mM.
number. parent. present. absent. number. parent. present. absent.
DS Ogee x) de 2 2 Bia x sige 2 2
622° =X “9. 3 3 397 fies Lp n> 1 1
G32 XG: 1 4 Bia. x ge 1 i
64.10 x 4g. 3 4 553) ox MG: 1 4
65.2 éx* 4g. 2 1 Olt b, GXe BOs if a
65.6 x 4g. 2 2 G31e xi) 5g: 3 2
6518) Tux, J. 2 3 --
66.4 ©“ g. 1 2 5 9 14
66.8 x 4g. 5 2
9 21 23

* © Archiv fiir Rassen- und Gesellschafts-Biologie,’ vol. 2, p. 672, 1905.

Certain Invisible Characters in Peas.

F,. ; F,, F;.
(a ‘ White a_i
; parent Coloured = p. Pp. parent Coloured m. m.
number. parent. present. absent. number. parent. present. absent
Bae xg. 2 — 638.4 ©“ 4g. 2 _—
wa x 9g. 4 aa 63.9 iaxtnecag. 3 —
5 a Zare P 2 — 64.2 §Xoucy, 3 —
Bing x “ g. 5 — 64.3 x. g. 5 —
57. 5 x g- 3 — 64. 4 x - Gg. 3 a
eh, xk. Gg. 5 = 64.5 x“ 4g. 6 —
57.10 x 4g. 2 = 64.7 x«K 4g. 3 —
Dae2 =X Gg. 1 =* 64.8 ©. o+g. 3 =
COrl x 4g. 2 as 64.9 ‘* 4g. 6 —
COZ x Gg 4 = 64.10 x 4g. vi Sas
60.3 x“ 4g. 4, Bee 65,1 xo; g. 2 —
61.38 ¢*“ 4g. 5 a Gos 2a Xe ag. 3 =
62.4 ™‘“ 4g. 5 = 65.3 ™‘ 4g. 3 —
G24 x“ 4g. 2 = Ghee. gs 7 —
62.7 © 4g. 6 <= Gb:6 1x 6g, a a=
G28 xX 4g. i — 65:8 x. g. 5 =
63. 6 xX Gs 2 == 66. 2 Ken ge 3 —
63. 9 xe g. 3 = 66. 3 se og 4, ee
64.2 ‘* g. 3 — 66.4 ™%“ 4g. 3 ae
64. 3 x ¥g. 5 = 66. 5 % gh 5 —
64.4 ‘%“% gp. 3 == 66:6 x 4g. 3 —
G45 x 4g, 6 a 66.7 |) og. 4 —
64.9 ™“ 4g. 6 a 6658 «x! + g. 7 Lae
G6.5° x 4g. 5 === 6659) -axdl> og; 2 —
GG;9- x 4g. 2 == 69. 1 > a 2 ~~
69.5° sal) g. 4 —
23 63.6." *%') Gg: 4, —
692.9 x 4g. 4, —
58. 3 x g. — 2
58. 4 Kit Gi — 2 Ze
bB.5 =X ~~ g. — 6

Bost - xX, g; —- 1 DAGUt \. Xe rege = 3
Boo xX 86 Ge — 4 SiO ox eG: — 2
59.38 ©“ 4g. — 4, BG; 2 = 4 9. — 1
69.4 x -g. = 5 58.38 ©“ 4g. = 2
59.5 ™*“ 4g. == 3 8.4 | Ki: ge = 2
UG etx gg. — 2 58. 5 Ko, — 6
Oo =x 4g. — 6 58.6 x: 4g. — t
59. 8 x I: — 2 59. 1 x 9g: — 1
59.9 x On — 3 59. 2 x Gh — 4
G21 x 4g, — 3 59.3 x 4g, = +
6253 * 4g. — 4 59.4 ™%“ 4g, — 5
638.4 é« 4g. — 2 59.5 x“ g. — 3
64. 7 x gs — 3 59. 6 Ken Ig? a 2
64. 8 x g. = 3 59. 7 x 9g. —_ 6
Gai x . g. — 2 5S «x — g. = 2
G5.3. xg. — 3 59:9 ox . Gg, _ 3
65.5 ™%“ 4g. — ih COs x. 6, —- 1
66. 2 x g: — 3 60. 2 x g- — 4,
GOs3. X | Gs = + 60;3° x g. = 4,
GGG" x 4g. — 3 GIs x ge — 5
GONs =) x |g. = A G25 x) ig — 3
Oot. x 4g, — 2 6252. xX == 6
69.5 x 4g. — 4 6256 xX 4g. — 4
G76. <%=: g. — 4 62.4 ™%“ 4g. = 5
Sooo x | Gg. — 4 62.4 éK“ 4g. — 2
62:7) x. g: — 6
2 28 62585 XG, = 1
Fs 63. 6 x g. = 2

‘ 27

VOL. LXXIX.—B,

34 On the Inheritance of Certain Invisible Characters in Peas.

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35

On the Occurrence of Enecystation in Trypanosoma grayi Novy,
with Remarks on the Method of Infection in Trypanosomes
generally.

By E. A. Mincuin, M.A.

(Communicated by Professor E. Ray Lankester, F.R.S. Received November 3,—
| Read November 8, 1906.)

In a tsetse-fly dissected and examined by me in the laboratory of the
Sleeping Sickness Commission at Entebbe, Uganda, it was found that not
only was the intestine swarming with 7rypanosoma gray, but the proctodeum
also contained vast numbers of trypanosomes. Under moderate magnification
they could be seen in dense clumps attached to the wall of the proctodeum,
each clump having a superficial resemblance to a patch of mould, the whole
mass, however, vibrating with the movements of the flagella. It was rather
uncommon to find these parasites in the hind gut, and I at once made smears
from different regions of the digestive tract, and carefully preserved and
stained them. As it was but a short time before my departure from Entebbe,
I was not able there to do more than glance at my preparations, but I noticed
at once an important fact, which since my return to England I have been
able to confirm and extend by careful study of my slides: namely, that in
the hind gut the trypanosomes are in process of becoming encysted. Before
proceeding to describe the encystment, I will say a few words about the
conditions of the occurrence of these trypanosomes, and the manner in which
they were preserved.

The tsetse-fly in question was one of a batch caught by our fly-boys
at Entebbe on November 2, and fed the next day on a monkey infected with
Trypanosoma gambiense from the cerebro-spinal fluid of a Sleeping Sickness
patient. On every subsequent day these flies were fed on a healthy guinea-
pig, and a certain number of flies were dissected daily and examined, until the
batch was used up. Without going into details, it is sufficient to say that
I’. gambiense was found sparingly in the flies dissected up to 96 hours, that is
to say, on November 4, 6, and 7. After this date 7. gambiense disappeared
completely and could not be found in any of the flies dissected. The fly in
which the encystment was discovered was dissected on November 14. It
must, of course, have been infected with 7. grayi when caught, and, as I
have stated, it had been kept 11 days in the laboratory and fed daily. At
the autopsy it was found to be gorged with blood, and with all its organs
perfectly normal and healthy in appearance. I examined the salivary
glands and genital organs (testes and vesicule seminales) without finding

D2

36 Mr. E. A. Minchin. On the Occurrence of — [Nov. 3,

anything resembling a trypanosome, Finally the digestive tract was
exainined, with the results stated. No trypanosomes were found in the
proventriculus. |

The films were made in the usual way by drying the smears, which were
then fixed with methyl alcohol and stained with Giemsa’s stain. A few were
kept unfixed and have been fixed and stained for me recently by my assistant,
Dr. J. D. Thomson, with excellent results. It is a matter of great regret to
me now that none of my smears of this fly were fixed by the osmic-vapour
method. The method of drying smears is a drastic one, which is likely to
deform finer details, and in the present instance the cyst-wall is often injured,
being evidently of a soft consistence.

The gut was divided into four regions and smears made from each. In the
anterior region of the intestine the ingested blood is of red colour, very
thick, and jelly-like and difficult to smear out nicely. Further back it gets
more fluid and begins to turn black. In the hinder part of the intestine the
blood is black and fluid. I refer to these three regions briefly as the red,
red-black, and black blood respectively. The black blood stops sharply and
suddenly at the point at which the Malpighian tubules enter the gut. The
proctodeum contains no blood, but only a yellowish fluid containing
innumerable coarse granules.

I proceed now to describe briefly the process of encystation observed by
me in the fly. A glance at the preparation shows many different stages of the
process side by side. In the first place we find individuals in which encystment
has not begun (figs. 1,2). These are forms for the most part very slender and
smaller than any of the forms of 7. grayi found in the intestine of this or
other tsetse-flies ; but their most striking feature is the absence of any distinct
undulating membrane, so that they bear a great: resemblance to the genera
Herpetomonas and Crithidia, especially the latter. The flagellum is long and
appears to run down the side of the body. The blepharoplast has the large
size and rod-like form characteristic of 7. grayi. The nucleus is either
compact or broken up into granules of chromatin. Division stages have been
found, but are very rare.

In the first stages of encystment the flagellum becomes shortened and stains
more deeply, suggesting that as it diminishes in length it becomes thickened.
At the same time the cyst begins to appear as a layer of substance which
stains reddish with Giemsa, forming a cap at the hinder pole (figs. 3, 4).
These two processes continue until on the one hand the flagellum is com-
pletely retracted and on the other hand the body is enveloped in a pear-
shaped cyst, at first incomplete towards the pointed end (figs. 5,6). The
flagellum appears to become retracted into a pink-staining vacuole (fig. 5,

>.
2
*.

1906. | Encystation in Trypanosoma grayi Novy, etc. 87

ji. v.), which reminds one of the flagellar vacuole described by Leishman
during the formation of a flagellum in the Leishman-Donovan bodies in

cultures, but here the sequence of events is inverted, as it were. Finally

the pink vacuole disappears, but a streak, which gradually fades away, can be
seen for a time in front of the blepharoplast (fig. 6). The cyst closes up
round the pointed end of the body, and then changes in form, becoming
first oval (fig. 7) and then irregularly circular in outline (fig. 8). In the
circular cysts, which are the last stage, it is difficult to make out much detail,
but in favourable examples they can be seen to contain hyaline protoplasm,
staining faintly bluish, with the dark purple-stained blepharoplast and the
nucleus generally represented by irregularly scattered granules, staining a
purplish-red tint. The cyst-wall also stains strongly and appears slightly
redder than the nucleus.

Fie. 3. Fia. 4.

Fia. 5. Fic. 6.

Fig. 8.

I will now state briefly the conditions observed in other parts of the gut
of the fly in which the cysts were found. The red blood was found teeming
with trypanosomes, nearly all large forms of indifferent type. Very few
young forms were found, and male forms were very scarce. No pronounced
female types were found in this region, though many of the large indifferent
forms approach the female type in the shortness of their free flagellum. In

38 Mr. E. A. Minchin. On the Occurrence of [Nov. 3.

the red-black blood the conditions were similar, but young forms are becoming:
commoner. In the black blood young forms predominate, and large individuals
are comparatively infrequent. Thus in the gut of this fly as a whole we find
great rarity of differentiated sexual forms, but a swarm of indifferent forms
which in the hinder part of the intestine give rise to very numerous young
forms, and these in their turn would appear to pass into the small,
Herpetomonas-like forms found in the proctodzum, starved-looking creatures
which, in a medium where there is probably no nutriment, go through the
process of encystment already described.

Having so far confined myself strictly to matters of fact, I will now offer
a few suggestions and speculations as to the probable significance of the
encystation in the economy of the life-cycle of the parasite. In the first
place, the resemblance of these cysts, and especially the pear-shaped forms,
to the “Schleim-Cysten ” described by Prowazek* in Herpetomonas musce-
domestic, Burnett, is very marked, and has struck everyone to whom I have
shown both my preparations and Prowazek’s figure. I think there can be no
doubt that they are similar bodies, and have a similar function, that is to say,
that they are destined to pass out of the gut of the fly with its dejecta. Very
numerous analogies in support of this inference could be cited from other
parasitic Protozoa. The question which interests us most is: what becomes
of them after being cast out from the fly? In the absence of any observa-
tions or experiments upon this point, one can only draw conclusions from the
analogy of what is known in other cases. In the case of H. musce-domestice
the cysts are scattered about everywhere, and, as house-flies are far from
being particular in their feeding or sanitary in their habits, a fly runs every
chance of infecting itself with the Herpetomonas by swallowing with its food
cysts dropped by another fly. There can be no doubt whatever, it seems to
me, that this is the manner in which the house-fly acquires the infection of
the Herpetomonas. ‘The case is, however, very different with the tsetse-fly,
which does not haunt the enclosed spaces and insanitary surroundings of the
house-fly, but lives a free, open-air life by lake and forest. Nor is the
tsetse-fly a foul feeder like the house-fly. My assistant, Mr. Degen, and
myself made many attempts to feed tsetse-flies on all kinds of food, but
always without the least success. On the other hand, the tsetse-fly is
a greedy blood-sucker, and will attack anything from a frog, lizard, or bird
to a hippopotamus, but, in my opinion, it does not feed in any other way.
Further, the tsetse-fly is not like the Stomoxys, which lays its eggs in dung;
it is viviparous, as is well known, and nourishes its larva in the uterus until’
full-grown. )
* ¢ Arbeiten a. d. k. Gesundheitsamte,’ Berlin, vol. 20 (1904), p. 446.

—
s

1906.] - Encystation in Trypanosoma grayi Novy, etc. — 39

' For all these reasons it seems to me in the highest degree improbable,
indeed I may say impossible, that a tsetse-fly would ever infect itself by
sucking up cysts dropped by another fly, or that a parasite which had to
depend on this method of dissemination could maintain its existence in the
tsetse-fly. The only possible destiny I can imayine for these cysts is to be
swallowed accidentally by some vertebrate, the (as yet unknown) host of
Trypanosoma gray, in order to germinate in its digestive tract, to pass
thence into the blood, and to be taken up again with the blood by the
tsetse-fly. A cycle of this type is as yet unknown, but there are abundant
analogies for all parts of it. In the first place, 1t is a common thing for
animals to have protozoan parasites in the gut, which they take up in the
encysted condition after they have been dropped by another individual.
Without multiplying instances unnecessarily, I may point out that Schaudinn
proved the infection of Ameba coli to originate in this way, and that it is
a common human parasite in regions where sanitation has not advanced
beyond the primitive condition of ¢pandage par terre. In the second place,
there are many instances among Sporozoa of cysts germinating in the
intestine and liberating motile forms which then pass through the wall of
the gut into other organs of the body.

In a former communication by my colleagues,* Lieutenants Gray and
Tulloch, and myself, we were able to confirm Bruce’s results as to the
existence of direct mechanical infection by means of the tsetse-fly, which, if
it stabs its proboscis first into an infected animal and then soon after into ~
a healthy one, can infect the latter. We were not able to demonstvrate,
however, what I may term cyclical infection, which at present has not been
shown to exist. I suggest that there are two possible modes of cyclical
infection, in the dissemination of protozoan blood-parasites by biting insects
generally. In one method, which I may term inoculative, the parasite, after
going through developmental changes in the insect, passes back again into
a second vertebrate host through the proboscis, as in the case of malaria
transmitted by a mosquito. In the other method, which I propose to term
contaminative, the parasite taken up by the biting insect, after going through
developmental changes within its gut, would pass out of it through the anus,
and infect the vertebrate host by contaminating its food or drink. We have
all of us (I speak for myself) been imbued hitherto with the idea that the
cycle of the trypanosome in the tsetse-fly must be of the inoculative type,
and have failed to find it. I wish to suggest strongly to those working on
the subject of trypanosome-infection the desirability of making experiments
and observations to prove or disprove the existence, in the insect which

* © Roy. Soc. Proc.,’ B, vol. 78 (1906), p. 242.

40 Occurrence of Encystation in Trypanosoma grayi Novy, ete.

disseminates the parasite, of a life-cycle which results in a contaminative
infection of the vertebrate host.

A full account of the trypanosome-cysts described in this note, and of
other points relating to Trypanosoma grayi, will be published, with
illustrations, in the ‘Quarterly Journal of Microscopical Science.’

DESCRIPTION OF THE FIGURES.

Figs. 1 and 2.—Free-swimming Herpetomonas-like forms of Trypanosoma grayi from the
proctodeum of the tsetse-fly, before encystation has commenced; W., principal
nucleus ; 2., blepharoplast ; 7., flagellum. x 2000.

Fic. 3.—Commencement of encystation. The flagellum is becoming retracted, and the
first appearance of the cyst-secretion is seen at C. x 2000.

Fig. 4.—Similar stage, slightly more advanced. The cyst-wall (C) is damaged at one
point. x 2000.

Fic. 5.—Flagellum completely retracted, represented by a vacuole (77. v.). x 2000.

Fie. 6.—The flagellar vacuole is absorbed and the flagellum is represented only by a
delicate streak (fl. s.). Cyst much damaged. x 2000.

Fic. 7.—The cyst-secretion now extends all round the body. x 2000.

Fie. 8.—Rounded-off cyst, the final stage. Letters as before. x 2000.

41.

On the Transpiration Current in Plants.

By Henry H. Drxon, Se.D., Professor of Botany in Dublin University, and
Director of Trinity College Botanic Gardens.

(Communicated by Professor J. Joly, F.R.S. Received November 7,—Read
December 6, 1906.)

In a paper* published in 1905, Professor Ewart investigates the question
as to what force is required to move water through the waterways of plants
at the same velocity as the transpiration current. His general conclusion
is that the resistance is so great that neither are sufficient forces generated
in the leaves to raise the water at the required velocity, nor is the tensile
strength of water adequate to transmit these forces downwards, if such
existed. In a subsequent paper he seems to have modified this latter view
and quotes Berthelot as stating that air-free water may support a tension of
over 200 atmospheres.t
It is needless to criticise Ewart’s calculations of the resistance based on
Poiseuille’s formula, which he himself admits is quite inapplicable to the case,
owing to the presence of cross-partitions and irregularities in the cross-
sections of the tracheal tubes. It may be noticed, however, that when care
was taken that discontinuities were not present in the water columns of the
wood experimented upon, the flow observed approximated to the flow
calculated by the formula. In an experiment on a piece of yew wood the
approximation was very remarkable. The actual amount transmitted} through
a length of 15 cm. was 4:2 c.c. per hour, while the calculated amount was
98 cc. The average distance from one another of the cross-partitions in the
fine tubes composing the wood is 0°25 cm. Therefore about 60 partitions
must be traversed in passing through a length of 15 cm. This indicates that
the resistance offered by the walls, or rather by the pits in une walls, to the
passage of water is very slight.

Ewart also endeavours to find experimentally the resistance offered to the
transpiration current. His first method, that of Janse and Strasburger,§ was
as follows: “Leafy branches 4 to 8 feet in length were cut under water and
kept in darkness for half an hour. Clean ends were then cut under water,
placed in freshly filtered eosin solution, and at once exposed in the open on
_ * & Ascent of Water in Trees,” ‘ Phil. Trans.,’ 1905.

+ “The Resistance to Flow in Wood-vessels,” ‘Ann. of Botany,’ vol. 19, No. 75, July,
1905.

{t “ Ascent of Water in Trees,” ‘ Phil. Trans.,’ 1905, p. 50.
§ Strasburger, ‘ Histologische Beitrige, 1901,’ Part ITI, p. 778.

42 Prof. H. H. Dixon. [ Nov. 7,

bright cloudless breezy June days between 10 a.m. and 1 p.m., with a shade
temperature averaging 18° to 22°C. The conditions for transpiration were,
therefore, optimal.

« After a timed period the stem was removed and rapidly sectionised from
apex downwards until the eosin solution was visible in the wood, the length
of the remaining portion of the stem giving the rate of flow during the period
of observation. Portions of the same stems and also similar ones from the
same plants were then subjected to varying water pressures until closely
corresponding rates of flow were reached.”*

By this method, and by assuming that the velocities shown in these

branches are maintained throughout the stem, Ewart obtains results which
indicate that, in order to move water in the stems of plants at the velocity of
the transpiration current, pressures equivalent to a head of from 6 to 33 times
the height of the plant are required.
_ There are several reasons why this unexpected result of Ewart’s must be
regarded asincorrect. (1) The velocity of flow given in Ewart’s experiment is
probably far in excess of even the maximum velocity of the transpiration
current in the intact plant. (2) The velocity cannot be assumed to be
uniform throughout high trees; but may fal] off from below upwards.
(3) Ewart’s results for the resistance to flow in stems are not in agreement
with a large body of experiment to be quoted presently, but appear to be
excessive. |

First, with regard to the velocities in Ewart’s experiment and in intact
trees. In intact trees the lifting forces generated in the leaves must do work
against the resistance to flow all along the path of the current, and, if the supply
is inadequate from the roots, against other opposing forces in addition, and
against the whole hydrostatic head. In Ewart’s experiment not only are the
resistance of the lower part of the conducting system, the other opposing
forces and the hydrostatic head removed, but they are replaced by the
atmospheric pressure acting as a vis a tergo, urging the water upwards.
Naturally, then, a much greater velocity is attained in the latter case than
when the branch is still attached to the tree.+

The subsequent wilting of the leaves of cut branches shows that clogging
afterwards greatly reduces the flow; but, of course, in the experiment quoted
it is only the initial stage which is recorded.

* Ewart, loc. cit., p. 24.

+ It may be mentioned that Janse’s method of observing the amount of transpiration
by successive weighings of a severed branch is not falsified by this error, but probably
gives too small an amount after transpiration has proceeded for some time. Strasburger

considers that this is corrected more or less accurately by his over-estimate of the resistance:
(Strasburger, loc. cit., p. 779).

| 1906.}. On the Transpiration Current in Plants. 43

The assumption that the velocity in the terminal branches is as great or
greater than in the trunk is also not justified. The lower leafy branches
have to do work against a smaller hydrostatic head, and against a smaller
- resistance than the outer and upper branches, and consequently the flow will
be faster through the lower parts than above. Furthermore, in many cases
the effective cross-section supplying unit transpiring area is greater above’
than below. A good instance of this was brought under my notice by
Dr. J. Joly last summer, in a young specimen of Abies excelsa, which had just
been felled. The tree was 550 em. high. The section at its base showed
17 year rings. The lateral branches were almost uniformly clothed with
leaves, so that the leneth of the branches was approximately proportional to
the leaf-area it supported. At 52 cm. from the apex the area of the cross-
section of the wood was 0°63 sq. cm. and the sum of the length of the leafy
branches above this was 170 cm. At 134 cm. from the apex the cross-section
was 4:29 sq. cm. and the sum of the lengths of the leaf-bearing branches
above was 1500 em. At the higher level the cross-section of the supply
conduits was 0°37 sq. cm. per 100 cm. of leafy branch, at the lower
point the cross-section of the supply was 0°286 sq. cm. per 100 cm. of
branch. Therefore, in the case of this tree, if all the leaves were transpiring
uniformly the velocity at the lower level must be greater than above. Hence
in Ewart’s experiments it is quite possible that the velocity below was
considerably in excess of that in the upper parts of his severed branch, and
certainly without special measurements it is not legitimate to assume that the
velocity throughout the length of the branch was uniform. This is equally
true with regard to intact large trees.

With regard to the resistance it will be seen later on that Ewart’s results
are considerably too high.

- The second method of determining the maximal velocity of the transpiration’
current employed by Ewart* is also open to objection. It is described by
him as follows: A small branch bearing a small number of leaves, while still
attached to the tree, is led through a split rubber cork into an air-tight glass
chamber containing a weighed quantity of calcium chloride. The gain in
weight of the latter gives the amount of water transpired by the leaves in a
given time. Assuming, then, that all the leaves of the tree may act like those
in the closed glass chamber, the number of leaves on the whole tree will give
the weight of water transpired by the tree and consequently the amount of
water which passes up the tree in a given time. Then, by measuring the
effective cross-section of the trunk, the velocity of the transpiration current
may be estimated. |

Ls yi: * Loe. cit., p. 56.

44 | Prof. H. H. Dixon. [ Nov. 7,

The objections to this method are obvious.

1. When the branch is first introduced into the desiccated chamber it will
lose water more rapidly than when it was transpiring into the less dry
external air. It will continue this abnormally rapid rate of transpiration
until the concentration of the vacuoles of the evaporating cells reaches a
steady state, depending on the freedom of supply from the water conduits and
the vapour pressure in the chamber, but, until this steady state is attained
the amount of water entering the calcium chloride may be largely in excess of
that passing up the stem.

2. But a more serious source cf error is the assumption that all the leaves
of the tree can transpire at the same rate as those on the single desiccated
branch, whereas the supply, which under the conditions of the experiment
is available for the very actively transpiring branch, would be largely
encroached upon if all the branches were under equally favourable conditions
for transpiration. In fact, the single branch in the desiccated chamber and
still attached to the rest of the tree, which 1s under normal conditions of
moisture, is under conditions of supply approximating to those of a cut
branch set in water and, for the same reasons, cannot be assumed to give a
correct estimate of the velocity of the transpiration current throughout the
whole tree.

The validity of this objection may be demonstrated experimentally by
weighing the amount of water given off by a given number of leaves in
a desiccated chamber, and comparing this amount with the quantity of water
transpired by the same number of leaves on the same tree exposed to normal
conditions of maximal transpiration.

I have made several of these experiments: A small yew-tree was removed
from the flower-pot in which it had been grown, and the roots, and their
surrounding soil, enclosed in a rubber bag ; to prevent loss of water, except from
the leaves, the opening of the bag was tied tightly round the stem. Periodic
weighings gave the amount of water transpired. At the same time a branch
still attached to the tree was introduced into a hermetically closed flask
containing calcium chloride. The flask could be removed and weighed
periodically. A rubber bag, similar to that enclosing the roots, filled with
moist earth and closed, was exposed to the same conditions and weighed
simultaneously, thus giving a small correction for loss through the bag. In
one of these experiments the unenclosed branches supported approximately
9500 leaves, the enclosed branch 520, 2.¢., the proportion of leaves on the
single branch to those on the whole tree was 1:18. When the tree was
exposed in a hot sun and brisk breeze, air temperature varying between 31°
and 24° C.in the month of July, the amount transpired was 5°171 grammes

1906.]. = On the Transpiration Current in Plants. A5

per hour, or 0°544 gramme per 1000 leaves. The amount transpired by the
desiccated branch, on the other hand, was 0°781 gramme per hour, or
1502 grammes per 1000 leaves per hour. Hence the leaves in the
desiccated chamber transpired nearly three times as much as the others
under normal conditions of maximal transpiration. In this experiment the
loss of water from the enclosed branch does not represent any temporary
desiccation of the surface tissues, for before the weighings were made the
branch had been enclosed-in the flask for a day. If this initial desiccation
had been included, as it was in Ewart’s experiments, the difference between
the amounts given off by the enclosed and unenclosed leaves would have
appeared greater.

In diffuse light the difference is not so marked. The unenclosed leaves of
the tree used in the last experiment in diffuse light with a temperature
of 21° C. transpired 1:°250 grammes per hour, or 0°130 gramme per 1000
leaves; the enclosed leaves simultaneously transpired 0:099 gramme, or
0-192 per 1000 leaves per hour. The desiccated leaves are nearly 14 times
as active in transpiration as those under the normal conditions. 7

These experiments show that it is not justifiable to assume that the rate
at which water is given off by an isolated branch under conditions of abnormal
desiccation is attained by all the branches when all alike are exposed to
conditions most favourable to transpiration. The excess evaporation from the
desiccated leaves will be greater when the bulk of the isolated branch is but
a small fraction of the bulk of the whole tree ; for the greater the preponder-
ance of the latter the larger will be the supply available for the branch and
consequently the less the resistance to transpiration. In Ewart’s experiment,
then, where the branch had only 500 leaves, while the whole tree had 9,000,000
(1: 18,000), 1t is probable that the effectiveness of the former was much greater
than that of the remaining leaves of the tree. Such an over-estimate in the
amount transpired involves, according to the method, an exaggeration in the
velocity of the current in the trunk.

The control of transpiration exercised by the freedom of supply may be
easily observed by means of the weighing method. The amount transpired
will be found to fall off as the plant exhausts the water in the soil round its
roots, and to rise when the soil is again rendered moist. The following figures
illustrate this fact in the case of a small yew-tree which was exposed to
conditions favourable to transpiration on seven successive days. The
conditions were fairly uniform, as throughout the experiment the sky was
lightly overcast and a light east wind blew.

46 , Prof. H. Hf. Dixon. [ Nov. 7,

Table I.
Transpiration per hour. Date. | Temperature.
grammes. rows
2°65 July 24 18 °5
2°14 25 20 °5
3-25 » 26 22-0
0 ‘87 ne ONY 19°5
0:97 : » 20 21 °5
Watered—
2°23 ess) 24 °O0
Watered—
1°59 » 30 19-0
3-05 80 22-5

The dependence of transpiration on the supply is prettily illustrated by
Darwin’s experiment with the horn hygroscope.* Darwin records that when
the hygroscope is applied to the leaves of a branch severed from a plant, it
indicates a gradually diminishing rate of transpiration as the store of water
in the branch is gradually exhausted. A diminished rate of transpiration is
also indicated by the hygroscope when the supply to a branch is reduced by
the application of a clamp constricting the water conduits leading to its leaves.

The diminution in the rate of transpiration in these experiments is no
doubt due to a rise in the tension of the water columns supplying the
evaporating cells. The tension rises in the first case as the limited water
store is drawn upon, and the water surface is dragged into the cut surface of
the branch. The surface-tension forces developed there then oppose the
transpiration current. In the second experiment the tension is increased by
the rise of the resistance brought about by the reduction of the calibre of the
conduits. Further reduction of transpiration is occasioned by the closing of
the stomata and by other phenomena, which are themselves direct or indirect
consequences of the rise of tension in the water.

According to this view an interesting observation of Darwin’s} receives a
ready explanation. There is a momentary increase of transpiration in these
experiments immediately after the separation of the branch and immediately
after the application of the clamp. In the first case we may assume that the
tension in the water supply is reduced by fracture, in the second case by the
compression applied when screwing up the clamp. Transpiration obviously
soon re-establishes tension, which becomes greater in both cases than it was
originally.

* Darwin, “ Observations on Stomata,” ‘ Phil. Trans.,’ B, 1898, pp. 539 ff.
+ Darwin, loc. cit., pp. 548 ff.

1906.]. On the Transpiration Current in Plants. 47

In many ways, then, we see it is established that supply largely controls

transpiration, and in neglecting this factor Ewart considerably exaggerated
the maximal velocities of the transpiration through the stems of trees.

We come now to consider the amount of resistance experienced by the
transpiration current 1n its passage through the water conduits.

To obtain the resistance, Ewart forced water through lengths of branches
under various pressures. The pressure which gave the same velocity in the

branch as that estimated by his methods for the transpiration current he took
to be equivalent to the resistance experienced by the transpiration current.

Ewart does not mention how he prepared the pieces, or how he cleansed the
water for the experiment. The introduction of air-bubbles or of any clogging
substance at the cut surface would materially exaggerate the pressure needed
to obtain the observed velocity. Indeed, if the branch were transpiring
actively when the experimental pieces were removed, it would not be
sufficient to cutit under water; for the liquid in the branch being in tension,
bubbles would be formed at the surface of the cutting knife. These bubbles
would require some time to dissolve and disappear. The slimy materials
exuding from the injured cells also clog the branch and raise the apparent
resistance of its conduits. The comparatively high pressures with which
Ewart worked would render the clogging from this source and from any
impurity in the water more marked. Fig. 1 illustrates this point. The

& Head £\ um

6
, .
\.
é
» a
yy i
: O
&
‘s
: .

Ingrs. per Aeeorel,

48 Prof. H. H. Dixon. [ Nov. 7

ordinates of the curves there shown indicate weights of filtered tap-wate1
transmitted through 3 cm. of wood of Abies pectinata per second. The
abscissee indicate time in minutes. The curves show the diminution in the
rate of transmission for each pressure, the fall-off in the amount being much
more rapid for the higher pressures. From these it is seen that it is desirable
(especially when using colour solutions, which from the nature of the case
cannot be distilled) to employ low pressures in order to determine the
resistance of the conduits apart from the surface resistance. For this reason,
then, when I began to determine the resistance of the tracheidal tubes I
preferred to experiment with very low pressures, assuming that the flow for
higher pressures would be proportional to the head. This point was examined
later.

The wood used in these experiments was that of Z'axus baccata. The
uniformity and comparative homogeneity of structure (resulting from the
fact that it is composed solely of tracheids and is not penetrated by resin
ducts) recommend it as far the most suitable wood for experiment, when
the qualities of the water-conducting tissues are being examined. The
tracheids composing the wood of Taxus baccata are elongate spindle-shaped
chambers 1 to 5 mm. long and approximately square in cross-section. The
cavity of these chambers is comparatively small. In cross-section the
lumen forms about a quarter the entire area of the tracheid. The only
other constituents of the wood are the cells of the medullary rays, which
are radial tiers of cells 0-4 to 0°02 mm. in height. They do not materially
differ in percentage from one level in the stem to another, and consequently
do not introduce differences of serious magnitude. The fact, however, that
they exude small amounts of slimy materials sometimes introduces
difficulties. But inasmuch as there are approximately the same amount of
medullary ray-cells per unit area in every cross-section, the clogging
introduced in this manner is uniform and, it is to be noticed, tends to
increase the apparent resistance. The presence in other woods of large
medullary rays, of wood-parenchyma, of vessels of variable size, or even of
resin ducts, renders them unsuitable for experiment.

Certain precautions are necessary in preparing the wood for these
transmission experiments. It is best to cut away the transpiring leaves
from the selected branch, and then to cut off a short length of it (say 15 to
25 cm.) under water. Five to ten centimetres are now cut from each end,
and the remainder, after lying in water for at least 30 minutes, may be used
directly or reduced to smaller lengths for experiment. These precautions
are necessary, so that bubbles shall not be generated in the conduits. It
seems possible that Ewart’s high estimate of the resistance was, in part, due

1906. | On the Transpiration Current in Plants. 49

to bubbles being generated in the conduits while preparing the wood for his
resistance experiments. He certainly does not mention having taken these
or similar precautions in this connection, and his results are three to four
times as high as those given when these precautions are taken.

When determining the velocity of flow with colour solutions, I usually
worked with a head of water equal to, or less than, the length of the piece
of wood experimented with. When using unit head, a fresh surface was
cut with a razor on one end of a piece of a branch prepared as just described.
The piece was supported vertically with the fresh surface uppermost, and
a little vaseline smeared round the bark of that end. A drop of filtered
concentrated eosin solution was placed on the upper surface, and as the
drops formed at the lower end they were drawn away by lghly touching
them with bibulous paper. As the eosin sank through the wood it was
kept constantly replenished drop by drop above. After a definite time
the experiment was stopped, and longitudinal cleavage of the piece of wood
showed the distance travelled by the eosin solution during the time of
observation at unit head. The following are the results of some of these
observations :—

Table IT.
Length of piece. | Time of observation. | Distance travelled. | Velocity per hour.
centimetres. minutes. centimetres. centimetres.
: 15 ‘0 le: 6
6-0 10 ‘0 1°2 72
4 °O 15 ‘0 Le Uh 6°8
3°5 15 0 8 72
10 ‘0 20 ‘0 2°2 6°6
3:0 25 °5 3 °0 6°9

— t os Woe ce es

In the last experiment the fluid transmitted was a solution of ferrocyanide
of potassium. Its presence below was detected by ferric chloride. The
mean of these experiments gives a velocity of 69 cm. per hour under a
pressure equivalent to a head of water equal in length to the experimental
piece of wood.

_ Experiments were also made at lower pressures. For these the water-
pressure was applied at the lower end of a vertical piece of wood, through
a rubber tube bent into a U-shape. This tube was filled with eosin solution
and the surface of the solution was raised to the desired height above the
upper surface of the wood.

The mean of these observations is 8°5 cm. per hour, calculated to unit
head. If we exclude the two extreme observations we get 7°6 cm. per hour

VOL. LXXIX.—B. E

50 Prof. H. H. Dixon. [ Nov. 7,

Table ITI.
Length of piece. Head. Distance traversed. Time. Velocity per hour
per unit head.
centimetres. centimetres. centimetres. minutes. centimetres.

8 Hf 20

6 2 0°8 20 Ther
6 3 1°8 15 14 °4
6 3 0:9 15 72
6 33 0°7 15 56

as the velocity under a head equal in length to the stem. The last
observation of the series was made on a piece of a narrow branch about
0-5 cm. in diameter, the others on pieces about 1 cm. in diameter. This
difference appeared almost constantly in my experiments. The thin distal
portions of the wood in almost every case offer a greater resistance to flow
than the thicker parts. The high estimates of resistance are almost always
obtained with the former. This fact is probably of importance in
determining the total resistance in the intact plant. JI have included
the third observation in the table, although it diverges so markedly from the
mean, because I could see no error in the experiment, and it is quite possible
that a maximal result like this is the nearest to the actual flow in the
uninjured tree.

The higher mean in the second series of experiments for the velocity of
transmission is probably due to the fact that clogging substances are less
likely to accumulate owing to the actually slower flow and to the position
of the surface of application.

With care, good results may be obtained with higher pressures, if the
supply is from below. In the following experiments (Table IV) the cylinder
of wood was fixed in the short arm of a vertical J-tube filled with a repeatedly
filtered solution of ferrocyanide of potassium. The moment of penetration
through the wood, which was 3 cm. long in each case, was determined by its
reaction with ferric chloride applied in a piece of bibulous paper to the upper
surface of the cylinder.

The mean of the entire series gives 6°9 cm. per hour as the velocity at
unit head. As all the known errors, such as the introduction of bubbles,
clogging and injury of the tracheidal tubes, tend to reduce the result, it is
probable that the velocity in the intact tree would be at least 7 to 8 cm.
per hour under the same pressure. The occasional high results obtained
indicate a still higher figure as the probable velocity.

In fig. 2 I have plotted these results. The ordinates represent’ the length

1906. | On the Transpiration Current in Plants. 51

traversed in one hour, while the abscisse indicate the pressures, considering
a head equal to the length of the branch as unity.

Table LV.
Time Velocity per hour
fe, traversing 3 cm. at ay head.
centimetres. minutes. centimetres.
30 2°0 vee
30
27 1
27
24 5
24,
24
21

or

Or

a
Or Or

on
SESHNIBDNHONSSHANANELKBSOSSSC

RWNWOWSOANTMAEEEERTRWOWWNDNH
HROETAIV AS AAKAATANIAACAIAdSES
I ADIATAWAWD DMO AANAAAABRWHOAD

bo

sol}
ye
Q
40 :
Wh: ¥
C)
By
: Aira
a4
9
Za
C)
ye

° ‘ a pe | 5 6 7 8 9 Te)
Clnife seal, ‘

Fig. 2.

Cm

30

52 Prof. H. H. Dixon. | Nov. 7,

It may be noticed that the most divergent observations are those made
at the higher pressures.

When we compare these results with Ewart’s* a very wide discrepancy
is apparent. The results of three of his experiments allow themselves to be
readily compared with my figures.

(1) He found that water travelled in a piece of yew stem, 35 cm. long,
at the rate of 11°7 cm. per hour under a head of 3 metres. The head here
is nearly 86 times the length of the transmitting wood. Assuming the
velocity proportional to the pressure, at unit head the water would travel
at 1:36 cm. per hour. This rate is stated to be maximal.

(2) On pp. 51 and 52 it is stated that the rate of flow in a piece of
yew wood, 25 cm. long, under a head of 3 metres is 26 cm. per hour.
In this experiment the head is equal to a column 12 times the length of the
transmitting branch; when reduced to velocity under unit head the result
is 2°17 cm. per hour.

(3) Again, on p. 55 an experiment is recorded which is suitable for
comparison. A velocity of 19 cm. per hour was observed in a branch 25 cm.
long under a head of 4 metres. This becomes 1:19 cm. per hour under
unit head.

The mean of these three observations gives 1°57 cm. per hour as the
maximal velocity of flow in the yew wood under unit head.

The results of my own numerous observations, on the other hand, made
under very various conditions of pressure and by different methods, point
to a velocity exceeding 7 cm. per hour with the same head.

The only explanation of this discrepancy which appears possible is that
in Ewart’s experiments sufficient care was not taken to prevent bubbles
forming in the opened conduits and to obviate clogging at the surface.
This last effect would be exaggerated in his experiments, as he worked
apparently in every case at such high pressures. Reference to fig. 2
illustrates this point. There it appears that the erratic observations are
those made at high pressures, although at both high and low pressures
similar precautions were taken. It is in only the first of Ewart’s experiments
quoted above that it is mentioned that the experimental branch was cut
under water. Other precautions are not mentioned.

Whatever is the cause of the discrepancy, it is certain that if Ewart had
obtained my results, the difficulty of resistance, which he finds to be fatal
to the cohesion-theory of the ascent of sap, would not have presented itself
to him, for the velocity of 7 cm. per hour, which he demandst in the stem

e LOCH Ciao:
t Loe. cit., pp. 56 and 57.

1906. | On the Transpiration Current in Plants. 53

of the yew, would not require a pressure equivalent to a head of 65 metres
of water, as he supposes, but only to 11 metres. The recognition of the fact
that unit head produces a velocity of about 7 cm. per hour invalidates his whole
calculation, intended to show that to raise the sap in trees 150 metres high
would require a pressure approaching 100 atmospheres. The facts of the
case would be more correctly stated by saying that if the amounts
transpired by isolated branches, under exceptionally favourable conditions
for transpiration, were transpired by the remaining branches of the yew, and
if we further assume that the velocity in the trunk is maintained out into the
finest branches of high trees, then the resistance to flow in the conducting
tracts would be about equal to a head of water the same height as the tree.

My results for the resistance, which were obtained as described above by
directly measuring the velocity of flow under a given head, were fully
confirmed by other experiments in which the amount transmitted under
a given head was observed. An estimate of the cross-section effective in
transmitting the current then gives the velocity.

A piece of a branch of Taxus baccata, 4 cm. long and having a woody
cylinder 1°35 cm. in diameter, was placed with its long axis vertical.
Water was supplied at its upper surface just as quickly as it percolated
through the wood, so that the upper surface of the wood was continually
wet, but the water was never appreciably piled upon it. The amount of
water transmitted in this manner under unit head was 1°356 gramme per
hour. In order to find the effective cross-section, after this observation was
made, a solution of eosin was supplied under similar conditions. By this
means the transmitting portions were coloured, and the area of their cross-
section easily estimated. A mean of three such estimations gave the
effective cross-section as 0°7 sq.cm. From this it appears that under unit
head 1:93 grammes 1s tgansmitted per square centimetre per hour. Assuming
with Ewart that the lumina occupy about 0:25 of the cross-section, the
velocity to secure this rate must have been 7:5 cm. per hour. This agrees
well with the results obtained with the other method.

In order to determine the amount of flow without danger from the error
of clogging at the surface of supply, the flow was measured in a lateral
branch springing from a stout stem. Water under pressure was supplied at
the two cut ends of the stem. With this arrangement, owing to the
relatively large surfaces of supply, the clogging taking place at these does not
encroach upon the amount necessary for the supply of the small lateral branch
for several hours, and consequently the rate of transmission in the lateral
branch remains constant for several hours. To quote one of these:
experiments: A piece of yew stem 7°3 cm. long, having a straight lateral

54 Prof. H. H. Dixon. [Nov. 7,

branch at its middle length, was selected. The diameter of the stem was
0°85 cm. and 1:05 cm. at each end respectively. Its wood was also laid bare .
by an oval scar where another lateral branch was removed. This scar was
13cm. by 1-1 cm. The smal! lateral branch was cut to a length of 10°5 cm.,
and its wood had a diameter of 0°47cm. Not more than a quarter of its cross-
section was in a state suitable for transmitting water—the greater part being
occupied by blackened duramen. This lateral branch was fixed water-tight in
a rubber bung in an orifice in the bottom of a tank in such a manner that the
cut end of the lateral branch projected from the tank, while the supporting
stem was immersed in the water in the tank. With the head of 30 cm.
0300 gramme were transmitted per hour. The transmitting cross-section
was about 0:°043 sq.cm. One-quarter of this area would be lumen: therefore
the velocity of flow must have been 27:2 cm. per hour. If we reduce this to
unit head the velocity becomes about equal to 9-4 cm. per hour, which again
falls within the limits of the results obtained by the previous method.

Throughout this discussion it has been assumed that the velocity 1s
proportional to the pressure. This is usually done. Fig. 3 shows that this is
certainly approximately true for pressures up to 16 units head. On fig. 3
are plotted the results of many experiments, the object of which was to
determine if the amount transmitted in a given time is proportional to the
pressure, as the former assumption would involve. The utmost care was
taken to obtain clean water to supply to the wood and to free the latter from
clogging material as much as possible. The water used was carefully
distilled, the vessels and tubes of supply were repeatedly washed with
distilled water before using, and the surface of the wood freshly cut and
rinsed with distilled water immediately before the experiment began. The
woody cylinder was 3 cm. long. It was supported horyontally, and a tongue
of bibulous paper was applied to the outer surface of the wood and hung down
vertically into a little phial which received the drops of transmitted water.
The time required for five drops to fall from the tongue was noted by means
of a stop-watch, and the weight of these five drops determined by weighing
the phial. The mean of three such observations is given below (Table V) for
each pressure.

These results confirm the received view that the amount transmitted varies
directly with the pressure. The slight bending over of the line joining the
observations at the high pressures is amply explained by the gradual clogging
which takes place despite all precautions; for the experiments giving these
results were made with the same piece of wood after those at lower pressures

had been carried out.

On the Transpiration Current in Plants. 55

ii rs
|
ay
} t:
‘ per
° 2 4 6 8 fo (2 14 1G
Zinits Head
igs 3.
Table V.
Head Milligrammes Te Milligrammes per |
transmitted. second.
‘oe |
centimetres. seconds.
6 AA -O 246 0-182
12 50 ‘0 137 O °365
18 53 °0 99 0 °5385 |
24 50-0 ae O °685
30 52-0 63 0 °825 |
36 58 :O 60 O 966 |
48 59°5 AT 1-266 !

The proportionality of head to flow may be indirectly investigated in another
; manner. A straight branch fixed in a horizontal position is connected to a
large vessel of distilled water. Precautions are taken to clean the connections

56 Prof. H. H. Dixon. | Nov. 7,

and the freshly cut inner end of the branch. A tongue of bibulous paper is
applied to the outer end to draw off the transmitted water. The head once
adjusted is kept constant; but after each determination the branch is
shortened. Fig. 4 records such a series of experiments. The head through-
out these was 100 cm. The initial length was 25cm. At that length the
flow was 1:18 mg. per second. Five centimetres were then cut off the outer
end and the flow rose to 1°66 mg. per second. A shortening to 15 ecm.
increased the flow to 2°33 mg. When the branch was 10 cm. and 5 em. long,
the flow was 3°79 mg. and 6°70 mg. respectively. The curve plotted in fig. 4

is a rectangular hyperbola in which M = K//; M being the number of
milligrammes transmitted per second, J = length of wood, and K = average
value of the product M/ observed. The observations, it may be seen,
approximate fairly closely to this curve. When the weights of transmitted
water are plotted against units head the curve shown in fig. 5 is obtained.
Here the proportionality of flow to head, or, rather, the inverse proportionality
of flow to length, is immediately apparent. Up to 10 units the curve is
almost a straight line. The bending over which occurs after that point is to
be attributed to the clogging, which is practically unavoidable when the flow
is rapid.

In conclusion, it seems to me that Ewart’s objections to the cohesion-
theory, based as they are on an excessive estimate of the velocity of the
transpiration current and of the resistance to this current in the conduits,

1906. | On the Transpiration Current on Plants. 57

noe
7
.
7
7
Ae
.

Mgrs. per second.

Units Head.
Fie. 5.

fall to the ground. With regard to the soundness of the cohesion-theory
itself, apart from the weighty evidence which has elsewhere been adduced in
its favour,* the fact that other theories, both old and new, have to assume
properties for the water-ways of plants which are either in the highest
degree improbable according to received scientific views, or are even directly

negatived by experiment, seems to support the theory by a process of
exclusion.

* Dixon and Joly, “ Ascent of Sap,” ‘ Phil. Trans.,’ B, 1895 ; Dixon, ‘‘ Physics of the

Transpiration Current,” ‘Notes from the Botanical School, Trinity College, Dublin,’
May, 1897.

:

08

Nitrification of Sewage.
By Georce Rep, M.D., D.P.H.

(Communicated by Professor Gotch, F.R.S. Received May 7,—Read
November 1, 1906.)

My special object in this paper is to direct attention to certain observations
I have recently made bearing upon depth of filters and grade of particles as
affecting both the quality of the work done and the capital outlay involved.

To effect the necessary changes it is essential that the sewage in a fine film
shall be brought into intimate contact with the nitrifying organisms in the
presence of an adequate supply of oxygen. It follows, therefore, that the
larger the number of organisms employed, so long as they are maintained in
a healthy active condition, the greater the amount and the better the quality
of the work done. How then should a filter bed be constructed, so that,
within a given cubic space, it shall afford the largest possible surface for
bacterial growth under healthy conditions? Clearly, I suggest, by reducing
the particles composing the filter to the smallest size which is found to be
compatible with free aeration.

Compare, from the point of view of effective working area, a filter formed
of 2-inch cubes—a grade in common use at present—with one formed of
+-inch cubes, and it will be found that, bulk for bulk, the area in the latter
case is 16 times greater than in the former, but taking into account the
relatively larger space occupied by the smaller particles, the actual gain is as
1 to 14°7.

The question is, then, to what extent in actual practice is it possible to
reduce the size of the filter particles? Some four years ago I had an
opportunity of conducting an experiment on a large and practical scale at
Hanley with a plant which complied with all the conditions which previous
experience had shown me to be essential, including, for the first time I
believe, perfect and uniform distribution of the sewage on to the filter by
means of a power-driven apparatus.

Shortly, this plant, which dealt with 500,000 gallons per day (1,000,000
gallons per acre), consisted of :— |

1. A detritus tank and strainer of the usual type.

2. Three subsidence tanks having a total capacity equal to one-eighth the
daily dry weather flow.

3. An open septic tank with a capacity of seven-eighths the daily flow

Nitrification of Sewage. 59

(giving, with the subsidence tanks, a capacity for quiescence equal to a day’s
flow). |

4, Two quarter-acre “percolating” filters 4 feet 9 inches deep, the one
circular and the other rectangular, and each having a different type of power-
driven distributor. I may here mention that I have long since discarded the
“contact” method of working bacteria beds in favour of what is termed
“ percolating ” filtration.

The filtering medium in this case consisted of broken “ saggars,” the
discarded hard-burned clay vessels in which china is packed for firing, and
the filters were constructed in four separate sections, each having different
eraded material. This material ranged (in separate sections) from 14 inch to
4 inch. I have elsewhere given a full description of the working of this
plant during a period of about eighteen months, and the results proved that
such filters, working at the rate of 200 gallons per square yard per 24 hours
(1,000,000 gallons per acre) continuously, produce a high-class effluent, the
best results being obtained from the section of the filter composed of the
finest grade particles (4 inch).

So much for the preliminary work which led up to the present investiga-
tions, the results of which seem to me to be of scientific interest and of
practical importance.

It would seem to be established that the nitrification of hydrolysed sewage

_ takes place in stages, the nitrous change being effected by one set of organisms

as a preliminary to the nitric | change brought about by another set, and the
theory is that the two sets of organisms are located in different layers of the
filter. With the view of verifying this, and, at the same time, ascertaining
the degree of purification effected at different depths, I determined to tap the
filter which had been used for the purpose of the experiment already referred
to, in such a manner that samples might be collected at different levels,
and I selected the finest grade section for this purpose (4-inch particles).
Accordingly, four longitudinal shallow trays, with perforated covers, were
placed in the body of the filter, the first at 1 foot from the surface, the second
and third at 1-foot intervals downwards, and the lowest 18 inches below the
third one, pipes being carried from the various collecting trays through the
wall of the filter, to allow of the collection of the respective samples. In
order that the results from the lower depths might not be affected by the
presence of the trays above, the trays were placed obliquely from above
downwards, so that no tray had another in the vertical line above it. As the
filter at this time had been constantly at work for two and a-half years, in
replacing the filtering material which had been disturbed in order to intro-
duce the trays care was taken to ensure as far as possible that each portion

60 Dr. G. Reid. [ May 7,

was placed in the position it had previously occupied in the depth of the
filter, and seven days were allowed to elapse for recovery before collecting the
first samples.

As regards analytical methods, the free ammonia was determined in the
usual way by distillation and Nesslerising, and the albuminoid ammonia by
Wanklyn’s process, the quantities taken respectively being—sewage, detritus
tank, and septic tank, 50 cc., filter effluents, 500 cc., the former being
made up to 500 cc. by ammonia-free water. The oxygen absorbed was
determined by Tidy’s process, the nitrous nitrogen by Griess’ method, and the
nitric nitrogen by Sprengel’s method.

In order to discount possible subsequent changes from the lower to the
higher oxide, and vice versd, the tests for nitrous and nitric nitrogen were
made at the works immediately on the collection of the samples, and again in
the laboratory the following day, when the complete analyses were made.

The following figures represent the mean results of a series of analyses, the
individual figures of which are given in the detailed tables attached. The
samples were collected at intervals extending over a period of about six
months, the filters being steadily worked at the previous rate, namely,
200 gallons per superficial yard per 24 hours, and the delivery being uniform
and continuous day and night.

In the first place, before commenting generally on the conclusions which
may be drawn from the results as a whole, I propose to refer shortly to
each analytical record separately :—

1. With regard to the suspended solids it will be noticed that a reduction
of 73 per cent. 1s effected in the detritus tanks, and that a further reduction
of 15 per cent. takes place in the septic tank, making a total reduction of
88 per cent., and resulting in an effluent being passed on to the filter
containing 76 parts per 100,000, exactly one-half of which is mineral
matter. This suspended matter, it will be seen, is practically all retained
in the top layer of the filter, where the organic portion is liquefied, in all
probability, by aerobic organisms. The mineral matter, however, must
remain in the filter, and in time, no doubt, it will be found necessary to
remove the filtering medium to a depth of a few inches for the purpose of
washing it, but so far, after over three years’ constant working, no such
necessity has arisen. As a matter of fact, if the total mineral suspended
solids passing on to the filter during the three years were deposited ina
uniform layer over the whole surface, the depth of the coating would be less
than 14 inch.

2. The reduction of the free ammonia at a depth of 1 foot is remarkable,
especially considering the fact that the change has been effected in about

Mitrification of Sewage.

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62 Dr. G. Reid. [May 7,

12 minutes—the time occupied by the sewage in passing downward through
the first foot of filter. In my experience it is not unusual to find the free
-ammonia figure reduced almost to an equal extent in effluents from fine-
grade filters, but, hitherto, I had no conception that the change was brought
about by so shallow a depth of filter.

I may here mention that the rate of travel downwards of the sewage
through the filter was found, as the result of several observations, to vary
in accordance with the depth as follows :—

Hrommcurtace to (ibs 0 see 12 mins.
ee ak Ete 1 2 a ee eee 1 es
she dey SOs soeaiede re lace eee .
engl ler 7 4 1U AG naan HERTS
Totalliinc sees verge 20! is;

This is a slow rate of travel compared with the rate in the case of large
particle filters, which, in my experience, in some cases allow the sewage
to pass through the entire depth of from 4 to 5 feet in about four minutes.

The rate of travel was ascertained by a series of observations as follows :—
The delivery on the filter was stopped until the discharge pipes from the
respective trays showed unmistakable signs of diminished flow, when it was
started again, the period which elapsed before the discharge from the pipes
was restored being taken as the time occupied in the passage downward of
the sewage to the different depths.

The free ammonia figure presents one other interesting feature. It will
be noticed that the progressive reduction which takes place during the
passage downwards through the first 3 feet is suddenly interrupted, a
considerable increase in the amount being recorded at a depth of 4 feet
6 inches. Accidental error in analysis does not explain this, for it will be
seen on referring to the detailed tables that this increase is invariably
recorded, and, as a matter of fact, I verified the accuracy of the results by
a second analysis in the case of the first few samples until it became quite
obvious that no such explanation of the occurrence was feasible. This
phenomenon will be referred to later in dealing with the general arguments
and conclusions.

3. As regards the albuminoid ammonia figure, it will be seen that a
highly satisfactory reduction is effected by filtration through 1 foot only.
In fact, this figure and the oxygen absorbed and nitric nitrogen figures
considered together indicate a very high degree of purification, which
precludes the possibility of subsequent putrefactive change.

4, It will be seen that the reduction in the oxygen absorbed is, stage by

1906.| Nitrification of Sewage. | 63

stage, proportionate to the albuminoid ammonia reduction, and the rapidity of
the change in this case also is equally marked.

5. With reference to the oxidised nitrogen the figures are very startling,
for not only are they indicative of extremely heaithy and active biological
conditions, but, considered in relation to the other figures, they also
demonstrate conclusively, in the case of the sewage in question at any rate,
that the work of purification is practically completed within a few inches
of the surface of the filter. Within 12 minutes a foul and offensive liquid
is rendered not only clear and non-odorous, but also absolutely stable as
regards putrescible qualities, as is proved by the oxygen absorbed figures
before and after incubation. I must say that this result surprised me very
much. Theoretically, as I have said, one expected to find that the process
was a more gradual and progressive one, nitrites being formed chiefly in the
superficial layers of the filter and nitrates in the deeper layers, but this
did not prove to be the case. At the same time I do not suggest that the
change is not gradational, but rather that the two sets of organisms are
at work side by side, and in this case the absence of more than a trace of
nitrous acid in the 1-foot effluent, although the tests were applied at the
moment of collection, may be accounted for by the preliminary stage of the
oxidation process being an extremely evanescent one owing to the highly
efficient working conditions.

6. The column headed “depth of column necessary to obscure test lines ”
requires explanation. It is a standard opacity test which I adopted some
years ago for Staffordshire, and which has since been adopted by some
other river authorities. The lines referred to are engraved on a white
porcelain disc, which is attached by means of a socket and rubber washer to
a 2-foot glass tube, graduated in half-inches. The exact mesh and depth of
lines are as shown in the drawing, and in fixing the scale
as regards mesh and length of tube, in the first instance,
I was guided by my experience at the time as to the
opacity of well-clarified effluents. J have since found,
however, that the clarification effected by fine-grade
filters and efficient distribution is so perfect that the
lines are not usually obscured by a depth of 24 inches, and this was the
case in the present instance. It will be noticed that while the mean
depths of fluid necessary to obscure the lines in the case of the crude sewage
and the detritus and septic tank effluents were 0°5, 1:6, and 1°5 inches
respectively, the filter effluents, even those from the 1-foot tray, were so
highly clarified that the full 24-inch column did not suffice to obscure the
lines. It is true that the opacity test in itself is not a reliable index of purity ;

64 Dr. G. Reid. [May 7,

at the same time, such a record, considered in conjunction with that of the
suspended solids, is useful for comparative purposes.

For convenient reference, I have shown in the form of a diagram the
percentage reduction in free ammonia, albuminoid ammonia, and oxygen
absorbed at different stages, the figures for the sewage being taken at 100,

and also the actual nitric nitrogen figures in parts per 100,000.

ae Free Ammonia

Percentage } Albuminoid D2 E

reduction of | Oxygen absorbed C__])
in 4 hours at 80°F.

Nitric Nitrogen
in Parts per 100000.

MMMM

VM
WY

CMLL

CMMMM@@TC_@C/ V@HT=@#_HWTMHV@T@H@_@_ M00,

WWAMMM@C@C=V=@H@ste@ br
WHMMMHZZ@@="|"M|'€6ta

SS BS | mS BS i a_i | NIB! a

sewage |Detritus| Septic | 1fool | zfeet | afeet j4asfeet
tank tank

J ROKO|C

zfeetx

afeet
4.5 feet

|

So much, then, for the detailed results of the working of this plant.
Whether what has been proved to be possible in this case will be found to be
equally possible in other cases remains to be seen, and if the answer be in the
affirmative, it 1s unnecessary to point out that the resulting economy in the
construction of filters would be very material. Personally I am extremely
optimistic regarding future possibilities in this direction, and only the other
day I had an opportunity of practically applying this recent experience with,
so far, most gratifying results, in the case of a stronger sewage and one which
contains a considerable amount of brewery waste.

In this case the authority had some years ago constructed works which
provided for the nitrification of the sewage by means of “contact” beds, and,
being lately threatened with proceedings on account of pollution, on the
advice of their engineer an experimental plant was constructed, with the
view of determining whether better results could be obtained by means of
“percolating” filters. Having worked these filters for about seven months,
the engineer came to the conclusion that he might safely recommend the
adoption of a scheme on similar lines, but with 5-ft. 6-in. filters, to dispose of

1906. | Nitrification of Sewage. 65

the whole of the sewage of the town. Before assenting to this proposal,
however, the authority decided to seek further advice, and this led to my
being consulted.

Shortly, I may state that the experimental filter in this case had a mean
depth of 4 feet, and was formed of clinker broken to sizes varying from
2 to d inch, the sewage, after having undergone preliminary treatment, the
nature of which need not here be described, being distributed on to the filter
by means of a series of fixed sprays. The results obtained were fairly satis-
factory as regards nitrification, but the organic matter remaining uncon-
verted was considerabie, and the effluent, so far as appearance went, was a
poor one, owing to the amount of suspended matter it contained.

In order to determine whether and to what extent the imperfect results
were attributable to the comparatively large-sized medium composing the
filter, and with the view of ascertaining whether, in the case of the sewage
in question, a shallower filter would answer the purpose, I suggested that:
a section of the filter should be partially reconstructed by removing the top
medium to a depth of 12 inches and replacing it by granite chippings broken
to 4 inch, and also that a collecting tray should be introduced at a depth of
3 feet in the reconstructed section. This alteration was made, and we
thus had the means of comparing the results obtained by the altered section
at depths of 3 feet and 4 feet 6 inches with those from the undisturbed section
at a depth of 4 feet 6 inches under identical conditions.

Having resumed the working of the filter, at a rate of flow of 200 gallons
per square yard per day, three weeks were allowed to elapse for ripening
before the first set of samples were collected, and up to the time of writing
this paper the experiment, under the altered conditions, had been in
operation for four months. :

So far I have had an opportunity of analysing two sets of samples from
the two sections of the filter, and the following are the means of the more
significant results obtained :—

Parts per 100,000.

Unaltered Altered altenea
ahs section of section of eae oe f
ee filter at filter at Aisa:
See sta eC eke es a rar
deep. deep. pet
Solids in suspension ...............00000 9-10 2°10 1°15 1-00
MSOMAMIMONIA © bck se ca sdec dass 1 067 0-710 0-250 0 :067
Albuminoid ammonia .................. 0 558 0 154 0-107 0 044,
Oxygen absorbed in 4 hours at 80° F. 3 °157 0 °818 0 ‘680 0 °388
PMG CT fons vied vinwieasiiee voesasiens Nil 1-04 1°16 2°58

VOL. LXXIX.—B. F

66 Dr. G. Reid. | May 7,

It will be seen that the improvement effected by apparently so trivial an
alteration is remarkable. The improved quality of the effluents from the
altered section of the filter at a depth of 3 feet compared with those from
the same section and the unaltered section at a depth of 4 feet 6 inches is
most striking.

With reference to the work done at different depths in filters, Scott-
Moncrieff has added very much to our knowledge of the subject by his
well-known experiments: on a small scale at Ashtead some eight years ago,
and, later, on a larger scale, at Caterham Barracks, The filters were built up
of nine trays, each 7 inches deep, with perforated bottoms, and supported
vertically over one another with about 3-inch interspaces. The depth of the
filtering medium in each tray was 6 inches, giving a total effective depth of
4 feet 6 inches. The medium consisted of coke, broken to pass through
a ring 1 inch in diameter, and the rate of delivery was about 200 gallons
per square yard (1,000,000 gallons per acre).

The object Scott-Moncrieff had in view was a two-fold one: first, to secure
the freest possible aeration of the filter; and, secondly, to separate, as far as
possible, the various stages of the process by providing facilities for the
differentiation of the colonies of nitrifying organisms. Incidentally the
arrangement also allowed of the collecting of samples of effluents for
analysis from different depths, for comparative purposes as regards the
oxidising changes.

Without going into detail, 1 may mention that while the nitrification
effected by the tray filters was exceptionally high, the changes, notwith-
standing the free aeration which the arrangement provided for, did not take
place so rapidly, nor at so shallow a depth, as in the case of the Hanley
experiments. The nitrous change was more marked, and was continued to
a greater depth, and the reduction in the free ammonia, as well as in the
oxygen absorbed, was far more gradual. It is true that the sewage was
a much stronger one, but, making allowance for this, the work done per foot
deep of filtering medium was relatively considerably less than in the case of
Hanley. I feel pretty confident that had a much finer filter medium been
used by Scott-Moncrieff the high oxidising changes would have been effected
at a shallower depth, and practically the whole of the suspended solids
would have been liquefied and nitrified within the filter.

Dr. Harriette Chick, in a paper published in the ‘ Proceedings of the Royal
Society,’ 1906, B, vol. 77, gives an account of certain laboratory experiments
she conducted in 1901, and again in 1903, having reference to the nitrifi-
cation of sewage. Three cylindrical filters were employed, each 4% inches in
diameter, and approximately 14, 33, and 64 feet in depth respectively. The

1906.] Nitrification of Sewage. 67

filtering material used was coke of practically the same grade as that used at
Hanley, and the sewage was delivered on to the filters at the rate of
300 gallons per 24 hours by means of a syphonic discharge on to a per-
forated distributing disc at intervals of 38 minutes. The sewage dealt with
was artificially prepared by diluting liquid cow manure with water, the dilution
in the first experiment being 1 in 20 and in the second 1 in 50 to 1 in 100.
The suspended solids were removed by rough filtration through glass wool.

In this case, the filters being new, it was possible to determine the stages
in the ripening process, and it was found that while a marked amount of
general oxidation was apparent in the effluents from all the filters in two
weeks, no trace of oxidised nitrogen appeared until 10 days later, and until
then the ammonia in the effluents was the same as in the sewage. The first
trace of oxidised nitrogen in the effluents appeared in the form of nitrous
nitrogen on the following dates, all three filters having been started on

February 20th :—
Deep filter on 18th March.

Medium , 21st March.
Shallow ,, 28th March.

For some time the formation of nitrous nitrogen continued in increasing
amount, and later, when nitric nitrogen made its appearance, the nitrous
nitrogen gradually diminished until ultimately the effluents from the medium
and deep filter showed no trace of it. For some reason it would appear that
the effluents from the shallowest filter ceased to be recorded before this period.

Regarding the absence of nitrous nitrogen in the effluents after the filters
had matured, the results accord with those at Hanley. In one other respect,
namely, the reduction of general oxidisable matter, the results also agree
with those at Hanley in the fact that practically the reduction was as marked
in the shallow as in the deep effluents.

As regards the degree of nitrification at different depths, Dr. Chick’s
results do not quite compare with those of Hanley. It must be remem-
bered, however, that the conditions differed widely. The volume of sewage
dealt with in the former case was relatively greater, and its ammoniacal
strength was very much higher. Also the intervals of delivery on to the
filter were much longer, namely, 38 minutes compared with 5 minutes in the
case of Hanley, and in addition all the disturbing influences incidental to a
laboratory experiment of this character have to be taken into account, such
as the effect of peripheral capillarity upon the uniformity of the downward
flow in a filter of such small diameter. At the same time, the nitrification
effected at the medium depth was very considerable, and it would have been
interesting to have had another record between that and the 6-ft. 6-in. depth.

F 2

68 Dr. G. Reid. [May 7,

It may now be convenient to sum up the arguments which may be
adduced, and the conclusions which may be drawn, from the recent observa-
tions at Hanley. In the first place, two important factors undoubtedly
contributed in no small degree to the high quality of the work done: (1) the
small amount of suspended matter in the effluent applied to the filter ; and
(2) the very efficient means of distribution provided.

The oxidation of organic matter already in solution is a comparatively
simple process, but to bring about the liquefaction of suspended organic
solids takes longer time, hence the importance of providing, as far as possible,
for their removal by mechanical subsidence or other methods, and their lique-
faction by anaerobic organisms as a preliminary process. When septic tank
treatment was first introduced, its advocates, in their enthusiasm, predicted
that the difficult question of sludge disposal would thus be solved, but
experience does not quite corroborate this prediction. In the case in point I
attribute the satisfactory reduction of suspended solids in the septic tank
effluent to the large capacity of the detritus tanks. These were divided into
three, two of which were always in use, while the third was emptied for
cleansing weekly, and so on in rotation, the whole three being brought into
use during rain periods.

As regards the distribution of the sewage on the filter, it is obvious that
uniformity is all-important, otherwise certain sections might be greatly over-
taxed, while other sections were working much below their capacity. The
distribution in this case was so perfect that frequent tests failed to establish
any appreciable difference between one square yard of filter and another.

Now, as regards the rapidity of the oxidising changes, and the remarkable
purity of the effluent after filtration through 12 inches only. The three
essential factors in the final changes are time, air, and organisms, and, given
a sufficiency of air, the greater the number of organisms present the larger
the amount of work done, provided the organic matter both in solution and
suspension is brought into intimate contact with the organisms. The factor
which governs the bacterial population is the area available for growth, and
this may be increased by two methods, either by enlarging the cubic capacity
of the filter or by subdividing the filtering medium. In the case in point,
the latter was the expedient adopted, and the subdivision was carried as far
as it was thought possible to carry it without preventing the superficial
penetration of the suspended solids into the interstices. In view of the
results, it is needless to discuss whether the reduction in the size of particles
resulted in an inadequate air supply.

The relative amount of carbonic acid in the air of the filter at different
depths also shows the highly active oxidising changes which take place in

1906. | Nitrification of Sewage. 69

the superficial layers. A series of samples of air aspirated from different
depths, by means of iron tubes driven vertically into the body of the filter
while in continuous use, yielded the following mean results :

Carbonic Acid in parts per 1000.

LEE, 2 ft. 3 ft. 4 ft.
19°5 215 20:0 20:0

An interesting fact bearing upon the aeration of the filter was incidentally
made apparent by the method at first adopted of collecting the air. The
distributor passing backwards and forwards over the filter was found to
interfere considerably with the collection of the samples, and, to obviate this,
the apparatus was periodically stopped for varying periods during the
operation. Owing, however, to extraordinary discrepancies in the results
thus obtained, measures were taken to overcome this difficulty and enable the
air to be collected without interrupting the regular flow of sewage on to the
filter. The results, of which the figures just given are the means, were then
found to be remarkably uniform, whereas in the case of the earlier samples
the carbonic acid varied in amount according to the intervals which elapsed
between the stopping of the distributor and the collection, short though these
intervals were, from 2°8 to 26:1, showing how free was the current of air
through the filter.

If, then, by using fine-grade particles the depth of filter may be greatly
reduced, the resulting economy would dictate such a course, but there is another
important consideration which, other things being equal, tells in favour of
shallow filters from the point of view of aeration. The air travels through a
filter from above downwards, the direction of the current probably being
mainly due to the percolation downwards of the sewage, and its more rapid
flow along the effluent drains. The air, therefore, as it passes downwards,
carries with it the products of the combustion which has taken place above,
and thus has an asphyxiating effect upon the organisms below, and it is
possible that even anaerobic fermentation may be revived in the bottom
layers. The sudden increase in the free ammonia figure, noted in the case otf
the effluent from the lowest tray, may possibly be accounted for in this way,
because the albuminoid ammonia figure does not represent the total organic
nitrogen present, therefore there is an unrecorded margin of nitrogenous
organic matter available for the revival of the ammonia change should this
explanation of the phenomenon be the correct one.

Be this as it may, however, the phenomenon does not appear to be
accounted for by fouling of the deeper strata. At the end of the
observations the filter was opened and carefully examined throughout its

70 Dr. G. Reid. [ May 7,

depth, when it was found that the dark discoloration from deposit was
confined to the surface, as was evident from the untarnished appearance
of the light-coloured filtering medium below the top 14 to 18 inches. Also,
the relative amount of organic solids in the interstices at different depths,
ascertained by drying and igniting 10 grammes of the filter particles in each
case, supports this contention, as the following figures show :—

Percentage Loss on Ignition of Filter Particles at Different Depths.

6 in. 1 ft. 2 ft. 3 ft. 4 ft.
3°25 0:99 0°65 0:53 0:53

I may mention that on ignition there was practically no smell, except in the
case of the samples collected at the 6-inch and 1-foot depths, and that in the
case of these only an odour such as that of burning soil could be detected.

Apart from all theory, however, the fact has been established beyond
all doubt that, in the case of the sewage in question at any rate, the lower

or 3 feet of filter medium is absolutely unnecessary, and, so far as the
cost of construction of the filter is concerned, the expenditure might be
reduced by about one-half. Again from the point of view of cost, another
important consideration comes in. It frequently happens that the absence of
2 or 3 feet of available fall is the determining factor between a gravitation
and pumping scheme, and in this respect the reduced depth of filter might
lead to further economy, not only in capital outlay, but in maintenance
charges.

Now, the question may be asked whether this experience acquired at
Hanley may be applied in other cases where the sewage may be of a stronger
character? I am not at present in a position to give a positive answer
to that question, but if, as is probable, such should prove not to be the case,
the observations I have recorded clearly point to the conclusion that the
extra filtering capacity should be provided for by increasing the area rather
than the depth of the beds. The three factors in the nitrifying process being
a given time, a given volume of air, and a given bacterial population, all these
would be supplemented by extending the area in accordance with the
combustion which has to be effected, and thereby diminishing the delivery per
square yard of filter. Of course, I do not suggest that the depth of filters
may be reduced to 1 foot, because we must allow for the eftluent drains,
and the few inches of large material immediately on the top of them, but
I do suggest the practicability, if a fine medium is used, of reducing the
total depth to, say, 2 feet 6 inches.

The tables appended give the details of the analyses and other determina-
tions made during the investigations which have been described.

71

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75

The Relation of the Kidneys to Metabolism.—Preliminary
Communication.

By F. A. BatnsripG£, M.A., M.D., and A. P. Bepparp, M.A., M.D.

(Communicated by Professor E. H. Starling, F.R.S. Received November 20,—
Read November 22, 1906.)

(From the Gordon Laboratory and Physiological Laboratory, Guy’s Hospital.)

The effects following on the removal of large portions of the kidneys of
dogs were studied by Rose Bradford in 1892. In the first place, he found
that animals deprived of three-quarters or more of their total kidney weight
rapidly wasted, and died in two or three weeks, or even less; and, although
they often refused food, they passed daily an amount of urea almost or quite
equal to that passed by the same animals on full diet before operation.
Secondly, he observed that after the removal of a portion of one kidney, the urine
became more abundant and more dilute, and the dogs were apparently unable
to excrete a concentrated urine; this effect was intensified by the subsequent
removal of the opposite kidney. Bradford concluded that the kidneys in
some manner normally control the nitrogenous metabolism, and that in the
absence of sufficient kidney substance this metabolism becomes excessive.

In view of their extreme importance, a repetition of these experiments
appeared advisable ; it was hoped, too, that more complete analysis of the
urine might throw some light on the course of nitrogenous metabolism as
a whole. This paper contains a preliminary account of our observations.

Methods.

Cats were used for these experiments. They were anszesthetised with
chloroform and ether, the anesthesia being maintained by ether throughout
the operation.

The abdomen was opened in the middle line, and the kidney drawn up into
the wound ; the capsule was opened, and stripped off the kidney. The renal
vessels were digitally compressed, a wedge cut out of the kidney, and the
cut surfaces brought into apposition and kept together by two or three
sutures passing deeply through the kidney substance. The kidney was then
replaced in its capsule and the latter was closed by a continuous suture ; the
kidney was returned to its place and the abdominal wound closed. No
vessels were ligatured, since forceps are so apt to damage the kidney
substance: a little blood collects between the kidney and capsule, where it
clots and checks further loss of blood.

76 Drs. F. A. Bainbridge and A. P. Beddard. [Nov. 20,

Two or three weeks later the opposite kidney was removed under ether
anesthesia through an abdominal incision.

The animals were kept in cages whose floor consisted of a wire netting ; the
cages stood upon a zine plate, sloping towards the centre, which was
perforated by a small hole. The urine ran down the zine sheet through the
hoJe into a basin containing a little chloroform. The urine and feces were
collected every 24 hours, and very great care was taken to keep the urine
free from food or feces.

The cats were kept at a fairly even, moderate temperature, and were
allowed some exercise daily in the laboratory.

The food was measured daily, and any food left over was also measured.
Their diet consisted of minced meat, milk and water; the amount of the
latter, of course, was not restricted.

The total nitrogen in the meat and milk was estimated by Kjeldahl’s
method. A considerable amount of meat was minced, and mixed into a
uniform mass; a sample of this was analysed, and the rest kept in an ice
chest ; the cats received a definite quantity each day.

The urine was analysed daily; the feces were collected for several days,
and the total nitrogen present estimated. The total nitrogen of the urine and
feeces was estimated by Kjeldahl’s method, the urea by a modification of
Folin’s method, the ammonia by Schaffer’s method, and the creatinin by the
colorimetric method of Folin. All the analyses were made in duplicate.

Results.

The effects of removing portions of the kidneys may be grouped as
follows :—Firstly, the general conditions of the animals including changes in
body weight; secondly, the nitrogenous metabolism ; thirdly, alterations in
the amount and concentration of the urine.

I. The Condition of the Animals—After the first operation, the cats
usually drank a little milk on the same day, but for several days their
appetite was poor, and they lost weight. Eventually they ate very well,
and appeared to be more hungry than normal cats. For a day or two the
urine contained blood, and for another two or three days albumen was
present.

Three cats survived the second operation for some time; they rapidly
recovered from the anesthesia, and showed very little shock.

Two of the three cats ate very little after the second operation, and
refused food altogether during the last few days of life; the third cat ate well
for 10 days, when its appetite also failed.

Vomiting and diarrhoea were observed in all the cats, and they seemed

1906. | The Relation of the Kidneys to Metabolism. 77

very susceptible to cold. They acquired a very foul breath, and soreness of
the lips and gums; their fur came off very rapidly.

Cat I lived six days, Cat II lived seven days, and Cat III lived 17 days
The rectal temperature was maintained at 100°

after the second operation.
or more until the last few days of life.

The Body Weight.—It will be seen from the following table that normal
cats lose weight when kept in confinement, notwithstanding some exercise
daily. The loss of weight, however, is less than that observed in Rose
Bradford’s dogs, most of which lost 10 per cent. or more of their initial
weight before the first operation. In our experiments, the cats were not put
into the collecting cage before the first operation, so that this initial loss of
weight was avoided.

Table I.
| Initial weight. Final weight. | Loss Time in cage.
grammes. grammes. Per cent. days.
Normal cat......... 3650 3580 1°9 19
Normal cat......... 2300 2140 6 °9 8

The effect of the two operations upon the weight of the cats is shown in
the following table :—

4

Table IT.
leith | sere | Life after
: time of Final
weight, first Loss. . Loss. second
; second weight. :
operation. . operation.
operation.
grammes. grammes. | Per cent. | grammes. | Per cent. days.
Cin) rae 24.20 2120 12 °3 1600 33 °8 6
Oo, 8 eee 2880 2550 11 *4 1840 36 ‘1 7
Cay INT... ....:. 2500 2220 1640 34 *4 17

| I 2

The final percentage loss of weight is much the same, and was reached in
Cat III in 17 days, and in a much shorter time in the other cats, because
Cat I ate well for 10 days, whereas the others ate very little after the second

operation.

These figures correspond closely with those of Rose Bradford ; he

also found that the animals lived much longer after the second operation

when they took food.

II. The Nitrogenous Metabolism.—tThe total nitrogenous metabolism was
determined in three cats before and after the second operation; the urinary
nitrogen was also estimated in one of these cats before the first operation.

The results are summarised in the following protocols :—

78 Drs. F, A. Bainbridge and A. P. Beddard. [Nov. 20,

Cat I—October 12. Weight 2420 grammes. Removed part of left kidney,
weight, 6°3 grammes.

: Total N in N as N as :
Date. Weight. Na, N as urea. Saniacnal eee N in food.
grammes. | grammes. | Per cent. | Per cent. | Per cent. grammes.
October 26...... | 2130 | 169 | — | 3°9 1°6 3°0
a a hi lol 2160 3°44 90 | 4°3 1h 4 3 °0
. AB a sels 2090 1:09 — 2:2 1°5 2°6
. VAS Eve Removed right kidney: weight 10°5 grammes.
by BOR... 2050 | 0°94 74: 2°6 2°3 }
alee. | 1950 0°86 90 1°6 2 °4: |
November 1 Pe 8ZO 0°84 — 3 °6 2 °7. © 0r8e
, 24 nae Puelley(edO) 1°73 | — 2°0 2°0 |
i 3...) L660 1-02 | 70 6:0 6 5)
a 4 | 1600 Found dead.
N intake. N output.
‘ eR TT Feces.
Total. : Average. Total. Average.
After first operation— grammes. grammes. grammes. grammes. gramme.
Sidays’ period...0.0..2.. | 8°5 2 84, 6°22 2°07 | 0°59
After second operation— | | |
claw aa ee 1-2 0-2 5-71 0-95 os2 |
| |

Post mortem.—No evidence of sepsis. Some subcutaneous fat still present.
Remainder of kiduey weighed 3°8 grammes.

Moralgkidney, weight. 2f20" 3. mesceme eet .. = 20°6 grammes.
Amount after second operation (3°8 grammes) = less than } total kidney
| weight.

= 1°5 grammes per kilo.

Cat I].—October 19. Weight, 2880 grammes. Removed part of left kidney,
8:8 grammes. |

Total N in | N as N as

I TEI urine. Pease ac ammonia. creatinin.
: as.
grammes. grammes. Per cent. Per cent. Per cent.
MctoberyAG ine. ccs: 2600 2°65 73 °9 3°8 1 ‘4
November! U0 /.s..05..; 2510 3°08 92 °8 3°3 Lee
: Be ur | 2550 3°43 79:9 Mi 12
. Ey aN glee 2550 3°59 | = 3°8 1°4
a Dinara | Removed right meer weighing 141 grammes.
(25 a ami ¥0) 10 88 -2 Rid, |) 1) eee
i Pedder sl): BEBO i 69 84-0 16) MARS
SO Minette 420018 he OOOO 1:79 89-9 2-0 1°2
iS 12 ............| Catmoribund. T.95°°0. Killed by chloroform. Weight 1840 ers.

1906. ]

The Relation of the Kidneys to Metabolism. 79

N intake. N in urine.
; Total feces.
Total. oe Total. anes
average. average.
|
|

After first operation— grammes. | grammes. | grammes. | grammes. | grammes N.

Period of 10 days ............ 39 °7 3°9 29 ‘Ol 2°9 2-26
After second operation —-

Le ee eee 4°46 0-74 8°78 1°46 1 ‘0 (approx.)

Post mortem.—Remainder of kidney adherent to spleen, normal in appear-
ance, very little omental fat; none visible elsewhere.
wasted. No evidence of sepsis.

Muscles not obviously

Total kidney weight......
Weight found post mortem =

= 29°6 grammes.

6-7 ‘3

= 2 total kidney weight.
= 2°3 grammes per kilo.

Cat I[].—September 4. Weight, 2500 grammes.
kidney—4 grammes.

Removed part of left

Date. Weight. Joel Bean | N as urea.| N as NH. a md, N intake.
urine. | creatinin.
grammes. grammes. | per cent. | per cent. | percent. | grammes.
September 11...... 2140 3°52 2 3 ‘1 1°3 10 °4
5 ore 2120 4°51 88 °8 45 ras 7°6
6 Gece ane 2220 5°12 90%:1 3 °6 1°3 7°3
NO se Removed right kidney, wt. 10°25 grammes ; wt. of cat, 2220 grammes.
i 2p Se 2020 3°26 75 0 9°9 2°0 | 9 °0
October 2 ......... 1880 1°13 83 ‘1 10 ‘0 Le nil
LS RSA se Saas 1670 0°89 = 2°0 Pl nil

After the second operation, the cat ate well for 12 days; after that it ate
very little and wasted rapidly. It was moribund on October 6, and was killed
by chloroform. Weight at death 1640 grammes.

Post mortem.—There was no sepsis; very little fat was visible in the

subcutaneous tissues.
Total kidney weight...... = 20°65 grammes.
Amount left post mortem = 6°4 es

= less than 4 total kidney weight.
= 2°5 grammes per kilo.

80 Drs. F. A. Bainbridge and A. P. Beddard. [Nov. 20,

: : Total N in .
Total N intake.| Daily average. Rin ¢ Daily average.

After first operation— grammes. grammes. grammes. grammes.

Says) POROGsee tess cs. 25 2 8 °4 11°3 3°77
After second operation—

HirstaGidaysisoeureeas vee 23 °4 3°9 12 ‘86 2°14

Second:6 Gays i0.4..:.0: 24 °2 4°03 16 °0 2°6

Hast A dayiss os 2ecccccra: 0 0 3°72 0°93

The nitrogen intake for Cat III is not absolutely exact, since the meat and
milk were not analysed every day.

The clinical condition of these cats was apparently identical with that of
Rose Bradford’s dogs; in both cases, the second operation was usually
followed by rapid wasting. In two of the three cats considerably more than
three-quarters of the total kidney weight was removed; in the third cat
rather less than three-quarters was removed, but since the animal died, one
may conclude that cats need rather more: kidney than dogs in order to main-
tain life. In this connection it may be noted that in cats the average kidney
weight is 91 grammes per kilo., as compared with 6°7 grammes per kilo.
in dogs.

There is no doubt, then, that the experimental conditions of Bradford’s
dogs were strictly reproduced. Yet one of the cats showed no increased
output of nitrogen after the second operation; in one cat the increase of
nitrogen first occurred five days after the operation, when the cat had lost
28 per cent. of its weight, and was almost moribund ; and in the other cat
the increase occurred four days after the operation, when the loss of
weight was 22°5 per cent.

In none of the cats did the nitrogen output after the second operation ever
reach that observed after the first operation, and it never greatly exceeded
that found in normal fasting cats. The greatest daily output of urinary
nitrogen after the second operation, when the cats refused food, was 1°79 in
Cat II on the last day of life. For normal cats kept without food for 24 hours
the output of urinary nitrogen is about 1 gramme and the loss of weight
about 100 grammes.

The protocols, moreover, show that the second operation has no appre-
ciable effect upon the percentage of urea, ammonia and creatinin relatively
to the total nitrogen. There is no increase in the output of creatinin (on
a milk diet) such as might be expected if muscular metabolism were
excessive.

We find, therefore, that the removal of three-quarters of the kidney weight
of cats has no influence upon their nitrogenous metabolism until the last

1906. | The Relation of the Kidneys to Metabolism. Sp"

two or three days of life. This terminal increase of nitrogen only occurs
when the cats have lost 22 per cent. or more of their body weight, and are
obviously in a state of inanition. The two cats which showed this terminal
increase of urinary nitrogen ate very little after the second operation, and
wasted rapidly.

The effect of starvation upon the body weight and urinary nitrogen of
normal animals has been investigated by Voit, Schondorff, Kaufmann, and
others. It ‘is clear, from a comparison of their results with the condition:
of our cats, that the condition of starving animals is identical with that of
cats which refuse food after the second operation on the kidneys.

There is in both cases a progressive loss of weight, a fall of temperature
during the last few days of life, and usually a terminal rise in the output
of nitrogen in the urine. E. Voit and others have shown that, in starvation,
the nitrogen output may increase several days before death, and that the time:
of its onset is directly related to the amount of fat originally possessed
by the animal. A very fat animal may never exhibit the increase of
nitrogen, whereas, if the original store of fat is small, a very few days of
starvation suffice to evoke an increased output of nitrogen. For example, a
starving dog, observed by Schéndorff, showed an increased output of nitrogen
when it had lost 20 per cent. of its weight; in our cats the nitrogen rose.
coincidently with a loss of 22 per cent. or more of the body weight.

We consider, therefore, that the wasting, fall of temperature and final
increase of urinary nitrogen occur simply because the cats refuse food after
the second operation; they die from inanition, and the rise of urinary
nitrogen, when it occurs, merely means that the animal’s store of fat is much
diminished, and that more energy must be supplied by proteid breakdown.

We consider, further, that the same explanation fully accounts for the
results obtained by Rose Bradford, and that there is no evidence from his.
experiments that the kidneys directly influence nitrogenous metabolism.

It may be added that Dr. Pembrey has investigated the gaseous exchange:
of these cats; he finds that his results are such as would be given by animals
in a state of inanition.

Ill. The Amount and Concentration of the Urine-—The amount and specific
gravity of the urine of cats varies widely according to their diet. On a meat
diet the urive is concentrated; on a milk diet it is abundant and dilute.
Making allowance for variations of diet, we find no evidence that the amount
and specific gravity of the urine are necessarily modified by either the first
or second operation. Only one cat almost invariably passed abundant
quantities of dilute urine after the first operation; the others often passed
a concentrated urine. In this respect our results are at variance with those-

VOL, LXXIX.—B. G

82 The Relation of the Kidneys to Metabolism.

of Rose Bradford ; and it may be pointed out that the effect, even in dogs, is
not a constant one, since in half of Bradford’s experiments the urine was no
more abundant after the second than after the first operation.

It may be concluded, therefore, that the increase in quantity and decrease
in concentration of dog’s urine, after operations on the kidneys, is an
incidental rather than an essential result.

Table IIL.
| | Urine.
| Nature of food. |- mavGs ico
| Amount | Sp. gr. | Total N.
c.c. | grammes.
Neel at it Meat! oo ecceerteee 112 1046 5 °4
AT er) a ae Bread and milk 310 1007 1°6
After first operation— |
Cs Meat Uthie, aaa: 128 1040 5:1
Cr aay sat tea Bread and milk ... 435 1005 1°55
One Tal Meatinsecccece 164 | 1038 3°44
TS gene: Par Beane re a Mik) ost tae once 124 1020 2°1
ate ears eeccsneecat 8 Meat and milk ... 68 1042 2 °65
Cab MIVA ites sh adh tans Meat? 29) ee 110 1040 ?
Meat cnet 43 | 1054
| CENT Ey an i oe Milk oan alee 150 | 1015
| After second operation—
Prater ucla Mille, eee tee 90 1020 1-49
| ib he diy eae eT ey Meat: rig oe 182 1018 3°02
78 1030 0 94
Cap iis ae eee Vk yee { ie hes tae
: 100 1019 1°10
| (OP g LOIN, esc eee nan ane aie ae reas Meat and milk { 208 1018 3 4A
|
Conclusions.

1. We confirm Bradford’s observations that removal of three-quarters or
more of the total kidney weight in cats is followed by loss of appetite, wasting,
and death within a few days or weeks.

2. We find that in such cats an increased output of nitrogen is not of
constant occurrence ; and that it takes place only in cats which have lost
22 per cent. or more of their initial body weight at the time of its onset.

3. We conclude, therefore, that the kidneys have no direct influence upon
nitrogenous metabolism, and that the increased output of nitrogen is simply
the result of inanition, andis of the same nature as that observed in starving
animals.

4, We find that, after removal of a portion of one kidney, and also after
subsequent removal of the opposite kidney, cats are still able to pass a
concentrated urine, and that the amount of the urine is not necessarily
increased beyond the normal.

7
=> ics

nas y ;
eT

enses of this research were partly defrayed by a grant from the
Sommittee of the Royal Society, to whom we desire to express our
ndebtedness

REFERENCES.

: Bradford, ‘ Journ. Physiol.,’ vol. 23, p. 415, 1898.
a Folin, ‘Journ. Phys. Amer.,’ vol. 13, 1905.
Kaufmann, ‘ Zeitschrift f. Biol.,’ vol. 41, 1901.
Schondorff, ‘ Pfltiger’s Archiv,’ vol. 82, p. 60, 1900.
Voit, E., ‘ Zeitschrift f. Biol.,’ vol. 41, p. 550, 1901.

Anniwersary Address by Lord Rayleigh. 83

The expenses of this research were partly defrayed by a grant from the
Grants Committee of the Royal Society, to whom we desire to express our

indebtedness.

REFERENCES.

Bradford, ‘ Journ. Physiol.,’ vol. 23, p. 415, 1898.
Folin, ‘Journ. Phys. Amer.,’ vol. 13, 1905.
Kaufmann, ‘ Zeitschrift f. Biol.,’ vol. 41, 1901.
Schoéndorff, ‘ Pfliiger’s Archiv,’ vol. 82, p. 60, 1900.
Voit, E., ‘ Zeitschrift f. Biol.,’ vol. 41, p. 550, 1901.

Address of the President, Lord Rayleigh, O.M., D.C.L., at the
Annwversary Meeting on November 30, 1906.

Since the last Anniversary the Society has sustained the loss of twelve

Fellows and two Foreign Members.
The deceased Fellows are :—

Professor Lionel Smith Beale, died March 28, 1906.

Sir Walter Lawry Buller, K.C.M.G., died July 19, 1906.

Charles Baron Clarke, died August 25, 1906.

Right Hon. Sir M. E. Grant Duff, G.C.8.1, died January 11, 1906.
Professor Charles Jasper Joly, died January 4, 1906.

Colonel Sir Alexander Moncrieff, K.C.B., died August 3, 1906.
George James Snelus, died June 18, 1906.

Professor Hermann Johann Philipp Sprengel, died January 14, 1906.
General Sir Henry E. L. Thuillier, C.S.1.

Rev. Canon H. B. Tristram, died March 8, 1906.

Professor Harry Marshall Ward, died August 26, 1906.

Professor Walter Frank Raphael Weldon, died April 13, 1906.

The Foreign Members are :—
Professor Ludwig Boltzmann.
Professor Samuel Pierpont Langley.
On this list are to be found the names of veterans distinguished in many
branches of science and in public affairs. One name is a household word in

7Ou, LXXIX.—B. H

84 Anmversary Address by Lord Rayleigh. —[Nov. 80,

every physical and chemical laboratory. It would be difficult, indeed, to
enumerate the investigations which have owed their success to the invention
of the Sprengel mercury pump. In other cases, scientific careers still in full
activity have, unhappily, been cut short. I allude especially to Joly, Marshall
Ward, and Weldon. Even within my term of office our discussions have
been enlivened by Weldon’s scientific enthusiasm and vigorous polemics.

On the Foreign list are two distinguished names. Professor Ludwig
Boltzmann, of Vienna, was perhaps the first Continental physicist to take
up the ideas of Maxwell’s electric theory of light, of which he had early
grasped the scope, and became for many years one of its most emphatic
supporters. One of his earliest series of experiments was a determination of
the influence of the crystalline quality on the dielectric constant of sulphur,
with a view to comparison with its optical double refraction. In the theory
of gases he is to be classed along with Clausius and Maxwell as one of the
creators of the dynamical theory, on which he became the highest authority.
By developing an idea originated by Bartoli he placed Stefan’s law of
intensity of natural radiation on a theoretical basis, and thus became the
pioneer in the modern thermodynamics of radiant energy. He contributed
to the advance of physical science by many other investigations, and by his
books on Gas Theory, on Electrodynamics, and on Mechanics. I may
perhaps be allowed to add that at the time of his unhappy death, Boltzmann’s
name was before the Council as proposed for one of our medals.

Professor Langley’s work was more on the experimental side of physics.
In his bolometer he applied electric resistance thermometry to radiation, and
was thereby enabled to penetrate further into the important and mysterious
region of the ultra-red than had before that time been possible. For this
work he received the Rumford Medal in 1886. During the later years of
his life his attention was largely occupied with the mechanical problem of
flight, and his models attained a considerable measure of success. As
Secretary to the Smithsonian Institution at Washington he did much to
forward, by his co-operation and advice, all kinds of scientific investigation.

In the Report of the Council there has been laid before you an account of
the work of the Council and of various Committees in a very wide field.
The investigation of the terrible disease known as Sleeping Sickness has
unhappily been marked by the tragic death of Lieutenant Tulloch, who has
fallen a victim to his zeal in studying the disease in Uganda. Vigorous
efforts are being undertaken to discover some therapeutic remedy for the
malady. In the case of Malta Fever, too, the investigation of which was
entrusted to the Royal Society by the Colonial Office, good progress has been

1906.| Annwersary Address by Lord Rayleigh. 85

made. It has been ascertained by the Society’s Commission in Malta that
the main source of propagation is the milk of infected goats. When this
discovery was made the authorities of the island were at once warned of the
danger in the milk supply, and the necessary precautions were taken. Since
then the number of cases of fever in the hospitals has so greatly diminished
as to afford good hope that this disease, which has been so great a scourge in
Malta, may ere long be reduced to insignificant proportions or altogether
exterminated.

I observe that a movement has been been started in this country in aid of
the Greek Anti-Malaria League. Professor Ronald Ross, than whom there
is no higher authority, bears witness to the unexpected prevalence of the
infection in most of the localities examined, and he is confident that
practical results of the highest value would follow expenditure in combating
the disease on lines already laid down. Although I speak only from general
knowledge, I cannot let this opportunity pass without emphasising my sense
of the enormous importance of this class of work. If men knew where their
real interests le, our efforts in this direction would be doubled or quad-
rupled. In this way discoveries, which the future will certainly bring,
might be accelerated by decades, giving health and life to thousands or
millions who now succumb. Willing and competent workers would soon
offer themselves ; the principal obstacle is the want of means.

The preparation of the ‘ Royal Society Catalogue of Scientific Papers’ for
the remaining portion of the 19th century, which has proved a task so much
more gigantic than can have been contemplated by the originators of the
Catalogue nearly half a century ago, has been actively pushed forward. In
consequence of the increased expenditure, now at the rate of nearly £2000
a year, the funds available are again approaching exhaustion. The difficulties
of the President and Council and of the Catalogue Committee on this subject
have once more been promptly resolved by the action of our Fellow, Dr. Ludwig
Mond, who, after consultation with the Officers, has again made himself re-
sponsible for a further subsidy amounting to £2000 a year for three years. It
is hoped that with the balance in hand and other sources of income, including
the Handley Fund of the Royal Society, the income of which is devoted
to this purpose, this subvention will suffice for the preparation of the work
and for passing it through the press. Since the Royal Society took this
great national task in hand there has already been spent on it over £23,500,
while on each. occasion of financial stress Dr. Mond has come forward with
the means of relief, his direct contributions, including that just promised,
now amounting in the aggregate to £14,000. This great work when published
will thus be a tangible memorial of Dr. Mond’s practical insistence on

H 2

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

the importance of adequate indexes of the vastly increasing literature of
science.

Of the activities working under the Royal Society the one with which
I have been especially connected is the National Physical Laboratory.

In their Report for the past year the Executive Committee call attention
to the loss they have sustained by the deaths of Sir Edward Carbutt and
Sir Bernhard Samuelson, both members of the Committee and warm
supporters of its work.

The Report shows continued progress. As a result of a memorial to the
Chancellor of the Exchequer, signed by about 150 Members of Parliament,
the Grant for building and equipment for the year was increased from
£5000 to £10,000, and this has enabled the Committee to take in hand —
some urgently needed extension. 2

Buildings are now in course of erection for Metrology and for Metallurgical
Chemistry, while the Engineering Laboratory is being doubled in area.
The two last additions were called for in great measure in consequence of an
arrangement with the India Office whereby the testing work required for the
Indian Government, hitherto carried on at Coopers Hill, is to be transferred
to the Laboratory. The Indian Government provide the testing machine
and other appliances required for the work, and, in addition, have intimated
their intention of placing in the charge of the Committee the very admirable
electrical equipment now at Coopers Hill.

Towards the equipment of the Metallurgical Laboratory the Goldsmiths’
Company have made a very generous donation of £1000, while the Govern-
ments of New Zealand and Western Australia have contributed £100 each
to the equipment of the Metrological Laboratory.

The buildings of the Electrical Laboratory have been completed, and were
formally opened by the Right Hon. R. B. Haldane in June last. On this
occasion many representatives of electrical industry from various parts
of the world, who were attending the special conferences of the Institution
of Electrical Engineers, were present, and joined in the inspection of the
Laboratory.

After the meeting Sir John Brunner announced his intention of subscribing
£5000 towards the equipment of the various buildings now in hand.

A number of important researches, a lst of which is given in the Report,
have been published during the year, and others are in a forward state
of preparation for publication. Dr. Carpenter and Mr. Edwards have com-
pleted the first part of their research into the properties of the copper-
aluminium alloys for the Alloys Research Committee. Dr. Stanton and
Mr. Bairstow have read a paper before the Institution of Civil Engineers on

1906. | Annwersary Address by Lord Rayleigh. ee

the effect of alternating stresses on steels. The work on electrical standards
with the Ampere Balance, which has a special interest for myself, is well
advanced. In the hands of Mr. Smith the measurement of the electrolytic
deposits of silver, which represent the whole passage of electricity through
the apparatus, has attained a remarkable precision and, what is perhaps
more important, some anomalies met with at first seem to be on the road to
elucidation. Mr. Campbell has made progress with his research on
hysteresis in steel sheets, and Dr. Harker is engaged with interesting
investigations in high-temperature thermometry.

Changes in the staff have taken place. Dr, Caspari resigned his position
in order to take up private practice, while Dr. Carpenter has been appointed
the first Professor of Metallurgy in the Victoria University of Manchester.
The arrangements with the Jndian Government have necessitated an
alteration in the metallurgical division, and the Committee have thought it
right to constitute a Department of Metallurgy and Metallurgical Chemistry,
with a Superintendent in charge of the work. They have been fortunate in
securing the services of Mr. W. Rosenhain as the first occupant of the post.

A question of importance has arisen as to the performance at the Laboratory
of tests, partaking of a routine character, on the physical and mechanical
properties of specimens of material. To this objection has been taken on
the ground of competition with the work of private establishments. In one
of its aspects the question is financial. But the Executive Committee are of
opinion that, even if the pecuniary loss were recouped, the efficiency of the
Laboratory would suffer from the abandonment of this work. While anything
like unfair competition with private establishments should be avoided, the
execution of tests is good practice for the staff, and tends to keep them in
touch with the manufacturers and with the practical problems which may
demand examination. In view of the difference of opinion that has manifested
itself, the Treasury has decided to appoint a small committee to inquire into
the working of the Laboratory, with a special reference to this question.

On a former occasion, my distinguished predecessor, Sir William Huggins,
called attention to some of the more important matters on which the Society
in the past had initiated, supported, or given advice about scientific questions
in connection with the State, and in other ways had made its influence felt
strongly for the good of the country. It would hardly become me, with my
short experience of the working of the Society, at least in recent years, to
pursue this theme. The function of the Society which lies most open to the
observation of an incoming President is that exercised at the ordinary
meetings. I am impressed with the difficulty, arising out of the ever-

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

increasing specialisation of science, in rendering really useful the reading of
papers and discussions thereupon. It is, of course, felt more severely in a
Society like our own, which embraces within its scope the whole scientific
field. It not infrequently happens that a paper is addressed to an audience
among whom there is no one competent to follow the detailed observations
and reasonings of the author. I am sometimes reminded of a saying of
Dalton’s on similar occasions at Manchester, quoted by Sir Henry Roscoe in
his genial and entertaining ‘ Reminiscences’: “ Well, this is a very interesting
paper for those that take any interest in it.” A little more discretion on the
part of readers of papers in having regard to the composition of their actual
audience would be helpful here. In some cases experimental illustration
would bring home to a larger number what is followed with difficulty from a
merely verbal statement. But I am afraid that no complete remedy is
within reach. |
Increase of specialisation, however inconvenient in some of its aspects,
is, I suppose, a necessary condition of progress. Sometimes a big discovery,
or the opening up of a new point of view, may supersede detail and bring
unity where before there was diversity, but this does not suffice to com-
pensate the general tendency. Even in mathematics, where an outsider
would probably expect a considerable degree of homogeneity, the movement
towards diversity is very manifest. Those who, like myself, are interested
principally in certain departments, and can look back over some 40 years,
view the present situation with feelings not unmixed. It is disagreeable
to be left too far behind. Much of the activity now displayed has, indeed,
taken a channel somewhat remote from the special interests of a physicist,
being rather philosophical in its character than scientific in the ordinary
sense. Much effort is directed towards strengthening the foundations upon
which mathematical reasoning rests. No one can deny that this is a
laudable endeavour; but it tends to lead us into fields which have little
more relation to natural science than has general metaphysics. One may
suspect that. when all is done fundamental difficulties will still remain to
trouble the souls of our successors. Closely connected is the demand for
greater rigour of demonstration. Here I touch upon a rather delicate
question, as to which pure mathematicians and physicists are likely to differ.
However desirable it may be in itself, the pursuit of rigour appears some-
times to the physicist to Jead us away from the high road of progress. He
is apt to be impatient of criticism, whose object seems to be rather to pick
holes than to illuminate. Is there really any standard of rigour independent
of the innate faculties and habitudes of the particular mind? May not
an argument be rigorous enough to convince legitimately one thoroughly

1906. | Anmwversary Address by Lord Rayleigh. 89

imbued with certain images clearly formed, and yet appear hazardous or
even irrelevant to another exercised in a different order of ideas? Merely
as an example, there are theorems known as “ existence-theorems ” having
physical interpretations, the object of which is to prove formally what to
many minds can be no clearer afterwards than it was before. The pure
mathematician will reply that even if this be so, the introduction of
electrical or thermal ideas into an analytical question is illogical, and from
his own point of view he is, of course, quite right. What is rather surprising
is that the analytical argument should so often take forms which seem to
have little relation to the intuition of the physicist. Possibly a better
approach to a reconciliation may come in the future. In the meantime we
must be content to allow the two methods to stand side: by side, and it will
be well if each party can admit that there is something of value to be
learned from the point of view of the other.

In other branches, at any rate, the physicist has drawn immense
advantage from the labours of the pure analyst. I may refer especially to
the general theory of the complex variable and to the special methods which
have been invented for applying it to particular problems. The rigorous
solution by Sommerfeld of a famous problem in diffraction, approximately
treated by Fresnel, is a case in point. We have moved a long way from
the time when it was possible for the highest authority in theoretical optics
to protest that he saw no validity in Fresnel’s interpretation of the
imaginary which presents itself in the expression for the amplitude of
reflected light when the angle of incidence exceeds the critical value. In
this connection it is interesting to remember that, in his correspondence
with Young, Laplace expressed the opinion that the theoretical treatment
of reflexion was beyond the powers of analysis. The obvious moral is that
we are not to despair of the eventual solution of difficulties that may be too
much for ourselves.

As more impartially situated than some, I may, perhaps, venture to say
that in my opinion many who work entirely upon the experimental side
of science underrate their obligations to the theorist and the mathematician.
Without the critical and co-ordinating labours of the latter we should
probably be floundering in a bog of imperfectly formulated and often
contradictory opinions. Even as it is, some branches can hardly escape
~ reproaches of the kind suggested. I shall not be supposed, I hope, to under-
value the labours of the experimenter. The courage and perseverance
demanded by much work of this nature is beyond all praise. And success
often depends upon what seems like a natural instinct for the truth—one of
the rarest of gifts.

90° Annwersary Address by Lord Rayleigh. [Nov. 30,

MEDALLISTS, 1906.

The Copley Medal is awarded to Professor Elias Metchnikoff, For.
Mem. R.S., on the ground of his distinguished services to zoology and
to pathology, particularly for his observations on the development of
Invertebrates and on phagocytosis and immunity. From 1866 to 1882
Professor Metchnikoff’s work was exclusively zoological, and mainly during
that period he produced a series of brilliant memoirs dealing with the early
development and metamorphoses of Invertebrates.

Although his name stands in the first rank of investigators of these
subjects, the most celebrated of his discoveries are those relating to the
important part played by wandering mesoderm cells and white blood-
corpuscles in the atrophy of larval organs, and in the defence of the organism
against infection by Bacteria and Protozoa. It was on these researches
that he based his well-known ‘Phagocyte Theory.’ Metchnikoff’s funda-
mental observations were made in Messina in 1882, and were published in
the following year. In these he showed that the absorption and dis-
appearance of the embryonic organs of Echinoderms were effected by
wandering mesoderm cells, which devoured and digested the structures
which had served their purpose and become effete. The observation that
white blood-cells accumulate in an inflamed area after infection by Bacteria
suggested that these cells might also devour and thus destroy the invading
microbes, and that the process of inflammation was really a physiological
and protective reaction of the organism against infection. The study of
the infection of Daphnia by Monospora bicuspidata entirely justified this
prediction. The account of the phenomena of infection as seen in this
transparent Crustacean was published in ‘ Virchow’s Archiv’ (vol. 96) in
1884, while, later in the same year, Metchnikoff published another paper
extending these observations to Vertebrates, and showing the universal
applicability of his generalisation as to the essential character of the
inflammatory process.

During the twenty years which have elapsed since the publication of the
‘Phagocyte Theory, Metchnikoff, with the assistance of a host of pupils
and disciples from all parts of the world, has been continuously engaged in
the study of the reaction of the organism against infection, and in investi-
gating the essential features of immunity in the light of the illuminating
generalisation laid down in 1884. :

Though of limited range, and therefore inferior in scientific importance
to the more fundamental researches carried out by him _ previously,

1906. | Anniversary Address by Lord Rayleigh. 91

Metchnikoff’s recent work on infection by the micro-organism of syphilis
and the attainment of protection and immunity against this disease may be
mentioned on account of its important practical applications.

It is not too much to say that the work of Metchnikoff has furnished
the most fertile conception in modern pathology, and has determined the
whole direction of this science during the last two decades.

The Rumford Medal is awarded to Professor Hugh Longbourne Callendar,
F.R.S., for his experimental work on heat.

Professor Callendar has devoted his attention chiefly to the improvement
of accurate measurement in the science of heat by the application of electrical
methods. His first paper “On the Practical Measurement of Temperature,”
‘Phil. Trans.,’ 1887, paved the way for the application of the electrical
resistance thermometer to scientific investigation. Ina later paper, written
in conjunction with Griffiths, “On the Boiling Point of Sulphur, ete.,” ‘ Phil.
Trans., 1891, the application of his method was further extended, and a
simple method of standardisation was proposed. In continuation of this
work Professor Callendar has written a number of subsidiary papers dealing
with details of construction of instruments, and applications to special
purposes. The results of this thermometric work have since been confirmed
by Chappuis and Harker, ‘ Phil. Trans.,’ 1889, at the Bureau International,
Paris, and by other observers, and are now generally accepted.

More recent developments in accurate electrical thermometry have been
described by Professor Callendar in later papers. He has also devised a
special type of “gas-resistance ” thermometer, depending on the increase of
viscosity of a gas with temperature, which is the exact analogue of the
electrical resistance thermometer, and possesses peculiar advantages for high-
temperature measurements.

The application of electrical resistance thermometers and thermo-couples
to the observation of rapid variations of temperature has been utilised by
Professor Callendar in the study of the adiabatic expansion of gases and
vapours, and in the observations of the cyclical changes of temperature of the
steam and of the cylinder walls in a steam-engine. The latter research
was undertaken in conjunction with Professor Nicholson, with a view to
elucidate the theory of cylinder-condensation.

The researches of Rowland and other experimentalists on the specific heat
of water, and the mechanical equivalent of heat, had shown that grave
uncertainties affected the value of this most fundamental physical constant,
which could not be removed satisfactorily without a complete investigation of
the variation of the specific heat of water between 0° and 100° C. Professor

92 Anmversary Address by Lord Rayleigh. [Noy. 30,

Callendar devised a continuous electrical method of attacking this problem,
possessing many important advantages as compared with older methods. He
was assisted by Dr. Barnes in carrying out this work, the results of which
form the subject of papers by Callendar and Barnes in the ‘ Phil. Trans. Roy.
Soc.,' 1901. As an illustration of the probable accuracy of their results it
may be observed that, whereas by any of the older formule accepted for the
variation of the specific heat of water the values of Rowland and of Reynolds
and Moorby for the mechanical equivalent are seriously discordant, they are
brought into perfect agreement by the work of Callendar and Barnes.

In the subject of conduction of heat Professor Callendar has contributed
many original methods described in various minor papers, and, in addition to
the thermal investigations with which his name is chiefly associated, has
carried out some purely electrical researches.

One of the Royal Medals has been awarded, with the approval of His
Majesty, to Professor Alfred George Greenhill, a Fellow of the Society,
on account of the number and importance of his mathematical investi-
gations produced between the year 1876 and the present time. They
embrace a variety of mechanical and physical subjects, including dynamics,
hydromechanics, electricity, and gunnery. He is the author of two treatises
on hydromechanics, both remarkable for originality of treatment.

The subject, however, to which he has devoted most time and attention is
the theory of elliptic functions. His work on this subject may be placed
in two classes: (1) Investigations in which he has extended the subject into
new fields, as in the series of memoirs on the “ Transformation and Complex
Multiplication of Elliptic Functions,” contributed to the ‘Proceedings of
the London Mathematical Society’ (vols. 19, 21, 25, 27), and in the memoir
on the “Third Elliptic Integral and the Ellipsotomic Problem,” in the
‘Phil. Trans.’ (vol. 203). (2) Applications to Mechanical Problems, mainly
dynamical, for purposes of calculation or illustration. In this class may be
placed his treatise on the Elliptic T'unctions, as well as numerous papers in
journals and the proceedings of scientific societies.

All Professor Greenhill’s work is characterised by much originality, and
by a rare power and skill in algebraic analysis.

His Majesty has also approved the award of a Royal Medal to
Dr. Dukinfield Henry Scott, also a Fellow of the Society, for his investi-
gations and discoveries in connection with the structure and relationship
of fossil plants. Dr. Scott began the very important work which he has
accomplished in this subject by helping the late distinguished paleo-

1906. | Annversary Address by Lord Rayleigh. 93

botanist, Professor W. C. Williamson. In this co-operation he greatly
enhanced the value of Williamson’s work. He not only added many new
discoveries, but, what was more important, demonstrated the value of the
work in relation to phylogeny.

Dr. Scott has since added much of first-rate importance. He has discovered
and elucidated many important types, his work constituting a most valuable
acquisition to botany from the evolutionary point of view. It is not only in
the accurate investigation of difficult structures that Dr. Scott has been so
successful; not the least of his merits les in the philosophical treatment of
the problems suggested by his discoveries. His position as one of the
leading palzobotanists in the world is well recognised. He has, both by his
personality and by his writings, exercised a well-marked and widespread
influence on the work of other botanists. The fact that he has created in
this country a vigorous school of paleobotanists may be regarded as an
additional claim for the honour now conferred upon him.

The Davy Medal is given to Professor Rudolf Fittig, Professor of Chemistry
in the University of Strassbourg, who began to publish scientific work as
early as 1858 and in 1864 discovered the method for the synthesis of
hydrocarbons homologous with benzene, which has ever since borne his name.
‘Up to about 1880 he worked chiefly on benzene derivatives, but his attention
was gradually attracted to the study of lactones and acids, both saturated and
unsaturated, which has largely formed the subject of his numerous published
papers down to the present day.

F ittig has been a remarkably active worker. The Royal Society Catalogue
contains under his name alone 96 papers, and, jointly with students and
others, 71 more down to 1883. Since that time a number about equally large
has been recorded in the indexes of the chemical journals. The work of
Fittig and his students on lactones and acids, and particularly the inter-
molecular changes which many unsaturated acids undergo, may be said to be
¢lassical, and it has had an important influence on the progress of theoretical
chemistry. |

The Darwin Medal has been awarded to Professor Hugo de Vries, For.
Mem. R.S. Professor de Vries has made a series of important discoveries in
connection with the manner in which new races of organisms may originate,
and he has materially extended and systematised our knowledge of the laws
affecting the results of hybridisation. His work is the outcome of very
extensive experiments that have been carried on for many years. He has
stimulated numerous investigators, both in Europe and in America,

94 Anmversary Address by Lord Rayleigh.

to extend these enquiries; and the results already obtained are of great:
importance, both from a theoretical and from a practical point of view.
De Vries’ work has exercised considerable influence on other branches of
biology, and has suggested new lines of investigation in many directions.

Mrs. W. E. Ayrton is the recipient of the Hughes Medal, which is awarded
for original discovery in Physical Sciences, particularly electricity and
magnetism, or their applications. Her work on the Electric Arc has been
described in a paper published in the ‘ Philosophical Transactions, and in.
various other publications.

Mrs. Ayrton’s investigations cover a wide area. She discovered the
laws connecting the potential difference between the carbons of an arc with.
the current and with the distance between them, and proved these to apply
not only to her own experimental results but to all the published results.
of previous observers. Dealing with the modifications introduced into the
arc by the use of cores in the carbons, she found the causes of these
modifications. The peculiar distribution of potential through the are was.
traced, and its laws were discovered by her.

Having found the conditions necessary for maintaining a steady arc, and
for using the power supplied to it most efficiently, she was able to explain
the cause of “ hissing,” and the causes of certain anomalies in the lighting:
power of the arc.

For the past four years Mrs. Ayrton has been engaged in investigating.
the causes of the formation of sand ripples on the seashore.

95

Further Observations on the Effects produced on Rats by the
Trypanosomata of Gambia Fever and of Sleeping Sickness.
By H. G. PLimmer, F.LS.

(Communicated by Dr. C. J. Martin, F.R.S. Received November 21,—Read
December 13, 1906.)

[PLATE iE]

In January, 1905, I published* a note of some experiments with the above-
named strains of trypanosomata, and I have continued the experiments up
to the present time, and have embodied the total results in this paper.

1. Gambia Fever.

The number of rats inoculated with this strain of trypanosomata was
altogether 129 in 90 successive inoculations. In all of these rats, with the
exception of the five mentioned below, trypanosomata were present in the
blood from two to three weeks after inoculation (average time 18 days)
until death ; generally in good numbers, and increasing to large numbers for
some days before death. The spleen was, in these cases, very much enlarged,
and there was considerable blood destruction, with large numbers of poly-
chromatophile corpuscles, these being the most obvious gross signs.

In the first 14 of these rats, as noted in the paper referred to above, no
paralytic symptoms were present, but out of the total number inoculated,
five became paraplegic, and the course of the disease in these animals
differed from that in the remaining 124.

In the series, the numbers of the paraplegic rats were 35, 42, 68, 101,
102, and there were no perceptible differences in their conditions and those
of the other rats used.

The first rat which became paralysed (No. 35, of the 25th inoculation) was
inoculated on March 17, 1905, and on the 31st of the same month both hind
legs became paralysed. No trypanosomata were found in the blood until
April 18, and from that time till death, which took place on April 28, only
a few were to be found in ordinary blood films. The second rat (No. 42, of
the 28th inoculation) was inoculated on April 13, 1905, from the blood of
another rat which had been 20 hours in the stomach of a leech, in which
some of the trypanosomata will live for at least 36 hours. The rat became
paraplegic on June 15, at which time no trypanosomata could be found in
ordinary blood films. The rat was killed on June 16, and a few trypano-

* ‘Roy. Soc. Proc.,’ vol. 74.

96 Mr. Plimmer. Effects on Rats of the Trypanosomata [Nov. 21,

somata were found both in the centrifugalised blood and in smears made
from the spinal cord. The third rat (No. 68, of the 42nd inoculation) was.
inoculated on September 26, 1905, and became paraplegic on December 13.
No trypanosomata could be found in ordinary blood films in 17 examinations
made during the disease, nor at the time of death. The animal died on
January 3, 1906, when a few trypanosomata were found in the centri-
fugalised blood, and in smears from the spinal cord. The fourth and fifth
rats (Nos. 101 and 102, of the 70th inoculation) were inoculated on May 26,
1906, and on July 17 No. 101 became paralysed in the right hind leg, and
on July 18 No. 102 also became paralysed in the right hind leg. Both of
these rats had a good number of trypanosomata in their blood of the ordinary
long, very motile kind. On July 19 No. 101 became completely paraplegic,
and died on the 22nd. No. 102 died on July 23, without the other leg
becoming paralysed.

The following table gives a comparative outline of these five experi-

ments :—
Rat. Diavacontota ence Appearance of Trypanosomata in blood during
paralytic symptoms. life.
35 Al: ays eee eienedas TACHVGBY Meck ascds.t sek. Very scanty.
42 64 days, rat killed...| 68rd day ............... Not found; few found post mortem
in centrifugalised blood.

68 102 days yee esse (Sthidayebn peeve Ditto.

101 SAAS os Scere DANGLY ae sc ate Good number.

102 DSidays (fae. capeeeee Sordiday ic. Att. ..8s | Ditto.

It will be noted that, in Rats Nos. 101 and 102, trypanosomata were
present in good numbers in the blood, but in those cases (Nos. 42 and 68)
in. which the paralysis was most marked, and which, to all appearances, were
exactly like rats paralysed after inoculation with the Sleeping Sickness strain
of trypanosomata, they were absent, or at any rate so few in number that
they could only be found in centrifugalised blood after death. From all of
these rats inoculations were made, with the constant result that the disease
in the inoculated animals reverted to the generalised form mentioned above
as characteristic of this strain of Gambia Fever.

This strain of trypanosomata was also inoculated into a monkey, which
lived 33 days after inoculation, this being three days longer than the
average time of the 14 monkeys I have inoculated with the Sleeping Sickness
strain. Trypanosomata appeared in the blood 10 days after inoculation, and
were of the type of the Sleeping Sickness strain. Rats inoculated from this
monkey did not become paralysed; trypanosomata of the Gambia Fever

~~

1906.| ofGamlha Fever and of Sleeping Sickness. 97

type were plentiful in their blood, and the disease ran the course of that
produced by this strain.

2. Sleeping Sickness.

In all, 82 rats have been inoculated with this strain of trypanosomata, in’
59 direct series of inoculations, and, of these, 22 have been paralysed.
In these cases trypanosomata could be found only at intervals and rarely,
and sometimes not at all, in ordinary blood films, and in some cases none
could be found in the centrifugalised blood post mortem; but in cases where
prolonged search was made a few were always found. The paralysis occurred
most generally in those rats which were inoculated direct from monkeys, and
it occurred less and less frequently as the passages got more remote, firstly from
the monkeys and secondly from the original monkey. I have succeeded
in producing paralysis once as far as the fourth passage from the monkey ;
generally after the second, in five instances after the first, passage the
trypanosomata have become generalised in the blood, and the symptoms and
course of the disease have been those of the Gambia Fever strain.

The following table gives an outline of the instance mentioned above, in
which paralysis occurred in four successive passages :—

Duration | Appearance of Pry patiosoriatasia

Rats. | Inoculated from of paralysis after reg Spleen.
disease. inoculation. spe
) mths. days.| mths. days.
7a) | Monkey which was | ( 6 7 5 15 INONG SECM .0.2 cee. Not enlarged.
inoculated from the
. monkey brought !
7b home by Col. Bruce| 3 14 2 2 None 'seeni... 2.2.20 Not enlarged.
8 BEB AG abso hxa £500 ss, s.0 6 0 5 2 Wone Seen i...2505 +0 Not enlarged.
rea Rat 8 { 3 623 3 1 OW?) eleaba dee dese ade Enlarged.
WOUNP EG ine? 4. 6 3 0 OWA ougt anctuernelsas Enlarged.
A tTOG « hca-cc. ion.; 3 3 | Not paralysed | Few in every field | Large.
136 2 0 1 20 WOW: 11500 £9454 sdate sao Large.
16 AULD © 0 cskiscvenseels 2 15 | Not paralysed | Plentiful ........... Very large.

The rats Nos. 7a and 76 at the head of the above series were inoculated from

a monkey which had been inoculated from the monkey Colonel Bruce, F.R.S.,
brought home from Uganda, this monkey having been inoculated on the spot
with cerebro-spinal fluid from a case of advanced Sleeping Sickness.

In rats inoculated from the 10th monkey done in direct succession from
Colonel Bruce’s monkey, the paralysis occurred only in those inoculated
directly from the monkey, the next passage producing trypanosomata and
symptoms of the Gambia Fever type, as set forth in the following
table :— :

98 Mr. Phmmer. Effects on Rats of the Trypanosomata [Nov. 21,

| : Appearance of
Rat. Inoculated from aes OE paralysis after yeas ei Spleen.
isease. RS Sc in blood.
|
66a ] Tenth monkey in | Killed at 2 mths. 22 days None seen Not enlarged.
direct succession ) | 8 mths. 25 days |
665 i from Col. Bruce’s) | 4 mths. 2 days | 38mths.1lldays! Noneseen | Not enlarged.
66e monkey 4 mths. 6 days | 3 mths. 22 days None seen Not enlarged.
68 EVO Gears se ccs cece nice | 2 mths. 26 days None Few from 15th | Large.
day after inocu-
| lation, increasing
| till death
69 RAE IOOD:L20 Jes cenoeson eee] oo MONS aay, None Plentiful Very large.
70 RABIGGO (cae secnaeds «cet Killed at None Fair number | Large.
1 mth. 20 days
aA HRD ROO Mi ein clea tales 2 mths: 18 days None Plentiful Very large.

3. Paralysis after Inoculation with Trypanosomata.

With regard to the question of paralysis occurring in the rat after
inoculation with the above-mentioned particular strains of trypanosomata, it
does not appear to have come within the experience of all those who have,
worked with these particular organisms. For instance, neither Dr. Thomas,
of the Liverpool School of Tropical Medicine, nor Dr. Laveran, of Paris,
appear to have encountered it in their work with these trypanosomata.

But Lieutenants Tulloch and Gray, in Uganda, have made a number of
experiments to test the statement made in my Preliminary Note, and have
recorded the same results in a paper published in No. VIII of the Sleeping
Sickness Reports of the Royal Society.

In four rats inoculated with the blood of a monkey which had _ been
previously inoculated with cerebro-spinal fluid from Sleeping Sickness
patients, paralysis of the hind limbs occurred 10 or more days before death ;
and in three out of the four rats no trypanosomata could be found in the
peripheral blood at the time of death. They also found that rats did not show
any signs of infection after inoculation with the blood of Sleeping Sickness
patients, which has also been my experience. I inoculated two rats with
blood from a case of Sleeping Sickness, in University College Hospital, a year
before death, and two more shortly before death, on both of which occasions
trypanosomata were present in the patients’ blood; the results in both series
were negative. |

The site of the inoculation apparently determines the locality of the
paralysis. In all my experiments, with the exception of four, inoculation
was made at the inner side of the hind leg, and the paralysis occurred in the
lower limbs. Only in one rat, which lived nearly 11 months, did any weak-
ness of the forelegs show itself; and this was due to extensive degeneration

1906. | of Gambia Fever and of Sleeping Sickness. 99

ascending the spinal cord from the lumbar region. The lesions in the spinal
cord were also confined more or less to the lumbar region, depending for
their extent upon the duration of the disease. In two cases I inoculated the
animals on the inner side of the foreleg, and both these animals became
paralysed in the front legs, and the degenerative lesions were most marked in
the corresponding parts of the spinal cord.

I inoculated two rats in the peritoneum, but in neither did any paralysis
occur.

4. The morphological characters of these two strains of Trypanosomata.

It is impossible at present to insist that any differentiation based solely
upon microscopical observations should be sufficient in the case of organisms
so much alike as the trypanosomata. Our methods and observations are
neither uniform nor good enough to enable us to make, at the present time
by the microscopical method alone, a sufficient differentiation. Until all
observers use the finer zoological methods of fixation, etc.,* instead of the
barbarous method of drying blood films at present almost exclusively in use,
we cannot look for much certainty in the microscopical differentiation of very
similar organisms. This must be supplemented by observing the differences
in their pathogenic action.

We must not forget, either, the variability of organisms in the same
species of animals in the same country, and their still greater variability in
different animals in the same, and in other countries ; and we must remember
the great differences in the physical conditions of an organism which are
due to environment. So I would not insist, with our present knowledge, too
strongly upon the differences in form in the trypanosomata mentioned
above, under the different conditions which have been described. But if
preparations are made under the same conditions, and with the same care,
it can be seen that the trypanosomata from the cases in which paralysis has
occurred (in which very few are found in the blood, the greater number
being in the central nervous system) are short and thick, with the flagellum
extending very little beyond the body of the organism; they contain a
varying number of vacuoles, and sometimes many granules; they move
slowly, even when the smear of cord or brain is diluted with normal citrate
solution (fig. 3). On the other hand, those found in the generalised cases (the
Gambia Fever type) are long, with a long flagellum, and are not vacuolated,
and move quickly (fig. 4). I have not found the other form in any of these
cases, even when the animal has been long dying. This short, badly staining,
vacuolated form is probably due to some condition of environment about

* See Note on methods.
VOL. LXXIX.—B. I

100 Mr. Plimmer. Effects on Rats of the Trypanosomata [Nov. 21,

which we know nothing, which produces not only changes in the morphology
of the organism, but also in its pathogenic effects, for, from the above
experiments, it can be seen that the inoculation of these short forms may,
in the first or subsequent passage, give rise to the long form in the inoculated
animal, with quite different symptoms (c¢/. figs. 3 and 4: fig. 3 photographed
from a smear of spinal cord of Rat 66 0, and fig. 4 from a blood film of Rat 69,
which was inoculated from Rat 66 0).

5. Pathological Anatomy.

The examination of these two series of rats has shown that the pathological
effects are fairly uniform in each of the two types of cases.

In those animals in which paralysis occurred, the principal lesions were
found in the spinal cord. There was invariably congestion of the vessels, and
in eight cases well marked hemorrhages around and in the substance of the
cord. There was the characteristic exudation around the blood-vessels of
mononuclear cells, and of plasma cells, such as Dr. Mott has described in the
brain in cases of Sleeping Sickness; and in all the cases a varying amount of
glia proliferation (vide figs. 1 and 2). The nerve-cells in the affected areas of
the cord were degenerated in varying degrees up to the disappearance of the
processes, and even of the nucleus with vacuolation of the protoplasm.

The brain showed much less change, and only in the chronic cases was
any exudation found around the vessels. Small hemorrhages in the
meninges were seen in five cases.

Trypanosomata, mostly in stages of degeneration, were found scattered
through the substance of the cord and brain in varying numbers ; none were
found in the vessels, which were generally distended with blood.

The other organs showed very little change; they were anemic and firm ;
the spleen was not enlarged in the chronic cases with marked paralysis, and
was only slightly enlarged in the transitional cases. There was no enlarge-
ment of the superficial or deep lymphatic glands, and the eyes and genitals
were normal.

In the cases in which the trypanosomata were plenteous in the blood, and
which had no paralytic symptoms (the Gambia Fever type), the brains and
cords were very congested, and the blood in the distended vessels contained
large numbers of trypanosomata. None were seen in the substance of the
brain or cord. In six cases small hemorrhages were found on the meninges.
There was no degeneration of the brains or cords.

The superficial lymphatic glands were often enlarged. The abdominal
glands were always enlarged, and some of both superficial and deep glands
were reddish to brown in colour. The enlarged glands showed changes of a

1906.| of Gambia Fever and of Sleeping Sickness. 101

hyperplastic nature, and the reddish ones showed sinus formation, extreme
congestion, and often hemorrhages.

The lungs were always very congested, with sometimes subserous
hemorrhages in the lower lobes. Small hemorrhages were found in
61 cases in the pleure.

The livers were always extremely congested, and were very soft in texture,
and showed extensive parenchymatous degeneration, and often fatty infil-
tration and degeneration. The vessels were very dilated and distended, and
often showed proliferation of their endothelium.

The stomach in 13 cases was ulcerated ; generally one small ulcer filled
with clot was found, but sometimes as many as three or four. They were
always found on the greater curvature. In two cases death occurred from
perforation.

There was generally a diffused congestion of the lining membrane of the
intestines, and on the peritoneal surface ecchymoses dotted about, either
singly or in groups, were often found. The kidneys were congested, and had
always macro- or micro-scopical hemorrhages.

The spleen in every case was enormously enlarged, with great congestion
of the pulp, and considerable hyperplasia of the follicles. There was generally
proliferation of the endothelium of the distended vessels, and a great deal of
phagocytosis of red blood corpuscles and trypanosomata.

The bone marrow (femur) was profoundly altered, having the appearance of
red marrow. It was very cellular, with scarcely any fat, and there was
always considerable phagocytosis, and many nucleated red corpuscles.

6. Conclusions.

From the above summary of 211 experiments, extending over a period of
nearly three years, it will be obvious that the tentative deductions which I
made in my Preliminary Note from the few experiments therein recorded,
that Gambia Fever and Sleeping Sickness are two distinct diseases, cannot
be maintained.

This extended series of experiments and abservations go to show that each
of these two strains of trypanosomata has produced two different effects
in the same class of animals, under conditions of which we at present know
nothing; and that these effects are alike for the two organisms; and that
the trypanosomata found in these two types of disease are one and the same
organism, modified by passage from man through monkeys to rats, and

perhaps in the strains I have used, by transplantation into animals of, and
in, another country. ~

102 Effects on Rats of the Trypanosomata of Gambia Fever, ete.

NOTE ON METHODS.

With regard to the mention made in Section 4 of methods, the following, which has
been used by the writer for some years, gives uniformly good and, as he believes, accurate
results. The specimen is never allowed to dry, and there is no shrinkage of the cells, and
the finest cytological details can be observed.

1. Expose a cover-glass to the vapour of

Osmic acid, 1 per cent. ....... 1 ce.
Glacialbaceticiacid ioctae. 3—5 drops
for 2 minutes.

(This can be most conveniently effected by using a block-dish covered by a thin glass
having a hole in it rather smaller than the cover-glass used.)

2. Place a drop of fresh blood in one corner of the cover-glass, and expose again to the
vapour for 30 seconds.

3. Spread the film carefully, and expose again for 15 to 30 seconds to the vapour until
the surface appears no longer moist. (The film will not be really dry, and can be easily
smeared off the glass with the finger: a really dry film will be much lighter in colour,
and cannot be rubbed off with the finger.)

4, Place the cover-glass in absolute alcohol for 10 minutes.

5. Place the cover-glass in a faintly rose-coloured solution of permanganate of potash
for 1 minute. (Two or three drops of a 1-per-cent. solution to 50 c.c. of water.)

6. Wash in water for 5 minutes.

7. Stain in a modified Romanowsky’s stain, made by mixing just before use—

Azar ts) 1 perscenty aise: nsec tenestes oye:
Kosin, (BA; 11000) bac. sbeneeee cites Dien.
Water: cretads a ae tena. oes semaine sent 8 fix

for 15—30 minutes.

8. Wash.

9. Differentiate in orange-tannin, 30 seconds.

10. Wash well and drain.

11. Absolute alcohol for a few seconds.

12. Alcohol—xylol (in proportion of 2:3), two or three changes.

13. Xylol, and mount.

Instead of 7—13, any other method of staining can be used, according to what structures
it is desired particularly to show.

DESCRIPTION OF PLATE.

Fig. 1.—Section of half of spinal cord of rat which had been paralysed for 2 months

before death. X40. Shows meningitis, the exudation around the vessels, and.
, a large area of degeneration.

Fig. 2.—Longitudinal section of vessel in spinal cord of rat which had been paralysed for
6 weeks before death. x220. Shows the cellular exudation around the
vessel, and on the right an area of degeneration.

Fig. 3.—Trypanosomata from a mash of spinal cord of rat, from which cord fig. 1 was
photographed. x1000. Stumpy forms, with very short flagella, staining
badly, with very little differentiation.

Fig. 4.—Trypanosomata in blood of rat which was inoculated with the mash of spinal
cord from which figs. 1 and 3 were photographed. Long, well-differentiated,
easily staining forms. This rat was not paralysed.

Roy. Soc. Proc., B. vol. 79, Plate 1.

Plimmer.

x 1000.

4

Fic.

»

x 1000.

ie. 3.

' - OBITUARY NOTICES

OF

FELLOWS DECEASED.

CONTENTS.

PAGE
Sir JoHn Scorr Burpon-SanDERSON (with plate) ... as a sek lil
Henry Benepict Mepuicotr Kes Lis wae ort Bee aie DS
WILLIAM THoMAS BLANFORD ne ane i ape aoe od, XRT
Tuomas GEORGE Bonp HowEs ... a ae fits ce Sacro
FERDINAND Baron von RICHTHOFEN are as oan oe cca, | RN
FREDERICK WoLLASTON HUTTON ... Ose ber a bee Be xli
GEORGE BowpLER BucKTON nn an Be ue oa wae, LN
CHARLES BARON CLARKE... on lt ae aiete me ce lx

LIonEL Smith BEALE hes ee ate a ae ae ee lvii

SIR JOHN BURDON SANDERSON.

103

On Intravascular Coagulation in Albinoes and Pigmented
Animals, and on the Behaviour of the Nucleo-proteids of
Testes in Solution in the Production of Intravascular
Coagulation.

By Grorce P. Mupas, A.R.C.Se. Lond, F.Z.S., Lecturer on Biology at

London Hospital Medical College (University of London), and at the
London School of Medicine for Women (University of London).

(Communicated by Dr. A. D. Waller, F.R.S. Received April 6,—Received in
revised form October 16,—Read November 1, 1906.)

CONTENTS.
PAGE
Part [-—
Mart ECHO AMO. EVISLOLICAL. .c50) ccc. ceccecssocccenccedavsdcceteccceccscocucesevechisbestivansecy« 104
Part [I—
The Relation of Albinism and Pigmentation to Injected Nucleo-proteids ......... 105
(A) Albinoes Injected with Nucleo-proteid derived from Pigmented Animals 105
(B) Albinoes Injected with Nucleo-proteid derived from Albinoes............... 106
(C) Pigmented Animals Injected with “ Albino” Nucleo-proteid ............... 108
(D) Pigmented Animals Injected with “ Pigmented” Nucleo-proteid ......... 109
Part ITI—
(A) The Relative Resistance of Albinoes and Pigmented Animals Quanti-
BTEC lye DELORTIMMECGN caters ee Nos tecev eee tab sneasce coceectOeen bese sacastewenameecees 110
(B) The Relative Resistance of Albinoes and Pigmented Animals to the
Nucleo-proteids derived from the two Alternative Sources ............... 113
Part [V—
(A) Variation in the Activity or Quantity of Nucleo-proteids derived from
Testes in Different Stages of Maturity ....... i ts eee Deepa ae ne 116
(B) Variation in the Activity of Solutions of Nucleo-proteids with Increasing
PE Me Neb N ica ter tite RG Wek dua he it oN et oe 119
ENV AOR ONMCLUISIONS, cc cccscce iiss satecntics snuciclecseaternsocieatevdaddve vest cscwosveredesedetaes 121
MERE LOT SOMMUION SUSE. 2.5 ache beynnnd coil oars dere daie vis beaeinieeidee slewing sdsededclgeccvestaeetes 122
NE OMe eae ea ean dees 8 <hcea Wi iudalt esos sen ectned ade teedesed soe ceaddee deadag 125

The experiments described in this paper have been performed in the
physiological laboratories of the University of London, and my best thanks
are due to Dr. Augustus Waller, the Director, and to Mr. E. Legge Symes,
the Demonstrator in Physiology, for the many facilities that were kindly
placed at my service. Especially would I like to add my testimony to the
value of the Dubois chloroform apparatus as a means of easily and safely
administering chloroform to animals.

VOL, LXXIX.—B. K

104 Mr. G. P. Mudge. Intravascular Coagulation [Oct. 16,

The cost of these experiments has been defrayed from a grant of the
Government Grant Committee of the Royal Society.

Part I.—INTRODUCTION.

The work described in this paper was undertaken in order to ascertain the
differences in relation to intravascular coagulation which distinguish the
albino and pigmented races of a species.

There exists a considerable body of evidence tending to show that in
certain qualities albinoes are less constitutionally vigorous than pigmented
individuals (Darwin, 1899; Heusinger, 1846 ; Mudge, 1907; Twining, 1845 ;
Prichard, 1855). Not a few cases of the converse kind are known, however,
and are cited by Darwin (1899). Farabee (Farabee and Castle, 1903) has
stated that albino negroes are taller and broader than their pigmented fellows,
and we may interpret this difference as meaning some sort of physical
superiority.

Quite recently we have had the experiments of Halliburton, Brodie, and
Pickering (1896, 1897), which show that while the albinoes intravascularly
injected by them with nucleo-proteids do not clot, their pigmented fellows
do. Halliburton and Pickering (1897) further proved that the three
synthesised colloids, A, B, and C of Grimaux, similarly clotted pigmented
animals, but failed to do so with the albinoes used by them. Their experi-
ments, while thus showing that albinoes may be more resistant than
pigmented individuals, also apparently demonstrate the existence of a
fundamental distinction between albinoes and pigmented individuals in their
reaction towards injected nucleo-proteids.

But the results of the experiments described in this paper iead to a
modification of such a conclusion, and show that two different sub-races of
albinoes exist; one resembling pigmented animals, since its individuals clot
upon the intravascular injection of nucleo-proteids, while the individuals of
the other sub-race fail to clot.

Pickering (1897, 1899) further stated that the Norway hare in its winter
coat reacts like an albino, but in its summer coat like a pigmented individual.
The Norway hare, however, is not a complete albino when in its winter coat,
for it has a patch of colour on its nose, at the tips of the ear pinne and the
extremity of the tail, as well as possessing black eyes. The Himalayan
domestic breed of rabbits, with which I have experimented, are similar to
the Norway hare while in its winter coat, but they have pink and not black
eyes. They are, therefore, not wholly comparable to the Norway hare. I
find the Himalayan rabbits react like true albinoes.

1906. | in Albinoes and Pigmented Animals, ete. 105

Part IJ.—THE RELATION OF ALBINISM AND PIGMENTATION TO INJECTED
NUCLEO-PROTEIDS.

(A) Albinoes Injected with Nucleo-proterd derived from Pigmented Animals.
Table IT.

The first question I investigated was whether albinoes invariably failed
to clot when injected with a nucleo-proteid derived—like that used by
Halliburton and Brodie—from pigmented animals. I injected (Table IT)
43 albinoes, of which five were rats and the rest rabbits, with such a
nucleo-proteid. I also injected eight of the partial albinoes known as
Himalayan rabbits (p. 107 and Table V). Nine different solutions,* 7.¢., B, H,
D, I, J, K, N, O, and T, were used, most of them while freshly made (on
day of preparation), but some both when freshly prepared and at stated
intervals of two, five, seven, or more days after preparation, exclusive of the
day on which they were made. The solutions were kept in an ice safe during
these periods, and their physical characters, such as colour, odour, alkalinity,
and viscosity were normal on each occasion of using. The results are shown
in a tabulated form in Table II for the pure albinoes, and in Table V for the
Himalayan rabbits.

The majority of pure albinoes manifested more or less extensive intra-
vascular coagulation, the general intensity of which is shown in the sixth
column of the table. In four albino rabbits, the blood absolutely failed to
clot (experiments 26, 53,71, and 173); while three gave a qualified failure of
such a kind that we may, I think, regard it as of the same nature as the
absolute failures (experiments 29, 30, and 72). In the case of the four
absolute failures, as in all others, the solution was injected by one of the two
methods described on p. 110 until death resulted, and an immediate post-mortem
examination failed to reveal any clots anywhere, though I very carefully
examined all the smallest pulmonary vessels that could be seen with the
unaided eye, in addition to the main systemic and portal veins. In the three
qualified cases, no clots comparable to these produced in a typical intra-
vascular coagulation were found ; for these latter clots in a typical case are
strands of the form of the containing vessels and are red in colour. The clots
which were found in these qualified cases were not red, but colourless, and
they were not of the form of the containing vessel, but mere flocculi or flecks.
Moreover, they never occurred in the veins, but always in the cavity of the
right ventricle, and in two instances attached to the chorde tendinez, and in
the other lying in the apex.

Three other qualified cases (experiments 6, 65, and 70) also occurred, but

* As to mode of preparation of these solutions, see p. 122.

i 2

106 Mr. G. P. Mudge. Intravascular Coagulation [Oct. 16,

these, I think, must be regarded rather as very limited coagulations than as
failures, because the clots were red. In experiment 70, the clot was red and
very small, and it was attached to a few of the chorde tendinez where they
joined the tricuspid valve. In experiments 6 and 65 no clots occurred
anywhere, except that in the former case a very small red one was found in
the postcaval vein just before it entered the heart; while in the latter case,
in addition to a few colourless fibrin specks in the right ventricle, there was
a semi-liquid red clot about 2 inches long formed in the postcaval vein just
posterior to the liver.

(B) Albinoes Injected with Nucleo-proteid derived from Albinoes. Tables I
and V.

I next ascertained whether any failures of coagulation occurred when
albinoes are injected with a nucleo-proteid derived from albino animals. As
is shown in Table I, 24 albinoes were injected with albino nucleo-proteid, and
of this number four were rats and the remainder rabbits. Two Himalayan
rabbits were also similarly injected (Table V), but these are considered later
(p. 107). Seven different solutions were used, 12., A, CO, F; UL) MP fandss
(p. 122). In most of these experiments the solution was freshly made, but
in a few of them it was two or seven days old. In no case was there any
condition which may be termed an absolute failure of coagulation. In
experiment 140 there is the nearest approach to a qualified failure, for the
only definite clotting which occurred was in the right ventricle, where a small,
colourless, fibrin flocculus was formed. Careful examination of the smallest
pulmonary vessels, 2.¢., those just visible to the naked eye, showed, however,
that the blood appeared to be viscous and forming a kind of liquid clot. It
was impossible to decide definitely, owing to. the smallness of the vessels.
Certainly, in the vessels large enough to allow of a definite observation, the
blood was neither clotted nor viscous. Experiment 165 helps us in a measure
to interpret the results of the experiment just described. In this rabbit a
few colourless fibrin clots were found in the right ventricle, as in rabbit 140,
but, in addition, there were also a few red clots in the smaller branches of
the pulmonary vessels, though the main vessels contained none. In experi-
ment 80, the pulmonary vessels contained no clots, neither did any of the
systemic or portal veins, but in the right ventricle there was a mixture, not
intermingled, of colourless fibrin clots with the ordinary red clots.

These experiments justify the conclusion that when albinoes are injected
with nucleo-proteid derived from albino animals, no absolute failure of
coagulation occurs, such as happens in a certain percentage of cases when
they are injected with nucleo-proteids derived from pigmented animals.

1906. | in Albinoes and Pigmented Animals, etc. 107

Doubtless, in the case of experiments 80 and 165,a much more extensive
coagulation would have resulted had larger doses been injected; and the
fact that coagulation occurred at all, with the formation of the ordinary red
clots, with the minute doses used, is evidence that the animals were susceptible
to the coagulative properties of the nucleo-proteid, and possibly the same
consideration holds for experiment 140; the doubtfully viscous nature of the
blood may have become a definite coagulation had a larger dose been injected.
But even if we assume that in this rabbit the pulmonary blood in the smallest
vessels was not viscous, the case still remains one of qualified and not of
absolute failure; for the right ventricle contained colourless fibrin clots.

Thus, when albino rabbits are injected with “ pigmented” nucleo-proteid,
about 9 per cent. of absolute failures occur, and also about 7 per cent. of
qualified failures. But when they are injected with “albino ” nucleo-proteid
no absolute failures occur, and it is very doubtful if any qualified ones do.

Partial Albinism (Himalayan Rabbits). Table V:

The characters of Himalayan rabbits are described on p. 104. The only
feature concerning them which it is necessary to note is that, with the excep-
tion of the pink eyes, they externally resemble the winter condition of the
Norway hare, in which Pickering failed to obtain intravascular coagulation. I
injected 10 of these rabbits, eight with “ pigmented” nucleo-proteid and two
with “albino” nucleo-proteid (Table V). With respect to the eight rabbits,
in experiment 28 there was an absolute failure of coagulation, and in
experiment 8 a qualified failure, only a few colourless fibrin specks being
detected in the right ventricle. In the remaining six cases, coagulation,
though limited, was produced. Thus about 12 per cent. each of absolute and
qualified failures occur.

The two which were injected with “albino ” nucleo-proteid both clotted in
a pronounced fashion, though the dose injected in one case (Table V,
experiment 79) was very small. The number of experiments is too few to
enable one to positively state that none of them when thus injected fail to
clot. But if this should subsequently prove to be the case, and remembering
that a certain percentage of them fail to clot when injected with “ pigmented ”
nucleo-proteid, it is evident that Himalayan rabbits behave like pure
albinoes.

The Cause of Fatlure to Coagulate is not due to the Solutions.

In those cases, both with the complete and partial albinoes, where
coagulation failed to occur, the cause of failure is due to inherent qualities of

108 Mr. G. P. Mudge. Intravascular Coagulation [Oct. 16,

the individuals and not to the solution or to environmental changes. The
failures have occurred with both fresh (experiments 26 and 173) and kept
(experiments 53 and 71) solutions. Moreover, fresh solutions may cause
coagulation in some rabbits (experiments 24, 25, 27, 171, 172, and 174) and
the same solution may yet fail in others (experiments 26 and 173). Smaller
doses may suffice to clot in some cases (experiments 37, 57, 68), and larger,
or sometimes very large doses of even the same solution fail in others
(experiments 30 and 71). In all cases the solution is injected until death
results. It is obvious that the failure to coagulate is an inherent quality of
the organism and is independent of any possible changing conditions of the
solution.

(C) Pigmented Animals Injected with Nucleo-proterd derived from Albinoes.
Table ILI.

- I next ascertained if when pigmented animals are injected with “ albino”
nucleo-proteid any failure of coagulation would occur, such as is the case
when albinoes are injected with “ pigmented” nucleo-proteid. Twenty-nine
experiments under these conditions were performed and no failures of any
kind occurred. There are, however, three instances, 2.¢., experiments 136,
137, and 163, where the coagulation is very limited. In the first of these
only one small clot in one of the smallest pulmonary vessels was found,
though the shed blood clotted in 25 seconds. In’the other two cases only afew
clots could be found in the smallest pulmonary vessels. In experiment 92 the
smallest pulmonary vessels contained thin axillary threads of clot, and there
was a red clot in the apex of the right ventricle, but none elsewhere. In
experiment 89, the coagulation, though very limited in extent, was more
pronounced, for the right auricle and ventricle were full of red clot and the
left ventricle contained some colourless fibrin clots, but none of the veins
contained any. In case of the limited coagulation occurring in the rabbits
(136 and 137) just described, it is to be noted that death occurred when
a very minute dose only was injected. It is, therefore, probable that had not
death occurred when it did, a larger injection would have caused more
extensive coagulation.

In experiments 145, 151, 162, and 169, the chief object was to ascertain
the degree of variation in the activity of the solution with increased age, and
as my solution was limited in quantity, I could not with these particular
individuals carry the injection far enough to produce either coagulation or
death, owing to a marked reduction in the strength of the solution or to idio-
synerasy of the individuals. In experiments 145 and 162 the animals were
idiosyncratic to a marked degree. In the four experiments mentioned, the

1906. | in Albinoes and Pigmented Animals, ete. 109

animals were not therefore killed by the injections, but doubtless had it been
possible to carry them far enough, coagulation would have occurred. In
experiment 170, the anima! died as the result of the injection, and with the
typical symptoms of intravascular coagulation, but owing to the lateness of
the hour, a post-murtem examination could not be made. These experiments
do not, therefore, invalidate the conclusion that failure to clot does not occur
when pigmented animals are injected with albino nucleo-proteids.

(D) Pigmented Animals Injected with Nucleo-proteids derived from Pigmented
Animals. Table LV.

Table IV shows the result of injecting 63 pigmented rabbits with “ pig-
mented” nucleo-proteid. No failure, absolute or qualified, occurred. One or
two explanations are necessary. In experiment 34, the blood in the vessels
escaped coagulation, but a large red clot was present in the right ventricle ;
the clotting was therefore of the typical nature, though very limited in extent.
In experiment 157, the solution was used on the second day after its
preparation, and a comparison of experiments 158, 159, and 160, where the
solution was of the same age, with experiments 152 and 153, where it was
used fresh, showed that a small fall in activity had occurred. But in
experiment 157, a very large dose was injected without death resulting. I
did not carry the injection further, because my solution was running low.
Doubtless, had the injection been carried far enough, coagulation could have
been induced ; for it was not an infrequent phenomenon of these experiments
that animals possessing an idiosyncratic resistance to injected nucleo-proteids
now and then appeared and required abnormally large doses before coagulation
could be brought about. Experiments 168 (Table IV), 54, 71, 73, and 74
(Table II) were with rabbits of this kind. In some instances, as in
experiments 169, 170 (Table III), 75, and 76 (Table II), the large dose .
required was due to a falling-off in the activity of the solution through age ;
but such cases can be distinguished from the idiosyncratic ones by the fact
that large doses were uniformly required in all the experiments in which the
particular solution of a certain age was used. And in the experiment now
under consideration, #.¢., 157 (Table IV), there is no doubt that the failure to
kill is due to idiosyncrasy and that a sufficiently large dose would have
produced coagulation. For larger doses than 16 c.c. per kilogramme of body
weight have been necessary to produce such a result, as in experiments 1, 74,
and 170, where 21 ¢.c., 23 c.c., and 20¢.c. respectively were required to clot. It
may, therefore, be assumed that experiment 157 does not invalidate the
conclusion that pigmented animals are always clotted upon the injection of a
nucleo-proteid derived from pigmented animals.

110 Mr. G. P. Mudge. Intravascular Coagulation [Oct. 16,

With respect to experiments 166, 167, and 168 (Table IV), the injection
resulted in death, with the usual symptoms accompanying death from intra-
vascular coagulation, but I had not time to make a post-mortem examination
in these cases. These experiments, in fact, were made for another purpose,
ae. to ascertain the variation in strength of the solution with age.

Part III.

(A) Relative Resistance of Albinoes and Pigmented Animals, as Measured by
the Mean Quantity Injected Required to Produce Intravascular Coagulation.
Tables I to V.

In all my experiments the quantity of nucleo-proteid injected was measured
and the body weight of the animal determined before the experiment
commenced ; with but few exceptions the animals were obtained some days
before the experiments were made and were kept under uniform conditions
with regard to time of feeding; so that differences in body weight were not
due to relative differences of time between feeding and weighing. All the
animals injected with Solution I, 2.2, 14 albinoes and 11 pigmented rabbits,
were kept under the same conditions for an interval of six weeks.

The experiments were divided into two series. In the first, the injections
were continuous though slow, and were continued until death resulted, the
dose required and the time occupied being noted ; the mean time is approxi-
mately 1 minute 6 seconds for every cubic centimetre injected. This method
of injection I shall speak of as Method A. In the second series, very small
doses, usually 0-1 c.c., were injected every two minutes until death resulted,
and the total quantity injected then noted. As a rule death occurred at from
50 to 60 seconds after the lethal quantity had been injected. This method I
shall name Method B, and it is sometimes alluded to as the method of
incremental doses. In all cases the injection was made through one of the
marginal ear veins, by a syringe capable of containing 11 c.c. of solution.
The results of these experiments and the experimental data are given in a
tabulated form in Tables I to V, pp. 125—129.

In making the comparisons between the mean dose required to kill
pigmented and albino animals, in either of the two series («e., in the series of
injections of albino and pigmented animals with “albino” nucleo-proteid and
in those with “pigmented” nucleo-proteid), we can either take the results
derived from each separate solution (group comparisons), or they can be
compared in the aggregate (final comparison). Neither one of these two
modes of comparison is preferable to the other; since while in the former
the factors in each group are homogeneous, it follows that in the latter each

1906. | in Albynoes and Pigmented Animals, ete. 111

pair of groups (that for the albinoes and pigmented individuals) are also
homogeneous. The sequel will show that the conclusion is the same by either
method. All group comparisons must be made not only with animals injected
with the same solution, but with the same solution of the same age, because
the strength of the solution varies with its age (infra, p. 119). Below, in
Table VI, I give the result in a tabulated form, of obtaining the mean
dose required to kill albinoes and pigmented animals respectively with each
of the solutions used (group mean doses). The means are calculated from the
data given in Tables I to V, pp. 125—129.

Table VI.
Pigmented animals. Albinoes.
. Age of
ealucion. solution.* Mean dose | Mean dose
per kilogramme Experiments. | per kilogramme Experiments.
of body weight. | of body weight.
Series 1.—Continuous but slow injections until death results (Method A).
A ee O | 3 68 | 19—23 4, °, 24—27
i 1°18 | 82, 84,36 | 1°41 29, 30, 31, 33, 35,
37
G5 0°76 | 43 4°28 40—42
meat 2 | 0-52 | 57 3°21 or 7 ‘37+ 53, 54, 56
Series 2.—Incremental doses, usually of 0°1 ¢ c. at 2’ intervals until
death results (Method B).
Oe eee 0 0-484 or 0 ‘92+ 59—64, 63 0 °705 or 1 ‘83+ 65—67
ae 0) 0-345 83, 84 0-458 77, 78, 8082
ae 0 0-26 86, 89, 90, 92, 94, 0-38 85, 87, 88, 91, 93,
96, 98 95, 97, 99, 100
INGA Ser. 0) 0 206 102, 104, 106 0°33 101, 103, 105, 107
OUR eee O 0°22 108, 110, 118, 114 0 286 109, 111, 115
2 0 38 116—120, 122 0 58 121
PY E eslisiaens 0) 0 °188 136—139 0 :235 or 0 °437+ 140—747
esc serrs: 2 0°45 or 2 40+ 161, 762, 168 1:0 164, 165
Me tsk. 0 0°41 or 1°75 171, 172 0-42 or 2‘1¢ 178, 174

* The figures under column “ Age of solution” have the following meaning :—O = solution.
used on day of preparation ; 2, 5, 7, etc. = solution used on the 2nd, 5th or 7th, etc., day after
that of preparation and exclusive of it.

+ The alternative figures are rendered necessary by the appearance in certain experiments
(indicated by their reference number being in italics) of individuals of marked idiosyncrasy.
The lower figure is the result of excluding these experiments from the calculation.

In arriving at the results tabulated above (Table VI, +) I have, in the
ease of certain experiments where the influence of idiosyncrasy manifested
itself, given two alternative mean results, in one of which the idiosyncratic
animals have been excluded and in the other included. But whichever of

i12 Mr. G. P. Mudge. Intravascular Coagulation [Oct. 16,

the two means is accepted, as the alternative ratios given show, the general
result is the same, «¢. the albino is markedly more resistant than the
pigmented animal.

An inspection of the figures in Table VI shows at once that in each group
of experiments, and with each solution used, whatever its age, the albinoes
require a larger mean dose per kilogramme of body weight in order to
produce death by intravascular coagulation. If the mean of all the means in
the above table is ascertained, it is found that for the pigmented animals it
is 0°69 cc. or 0°98 cc. per kilogramme of body weight, and for the albinoes
it is 1°36 cc. or 1°91 cc. The alternative figures depend upon whether the
lower or higher mean under Solutions J, K, P, and T for the albinoes, and
K, S, and T for the pigmented animals is included in the calculations.

The albinoes thus appear to be about twice as resistant to the coagulating
influence of nucleo-proteids than do the pigmented animals, if the idiosyncratic
individuals are excluded, or rather less if they are included in the calcula-
tions. And, in whichever way we deal with the figures, the same result
comes out. For if, instead of taking the mean of the means included in both
Series 1 and 2, we ascertain the mean of the means in each series separately,
we reach the following figures:—The mean of all the means in Series 1 is
for pigmented animals 1°53 c.c. and for albinoes 3°32 or 4°36 c.c. per kilo-
gramme of body weight. The mean of the means in Series 2 is for pigmented
rabbits 0°320 or 0°735 c.c., and for albinoes 0°488 or 0°822 c.c. per kilogramme
of body weight.

Thus by the Method A of injecting, the relative resisting power of
albinoes is about double that of the pigmented animals, the ratio being
2°16 or 2°8, and by the Method B it is less than double, the ratio being 1°52
in favour of the albino. But in the alternative results, under Method B,
2.¢., those in which the idiosyncratic individuals are included, the pigmented
and albino individuals are about equally resistant, the ratio being 1:1 in
favour of the albino individuals. This result is apparent and not real,
and is due to the markedly idiosyncratic pigmented individual, 162, under
Solution § and, as it has been pointed out on p. 115, Part III (B), there can be
no doubt that it should be rejected. If the result of the S solution be rejected
altogether on both sides—for both the pigmented and albino series—the
result is a mean dose for the pigmented animals of 0°527 instead of 0°868,
and for the albinoes of 0°80 instead of 0°822; the ratio thus becomes
1-5 in favour of the albinoes. This ratio is the same as that for the whole
series under Method B, when all the idiosyncratic animals are excluded.

The difference in the ratios of Series 1 and 2, 2.e., 2:16 for the former and
1°52 for the latter, is partly due to the fact that Method A of injection

1906. | on Albinoes and Pigmented Animals, ete. 113

(Series 1) is not so refined as Method B (Series 2), and partly to the fact
that in Series 1 a certain number of experiments in which the solution was
used on the second, fifth, and seventh days after preparation are included in
the mean results, while fewer of such experiments are included in the second
series; the reason for the operation of this latter influence is given infra,
p. 113. That the ratio of the two series should be in the same direction
is corroborative proof of the truth of each.

One possible source of error, arising from a want of complete homogeneity,
remains to be considered. It will be seen from an inspection of Tables IT
and IV, or of Table VI, that the I solution, when of seven days’ standing
after its preparation, was injected into only one pigmented rabbit (43) but
into three albinoes (40 to 42), and that the dose for the pigmented animal is
very small. It is possible that had another two pigmented rabbits been used,
the mean dose of the solution of this age would have been raised for the
pigmented animals. The same consideration holds for the J solution on the
second day after its preparation ; for only one experiment was performed on
a pigmented individual (57), and two on albinoes (53 and 56). But it is
obvious from a review of the means of these two solutions at other ages and
of the other solutions, that had the number of pigmented and albino
individuals been more nearly equal in these particular experiments, the
mean result would still have been higher for the albino and the ratio would
have been near that arrived at. That it would have been so is rendered all
the more certain by the fact that the ratio of resistance of albino and
pigmented animals is increased as the age of the solution increases; this fact
alone explains the large difference between the albino and pigmented races
in these two experiments, 2.c., those of the I and J solutions of seven and of
two days’ standing.

The greater resistance of the albinoes is graphically shown in fig. 1,
where the upper line, 7.¢., line of larger dose is the albino line. Cp. p. 117.

(B) The Relative Resistance of Albinoes and Pigmented Animals to Nucleo-
Proteids derwed from the Two Alternative Sources. Tables VI and VII.

The results arrived at above show that albinoes are more resistant to
injections of nucleo-proteids than are pigmented individuals, and that this is
the case when the nucleo-proteid is derived from either pigmented or albino
individuals. The experiments, however, enable us to ascertain whether
albinoes and pigmented animals are more resistant or less resistant to
nucleo-proteids derived from their own source, or from the alternative
source. For, by comparing the mean results (p. 115) of injecting albinoes
with “albino” nucleo-proteid with those obtained by using “pigmented ”

114 Mr. G. P. Mudge. Intravascular Coagulation [Oct. 16,

nucleo-proteid, any difference which may exist in the reaction of albinoes
towards nucleo-proteids derived from these two sources, can be ascertained.
And the same fact can be similarly ascertained for the pigmented animals.

The mean doses required to kill albino and pigmented animals when both
kinds of nucleo-pruteids are used in each instance may be obtained by calcu-
lation from Tables I to IV, or more quickly from the mean results of
Table VI, p.111. Those experiments only in which the injections were made
by the incremental or B method, can be utilised for this purpose, because no
albinoes were injected by the A method with nucleo-proteids derived from
albinoes. And also, with but one exception, only those experiments have
been included in the calculations in which the solutions were used on the
day of their preparation ; the number of experiments in which older solutions
were used are not sufficient to enable more comparisons to be made. Owing
to the existence of a few individuals which manifested more or less marked
idiosyncratic resistance, it is necessary to obtain, both for the albinoes and
the pigmented animals, two mean results instead of one. One of these two
results in each case will include the idiosyncratic animals and the other will
not. The animals which are regarded as idiosyncratic are those in experi-
ments 63 and 67, Sol. K; 141, Sol. P; 172 and 174, Sol. T; and 162, Sol. 8;
Tables I to IV and VI.

If we now ascertain the mean dose of the “albino” nucleo-proteid
(Solutions L, M, P, and $8) required to kill albino animals, it is 0°518 c.c. per
kilogramme of body weight, if the idiosyncratic animal 63 is excluded, and
0-575 if included (Table VII). If we similarly determine the mean dose of
nucleo-proteid derived from pigmented animals (Solutions K, N, O, T) neces-
sary to kill albinoes, it is found to be 0°435 c.c. (excluding experiment 67), or
1/136 c.c. including it. Thus, by rejecting the idiosyncratic individuals, the
mean dose of “ pigmented” nucleo-proteid necessary to kill albinoes appears
to be smaller than that obtained from albinoes. The albino, therefore, is
more resistant to its own nucleo-proteids than to those obtained from
pigmented animals.

The mean dose required to kill pigmented rabbits by the injection of
“albino” nucleo-proteid is 0°298 e.c., or 1:08 cc. per kilogramme of body
weight ; the great increase due to idiosynerasy occurs under the S solution in
relation to one animal only (162), and it must obviously be rejected. The
mean dose of “ pigmented ” nucleo-proteid required to kill them is 0°330 c.c.
if we reject the idiosyncrasies (experiments 63 and 172), and 0°777 c.c. if we
include them. Thus, disregarding the idiosyncrasies, the pigmented animals
appear to require a larger mean dose of their own nucleo-proteid to clot their
blood than they do of that obtained from albinoes. They are thus more

1906. | in Albinoes and Pigmented Animals, ete. 115

resistant to “ pigmented ” nucleo-proteid than they are to “albino” nucleo-
proteid.

If the idiosyncratic animals are included, however, the results are in both
cases reversed. The rejected experiments are 63 and 172 (Table IV), 162
(Table IIi), and 141 (Table I). An inspection of the figures given in the
tables will show that these individuals were markedly idiosyncratic, for
rabbits Nos. 141, 63,172, and 162 were respectively three, six, eight, and
twelve times more resistant than the average of their fellows injected with the
same solution at the same time. They clearly stand above their fellows as
marked mutations, and must therefore be excluded in any statement of a
mean result.

Stating the results described above in a generalised form, they may be
expressed as follows: that both albino and pigmented individuals are more
resistant to nucleo-proteids obtained from individuals of their own race than
they are to those obtained from the alternative source.

In the following table (VIL) the group mean doses extracted from Table VI
are arranged to illustrate the results just stated; and, in addition, for the
purposes of a later discussion, there is also included the weight of the testes
employed in the preparation of the different solutions used. The idiosyn-
cratic animals are not included.

Table VII—To Illustrate, in the Case of both Albino and Pigmented Animals,

the Relative Resistance to “ Albino ” and “ Pigmented ” Nucleo-proteids,

Mean dose when | Mean dose when
Solution | Weight of injected with Solution | Weight of | injected with
used. testis. “ albino ” nucleo- used. testis. ‘* pigmented ”’
proteid. nucleo-proteid.
|
Albinoes.
grammes. 6.C. grammes, | Ce.
Ey cso: 1°0 0 °235 Dee. ; 0 330
1D ages le 3 °2 0 458 LS aire 2°9 0 °286
WE ici 4, °2 0 °380 Lane ee 5°2 0 °420
ee 5°0 1-000 ere 5°5 0 “705
Mean weight]... Mean of the i Mean weight 6 Mean of the St
of gland }s 2 means bo pis of gland }s 7 means bo Ber
Pigmented Individuals.
| eee 1°0 0 °138 IN), ‘sicp:-ss 0°6 0 *206
MD otis 3 °2 0 °345 ON nsec. 2°9 0 220
1 4 °2 0 °260 NO See 5°2 0 °410
2 ee 5 °0 0 °450 Tey sae 5°5 0 °484,
Mean of the means 0 ‘298 Mean of the means 0 ‘330

eee
®

116 Mr. G. P. Mudge. Intravascular Coagulation ([Oct. 16,

An inspection of the table will show that the conditions of the experiments
were nearly, but not quite, equal. A comparison of the relative resistance of
albinoes and pigmented animals towards nuclev-proteids derived from both
albino and pigmented individuals has been made. It is obvious that the
nucleo-proteid solutions must be equal in every respect, except in their origin,
if the comparison is to be fair. But the mean weight of the glands used in
the preparation of the “pigmented” nucleo-proteid is 3°7 grammes, while
that for the glands used in the preparation of the “albino” nucleo-proteid is
3°35 grammes. If there should exist any difference in the activity of solutions
made from spermatic glands at different stages of maturity, it is obvious that,
under the conditions of the experiments, the conclusions stated above would
possibly require slight modification. This possibility is discussed (Part IV, A,
p. 118). It may be stated, however, that such a modification is probably not
needed.

Part LV.

(A) Variation in the Actiunty or Quantity of Nucleo-proterds derived from
Testes in different Stages of Maturity.

I was led to investigate, through the suggestion of other facts which I had
previously ascertained, and which are described in Section B, what influence
weight of gland (and presumably, therefore, degree of maturity) had upon the
activity of the solutions made from them. In Table VII are given the
weights of the testes (p. 115) from which the solutions used were made, and
it will be seen that the mean weight of all the glands used in the pre-
paration of the “albino” solutions was 3°35 grammes, and that of glands
used in the preparation of the “pigmented” solutions was 3°7 grammes. If,
then, the heavier glands should be either more or less active in the pro-
duction of nucleo-proteids than the lighter ones of the younger individuals,
it is obvious that this slight difference in the mean weight of the glands
used from the albino and pigmented animals respectively, might introduce a
disturbing factor, and the arrangement of the experimental data available
(as in Table VIT) in a form to show the relative resisting powers of albinoes
and pigmented individuals towards the two classes of nucleo-proteids, might
give apparent and not real results.

Accordingly, the experimental data were arranged in a way to show what
was the influence of the weight of gland upon the activity of the solutions of
nucleo-proteids. I had no data as to the age of the rabbits from which the
glands were extracted, but as every gland used had been carefully weighed,
and the colour of the individuals from which they were obtained had been
recorded, I knew the weight, origin, and number of glands used in the pre-

g

1906. | mn Albinoes and Pigmented Animals, ete. 117

paration of each of the solutions (p. 122). To an approximate degree of
accuracy, it may be assumed that weight of gland will coincide with the pro-
portional age of the animal from which it was extracted.

If the results (only those obtained from the Method B of injection
are used) are plotted out, as in fig. 1, in such a way that the abscisse indicate
the weight of the glands, and the ordinates express the measure of the mean
dose of the solutions prepared from glands of different weights required to
clot the blood of either albinoes or pigmented individuals, one fact stands out
prominently. An examination of the figure shows that on the whole there
is a fall in strength of the solution as the gland increases in weight (age),
since a solution made from a heavy gland requires to be used in a larger mean
dose per kilogramme of body weight to produce intravascular clotting than
does one made from a lighter (younger) gland. The curves, while on the
whole gradually trending upwards, show, however, that the fall in strength is
not one without any fluctuations, and the nature of these will be considered
later.

The two curves in fig. 1 very clearly show the greater resisting power of the
albinoes towards injected nucleo-proteids than that of the pigmented
individuals, for the upper curve represents the former, and the lower the
latter, and practically through the whole of its range, the “ albino line” is

U1 o DUE OSes EEO EGR Ae SREB
CHk0U SRE ee ee sees eee BEES eens
os BRERA PRRs ttt titty Lt BEE EEE CEE
ea ea a [a | a | | ESR eee s Pt
08 A a lee Pngee== aN Seo
ane He RSET

07 LOCC EEE Pee ercine meee. te epee eye
SEER CCE EEEC ECE

Fig. 1.—Showing the progressive fall in activity of solutions of nucleo-proteids derived
from testes of increasing weight (age). The upper line is for albino animals, and the
lower line for pigmented individuals. The nature of the solution used (p. 122) is
indicated by the large letters, and its origin, 7.e., whether from glands of pigmented
or albino individuals, is shown by the small index letters P and A respectively. The
abscissee show the weight of the glands in grammes, and the ordinates the amount
of injection in cubic centimetres per kilogramme of body weight necessary to kill by
intravascular coagulation.

higher than the “pigmented line.” The solutions are the same for both
varieties of rabbits throughout. The two curves are constructed from all the
results ; that is, the mean doses of the solutions of both “pigmented” and
“albino” nucleo-proteids are used together. In the figure, the solutions are
indicated by their reference letters, and the small letter to the upper and
right side of them, shows whether it is a solution of a “pigmented” or
“ albino” nucleo-proteid.

118 Mr. G. P. Mudge. Intravascular Coagulation [Oct. 16,

In fig. 2, however, the series of solutions are separated, so that four curves
are shown, two each for the “albino” and “pigmented” solutions. The
existence of two curves for each solution is due to the fact that the two
kinds of solution were injected into both albinoes and pigmented
individuals. The higher resisting power of the albino is again well shown

o's Cer ea

O3 THT SS CCIE
f aie a

01 Senessesueeaueesnuebennsraa sens aegeegeueseeeee

a
EEECEEEE TEEPE EEE Eee EEE EE EEEE EPP EE EEEEEEE EEE EE EEE

O71

Fie. 2.—The combined results shown in fig. 1 are here separated. In the upper figure
the albino individuals only are considered, and in the lower one the pigmented indi-
viduals only. The existence of two curves for both albinoes and pigmented
animals is due to the fact that both races were each injected with “albino” and
“pigmented” nucleo-proteid. The “albino and pigmented solution” lines are indi-
cated by A and P respectively. The other reference letters and numbers have the
same significance as those in fig. 1.

and towards both kinds of nucleo-proteids, for the upper line in the two
pairs uf curves is the “albino” one. And it is obvious that in the general
trend, @.¢., increasing loss of activity with increasing maturity of gland, the
testes of both the albino and the pigmented animal behave much in the
same way.

But there is a difference of behaviour at different stages of maturity, and
fig. 2 shows that the somewhat abrupt fall in strength at 3:2 grammes
weight of testis in fig. 1 is due to the albino gland; but, on the other hand,
the increase of strength at 1 gramme and at 4:2 grammes is mainly
due also to the albino gland. At the 5 gramme weight stage of maturity
the testes of albinoes and pigmented animals appear to be equal in this
respect.

The same general trend of variation in activity is shown by solutions on
the second day after their preparation, for, as far as the observations go, the
maturer glands give less active solutions, and the same may be said of
solutions used on the fifth day after their preparation.

Reverting to the conclusion stated on p. 115, that both albino and pig-
mented animals are more resistant to nucleo-proteids derived from glands of
individuals of the same race, and less resistant to those cbtained from the
alternative source, it must be borne in mind that this is a mean result.

1906.]|) in Albinoes and Pigmented Animals, etc. 119

But an inspection of the curves in fig. 2 (upper and lower), shows that upon
analysis this result apparently holds true for a part only of the range of
gland-weight. If the conclusion were generally true, the curve for pigmented
animals injected by “ albino” nucleo-proteid should be lower throughout its
range than for those injected by “ pigmented ” nucleo-proteid ; and the curve
for albino individuals injected with “ pigmented” nucleo-proteid should also
be lower throughout its range than that for those injected by “albino”
nucleo-proteid. But this is not wholly the case. With the albino individuals
(fig. 2, upper) the two curves are relatively as they should be throughout the
greater part of their range, the lower one crossing the upper one towards the
left. But this crossing obviously may be due to the absence of any data for
“pigmented ” testis weighing 1 gramme, and to that for “albino” testis
weighing 0°6 gramme. The same considerations hold for the pigmented
animals (fig. 2, lower); had data been available for “pigmented” testes of
1 gramme and 42 grammes, the “ pigmented” curve may have been lowered
at these two points, and they would then have followed a uniform course
throughout their range, the “albino” line being the higher.

(B) Variation in the Activity of Solutions of Nucleo-proteids with
Increasing Age. |

In the experiments described in this paper, most of the injections were
made on the same day that the solutions of nucleo-proteid were prepared,
some of them two days later (so that the solution, inclusive of the day of its
preparation, was then three days old), and some of them five days later (the
solution then being six days old), and a few at still later periods. If we
examine the results obtained by using the same solution on different days it
is found that the mean dose per kilogramme of body weight required to
clot varies with the age of the solution. There were seven different
solutions, z.e., I, K, O, P, R, 8, and T, each of which were used on different
days. Four of these, ze. R,S, T, and K, which were derived from heavier
glands (cp. p. 122 and figs. 1 and 2), gave a continuous fall of strength with
increasing period of keeping, but three of them, ze. I, O, and P, which were
derived from lighter glands, gave different results (fig. 3).

The Rk, 8, and T solutions were tested on the day of preparation and on
the second and fifth days after, and the K solution on the day of
preparation and on the second day after. All these solutions fell in strength
with each successive period, but the S solution fell markedly on the fifth
day. With respect to the T solution, experiment 172 has to be eliminated,
owing to idiosyncrasy ; how marked it is can be seen by comparison with
experiment 171. |

VOL. LXXIX.—B. L

120 Mr. G. P. Mudge. Intravascular Coagulation [Oct. 16,

(he al
) Seed eran esesees
PTT Pe Nt tt
te ty |

eal Ee ea a
Bail i

iene
(fo eS

zi
<
|_|
ce
fe) a
SEES
ise | et
eae
-
i
es | |
Br a a || | i
ball bol SS eS Ee

alee

Se PN

YG we SCwRe seo aeL)
6 ee ee PF
2 3 /q

Fic. 3.—Curves showing the fluctuation in activity of solutions of nucleo-proteids with
increasing period of keeping. The abscissee show the age in days of the solutions,
and the ordinates the amount of the injection in cubic centimetres of the solution per
kilogramme of body weight to produce death by intravascular coagulation. The
solutions used (cp. p. 122) are indicated by the letters attached to the curves. The
curves marked I4 and I are for albinoes and pigmented individuals respectively.
The curves for O and P are for pigmented individuals only.

The behaviour of Solutions I, O, and P is graphically represented in
fig. 3. With Solution O, the mean dose required to clot on the day of its.
preparation was 0°24 c.c. per kilogramme of body weight; on the second
day afterwards (third day old solution) the activity had fallen, and a mean
dose of 0°38 c.c. per kilogramme of body weight was required to clot; on
the fifth day after preparation the strength had increased to 0°252 ec. 3.
this increase continued until the seventh day of preparation, when it was

1906. | in Albinoes and Pigmented Ammals, ete. 121

slightly stronger, 7.¢., 0°236 c.c., that is, nearly as active as on the day of
its preparation; on the ninth day it had fallen in activity considerably ,
1.¢., to 0°9 c.c., and on the 11th day the loss of strength was very great,.
to 436 cc. The same fall on the second day, with a subsequent rise in
strength on the fifth, is shown in a yet more marked manner by the curve:
for Solution P.

In the case of the I solution, for which two curves are shown, one for
the albino animals (I) and one for the pigmented animals (I), no
examination of its strength on the second day was made. But for both
pigmented and albino animals there is a very marked rise in activity on.
the fifth day after the preparation of the solution. In the case of the
albinoes the strength of the solution falls on the seventh day, but rises for
the pigmented animals; this difference may be due to the fact that only
one pigmented rabbit was injected on the seventh day ; but, bearing in mind
the curve for Solution O, it is possible it may have still retained its present:
form if more experiments had been performed. On the 19th day the
activity of the solution, when used on pigmented animals, fell very
markedly. It is very probable that had Solution I been tested on the
second day, that it would have shown, like Solutions O and P, a fall in
activity. My experiments, however, at this period were not designed to
investigate this problem, and I did not consequently test the solution on the
second day.

SUMMARY OF CONCLUSIONS.

1, When albinoes are injected with a solution of nucleo-proteid derived
from a pigmented animal, a certain number of them, about 9 per cent.,
absolutely fail to clot, while about 7 per cent. give a qualified clotting, the
remainder giving a typical intravascular coagulation of more or less.
extensive development.

2. When albinoes are similarly injected with a solution of nucleo-proteid,.
but derived from albinoes, no absolute failure of coagulation occurs, and it.
is very doubtful if any qualified ones do. The great majority clot as
distinctly as do pigmented individuals.

3. When pigmented rabbits are injected with solutions of nucleo-proteids,
derived from albinoes or with those derived from pigmented individuals, no-
failures of coagulation occur.

4. The Himalayan rabbit, in respect of its reaction to injected nucleo-
proteids, behaves like the complete albino. This rabbit, though resembling
the Norway hare in its winter coat, in which condition Pickering failed
to obtain intravascular coagulation, differs from it in having pink

L 2

122 Mr. G. P. Mudge. Intravascular Coagulation [Oct. 16,

(unpigmented) instead of pigmented eyes and in never becoming periodically
wholly pigmented. It cannot, therefore, be used as corroborative evidence of
Pickering’s conclusion with respect to the Norway hare.

5. That failures to coagulate, when they occur, are due to inherent
qualities of the individuals and not to weakening in the activity of the
solutions used.

6. Albinoes require a larger mean dose per kilogramme of body weight of
injected nucleo-proteid to cause death by intravascular coagulation than do
pigmented animals, the relative resisting powers of the pigmented and
albino individuals being as 1 to 1°5 respectively.

7. Both albino and pigmented individuals are more resistant to nucleo-
proteids, obtained from individuals of their own race, than they are to those
obtained from the alternative source.

8. The activity of a solution of nucleo-proteid, prepared from spermatic
glands, decreases (but not quite uniformly) as the maturity (weight) of the
gland increases. : )

9. Solutions of nucleo-proteids, prepared from heavier (maturer) spermatic
glands, undergo a progressive loss of activity with increasing period of
keeping, 7.2, from 1 to 20 days. But solutions derived from lighter
(immature) glands undergo a fluctuating variation in activity, falling off on
the second day after preparation and rising again on the fifth to seventh,
and thence exhibiting a progressive fall.

NATURE OF SOLUTIONS USED.

W.G. = weight of gland ; D. = dissolved in 1-per-cent. solution of anhydrous Na,CO,.
The amount of solution used in each case is indicated in cubic centimetres. Unless other-
wise stated, all the solutions were prepared by pressing the glands through finely meshed
iron wire gauze into 1-per-cent. solution of Na,CO..

Sol. A.—(1) Thymus glands from four albino rats, 6 weeks old, descended from albino
parents. (2) Testes from albino rat of unknown parentage. (3) Thymus from
albino rat, 8 weeks old, from black and white parents. W.G. = 4:2 grammes,
D. in 65 c.c. = 1 gramme in 15°5 e.c.

Sol. B.—Thymus gland of a black and white calf. Portion of organ minced and ground
in a mortar with an equal volume of sodium chloride. Separated the nucleo-
proteid by addition of distilled water, and collected into 1-per-cent. solution of
Na,CO,. Strength of solution was not ascertained, but was probably nearly
saturated.

Sol. C.—Testes from albino rabbit. W.G. =6 grammes, D. in 100 cc. = 1 gramme in
15'5 c.c.

Sol. D.—Thymus glands from four young pigmented rabbits, with much unpigmented
coat. Glands crushed in distilled water, macerated for 36 hours, and the nucleo-
proteid then precipitated by 0°2-per-cent. acetic acid. Dissolved in 70 c.c. of
1-per-cent. solution of Na,CO,. .

1906. | m Albinoes and Pigmented Animals, etc. 123

Sol. F.—Testes from two albino rats of albino parentage. W.G. = 3°6 grammes, D. in
50 c.c. = 1 gramme in 14 «cc.

Sol. G.—Testes from Himalayan rabbit. W.G. = 2°2 grammes, D. in 32 c.c. = 1 gramme
in 14°5 ce.

Sol. H.—Thymus glands from three black and one brown rabbit. Dissolved in 1-per-
cent. solution of Na,COs.

Sol. I.—Testes from five brown (grey), one black, one silver grey, one fawn and one
brown and white rabbits. The glands from the five brown rabbits weighed
8 grammes, and those from the remaining four rabbits 19 grammes. Total

W.G. = 27°2 grammes, D. in 423 c.c. = 1 gramme in 15°5 c.c.

Sol. J.—Testes from a black and white rabbit, a blackish-grey, and a brown one.
W.G. = 11 grammes, D. in 170°5 c.c. = 1 gramme in 15°5 c.c.

Sol. K.—Two testes from a brown rabbit. W.G. = 5°5 grammes, D. in 85¢.c. = 1 gramme

in 15°5 c.c.

Sol. L.—Testes from albino rabbit. W.G. = 3°2 grammes, D. in 49 cc. = 1 gramme in
15°5 ce. :

Sol. M.—Testes from albino rabbit. W.G. = 42 grammes, D. in 65 cc. = 1 gramme in
15°5 c.c.

Sol. N.—Testes from two grey (brown) rabbits. W.G.= 1:2 gramme, D in 18°6 c.c.
= 1 gramme in 15°5 cc.

Sol. O.—Testes from a grey (brown) rabbit. W.G. = 2:9 grammes, D. in 45 ce.
= 1 gramme in 15'5 c.c,

Sol. P.—Testes from an albino rabbit. W.G. = 1 gramme, D. in 15°5 c.c.

Sol. R.—Testes from one fawn and one grey rabbit. W.G. = 7°8 grammes, D. in 121 cc.
= 1 gramme in 15°5 «ec.

Sol. S.—Testes from Himalayan rabbit. W.G. = 5 grammes, D. in 77°5 c.c. = 1 gramme

| in 15°5 cc.

Sol. T.—Testes from black rabbit. W.G. = 5:2 grammes, D. in 80°6 c.c. = 1 gramme in

15°5 c.c.

_ EXPLANATIONS OF THE ABBREVIATIONS USED IN THE TABLES I—V.

“Albino” solution and “pigmented” solution mean a solution of nucleo-proteid
derived from albino and from pigmented animals respectively.

The capital letter in the column headed “Solution used” refers to the nature of the
solution used. A description is to be found on p. 122. The small numeral at the top
right-hand side of the capita] letter indicates the age of the solution when used. Thus L°
means that it was prepared and injected on the same day: and I> that it was injected on
the fifth day after its preparation, exclusive of the day of preparation. The solutions
were kept in an ice safe, gave no signs of putrefaction, and retained their physical
characters at the time of using.

V.P. = Very pronounced. Ali the vessels examined ; 7.¢., right and left precavals, right
and left external jugulars, the whole length of the postcaval (that lying in the
thorax, and for brevity called “thoracic portion,” and that lying in the abdominal
cavity and called “abdominal portion” or “abdominal postcaval”), and the
hepatic-portal veins, being filled solid with continuous axillary strands.

P. = Pronounced. The clot in the vessels either fills it solid, as above, or does not quite
fill it, but in either instance is absent from the “ thoracie postcaval,” or from the
portal vein, or from both.

124 Mr. G. P. Mudge. Intravascular Coagulation [Oct. 16,

F.P. = Fairly pronounced. The clot is axillary and continuous, but thin, and is absent
from the “ thoracic postcaval” or portal vein, or from both.

F. = Faint coagulation. The clot is a mere cotton-like thread, and is more or less
continuous and axillary, but is absent from the “ thoracic postcaval” and portal
vein.

V.F. = Very faint coagulation. The chief feature of this condition is in its limitation, for
the clot may be a mere cotton-like thread or fairly thick (nearly as thick as in P).
The clot is limited, however, to one or other of the two jugulars and associated
precavals, or is confined to any other one vein; and small clots may or may not
be present in the chambers of the heart.

A.B. = Coagulation almost absent. Only a few colourless clots of fibrin specks or fila-
ments are present in the ventricles of the heart, and usually attached to the chordze
tendineze. There are no clots of any kind in any of the veins.

A.B’.= Like A.B., but the clot in the chambers of the heart is red (not colourless) and is
fairly large, varying from a tenth of an inch to a clot which fills the chamber.

A. = Absence of coagulation absolute.

The above definitions of conditions are based upon actually occurring states, and are not
therefore wholly arbitrary; but in a few of the experiments it is difficult to assign the
observed state to any one of the above defined groups. Such instances are indicated in
the tables by the interrogative sign.

No result = Means that as much of the solution that could be spared was injected, but
was not sufficient to kill the animal. It was then killed by an overdose of
chloroform.

No P.M. exam. = A post-mortem examination was not made. In the few cases in which
the examination was omitted (owing to the lateness of the hour) the symptoms
were typically those of death by intravasculation coagulation. The object of
the experiment in these cases was not to ascertain the extent of coagulation, but
the amount of the minimum lethal dose.

#y

1906.]

Dose per
kilo-
gramme
of body
weight.

et

SSeSeoceooeooeoocoene
To)
=

33 ‘0

mn Albinoes and Pigmented Animals, ete.

Weight | Total
of dose
animal. | injected.

Table I.

Method of injection.

(A) Albino rabbits injected with “ albino” solution.

(B) Albino rats injected with “ albino” solution.

kilos. | ce.
0°6 10-0
0°8 10 °0
1°75 0°5
0°5 0°3
0°9 0°25
0-8) | . O25
06 | 0-50
0°55 | 0°25
E70" |) =< 0-50
O'S * | .0°25
2°2 1-0
0°5 0°2
rf 0°5
0°43 | O71
1°55 0°5
0°5 0°25
Ls 0 °4
0°85) |, 10-55
0°55 | 0°50
0°45 0°50
0°5 10 ‘0
_ 8:0
= “0
0°12 4:0

ere resort ees esa oes ese esses

33 ”
- i Pe RY eT Ore
In 0°25 c.c. doses at 2’ intervals...
Im one dose; slowly. ~..as.ecust<-s4-<¢
In 0°25 c.c. doses at 2’ intervals...
iim: OnerdoseySlOWLY 0. acraclnccsdaes
In 0°25 c.c. doses at 2’ intervals...
Invone dose, slowly -....cscee.-.4.0--:
In 0°25 c.c. doses at 2’ intervals...
MCONCLGOSE ssc enacted rocstsetacaes.
In 0°25 e.c. doses at 2’ intervals...
In two 0°1+ one 1°5 c.c. doses at
2’ intervals
In 0°2+01+0°1 c.c. doses at
2’ intervals
In one 0'1+ three 0°15 c.c. doses
at 2’ intervals

nis ONE CORES hc tho asec Motes at ok |
In 0°1 ¢.c. doses at 2’ intervals ...'

nome GOSelL usc eee tee |

Solution Result.

used.

S2
S2

PayPal Fp fy FO fy by ty

125

No. of
experl-
ment,

126
Dose per
kilo- Weight
gramme of
of body animal.
weight.
kilos,
5°0 2°0
4°0 2°0
2°5 2°8
6-1 1°8
1°0 1°5
2°0 | 1°3
2A 1°9
1°25 1°6
1°15 1°3
1°0 17
4°66 dee
0°5 2°0
7 “69 Leg
0 52 1°9
ab 7, Ors,
5°9 1°95
0°41 1
1°0 15
4:1 S37.
2 82 0°85
6°8 0°8
5-7 0°7
21 °4 0°7
4°28 On,
12 °5 06
23 °6 0°55
10°0 0°6
9-0 0°5
0°31 0°8
0°13 8
0°38 1 Ra
0°5 10)
0°41 1°2
0°14 0:9
0°31 0°8
0°58 0°6
0°40 0°5
3°80 . 2°85

Total
dose
injected.

a
Os
66°

ra

a
HOH HE HOROHENERN HEY

—

OTR OT bo ~“

a
Kn®e 60m FB SG AARURSSSSANASSAUASS

8 of

20000 coon aA AH
pd EG AN wad
Or OU OF

(B) Albino rats injected with ‘‘ pigmented ” solution.
Not weighed | Not recorded

40
5:0
6:0
8 0

+5of J

Table II.

Method of injection.

Tn ‘one dose, slowly. ......+00..0cese+0«
ps Pn Pathbbecdonsdcdscoouss
3 Tica Do toee nccceiicananeced
9 sett DM sa nee oat nseemnteeyetis
; Sie rose eee aan een aaice
» Montel Reseicecth tseenan
9 Sy Maken sacle sunerne saere
9 Pepe hj, dod sane nodndanacoude
3 PHM Mk csc tos ONonHd dodabe
% PEI ny busuoadaacudannSanadat

eeevceeoseerevocvce ves
Coc eeeoeeecocecos veeose
eee ere cee eee ees oes vce

eecersecerees eos ecevee

In 1 ¢.ec. doses at 2’ intervals ......

ENVONS MOSER A et feet case clon

In two 0°5+o0ne 0'25+three 0°75
c.c. doses at 2’ intervals

In 0°25 and 0°5 ¢.c. doses at 2’ in-
tervals

In 0°3 and 0°5 c.c. doses at 2’ in-
tervals

In 0°5 c.c. doses at 2’ intervals......

eeevece

tervals
In 20+ 1:0 c.c. doses at 1’ intervais
In 15 and 1:0 c.c. doses at 1’ in-

tervals
Continuously, but very slowly ......
” spe! Og OO
imione dose; quickly cen. cr..cis.
9 SLOW Yad. wsroinscnene seas’

Seeeeceeserceseeoes

tervals
In 0°25 c.c. doses at 2’ intervals ...

93 eee
TnFONECIMOSE As conan cee csee ene

Coe eee ere ses oereses ove oeeeseore

tervals
ATONE OSE Sosa escceeo ere
In 0°25 c.c. doses at 1’ intervals ...

Tm One ose iiss ch reece eee

Oy

eve ese cee eee eosereeoeeeeeseosoee

Cr oad

Solution
used.

(A) Albino rabbits injected with “ pigmented ” solution.

kK? & J’

mo
mo

Be

Mr. G. P. Mudge, Intravascular Coagulation [Oct. 16,

Result.

_
wa

<4q <>
ijt ps

<
ac)
B
wo
oe

“~~

Fy Ff Ff fo or] ej Ey =

ddd dj

<
ty

a

No. of
experi-
ment.

1906. | im Albinoes and Pigmented Animals, ete. 127

Table III.—Pigmented Rabbits injected with “ Albino” Solution.

| Dose per
kilo- Weight | Total Soluti No. of
| gramme of dose Method of injection. ve es Result. experi-
| of body | animal. | injected. be 7 ment.
| weight.
kilos. €.6.
21°0 2°0 42°0 | In doses of 10+10+11+11 c.c., quickly 3 EP, t
9-09 -y 10-0 WEITIOME COSC. he want icedclacte ce nehenngsPanneds Cc? V.P. 4,
12°5 0-4 5:0 Sage Nd Ae ein F VP. 11
0°35 1°4 0°5 In 0°25 ¢.c. doses at 2’ intervals............ IK Pp? 83
0°34 1-45 05 i Fs Saige at, cane L° VF. 84,
0°31 . 0'8 0°25 | In two equal doses oN aden nee ES ee M° V~.E. 86
; 0 °185 1°35 2b MPH ONG COKE! ©. 34% so5s< cane. cosas deswnas eeeseaues M° A.B’. 89
0:28 0 ‘85 0°24 | In two equal doses at 2’ interval ......... M° E.P..? 90
0°19 1°3 Opaomen WW dine AOSC! os cas. cearscces aacadanler vecnee vaste M? ABI 92
0:2 2°5 0°50 | In 0:25 c.c. doses at 2’ intervals............ M° V.E. 94
0:19 13 OL Z OMEN O16 'COSE We fienconiseae dawns sunsindesandechion M? V.F. | 96
0°5 0 0°5 In 0°254+1:'25+1°25 c.c. doses at 2’ M? VBS | 98
intervals
0 089 1°12 O°l PMG OsGORE, os2sginessiayncinenedwoatchnnseuabiens Pe Nearly A | 1386
0 083 12 0:1 MONE Sanat eae at baie snared a aonetas jew Nearly A 137
0°19 1-3 0°25 | In01+0°15 c.c. doses at 2’intervals ...| P® VE: 138
0°19 1°05 0°20 | In 01 ear he RE Meee ipo VeRS2 139
2°0 1°5 3 °0 es vs op a ee P? No result 145
1°3 0°75 1-0 i ‘ i pn ears rie Pp? V.F.? 146
1°56 0:8 1:25 | In 0-25 : Sees aint. ps F.? 149
0°57 1°3 0°75 .e ers. nei pe V.E. 150
4.°5 1°0 4°5 In 0°254+0°2 ,, PR Re ips No result 151
0°13 Bel ORR GGA TMVOME CORE: 125. costes ods 8e ccdenceliadlesetces cvee gs? VE. 154
1°77 2°25 40 In 0°2 c.c. doses at 2’ intervals ............ s° VE? 155
0-7 10 0-7 |Inol " hed ee go VF. 156
0°5 0°6 0°3 Ps - Ba Me tiaaines ok S? V.F.? 161
6°3 0°95 6:0 In 0°1 c.c. and then 0°5 ¢.c. doses at 2’ a No result 162
intervals
0°41 0°6 On Zora MMeOne NOB! -cccssnedeces detecee sniineiesae eet S? Nearly A 163
11 °0 1°8 20 ‘0 In 2°0 c.c. doses at 2’ intervals ............ s° No result 169
20-0 0°55 11°0 In 1:0 - ON Fete Te S® No P.M. exam.| 170
Table IV.—Pigmented Rabbits injected with “ Pigmented” Solution.
Dose per
kilo- Weight Total Soluti No. of
| gramme of dose | Method of injection. [Seals a Result. experi-
_ | of body | animal. | injected. | eee ment.
weight. |
kilos. Ce:
| 9:0 0°5 45 Ear Omer OSE) detected nacsn tee Sucn wet cos nociaet 1B F.P. ? 16
28. 2°1 6:0 ie Slowilivay acta ccdansceactcd ene nis yo V.F. 19
| 46 1°5 70 ¥ Eee che 1° P.? 20
| 4:0 2°5 100 e TOY ioc aaa ae T° V.F.? 21
| 4:0 2°7 11:0 5 Ree inate ot sonisnetaa saan: Jo Pp: 22
| 3-0 20 6-0 ‘ SELES STE Oe ene ek 10 FP. 23
| 1:07 1-4 1:6 yi Wi Macatee a lc Net 15 E.P. 32
y LS 2°0 3°0 33 eee eee er ie A.B’, 34
0°97 1 ‘85 1°8 is ME, doce (Oey eee on I> BPs? 36
0°76 1:3 1-0 « SARTRE ONG Lt li P. 43
3°8 1°3 5°0 - WA lier as ueaiies Clase nae a8 EP: 46

102
104:
106
108
110
113
114
116
17
118
119
120
122
128
129
130
131
132
133
134
135
142
143
144,
147
148
152
153
157

158
159
160
166
167
168
171
172
175
176
177
178
179

128 Mr. G. P. Mudge. Intravascular Coagulation [Oct. 16,
Table [V—continued.
| |
Dose per | |
kilo- | Weight Total Soluti
gramme of dose Method of injection. os al Result.
of body | animal. | injected. Wee.
weight.
kilos. G:C; |
4°6 1°5 7°0 Im one ’dose,:slowly c.ta.2.seeee ee erence le ame 22 Ver
| 0°52 1°9 Pcp Treks aur RATE ene OR 2 V.F.
| 4°28 2:1 9-0 In 1 c.c. doses ab 2! Intervals .......s.0s000 eae V.E. ?
Oro 0) 0°5 In: one-dose, ‘slowly <2. 22.ttaccseso.t sean | aie to
0°55 0°9 0°5 ut Fir beech eee K° Vie
0°5 1:0 0°5 ie vcle Hethun sits Geta A Ee ko Le SA Ve
0°37 0°8 OES rN’ nat: ts cout ANN OR A MGR CS ceN EO Said ac Oe eee K? V.F.
3°1 0°8 25 In 0° 25 +0°5 ¢. c doses at 2’ intervals ...; K® V.F.
0°5 1°0 Oc | Im one dose, slowly pets .ne ete traces ko PB:
0°12 2°05 (Oy inl Me POR CR CRG RR A) Maat Ee a N°? Vn
0°25 0°8 0:2 In 0° ‘1 C.c. dorest at2’ imtervals ....:/...... N? V.BE?
0°25 1:0 0°25 | In 0'14+1°'5 c.c. doses at 2’ intervals ...... N° V.F.?
0 ‘26 1°9 0°5 In 0°25 c.c. doses at 3’ intervals............ O° Vile?
0-30 <2 0°37 | In 0:25+4+0°12 c.c. doses at 2’ intervals ... 0° VE
0 :20 1:0 0°20 | In 0:1 c.c. doses at 2’ intervals ............ O° V.F.
0°12 0°8 OL oh. 5 Lene SAO et hes eo Mind OR le a oRae SU hee ee, 2 een O° VF.
0°45 0°55 0°25 |In0: re i 0°15 ct oe closes a5 cy intervals ... O? V Ere
0°24 1 °25 0°30 | In 0°15 e.c. doses at 2’ intervals............ 2 VEEP
0°41 0°6 0°25 | In 0'1+0°15 c.c. doses at 2’ intervals O- VEL?
0-38 0°65 0°25 ts , . il, 12 V.F.?
0°5 0°6 O«3 In 0'1 c.c. doses at 2’ intervals ............ O? V.F. ?
0°31 0°8 0°25 | In 0:1+0°15 «.c. doses at 2’ intervals 2 VF.
0:41 0°6 0°25 ‘ i ‘ 0° VF.
0°27 0:9 0°25 3 ‘ i O* V.E.
0°173 1°15 0-2 |In01+01 . i el 118.3 V.F.
0 °158 0 ‘63 Onl Wi OMe {OSC ae ahaha ce a eee O° V~.F.
0°25 2°0 0°5 In O° ¢.c. dosesmat-2” intervals (:.5.0...5.28 O° V.F.
0-28 1-25 0°35 | In two equal doses at 2’ intervals ......... O% F.P.
0178 1°4 0°25 . hs, WE 7 FP.
0°25 0°6 Ocls> len one:dose-n. cette eee Of V.F.
0°8 0°5 04 In three equal doses at 2’ intervals ...... O° VF.
1°0 0°6 0°6 In five - ” ee a OR Pe 2
0-91 0°55 0°5 In three do Veet a oie Sehr pete a O° VEO?
1-22 1°43 1°75 | In 0°25 c.c. doses at 2” intervals We amc Ol V.F.
7-5 1-0 75 | E Welt 7 Ma a Uy ou V.E
0°37 1°35 05 |Inol i Ames 70h R9 ie D
0 26 0 ‘95 O25 2 pln OAs 5 ee ean en eee re R° V.F.
16°8 1°48 25°0 |In 0140°25+054+1° 0° 16. c. doses at R? No result
| 2’ intervals
Ol 0°7 0°5 In 0°25 c.c. doses at 2’ intervals............ R* 1) aes
0°73 iy 1°25 = a CN eee as « R? V.F.
1°3 0°75 1:0 In 0:1 sa ee Ween apes Rez V.E,
1°29 0°85 Lei “ rs ipo Spence eee R®_ | No P.M. exam.
2°9 1°7 5:0 | In 0°25 . stl Aron R? 2
13 °3 0°6 8:0 In 0°140°25 _,, cn Ot etek aneee: R° i.
0°41 1-95 08 |Inol f SOE ee Ae [ P?
rae hl 0°86 2°75 i ON TD Mca oe T° V.F.?
1 ‘18 1°9 2°25 | In 0:25 a aay pees Ty T? P?
1°6 1 °*4 2025 i A asad LN i eras Aen Gye) VRE
2°32 1°4 3°25 D e PE Ria eine T> |No P.M. exam.
1°13 1‘1 1°25 if i et bk i fae tes TS | é
2-6 1-05 2°75 i ¥ San aE a TS 7
3°0 0°75 DeDie Ul oy LE MAUVE ERASE) Gh N50 55 RED Oe ne er Té V.E

180

‘ | Dose per

'
|
t
:

kilo-

| gramme
| of body

weight.

COPRrFOBBO
DE OH Cd
NONNN OK &

1889.

1899.

1903.
1880.
1893.
1895.
1896.
1897.
1897.
1846.
1894.
1874,
1896.

1907.

1897.
1899.

1855.
1897.
1898.
19083.
1845.
1886.
1888.
1891.

1906. | in Albinoes and Pigmented Animals, etc. 129

Altmann. ‘ Arch. f. Physiol.,’ Leipzig, 1889.

Darwin, Charles. ‘Variation of Animals and Plants under Domestication
2nd edit., vols. 1 and 2, London, 1899.

Farabee and Castle. ‘Science,’ N.S., vol. 17, New York.

Foa and Pellacani. ‘Riv. clin. di Bologna,’ 1880.

Halliburton, W. D., and Brodie, T. G. ‘Journ. Phys.,’ Cambridge and London,
vols. 14, 16, 17, and 18.

Halliburton and Pickering. ‘Journ. Phys.,’ vol. 18.

Heusinger. ‘ Wochenschrift fur die Heilkunde,’ Berlin, 1846.

Martin, C. ‘Journ. Phys.,’ Cambridge and London, vol. 15.

Miescher. ‘ Verhandl. d. naturf. Gesellsch. in Basel,’ 1874, Part 1.

Miescher and Schmiedeberg. ‘ Arch. f. exper. Path. u. Pharmakol,’ Leipzig, 1896
vol. 37.

Mudge, George P. ‘ Encyl. Brit.,’ art. “ Albino,” 10th edit.

} Pickering, ‘Journ. Phys.,’ vols. 18 and 20.

Prichard, Jas. C. ‘Natural History of Man,’ London, 1855.

Ringer. ‘Journ. Phys.,’ Cambridge and London, vol. 18.

Schafer, E. A. ‘Text Book of Physiology, London, 1st edit., vol. 1.
Stephens and Myers, ‘Journ. Phys.,’ vol. 23.

Twining. ‘ Brit. Assoc. Reports,’ 1845.

Wooldridge. ‘ Proc. Roy. Soc. London,’ 1886.

Wooldridge. ‘Arch. f. Physiol.’ Leipzig, 1888.

Wright. ‘Journ. Phys.,’ vol. 12.

Table V.
Weight Total Solukion No. of
of dose Method of injection. used, | result. | experi-
animal, | injected. ; ment.
(A) Himalayan rabbits injected with “ pigmented ” solution.
kilos. c.c. ;
2°3 1°0 | Enger: (Glo ope Micr ete ee 6p ROCRIC ICM OC ae TRC R EE EC Oeee | iL, A. 28
2°3 100 ZUR) Uc Sih Sgt en hail eee Ne ean (BS AB 8
1°7 7°0 In 0°7 c.c. and in 0°5 c.c. at 2’ intervals ......... | J Vikv?! | 49
1°9 1°0 TOME OSE 2s tans hcaitatle conse SF aes ae a agate qoide ses J ° 50
1°9 3 °0 In one 1 ¢c.c. and in 0°5 c.c. at 1’ intervals... J* 51 |
1°6 30 In one 0°8 c.c. and in 0°2 ¢c.c. at 2’ intervals ... J- A 52
2°7 0°5 In O: 25: 2c, at 2’ imtervalls 2....2s00 unm ee son seen fe @)= Wao okt
0 °4 0°25 | In two 1:5+1 ¢e.c. at 2’ intervals ..............0004 O? VEY? 123
(B) Himalayan rabbits injected with “ albino” solution.
2:1 10 ‘0 RONG AGO an. Reds athe dinacnaassopetogeea since et (ame bie Nee 9
0°5 0°25 cl aac a ASA de I ames JANE He ae | TL? | By ee
REFERENCES.

130

The Chenustry of Globulin.

By WILLIAM SUTHERLAND, M.A. Melbourne.

(Communicated by Dr. C. J. Martin, F.R.S. Received July 26,—Read
December 6, 1906.)

The object of the present paper is first to establish simple formule for the
more important of the experimental results obtained by Hardy* and
Mellanby,+ then to interpret these in their bearing upon the chemistry of
globulin in connection with a theory of colloids, and finally to find the
molecular mass (weight) of globulin. The work will: be taken according to:
the following table of contents.

1. Formula for the solution of globulin in solutions of neutral salts, and.
the laws associated with this formula.

2. Solution of globulin in acids and alkalies.

3. Formule for the precipitation of globulin by salts and by acids.

4, A theory of the colloidal state.

5. The electric conductivity of globulin solutions.

6. The molecular mass (weight) of globulin.

1. Formula for the Solution of Globulin in Solutions of Neutral Salts, and the
Laws Associated with this Formula.

Mellanby sets forth his results in graphs, from which he makes some broad
deductions, to be discussed later in this section. _ His most striking result,
obtained also by Hardy, is that the fraction (or percentage) of a globulin
Suspension which is dissolved by a salt solution of given strength is nearly
independent of the original strength of the suspension. In other words, the
amount of globulin dissolved by a given salt solution is proportional to the
amount of globulin presented for solution in the form of a suspension. This
fact is of fundamental importance for the colloidal globulin of the suspension.
For example, a 0°3-per-cent. NaCl solution dissolves from a 3:04-per-cent.
suspension of globulin in 100 cc. 112 grammes, from 1°68 per cent. 0°64,
from 1:06 per cent. 0°39, and from 0°71 per cent. 0°30.

Let G be the original strength of a globulin suspension when it is.
instantaneously distributed through a solution of a salt of concentration e,
G and ¢ being given in grammes per cubic centimetre. Let g be the
grammes of globulin per cubic centimetre dissolved from the suspension
when saturation is reached, and let C be the concentration of the salt

* ‘Journal of Physiology,’ 1905, vol. 33, p. 251.
t+ Loe. cit., p. 338.

The Chemistry of Globulin. 131

required to just dissolve the whole of G. Denote g/G by », and ¢/C be q,
then Mellanby’s results are expressed by the equation.

p(i—q) = Ag(l—p), (1)

in which A is a parameter having characteristic values for each type of

neutral salt. To illustrate the applicability of this equation, the following
comparison is made of the values of p(1—q)/q(1—p) for the different values
of c and p given by Mellanby for his first set of data for NaCl. C was not
actually measured by Mellanby, but is obtainable from his first graph with
only a small extrapolation as 0:0052. Hence, from Mellanby’s tabulated
values of c, we obtain the values of gq, given in the table along with his
values of p. The last row contains the ratio p(1—q)/¢(1—>p).

Table I.
an 50 45 | 40 35 30 25
MN ca tecasncesss--|- 962 865 | 769 673 577 481
"5 en a 910 830 «665 555 450 356
19 EOD Bt is bag 76 | 60 61 60 60
q(l—p) —
|

The first two values are aberrant, because 1—q and 1—p being
small are powerfully affected by experimental uncertainty. If we take
p(1—q)/¢(1—p) = 0°60 = A, and use the equation to calculate p from g, we
find for p in these first two cases 0°938 and 0°794 in place of 0°91 and 0°83,
the differences being within the limits of experimental error.

Mellanby shows that the graphs for NaCl, KCl, and NH,Cl coincide when
¢ expressed as gramme equivalents is made abscissa and » ordinate. Hence
the value of A just found for NaCl, namely 0°60 holds for KCl and NH,Cl.
Mellanby generalises this result for all neutral salts composed of two
monovalent ions, but in the appendix to his paper he points out that the
graphs for KA, KBr, and KI do not coincide with that for KCl, a result
previously found by T. B. Osborne, and given also by Hardy. Mellanby
finds that a single graph will suffice for Na2SOu, K2SO.4(NH,)2S0., MgCl,
BaCle, and CaCla For this type, namely, a divalent and two monovalent
ions, I find A=0°613, say 0°61, and for the type MgSO, A = 0°66.

One of Mellanby’s discoveries leads towards a useful generalisation in
connection with A. He measures the average solvent power of any salt for
globulin up to the point when the whole of a suspension is dissolved, and
finds this proportional to the sum of the squares of the valencies of its ions.
For example, if the solvent power of Na is 1? and of Clis 1?, that of NaCl
will be 2, that of MgCl, will be 2?+4 2 or 6, and that of MgSO, 27+ 2? or 8.
It seems simpler to measure solvent power by the reciprocal of the con-

i32 _ Mr. W. Sutherland. [July 26,

centration required just to dissolve the whole of a given globulin suspension.
From Mellanby’s graphs we find this concentration C to be 0:000094 E for
salts of the type NaCl, E being the gramme equivalent. For the type CaCle
and NagSO, it is 0000065 E and for MgSO, 0:000056 E. Hence, for 1/C
for these three types, we get the values 10600/E, 15400/E, and 17850/E.
If we use gramme molecular mass M instead of gramme equivalent E, these
become 10600/M, 30800/M, and 35700/M, in which the numbers are as.
2:6:7, corresponding to the ratios 2:6:8 found by Mellanby when using
average solvent powers. We can regard this result in a different way. Let.
us write (1) in the form

p (1-4) =4c(1-p), (2).

and study A/C for each type, thus making the absolute concentration ¢
prominent. The values of A/C for the three types are 6400/M, 18800/M,
and 23600/M, the numerical parts of which are MA/C, and are more nearly
as 2:6:8. From this point of view, we find Mellanby’s law apply better to.
MA/C than to M/C, to an average value of which Mellanby applied it. In
Section +, I shall try to give a theoretical explanation of Mellanby’s law
applied to MA/C. Meanwhile, to complete the discussion of experiments

bearing on A, we shall consider his results concerning the influence of |

temperature on the solvent power of NaCl for globulin. He comes to the
conclusion that the solvent power is proportional to the electric conductivity
or summed ionic velocities of the solution of NaCl at the temperatures.
used. But it will be better to investigate the effect of temperature on A and
A/C, and then follow Mellanby in taking account of ionic velocities in this
connection.

The following table contains the values of A which I have calculated from
Mellanby’s data* at the five temperatures, also the values of C/E derived
from his graphs. The values of EA/C are also tabulated to give relative
values of A/C. The column marked 7 gives the viscosity of water at the
five temperatures, and the last column 7EA/C :-—

Table II.
| Temp. 107A. 10°C/E. EA/C. 1047. nH A/C.
aa OF |
1°5 860 168 5120 172 88
20 600 102 5860 101 59
| 30 5386 84 6310 80 | 50
Gane 40 512 75 6790 66 45
| 50 605 71°5 8470 55 47
| | |

* Loe, cit, p. 353.

' =

1906. | The Chemistry of Globulin. 133

The value of A attains a minimum between 30° and 50°, probably between
30° and 40°. It is worth noticing that this minimum comes near to the
temperature of warm-blooded animals. The viscosity of water controls the
velocities of an ion at different temperatures and is inversely proportional to
them. So the column headed nEA/C gives relative values of the ratio of
EA/C to the velocities of Na and Cl at the five temperatures.

These show a minimum about 40° and can be expressed by

nEA/C = 4540-031 (t—40)2. (3)

This equation makes the value of A/C at different temperatures depend
not only on the viscosity », which determines the velocities of the ions, but.
on specific properties of the globulin, which are a function of the temperature.
Certainly one would expect, a priori, that the profound dependence of the:
chemical and physical properties of proteid on temperature would appear in
such an equation; one would expect the approaching coagulation at 75° to
be foreshadowed. It is again striking that the temperature of minimum
nHA/C is about 40°, near that of warm-blooded animals. These experiments.
have yielded in equation (1) the result that the chemical equilibrium
between globulin and neutral salts is of a simple type, dependent, as far as.
the salt is concerned, upon ionic electric charges and velocities, but also.
dependent upon special properties of globulin.

It has already been mentioned that KA, KBr, and KI are exceptional to
a certain extent and show that other properties of salts than ionic charges.
and velocities have an influence on their chemical equilibrium with globulin.
Moreover, the solvent powers found by Hardy* for globulin from ox blood do:
not agree closely with those given by Mellanby for horse globulin. For
instance, Hardy finds MgSO, less powerful than K2SQ,, whereas Mellanby
finds its power 8/6 of that of K.SO, Hardy finds KNOs:, NaNOs, and
NH,NO3 1°5 times as powerful as KCl and NaCl. Yet Hardy’s data on the
whole show that valency is the chief factor controlling solvent power. He
finds K and NH, oxalates to have the same power as (NH4)oSO, and an
equivalent of Na citrate 3°3 times as powerful as NaCl, so the gramme:
molecule would be 9°9 times as powerful as that of NaCl. On Mellanby’s.
principle it would be (3?+3)/2 or six times as powerful. Further investiga-
tion must clear up these points.

2. Solution of Globulin in Acids and Alkalies.

Hardy’s experiments show that the reactions between globulin on the one:
hand and acids and alkalies on the other take place under the usual chemical
* Loe. ctt., pp. 308 and 309.

134 Mr. W. Sutherland. [July 26,

conditions of combination. Whereas the amount of a globulin suspension
dissolved by a certain strength of NaCl is proportional to the original
concentration of the suspension, the amount dissolved by HCl depends only
on the total amount of acid which has access to the suspension. Hence, for
acids and alkalies in place of (1), we have the relation

gj = Be, (4)
where B is a parameter characteristic of each acid and alkali. Mellanby
established the same result and shows that B does not vary with temperature.
The contrast between solution in neutral salts and in acids or alkalies is
complete. Hardy proves that a given amount of globulin reacts not with
equivalents, but with molecules of such acids as HCl, H2SOu, and H3POx,.
Similarly he finds that the molecule Ba(OH),. has the same solvent power as
KOH, NaOH, and NH,OH. To dissolve one gramme of globulin 10 x 1075
gramme equivalent of alkali is required, and 18x10~-° gramme equivalent
of the strong acids. These results will be used in Sections 4 and 6.
T. B. Osborne, from 1899 to 1901, obtained similar results for the vegetable
globulin edestin. :

3. Kormule for the Precipitation of Globulin by Salts and by Acids.
The first case is that in which globulin is precipitated by excess of
a solvent neutral salt. Let ¢ be the concentration of the salt and p the
fraction which the precipitated globulin is of the total originally in solution,
then Mellanby’s graph for (NH4)2SO.* is given by the equation
p(lt+p) = 28°8 (¢«—0152). (d)
The values of ¢ given by this for the experimental values of p are compared
with the experimental values of ¢ in the following table :—

Table III.
LOR py tet aa ee: 315 | 447 | 5383 | 695 | 787 | 858 | 1000
L0%c exp......:... | 166 | 175° 91837" 192" || 200") 208 aaets
108¢ cale.......... 166 | 174 | 180 | 198 | 202 | 207 | 221

To interpret this equation, we start with the consideration of the value ¢ of
¢ at which precipitation just begins. ‘Two actions of the salt just balance
here, the solvent action and the precipitant. If, then, by increasing the
concentration to ¢, the fraction p of the globulin is precipitated, an amount
of solvent power proportional to cop is liberated, so the solvent power, which
at ¢) was proportional to ¢, at ¢ is proportional to (1+p)¢. This implies

* Loc. cit:, pico k

1906. | The Chemistry of Globulin. 135

that the whole of the additional salt c—c) is occupied in keeping the
precipitate down. This solvent power proportional to (1+) ¢ acting on the
precipitate » will give a rate of solution of the precipitate proportional to
p(1+)c, and this must be equal to the rate of precipitation when
equilibrium is reached. But as’ c—cp is occupied in keeping the precipitate
down, the simplest assumption to make is that the rate of precipitation is
proportional to c—c¢. Hence, for equilibrium,

p(i+p) = Fe), (6)
where F is a parameter eharacteristic of each salt.

This equation of equilibrium is of the same form as (5). The precipitate
in this case is probably not globulin, but a compound of globulin and the
salt. It begins to come down at a concentration about 40 times that which
produces complete solution of the original globulin. But Mellanby’s data for
MgSO, cannot be expressed by an equation like (5). They could be represented
by portions of two straight lines.

When a salt of a heavy metal is added to a solution of globulin in a neutral
salt, the strength of the neutral salt being kept constant in all the comparisons,
the fraction of the total globulin precipitated is proportional to the amount
of the salt of heavy metal up to a certain point.

Thus, with globulin dissolved in 0°6 per cent. NaCl and then precipitated
with ZnSO,, also dissolved in 0°6 per cent. NaCl, so that the total amount of
globulin in 10 cc. was always the same, the fraction p of the globulin precipi-
tated is given by

p = 2730e, (7)
up to ¢ = 0:000125 gramme per cubic centimetre, ¢ being here the grammes
of ZnSO, per cubic centimetre of the 10 e.c. used. From ¢ = 0:000125 to
e = 0000225 the formula is

p—0°35 = 1030 (c—0:000125). | (8)
Mellanby converts these two lines in his graph into a single curve, but
with some violence to the experimental data. For a solution of globulin
containing 0°0027 gramme NaCl per cubic centimetre and ZnSQ, as
precipitant, p = 236c¢ up toc = 0-001, while from ¢c = 0001 to ¢c = 0:0035
we can write p — 0:15 = 82 (c — 0-001).

For CuSO, the corresponding equations are p = 3660 up to ¢ = 0:00015,
and p — 0°55 = 2000 (¢ — 0:00015) up to ¢ = 000025.

In this connection it is interesting to review the considerable amount of
analytical work done on the albuminates of the heavy metals.* Harnack
found, in 1881, that copper albuminate precipitated from excess of albumin

* See F. N. Schulz, ‘Die Grésse des Eiweissmolekiils,’ Jena, 1903.
VOL. LXXIX.—B. M

136 Mr. W. Sutherland. [July 26,

contains 1°35 per cent. of Cu, and by excess of Cu 2°64 percent. In 1877,
Morner prepared a copper albuminate with 1:2 per cent. of Cu, and another
with 1°85 per cent. In 1887, Chittenden and Whitehouse obtained copper
albuminate containing from 0°71 to 1:21 per cent. of Cu, but found that by
redissolving and reprecipitating the percentage could be raised to 2°19.
Now with our formula for CuSQ,, and the fact that Mellanby’s globulin
solution contained 0:024 gramme of globulin per cubic centimetre, we get
0:024 p= 88c¢, that is to say, that at the earlier stages of precipitation
CuSO, throws down 88 times its own weight of globulin. Thus, neglecting
the small amount of Cu left in solution, we can calculate that the copper
globulinate of Mellanby’s experiment contained 0°45 per-cent. of Cu. If all
the globulin is precipitated, our equations give that 0°55 of the precipitate
contains 0°45 per cent. of Cu, and 0°45 of it contains 0°83 per cent. of Cu, so
that the precipitate as a whole contains 0°62 per cent.

These are only about one-third of Morner and Harnack’s vals for the
albuminates. But the larger value, 0°62 per cent., is close to the smallest of
Chittenden and Whitehouse’s, namely, 0°71. In the same way, from our
equations for Mellanby’s ZnSO, data, we find that the first precipitate of zinc
globulinate contains 0°61 per cent. of Zn, and that if all the globulin is
thrown down the compounds average 1°22 per cent. of Zn. Now Chittenden
and Whitehouse found 0°91 per cent. of Zn in their zinc albuminate. So
there is a fair correspondence between the composition of Mellanby’s
globulinates and Chittenden and Whitehouse’s albuminates of Cu and Zn.
Thus it bears upon the present inquiry to look more closely into the
albuminates of these investigators. The following table contains, in the first
row, the fraction / of metal in unit mass of its albuminate, in the second the
equivalent mass E of the metal, and in the third the ratio //E.

Table IV.
Cu. | Zn. | Fe. U. Pb. | Hg. | Ag.
HOHE. v1 | 91 95 | 460 | 250 | 289 | 409
Mee cas ciaele ssiieielen 32 32 °5 28 120 104 100 108
HOW ecsscsss: 22 | 28 34 38 24 29 38

From the mean value of the last row we find the percentage of heavy
metal in its albuminate to be 0°03 E. Hence the minimum molecular mass
for albumin in the albuminates is 100/003 or 3300 nearly. Hence, we infer
from the above argument that Mellanby’s data would agree with the
altogether independent data for the albuminates in making the minimum

1906. | The Chemistry of Globulin. 137

possible molecular mass for globulin about 3000. This result will be used in
Section 6.

We shall now discuss the precipitation by acids of globulin dissolved
in neutral salts. This is a very characteristic point in the chemistry of
globulin. While a little acid dissolves globulin, it precipitates it from
solution in salts. One would expect a priori from the similarity of salts and
acids that they would help one another in dissolving globulin. Instead
of that they are antagonistic. Hardy has shown that the solvent power of
two neutral salts for globulin is the sum of their separate powers. All the
more striking, therefore, is the antagonism of neutral salts and acids. It
seems to me that the explanation of this fact is to be found in this, that the
acids enter into the globulin molecule in different places from the neutral
salts, because the H atom can play a different ré/e in organic compounds from
that possible to metallic atoms. The ordinary organic compounds are far more
stable than the organo-metallic.

Moreover, the molecule of acid and of neutral salt enter the globulin not
only in different places, but in places which make them incompatible with
one another, unless special provision is made to keep them in position. Each
is stable in its own place. Together they tend to instability. Hence, acids
and neutral salts inhibit one another’s action in dissolving globulin. We can
apply this idea quantitatively to Mellanby’s experiments in which acid is
made first to precipitate globulin from solution in salts, and then to redissolve
the precipitate. He used his solutions in such a way that the total amount
of globulin and of neutral salt in the 10 cc. of mixture under observation
remained constant in a set of experiments. The strength of the neutral
salts was 0°005, or 0:°003 gramme per cubic centimetre, and of the globulin
sufficient to saturate the salts. The amount of acid present in any case in the
10 cc. is specified by the number of cubic centimetres of N/100 acid from
which it could be obtained. The results show that up to maximal precipita-
tion the fraction of the globulin precipitated is practically proportional to the
amount of acid present. In fact, Mellanby’s graphs, in which p the fraction of
globulin precipitated is ordinate and amount of acid is abscissa, could be
replaced by two straight lines, one running up from the origin to the point
of maximum precipitation, and the other running down again to a point of
zero precipitation or complete solution.

‘To discuss the theory of the whole action, let us denote by a the cubic
centimetres of acid which in the absence of salt would just dissolve all the
globulin. Let 6 be the number producing maximal precipitation in a given
salt solution containing the globulin. Then 0 would dissolve the fraction b/a
of the globulin in the absence of salt. But the salt present is able just
| M 2

138 Mr. W., Sutherland. [July 26,

to dissolve all the globulin by itself, hence we may say that of the total
solvent power of the salt and the acid 0, the salt and acid possess the fractions
1/(1+6/a) and (6/a)/(1+6/a) respectively. So if p» is the maximum

fraction of the globulin precipitated by 6, then 1—p, is the fraction
held in solution by acid, and the fraction held in solution by the salt is
= Pin

14+ b/a-

Hence, by the presence of the acid 6 the salt is deprived of
1—(1—pm)/(1+6/a) of its solvent power, and this gives us a measure
of the inhibition. The amount of acid required to inhibit all the salt will
be b+{1—(1—p,)/(1+6/a)}. But to dissolve all the globulin as well as
to inhibit the salt we shall need an additional a of acid. Let 7 be the
total experimental amount of acid that will just give a clear solution again,
then

b

I= payer ="

This is an equation for calculating a from the observed values of Pm, 0,

and r. For a solution containing 0:005 gramme of NaCl per cubic centi-

metre, with HCl and H.SO,.as acids, Mellanby found p, = 015 and

b = 2:5, while r= 7°3. Solving (9) for the positive root of a, we get

a = 2'8. From all his data the following table has been prepared for four
salt solutions of the strength specified :—

Table V.
SanSERENTSTEaIIaInN SOUR
| NaCl Na.SO, MgSO, MgCl,
0°5 0°5 0°3 0°3
| per cent. per cent. per cent. per cent.
WO koe 15 9 17 7
VOOtE ec sosat 25 45 40 39
Sacked Ogi cae awie tes 73 90 100 67
| NOG. ose ee 28 25 39 15

These strengths of solutions dissolve nearly equal amounts of globulin
in 10 cc. but not quite equal. If we calculate the relative amounts of
globulin by the principles of Section 1, we find that they are as 1°7, 2:1, 2:0,
and 1°9. Hence, relative values of a, referred to equal amounts of globulin,
will be derived from the values of 10a in the table by dividing by these
numbers, giving 17, 11:4, 17°5, and 9. Now these are to one another
nearly as the values of 10%p,, in the table, a fact which confirms in a general
way the above reasoning and calculation, though, strictly, instead of comparing

a

1906. | The Chemistry of Globulin. 139

with pm, we should compare with pm referred to equal mass of globulin for
each salt. In a broad way a tends to be proportional to pm.

It is a striking fact that though the acids HCl and H2SQ, act by
equivalents in each case, the same number of equivalents is not required
in different cases; in other words, a for different solvents and a constant
amount of globulin is not a constant, although with pure globulin these
acids react in definite proportions. It seems, indeed, as_ though the
precipitate we are now discussing cannot be pure globulin. So it is note-
worthy that the amounts of acid required to yield pm are nearly proportional
to the cube of the total globulin strength in each of the above four cases,
for, taking the values of 105 from Table V and dividing them by the cubes
of the relative globulin strengths given above, we get 25/1°7°, 45/2:1°,
40/28, and 35/1°98, or 5:1, 4:9, 5-0, and 5-1. It would appear, then, that the
equation of chemical equilibrium between acid 6 and total globulin G,
dissolved in neutral salt, is of the form

G3 = kb. (10)

Here, evidently, G? represents the mass action amongst three globulin
compounds, whose amount in each case at the point of maximal precipitation
is proportional to the total amount of globulin present. Of these three we
know that two are probably the compound of globulin with acid alone and
the compound with neutral salt alone. The third, then, is probably a
compound containing both acid and salt, stable only when the other two
are present in excess. These compounds form by their joint action the
precipitate, whose rate of formation is measured by G* Then in (10) kd
must represent the rate of action of the acid tending to dissolve the
precipitate, which acts in the usual way with a constant active mass.

It is worth noting that when NaCl and MgSO, are the solvents, acids
precipitate globulin nearly equally, while when Na2SO, and MgCl, are the
solvents, acids precipitate nearly equal fractions, but smaller than in the
previous case. It seems as if ions of the same valency, like Na and Cl or
Mg and SOx, can co-operate better than dyad with monad as found in Na2SO,
and MgCle This recalls the observation of Picton and Linder, that ions of
different valency inhibit one another in precipitating colloids, while ions
of the same valency co-operate.

4. A Theory of the Colloidal State.
The characteristic of the colloid state is that the molecules cease to have
a separate existence; they link on to one another by means of the atomic
electric charges, thus forming the meshes so characteristic of colloids

140 Mr. W. Sutherland. [July 26,

Each particle in a suspension of a globulin might, therefore, be called a
molecule, but with no advantage. In each such particle, however, a certain
pattern is repeated in three-dimensional space. In a colloid this pattern
is the real subject of the chemist’s study, just as the molecule is in
crystalloids. Because of its importance, I propose to call this pattern a
semplar. An imaginary case will enable the theory to be presented in a
definite form. The nitrogen of NHz acts trivalently, but it can act also
with pentad valency. In the old phraseology two of the bonds of N in
NHz3 were said to satisfy one another. Now, in Helinholtz’s theory, valency
is the number of electrons associated with an atom. I have proposed
to consider variable valency as due to the existence of opposite electrons
in the atom showing it,* and for convenience of notation and printing
I have proposed to denote the positive electron by #-and the negative by b.
Thus both the triad and pentad valencies of N are expressed by writing it
ENbu, because and 4 can coalesce to form a doublet th in which each
electron almost completely neutralises the chemical activity of the other.
On the same principle the symbol of the hydrogen ion is H#, so that in
NHs3 there are three doublets #) chemically effective and one ineffective.
I have shown that these doublets and others are the cause of cohesion and
rigidity. In a crystal of frozen NHs; the molecules are all separate, each
affecting its neighbours by the action of these doublets upon one another.
But imagine each ineffective doublet of N to be made effective, so that
¢reaches out to unite with } of a neighbour N, then every molecule of
NHs becomes chemically linked with two of its neighbours, it becomes a
semplar, and the whole crystal becomes a mesh of such semplars. Under
certain conditions it could retain its crystalline form. This imaginary
example explains the nature of a colloid on the present theory, and shows
how the transition from crystalloid to colloid can be continuous with
occurrence of crystalline colloids. The formation of polymers is due to
ineffective doublets becoming effective, though generally the linking up of
molecules is restricted to groups of m molecules, where m measures the
amount of polymerisation. The most important case is that of water, which
I have shown} to be a mixture of (H20O)s trihydrol, found pure in ice, and
of (H20). dihydrol. In water and other polymers we have the simplest
instances of semplars. When m, the measure of polymerisation, becomes
large and indefinite, it gives us the colloid state, in which chemical forces
between neighbour semplars produce effects of the same order as those
between the separate doublets of molecules in the solid state. Before

* * Phil. Mag.,’ [6], vol. 3, p. 161.
+ ‘Phil. Mag., [5], vol. 50, p. 460.

1906. | The Chemastry of Globulin. 141

applying this theory to the chemistry of globulin, it will be helpful to discuss
with its aid the remarkable connection, discovered by Schulze and investi-
gated by Picton and Linder, between the valency of ions and their coagulating
power. Hardy has shown that a similar relation holds for certain organic
colloids. A theoretical explanation of it has been offered by Whetham,*
based upon considerations as to the probability of electrolytic ions of like
sign coming together. Though suggestive, this theory seems to me inade-
quate, because the electric repulsion between ions of like sign tends
powerfully to keep them apart. On the present theory, coagulation
consists in opening out ineffective doublets $b so that they link together
neighbour molecules of the coagulating substance. Hence ionic charges must
sometimes be instrumental in coagulation, because a free # tending to
attract bin the doublet and to repel b is an agent for opening out ineffective
doublets. Suppose that a free # unites with b from an ineffective #) of a
molecule of coagulee, then it liberates #, which will grasp the b of 4 neigh-
bouring ineffective $b, and so the coagulation spreads until perhaps the other
ion of the electrolyte with its electron b steps in and arrests the process. But
there is another way in which coagulation can be propagated from an ion as
centre. The electric force of the ion will act. on the ineffective doublets out
to a considerable distance from itself, and, moreover, every ineffective doublet
which is made chemically effective alters the local electric equilibrium by the
change. Each coagulated molecule is a centre of coagulation. Hence we
have the speed of coagulation due to an ion proportional to the electric
charge of the ion and to the amount of coagulation already produced by it.
Let ¢ be the coagulum produced by electrolyte of concentration s, then we
have, with v for valence of the effective ion and & a constant,

7 (6) = y= +. log eft = ky (t—t), (11)

S

where ¢ is the amount of coagulum produced at a certain short time ¢ after
the beginning of the process. This small initial coagulation is produced
mostly by the first process of direct linking, and is proportional to vs the
total number of electrons which start the linking up, so we may write
log c/vs = kv (t—t’9). (12)
Now, in the experiments, the number of equivalents vs of electrolyte
required to produce a certain amount of coagulation ¢ in a very short time
are measured. We may neglect 7¢’) and take ¢ as well as ¢ to be the same
for each electrolyte. If we call 1/vs the coagulating power of the electrolyte,
_ equation (12) makes its logarithm linear in the valency v. Schulze found
* ©Phil. Mag.,’ [5], vol. 48.

142 Mr. W. Sutherland. [July 26,

the coagulative powers of chlorides of mono-, di-, and tri-valent ions on
arsenious sulphide to be as 1:30:1650, the common logs of which are
0, 1477 and 3°217, which fall nearly into the linear form 1:°564 (v—1).

The data of Linder and Picton for the corresponding sulphates on arsenious
sulphide are as 1:35:1023, whose logs are 0, 1:544, and 3:01, which may
be written 1°515 (v—1).

The present theory, then, lends itself well to an explanation of this
remarkable law.

In the chemistry of globulin, the prominent fact is that salt solutions
dissolve from a suspension an amount nearly proportional to the original
total strength of the suspension. According to the present theory, the
explanation is as follows: Just as ions by their elective force draw out the
ineffective doublets of molecules, which they cause to join up as semplars,
they can, under suitable energy conditions, break the doublets joining the
semplars, which they liberate as molecules. For example, if a crystal of
our imaginary colloid NH: were dissolved in HCl, the action would be one
in which the ions H and Cl would break the doublets joining the semplars
NHs3, and would join on to form NH,Cl. This I take to be a representation
of the solution of globulin in acids, the action of salts being not so simple.
It is a corollary of the coagulation of colloids by electrolytes. The same
electric actions that build up a coagulum can sometimes pull it down,
especially if the operating ions can step into places which make the total
chemical potential energy smaller-than before. Now the number of semplar
doublets in a globulin suspension which the ions can break will either be the
total thereof or a certain fraction of the total. In the former case we should
have solution of the colloid in the ordinary way, as by acids. In the latter
case the ions will only break those doublets which can be made unstable by
the particular attacking force of ions used. Hence the fraction of a globulin
particle dissolved by a given solution will have a definite value, and the
amount of globulin dissolved will be proportional to the total number of
particles, that is, to the original concentration of the suspension. This is the
explanation of the most striking experimental result brought out by
Hardy and Mellanby. We now proceed to give the corresponding interpre-
tation of equation (1) deduced from Mellanby’s experiments. Let us denote
the semplar of globulin by G. The simplest possible formula for the semplar
at the moment of its detachment is bG#. Then either # and b can coalesce
to form an ineffective doublet, leaving G as a molecule, or if ions like Na and
Cl are present, their electric charges will link them on, giving Na#bG#5Cl, or,
more briefly, NaGCl. Now the fact that about 150 times as many equiva-
lents of NaCl as of HCl are required to keep a given amount of globulin in

1906.] The Chemistry of Globulin. 143

solution, shows that NaGCl must be relatively very unstable; a large excess
of Na and Cl ions is required to keep it in existence. Its existence depends
on the breaking of doublets and keeping them broken. The number of bonds
broken per second by the Na and Cl ions will be proportional to the concen-
tration c of these ions and to the number of breakable bonds remaining to
be broken, that is to G—g. The rate of solution can be written kic(G—gq).
Now the rate at which the dissolved g tends to go out of solution will be
proportional to g and to a function of c, which just vanishes when c = C, the
concentration which dissolves all the globulin. The simplest such function
is C—c, so we write the rate of precipitation as keag(C—c). So, for equili-
brium,

Pec) = iyc(G<o), therefore »(1—9)—= Ag —p) = a e(1—p),

which is identical with (1) and (2). Now A is the ratio of A, the velocity
of solution of the globulin, to ‘2, the velocity of precipitation. If instead of
e we use the number of gramme molecules c/M in these equations, its
coefficient becomes MA/C, which gives a measure of the ratio of the velocities
of solution and precipitation when gramme molecules of different salts are
compared as to their actions on globulin of a given concentration. Now the
velocity of solution of globulin by a molecule will be proportional to the
rate at which the doublets #) are broken by ionic charges, so it will be pro-
portional to the velocity of the ion and its charge, that is to say its valence.
But in the precipitation of globulin the ions have to be extracted from com-
bination with the globulin, so the number of doublets to be broken will be
proportional to the valence of the ions. It is assumed here that in salt
globulins an ion is loosely attached at the semplar doublet which it breaks,
and that as breaking power is proportional to valence, so also is the strength
of attachment. The velocity of precipitation will be therefore inversely as
the valence. Hence MA/C will be proportional to the square of the valence,
which is the modified form given to Mellanby’s law in Section 1. As regards
the dependence of the velocity of precipitation on velocities, it will arise
mostly from motion of the combined ions in the globulin compound. We
find, then, that MA/C must be proportional to the velocity of the ions and
to a velocity depending on the globulin. Now the velocity of the ion must
not be confused with the quantity defined in physics as the ionic velocity,
which is a velocity generated under a certain unit external electric force.
Apparently, in reacting with globulin, the different ions move with about the
same speed. But as regards the effect of change of temperature on the velocity
of ions, we know the velocity to be inversely as the viscosity of water at

144 Mr. W. Sutherland. [July 26,

different temperatures. Hence, our equation (3) expresses both this
dependence of MA/C on the viscosity of water and on velocity inside the
globulin compound. Thus the present theory of colloids is able to give a
consistent account of these important features of the chemistry of globulin.
The precipitations of globulin hardly need review in this connection, as they
have been pretty fully considered in Section 3.

5. The Electric Conductivity of Globulin Solutions.

Hardy, by overcoming the experimental difficulties of measuring these
conductivities, has obtained data of great importance, which I propose
to make more available by expressing them in interpretable formule. With
HCl 1 gramme of globulin was dissolved in 9°32 x 107° gramme equivalents of
acid, and the solutions prepared from this by successive dilution have their
strengths given by v, the volume of solution containing 1 gramme equivalent
of HCl. From the measured specific conductivity of these solutions the
molecular conductivity of the HCl is calculated in the usual way, but it
should be noticed that it is expressed with reference to a unit of resistance
10 times as large as that often used, so that conductivities are made
10 times as large. For HCl globulin solutions the molecular conductivity p
of the HCl given by Hardy* is expressed by—

1/ = 0:00026 +.0:0090-3, (14)

as the following comparison shows :—

Table VI.
Oceecl Se ees cee 107 | 217 | 484 | 868 | 1735 | 3470
je Gales ee 465 | 568 | 690 | 830 991 | 1170
fitexqp ace ees 439 | 596 | 706 | 884 | 991 | 1167

|

At infinite dilution (14) gives w = 3846, the value for pure HCl being
3840. It appears, then, that at infinite dilution HCl globulin is entirely
dissociated, yielding completely ionised HCl. The equation having so
extensive a range must have fundamental theoretical significance, which is.
easily brought out. The occurrence of v~? reminds us of Kohlrausch’s.
discovery that the molecular conductivity of dilute electrolytic solutions is.
a linear function of v~3,a fact which I have sought to trace back to the
3 occurring in the formula (H,O)s3 for trihydrol.t

For the molecular conductivity of an ion of radius a, valency », dielectric:

* Loe. cit., p. 274.

+ See’ “The Molecular Constitution of Water,” and « Tonisation, Ionic Velocities, and
Atomic Sizes,” ‘ Phil. Mag., [5], vol. 50, and [6], vol. 3.

1906.] — | The Chemistry of Globulin. 145

capacity K, and charge ¢, moving through water of dielectric capacity Ky and
viscosity 7 the formula is—

pe = ve?Ko/6irnak ; (15)
that is to say, the ionic velocity is inversely as a, so that the time taken by
an ion under standard electric force to travel 1 cm. is proportional to a.
Now in the case of HCl globulin the corresponding time is proportional
to 1/, and therefore to 0:00026+0-009v—%. Here 0:00026 represents the time
that would be taken by the H and the Cl ions to increase their distance
apart by 1 cm., if they were always dissociated from the globulin. The term
0-:009v-* represents the time lost on account of the presence of the globulin.
The way in which this time is lost is the following: Suppose that HCl
globulin is HGCl, yielding two sets of ions HG# and Clb, and also GClb and
- H#. So the H ion travels part of its path as H#and part as HG#, while
the Cl ion travels part of its path as Clband part as GClb. Let 7 be the
fraction of each second which H# and Cl> spend in the forms HG and GCl.
During this fraction each moves with the smaller velocity wu due to the large
radius of G, for the rest of the second it moves with the ionic velocity wo
appropriate to H# or Clb. The average velocity of the ion is w(1—7r)+ur.
Hence the time taken by the H and Cl ions to separate by 1 cm. is

proportional to—

1 1 tT (1—u/u)

ee ae ee 16

Up (1—7T)+ur al See ea ATS) ey

If, then, we write 0:00026+0-:009v-: in the form 0:00026 (1+3460-5),
we have

Tilia ons sy
eh (17)

Denote 7 (1—u/uo) by «, then we have «/(1—«x) proportional to uv, and
therefore to c?, where ¢ is the concentration of the HCl. If w/w is small,
as Hardy directly proved it to be in the way we shall discuss in the next
section, then x stands almost for 7, the fraction of the total H or Cl existing
at any moment as HG#or GClb, while 1—7 stands for the amount of free
HCl. In short, 7 represents the fraction of the HCl which is combined, and
1—v that which is free. Hence the proportionality proved above, which
may be written

z= k(1l—z)e, (18)
is an equation of chemical equilibrium expressing that the amount of
combined HCl existing as HGCl in the form of ions HG and GCl is propor-
tional to the amount of free HCl and to the amount of something else. In

“ Tonisation, etc.”* it is suggested that this something else in ordinary
* Loe. cit., p. 173.

146 _ Mr. W. Sutherland. [July 26,

electrolysis is $H,0b broken out of trihydrol. It is shown how the number
of these in a solution is proportional to c—?, so that when they act upon the
whole solute ¢ they produce an agent of amount proportional to ¢~%e,
that is ¢*, which, in the present case, acting on the residue (1—2) of free
HCl keeps the amount # of combined HCl formed. Hence, for equilibrium,
we have w = & (1—z) ct as in (17) and (18). If in (16) we neglect u/um and
put v= 1000, we find + = 0°78, that is to say, that N/1000 HCl, which
is Just saturated with globulin, contains 78 per cent. of the H and Cl ions in
combination with globulin, and only 22 per cent. free. The experiments of
Hardy, when summarised in (14), give a pretty complete insight into the
chemistry of HCl globulin.
As regards alkali globulins, the data of Hardy can be expressed by

(1 gramme glob. diss. in 9°7 x 107° gramme equiv. NaOH)
fe = 220+1/(0:0005 + 0-031x-8),

( ms ° 18 x 10-5 gramme equiv. NaOH)
pw = 290+1/(0:0005 + 0:0310-5),
(elobadiss. in IN HyO)iee eee eee = 290+1/(0:00048 + 0:023v-),

(19)

We shall at first discuss only the first and third of these as characteristic
of NaOH and NH,OH. When v is infinite, the first gives « = 2220, while
in the same units the standard data of Kohlrausch would give 444 for Na
and 1740 for OH, that is w= 2184. The formula gives complete dissociation
of the alkali globulin at infinite dilution. It is noteworthy that the first
term in (18) for NaOH globulin is 220, which is half of the standard value
for Na. It appears, then, that half the Na unites with globulin to form
a globulinate, which dissociates completely at all the dilutions of Hardy's
experiments, giving Na ions, and globulinic acid ions whose velocity is too
small to make itself apparent. As the OH of this half of the original NaOH
does not contribute to the molecular conductivity in the same way as the Na,
but yet appears in the conductivity at infinite dilution, we see that it joins
on to the globulin in a similar way to that of the other half of the NaOH.
The H of the globulin displaced by the Na atom must also join on to
the globulin in a different function. The other half of the NaOH seems
to combine with globulin in a way similar to that of HC]. A compound
such as NaGOH seems to be formed (with H and OH attached from the first
half of the NaOH), yielding ions NaG#:and GOHb which may be practically
two varieties of the globulinic acid with which the other half of the Na
combines. From the second of equations (19) it appears that if twice as
much NaOH is used as suffices to dissolve all the globulin, its molecular con-

1906.] _ ~The Chemistry of Globulin. 147

ductivity is but little changed. So in this case a quarter of the base reacts in
quite a different way from the remaining three-quarters. The same considera-
tions apply to the case of NH,OH. The first term 290 is close to the 311
which the standard datum of Kohlrausch would give if half the NH* existed
as basic ions taking the place of the H ions of globulinic acid. The other
half of the NH4OH combines additively like the second half of the NaOH.
Jn the alkali globulins we must distinguish between the basic half of the
alkali and the additive. To the additive half exactly the same considerations
apply as to the additive HCl globulinate, and the degree of dissociation of the
additive alkali hydrate can be determined as in the case of HCl globulin.
Hardy found that to neutralise globulin to phenolphthalein with NaOH the
amount of alkali required is twice that needed to dissolve the globulin. Here
we have additional evidence that the alkalies act in two ways. Hardy finds
that 1 gramme of globulin is dissolved by 10~* gramme equivalents of alkali,
and by 2x 10-4 of acid. Now only half the alkali displaces acidic H, hence
the acidic H atoms are only one-quarter the number of places where HCl can
add itself to the globulin molecule. Mellanby’s measurements on horse
globulin do not agree with Hardy’s distinction between the number of
equivalents of acid and alkali needed to dissolve globulin. He finds a
gramme of globulin dissolved by about 10~* gramme equivalents of HCl and
about the same number of NaOH. Globulins of different origin and history
appear to differ to an extent that limits us at present to a discussion only of
orders of magnitude in this connection.

Concerning the molecular conductivity of neutral salt solutions saturated
with globulin, Hardy made some measurements. NaCl of concentration 0:16 N
had its conductivity depressed 2:4 per cent., of 0-11 N by 2°3 per cent., and
of 0°09 N by 2:1 percent. While of MgSO, 0°3 N the lowering of conductivity
was only 1:4 per cent.

Hardy does not give the strength of the globulin suspension with which he
saturated his salt solutions, but states that a gramme of globulin was dissolved
by from 10~? gramme equivalents up to 2x107~%. About 100 times as many
equivalents of NaCl are required to keep globulin in solution as of NaOH, but
as the globulin reduces the conductivity of NaCl about 2 per cent., we may
take 2 per cent. of the salt to be combined with globulin, so two equivalents of
NaCl actually unite with globulin for one of NaOH. Thus it appears that
NaCl in its action on globulin is liker to HCl than to NaOH. The con-
ductivity measurements fully confirm the conclusions arrived at from
the solvent powers of neutral salts, namely, that the compounds formed
are very unstable and require a large excess of salt to maintain their
existence. |

148 Mr. W. Sutherland. [July 26,

6. The Molecular Mass of Globulin.

In “A Dynamical Theory of Diffusion for Non-Electrolytes and the
Molecular Mass of Albumin,”* I have sought to show how the molecular
radius of a molecule can be found from its coefficient of diffusion, having
previously obtained the corresponding relation reproduced as (15) in the
present paper between molecular conductivity and radius. These two
definite and accurately measurable velocities, the ionic and that of diffusion,
are convenient aids in the measurement of large molecular masses. From the
Graham-Stefan coefficient of diffusion for egg albumin the radius of a gramme
molecule of albumin was found to be 30 cm., whence, from the percentage
composition, the following formula is obtained for albumin, Cy43g Ho3¢4N 3590482815
with a molecular mass 32,814. Now Hardy has obtained a _ direct
measurement of the ionic velocity of globulin, namely, about 10~* cm. per sec.,
which corresponds to a molecular or rather ionic conductivity 100 in the
units used by Hardy, and 10 in the units of Kohlrausch. With the latter
units (15) is transformedf to

Bs = 280v/pK, (20)
in which K is the dielectric capacity of the ion, having for globulin the value
2 nearly, because for proteids the index of refraction which equals K? is about
1:4. Bis the volume of the molecules in a gramme molecule of the ion, and v
its valence. We are going to use this equation to find B for globulin, using
Hardy’s value 10 for » But so far vy is unknown. It is the basicity of
globulinie acid which is easily determined approximately by the facts we have
in hand. Thus it has been shown that when NaOH dissolves globulin, half
of it acts as a relatively strong base. Let M be the molecular mass of
globulin, then v gramme molecules of NaOH act as a strong base with M
grammes of globulin. But Hardy finds 10~* gramme molecule of NaOH
just dissolves 1 gramme of globulin, so 10~4/2 gramme molecule satisfies the
y-basicity of globulin

2 104y = M. (21)
With w = 10 equation (20) gives
Bi = 14». (22)

For the percentage composition of globulin, we have Hammarsten’s
determinations, with which I shall give the limiting volume of a gramme atom
of the elements taken from my ‘ Phil. Mag.’ papers on molecular force.

Chibi: is banc iSE O. S.
Per cent. 59°71. TO WS Sb 23:32) ule
Atomic B 22.8: 8 4 8 6 18

* ¢Phil. Mag.,’ [6], vol. 9, 1905.
+ See “ The Dielectric Capacity of Atoms,” ‘Phil. Mag.,’ [6], vol. 7.

1906. | The Chenustry of Globulin. 149

Let z be the number of atoms of C in the molecule of globulin, then

12” = 0°5271M, (23)
4 and also

:

12 701 15°85 23°32 ds 8 ai

These four equations (21, 22, 23, 24) give us the four unknowns », 2, B, and
M. The best way of solving them is to give v integral values, and find which
value makes the four equations as consistent as possible within permissible
limits of error, With »y=1 (21) gives M = 20,000, and then (22) gives
B = 2744, which in (24) yields « = 147-7, and this in (23) makes M = 3362.
As they stand, the two values of M, namely 20,000 and 3362, are inconsistent.
If vy = 2, the first becomes 40,000 and the second 8 x 3362 or 26,896. For
y = 3 the comparison is between 60,000 and 3362 x 27 or 90,774. No value
of vy above 3 is permissible. Hence the choice for the formula of globulin is
between one which makes it a dibasic acid of molecular mass about 40,000
and another which makes it a tribasic acid of molecular mass about 60,000.
Let us inquire what changes in the experimental data would make the four
equations strictly consistent for y= 2 and y=3. The most uncertain
number is Hardy’s valuable datum «4 =10. For a transparent opalescent

dialysed solution of NaOH globulin he found » =7°66 and estimated 10 as
the probable limit to which the value would rise with complete ionisation of
the compound. But his solution being opalescent, there must have been some
electric endosmose such as he found with HA globulin, for which it raised
up to 23. In the observed 7:66 for NaOH globulin there is already some
compensation of errors, and it is an open question whether the limit is more
or less than 10. It is easy to calculate the value of w in (20) which would
make vy = 2 and M = 40,000 satisfy (21, 23, 24). It is 8°76. Soforv=3
and M = 60,000 we should need «» = 11°50. Hence the decision between the
two alternatives should be reached by a sharper determination of the ionic
velocity of globulin or by its coefficient of diffusion, or preferably by both of
these methods. Probably 8°76 is the better value for w. With this and
v=2 and M = 40,000 we get x = 1756. Hence the formula of globulin is
Ci75¢HogoaN 4520 sg4Su4-
_ This formula of the globulin molecule furnishes a molecular mass 1:22 times

_that given above for egg albumin obtained from the Graham-Stefan
coefficient of diffusion. That formula can be greatly simplified for discussion —
if we replace the number of carbon atoms 1436 by the round number 1440,
the 2364 of H by 2400, the 359 of N by 360, and the 482 of O by 480, and
neglect for a while the 15 for S. These changes keep the percentage com-

150 Mr. W. Sutherland. — [July 26,

position of egg albumin true within the limits of analytical error, and enable
us to write the main part of the egg albumin molecule as 120 (Cyz2H2)N30,).

The formula of globulin cannot be quite so much simplified, but we can
write it 146(CizHigN304)+14N, neglecting S meanwhile. The group
CizH2N30.4, which is repeated 120 times in the albumin molecule, is closely
related on the one hand to the polypeptides synthesised by E. Fischer, and to
the pure peptones prepared by Siegfried. For the derivation of dipeptides
and tripeptides from the amino acids by abstraction of water we have the
equations*

2NH.(CH2),COOH—H,0 = NH2(CH2)2,NHCOCOOH, dipeptide ;

If, now, in this general formula for a tripeptide we put » = 3, we obtain
Ci2Ho3N304, a group containing 3H more than our typical albumin group
above. If, then, we remove 3H from this polypeptide, we obtain a trivalent
radicle, CizH2)N30., which we find repeated 120 times in the molecule of egg
albumin. It hardly matters at present how many times CH2 may be removed
from one of these groups and inserted into others, for we are considering only
the broad plan on which the albumin molecule is built.

Similarly, the properties of globulin depend on a certain preponderating
group whose average composition is close to Cy2zHi9gN30,. These groups are
almost as closely related to the pure natural peptones prepared by Siegfried
as to the synthesised polypeptides of E. Fischer.t These are the antipeptones
a and £, or trypsin-fibrin peptones Cy)Hi;N30; and Ci,;HigN30;, of which the
next homologue would be Ci2HaN3Os, differing from our albumin group by
OH and from our globulin group by OH». Even if these formule need to be
doubled, the relation would not be altered. Then come the pepsin-fibrin
peptones « and PB, CaHssNeOo, CaiHssNeOi. It is significant that the latter
could be produced by the additive union of the antipeptones « and £.

Now, the experiments of W. Neumann and Siegfried Rave yielded the
result that in the antipeptones the H ions are to the OH ions as 2:1,
and in the pepsin peptones as 3:2. But the number of H ions gives the
number of COOH radicles in the molecule, while that of the OH ions gives
the number of NHgz radicles. Hence we infer that the group Cy2HigN30,,
when converted into an antipeptone, contains COOH twice and NH once.
Hence, in the joining up of such groups to the number of 146 to form
globulin, there must be a large amount of internal salt formation, since
COOH occurs only two or three times in the complete globulin molecule. It

* See Cohnheim’s ‘Chemie der Eiweisskorper,’ p. 59.

+ Siegfried, ‘Ber. d. Deutsch. chem. Ges.,’ vol. 33; ‘ Zeitsch. f. Physiol. Chem.,’ vols, 35
38, 45.

°1906.] The Chemistry of Globulin. 151

is most probable that the internal salt formation goes on in such a way that
COOH and NH, neutralise one another.* Thus in the extreme case of all the
oxygen existing as COO in Cj2Hi9N30., two-thirds of the total nitrogen
would be present as NHg, joined up with COOH in the globulin molecule.
When the globulin molecule is made an ion, it has probably only two COO
radicles showing the acid character. Hence we infer from our discussion of
Hardy’s conductivity measurements that the globulin molecule contains NH,
eight times in such a form that HCl can join on as it does with NHs3 to form
NH,Cl. These eight NH» and the two COOH are the only ones left free out
of about 290 of each sort existing in the globulin molecule. By more or less
drastic treatment the latent NH, and COOH radicles may be called into
partial action, and so globulin and albumin may be made to show apparently
higher acidity and basicity than the eight and the two determined above. In
the same way arise the discrepant results of different experimenters, discussed
in Section 3, as to the percentage of heavy metals in their compounds with
albumins. Different treatments open out or break up the albumin molecule
differently. But yet a rather striking agreement appears in these discordant
results if we regard them in the following way. The range in the percentages
of Cu in albuminates and globulinates is 0-45 in Mellanby’s globulinate,
0°71 in the albuminate of Chittenden and Whitehouse, 1:2 Morner, 1°35
Harnack, 1°85 Morner, 2°19 Chittenden and Whitehouse, 2°64 Harnack, and
11, the average of Ritthausen’s measurements with vegetable albumins. For
the weight of proteid combined with a gramme equivalent of Cu (31°6) these
give the numbers—

7020 4450 2630 2340 1710 1440 1200 287

241 153 905 804 587 496 410 0:987
which are expressed in the second row as multiples of 291, these multiples
being all nearly exact integers 1, 4, 5, 6, 8, 9, 15, and 24. Here, then,
we have evidence of the splitting up or opening out of the albumin
molecule into parts, each of which contains an integral number of a group
whose molecular mass is about 291, which is quite close to the 270 of our
CieHaN304 This group with HO would give 288. This seems a fairly
conclusive proof that there is a large amount of internal salt formation in
albumins. The 3300 found in Section 3 for the mean mass of albumin combined
with an equivalent of heavy metal in Chittenden and Whitehouse’s experi-
ments represents about 12 of our groups. It is interesting to note that
the treatment of a vegetable albumin with copper breaks up the albumin
molecule into parts of about the size of a peptone molecule. This probably

* See Mann, ‘Chemistry of the Proteids,’ pp. 210 and 211.
VOL, LXXIX.—B. N

152 Mr. W. Sutherland. [July 26,

accounts for the powerful toxicity of copper towards those lower plants to
whose protoplasm it gains ready access. |

The large molecular mass of about 40,000 found for globulin accounts for
the fact emphasised by Hardy that the polybasic acids H2SO., HsPO,, ©
combine by molecules and not by equivalents with globulin. The eight
NHp radicles are so widely distributed over the large molecule that only one
can come within the reach of an acid molecule. Thus only one OH radicle
in H.SO, and H3PO, can operate. In the same way, since globulin contains
only two COOH radicles, these are probably far apart, and a divalent atom
like Ba can replace only one H. It would be possible for Ba to unite two

“globulin molecules through a COO radicle in each, and for SO, to unite two
through an NH, radicle in each. Probably the colloidal state of a globulin
suspension is due to the ordinary ineffective doublet of trivalent nitrogen
becoming effective, thus enabling the globulin molecules to grip each other
by means of the eight free NH, radicles in each. Solution in acid occurs
when the acid satisfies the extra valencies of the NHz. radicles, thus
breaking their hold on one another. The semplar G of globulin escapes into
the freedom of solution as part of the molecule G(HCl)s. In edestin,
T. B. Osborne has found what he calls the monohydrochloride insoluble,
while the dihydrochloride is soluble. The explanation of this is that in the
monohydrochloride sufficient junctions amongst the semplars are still left
intact to keep the colloidal monohydrochloride from going into solution.
The equivalent weights, 14,300 to 7000 given by Osborne for edestin, are
1/3 and 1/6 of the 40,000 found above for globulin.

As to solution of colloidal globulin in bases, we have seen that in the case
of Hardy’s ox globulin it was not sufficient to supply merely enough NaOH
to form the easily dissociable globulin salt, but twice as much was required
for complete solution. Here the extra NaOH is required along with OH and
H due to the first half of the NaOH to break the colloidal junctions between
the semplars. How it does this as efficiently as four times the equivalent
HCl forms an interesting chemical question. The probable mode of
operation of neutral salts in dissolving colloidal globulin has already been
discussed.

Summary.

By expressing the experimental results of Hardy and Mellanby in simple
formule, it is shown that the solution of globulin and its precipitation take
place under simple conditions of chemical equilibrium. For example, if p is
the fraction of a globulin suspension dissolved in a salt solution whose
concentration is the fraction g of C that is required just to dissolve the whole

1906. | The Chemistry of Globulin. 153

of the suspension, we get equation (1) p(1—g) = Ag(1—p), in which A is the
ratio of a velocity of solution to a velocity of precipitation. Mellanby’s
discovery of the dependence of M/C on valency and ionic velocity is applied
to MA/C, M being molecular mass, and it is shown that when temperature
varies, not only does MA/C depend upon the viscosity of the solvent water,
but also on a function of temperature given in equation (38) which expresses
the part played by globulin. It is noteworthy that this function has a
minimum value about 40° C., near the temperature of warm-blooded animals.

For the precipitation of globulin by excess of (NH4)SOu, equation (5) 1s
established, namely p(1+p) = 28°8(¢ — 0°152), p being the fraction which
the precipitated globulin is of the whole and ¢ the concentration of the.
(NH,)2SO, solution in grammes per cubic centimetre.

Then follow formule for the remarkable precipitation of globulin by acids
from solution in neutral salts. From these it appears that three compounds
of globulin react in producing this precipitate.

Section 4 is devoted to a theory of the colloidal state, namely, that a
colloid consists of molecules which are chemically united neighbour to
neighbour by the action of valencies which are usually latent. Cases of
multiple valency like that of nitrogen and oxygen are best accounted for by
the electron theory of valence due to Helmholtz, with the assumption that
a single atom can contain both negative and positive electrons. Thus the
nitrogen atom has four negative electrons and one positive. In trivalent
nitrogen the positive electron neutralises one of the negative, so that the
atom has only three effective negative electrons along with an ineffective
doublet. Butif this doublet is opened out, it can link on its atom to two
neighbour atoms, the negative electron grasping a positive, and the positive
a negative.

According to this chemical theory of the colloidal state, the term molecule
ceases to have a useful meaning when applied to a colloid, so the term
semplar is used to name that structure which is repeated like a pattern in
three dimensions through a colloid. By suppression of the colloid-producing
valencies of doublets, a mass of semplars is caused to fall into a collection
of separate molecules. In illustration of the usefulness of this theory, it is
applied to show the dependence of the coagulating power of an ion on its
valence. It is then applied also to explain the remarkable fact that the
amount of globulin dissolved by a given salt solution from a globulin
suspension depends on the concentration of the suspension. The action of
the ions of a neutral salt in dissolving globulin is treated as only another
manifestation of the same electrical effect which enables them to coagulate
arsenious sulphide. This theory of the colloidal state leads to a theory of

N 2

154 The Chemistry of Globulin.

equation (1) and of the laws of A in it, especially that MA/C is proportional
to the sum of the squares of the valencies of the ions of the electrolyte.

In Section 5 the experiments of Hardy on the conductivities of globulin
solutions are expressed by formule which admit of very simple interpreta-
tion, and connect these conductivities with those of ordinary electrolytes.
At infinite dilution the HCl compound of globulin is completely dissociated,
its conductivity being that due to the completely ionised HCl, the part due
to the globulin being then too small to appear. For any other dilution the
amounts of combined and free HCl can be calculated by equations (17) and
(18). In the compounds of NaOH and NH,OH with globulin the con-
ductivities show that half of the base combines with the globulin in a way
which proves it to be an acid, the resulting salt being completely dissociated
and showing the conductivity due to Na or NH, alone, that of the globulinic
acid being too small to appear. The other half of the base which is required
to dissolve globulin completely combines with it in a way similar to that of
HCl, namely, by addition.

In Section 6 globulin is shown to have probably a molecular mass 40,000
and a basicity 2, the alternative being a mass 60,000 and basicity 3.
Further experiments like those of Hardy on the ionic velocity of globulin
and also on the coefficient of diffusion of globulin solutions would decide the
matter, though doubtless various globulins differ in mass and basicity. A
group, CisH2N3O0,, related to polypeptides and peptones is shown to be the
predominant structure in aloumins. The discrepant results of different
experimenters on the precipitation of albumin by heavy metals fall into
harmony when it is proved that they precipitated different integral
numbers of a group such as this in combination with an equivalent of

heavy metal.

155

Observations on the Life-history of Adelea ovata, Aimé Schnerder,
with a Note on a New Gregarine from the Gut of Lithobius
forficatus.

By C. C. DoBELL, B.A., Scholar of Trinity College, Cambridge.

(Communicated by J. J. Lister, F.R.S. Received November 19, 1906,—
Read January 17, 1907.)

[PLATES 2 AND 3.]

At the suggestion of Mr. J. J. Lister, F.R.S., I recently undertook a
reinvestigation of the sporozoan parasites occurring in the gut of the
centipede, Lithobius forficatus. The present paper is the result of these
investigations. It is with pleasure that I take this opportunity of expressing
my thanks for the advice and assistance I have received not only from
Mr. Lister, but also from Mr. Adam Sedgwick, F.R.S., and from Mr. W. S.
Perrin.

No less than six protozoan parasites have been recorded from the gut of
LInthobius forficatus—four Coccidia and two Gregarines. The Cambridge
centipede appears to harbour but a single one of the former—namely,
Adelea ovata, A. Schn.—whilst both the latter* have been observed in rather
less than 1 per cent. of the animals examined. It is with the sexual phases
of the life cycle of A. ovata that the present paper is chiefly concerned.

Previous Work.—Adelea ovata was first described+ by Aimé Schneider
in 1875 [12], and although a part of the sexual process was observed, it was
not recognised as such. Schneider published some further particulars
in 1892 [14]. In 1897 Schaudinn and Siedleckif published a preliminary
account of the Coccidia of the centipede, and later—in 1899—Siedlecki [16]
gave a detailed description of the whole life-history of Adelea ovata.
Subsequent writers have described other species of the genus Adelea, and in
all cases the life cycle appears to correspond closely with that recorded
by Siedlecki. I may say that I am able to confirm most of the results
obtained by this writer, but I have come to different conclusions regarding
one or two points. The most important of these deals with the formation of
the microgametes.

MetuHops.—The best results have been obtained by adopting Schaudinn’s
methods[11]. The entire gut was removed, and the epithelial cells and gut-
cgntents spread out upon a coverslip. The films thus obtained were

* Viz., Actinocephalus dujardini, A. Schn., and Hchinomera hispida, A. Schn.

t As a gregarine.
{ Schaudinn and Siedlecki, ‘Sitz. ber. d. d. zool. Gesell.,’ 1897.

156 Mr. C. C. Dobell. Observations on the [Nov. 19,

instantly fixed by immersion in hot sublimate-alcohol containing a trace of
acetic acid (Schaudinn). After fixation the films were treated with alcohol
containing iodine, and stained in Biitschli’s modification of Delafield’s
hematoxylin.* This I prepare by adding 1-per-cent. acetic acid solution to
a 0-5-per-cent. solution of the ordinary concentrated Delafield’s hematoxylin
in water until a pink colour is produced. Staining, for all stages except
spores, is complete in from 15 to 30 hours.

Giemsa’s modification of the Romanowsky-Nocht stain was tried, among
others, but it proved to be of little use for delicate nuclear structures, owing
to the drying which necessarily takes place.

Transverse sections of the gut were made, but they were found to be
unsatisfactory. Moist films are far more useful for examining the Coccidia.

I will now proceed to describe the stages which I have observed and which
differ from those hitherto recorded.

(1) AssoctaTion.—The entire genus Adelea is characterised by a precocious
association of the gametocytes. This was observed in A. ovata as long ago
as 1875, although, strangely enough, it was not even suspected of being
connected with any sexual process. Association, it may be noted, takes
place in the lumen of the gut, and not in the epithelium. Siedlecki, who
ultimately interpreted this phase correctly, states that but a single micro-
gametocyte ever attaches itself to a macrogametocyte. Only once did he
observe two microgametocytes attached to the same female individual. My
observations, on the contrary, lead me to believe that this condition is not
uncommon. I have seen upwards of a dozen instances in which two micro-
gametocytes were attached to the same macrogametocyte. Apparently,
these microgametocytes do not always develop or become attached
simultaneously, for they may be found in different stages of maturity
(cf. Plate 3, fig. 3). Association in which more than one microgametocyte
takes part has been described by Laveranf in the closely allied form Klossia
helicina.

An association of two microgametocytes with one another as described
in A. mesnili (Pérez) and A. zonula (Moroff), has never come under my
observation.

(2) FoRMATION OF MicroGAMETES.—The only detailed account of the
genesis of the male gametes of A. ovata is that given by Siedlecki. He
states that the nucleus of the microgametocyte divides into two daughter-
nuclei, each of which then divides in a plane at right angles to that of the
first division, so that four nuclei result. These nuclei then develop into the

* Biitschli, ‘Zeitschr. f. wiss. Mik.,’ ix, 2, 1892, p. 197.
t Laveran, ‘C. R. Soc. Biol. Paris,’ 1898 (Novembre).

1906.] Life-history of Adelea ovata, Avmé Schneider. 157

four microgametes, one of which is destined to fertilise the macrogamete ;
the remaining three perish. In the first nuclear division it was believed
that the quantity of chromatin was reduced, whereas the number of chromo-
somes was reduced in the second division. I am convinced that this is not
: a correct interpretation of this particular gametogenesis ; and the inference
that the maturation of these gametes approaches the metazoan type is, to my
mind, quite unjustifiable.

To follow the formation of the male gametes from the beginning, it is
necessary to describe in detail a merozoite of the male type. Such a
merozoite is shown in the accompanying figure (text-fig. 1).

It is seen to consist of a somewhat fusiform (or sometimes falciform) body,
containing a nucleus (n.) and a large peripheral karyosome (4.). Sometimes
two or even three karyosomes are present, but the usual number is one.
Occasionally it appears to be extranuclear. At one end of the merozoite—
usually that remote from the karyosome—a number of granules is usually,
though not always, present. These are the so-called “ pigment granules ”
(p. gr.). Iam inclined to believe that they are really chromatin granules.
They stain deeply with hematoxylin, and are coloured red by Giemsa’s stain.
They are never present in macromerozoites.

a) 10.
Fic. 2.—Semi-diagrammatic Representation of Formation of Microgametes. The
macrogametocyte is not shown.

/

158 Mr. C. C. Dobell. Observations on the [Noy. 19,

A micromerozoite, such as that just described, may increase in size and
become a microgametocyte (text-fig. 2 (1)). It then becomes attached to
a macrogametocyte in the usual manner. During its transformation into
a microgametocyte, the karyosome travels to the centre of the nucleus,
and is apparently reinforced by the nuclear chromatin. <A part of this
latter, however, is usually to be found lying free in the clear nucleus in the
form of granules.

The next step towards the formation of the microgametes consists in
a dispersal of the karyosome and the formation of a chromatic network in
the nucleus. This is shown in (2) text-fig. 2, and may take place before
association occurs. Further breaking up* of the karyosome now ensues—
provided that association has taken place—the chromatin particles invading
the cytoplasm and there forming a chromidialt net (3). When the
karyosome is completely broken up, and the outline of the nucleus has
completely disappeared, the microgametocyte is seen to contain a chromidial
net completely filling it from end to end (4). The foregoing stages (1 to 4)
may be conveniently termed the analytic phase. Those which follow
constitute what I may call the synthetic phase, in which four new nuclei
are built up from the chromatic elements of the old nucleus. Synthesis
begins with the gathering up of the chromidial net into a more or less
rounded mass (5) which soon becomes constricted in the middle, giving rise
to a dumb-bell shaped reticulum (6). If deeply stained, it has the appear-
ance of a dividing nucleus. The ends of the dumb-bell now extend
transversely, and the chromatin becomes localised at the four poles so
formed (7). At the same time, the intermediate portion becomes diffuse ;
it finally breaks up, undergoes degeneration, and is left behind—with a
small quantity of pigment—in the residuum of the microgametocyte
when the microgametes forsake it.

Four special points, we have seen, have now become centres for the
ageregation of chromatin. Chromatin continues to travel to each point, and
at first arranges itself in the form of a loose net. The net then resolves

* The exact manner in which the breaking up of the karyosome takes place is unknown
tome. I have never been able to watch the process in the living animal. That breaking
up does occur is inferred from the fact that the karyosome becomes smaller and smaller,
whilst the chromatin granules increase in number.

t+ See, further, under “Chromidia,” next page.

t I should like to mention here the fact that published figures purporting to show two
nuclei in the microgametocyte really show what I believe to be the stage just described.
All the figures given by Siedlecki show two “nuclei” connected together—in no case are
they completely separate. Siedlecki’s ‘“corpuscule intermédiaire” is,-I believe, a more
deeply stained portion of the intervening network.

1906.]. Life-history of Adelea ovata, Avmé Schneider. 159

itself inte a stellate mass of chromatin. Hence the highly-characteristic
figure of a microgametocyte containing four star-shaped nuclei is presented (8).
The stars finally become rounded off (9)—the spherical nuclei so formed
afterwards elongating to form the microgametes, which apparently consist
entirely of chromatin (“chromatozoites ”) (10—11). Many of the micro-
gametes enclose a clear space resembling the vacuole described by Schaudinn
in Coccidium schubergi. A mature microgamete is shown on a larger scale
in text-fig. 2 (12).

It will be seen from the foregoing that my account of the formation of the
male gametes differs considerably from that of previous writers.

It now becomes an interesting point to determine whether a similar
chromidial condition occurs at any period in the life-history of the female
forms. So far, I have been unable to reach any satisfactory conclusion with
regard to this. However, the following observations seem worth recording
in this connection :—

(a) A single instance has occurred in which the individuals of a group of
macromerozoites were found to have their nuclei in a chromidial condition
(see Plate 3, fig. 6).

(5) Since the stellate nuclei of the microgametocyte are formed by
synthesis from chromidia, it is possible that the stellate nuclei of the macro-
schizont may have a similar origin. Although, up to the present, I have not
been able to demonstrate that this is the case, nevertheless, 1 think that the
macroschizont, of which fig. 5, Plate 3, is an exact copy, probably shows a
stage in which neuclei are being synthesised by the concentration of
chromidia at various points. It should be noticed that a considerable
quantity of chromatin is scattered through the cytoplasm. Iam, of course,
ready to admit that this may be quite a wrong interpretation of the
appearances. Possibly they are due to degenerative changes, Nevertheless,
the phenomenon of chromidia-formation has assumed such importance, owing
to recent work in other groups of the Protozoa, that I believe the conditions
described may be found to be not altogether insignificant.

Chromidia.—So many new facts regarding chromidial structures have
recently come to light, and so little notice has been taken of them in
England, that I think a few words on chromidia in general may not be out
of place here. As it is obviously impossible to treat the subject fully in the
present paper, I have given a short list of papers bearing on the subject in
the literature list (B) at the end; for a more detailed list of references
I refer the reader to the writings of Hertwig [21] and Goldschmidt [18, 19].

The conception of chromidia was first formulated by Richard Hertwig in
1902 [20], and resulted from his observations on Actinospherium, Arcella, etc.

160 Mr. C. C. Dobell. Observations on the [Nov. 19,

Since then, many other “chromidial” nuclear phenomena have been
described. Indeed, it is now a matter of some difficulty to give a definition
of chromidia which will embrace all conditions so described. In its widest
sense, the term is applied to all chromatin substance (2.¢., all matter which
takes up nuclear stains) which lies within the cell and which is not gathered
together in the form of a definite nucleus. Chromatin granules of diverse
shapes and sizes, strands, networks—all these come in this category; and,
moreover, the physiology of these structures seems to be no less heterogeneous
than their morphology. For chromidia seem to be formed sometimes as the
result of degeneration, sometimes as a form of nuclear division, sometimes as
katabolic products of nuclear activity—sometimes (as in bacteria), being the
normal vegetative stage of the neucleus—and so on.

Many new terms have been introduced for these various forms of
chromidia. Since many of these have not received general acceptance, and
since many are, I think, quite unjustifiable, owing to our present ignorance
of the causes underlying the different phenomena, I have used but two terms
in my description of the nuclear changes in A. ovata,— chromidia” for
separate chromatin granules lying in the cytoplasm, and “chromidial net” for
a collection of chromatin granules united by a reticulum. Amongst the
terms which are more frequently met with in papers on this subject the
following may be mentioned:—The name “ Sporetiwm” (Goldschmidt) has
been given to chromidial structures formed previous to gamete formation.
“ Protogonoplasm ” (Calkins) and “ Jdiochromidium” (Goldschmidt) are other
terms applied to the same structures. Then, again, we have such terms as
“ Trophochronidium” and “ Chromidium” (sensu stricto) applied by Gold-
schmidt to chromidial structures of a vegetative (as opposed to reproductive)
nature. Other terms, such as “ distributed nucleus ” (Calkins), etc., require
no special explanation.

A chromidial condition of the nucleus may occur, apparently, not only in
all the chief subdivisions of the Protozoa, but also in some Metazoa and in
Bacteria.

The great interest which attaches to chromidia arises from the fact that
investigation of these structures has revealed a “ Doppelkernigkeit,” or
nuclear dimorphism, in many Protozoa. That is to say, two distinct elements
can be recognised in many protozoan nuclei—one, a “ propagative” element,
the other, a “somatic.” The propagative seems specially liable to separate
from the somatic as chromidia (¢f Schaudinn, Neresheimer, etc.). The
heterokaryote condition of the Ciliata thus finds a parallel in many other
Protozoa. To the single nucleus, combining the chromatic elements proper
both to the soma and reproduction, is given the name “ amphinucleus.”

1906.] Jufe-history of Adelea ovata, Aimé Schneider. 161

From the foregoing remarks, I think it will be evident that the subject of

chromidia is one of great importance, but not one on which we are at present,

owing to our lack of knowledge, able to dogmatise. I will conclude by
quoting Mesnil. “Il faut,’ says this writer, “bien se garder de trop
généraliser, et chaque cas ou le protoplasme se charge de granulations
chromatiques doit étre étudié avec soin a ce nouveau point de vue.”

Note on a New Gregarine from the Gut of Lithobius forficatus.

Whilst examining centipedes for stages in the life-history of A. ovata,
I came across the trophozoites of a gregarine which, I think, has not. been
recorded hitherto. The trophozoites were very numerous, but no spores or
other stages could be found. Hence it is at present impossible to refer the
animal to its proper systematic position.

Only one centipede which I examined contained these gregarines, but in
this particular case they were very numerous. The Lithobius infected was in
a dying condition just before I examined it, possibly owing to the harmful
effects of the parasite.

The gregarine has an exceedingly simple structure (see fig. 3). There is

a aie ee

Fig. 3.

no epimerite, and the body is non-septate and usually slightly more pointed
at one end than at the other. A large nucleus containing a big karyosome is
‘situated more or less in the middle of the animal: its position varies. The
average length is 127 w, breadth 484. The measurements were made from
permanent preparations.

LITERATURE.

(A.) Relating to Adelea.

1. Biitschli, O. “Kleine Beitrige zur Kenntniss der Gregarinen: III, Uber eine
__ eiformige Psorospermie aus dem Darm des Lathobius forficatus” (‘Zeitschr. f.
wiss, Zool.,’ vol. 35, 1881).
2. Dofiein, F, « Die Protozoen als Parasiten u. Krankheitserreger, etc. ” Jena, 1901.
3. Labbé, A. “ Recherches escent ues, cytologiques et biologiques sur He Coccidies ”
(‘ Arch, de Zool. exp. et gén.,’ vol. 4, 1896).

4 Art. “Sporozoa,” in ‘ Das Tierreich,’ Lief. 5, 1899.

162 Mr. C. C. Dobell. Observations on the [ Nov. 19,

5. Léger, L. “Sur la morphologie et le developpement des Microgamétes des Coccidies ”

6.

Lyf.

18.

19.
20.

21.

22.

23.

24,
25.

26.

27.

28.

(‘ Arch. de Zool. exp. et gén., Notes et Revue,’ vol. 6, 1898).

“Essai sur la Classification des Coccidies et description de quelques espéces.
nouvelles ou peu connues” (‘Ann. de Mus. de Hist. Nat. Marseille-—Bulletin,”
vol. 1, fasc. 1, 1898).

‘““Sporozoaires parasites de ?PHmbia soliert, Rambur ” (‘ Arch. f. Protistenk.,”
vol. 3, 1904).

. Minchin, E. A. Art. “Sporozoa,” in Lankester’s ‘Treatise on Zoology,’ Part I,

fasc. 2, 1903.

. Moroff, T. “ Untersuchung tiber Coccidien. I. Adelea zonula, nov. sp.” (‘ Arch. f.

Protistenk.,’ vol. 8, pt. 1, 1906).

. Pérez, C. “Le cycle évolutif de PAdelea mesnili, coccidie ccelomique, parasite d’un

Lepidoptére” (‘ Arch. f. Protistenk.,’ vol. 2, 1903).

. Schaudinn, F. “ Untersuchungen tiber den Generationswechsel bei Coccidien” (‘ Zool.

Jahrb., Abth. f. Anat.,’ vol. 13, 1900).

. Schneider, Aimé. ‘Contributions a histoire des Grégarines des invertébres de Paris.

et de Roscoff” (‘ Arch. de Zool. exp. et gén.,’ vol. 4, 1875).
“‘ Coccidies nouvelles ou peu connues” (‘Tabl. Zool.,’ vol. 1, 1880).
“ Coccidies nouvelles, ete.” (Tabl. Zool.,’ vol. 2, 1892).

. Siedlecki, M. “Etude cytologique et cycle auolutt Ae la Coccidie de la Seiche ”

(‘ Ann. de l’Inst. Past.,’ vol. 12, 1898).
“Etude Srlosiane et cycle évolutif de Adelea ovata, Schneider” (‘ Ann. de
VInst. Past.,’ vol. 13, 1899).

(B.) Relating to Chromidia.

Calkins, G. N. ‘“ Evidences of a Sexual-cycle in the Life-history of Ameba proteus”
(‘ Arch. f. Protistenk.,’ vol. 5, 1905).

Goldschmidt, R. ‘“ Der Chromidialapparat lebhaft funktionierender Gewebszellen ”
(‘ Zool. Jahrb., Abth. f. Anat.,’ vol. 21, 1904).

“Die Chromidien der Protozoen” (‘ Arch. f. Protistenk.,’ vol. 5, 1905).

Gonder, R. “ Beitrage zur Kenntnis d. Kernverhaltnisse bei den in Cephalopodem
schmarotzenden Infusorien ” (‘ Arch. f. Protistenk.,’ vol. 5, 1905).

Hertwig, R. “Die Protozoen und die Zelltheorie” (‘ Arch. f. Protistenk.,’ vol. 1,
1902).

“Uber physiologische Degeneration bei Actinospherium eichhorni”
(‘Festschr. f. E. Hackel,’ 1904).

Lister, J. J. “The Life-history of the Foraminifera” (‘ Presidential Address, Sec. D,
Brit. Assoc.,’ 1906).

Mesnil, F. ‘“ Chromidies et questions connexes” (‘ Bull. Inst. Pasteur,’ vol. 3, 1905).

Neresheimer, E. “Die Zeugungskreis v. Opalina” (‘Sitzungsb. d. Gesell. f. Morph.
u. Physiol. in Miinchen,’ Part 1, 1906).

Schaudinn, F. “ Beitrag zur Kenntnis der Bakterien u. verwandter Organismen ”—
1. Bacterium butschlit (‘ Arch. f. Protistenk.,’ vol. 1, 1902); 2. Bacillus sporonema
(loc. cit., vol. 2, 1903).

: fornceecuce: tiber d. ranean einiger Rhizopoden” (‘ Arb. aus d. k.
Gesundheitsamte,’ vol. 19, pt. 3, 1903).

Ziilzer, M. “Beitrag zur Kenntnis von Diflugia urceolata, Carter” (‘ Arch. f.
Protistenk.,’ vol. 4, 1904).

Dobetl.

Ese

eo Le
FA. te

1906.| Life-history of Adelea ovata, Aimé Schneider. 163

DESCRIPTION OF PLATES.

[ All figures are drawn from permanent preparations stained with hematoxylin. Drawings
made under a 3-mm. apochromatic objective (Zeiss) with compensating-ocular 12.]

PLATE 2.

Fic. 1.—Microgametocyte associating with macrogametocyte (lying free in gut). Karyo-
some of microgametocyte has partially broken up to form a chromidial net.

Fig. 2.—In this figure the chromidial net is seen distributed throughout the whole of the
microgametocyte.

Fic. 3.—The chromidial net of the microgametocyte has become gathered together into a
dumbbell-shaped network.

Fic. 4.—Stage in formation of four nuclei. The developing nuclei are still connected
with the chromidial net.

Fie. 5.—The four nuclei are showing signs of becoming separated as star-shaped masses
of chromatin. Part of the network is still visible.

PLATE 3.

Fig. 1.—Association of two microgametocytes with a single macrogametocyte. A
chromidial net is seen in both the former.

Fig. 2.—Association of two microgametocytes (each containing four characteristic star-
shaped nuclei) with one macrogametocyte.

' Fie. 3.—Two microgametocytes associating with one macrogametocyte. In one, four
nearly ripe microgametes are seen; in the other, a dumbbell-shaped network is
present.

Fic. 4.—Four mature male gametes formed. A chromatic spindle is seen in the
macrogamete.

Fie. 5.—Macroschizont, the nuclei in the form of networks. Numerous chromatin
particles are scattered through the cytoplasm.

Fic. 6.—Group of macromerozoites with nuclei in a chromidial condition.

164

The Natural and Induced Resistance of Mice to the Growth of

Cancer.
By E. F. Basurorp, M.D., J. A. MurRAyY, M.B., and W. Cramer, Ph.D.

(Communicated by Dr. J. Rose Bradford, F.R.S. Received December 10, 1906,—
Read January 17, 1907.)

In the present paper we propose to give an account of experiments
conducted during the past three years on the means whereby mice may be
completely protected against the inoculation of transplantable carcinomata,
which grow readily in normal mice. At the same time we shall show that
these experiments throw fresh light on the nature of cancer. The Executive
Committee and the Pathological Sub-Committee of the Imperial Cancer
Research Fund have been informed of the progress of these investigations,
and some provisional results have also been laid before the General Com-
mittee at the Annual Meetings. The experiments are being continued,
but some of the results which have accumulated, make it advisable to give
an account of the stage at which they have now arrived. They are based
mainly on a study of 23 transplantable carcinomata of the mamma of the
mouse, and of other malignant new growths which could not be propagated
artificially.

In these experiments we have used, for the most part, the growth of
Jensen’s tumour in normal animals to bring out the changes induced in
protected animals and the refractoriness of insusceptible animals; but we
shall also employ another tumour (XX VII) of different histology, which
grows equally well under artificial propagation. We have employed Jensen’s
tumour as a standard for the following reasons :—(1) It was the only tumour
used to control some of our earlier observations ; (2) With proper precautions
it gives regularly 85 to 100 per cent. of successful inoculations in a large
number of animals; (3) Within ten days the inoculation of 0°01 to
0°02 gramme of tumour tissue gives large easily recognisable tumours, often
1°5 grammes in weight; (4) We have fully demonstrated that it may produce
large metastases, that it may extend by the blood or lymphatic streams, and
behave under experimental conditions typically as a malignant new growth ;
(5) Its rate of growth is not exceeded by any mouse tumour now being
propagated ; (6) It is in the hands of most investigators throughout the
world, who will be able to repeat our observations. It therefore fulfils
better than any other tumour the requirements of such a standard.

Resistance of Mice to the Growth of Cancer. 165

Historical.

Jensen stated in papers appearing in 1901, 1902, and 1903* that he had
observed the complete disappearance of tumours from mice that had been
inoculated successfully. He added, however, that the “results of attempts
to cure mice of tumours have hitherto been uncertain; at times they have
been positive to an unexpected extent, at other times they have been
entirely negative.” For this reason he postponed the detailed discussion
of his experiments till he had come to a clearer understanding of their
significance, and to more reliable results. Referring to the fact that
_ 40 to 50 per cent. of the total inoculations of his tumour had _ been
unsuccessful, Jensen interpreted this to mean that the mice were protected
by some natural means. It seemed to him impossible to explain the fact
that a tumour which was growing well at the time of its removal from one
mouse should be unable to grow in other mice, except on the assumption
that the tumour had been transferred to a more refractory group of mice ;
but he stated, “it appears that the nature of the inoculated tissue is of no
little importance for the success attained. So far as it is possible to form
an estimate, the results appear less certain when small young tumours are
used which are actively growing, and the percentage of successful inoculations
is higher if the healthy portions be selected from older and larger tumours.”
Jensen especially pointed out that mice which had been unsuccessfully
inoculated at the first essay were also refractory to subsequent inoculations ;
but he was careful to point out that it would be a mistake to assert that
the second negative result was solely the consequence of natural refractoriness,
since it would be wrong to assume that the material inoculated and absorbed
in the first instance had not contributed to the exemption subsequently
exhibited.

Borrel} drew attention to the difficulties attending primary transplantation
and recorded suggestive experiments directed to obtaining a polyvalent
immune serum against human cancer, on the assumption that distinct sera
might be necessary for tumours of different histological types.

In November, 1903, we were able to add the study of Jensen’s tumour
to that of tumours discovered by ourselves, and we at once began investiga-
tions along the lines indicated by Jensen. In our earlier experiments, the

* “Nogle Fors¢g med Kraeftsvulster,” Lecture, December, 1901; “ Forsgg med
Musecancer,” Lecture, April, 1902, ‘ Biolog. Selskabs Forhandlinger, K6benhavn,’

1901—02 ; “ Experimentelle Untersuchungen iiber Krebs bei Mausen,” ‘Centr. f. Bakt.,’
vol. 34, 1903.

+ “ Epithélioses infectieuses et Epithéliomas,” ‘Annales de l’Inst. Pasteur,’ vol. 11,
1903 ; ‘ Roy. Soc. Proc.,’ vol. 73, 1904.

166 Dr. Bashford, Mr. Murray, and Dr. Cramer. [Dec. 10,

successful inoculation of Jensen’s tumour fluctuated between 20 and 90 per
cent. of the number of animals inoculated.

In a series of papers* we have drawn attention to the greater susceptibility
of young than old mice, to the important influence on the success of trans-
plantation, of the character and phase of the tumours inoculated, the dosage,
and site of inoculation. In the course of a careful study of the process of
absorption of tumours after exposure to radium, we showed its identity
with localised changes in the majority of propagated tumours, changes
which were found to have become general in the only case we had then
noted where a tumour of three weeks’ growth and of 1 cm. diameter had
actually diminished in size. While recognising the importance of
hemorrhage as an integral part of the process of absorption both when
spontaneous and following exposure to radium, we pointed out that the phase
of growth of the tumour was also of great and probably decisive importance.
These observations harmonised well with Jensen’s experience, and tended
to explain the irregularities in the disappearance of tumours under his
observation. Our position was summarised as follows :—“ No conclusion has
yet been arrived at as to what are the essential and what the subsidiary
features in this prolonged proliferation. The variations make it difficult to
correlate different observations, and to be sure of their proper interpretation.
Efforts to influence growth by immune (cytolytic) serum and other means
have, in consequence, not yet led to any definite conclusion.”

Clowes,t working also with Jensen’s tumour, obtained 30 per cent. of
success in normal animals. He recorded spontaneous absorption in from
15—20 per cent. of his transplanted tumours. The animals in which
spontaneous absorption had occurred were stated to possess a specific anti-
body in their serum, which, on injection into animals with transplanted
tumours, exerted a curative effect. In only one case out of 20 mice so
treated had the beneficial influence of the immune serum been missed. In
view of the low percentage of success obtained, the slow development of the

* First and Second Scientific Reports of Imperial Caneer Research Fund, 1904, 1905 ;
‘ Roy. Soc. Proc.,’ B, vol. 78, 1906.

+ We recorded the precautions we had taken in searching for tumours undergoing
spontaneous absorption. Clowes has taken this criticism of our own procedure as imply-
ing that in our opinion his “spontaneous recoveries were ulcerations” (‘ Brit. Med.
Journ.,’ December 1, 1906). Regression of inflammatory swellings may take place without
ulceration, and our remarks apply to our own doubtful “tumours” of less than 14 days’
growth, within which time Clowes made no mention of tumours having appeared.

{ “Preliminary Communication regarding an Immune Body Capable of Inhibiting the
Development of Cancer in Mice,” ‘Johns Hopkins Hosp. Bull.,’ April, 1905 ; “ Further
Evidence of Immunity against Cancer in Mice after Spontaneous Recovery,” Clowes and
Baeslack, ‘ Medical News,’ November 18, 1905.

1906.] Resistance.of Mice to the Growth of Cancer. 167

tumours, and Jensen’s and our own experience, we then expressed scepticism
of the curative results claimed by Clowes. In his later papers these results
are quoted, but a repetition of the positive results has not been recorded.
Evidence is adduced to show that the animals which have recovered
spontaneously cannot be successfully inoculated. This is regarded as an
action of anti-bodies. The presence of anti-bodies is further supported by
experiments in which tumour material had been exposed, in the test-tube, to
the action of the serum of spontaneously cured mice. On inoculation, such
material gave 12 per cent. of success, while material similarly treated with
normal mouse serum gave 31 per cent. These three sets of observations are
progressively evidence of a more limited action of the reputed immune sera.
The age of the animals used is not stated. In March, 1906, Ehrlich*
remarks with reference to these experiments: “So berichtet Clowes, dass
Miause, bei denen Tumoren spontan zur Resorption gelangen, in ihrem Serum
carcinomfeindliche Stoffe enthalten. Allerdings lassen sich gegen die
Deutung dieser Versuche mancherlei Einwendungen machen.” Other papers,
in which these and other observations are used as evidence of the infective
nature of cancer, have also been published by Clowes, Gaylord, and Baeslack,
from the Buffalo Cancer Laboratory, U.S.A.

Michaelis} obtained success in the same proportion as in normal animals,
on inoculating mice previously treated with tumour material killed by
chloroform and other chemical agents.

Ehrlich§ found that from 60 to 90 per cent. of mice inoculated unsuccess-
fully with spontaneous (especially hemorrhagic) tumours were refractory
on subsequent inoculation with a tumour giving 75 to 100 per cent. of success
in normal animals. Further, animals unsuccessfully inoculated with a
tumour giving nearly maximal percentage of success in normal mice, were
highly refractory to subsequent inoculation. This resistance was manifest
whether the second inoculations were made with the same or other tumours
(pan-immunity). Animals already successfully inoculated with a quickly
growing tumour could not again be inoculated with a rapidly growing tumour

* “ Experimentelle Carcinomstudien an Mausen,” ‘Arbeiten a. d. Kgl. Inst. f. exp.
Therapie,’ 1906.

t “On Spontaneous Cure of Cancer,” ‘Surgery, Gynecology, and Obstetrics,’ June,
1906; “Incubation of Mouse Tumours,” ‘Journ. of Exp. Medicine,’ August, 1906 ;
“ A Study of the Influence Exerted by a Variety of Physical and Chemical Forces on the

Virulence of Cancer in Mice,” ‘ Brit. Med. Journ.,’ December 1, 1906 ; ‘ Evidences that
Infected Cages are the Source of Spontaneous Cancer developing among Small Caged
Animals,” zbid. ;
_ f ‘Zeitschr. f. Krebsforschung,’ vol. 4, 1906.
§ “Experimentelle Carcinomstudien an Mausen,” ‘ Zeitschr. f. arzl. Fortbildung,’
1906, and Joc. cit.

VOL. LXXIX.—B. : O

168 Dr. Bashford, Mr. Murray, and Dr. Cramer. [Dec. 10,

(atreptic immunity): the tumour already established withdraws special
nutritive substances (substance X) from the circulation, so that the new
graft cannot grow. A similar explanation is invoked for the rarity, or small
size, of metastasis In mice with large transplanted growths. Repeated
inoculation of negative mice leads to a very refractory condition in the
animals which never develop tumours.

Rats can be successfully inoculated with rapidly growing mouse tumours.
Growth proceeds for a time and then ceases (atrepsie), and the animals are
then refractory to further inuvculation (active immunity).

Summary of the Present Investigation.

After discussing :—

1. The distinction to be drawn between the conditions of origin of a
sporadic tumour and the conditions of growth of cancer in mice, and its
bearing on experimental investigations.

2. The importance of natural variations in the resistance of mice to
inoculation and of inherent variations in the energy of growth of the
tumour cells.

We shall adduce evidence that :—

3. Mice in which a growing carcinoma has been spontaneously absorbed
may be completely protected against subsequent inoculation of the same
growth, and to a lesser extent against other and different growths.
Similarly, protection follows absorption of tumours after exposure to radium.

4, Protection may be induced, or, when naturally present, enhanced by
the inoculation of tumour material when no growth has resulted. This
protection is specific, and has only been induced in mice by the inoculation
of mouse tumours, and not by those of strange species. This specificity has
an important bearing on the nature of cancer.

5. Protection can also be induced by the inoculation of normal mouse
tissues, and particularly by the inoculation of blood.

We shall discuss the probable nature of the protection conferred ; its action
by means of the body fluids, at any rate in part, leading to the death of the
cancer cells after short sojourn in the body of protected animals.

(1) THE DISTINCTION TO BE DRAWN BETWEEN THE CONDITIONS OF ORIGIN
OF A SPORADIC TUMOUR AND THE CONDITIONS OF GROWTH OF CANCER
IN MICE, AND ITS BEARING ON EXPERIMENTAL INVESTIGATIONS.

When the energy of growth of the cells of a propagable tumour is great
and a high percentage of all inoculations are successful, the variations in the

1906.] Resistance of Mice to the Growth of Cancer. 169

natural resistance of the mice become of subsidiary importance. On the
contrary, they are of great practical importance when the energy of growth is
low, and in the primary transplantations of sporadic tumours. As we have
shown, the growth of a sporadic tumour is probably to be regarded as
heterogeneous. Growth is proceeding rapidly in one part, and slowly or
actually ceasing in another. When a sporadic tumour is transplanted into a
large number of mice, according to our procedure, it is subdivided into
minute fragments, and not made into a general emulsion. Each fragment is
- separately emulsified before transplantation, and the result of transplantation
is that the tumour is distributed over a large number of animals. Success, as
a rule, follows only in a small proportion of the inoculations, and it appears
probable that it is determined by the segregation of groups of cells of higher
energy of growth in mice whose resistance to inoculation is below the
average. The energy of growth of the tumour cells and the resistance of the
mice together contribute to the positive result. In this way the cells most
likely to continue to grow are separated and, by the repetition of the process,
a tumour is propagated by transplantation from one series of mice to another.
We may illustrate this by a summary of the transplantation of some sporadic
tumours.

We have found that malignant new growths are encountered in mice of all
ages taken at random in a proportion of one tumour in 3500 animals. The
final results of transplanting 32 spontaneous tumours of the mamma show
that 22'78 of the mice inoculated survived a sufficient time* to permit of a
final estimation, and 72 tumours have developed, 2.¢., one inoculation in 31°1
is successful, or 3°2 per cent. The conditions for successful transplantation
are at least 100 times more frequent in mice than spontaneous tumours.

Fifteen out of 32 sporadic tumours transplanted gave negative results on
1073 inoculations. Many of these tumours were apparently very rapid in
their rate of growth in the animal spontaneously affected, and extensive
metastasis was present in the lungs after death.

Ehrlich records similar results on primary transplantation of 94 spontaneous
tumours: 1504 inoculations were made and 41 tumours were obtained in the
inoculated animals (2°8 per cent.). The percentage of success (82 per cent.)
of the primary transplantation of the tumours (14 in number) which gave
positive results at all, was much higher than in our experiments. Apparently
our method of primary transplantation by small grafts in a large number of
young animals, gives success with tumours which would be completely

* The results of the primary transplantation of sporadic tumours have been calculated
on the number of mice surviving three weeks later.

0 2

170 Dr. Bashford, Mr. Murray, and Dr. Cramer. [| Dec. 10,

negative when propagated in the way he has adopted (large doses and smaller
number of animals).

The following experiments were undertaken because of the difficulties met

with in obtaining a positive result in the primary transplantations of
spontaneous tumours. It was of importance to determine whether the
spontaneously affected animals were more suitable for transplantation, both of
their own tumours (on the analogy of metastasis) and of other spontaneous
tumours. The difficulty of transplanting many mouse tumours, especially
those with extensive hemorrhage, has led observers who had failed to trans-
plant them to conclude that those tumours were of low “virulence.” We
had already succeeded* in three instances where others had failed in trans-
planting these hemorrhagic tumours, the daughter tumours being also
hemorrhagic, and had observed that they produced extensive metastasis in
the animals primarily affected. We were, therefore, of the opinion that it
was wrong to conclude from the difficulty of transplantation that they were
of low virulence.
_ We have prolonged the lives of mice presenting spontaneous tumours by
removing the primary growth by operation, wholly or partially. This has
permitted us to study their suitability for transplantation, both for their own
and for other spontaneous tumours, and to compare it with the suitability of
normal animals.

The opportunities for such experiments are rare. Up to the present
13 animals presenting spontaneous tumours have been inoculated with their
own and other spontaneous tumours. The results for 11 of these animals are
now available, and are as follows :-—

Two mice (XXXIIT and XXXIV) could be successfully inoculated with -
their own tumours, and in the case of one of them (X XXIII) three normal
animals also developed tumours out of 97 inoculated and surviving three weeks
after inoculation. In the case of the second mouse, her tumour gave negative
results in 140 normal animals, although successfully inoculated into herself.

A third spontaneous tumour (XXXII) gave four tumours in 156 normal
animals, was negative in the mouse spontaneously affected, but positive in
another mouse (XXXIIT) with spontaneous cancer. A fourth spontaneous
tumour (XL) inoculated into 49 normal animals, failed to grow and was like-
wise negative in two mice with spontaneous cancer (XX XVII and XX XVIII).
A fifth spontaneous tumour (XXX) gave one tumour in 72 inoculations into
normal animals, but failed to grow in three other mice (XXV, XXVII and
XXVIII) spontaneously suffering from cancer, as well as in the mouse herself.
Three inoculations were made in each of these four mice.

* Second Scientific Report, 1905, Part II, pp. 19 and 30.

aan.

1906. | Resistance of Mice to the Growth of Cancer. 171

A mouse which had been repeatedly inoculated unsuccessfully with Jensen’s
tumour developed a spontaneous growth of different histological type
(XXXVIII) seven months later. This tumour, transplanted into 86 normal
animals, gave rise to 20 tumours, of which 18, after attaining a diameter of
5 mm. and over, disappeared spontaneously. The mouse in which this tumour
had developed remained protected against Jensen’s and refractory to four
Spontaneous tumours.

The experiments are very few in number, and only tentative conclusions
can at present be drawn from them.

Five spontaneous tumours were transplanted into 11 mice in all,
spontaneously affected with cancer. The results above recorded show that
transplantation does not reveal conditions exceptionally favourable for growth
in them, as compared with normal animals. The animal itself is only
exceptionally successfully inoculated.

Bearing in mind the small number of spontaneously affected animals
inoculated as compared with the large numbers of normal animals, these
experiments show that spontaneously affected animals are not greatly more
susceptible to cancerous inoculation than normal animals.* Tumours which
do not grow in normal animals rarely grow when transferred to other parts of
the animal’s own body, and not at all in other spontaneously affected animals.

These facts, together with the development of a tumour in a mouse pre-
viously inoculated unsuccessfully with Jensen’s tumour, further emphasise
the distinction to be drawn between the conditions of origin and the
conditions of growth. They also seem to render superfluous any subsidiary
assumption of a constitutional condition as necessary in addition to the
primary cancerous transformation in the development of spontaneous cancer.t

* The results of the above experiments might, on superficial examination, appear to
justify the opposite conclusion to that drawn by us. The tumours of mice XX XIII and
XXXIV were transplanted successfully in those mice themselves in single experiments,
as against only three successes out of 99 attempts in normal animals in the one case, and
with negative results in 176 attempts in the other. Of all the sporadic tumours trans-
planted into other mice suffering naturally from cancer, XXXII alone grew in a single
experiment, giving at the same time only four successes out of 156 attempts in normal
mice. It would thus appear that 100 per cent. of success had been attained on three
separate occasions when transplantation was effected into mice naturally suffering from
cancer, as against very low percentages in normal mice. The results speak for a greater
suitability for inoculation on the part of individual mice, but not for a greater suitability
on the part of all mice which are naturally the victims of cancer. The conclusion as to
individual susceptibility is only justified if the distinction be borne in mind between
transplanting a tumour into the mouse in which it grew primarily, and into any mouse
afflicted with spontaneous cancer. The two cases are not the same, as pointed out later
on. The fact that a mouse is suffering naturally from cancer does not imply a greater

suitability for transplantation generally. Added Feb. 8, 1907.
t Cf., however, p. 184, Summary.

172 Dr. Bashford, Mr. Murray, and Dr. Cramer. [ Dec. 10,

A clear conception is required of the necessity for interpreting the results
of the experiments about to be described as bearing directly on the condi-
tions of growth only and their artificial modification. By inference, they
have also important bearings on the nature of cancer; but we desire to
enforce caution in anticipating prophylactic or therapeutic actions as likely
to follow the use of those measures we have found efficacious in raising the
resistance of animals to transplantation.

(2) THE ImMpoRTANCE OF NATURAL VARIATIONS IN THE RESISTANCE OF MICE
TO INOCULATION, AND OF INHERENT VARIATIONS IN THE ENERGY OF
GROWTH OF THE TUMOUR CELLS.

In previous communications we have directed attention to variations in
the susceptibility of individual mice to inoculation with a number of different
mammary tumours of the mouse. The natural insusceptibility to inoculation
to which we particularly wish to draw attention in the present communication
is that which we have stated was still present after the disturbing influences
of race, age, dosage, and fluctuations in the powers of growth of the
cancerous cells, have been allowed for in artificial propagation of trans-
plantable tumours. In earlier papers we used the natural resistance of the
animals as an indicator of the variations of the power of proliferation of the
cancer cells. We shall now conversely use the tumour-cells as indicators of
the susceptibility of the inoculated animals. In such experiments the
number of animals necessary is inversely proportionate to the percentage of
successful inoculations attainable. Where the tumour gives a maximal
success in normal animals, the number necessary in each experiment can be
reduced, and must be correspondingly increased when the percentage of
success in normal animals is lower.

Experiments in which less than 60 per cent. of the normal animals develop
tumours are open to grave objections, and will not be employed in this paper.

Until we can experimentally determine the development of cancer de novo,
we are necessarily confined to a study of its growth and the conditions which
favour or hinder it when cancerous tissue is inoculated into mice. The
differences between mice of different susceptibility are expressed quantitatively
in differences in the percentages of successful inoculations.

In all these experiments we have to take account of two variable factors of
prime importance which are beyond experimental control :—

(1) The varying natural susceptibility of the mice ;
(2) The varying energy of growth of the tumour cells.

These vary independently of each other, and under normal circumstances,

1906.] Resistance of Mice to the Growth of Cancer. 173

either alone or in conjunction with one another, they determine the
phenomena of growth under artificial propagation. Hence, extensive control
experiments are necessary at every step in attempts to modify growth
artificially, for a low percentage of success may be due to a refractory
condition in the mice used, or to a lower energy of growth of the tumour
cells. In addition, other factors of less importance can introduce variations
which are detailed below, and must be excluded.

The following precautions have been taken to obtain results which may be
legitimately compared. The differences which have been elicited in these
experiments are in many cases slight, and dependent on very subtle altera-
tions in the animals. In order to measure them, it has been found advisable
to work on the margin of maximum success in transplantation. In order to
obtain comparable results under otherwise similar experimental conditions,
attention must be paid to (1) the dose of tumour material, (2) the age of the
animals, and (3) the site of inoculation. The inoculations must be carried
out in such a way that “tumour formation” or “no tumour formation” can
be determined by the conditions of the animals for the most part. We have
worked mostly with a “minimal tumour-forming” dose, although multiples
of this dose have been employed for special purposes. Large doses frequently
lead to the development of tumours in animals which are completely pro-
tected against, or refractory to, smaller doses. Then the refractory condition,
or relative protection, may only be indicated by the smaller size and less
rapid growth of the resulting tumours, as compared with normal mice. A
cleaner result is obtained when the dose, while sufficient to produce tumours
in normal animals within 10 days, fails to lead to any proliferation whatso-
ever, thereby indicating the presence of abnormal refractoriness in the animals
whose resistance is being tested. Our animals not being very highly protected,
many of the differences we shall record are only clearly elicited by the use of
small doses. The use of large doses obscures them when results are recorded
as percentages of successful inoculations. The difference between different
sites is best brought out by small doses, and obscured by the use of large doses.

Young mice are more susceptible to inoculation than old animals. The
difference in percentage of success may be as great as 60 per cent. between
animals of seven weeks, on the one hand, and those of one year old, on the
other hand. In the following experiments the control animals have always
been chosen of the same size (and therefore of approximately the same age)
as the animals whose resistance is tested. This precaution is necessary in all
such experiments, because the duration of the preliminary treatment whose
effect is under discussion, of itself entails an alteration in the susceptibility
of mice whose total duration of life is short.

174 Dr. Bashford, Mr. Murray, and Dr. Cramer. [Dee. 10,

During the continued propagation of Jensen’s tumour in the last three
years, series of inoculations have been obtained repeatedly with maximal
percentage of success. The method of repeated subdivision of the
parenchyma into the small grafts necessary for the analysis of growth, showed
that each such maximum was followed by a rapid and great diminution in the
percentage of success, on which again an increase ensued, till a fresh
maximum was encountered. The complication which these fluctuations
introduce has been in great part avoided in the following way.

By choosing a suitable interval for inoculation of tumours selected from
series with from 90 to 100 per cent. of success, and especially by increasing the

6neE
63M
641A

mq N
~
3 ©

=

40

960 1900 1050 f DAYS

Graphic Record of Propagation through ten Passages of a single Strain of Jensen’s
Carcinoma in which success has not fallen below 85 per cent.

initial dose introduced into each animal from 0-01 to 0:05 gramme, we have
been able to evade the diminution usually following each maximum for a
considerable number of transferences. This result is artificial. It does not
indicate an increased “ virulence ” of the tumour cells, or a continuous growth
of uniform energy on their part. It is in complete harmony with the results
of the experimental analysis of the growth of cancer as we have already
described it.

In the case of other tumours we have obtained the same result in the same
way, but most speedily in the case of spontaneous tumour XX VII, which at
the third transplantation gave 80 per cent. of successful inoculations, and at
the fifth 98 per cent., the success of the primary transplantation being only
20 per cent.

Separate strains of Jensen’s tumour have been propagated, as indicated in

1906. | Resistance of Mice to the Growth of Cancer. 175

the accompanying graphic record, through successive transplantations, without
the percentage of success in the direct line of propagation having fallen below
85 per cent. This is merely the result of an artificial selection of the tumours
obtained at each successive transplantation. It furnishes a special case of the
process already described in detail, and receives mention here because of
the great convenience of maximum success in inoculation for controlling the
experiments with which the present paper deals, and because other observers
have,not yet succeeded in attaining to this maximum of transplantability in
the case of Jensen’s tumour. These results, together with the other facts
mentioned on a previous page, entirely do away with the statements that
Jensen’s tumour is not virulent. We have sent our strain of Jensen’s tumour
to Professor Uhlenhuth and Professor von Dungern. The primary results they
have communicated to us show that, in English mice, Professor Uhlenhuth
obtained 90 per cent. of success and Professor von Dungern 85 per cent. In
German mice the results were 90 per cent. and 50 per cent. respectively.

The slight differences which suffice to determine the success or failure of
transplantation are exemplified in the following series of experiments, which
were devised to demonstrate the inherent fluctuations of nearly allied strains
of the same tumour under the same conditions at each transference.
Incidentally the experiments revealed the danger of comparing together
inoculations not strictly in the same site.

Two strains of the same tumour were propagated simultaneously on
opposite sides of the body of the same mice. The strains used were chosen
because of moderate powers of growth, and the fluctuations they had
exhibited. On a series of 11 passages, it was found that the axilla was
invariably a more suitable site than the dorsal subcutaneous tissue: 59 mice
out of 286 developed tumours both back and front, 18 mice had tumours only
on the back, and 59 only on the front. The difference between the dorsal and
ventral sites is in all probability comparable to that already recorded by us
between the dorsal subcutaneous tissue and the peritoneal cavity. The most
natural explanation of the difference would seem to be that the connective
tissue reaction, which we have shown is specific and without which the grafts
cannot grow, is more readily supplied by the connective tissue of the
mammary region.

In these experiments we also noted that whenever a tumour of the low
percentage strain developed on the back, it almost always had its fellow in the
axilla, the converse not holding good.

When the greater suitability of the axilla had been established beyond all
possibility of doubt, the experiment was continued through five successive,
passages in the right and left axilla. The results, as a whole, are more

176 Dr. Bashford, Mr. Murray, and Dr. Cramer. [ Dec. 10,

uniform in this series. Out of 95 mice inoculated, 59 developed tumours on
both sides, five on the right side only, and 11 on the left side only. The two
strains, propagated in the right and left axilla respectively, fluctuated
practically independently of one another, at one time a higher percentage was
obtained in the right axilla, at another time in the left. It is to be noted,
however, that whenever the strain with the lower percentage for the time
being was able to establish itself on its side, the tumour giving a higher
percentage always succeeded in establishing itself on the opposite side of that
individual mouse, so that a tumour of the strain with lower percentage never
occurred singly, each had its fellow on the opposite side in the more numerous
tumours of the high percentage strain. Hence the differences in percentage of
success between the two strains are considered to be due to fluctuations in the
powers of proliferation of the cells of each, and to be independent of the
susceptibility or resistance of the mice in which they are, or have been,
growing. If the resistance of the mice in the right or left axilla be assumed
to be similar, then this resistance really serves merely as a measure of the
varying qualities of the tumour cells. It plays the part of a sieve, as it were,
which keeps back the cells with less powers of assimilation and growth, and
permits those others to pass which can survive the hindrance to their
continued existence and multiplication. |

(3) MIcE IN WHICH A GROWING CARCINOMA HAS BEEN SPONTANEOUSLY
ABSORBED MAY BE COMPLETELY PROTECTED AGAINST SUBSEQUENT
INOCULATION OF THE SAME GROWTH AND TO A LESSER EXTENT
AGAINST OTHER AND DIFFERENT GROWTHS. |

Animals in which tumours have developed and then have been absorbed
are highly refractory to further inoculation. This protection may be
absolute. This fact is sufficiently evidenced by the following experiments.
The animals were re-inoculated in another part of the body after complete
disappearance of the first tumour. The control animals were inoculated in
the same place as the second experiment of the spontaneously-recovered

mice.

Protocout to ILLUSTRATE ABSOLUTE PROTECTION AFTER SPONTANEOUS ABSORPTION.

A. Experiment 61, ii M (1). Tumour taken from a mouse of transplantation 60,
Series V, was transplanted into the right axilla of 20 normal mice. Eight tumours
developed in the 12 animals surviving after 10 days (64 per cent.).

(2) The same tumour was also inoculated at the same time into 36 mice which had been
previously inoculated successfully in the right axilla, but in whom the tumours evident
*after 10 days ultimately disappeared. Two small tumours developed in the 33 animals
surviving after 10 days (6 per cent.).

1906. | Resistance of Mice to the Growth of Cancer. 177

B. (1) Experiment 62, ii K. Thirteen days after the inoculation recorded above, 15 of
those mice which had presented no tumours were re-inoculated in the left axilla, with
tumour of transplantation 61, Series ii N. No tumours developed, while 15 normal
animals inoculated with the same tumour presented 13 tumours after 10 days (87 per
cent.).

(2) Experiment 62, ii L. The remaining 15 mice of Experiment 61, ii M, were
inoculated in the left axilla on the same day with a tumour of transplantation 61,
Series 11 J. No tumours developed, while 15 normal animals inoculated with the same
tumour all presented tumours after 10 days (100 per cent.).

The protected animals were further inoculated unsuccessfully, and were then used to
test the properties of the blood.

Protection similar to that afforded against Jensen’s tumour by its
spontaneous absorption is also manifested by animals in which other
spontaneous tumours of different histology have grown for a time, and the
tumours been absorbed. Of the tumours on which such observations have
been made, XIX was an adeno-carcinoma with cystic spaces and widespread
hemorrhages; XX VII another adeno-carcinoma tending to become alveolar,
4.¢., approaching the histological type of Jensen’s tumour, and XXXVII
a malignant adenoma with little tendency to form solid alveoli, while
XXXVIII was also an adeno-carcinoma with small acini interspersed with
solid alveolar portions.

The animals in which tumours have disappeared after exposure to radium
are refractory in the same way and to a similar degree as those in which
absorption has occurred spontaneously. This is only what was to be expected
in view of the probable identity between the process of absorption in the
two cases, as described in a previous paper.

The general conclusion can be drawn that recovery from one form of
tumour of the mamma protects against subsequent inoculation with other
forms. When protection is absolute in the case of the tumour recovered
from, it is not necessarily also absolute for other tumours. There is thus
a suggestion of a specificity in the protection. The mice which have
recovered from experimental cancer have undergone a change conferring on
them a degree of protection as high as that presented by the most refractory
of normal animals. This alteration is not confined to the tissues in the
immediate neighbourhood of the spontaneously absorbed tumour; it must
have become general by means of the body fluids.

(4) PROTECTION MAY BE INDUCED OR, WHEN NATURALLY PRESENT, ENHANCED
BY THE INOCULATION OF TUMOUR MATERIAL WHEN NO GROWTH
RESULTS.

We have already pointed out that the animals which have been unsuccess-
fully inoculated with small fragments of a tumour giving a low or moderate

178 Dr. Bashford, Mr. Murray, and Dr. Cramer. [Dec. 10,

percentage of success may, on re-inoculation, give a positive result in a
proportion little inferior to a series of control animals. The negative result.
in the first instance was thus due not entirely to the inoculation of refractory
animals, but largely to the low powers of proliferation of the tumour-cells.

introduced.
PROTOCOL.

Tumour XV. Mouse with a large tumour of the fifth transplantation received from
Cologne (Dr. O. Schmidt), together with 10 normal mice.

We effected the sixth transplantation into 10 normal Cologne mice, and 147 normal
English mice. In the English mice, three tumours presented themselves in the 61 mice
which survived 10 days after inoculation (5 per cent.). Four tumours developed in the
10 Cologne mice, all having survived (40 per cent.).

One month later one of the tumours which had developed in a Cologne mouse was.
transplanted into 45 English mice and the three Cologne mice which alone had survived
out of the six negative animals in the previous experiment. All the three Cologne mice
developed tumours, 100 per cent., while only two tumours developed in the 20 English
mice surviving after 10 days.

Repeated re-inoculation of the negative animals with small doses of tumour
leads to the sifting out of a residuum which may possess a high degree of
resistance. As the end result of such a process of elimination by successive
inoculation, animals can be obtained in which, while similar control animals,
give 70 to 80 per cent. of success, only from 10 to 12 per cent. of successful
inoculations are obtained. When, however, the small number of animals
unsuccessfully moculated in an experiment with sub-maximal success are
re-inoculated, they evince a decided increased refractoriness as compared
with normal animals. Some change in the animals following upon the
absorption of inoculation material is not an unimportant factor in the
development of this condition, as already suggested by Jensen. That such
is the case seems more than probable from the result following, when the
initial and subsequent inoculations are made with large doses, more than
0°05 gramme of tumour material. Here the refractory condition is increased
in a much shorter series of inoculations, so that already at the third or even
at the second inoculation, a diminution of 50 to 60 per cent. may be
observed as compared with normal animals. This result confirms Ehrlich’s
observations on the same subject.

Protocon To ILLustRaTE IncREASED RESISTANCE AFTER A PRECEDING NEGATIVE
INOCULATION.
Nineteen normal mice were inoculated in the left axilla with 0°04 gramme of a tumour
of transplantation 62, Series ii C,* the experiment being labelled “63S.” All the mice
survived, but two only presented tumours after 10 days. Thirteen days after this

* Transplantation 62, ii C, had given 19 tumours in 19 mice (100 per cent.). The low
percentage obtained in 63 S, and the negative result in 63 T, the experiments providing

ON

1906.] Resistance of Mice to the Growth of Cancer. 179

inoculation, 13 of those 17 negative animals were re-inoculated in the right axilla with a

- tumour of transplantation 63, Series M, the experiment being labelled “64 W.” Of the

six animals surviving after 10 days, only one presented a tumour (17 per cent.). Of
15 normal mice which had been similarly inoculated among the 11 surviving after
10 days, eight presented tumours (72 per cent.).

Twenty-three normal mice were inoculated in the left axilla with 0°04 gramme of
another tumour of transplantation 62, Series ii C, the experiment being labelled “63 T.”
No tumours developed in the 20 animals surviving after 10 days. Thirteen days after
the preceding inoculation, 19 of the mice were re-inoculated in the right axilla with
0-03 gramme of a second tumour of transplantation 63, Series M, the experiment being
labelled “64 V.” Of the 18 mice surviving after 10 days, five presented tumours
(27 per cent.). Fifteen normal mice were similarly inoculated, 12 survived, and 10 pre-
sented tumours after 10 days (81 per cent.).

Differences in the size of dose of tumour probably explain the apparently
contradictory experience of others who have failed to produce protection in
this way, and of our own earlier experiments where the re-inoculation of the
negative animals gave a percentage almost as high as in the first instance.
The mass of tumour absorbed in the course of three or four negative
inoculations with 0°U5 gramme is so great that the animals, although never
developing tumours, cannot be regarded as comparable with animals in our
earlier experiments which, inoculated the same number of times, have only
received relatively insignificant doses (0°01 to 0°02 gramme).

Refractoriness to various spontaneous mouse tumours is apparently
reciprocal. Animals which have been unsuccessfully inoculated with large
doses of one spontaneous tumour, whether carcinoma or sarcoma, are less
suitable for the transplantation of other carcinomata than are normal
animals. This fact receives a natural explanation in the results of experi-
ments detailed below on the protection conferred by the injection of normal
mouse blood. On the other hand, the inoculation of mice with tumour
material from strange species—rat, cat, dog, man—has not induced a
refractory state. The protection induced in mice is therefore to be interpreted,
not as conferred by properties common to the malignant new growths of
different species, but only of the same species, by the absorption of substances
common to the tumours and tissues of the mouse.

(5) PROTECTION CAN ALSO BE INDUCED BY THE INOCULATION OF NORMAL
| TISSUES, AND PARTICULARLY BY THE INOCULATION OF BLOOD.

Early in 1904 we were enabled, through the courtesy of Professor C. J.
Martin, F.R.S., of the Lister Institute, to study the effects of immunising
the negative mice referred to, is a phenomenon to which we have previously drawn
attention (vide ‘Roy. Soc. Proc.,’ June, 1906).

The tumour tissue inoculated was in what we have designated, for convenience, the
negative phase of growth.

180 Dr. Bashford, Mr. Murray, and Dr. Cramer. [Dec. 10,

animals, including rabbits, guinea-pigs, rats, and mice against mouse tissues
(liver, kidney, testis) and tumour, all of which had been freed from blood by
washing out the circulation with normal saline, and ground to a fine powder
at the temperature of liquid air. The results were unsatisfactory, apart from
the exhibition of a distinct specific hemolytic action on the erythrocytes of
mice on the part of the serum of animals immunised against each of the
blood-free tissues and a precipitin reaction with mouse serum. In particular,
no satisfactory evidence of a specific action on the living cells of mouse
tumours could be obtained either in the test-tube or in the body.

We were led to investigate more fully the effects of the absorption of
mouse blood on normal mice, not only because of the difficulty of working
with the other tissues, but because of the following considerations : Hzemor-
rhage is frequent in the process of absorption of tumours after exposure to
radium, and during spontaneous absorption. A number of hemorrhagic
tumours successfully transplanted in the first instance were found to be
peculiarly liable to spontaneous absorption, and in their case the condition
was comparable to what is now well known to occur in human chorion-
epithelioma. In this connection, we are indebted to Dr. J. H. Teacher for
drawing our attention, early in 1905, to the frequency of the spontaneous
disappearance of the metastatic growths of chorion-epithelioma and their
hemorrhagic character. It was found that an injection of 0:3 to 0°5 ce. of
defibrinated normal mouse blood induced a definite refractory condition, even
in young animals. It is not so marked four days after injection as at 10 days,
and persists for at least three weeks. The repetition of the blood injection
after 10 days does not markedly increase the refractory condition. The animals
thus protected do not ultimately develop tumours when observed during a
period of three months.

PrRoTocoL To [ILLUSTRATE PROTECTION CONFERRED BY A PRECEDING INJECTION OF
Normat Mouse Buoop.

Experiment 61 T. Sixteen mice received an injection of 0°3 c.c. of normal defibrinated
mouse blood on the back. Five days later they were inoculated in the right axilla with
0-01 to 0°02 gramme of a tumour of transplantation 60, Series ii P. All the mice survived,
and seven presented tumours after 10 days (44 per cent.). At the same time 30 normal
mice were inoculated with the same doses of the same tumour, also in the right axilla.
Of the 26 animals surviving after 10 days, 19 presented tumours (73 per cent.). The
tumours in normal animals were throughout larger than those in the mice previously
treated with blood.

Experiment 62 Q. Twenty-eight mice received 0°3 cc. normal defibrinated mouse
blood on the back. Eleven days later they were inoculated in the right axilla with
0:01 to 0°02 gramme of a tumour of transplantation 61, Series S. Three tumours developed
in the 15 animals surviving after 10 days (20 per cent.). At the same time 30 normal
animals were inoculated in the same place with the same dose of the same tumour. All

Ne

1906. | Resistance of Mice to the Growth of Cancer. 181

survived, and 19 presented tumours after 10 days (66 per cent.). The tumours in treated
animals were smaller and grew less rapidily than those in the normal mice.

Experiment 62 Z. Fifteen mice received an injection of normal defibrinated mouse
blood. Ten days later they were inoculated in the right axilla with 0°02 gramme of a
tumour of transplantation 61, Series ii E. All the mice survived, and 10 presented
tumours after 10 days (67 per cent.). At the same time 15 normal animals were
inoculated in right axilla in the same way. Ten mice survived, and all presented
tumours after 10 days (100 per cent.). The same relation was again observed between.
the tumours in the two series.

Experiment 61, ii E. Thirty-nine mice received 0°3 c.c. of normal defibrinated mouse
_ blood on the back. Seven days later they were inoculated in the right axilla with
0°02 gramme of a tumour of transplantation 60, Series X. Of the 38 mice surviving
after 10 days, 15 presented tumours (39 per cent.). At the same time 20 normal animals
were similarly inoculated. All the 15 animals surviving after 10 days presented tumours
(100 per cent.). The same difference in the size and rate of growth was observed in the
animals treated with blood.

The difficulty of readily obtaining large numbers of young mice artificially
protected against carcinoma has been a hindrance to the studies of this kind.
We have depended on the selection of animals which had been negative to
repeated inoculations, or on collecting mice in which spontaneous absorption
had occurred. Both proceedings take up a large amount of time and lead to
experiments being conducted under unfavourable conditions, owing to the
greater suitability of young animals for growth (and therefore for control
experiments). By means of a preceding injection of blood, however, the initial
stages of the refractory state can be quickly induced in young mice, and the
condition then enhanced by suitable doses of tumour material. When once
the initial stages have been passed, there is little difficulty in increasing the
refractoriness.

Experiments conducted in this way appear to show that normal mouse
serum (0°3 to 0°5 cc.) free from corpuscles does not confer protection to
anything like the same extent, if at all. The blood-corpuscles, suspended in
saline after being freed from the serum by centrifuging, on the contrary, were
nearly as efficacious as full defibrinated blood. The biood was injected into
the dorsal subcutaneous tissue over the root of the tail. The inoculations
were then made into the axilla, either on one or both sides. The absence of
the same degree of protective action after the injection of serum appears to
show that it is not due to mere super-addition of protective substances
normally present in the blood of the mouse, but rather that the protection
results from the active intervention of the living tissues in dealing with the
injected corpuscles.

Although young mice are much more susceptible to inoculation, no differ-
ence could be discerned between animals treated with the blood of young and
old mice respectively.

182 Dr. Bashford, Mr. Murray, and Dr. Cramer. [ Dec: 10,

The blood of other nearly allied animals (rat, rabbit, guinea-pig) called
forth no such alteration, so that the action is strictly specific to the mouse.

These effects are best brought out when the inoculations are made with the
smallest convenient dose capable of giving rise to maximal percentages of
tumours and which lies in our experiments between 1 and 2 centigrammes.
When larger doses (5 centigrammes) are employed, the percentage of tumours
developing in the treated animals rose, although it did not reach that in
control animals, and the tumours which did develop also remained smaller.
In one experiment, however, in which massive doses were inoculated, tumours
subsequently developed in the animals in which no growth had appeared
within 10 days. Here, apparently, the larger dose of tumour had exhausted
the protective property, and subsequently growth was possible. The resist-
ance of animals in which spontaneous absorption has occurred has not been
overcome in the same way by increasing the dose of tissue inoculated.
Whether this points to a difference in degree only, or a qualitative difference
in the protection in the two cases, remains to be determined.

The nature of the action which the body fluids of protected animals have
upon tumour tissue in the days immediately following transplantation has
been approached in the following manner, after failure to obtain satisfactory
results in the test-tube. The failure signifies nothing particular, and may be
due either to low degree of protection or imply merely that a complex action
takes place in the body, and one we have not reproduced wn witro.

A number of animals in which tumours had spontaneously disappeared
were inoculated with large doses of tumour tissue, and at the same time twice
as many normal animals were inoculated in the same way. After varying
intervals the implanted tissue was removed from the protected animals and
transferred to young normal animals, and the results compared with those
obtained by re-inoculating the tissue removed from a similar number of the
control animals, sufficient of whom were left alive to prove the vitality of the
tissues inoculated in the first instance. These experiments are still in
progress, but there seems little doubt that, as compared with normal animals,
the tumour tissue rapidly dies in those protected by spontaneous absorption.

Summary.

Many apparently contradictory statements as to induced resistance of mice
to inoculation may be harmonised when due attention is paid to (1) the
susceptibility of young mice and the refractoriness of old mice, (2) to the
variations in success which depend on the size of the dose of tumour
inoculated, and (3) on the varying qualities of the cells of any one tumour

1906.| ° Resistance .of Mice. to the Growth of Cancer. 183

at different times according as they are in:the positive or negative phases of
growth.

When small doses of tumour, 0°01 to 0°02 gramme, are unsuccessfully
inoculated into young and susceptible mice, a second inoculation is often
successtul; similar doses will give negative results in a large proportion of
adult. animals at the first inoculation, and also on subsequent inoculation.
The differences between young and old mice and between the employment of
small and large doses explain contradictory statements to the effect that
apparently refractory animals of a primary inoculation can or cannot be
subsequently inoculated. When large doses of growing tumour are success-
fully inoculated into young mice, giving nearly 100 per cent. of success, the
same proportion is not always obtained in adult animals, although their
relative refractoriness can often be overcome by such an increase in dose,
provided the tumour tissue is in the positive phase of growth. Young mice
which prove refractory to such large doses also prove refractory on subsequent
inoculation, as also do adult and old animals.

The nature of the tumour tissue inoculated is of great importance. Where
small doses give a maximum success, large doses may also do so, producing
very much larger tumours in the same time; on the other hand, the larger
doses may be less successful than the small ones, this being explained by the
extent to which the simultaneous absorption of tumour tissue hinders growth.
The absorption of tumour tissue is Inversely proportionate to the number of
cells growing in the graft. Whereas a large dose of cells in the positive phase
leads to large tumours and no immunity, a large dose of cells in the negative
phase* yields very few tumours, permits of much absorption, and gives a
high proportion of resistant animals. There is much talk about the “virulent ”
and “avirulent” nature of different tumours, but the recognition of the fact
that the “virulence” of the cells of a single tumour fluctuates between
negative and positive phases will explain the contradictory statements of
those who have succeeded and those who have failed to induce immunity by
the injection of living tissue. It also explains the unexpected positive results
obtained in some curative experiments with immune sera and the absence of
any effect in others. Great care is necessary in accepting such positive results
as “cures” artificially effected, unless spontaneous absorption has been
excluded. For example, two tumours out of 12 disappeared after one or
two injections of rabbit serum. Spontaneous absorption occurred in the same
proportion of the control animals. The injections had no effect on tumours
of other series in which no spontaneous absorption took place.

The phenomena discussed in the preceding pages point clearly to the inter-

* Cf. protocol, p. 178, and footnote.
VOL. LXXIX.—B. P

184 Dr. Bashford, Mr. Murray, and Dr. Cramer. [Dec. 10,

action of two independent factors: (1) the variations from mouse to mouse
of the susceptibility to (or suitability for) the growth of cancerous grafts; and
(2) the variations in the energy of growth of the tumour cells.

The first variable fluctuates between fairly wide limits in individual
animals, but when large numbers of young mice are compared together, their
resistance acts like a sieve, with unequal meshes, it is true, but, on the whole,
of the same average fineness.

Spontaneous tumours vary considerably in the ease with which they can be
propagated, z.c., with which they can pass the sieve. These differences have
been regarded as depending on differences in “virulence.” There is some
evidence to show that the sporadic tumours fluctuate in energy of growth
just as do the propagated tumours,* and the direct conclusion as_ to
“virulence” from one primary transplantation may be upset when the
same spontaneous tumour gives an opposite result, 72, 1s again trans-
planted after recurrence. The term “virulence” is therefore unfortunate,
and must be used with so much reservation that it would be better discarded
altogether.

The influence of the individuality, ze, the sum total of changes
due to the past life of the organism, will be to make any mouse different
from all others, and these differences will increase the longer the animal
lives. The difficulty of obtaining success in the primary transplantation
of spontaneous tumours would be accounted for by supposing that the new
animals provide an environment to the cancer cells so strange, that they
cannot survive the interruption to their nutrition. Their failure to grow
does not necessarily imply that they would fail to proliferate in their new
hosts if the conditions to which they had been accustomed could be
immediately supplied in the experiment. Cells which have lived and
become accustomed to the body fluids of one mouse for, say, two years,
may easily die or fail to adapt themselves when transferred to the bodies
of new animals. The frequency, in our experience, of large metastases in
animals spontaneously affected, is in marked contrast to the difficulties in
obtaining growth in normal animals, and harmonises well with this view.
What has been said of the differences in transplantability between
spontaneous tumours appears to hold also with reference to separate strains
of propagated tumours. If the resistance of the mice be made the criterion
for the energy of growth of sporadic tumours, the same standard is equally

* Vide ‘Roy. Soc. Proce.,’ B, vol. 78, 1906, p. 220. Graphic record of transplantation of
a sporadic tumour (XIX) which recurred three times after partial excision.

+ Vide ‘Sci. Report II,’ Part 2, p. 40; Ehrlich, ‘ Berl. Klin. Woch.,’ No. 28, 1905, where
he also proposes “ energy of growth” as an alternative.

1906.] Resistance of Mice to the Growth of Cancer. 185

applicable to the continued propagation of a single tumour. It is impossible
to imagine that the differences brought out by our experiments on the
fluctuations in energy of growth are due entirely to variable susceptibility
of the animals. The simultaneous appearance of numerous spontaneous
absorptions, and associated therewith the rapid diminution in the percentage
of success in parallel experiments,* can only be referred to fluctuations
in the energy of growth of the parenchyma cells. Any other explanation,
and especially an explanation by assuming increased resistance of the
animals, introduces an arbitrary assumption which further complicates the
conceptions.

The resistance of the animals can be altered, and this change has been
described as an active immunity. We shall continue to speak of resistance or
refractoriness. The absorption of tumours which have grown for a time
undoubtedly calls forth this alteration in a high degree. The absorption
of inoculated material without obvious tumour formation has similar con-
sequences, Increased resistance results when blood injected subcutaneously
is absorbed. The refractory condition induced in this way is not so perfect as
that following the absorption of tumour material. It is, however, perfectly
definite, and removes any cogency which might attach to the argument that
the induction of a refractory condition by absorption of growing tumour or
living tumour cells indicates the existence of a hypothetical cancer virus.
The evidence we have obtained that the blood of other animals is unable to
induce such an alteration in mice, points to the same conclusion. The
absorption of the tumours of animals of alien species when inoculated into
mice is likewise devoid of effect. |

All these actions are curiously lmited to the production of an
insusceptibility to subsequent transplantation. We have not been able to
induce such an alteration in animals with growing inoculated tumours that
they should become unsuitable for growth to continue once it has started. It
would appear that the negative phase in growth of the tumour cells plays the
principal ré/e when spontaneous absorption occurs, the fall in the energy of
growth and the resistance of the mouse together contributing to the final
absorption. Once the cellular graft has been vascularised it is in a much
stronger position with reference to conditions which may be unfavourable to
it, than is a graft in the days immediately following transplantation. This is
much more vulnerable.

The biological reactions described in this paper are effective by means of
the body fluids. They are analogous to those that are now well known as

* © Roy. Soc Proc.,’ B, vol. 78, 1906. Graphic record on p. 206, and protocol, p. 209.

186 Resistance of Mice to the Growth of Cancer.

specific hemolytic, cytolytic, and precipitin reactions. As pointed out in
Second Scientific Report, 1905, pp. 32—33, they are of far vreater delicacy,
and have only been revealed by using living cells as indicators.

The experiments recorded in this paper give further evidence of the great
delicacy and the very narrow range of variations in the soil furnished by
the living animal, within which growth can proceed or is inhibited. Growth
has only been hindered by the intervention of mouse tumour or normal
mouse tissue, to an infinitesimal degree by the normal tissues of the rat,
and not at all by those of less nearly-related animals. We therefore feel
justified in concluding that these reactions are parallel to those others
whereby blood relationship has been established (Uhlenhuth).

When we attempt to formulate an explanation of the nature and
mechanism of the changes leading to inhibition of growth, it must be clearly
kept in mind that the evidence is almost entirely in the direction of showing
an alteration in animals whereby they are unsuitable for or inimical to the
establishment of grafts. There is no satisfactory evidence of an induced
action on growing vascularised tumours, but only one against newly-
introduced grafts. Spontaneous absorption, depending largely on alterations
from the side of the parenchyma, is sufficiently frequent to enforce caution
in assuming an artificial alteration in the resistance of animals already
carrying growing tumours. Complete absorption is necessary to induce
absolute refractoriness to subsequent inoculation. Hence the action of
various procedures in inhibiting the growth of grafts is not necessarily or
even probably due to a direct action on the parenchyma cells.

The connective tissue reaction, which a cancerous graft elicits, is taken
advantage of by the cancer cells and acquires characters specific for each
sporadic tumour. It is immaterial whether this reaction is protective on
the host’s part, and only not effective because the slight differences between
the tumour cells and normal mouse tissues* do not sufficiently stimulate the
protective process. The necessity which exists on the part of the cancer
cells for this reaction if they are to continue to grow, renders it possible that
a refractory condition may indeed not have anything to do with an action
against the cancer cells, but, on the contrary, be due to an alteration of the
connective tissue of the host which hinders it from supplying the necessary
connective tissue reaction. The possibility must also be entertained that the
alteration is directed against the chemotactic influence which may be pre-
sumed to be exerted by the cancer cells on the connective tissue of the host.
Our own experiments have not yet given satisfactory direct. evidence of an

* Once the tumour cells are dead they excite an energetic phagocytosis. Of figs. 483—48,
‘Second Scientific Report,’ Part 2, 1905. ;

Resistance of Mice to the Growth of Cancer. 187

anti-toxic or anti-cellular action, but we are far from denying the possibility
that such an action may be obtained. |

The phagocytosis of formed cellular elements plays an important réle in
inducing resistance ; serum is impotent to produce resistance, blood corpuscles
do so. The energetic phagogtosis which accompanies the spontaneous
absorption of transplanted tumours, and which occurs in absorption after
exposure to radium, speaks strongly for the conclusion that the processes are
the same in kind when blood or tumour cells, being absorbed, produce
resistance. But we are as yet unable to determine the extent to which
agencies directed against the tumour cells themselves may assist in
determining their early death in protected animals. Other experiments
still in progress may be expected to clear up the relative importance of the
parts played by the hypothetical inhibition of the specific stroma reaction, or
of an equally hypothetical direct lethal action on the tumour cells.

VOL. LXX1X.—B. Q

i ee
Ma fot

ar oe :
BM 16644 } “a

% ‘¥

; Combining Properties of the Opsonn of an Immune Serum. 187

anti-toxic or anti-cellular action, but we are far from denying the possibility
that such an action may be obtained.

‘The phagocytosis of formed cellular elements plays an important réle in
inducing resistance ; serum is impotent to produce resistance, blood corpuscles
do so. The energetic phagocytosis which accompanies the spontaneous
absorption of transplanted tumours, and which occurs in absorption after
exposure to radium, speaks strongly for the conclusion that the processes are
the same in kind when blood or tumour cells, being absorbed, produce
resistance. But we are as yet unable to determine the extent to which
agencies directed against the tumour cells themselves may assist in
determining their early death in protected animals. Other experiments
still in progress may be expected to clear up the relative importance of the
parts played by the hypothetical inhibition of the specific stroma reaction, or
of an equally hypothetical direct lethal action on the tumour cells.

On the Combining Properties of the Opsonn of an Immune
Serwm.*

By Rosert Muir, M.D., Professor of Pathology, University of Glasgow,
and W. B. M. Martin, M.B., Carnegie Research Scholar.

(Communicated by Dr. C. J. Martin, F.R.S. Received January 18,—
Read February 7, 1907.) :

In a recent publicationt we have studied the combining properties of the
thermolabile opsonins of normal sera, and have shown that various substances
or combinations of substances which absorb serum complements also absorb
the opsonins in question. Of special interest is the fact that red corpuscles,
and also heated normal sera, which by themselves have no appreciable
effect either on complements or on opsonins, absorb or fix both of these
bodies when combined with their corresponding anti-substances, immune-
bodies and precipitins respectively. Since the time of this publication we
have extended our experiments, and have always obtained the same result—
the opsonin is always fixed when the complement is fixed. Using the term
complement in the bio-chemical sense, we have said that the normal thermo-
labile opsonins belong to the group of complements. And we have also

* Towards the expenses of this research a grant was received from the Carnegie

Trustees, for which we have pleasure in recording our indebtedness.
+ Muir and Martin, ‘ Brit. Med. Journ.,’ 1906, vol. 2, p. 1783. -

VOL, LXXIX.—B. Q

188 Prof. Muir and Mr. Martin. On the Combining [Jan. 18,

found that just as complements in their combining affinities do not possess
specific properties, but are absorbed by a great many different substances,
so also these thermolabile opsonins show a corresponding community in their
combining relationships. We, however, expressly left out of consideration
the special opsonins of immune sera, and also the thermostable opsonins
of normal sera, of which latter the stable opsonin of human serum for the
diphtheria bacillus may be taken as an example. In the present communi-
cation we shall consider the thermostable opsonins of immune sera.

It is unnecessary to enter in detail into the literature of the subject,
as this has been already done by Dean.* It is, however, advisable for the
sake of clearness to refer to the chief facts which have been established and
to the chief points at issue. It 1s now a considerable time since Metchnikoff
showed that the establishment of active immunity towards various bacteria
was often accompanied by increased phagocytic action on the part of
leucocytes and other cells, or by the appearance of phagocytic action when
this was absent under natural conditions. Denys and Lecleff showed in the
case of rabbits immunised against streptococci that the increased phagocytosis
was due not to changes induced in the leucocytes, but to an alteration in
the serum, and pointed out that the leucocytes of the immune animal when
placed in a normal serum showed no greater phagocytic activity than
normal leucocytes did. Wright and Douglast were the first to show that
phagocytosis by leucocytes in the presence of normal serum depended upon
certain thermolabile substances—“ opsonins ”—in the serum which became
fixed to the bacteria in question and made them a prey to the leucocytes.
Their results were confirmed by Bulloch and Atkin,§ by Hektoen and
Ruediger,| and by others, and may now be accepted as established beyond
question. We have, on the other hand, a large group of observations which
show that in immune sera the substance which leads to the phagocytosis of
bacteria, or of red corpuscles, as the case may be, is thermostable, 2.¢., resists
a temperature of 55° C. for an hour. Among such observations may be
mentioned those of Savtchenko,{f Neufield and Rimpau,** Dean,fT
Leishman,{t and others. And Wright and Reid§§ have shown that in

* Dean, ‘ Roy. Soc. Proc.,’ vol. 76, p. 515.

+ Denys and Leclef, ‘ La Cellule,’ 1895, p. 177.

t Wright and Douglas, ‘Roy. Soc. Proc.,’ vol. 72, p. 357.

§ Bulloch and Atkin, ‘ Roy. Soc. Proc.,’ vol. 74, p. 379.

|| Hektoen and Ruediger, ‘Journ. of Infectious Diseases,’ 1905, p. 128.
4] Savtchenko, ‘ Annales de I’Inst. Pasteur,’ 1902, p. 106.
** Neufield and Rimpau, ‘ Deutsch. Med. Wochenschr.,’ 1904, p. 1458.
tt Dean, op. cit.

tt Leishman, ‘ Path. Soc. Trans.,’ 1905,

§§ Wright and Reid, ‘ Roy. Soc. Proe.,’ vol. 77, p. 211.

-1907.] Properties of the Opsonin of an Immune Serum. 189

certain cases the serum of patients suffering from tuberculosis may contain
a considerable proportion of heat-resisting opsonin, a circumstance which
may aid the diagnosis. The question therefore arises as to what is the
relationship between the thermolabile and the thermostable opsonins. Dean
thinks that the opsonin is of the nature of an immune substance, and that
in the case of normal serum it undergoes a large fractional destruction by
heat, whereas in an immune serum the portion which is thermostable is
increased. He, however, mentions the possibility that the opsonic effect
of a normal serum may in part be due to complement. Wright and Reid
consider that the opsonin in a normal and in an immune serum alike is
one and the same, and prefer to call it thermolabile. The question as
to the identity of such substances may be studied in other ways than that
of testing their powers of resistance to heat. One method is to test whether
cell receptors combined with their corresponding immune-bodies will take
up the opsonins of immune sera, as we have shown that they take up the
labile opsonins of normal sera and as they take up complements. Another
method is to test the degree of specificity in their combining affinities—
to test, that is, to what extent one bacterium will absorb the opsonins for
other bacteria. This has been done by Bulloch and Western* to a certain
extent, and also by Hektoen+ in the case of hemopsonins, and to these
experiments further reference will be made below. But the method has
not been carried out fully in a comparative way as between normal and
immune sera respectively.

We shall now give an account of experiments performed by us to elucidate
these questions in the case of an immune serum, giving in each instance for
comparison the effects on normal opsonins under the same conditions of
experiment. We have made use of two samples of anti-staphylococcic serum.
One of these was kindly given to us by Dr. Dean, for which we have
pleasure in recording our indebtedness, whilst the other was obtained by
us from a rabbit by repeated intravenous injections of dead cultures of
Staphylococcus aureus. As it was employed at various stages in the process
of immunisation, the degree of the opsonic effect varies considerably in the
different experiments. We may state that heating for several hours at
55° C. appears to have no appreciable effect on the thermostable immune
opsonin. The serum thus contrasts very markedly with the normal rabbit’s
serum, the opsonic effect of which is practically destroyed by heating for an
hour at 55° C.

- * Bulloch and Western, ‘ Roy. Soc. Proc.,’ vol. 77, 531.
+ Hektoen ‘Journ. of Infectious Diseases,’ 1906, p. 721.

Q 2

190 Prof. Muir and Mr. Martin. On the Combining [Jan. 18,

I. Tot ErFrFects oF ABSORBERS OF COMPLEMENT ON NORMAL AND
IMMUNE OPSONINS.

We have tested whether or not the three combinations of receptors +
immune-body which we formerly used, absorb the opsonin of the immune
serum.

The two following experiments bring out at a glance the differences in
the effects of treating a normal and an immune-serum in various ways, each
serum being afterwards tested on an emulsion of the Staphylococcus aureus

_

in the usual way :—

Opsonic index.*
Normal serum of rabbit—

Hreshvand wntreated:.....0.s.c cessor aceasee cee aete cecenee icone neee ee 18:1
Heated one hour at'5°°Co c2.55 suave. cosccateeceene see sceas eee ener eeee 0°6
Unheated and treated with red corpuscles + immune body ... 0°7
Unheated and treated with serum precipitate .................000- 1:0

Unheated and treated with emulsion of Staphylococcus aureus... 0°77

It is thus seen, in accordance with our previous results, that practically
all the normal opsonin is removed by these various methods of treating the
serum. To contrast with this we give the results in the case of an immune

serum.

Opsonic index.
Anti-staphylococcus serum from rabbit-—

Breshiand unheated 5.4.0 2d vecicdie «oat na casteeaiide bok toctie deme aaepnaeeeeae 12°3
Heated one hour’at:o0% Cs is. ew sccost score se ensgee ses see etree oeeOe 2°9
Unheated and treated with red corpuscles + immune body...... 31
Unheated and treated with serum precipitate................sceceees 3°5
Unheated and treated with emulsion of Staphylococcus aureus... O°7

It is thus seen that? treating the serum with the substances which absorb
complements has practically the same effect on the serum as heating has;
there being approximately the same amount of immune opsonin left. An
emulsion of staphylococcus, however, removes the opsonin almost entirely.

Other confirmatory examples may be given :—

Treatment by Precipitate.
Opsonic index.
Anti-staphylococcus serum—

Freshtand wnheatediei oe eee ee eee 18°2
Heated onevioura bop wee eee eeeeeee 69
Unheated and treated with precipitate......... 5'8

* The term “opsonic index” is used in the original sense to indicate the average
number of cocci contained in a polymorphonuclear leucocyte. The observations given in
each table were of course carried out at the same time.

+ The methods of treating a given serum are the same as those given by us in our
previous paper, quoted above.

1907. | Properties of the Opsonin of an Immune Serum. 191

In this case there was an enormous excess of precipitate, the actual bulk
a of the precipitate being three times that of the serum; there would, therefore,
| probably be some dilution of the serum. The treated serum has practically
the same opsonic value as the heated.

Treatment by Precipitate.*
Opsonic index.
Anti-staphylococcus serum --

PVCA TERA poy Oru wie cds 5 Shes tad duecs euaadeyencuswad 3

Heated and treated with precipitate............ 70
Normal serum—

Wrihkieabed, iu2 2 ol. cdustusvor a ccacuas esuuenetdedieades 24°6

Treatment by Red Corpuscles + Immune-Body.

Anti-staphylococcus serum—

HEAL Ab eO Oh Crs vonuten caters sceadondiceuwsuees es 8°3

Pleated, amd CLeated: cs cesasc peony seb cspeevecdscgagens 9°4
Normal serum—

WTEC ALCEL Piano aca nese evade cusvees Siguduewauevacess 20°8

Unheated and treated .....ccccicccscncotopecseveses 0°8

The opsonin of the normal serum is absorbed, whereas that of the immune
serum is unaffected.

Treatment by Emulsion of V. Metchnikovi + Jmmune-Body.

Opsonic index.
Anti-staphylococcus serum—

iEveated aloe ©. saat oe oi teahle ltd eindaae Lise

Heated and’ treabed oi... cc ccvesscesvsoves dasticesubes 175
Normal serum—

OHNE A TCO ase tatan cers somes canoes ssviea suis vacilesios' 25°0

Unheated and ‘treated .........c....cccesectceesees 2°36

The result is the same as before. |

We have also endeavoured to remove the opsonin of the immune serum by
treating it twice with a considerable quantity of serum precipitate, the pre-
cipitate being separated by centrifugalisation after each treatment, but no
diminution of the opsonin has resulted. The following is an example in
which the effects on the normal and the immune serum of the rabbit are
once more compared :-—

* In this and the two following experiments the anti-serum was the one which we
obtained from Dr. Dean.

192 Prof. Muir and Mr. Martin. On the Combining [Jan. 18,

Normal Serum.
Opsonic index.

Normal serum of rabbit (unheated)... ....5...<asceacstone repent renee ee eeeeeeee 17:0
" - treated once with staphylococcus emulsion... 1°6
” ” » twice ” ” os «=O
5 én »» once with serum precipitate ............ lt
” ” ” twice ” ” S00 0°3

Immune Serum.

Anti-staphylococcus serum (heated at 55° C.) .......ccccesceseseeeeeenenseeeee 23°3
FA a treated once with staphylococcus emulsion 23:0
” ” ” twice ” ” 2°2
“ Bs » . once with precipitate ..........2+00-0 25°0
es . » twice syle WLaEeY usiaaeielce ste seiemeeee 23°0

The result, again, is that the serum precipitate removes the opsonin from
the normal serum, but has no effect on the stable opsonin of the immune
serum.

Summary.—lIt appears, from the above experiments, that in the case of a
fresh anti-staphylococcus serum the effect of treating with substances which
absorb complement is practically the same as heating the serum, there being
a considerable residuum of opsonin in both cases. Further, after the ther-
molabile opsonin has been destroyed by heat, the stable opsonin remains
practically unaffected by treatment which removes normal complement and
normal opsonin. In other words, substances which we may call complement-
absorbers do not fix or combine with the thermostable opsonin of the immune

serum.

II. ON THE RELATIVE SPECIFICITY OF NORMAL AND IMMUNE OPSONINS.

An emulsion of dead bacteria is well known to have the power of absorbing
serum complements, as shown, for example, by hemolytic tests; it also
absorbs the opsonin of a normal serum as tested by the Staphylococcus aureus.
We have accordingly tested the effect on the opsonin of our immune serum.
In the following tables the comparative results are shown :—

Normal Serum.
Opsonic index.

Normal serum of rabbit (unheated) ............ siSieisate clelcolcaiars Jleeisiviaitanesteenete 380
m i treated with Staphylococcus Gureus .........00000+ 0°02
5 re a DSACUUUSICOLU ces cncebaedsins <sbomacates 33
ce 4, <3 VISIICLCNHUNOOD, ccncacees ss ccseeencetre 4°11
Tuberculosis Bacillus ......c0cceceee 2°6

” ” )
y ) .. Bacillus PyOCyANeus....ccsosveceveses 3°4

1907.] Properties of the Opsonin of an Immune Serum. 193

Immune Serum.

Anti-staphylococcus serum (heated at 55° C.) .......ceceeeceseeeereeeeceeeenes 78
a 3 treated with Staphylococcus aureus ......... 0°36
~ - TECHN COE sae cctun' cheap asyndven 8°8
Ms V., MCCCHRAOC critens cwnvnvensve 75
- a 3 Bacillus tuberculosis ..........+ 79
35 . : Bacillus pyocyaneus .....cc0000. 6°7

The tables show that emulsions of all the organisms tested produce a
marked diminution of the normal opsonin, whereas none of them, with the
exception of the staphylococcus, have any appreciable effect on the immune
opsonin.

In another experiment we treated the heated immune serum twice with large
quantities of B. coli, B. dysenteriae, Cholera spirillum, B. typhosus, Staphylococcus
pyogenes albus. In the case of the last mentioned, there was apparently a
slight diminution of the opsonic power, viz., from 26°7 to 20°3—a compara-
tively trifling effect in view of the close relationships of the two organisms ;
the serum was, however, practically without opsonic effect on the Staphylo-
coccus albus. In the case of the other bacteria mentioned, there was no
diminution whatever of the opsonin after treatment with the organisms.

These experiments, by the method of absorption, bring out a very marked
difference as regards specificity between the opsonins of a normal and of an
immune serum. With the exception of the possible slight effect in the case
of the Staphylococcus albus, we have failed to find any organism, except the
Staphyloccocus aureus, which absorbs the opsonin from the heated anti-
staphylococcus serum. In the case of the opsonins of a normal unheated
‘serum, the result is entirely different, as is shown by the table given above.
Every organism tested has absorbed large quantities of opsonin when the
Staphylococcus awreus is used as the test for opsonic action.* At the same
time, the staphylococcus appears to remove this opsonin more quickly than
any of the others. We have tested to what extent it is possible to remove
the opsonin of normal serum for Staphylococcus aureus by treating the serum
twice with an emulsion of another organism, ¢g., the B. coli. The results
are :—

Normal Serum.
Opsonic index.

CHMiMer pigs SCLUIM (UNNEATEC) ....5...00c-reccncanessconcencraesdvdssdeesencsenes 215
* $5 treated once with Staphylococcus aureus .........06 1:0

Pe a is twice os MP TS Pnaed Oo, gio = 0'4

oy " 95 Gnee, waite B., Cole, Vw anssevads Jaseeewallsdsscusewns ae a

re _ » twice se chciielos ae ace OMG aiihetouty 13

* These results, so far as the normal opsonins are concerned, are in harmony with those
of Simon Lamar and Bispham, whose paper (‘ Journ. of Exper. Med.,’ December, 1906
p. 651) has come into our hands since the present communication was written.

194 Prof. Muir and Mr. Martin. On the Combining [Jan. 18,

There is, therefore, only a slight difference as regards absorbing powers in
favour of the Staphylococcus aureus.

The immune serum was tested at the same time, and once more a great
difference is brought out. ‘

Immune Serum,
Opsonic index.

Anti-staphylococcus serum (heated at 55° C.) .......cccecesceeeseescereceeeees 26°7
55 Ms treated twice with B. colt ........ssscscsreeees 25°4
3 i : Staphylococcus aureus... 16

These results, as regards the specificity of the normal opsonins, appear to
be at variance with the results obtained by Bulloch and Western. They
found only a slight reduction of the tubercle opsonin of normal human serum
on treatment with the Staphylococcus awreus, and of the staphylococcus
opsonin on treatment with the tubercle bacillus. As stated above, we found
a great reduction of the staphylococcus opsonin on treating the normal
rabbit’s serum with the tubercle bacillus. The difference in the results
probably depends upon the amount of the bacterial emulsion employed. In
every case we used a large amount; in the tubes after centrifugalisation, the
volume of the deposit of bacteria would be about a tenth of the volume of
the serum, sometimes more. This is, no doubt, a large quantity, but it is to
be noted that the same amounts were employed in the case of the immune
serum, and no diminution of the opsonin was observed. The difference in
the two kinds of serum is, therefore, very remarkable.

We have stated above that the opsonin for a particular organism appears
to be more rapidly removed from a normal serum by an emulsion of that
organism than of any other, whilst at the same time any bacterial emulsion
will absorb large quantities of that opsonin. At present we are unable to
give the explanation of that fact. We use the term “normal opsonin” for
the labile substance which is destroyed by heat, but we do not know whether
or not another substance is present in small amount which acts as an
immune body. The problem is very much the same as in the case of
bactericidal action. A closely analogous phenomenon was demonstrated by
Bordet* in the case of hemolysis. Normal guinea-pig’s serum, after treat-
ment with rabbit’s corpuscles, is deprived of its lytic action for these
corpuscles; it still, however, produces lysis of pigeon’s corpuscles. IH,
however, the serum be treated by a more powerful absorber of complement,
viz., rabbit’s corpuscles treated with immune-body, it loses also its hemolytic
action on pigeon’s corpuscles. And it has been shown in our previous paper
that an emulsion of a bacterium treated with immune-body absorbs more

* Bordet, ‘ Annales de l’Inst. Pasteur,’ 1901, p. 317.

1907.| Properties of the Opsonin of an Immune Serum. 195

opsonin than the emulsion untreated, the amount of opsonin left being
practically the same as when the serum is heated. The following is an
additional example :—

Opsonie Action of Normal Human Serum on the Tubercle Bacillus.
Opsonic index.
Mormariserunt unheated): )i25.th.5. 5. warvciweadeds tbedleaveyeds 6°5

3 Be RCO ise we atniain is pure tench maaas sidunnis 0°2
a » treated with emulsion of V. Metchnikovi
MOLES WOU Y ..seciisevscenadacdevesesass 0°19

III. THE RELATIONS OF THE OPSONIN OF AN IMMUNE SERUM.

Our results, as above detailed, show that the opsonin of the anti-staphylo-
coccus serum corresponds as regards specificity and combining relationships
with true anti-substances developed in the process of immunisation. Does it
correspond with the type of an immune-body or of an agglutinin? Does it
in combination with its corresponding receptor lead to the absorption of
complement, or does it not? In the first place, it is certain that every
immune-body does not produce an opsonic effect. We have an anti-coli
serum, for example, which contains immune-body in considerable quantity,
but it has no opsonic effect. A given amount of emulsion of the B. coli alone
was found to take up 0°04 cc. of guinea-pig’s complement, while the same
amount of emulsion treated with the anti-serum took up 012 cc. the
absorption of complement being tested by the usual hemolytic methods. In
the case of the anti-staphylococcic serum the amount of immune-body
appeared to be considerably less. The sample with the greatest opsonic
power gave the following results :—emulsion of staphylococci alone took up
0:03 c.c. of guinea-pig’s complement, the same quantity treated with immune-
body took up 0:075 cc. This sample had a very powerful opsonic action,
giving, when heated, an average count of 20 cocci per leucocyte in the usual
test. The anti-staphylococcus serum which we got from Dr. Dean had a
weaker though still very decided opsonic action, yet it produced a scarcely
perceptible increase in the amount of complement absorbed; that is, it
contained a mere trace of immune-body. We are inclined to think from
these results that the opsonin in an immune serum may not be an immune-
body, but has the constitution of an agglutinin. But this is merely an
expression of opinion, as we know nothing with regard to the real amounts of
these substances. In order to say that the immune-body did not act as
opsonin, we would need to get an anti-serum with opsonic effect but contain-
ing no immune-body, and this we have not yet obtained.

If the opsonin has the constitution of an agglutinin, that is, possesses
merely a combining and an active (agglutinating) group, is the opsonin

196 Combining Properties of the Opsonin of an Immune Serum.

merely the agglutinin? To this question we can give no definite answer.
We have tested three anti-sera, viz., the Staphylococcus aureus, the B. coli, and
the Vibrio Metchnikovi, and have found that they all possess agglutinating
properties, while the anti-coli serum alone has no opsonic action. It is quite
possible that the same substances may act at the same time as agglutinin and
as opsonin, and that one of these properties may be wanting in any given
case ; but at present we have no facts to justify any expression of opinion.

So far as phagocytosis is concerned, the all-important factor in active
immunity would appear to be the development of an immune opsonin with
comparatively specific characters. In testing the opsonic effect of an
unheated immune serum the result will represent the sum of the actions of
the complement-like opsonin and the immune opsonin. As complements do
not appear to increase in amount during immunisation, a rise of opsonic
index will probably depend upon the development of immune opsonin, and
will thus have a certain specific character. Thus Bulloch and Western found
that inoculation of a patient with tubercle vaccine produced an increase of
tubercle opsonin but not of staphylococcus opsonin, and conversely inocula-
tion with staphylococcus vaccine caused an increase of the staphylococcus
opsonin but not of the tubercle opsonin. This appears to be in harmony
with the results which we have obtained. On the other hand, a fall in the
opsonic index might be due to diminution of the complement-like opsonin,
and thus be without specific significance. Further observations will be made
on this point. |

General Conclusions.

(1) The thermolabile opsonin of a normal serum and the thermostable
opsonin of an immune serum are two distinct classes of substances. In
addition to differing markedly as regards their resistance to heat, they differ
in their combining relationships.

(2) The thermostable opsonin of the anti-serum investigated is a true
anti-substance, and possesses the comparatively specific characters of anti-
substances in general ; it is left undetermined whether it has the constitution
of an agglutinin or of an immune-body, though certain facts point in favour
of the former.

(3) Emulsions of other organisms other than the organism used in immuni-
sation (Staphylococcus awreus) do not absorb the immune opsonin; on the
other hand, they absorb large amounts of the normal complement-like opsonin.

(4) Powerful complement-absorbers—red corpuscles or bacteria treated
with immune-body, or serum precipitate—have no effect on the thermostable
immune opsonin, whereas they remove almost completely the labile opsonin —
of the normal and the immune serum alike.

197

A General Consideration of the Subaérial and Fresh-water Algal
Flora of Ceylon. A Contribution to the Study of Tropical
Algal Ecology. Part I.—Subaérial Alga and Alge of the
Inland Fresh-waters.

By F. E. Frirscu, D.Sc., Ph.D., F.L.S., Assistant Professor of Botany,
University of London, University College.

{Communicated by Professor F. W. Oliver, F.R.S.,—Received June 26,—Read
November 1, 1906,—Received in revised form, January 2, 1907.)

Introductory Remarks.

During the year 1902 I was occupied with a study of the algal flora in the
hot-houses of the Royal Botanic Gardens at Kew, and certain observations
made then seemed scarcely to support the prevalent view of the very close
similarity between tropical and temperate algal vegetation. This stimulated
me to undertake a personal investigation of some tropical algal flora, and
Ceylon was chosen owing to the diversity of climatic conditions which it
presents. My object was rather to study the ecological and biological aspects
of the algal vegetation than to make a systematic collection of materials for a
flora, and I hope to be able to show that I have been in some measure
successful. A careful analysis, as I think the subsequent pages of this paper
will show, brings to light very considerable differences between algal growth
in the tropics and in our parts, although a certain similarity between fresh-
water Alge all over the surface of the earth (considerably greater than in the
case of the terrestrial flora) is indisputable. It is, in fact, a natural
consequence of the uniform character of the surrounding medium.

The island of Ceylon is peculiarly favourable for a study of the influence of
tropical temperature and moisture on the distribution of algal growth, since
the rainfall varies considerably in different parts of the island, while the
mountainous southern portion (attaining a height of 8000 feet and more)
produces a very considerable range of temperature, from the intense heat of
Colombo and other lowland places to the high-lying Nuwara Eliya, where, at
certain times of the year, the thermometer falls to below freezing point at
night. In fact, we have here confined in a narrow compass almost all the
different possibilities that can be realised in the tropics. It may be well at
this point to briefly outline the range of variation in this respect (see also
the map). The low country, with its high tropical temperature, shows great
diversity in the amount of rainfall; so that some localities are exceptionally

198 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

82°
CEYLON
Scale of English miles
10 (e) 19 20 O 40
Numbers printed thus:,25,etc = Tanks.
= Isotherms(Fahr® scale)

CO ——————— =Roads.

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AVERAGE ANNUAL RAINFALL.

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1907.] Fresh-water Algal Flora of Ceylon. 199

DESCRIPTION OF THE MAP.

The Map shows the different localities mentioned in the present paper, the distribution
of rainfall, and the isothermal lines (after Trimen and Wright). The bracketed numbers
placed below the place-names are amount of rainfall in inches. The unbracketed numbers
show the position of the tanks, which are as follows :—

1. Andankulam. 14. Kurunegalawewa. 27. Nuwarawewa.

2. Balaluwewa. 15. Madakanawewa. 28. Periyakulam.

3. Balankulam. 16. Madawachyawewa. 29. Punchikekirawa.

4, Basawakkulam. 17. Madokotaikulam. 30. Senadiniyagawawewa.
5. Borlasgamawewa. 18. Mahakadawellawewa. 31. Sigiri tank.

6. Dambullawewa. 19. Mahakekirawa. 32. Tibbotuwawewewa.
7. Habaranewewa. 20. Malawewewa. 33. Tirappanewewa.

8. Hiriwadunnewewa. 21. Mancadawewa. 34, Tissawewa.

9. Lake Kantelai. 22. Megaswewa. 35. Villamkulam.

10. Karambewawewa. 23. Lake Mineri. 36. Walikulam.

11. Kekunadurewewa. 24. Nalandewewa. 37. Wendrenkulam.

12. Kelawewa. — 25. Namoluwewa. 38. Yaka-anaguhuwewa.
13. Koilpuliumkulam. 26. Neravieawewa. 39. Kannia Hot Springs.

wet (eg., Kalutara, Labugama), others (under the prevalent tropical con-
ditions) very dry (eg., Hambantota, Vavoniya), whilst numerous intermediate
conditions between the two extremes are found (¢y., at Colombo, Matara,
etc.); the wettest place I visited was Labugama (166 inches). The amount
of rainfall at Peradeniya is not much less than at Colombo, but the lower
temperature, due to the higher altitude, has some influence on the algal
vegetation. In travelling upwards to Nuwara Eliya the gradual change of
temperature is very perceptible; the climate in the uplands is indeed
practically European, although not so cold in winter, and considerably hotter
in the middle of a sunny day (mean annual temperature a little over 60° F.).
The rainfall, however, is heavy (about 90 inches), and that produces an
abundant vegetation. If we further bear in mind that there is a coastal and
an inland region, we shall have roughly reviewed the diversity of climatic
conditions obtaining in the island.*

Since my stay in the island was of brief duration (August 21 to November
10, 1903) my account of the algal vegetation is necessarily very incomplete

* My travels in Ceylon may be subdivided into three distinct tours: (i) Along the
western and southern coasts as far as Hambantota, the time being devoted to a study of
the algal flora and Phytoplankton of the lagoons and estuaries at Negombo, Panadure,
Kalutara, Ambalangodda, Bentotta, and Matara, and to an investigation of the subaérial
Alge ; (ii) Into the northern plains as far as Vavoniya and Trincomalie, where I was
mainly occupied with a study of the Algee found in the inland fresh-waters (especially the
numerous tanks or irrigation-reservoirs) ; (iii) A brief visit to the uplands round about
Nuwara Eliya, which proved particularly interesting in comparison with the observations
made in the lowlands. In addition to that, I stayed intermittently at Peradeniya for
about a fortnight.

200 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

in places. I rarely stayed more than two or three days in any one locality,
so that although able to familiarise myself with a wide range of climatic
conditions and their influence on algal growth, I was unable to enter into
detailed study of any given problem. Moreover, my observations, of course,
merely deal with one phase of the algal vegetation, and it is possible that
many other peculiarities would come to light if the period of observation
could be extended over an entire year. I am, however, inclined to think that
algal growth in the tropics will not show so marked a periodicity as obtains
in our parts. The rice-fields alone are likely to be interesting from this point
of view (see p. 242), since the Algze in them are exposed to regularly
alternating periods of desiccation and inundation. The greater part of my
observations were carried out during the dry season, although I remained
long enough to see some of the effects of the heavy rains, which begin in
November. In many respects I was fortunate in being able to collect during
the dry period, since the rains would very soon make access to many of the
pieces of water studied exceedingly difficult. The dry season is, moreover,
the interesting one from our point of view, for we then have a very essential
factor influencing tropical algal vegetation in an acute stage, viz., the risk of
desiccation under the burning glare of the tropical sun.

The main purpose of the present paper* is to indicate some of the more
important differences between the subaérial and fresh-water algal vegetation
occurring in the tropics and that found in temperate regions, and also to point
out the essential variations in tropical algal vegetation under the influence of
diverse external conditions. Most of those who have written on the subject
of algal vegetation in the tropics—and the literature is practically confined to
a few short introductory remarks in the systematic floras—have emphasised
its similarity to that of temperate regions,t and little or no attempt has been
made at determining the differences ; this is mainly due to the fact that most
of the works on tropical fresh-water Alge are the outcome of casual, and on
the whole, probably very unrepresentative collections by botanical travellers
whose main interest lay in other directions. No one, as far as I am aware, has

* A large part of the expenses of the present investigation were covered by a grant
from the Government Grant Committee of the Royal Society, to whom I am much
indebted for the help thus afforded to me. I also take this opportunity of heartily
thanking all those who were kind enough to assist me in the course of these investigations,
either by their extensive hospitality or by direct help during the performance of the
work. I wish especially to return thanks to Dr. J. C. Willis, Myr. Carruthers, and
Mr. Wright, of Peradeniya, to Mr. K. Bamber and Dr. A. Willey, of Colombo, to
Mr. Nock, at Hakgalla, and Mr. Lewis, of the Forest Department. The entire illustration
of ee paper has been carried out by my wife.

+ Of, for instance, Lemmermann, “Ub. die v. Herrn Dr. Volz aa seiner Weltreise
gesammelten Stisswasseralgen,” ‘ Abh. Nat.-Ver.,’ Bremen, vol. 18, 1905, p. 148 e seg.

1907. | Fresh-water Algal Flora of Ceylon. 201

attempted to study the subject from the point of view put forward in the
present communication.* My object is thus to outline the general features of
distribution and biology, and I have rarely spent much time on specific
determinations. They would in most cases be quite valueless from the present
point of view, and would, moreover, have very considerably delayed publication ;
and it seemed well to write this general paper before one’s recollections and
impressions of the aspect of the algal flora had become dimmed by the lapse
of a longer period than has already passed since the work was undertaken.
Certain subjects, which are dealt with quite briefly in the present
communication, will be considered more fully later on. I hope ultimately to
be able to publish a complete systematic list of the Alge present in my
Ceylon collections, although I prefer to postpone such a publication until
I have had opportunities of further study of tropical algal floras.

Consideration of the Algal Flora.

The following special considerations are subdivided as follows :—

(a) Subaérial Alge, 7.2., those growing on trees, stones, walls, etc. (character
and distribution dependent mainly on temperature, moisture and degree of
shading) :—

(i) The lowland vegetation (p. 203).
(ii) The upland vegetation (p. 215).

(6) Algal vegetation of the inland fresh-waters (character and distribution
dependent mainly on temperature, aeration and composition of water) :—

(i) Alge of the tanks and other large inland masses of fresh-water
(p. 218).
(ii) Algze of roadside ditches and pools in the lowlands (p. 233).
(111) Algee of marshes and padi-fields (p. 241).
(iv) Algze of rock-pools (p. 242).
(v) Algze of wells and springs, «¢., aerated, but standing water (p. 250).
(vi) Algee of the small pools of the uplands (p. 252).+

This subdivision is in great part artificial, but it will be found that the
character of the algal growth in many of the collections of water mentioned is
on the whole sufficiently different to make such a separation useful. It must,
however, be pointed out, that in the case of Alge the delimitation of distinct

* Bohlin, in bis ‘ Etude sur la flore algologique d’eau douce des Agores,’ gives a some-
what analogous account of the algal flora of the subtropical Azores.

+ The second part of this paper, which I hope to publish in the course of the year, will
deal with the Algz of flowing water (rivers), and of the lagoons and river-estuaries, and
with the Plankton.

202 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

formations, except as regards the two fundamental groups of marine and fresh-
water Algz, is by no means so marked a one as in the case of the terrestrial
higher plants. It is often, only by a study of extreme cases, that we can draw
any definite limits between one kind of algal vegetation and another, for
generally numerous intermediate types are to be met with. Algae, and
probably water-plants generally, are much more plastic than terrestrial plants,
and many forms of aquatics are no doubt capable of adapting themselves to a
variety of modifications in their environment, But there are also amongst
the Alge certain forms which are much more susceptible than others to such
variations in their environment, and which are therefore of more limited
distribution, and these are the forms which will characterise certain prevailing
conditions in the surrounding medium. They are the character-plants, which
will help us to determine our aquatic formations, and it 1s requisite to study
them primarily, and to determine the exact conditions which influence their
presence or absence. Other species will then be found to be almost constantly
associated with these character-plants, and will make up the subordinate
members of the formation. Whereas amongst terrestrial plants each formation
probably always has one or more character-plants, which occur in this formation
only, and are absolutely distinctive of it, aquatic formations will often lack so
emphatic a character ; they will probably in many cases be distinguished by
the way in which the different members of the formation grow together,
rather than by any individual species. It even seems quite possible to imagine
two algal formations of almost identical specific composition, but differing
from one another biologically or physiologically ; as far as I am. aware, such
formations are not known to occur amongst terrestrial plants.

The preceding remarks are intended to indicate the scope for research in the
field of aquatic ecology, a subject on which practically nothing is known at
the present day, but the importance of which it is well to recognise at the
outset of considerations like those with which we are dealing. We are
almost entirely ignorant of the true factors which lead to the development of
certain algal forms in one piece of water and of others in a second. Certain
preliminary researches on this subject, which have been undertaken in the
past years on the British fresh-water algal flora, have given a number of
interesting results; some of these have been dealt with at another place, where
the subject of algal formations, briefly touched on above, is considered in
greater detail.* My time in Ceylon was, of course, far too limited to admit of
collecting more than a few meagre observations on possible tropical fresh-
water formations. Moreover it is quite impossible to fully characterise an

* See Fritsch, “ Problems in Aquatic Biology, with Special Reference to the Study of
Algal Periodicity,” ‘ New Phytologist,’ vol. 5, 1906, p. 149 e¢ seq.

1907. ] Fresh-water Algal Flora of Ceylon. 203

algal vegetation unless it has been studied, at least, throughout the course of

an entire year. So much seems probable, however, that a large number of the
algal formations of the tropics are quite different to those of our parts,
although certain of them may possibly be parallel in their nature (cf. especially
p- 240). On the whole, Phanerogams seem likely to be of more importance as
character-plants in the aquatic formations of our parts than in the tropics,
where the Alge will, probably in most cases, prove more distinctive; this
suggestion is, however, merely based on recollection of the aspect of the fresh-
water flora of the tropics, and careful investigation may quite disprove it.

(a) Subaérial Alge.

A consideration of the subaérial algal flora affords a good illustration
of the existence of important differences between tropical and temperate algal
vegetation.

(i) The Lowland Vegetation.—The first impression of an algologist after
landing in Colombo, which combines tropical heat with a fairly considerable
rainfall (88 inches), is the abundant algal covering on the walls of the houses,
on rocks, on exposed tree-trunks, etc. A rich growth of Alge on surrounding
objects is characteristic of every really moist locality, but it is only when heat
and moisture combine that we get so luxuriant a growth as in the tropics;
moreover, a few minutes’ walk about Colombo is sufficient to convince one that
the composititon of this algal growth is one quite unlike that of temperate
regions. One sees practically nothing of the Plewrococcus, Hormidiwm, etc., so
characteristic as epiphytes in our climates, green Algze being very inconspicuous
in all exposed situations (excepting Z'’rentepohlia, cf. below). Isolated colonies
of green unicellular forms are occasionally to be found in very subordinate
amount amongst unicellular Cyanophyceous (adhesive) growth, but pure green
coatings are completely wanting. ‘There are very few records in the literature
of the occurretice of green subaérial Algze in the tropics* (chiefly Plewrococcus
crenulatus, Hansg., P. Kiitzingu, West, and P. vulgaris, Menegh., Hormidiwm
murale, Kiitz),f and none of these records give us any idea of the quantity in
which these species occur; nor is it possible in the absence of accurate
descriptions of the habitat, to estimate the special conditions under which these
forms appear in the tropics.

The subaérialt Algee of the tropics nearly all belong to the Cyanophyceze

* Regarding terrestrial forms of Vaucheria, see p. 217.

_ + The existing data as to the composition of algal growth in the tropics are discussed
more fully elsewhere (‘ Annals of Botany,’ vol. 21, No. LX XXII, April, 1907, p. 239 ez seq.).

{ The term “subaérial” algal flora is used to denote all those forms not actually

growing in water. Epiphytic Algz constitute those growing on parts of other plants
(trunks of trees, leaves, etc.), lithophytes are those growing on rocky surfaces.

VOL. UXXIX.—B. , R

204 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

(Myxophycee) and, owing to this characteristic composition, the algal
covering is for the most part of a much more sombre hue than the bright
green one of our parts. This dark colour is only relieved here and there
by an occasional brighter patch, due either to a growth of Mosses, which,
however, do not apparently prosper very well in unsheltered situations, or to
the thin bright blue-green coating of an Oscillaria, or of some member of the
Chroococcacee (sensu Kirchner), which are often able to obtain a foothold on
very smooth walls, etc., which are unsuitable substrata for other forms.
Trentepohlia, with its reddish-yellow tufts, also forms a relatively frequent
and agreeable interruption to the dark Cyanophyceous growth.

Subsequent considerations (¢f. p. 224, et seg.) will show that the blue-green
Alge play a far more important part in the tropics than they do with us, and
a tropical fresh-water or subaérial* algal flora obtains its stamp from the
prevalence of this group. In order to realise the extent of development of
the subaérial Cyanophycez in the tropics, it is necessary to pay a visit to a
damp hot-house,t or to some hot spring,t where conditions of a similar nature
prevail. Here also we find the green Algee crowded out and superseded by
the blue-green forms.

Most of the Cyanophycee require two factors for abundant development ;
the first is plenty of moisture, the second a sufficiently high temperature.
These two factors determine the distribution and character of the blue-green
subaérial growth. The importance of the former factor is, of course, perfectly
obvious without much comment; it is also manifest in comparing the moist

* The only reference I am aware of that speaks of an abundant occurrence of subaérial
Cyanophyceous growth in tropical regions is one by Welwitsch (‘ Journ. Travel and Nat.
Hist.,’ vol. 1, 1868, pp. 22—36), on “The Pedras Negras of Pungo Andongo in Angola.”
The author describes the prolific growth of a species of Scytonema on the upper portions of
the mountains, and also refers to another species (Porphyrosiphon Notarisit) as covering
the sandy soil in the valley of the Cuanza River for considerable stretches (cf. also West
and West, in ‘Journal of Botany,’ vol. 35, 1897, p. 303; and Warming, ‘Okol.
Pflanzengeoor.,’ German Edit., Berlin, 1896, p. 215).

+ Cf. Fritsch, “ Algol. Notes, [V.—Remarks on the Periodical Development of the Algze
in the Artificial Waters at Kew,” ‘ Annals of Botany,’ vol. 17, No. LXV, 1903, p. 274; also
“ Alge,” in “The Wild Fauna and Flora of the Royal Botanic Gardens, Kew,” ‘ Bull.
Miscell. Inform. Roy. Bot. Gards., Kew,’ Addit. Series V, 1906, p. 187 e¢ seg. In the
Nepenthes-house at Kew the blue-green Algze even settle down on the leaves of the
cultivated plants (cf. also Schmidle, “ Epiphylle Algen,” etc., ‘Flora,’ vol. 83, 1897, p. 323
et seq.).

t See G. S. West, “On some Algze from Hot Springs,” ‘Journal of Botany,’ vol. 40,
1902, pp. 241248 ; W. H. Weed, “ Formation of Travertine and Siliceous Sinter by the
Vegetation of Hot Springs,” 9th Ann. Rep. U.S. Geol. Survey, 1887—88, p. 619 e¢ seq. ;
and F. Cohn, ‘“ Ueber die Algen des Karlsbader Sprudels,” etc., ‘ Abh. Schles. Ges. Vaterl.
Cultur,’ 1862, p. 35 e¢ seg. The vegetation in such springs is almost entirely composed of
Cyanophycez and Diatoms (see also Sect. 6 (v), of the present paper, p. 251).

1907.] Fresh-water Algal Flora of Ceylon. 205

lowlands of Ceylon with the drier regions having a lower rainfall. Thus at
Matara, on the south coast, and at Dambulla, in the Central Province, the
decrease in the extent of the algal covering is very marked; whilst in still
drier localities (¢.g., Hambantota and Negombo, on the coast, Anuradhapoora,
in the North Central Province), houses, rocks, etc., are practically clear of
any growth (or only bear small Mosses and Lichens), except in particularly
favourable positions. On the other hand, the heavier the rainfall at any
locality, under the same conditions of temperature, the more abundantly is
the blue-green element developed; the best example of this that I met
with was at Labugama (166 inches), where every available object is clothed
with blue-green forms. The importance of moisture is also well illustrated in
the colonisation of rugged vertical surfaces, since at first only the projections,
which are the points about which moisture mainly collects, become occupied
by Cyanophyceous growth.

The importance of a high temperature is also easily recognised. A visit to
a wet greenhouse in our regions, which is kept at a relatively low tempera-
ture, shows us that here green Alge and Mosses are the more successful
elements in the subaérial vegetation, whilst the Cyanophycee, where
developed, present a different aspect ; compare, for instance, the growth in the
Temperate and Tropical Fern Houses at Kew. The same observation can, in
some respects, be made in Ceylon, as we go higher and higher up the central
mountain range to Nuwara Eliya (see the discussion of the subaérial Alge of
the uplands).

Though temperature and moisture are in all probability the determining
factors in the distribution of the subaérial Cyanophycee within the tropics,
this group is provided with certain biological features, which must be
regarded as peculiarly suited to the dominant factors of the environment,
and enable it to exist so well under the prevailing conditions. In the first
place there is a well-marked alternation of a wet and dry period,* which the
Cyanophycee are well adapted to withstand, owing to the strongly developed
mucilage investments, which are so prominent a feature of the group, and
which enable them to withstand prolonged drought.t In this relation, it is
interesting to notice that forms with a sheath of semi-liquid diffluent

* See J. C. Willis, “The Royal Botanic Gardens of Ceylon as a Centre for Botanical
Study and Research,” ‘ Annals. Roy. Bot. Gards., Peradeniya,’ vol. 1, 1901, p. 17 ef seg. ;
also “Studies in the Morphology and Ecology of the Podostemaceze,” loc. cit., vol. 1, Part 4,
1902, pp. 278—279.

+ Hansgirg, “Ueber Bacillus muralis, Tomaschek, nebst Beitrigen zur Kenntnis der
Gallertbildungen einiger Spaltalgen,” ‘Bot. Centralbl.,’ vol. 35, 1888, pp. 54, 102;
B. Schroder, “ Untersuchungen tiber Gallertbildungen der Algen,” ‘Verhandl. Nat.-Med.
Ver. Heidelberg,’ New Ser., vol. 7, p. 183 e¢ seq., especially p. 185.

RZ

206 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

mucilage are far commoner in the uplands than in the lowlands, where the
risks of desiccation are much greater (see also below).* Secondly, we find
the subaérial growth of Cyanophycee developed in situations which are often
exposed to a strong hght and the presence of a second colouring matter
(phycocyanin) side by side with the chlorophyll, undoubtedly suits them to
an existence under such conditions. The presence of this additional pigment
shifts the assimilation-maximum,f but the phycocyanin, no doubt, also acts as
a kind of screen to the chlorophyll, although the exact modus operandi remains
an open question. The great variety presented by the colour of the cell-
contents in the Cyanophycee is familiar to all, yet we know practically
nothing about the meaning of these diverse shades of pigmentation.
Gaidukovt has experimented with Oscillaria from this point of view, and has
shown that the colour assumed by the cell-contents is complementary to that
of the supphed light, and that variation of the colour of the latter induces
corresponding changes in the colour of the Oscilaria-filaments. So that in
addition to the screening action§ of the phycocyanin it probably plays some
other important part in adapting the blue-green group to varying types of
illumination. The absence of any protective pigment is, no doubt, one of the
main causes of the lack of green algal forms in the subaérial vegetation,
although the rapid growth of the hardy blue-green element certainly
contributes very considerably to crowd them out. Mosses and Liverworts
are also mostly wanting in localities exposed to the strong light, although
they get on well enough as epiphytes on the tree-trunks in the moist shady
jungles and forests. It is interesting to notice in this connection that the
only member of the green Alge, which really competes successfully with the

* Cf. also Gomont, “ Myxophyceee hormogonee ” (in J. Schmidt., “ Flora of Koh Chang ”),
‘Bot. Tidsskrift.,’ vol. 24, 1901-2, p. 203. He remarks: “Si l’on examine le catalogue que
nous en donnons, on s’apercoit immédiatement que les Algues a gaines épaisses et colorées
V’emportent de beaucoup par le nombre des espéces et par leur fréquence. Ainsi, tandis que
les genera Oscillatoria, Lyngbya, Phormidium, Hydrocoleum, ne sont representés chacun
que par une espéce, les Scytonema et Stigonema en renferment treize 4 eux seuls
cette absence n’est pas purement accidentelle, mais, ainsi qu’on la maintesfois observé,
les plantes les mieux adaptées aux régions tropicales sont celles qui trouvent dans
Vépaisseur, la consistance ou la coloration de leur gaitne une protection contre les
sécheresses fréquentes et Vintensité de la lumiére.”

+ See Pfeffer and Ewart, ‘The Physiology of Plants,’ vol. 1, Oxford, 1900, pp. 348—344.

t “Uber den Einfluss farbigen Lichts auf die Farbung lebender Oscillarien,” ‘ Anhg. z.
Abh. Kgl. Preuss. Ak. d. Wissensch.,’ 1902; “‘ Weitere Untersuchungen tiber den Einfluss
farbigen Lichts auf die Fairbung der Oscillarien,” ‘ Ber. Deutsch. Bot. Ges.,’ vol. 21, 1903,
p- 484 e¢ seq.

§ In many cases the cell-contents no doubt obtain additional protection from the
intense light in the sheaths of the filaments, which are very frequently brown, yellow, or
red in colour. The colour of the sheath is also subject to much variation (cf the first
footnote on this page).

1907.) Fresh-water Algal Flora of Ceylon. 207

Cyanophycez in exposed situations, is the genus Z’rentepohlia ; the latter, as
is well known, is characterised by the presence of hematochrome in its cells,
and this probably also has a protective function against too intense illumina-
tion. Such an interpretation is suggested by the fact that the colour of the
filaments varies according to the intensity of the light, being more or less
green where shaded, and yellow to yellowish-red in the full light.* Species
of this genus are particularly common on the trunks of the cocoanut trees in
the plantations along the sea shore and on embankments by the roadside.

The subaérial Cyanophyceze in Ceylon exhibit a number of different
modes of growth, which are, no doubt, of the nature of adaptations to special
external conditions. My stay in the island was not sufficiently prolonged to
make it possible to analyse these conditions satisfactorily, but I am able to
put forward a number of probable suggestions regarding the factors favouring
the one or other mode of growth. The simplest and one of the commonest
habits assumed by the subaérial blue-green element may be styled the
“adhesive” mode of growth, the Alga being firmly adpressed to the
substratum ; this occurs in all the gelatinous} and encrusting unicellular forms
(Aphanocapsa, Aphanothece, Gleocapsa, Chroococcus, etc., fig. 1, B and D), and also
in certain of the filamentous genera (Nostoc, a few species of Calothrix, Oscil-
laria, Hypheothria, Lyngbya, especially the section Phormidiwm). On the whole
this type of growth rarely attains considerable thickness, a result probably of
the difficulties of interchange of gases in the inner layers of the stratum.
The only well-marked exception is the genus Phormidiwm, but here the inner
portions of the often thick lamelle merely consist of empty sheaths, while
only the outer parts contain living filaments. The forms exhibiting the
adhesive mode of growth are the first successful colonists of new ground, and
are especially adapted to grow on smooth surfaces, where they often form a
very thin, slippery film.

A second type of growth may be described as the “ tangled ” one; this (like
the remaining two) is confined to the filamentous forms, and is due to the fact
that the filaments are bound together in bundles, which are irregularly
interwoven with one another, much as in many of the submerged filamentous
Alge. The denseness of the tangles varies very considerably. In the majority
of cases, this growth was found to be relatively thin (although generally

* See also the statements on p. 248 of Oltmanns’ ‘Morph. u. Biol. d. Algen,’ vol. 1,
Jena, 1904.

t Species of Trentepohlia may become epiphyllous, like some of the Cyanophycee. I
did not observe any such cases in Ceylon, although that is probably due to the small
amount of time at my disposal. Other Chroolepidex are permanent leaf-epiphytes.

{ It should be pointed out, however, that these gelatinous forms are rather rare in the
lowlands, and far commoner in the moist uplands (cf. below, pp. 215, 216).

208 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

attaining greater thickness than an adhesive growth) before its surface became
colonised by other kinds of Algz (exhibiting tufted growth). In the few
cases in which a thick tangled stratum was observed, only the external portion
was generally found to be living, the inner part consisting largely of dead
sheaths. The tangled type of growth leads to the retention of a considerable
amount of air in the interspaces between the filaments, and this is probably
of advantage from a respiratory point of view in comparison to the adhesive
habit ; this suggestion has, however, first to be confirmed by experiment. On
the other hand, this mode of growth (like the adhesive one) is not well adapted
to the absorption of atmospheric moisture, since only few of the filaments
project freely into the air. Such growth is probably largely dependent for its
supply of moisture on the substratum on which it grows, and this is in
agreement with the fact that 1t prospers best on clay embankments or porous
rock-surfaces. As it gets thicker, it very often passes over into tufted growth
(see below and fig. 1, C) or becomes overgrown by the tufted growth of another
more successful form.*

The tangled type of growth is met with in subaérial forms of Zolypothria,
Hapalosiphon, Scytonema, Stigonema, etc., and in nearly all cases the filaments
are provided with firm consistent sheaths. In most of these genera (mainly
in Stigonema minutum, Hass.? and species of Tolypothrix) one occasionally
meets with tangles growing in protected situations, and exhibiting a
characteristic association with a film of air which gives their surface a bluish
grey or bluish colour and a silvery sheen, and not only occupies the inter-
spaces of the tangles, but covers their entire surface. The forms under
consideration seem to avoid direct moisture as much as possible, and grow in
situations which are more or less protected from the rain. ‘The air is held so
firmly in the interspaces of the tangle that it has not been dislodged by the
preserving fluid, but it seems probable that the impact of falling rain-drops
would be able to displace it, and that this is the reason why these forms are only
found growing in sheltered situations. The species exhibiting this association
with air, however, possess another peculiarity in the form of an irregular
incrustation on the sheath, completely absent at some points, but forming an
opaque covering which obscures the enclosed trichomes at others. In some

* It may be pointed out, however, that both from the point of view of respiration and of
absorption of moisture the various species and genera of Cyanophycex may differ markedly
among one another ; thus one form may be much better enabled to withstand the draw-
backs of an adhesive or tangled growth than another. That, under certain circumstances,
a tangled growth may be laden with moisture is evidenced by the fact that in damp
protected localities I occasionally found aquatic Alge (e.g., species of Ulothrix) growing
on the surface of the tangle. These Alge probably obtain the necessary moisture by
absorption from the tangle below.

SEZ
=

wr
SS
SSS SSE Sw
LIA Vis me IEE SK
SSE 7M

SSF.

>
LL

— > 7 Ss wy,
eae

oS
PS SSS
>

aments are shown). A (x 60).—Rather

209

a basal tangle, the former then
D (x 12).—Dense tufted growth

showing disorganised remains of the

upright branches arise from

4
3

C (x9).—Regular tufted growth of a Scytonema
branch abundantly, the ramifications being arranged to form a fairly regular palisade-like layer.

irregular tufted growth of a Tolypothriz. B (x60).—Tufted growth of a Tolypothrix,
of Schizothrix, arising from an adhesive base.

Fic. 1.—Illustration of various Types of Tufted Growth (only the sheaths of the individual fil
adhesive base.

bs
ys

210 Dr. F. E. Fritsch. On the Subaérial and -[Jan. 2,

cases this incrustation 1s due to carbonate of lime, but in other cases it is not
affected by acids, and it has as yet been impossible to determine its exact’
nature. It seems plausible to associate this incrustation with the presence of
the air, and possibly the latter may be a result of the formation of the
former. The phenomenon is certainly no casual one,* as it is to be met with
quite frequently in different parts of the island; the species exhibiting it are
in part common forms throughout the lowlands, and are by no means always
found with the associated air.

The third type of growth is the “tufted” one,t and is characteristic not
only of many of the Cyanophyceex, but also of the species of Zrentepohlia.t
In each tuft the filaments are firmly intertwined with one another to form
bundles (fig. 1), which stand out approximately vertically from the sub-
stratum. The free ends of the filaments thus project outwards, and are
occasionally considerably elongated and practically unbranched (frequently
in Scytonema and Tolypothriz); sometimes the projecting ends are of a
different (generally brighter) hue than the remaining parts of the filaments.
The tufts are often (notably in Schizothriz, fig. 1, D) so closely placed that, if
they all happen to be of the same length, it is difficult without the aid of a
lens to determine the composite character of the growth; on the other hand,
in the genus Symploca the tufts are generally quite isolated from one another.
Where a rich tufted growth occurs, the substratum presents a velvety
appearance owing to the numerous vertical tufts of intertwined filaments,
and this pilose covering is like a wet sponge in the rainy season. ‘The least
pressure squeezes water out of it. The tufted type of growth constitutes the
prevalent habit in Symploca, Schizothri« (fig. 1, D), etc., and a number of forms
which assume the tangled type of growth at first, subsequently raise their
filaments into tufts; such are Tolypothrix, Scytonema (fig. 1, C), Stagonema, ete.,
especially the species of the first-named genus varying very much in their
manner of growth,

* In a few cases, encrusting unicellular forms were found to exhibit the same charac-
teristic association with a film of air, although not growing in protected situations ; the
forms involved are species of Gleothece. Here also the thick outer lamellee of the sheaths
in most of the colonies were provided with a thin incrustation of a substance whose
nature I have been unable to determine. Under the microscope, one or more air-bubbles
are seen to be associated with each colony, being frequently drawn out intoa kind of beak,
the apex of which is in very close connection with the sheath of the Alga.

+ I must point out that the terms “tangled,” “tufted,” etc., refer to the macroscopic
appearance of the Alga. Under the microscope, many Algz of the adhesive type show a
tangle of filaments and, in the tufted form of growth, the filaments in each tuft are
entangled with one another to a varying extent.

t Some of the species of Trentepohlia exhibit a slight tangled growth at first, while the
basal disc (“sole”) is, of course, always adhesive.

1907.] ° Fresh-water Algal Flora of Ceylon. 211

These forms, and especially those exhibiting pure tufted growth («e., in which
there is little or no tangled base), readily absorb moisture from the substratum
by capillary attraction, whilst as long as the air is relatively dry (as compared
with the substratum), water will also be given off by evaporation from the
outer ends of the tufts, so that a constant circulation of water will take place.
On the other hand, when tufted growth occurs on a substratum which does
not furnish much moisture, the outer ends of the tufts will carry on the
reverse process, and absorb water-vapour directly from the air, provided the
latter is sufficiently moist. The tufted type of growth is thus liable to be
successful on all kinds of substrata provided the air contains the necessary
amount of moisture. These suggestions as to the biological value of tufted
growth seem to agree with the main facts of the distribution of the latter.
We should expect not to find tufted growth or to see it restricted to well-
protected situations in dry localities, in which both air and substratum furnish
little moisture ; and this is really the case, for at Anuradhapoora, Negombo,
etc., such subaérial growth as does occur is almost exclusively adhesive or
tangled.* On the other hand, the wetter the locality the more abundant does
this type of growth become; even at Kew it only occurs in the very damp
hothouses (¢g., the Nepenthes-house).

Atmospheric moisture thus seems to be the chief factor determining the
development of tufts, and there is good reason to look upon them as being
the result of a hydrotropic stimulus (¢f also below). The subaérial Cyano-
phycez under discussion, although they have left the aquatic habitat, are
still semi-aquatic in their requirements, and it is not surprising to find
moisture influencing them in various ways (z.¢., general distribution and mode
of growth). In moist localities (ie., where there is plenty of water-vapour in
the air) the advantage of the tufted as compared with the tangled type of
growth is obvious, especially in exposed situations where the surface layers
of the soil are very liable to be dried up by the heat of the sun’s rays, so that
absorption of atmospheric moisture is imperative. In damp localities, probably,
all tangled growth sooner or later passes over into a tufted one or becomes
colonised by some other species with tufted growth, but in dry localities the
latter affords too ready a means of evaporation and, consequently, is unfavour-
able. Without experimental study the conditions of respiration in this form
of growth can hardly be compared with those in a tangle; in all probability,
however, they are much the same in the two cases.

Some of the species of Zentepohlia quite conform to the above-described
method of growth (eg., 7. abietina, Hansg.), whilst others differ in the great

* These observations were, of course, made during the dry season ; possibly during the
wet period tufted growth may be more commonly developed at these localities.

212 Dr. F. E. Fritsch. On the Subaérial and ° [Jan. 2,

elongation of the filaments, which hang down in long tresses (eg., 7’. arborwm
(Ag.) De Willd.).

The last type of growth presented by the subaérial Cyanophycee is really
only a modification of the tufted one; it may be spoken of as “ stratified.” It is
found in the same species as exhibit the tufted habit, although on the whole it is
of rare occurrence—a result, probably, of the absence of the special conditions
which lead to its development. It was mainly observed in species of Tolypothrix
and Scytonema. The characteristic feature of this type of growth lies in the
fact that the closely-placed tufts are arranged in regular strata forming tiers
one above the other; each tier generally projects a little beyond the one
above it, and in this way a habit recalling that of some species of Hypnwm* or
one of Schimper’s “ Etagenbéume” is attained. Such growth is mainly to be
found on vertical walls or tree-trunks, where the species of Alga concerned
often formed huge coherent sheets, which with some little trouble could be
detached in their entirety. Since this type of growth occurs mainly in shaded
situations, it may be suggested that the projection of each tier beyond the
one above it enables the free growing ends of the tufts to obtain the maximum
amount of light ; in some cases these free ends were differently coloured to the
main inner mass, and these differences in coloration may be a result of the
illumination (see p. 206).. It seems possible, therefore, that “stratified ”
growth is the result of tufted growth on a vertical surface in a shaded
situation, and that stratification meets the requirements of illumination com-
bined with the largest possible extent of surface-development. But moisture
may also play some part in determining this type of growth, for, if one tier of
tufts is once established, others might gradually grow out below and elongate
sufficiently to catch up the drops of rain running off from the tier above ; in
this way tier upon tier might be formed. In this connection two points may
be noted: Firstly, the tufts of a tier in their entirety point downwards, so
that superfluous rain-water readily runs off one tier on to the next; and,
secondly, “stratified” growth occurs on substrata which probably do not
furnish much water in themselves. Both illumination and moisture may
thus be concerned in the development of the stratified mode of growth, but I
am inclined to regard the former as more important.

A few words may be added on the localities in which these different types
of growth seem to predominate, and on their inter-relation. In exposed
situations in the wet regions it is usual to find only one or other of the last
three types of growth (mainly the tufted one), so that ordinarily only such

* E.g., Hypnum (Hylocomium) splendens. Cf. Goebel, ‘Organographie d. Pflanzen,’ Jena,

1898—1901, p. 56, fig. 27. Such growth is, of course, of quite a different nature to the case
we are considering, but there is a considerable superficial resemblance.

1907 | Fresh-water Algal Flora of Ceylon. 213

substrata are colonised as are suitable for their development. Tree trunks
and smooth rock-surfaces, even in very wet localities, are often only provided
with a very poor algal covering, since the hard bark or rock is suitable only
for adhesive growth; the latter, however, probably meets with considerable
obstacles in the way of establishing itself owing to the very heavy downpours
of rain which scour the surface of the tree or rock. If these difficulties are
successfully overcome in early stages, adhesive growth may become
established, and will then, in course of time, prepare the way for other forms
of growth. Decayed trunks, of course, form a much more suitable sub-
stratum and generally bear rich algal growth of various kinds. In protected
situations adhesive growth becomes well developed, especially on walls,
rocks, embankments, etc., and is almost invariably the forerunner of tangled
and tufted growth. In many cases it is quite possible to detect the remains
of an adhesive growth at the base of tangled or tufted growth (fig. 1, B, D) ;
mostly, however, such adhesive growth is quite dead. It appears more
commonly to consist of unicellular encrusting forms than of filamentous
species, and the former by their decay probably afford a kind of soil for
further colonisation. In the dry regions, adhesive growth is predominant
everywhere. Heavy downpours of rain probably make it impossible for
larger gelatinous forms to obtain a foothold except in very sheltered
situations. Tangled growth, since it helps to bind the clay of an embank-
ment together, and to make it more suited to withstand a heavy downpour of
rain, is liable on such a substratum to be much more successful in exposed
situations than an adhesive growth; and such embankments may furnish
a foothold for tangled forms without any previous growth taking place,
although in many cases there is a passing preliminary growth of the adhesive
type. The tangles again give way to tufted growth, which generally gains
the upper hand ultimately. This tufted growth, as already mentioned above,
may belong to the same form as constitutes the basal tangle, or may be due
to a distinct tufted species. Thus Symploca is very commonly found growing
on a basal tangle of Yolypothrix, whilst in other places small Mosses are
found arising vertically from a similar base or even from a dense tufted
growth of one species or the other. In both cases I frequently found some
of the filaments of the basal tangle of Tolypothrix using the Symploca or the
Moss, as the case might be, as a kind of support round which they twined
themselves closely (fig. 2); they thus raise their filaments out into the air
and, in fact, form a kind of upright growth quite comparable to a tuft,* at

* This, no doubt, also illustrates the mode of formation of a tuft. One or two out-

standing branches first arise from a tangle, and these serve as supports for further
branches which grow up round about them.

214 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

Hy oaniese
EPROP) sity
Tey AES ED
s

Fie. 2 (x 72).—Tufted Growth of Tolypothrix, colonised by Bryophytes. The Alga is using
the latter as a support by means of which it raises its filaments up into the air. The
free ends of these filaments are appearing at all points. Only the sheaths of the
individual filaments are shown.

the expense of the Symploca or Moss. Such cases very clearly illustrate the

tendency of the filaments of a tangle to grow away at right angles to the

substratum, and the stimulus is here undoubtedly a hydrotropic one. The
ultimate effect of Moss, etc., being used as a support by the Alga on which it
first settles down will be to more or less kill the former, and it can only save
itself by growing out more and more as it gets entwined and killed off
behind. In this way a thick layer of humus will gradually accumulate and
serve as a base for the development of higher plants (small ferns, etc.).

This is probably the way (after preliminary adhesive and tangled growth) in

which bare rock-surfaces become gradually converted into substrata suitable

for all kinds of growth. The subaérial Cyanophycee play a great part in the
progressive colonisation of bare surfaces in the tropics, and to their agency
the wealth of vegetation on every conceivable object is primarily due.*

* This subject will receive fuller treatment elsewhere (see “The Adle of Algal Growth

in the Colonisation of New Ground, etc.,” in a forthcoming number of the ‘Geographical
Journal’); cf. also Treub, ‘Ann. Jard. bot. Buitenzorg,’ vol. 7, 1888, p. 213.

1907.] Fresh-water Algal Flora of Ceylon. 215

Gi) The Upland Vegetation—The following description is based on an
examination of the subaérial algal vegetation at Nuwara Eliya and
neighbourhood. Although only six days (in the early part of the rainy
period) were spent in this region, they were adequate for obtaining a general
conception of the character of the subaérial Alge, and I do not consider it
likely that other parts of the uplands will differ very materially. Nuwara
Eliya lies at a height of 6200 feet above the sea, and close at hand is
Pedrotallagalla, the highest point of the island (8296 feet). The rainfall is a
fairly high one (91 inches at Hakgalla) and, consequently, the subaérial |
Algee are well developed, nearly every rock, tree-trunk or embankment being
covered with them. This algal growth, however, bears a very different
stamp to that of the moist lowlands, whilst bryophytes and Lichens obtain
a foothold, and even attain an abundant development in situations which are
dominated by the blue-green Alge in the low country. In all shaded
localities, indeed, the greater part of the algal growth becomes completely
obscured by Mosses and Lichens; it is best developed on smooth rock-surfaces
or smooth clay-embankments in more or less exposed situations, which are
apparently not so favourable for the growth of the other plants mentioned.
A point which very soon strikes one with regard to the subaérial Algze of
the uplands is the great scarcity of the tufted mode of growth, so
characteristic of the wet lower regions. The majority of the Algz altogether
exhibit the adhesive type, and by far the most of them are of highly
gelatinous consistency. I have put together the following table with the
object of showing the relative frequency of the four types of growth in the
lowlands, in the country round about Peradeniya and in the uplands;
the percentages are calculated from the actual number of cases examined :—*

Type of growth. | Lowlands. | Peradeniya. | Uplands.
Per cent. Per cent. Per cent.
i AGNESIVE oo ceccsaks 25 AT °5 66 °5
Bir Marg led vai) 54 sinejees es 34 35 0 26 °5
Se ULEOC, © saiatsccidies aie aiviaaide 34, Al 17 °5 (Oe
4. Stratified ......cccess (A — —
No. of cases examined 56 40 30

The great prevalence of the adhesive habit is very manifest, and such
tufted growth as occurs rarely covers any great extent of surface; tangles
* The species of Trentepohlia, which always assume a tufted habit, are not taken into
account in the calculation of the above table ; their tufts are probably not dependent on

exactly the same conditions as the short dense tufts of the Cyanophycee, for they are just
as common in the uplands as in the low country.

216 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

are rather more frequent. Hmbankments are generally covered with a thin
slippery film of gelatinous forms (e.g., the whole embankment along one side
of Lady Horton’s walk at Nuwara Eliya), and only at a few points are
these overgrown by tangles or tufts. The Algze (Wostoc, Aphanocapsa,
(‘laocapsa, etc.) concerned in the formation of these gelatinous films are all
characterised by having very highly mucilaginous (almost diffluent) sheaths..
But itis amidst the thick growth of Mosses and Lichens on the trunks of
trees that these gelatinous Algze often attain their greatest development ;
both on the wooded slopes of Pedrotallagalla and in the jungle on the
Hakgalla rock the luxuriance of their development is remarkable. In most.
cases the Mosses are the more important epiphytes, but here and there
gelatinous Algee vie with them, either covering whole portions of the trunk
like the exuded resin or latex of an injured tree or hanging down in long,
jelly-like masses between the Mosses and Lichens.* Such growth would be
almost an impossibility on a bare trunk, whilst it easily obtains a foothold
and maintains its position amongst the growth of Mosses, etc. The rocks
occurring in these jungles are remarkably poor in algal growth, which is
probably due to the absence of forms with a type of growth suited to such a
substratum. An almost diffluent WVostoc is not well suited for attachment to
a smooth rock-surface in a rainy region, and such growth as does cccur on
these rocks is tangledt or very rarely tufted. The preceding remarks
indicate one of the chief factors in the uplands, viz., the competition of the |
Bryophytes, etc.; bearing this in mind, we can understand the scarcity of
tufted and tangled growth on moss-covered substrata, for it must inevitably
become overgrown sooner or later by the prolific Bryophyte and Lichen
vegetation.

There are thus very obvious differences between the subaérial Algze of the
lowlands and those of the uplands, but the exact factors leading to this
diversity are difficult to determine. As above indicated (p. 207, footnote),
gelatinous forms with semi-liquid, diffluent sheaths, do not get on successfully
in the lowlands, because they are probably too susceptible to desiccation.
In the part of the uplands studied, the relative humidity of the air is
considerably greater than in the lowlands, whilst the temperature is, of course,
lower, and these seem to be the chief factors determining the enormous

* The most important of these gelatinous forms are species of Gleocapsa, Glaothece,
Aphanocapsa, Nostoc,and Stigonema. Gleocapsa sanguinea (Ag.), Kiitz, possessing charac-
teristic red-coloured sheaths, was particularly common on some of the tree-trunks at.
higher altitudes. ;

+ In one or two cases J met with tangles associated with a film of air such as were

described for the lowlands on p. 208. They are, however, of rather rarer occurrence in the
uplands.

1907. | Fresh-water Algal Flora of Ceylon. | 217

development of gelatinous forms in these regions—in many localities to the
practical exclusion of other types of growth. But competition, encouraging
the growth of the more successful gelatinous forms, cannot always be the
explanation of the absence of tangled or tufted growth; such competition can
scarcely play a part on an embankment. Here it seems as though the
highly mucilaginous substratum afforded by the prevuiling gelatinous
adhesive growth is not a suitable base for growth of a higher type. A more
complete analysis of the prevailing conditions will probably cast more light
on the matter.

The subaérial algal flora of the uplands of Ceylon has a certain superficial
resemblance to that of a damp region in the temperate zone, but it differs in
its luxuriant development and in the important part played by the blue-
green element. The light-conditions in particular, but also the temperature
during the day (at least at most times of the year) are distinctly tropical—
witness the wealth of the Phanerogamic vegetation. The green algal
element, which is of considerable importance in the subaérial vegetation
of our parts, is still quite crowded out by the more successful blue-green
forms; their success is here again probably, to a large extent, due to adequate
protection from the strong illumination. It should be noted, however, that
I met with terrestrial species of Vaucheria (V. sessilis (Vauch.) DC.) at one
or two points round about Nuwara Eliya

forms which, as far as my observa-
tions go, are entirely wanting in the lowlands.

The subaérial algal vegetation at places like Kandy (rainfall, 82 inches)
and Peradeniya is approximately intermediate in its character between that
of the lowlands and uplands, although on the whole savouring more of the
former (cf. table on p. 215). In the wet regions of the lowlands we
have a profuse subaérial flora, which is probably in the main due to the high
temperature, whilst in the true uplands the rich algal growth is the result
of a rather high rainfall. At Peradeniya and neighbourhood both factors are
not so strongly developed, the temperature being low compared with that of
the low country, and the rainfall less than at Nuwara Eliya and neighbour-
hood. In correspondence with this we find a marked decrease in the extent
of development of subaérial algal vegetation at Peradeniya and in the
surrounding country. In the wet jungles on the hillsides round Peradeniya,
however (¢g., at Hantane), we meet with a state of affairs rather like that on
Pedrotallagalla, although on a less luxuriant scale. Otherwise the subaérial
algal vegetation in these regions stands behind both that of the lowlands
and uplands, and cannot even compare, for instance, with that at a place
like Matara (rainfall only 69 inches !). The character of the algal vegetation
is of particular interest. Tufted and tangled growths are fairly well repre-

218 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

sented (cf. table on p. 215)—especially the latter, which is no doubt a result
of the relative dryness of the atmosphere. Gelatinous adhesive forms are
much commoner than in the lowlands, although rarely exhibiting that
markedly diffluent character which is such a feature in the uplands; the
species of Vostoc, for instance, occurring round about Peradeniya, are more of
the foliose type with a fairly consistent investment. The arches of the
aqueduct in the experiment station at Gangarruwa, probably owing to the
abundance of moisture, are exceptional, since at most points they are densely
overgrown by a highly gelatinous JVostoc in all hues of brown and green to
almost colourless. |

A few words on the subaérial Algze found on the cliffs and rocks of the
sea-coast may be added, based on observations made at Matara, Ambalangodda,
Bentotta,and Trincomalie. In all cases the substratum is probably subjected
to occasional spray from the sea (in stormy weather), but otherwise the
conditions are identical with those influencing the lowland subaérial Alge
generally. Cyanophycee are the prevalent forms, and especially in the
higher zones their character 1s quite similar to that presented by the inland
forms above discussed. The lower parts of the cliffs and occasional rocks on
the sea-shore, on the other hand, tend to be colonised by a characteristic
assemblage of unicellular blue-green forms (species of Placoma, Radaisia,
Entophysalis, and Chroothece), which are more or less marine in character ;
their habitat is, no doubt, much more often reached by the sea-water, and
this factor excludes the ordinary inland algal growth to a varying extent,
whilst it calls forth the marine element just. mentioned. ‘The subaérial
aleal growth on the sea-coast finds an interesting parallel in the algal
vegetation of fresh-water pools, which are found in the sand of the sea-shore
at diverse points, and will be considered in Part II of this paper; in both
cases the occasional advent of sea-water brings in a modifying factor.

(b) Algal Vegetation of the Inland Fresh-waters.

In this section I propose to deal with the macrophytic algal vegetation
(both attached and floating) of the numerous pieces of inland fresh-water
(excluding rivers) I had the opportunity of studying. Since the essential
conditions influencing the character of the algal vegetation in these pieces of
water are much the same in all cases, I shall deal with them in detail in the
following discussion of the tanks, and merely point out the differences, when
speaking of the other pieces of fresh-water. Minor factors cause the
variation in the flora noticeable in the different collections of water.

(i) Alge of the Tanks and other Large Inland Masses of Fresh-water.—
The tanks are pieces of fresh-water of artificial origin, and often of very

1907. | Fresh-water Algal Flora of Ceylon. 219

ancient date; they occur all over the island, but are especially common
in the dry northern half. They have partly been made by the damming up
of broad river valleys, and partly by the collection of rain-water in specially
prepared hollows. In a few cases they occupy natural hollows in some
of the large basaltic masses which occur at many points (eg., Nalande
and Dambulla). Large numbers of small shallow tanks are found near
every village in the drier parts of the island. Tanks of medium size, and
apparently of some depth, are Madawachyawewa,* Kekunadurewewa,
Villamkulam,* Habaranewewa, Periyakulam, Kurunegalawewa, and we
may also include here the lakes at Kandy, Colombo, and Nuwara Eliya.
A few of the tanks are of very large dimensions; of these I was able to
examine the following (approximately in order of" size, beginning with the
largest): Lake Mineri, Kelawewa and Balaluwewa, Nuwarawewa, Tissawewa,
Basawakkulam, Lake Kantelai, the last named being shallow throughout.f
The algal flora is well developed in most of the tanks, and, as far as
the present state of affairs is concerned, their conditions of life are practically
natural. The special conditions may be briefly considered first. The illu-
mination is, of course, very intense, for the tanks are, as a rule, exposed to
the full glare of the sunlight.{ Trees and bushes only rarely come down to
the water’s edge, and the immediate neighbourhood of the bank tends
to be inhabited either by dead trees, whose bleached trunks and branches
look like spectres in the distance, or, at the best, only by a stunted growth,
owing to the frequent changes of water-level. The edges of the tanks
on the water-side are generally occupied by a rich growth of water-weeds

* The termination wewa is the Singhalese name for tank, kulam is the Tamil
designation.

+ It is almost impossible to obtain a boat on any of the tanks, and the presence of
crocodiles generally makes wading impossible. On the whole, therefore, my observations
are confined to the vegetation within a few feet of the water’s edge. It would have been
interesting to examine a few of the smaller tanks a little more completely, but the littoral
region is, after all, generally the most abundantly populated, and most likely to afford
important results from our point of view. In all cases I made a point of walking round
more than half the embankment of each tank, so as to obtain a good general idea of the
type of growth it contained. I was able to investigate Lakes Mineri (by the courtesy of
Mr. McPhail) and Kantelai more completely with the help of a boat, and also the lakes at
Colombo (by the kindness of the Colombo Rowing Club), and Kandy (by the courtesy of
Mr. Baird).

{ Oltmanns has recently pointed out (‘ Morph. u. Biol. d. Algen,’ vol. 2, 1905, p. 190),
that an important consideration with regard to the conditions of illumination influencing
aquatic algal growth is the quantity of light which gets reflected at the surface of the
water. Not only is the intensity of the light much greater in the tropics, but, owing to
the more or less vertical position of the sun, a far smaller percentage of the light is
reflected at the surface. I was only rarely inconvenienced by the glare of light from the
surface of the tanks (cf. also Ewart, in ‘ Annals of Botany,’ vol. 12, 1898, p. 379).

VOL. LXXIX.—B. =

22,0 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

(Myrophyllum iwmdicum, Trapa bispinosa, Nympheas, Neptunia oleracea,
Aponogeton monostachyon, Limnophytum obtusifolium, Chara, ete.), and
beneath these numerous algal forms generally find shelter. In some tanks
the muddiness of the water, in others an abundant Plankton (notably
Clathrocystis !) serve as a screen to the remaining life in the water. As
a general rule (which is, however, by no means without exceptions, cf. table
on p. 224), a tank with a poor Plankton is not rich in any kind of algal
growth (eg., Kurunegalawewa, Nalandewewa), whilst one with a fairly
abundant Plankton generally contains a good deal of other algal vegetation
(eg.. Kelawewa, Lakes Mineri and Kantelai). On the other hand, it seems
that, in some cases, the Plankton may become so extremely abundant that
it does not allow of the passage of sufficient light for any other noticeable
growth; this was the case in the tanks at Dambulla (Clathrocystis !) and
Habarane (Clathrocystis and Pediastrum!). The Plankton is, of course,
in no way the only source of shading in the tanks, and where there is
a well-marked growth of water-weeds, for example, these may shelter
abundant Algz, although the Plankton is poor.* The light to which
the aquatic Alge of the tropics are subjected, however, always remains
very strong, and it is, therefore, not surprising to find the Cyanophycee
again predominating (probably owing to their protective pigment, cf. p. 206),
although, perhaps, not to such a marked extent as in the subaérial vegetation

We have already, in the preceding section, learnt to associate the
Cyanophyceous element with a high temperature, and this is, of course,
the second condition to which the flora of tropical waters is subjected.
Leaving the lakes at Kandy and Nuwara Eliya out of consideration, the
approximate mean daily temperature of the water near the edge of the
tanks is 28°5 C.; the lowest temperature observed’(6 am.) was 25° C.,
which is only attained on hot summer days in our parts; the highest
temperatures (from 10 a.m. to 2 p.m.) range from 30° to 35° C.,f the latter
in shallow tanks like most of the smaller ones, as well as Kantelai and
Sigirl; in the deeper tanks (e¢g., Villamkulam, Dambulla tank) the tem-
perature does not seem to rise much above 30° C. In the shallow tanks
the range of daily temperature is very considerable (viz., from 25° to
34°°5 C. in Lake Kantelai, for example) and, in the small tanks, such a wide
range is probably the rule. Even in the deeper tanks the range is apparently

* Attention may be drawn to the fact that in the absence of water-weeds, the green
macrophytic algal growth of the tanks is almost invariably attached; floating green
patches only occur in those tanks in which the abundant growth of large aquatics
furnishes a considerable shelter against the strong light.

+ The temperature of a warm bath is about 35° C.

1907.] Fresh-water Algal Flora of Ceylon. 221

considerable, and not far behind that of the air (eg., m Villamkulam, water
from 25° to 30° C.; air from 24° to 30°5 C. in the shade) at least, as far as
the surface-layers of the water are concerned. These observations, which, of
course, refer to the temperature round the margins of the tanks only, and
apply also to all the different kinds of inland fresh-waters, show that a very
high temperature is frequently attained during the day, and that there
is a considerable, though gradual, cooling over night, which, however, never
results in a temperature which would be considered anything but warm
in our parts. It seems probable from what is known that a considerable
number of fresh-water Algez prefer rather cold water,* and this may be
one of the causes of the practical absence of a number of green forms from
the fresh-waters of the tropics (¢f. below). On the other hand, most of the
Cyanophycesz seem to flourish best in warm waters, and hence their pre-
ponderance in the aquatic flora of Ceylon. The relatively high temperature
of tropical waters, however, also involves the solution of a proportionally
smaller amount of oxygen and carbon dioxide. Especially the smaller
percentage of oxygen, and the consequent greater difficulty of respiration,
seems likely to be an important feature, although it remains to determine
its influence by exact experiment. I incline to asume that this is one of
the chief causes of the practical absence of certain temperate genera from the
waters of the tropics, and that it also accounts for certain peculiarities
of tropical fresh-water algal growth.t

The temperatures of the upland waters are, of course, in no way so high ;
thus lake at Kandy (10.30 am.) = 25° C.; lake at Nuwara Eliya (8 a.m.)
= 16° C., the latter being almost temperate, although on hot sunny days the
water probably becomes rather warmer than it ever does in our parts. The
range of daily temperature is certain to be very considerable here (cf.
introduction).

The third condition to which the algal vegetation of the tanks is subjected is
a frequent change of water-level, which probably takes place with a rapidity
unknown in our parts. During the dry period some of the smaller tanks
decrease to more than half their normal size, and are then surrounded by very
poor meadow-land with stunted grassy growth, the external portions of which
are often occupied by the dead trees above referred to; the limit of the tank in
the wet season is well marked by the fringe of dense jungle, which begins some

* Cf. Oltmanng, loc. cit., vol. 2, 1905, pp. 186 and 187.

t Cf. Warming, ‘Okologische Pflanzengeographie, Germ. Ed., Berlin, 1896, p. 121.
“Dass das Absorptionsvermégen des Wassers fiir Gase mit steigender Temperatur
abnimmt, ist vielleicht der wesentlichste Grund, weshalb gewisse Wasserpflanzen im
Sommer ... verschwinden” ; ¢f. also loc. cit., p. 124.

s 2

222 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

little way behind high-water mark. Towards the end of the dry season even
the larger shallow tanks are very much reduced, being surrounded by large
areas of meadow-land, marshy in character near the water but dry and
bleached beyond. In the deeper tanks (¢.g., Mineri, Nuwarawewa) the sinking
of the water is not so noticeable, although the bleached algal remains on the
exposed stonework of the dams bear testimony to the height of the water in
the wet season. The rise of the water-level can take place very rapidly, and
the change produced by a single night’s heavy rain is remarkable.* As soon
as the rains commence the shallower tanks are surrounded by a wide stretch
of shallow water devoid of any algal growth; this latter will, of course, put in
an appearance subsequently, but the few tanks that I was compelled to
examine in the (beginning of the rainy) season gave quite barren results in
this respect. That an abundant growth of Alge and other water-weeds does
often take place in the later portion of the rainy period is evidenced by the
decaying remains left behind on the sinking of the water-level in the dry
season. Where the irrigation-works are in working order they tend to
prevent the water-level from rising above a certain height.

More important than the actual variation in the height of the water-level
are the consequent sudden changes in the concentration of the matter dissolved
in the water; this cannot be without effect on a group so delicately constituted
as that of the fresh-water Alge, and possibly this is a cause for the poor
development of the algal flora in some of the tanks.

Whereas the three preceding conditions (light, temperature, and change ot
water-level) are practically the same in every piece of lowland fresh-water
(especially the last two), we have now to consider those factors which differ
from tank to tank and are in all probability frequently the cause of the minor
variations in the algal flora; these are: chemical composition of the dissolved
matter in the water, degree of movement in the water, nature of the sub-
stratum, and muddiness of the water. These (together with the conditions of
illumination) are just the same features as are probably the main cause of the
variation in the algal flora of temperate waters, but in the tropics the
dominant conditions (light, temperature) are the determining factors for the
general character of the fresh-water flora and, consequently, the minor
conditions have a rather different basis to work upon. My time was too
limited to enable me to collect many data regarding these minor factors and

* According to Mr. McPhail, the local engineer, the water-level in the huge expanse of -
Lake Mineri may rise as much as 10 feet in the course of one night. This tank is supplied
by a number of mountain streams, which, when the rainy season commences, pour large
masses of water into it. At Lake Kantelai I observed a rise of several inches in the
course of a single night.

1907. | Fresh-water Algal Flora of Ceylon. 223

their bearing upon algal vegetation in the waters of Ceylon, but I saw
enough to satisfy myself that a careful study would probably reveal somewhat
analogous algal associations in the tropics to those occurring in our parts of
the world.

I will now briefly consider the minor factors, and will begin with the little
I can say regarding the chemical composition of the water. Some of the
tanks must certainly be rich in dissolved organic substance owing to the
large amounts of water-weeds they contain ;* in other cases the feeces of the
abundant water-fowl must contribute to the organic contents. The frequent
presence of Characez probably means dissolved carbonate of lime.

Most of the smaller tanks are sheltered from the ordinary winds, and the
water, even at the edge, was usually quite undisturbed at the time of my visit.
In the larger tanks, on the other hand, a relatively large surface is exposed
to the wind, so that there are usually numerous small waves at the edge; the
littoral flora is therefore growing in water, which is frequently disturbed and
well aerated (especially noticeable in Nuwarawewa, Tissawewa, and Lake
Miner).

The substratum in the tanks is muddy or clayey except where the tank
occupies an excavation in the rock (cf p. 219). In Balaluwewa and
Villamkulam part of the substratum seems to be sandy. The dams are
often mere clay embankments, but in a number of the larger tanks are made
up of blocks of rock, and these rocks, of course, afford a suitable base for
lithophytes when under water.

In many of the smaller tanks the suspended particles of mud are of an
exceedingly finely divided nature, giving the water a yellow colouration ;
these particles are so fine that they often take several days to settle com-
pletely. The water of these tanks (e¢g., Punchikekirawa, Mancadawawewa)
is generally, but not always (eg., tank between milestones 101 and 102 on
road from Vavoniya to Madawachya), poor in organic life, and this latter
consists mainly of animals.

Briefly summarised, the conditions of life of the macrophytic Alge in the
Ceylon tanks are thus as follows :—Strong illumination; high temperature of
considerable daily range (varying from 6° to 10° C.); small amount of

* Thus, especially in Tank Basawakkulam and Lake Kantelai, the edge was fringed by
an accumulation of partly bleached and partly putrid remains of Chara, Myriophyllum,
etc., many yards in extent, and I have little doubt that such a mass of decomposing water-
weeds forms the bottom of the entire lake. Round about the edge of Lake Mineri I also
found Chara washed up in large amount. It is a well-known fact that especially
Cyanophycere prosper very well in a medium containing dissolved organic substance
(cf. p. 227, and also Kirchner, in Engler u. Prantl, ‘Die Natiirl. Pflanzenfam.,’ I Teil.,
Abt. 1a, 1900, p. 63). ana

224 Dr. F. E. Fritsch. On the Subaérial and — [Jan. 2,

dissolved oxygen in the water ; periodic (often rapid) change of water-level,
causing exposure for the attached forms of the higher zones and change in
concentration of the water for the forms of the lower zones; mostly a muddy
or clayey substratum with occasional rocks (detached from the dams); water
which is generally almost stagnant.

Looking upon the algal flora of the tanks in its entirety (we may even
for a moment include the Plankton in these remarks), one is struck by
the fact that in a considerable number of them it is ecmpletely dominated
by the blue-green element.* The table below may serve as an illus-
tration of this point; the various tanks studied are classified in three
series according to the composition of Benthos and Plankton. Those tanks
of the first series which are provided with an asterisk contain practically
nothing but Cyanophycee ; the remainder have a noticeable green element,
although still entirely dominated by the blue-green. It also seems of
interest to notice the relative amounts of Phanerogamic vegetation present ;

Table to show Essential Composition of Algal Growth in Ceylon Tanks.

Littoral growth, blue-green. Plankton only, blue-green. Plankton, green.

Plankton, blue-green. Littoral growth, green. Littoral growth, green.
Colombo Lake (!). Villamkulam. Pond in Peradeniya
Kandy Lake (1). Tirappanewewa.t d Gardens.

Nuwarawewa. Balaluwewa. ZS ( Kelawewa.t
3 ( Tissawewa (!). Periyakulam (near Trin- |S | Mancadawewat (animals
‘© | Basawakkulam. comalie). | } common).
< |} *Tank in Anuradhapoora 8 1 Punchikekirawa.f{
5 Gardens. H Ekiniyeawewa.t
z | *Town tanks, Anuradha- 4 | Malawewewa.t
r= poora. Balankulam{ (near Anu-
= = Madokotaikulam (near Va- radhapoora).
voniya). vhs saestils Ds Rv SUI A Pete a Madakanawewa.f
Madawachyawewa. p Mahakadawellawewa.
*Dambullawewa (!) Littoral growth only, Namoluwewa.
(Plankton only !). blue-green. Megaswewa (!)} (Plankton
*Neravieawewa (!). Plankton, green. only !).
Habaranewewa ('!) Wendrenkulamf (Plankton
(Plankton only!). only !).
Tank at Sigiri. Nalandewewa (!)f. Kurunegalawewa (Plankton
*Pokuna on top of rock| Walikulam (near Keki- only !).
Sigiri (!) (Plankton rawa). Senadiniyagawawewa (!)
only !). Karambawewewa. (near Habarane) (Plank-
Lake Mineri. Yaka-anaguhuwewa (near ton only !).
*Lake Kantelai. Habarane). Borlasgamawewa.t{
*Andankulam. Mahakekirawa. Tibbotuwawewewa.
Hiriwadunnawewa (near Kekunadurewewa (!)f. Koilpuliumkulam (near
Habarane). Vavoniya).
Malawewewa.{

* T, of course, exclude the green Phanerogamic vegetation, which is often abundantly
developed in tanks which are otherwise completely dominated by the blue-green forms.

+ Plant life very poorly represented.

{ With a noticeable admixture of blue-green forms.

1907. | Fresh-water Algal Flora of Ceylon. 225

where this latter is poorly developed, an exclamation mark in _ brackets
is added.

At first sight this table may not appear to bear out the above statement of
the dominance of the blue-green element very clearly, but a consultation of
the map will show that all the large and important tanks and lakes come
into the first or second columns, the only noticeable exceptions being
Kelawewa and Kurunegalawewa.* I may also point out that of the
tanks enumerated in the third column, five (all near Kekirawa) are
probably in connection with one another and with the waters of Kelawewa,
and that a considerable number of them (five) are very poor in
organic life altogether, which cannot be said of any of those in the first
column. I have endeavoured to bring out this latter point more clearly by
employing heavy type for those tanks which have an abundant algal
vegetation (either littoral or pelagic or both). It will be seen that whereas
nearly all the tanks with a dominant blue-green vegetation are rich in Alge,
this is only the case with respect to two of the tanks in the third column.
In further illustration of this point, I append lists of tanks in which the
green and blue-green forms respectively are negligible (the same
arrangement of type is preserved) :—

Green Element negligible. Blue-green Element negligible.
Basawakkulam. Mancadawawewa.
Tank in Anuradhapoora Gardens. Punchikekirawa.
Town Tanks, Anuradhapoora. Tibbotuwawewewa.
Dambullawewa (Plankton only !). Malawewa.
Madawachyawewa. _ Senadiniyagawawewa (Plankton only !).
Neravieawewa. Namoluwewa.
Pokuna on top of Sigiri (Plankton only !). _ Megaswewa (Plankton only !).
Lake Kantelai. Wendrenkulam (Plankton only !).
Andankulam. Kurunegalawewa.
Hiriwadunnawewa. . Borlasgamawewa.

Mahakadawellawewa.

As an outcome of these considerations, we may state that whenever there
is a well-developed algal flora in the considerable number of Ceylon tanks
and lakes I have examined there is always a noticeable blue-green element
which very frequently dominates the entire algal growth. If we now attempt
a comparison of the features just discussed with those of other tropical

* It is an interesting point that the dominance of the blue-green element is most
pronounced in the larger tanks. Thesmaller tanks probably present conditions somewhat
analogous to those prevailing in the smaller pools and ditches (viz., greater risk of sudden
desiccation, and stagnant water with less aeration ; cf. the next section of this chapter),

The smaller tanks thus gradually carry us over to the small pieces of water considered
subsequently.

226 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

regions, we meet with serious difficulties, for our existing tropical algal floras,
though doubtless of considerable systematic and phytogeographical value,
are practically useless from this point of view. I have nowhere found a
particular comment on the dominance of the blue-green element, although
Messrs. West and West* remark on the presence of well-developed
individuals of Clathrocystis in tropical Plankton. However, I do not hesitate
to suggest that in other parts of the tropics the blue-green element plays
the same important part that it does in Ceylon, in some cases to a less, in
others, perhaps, even to a more marked extent, according as the external
conditions vary. I shall return to this subject elsewhere,f and need here only
point out that in view of the very distinct aspect of the fresh-water algal
vegetation in the tropics it is certainly astonishing that its general character
has not previously been subjected to a critical analysis.

I will now endeavour to give a brief sketch of the essential character of
the blue-green macrophytic Ales (excl. Plankton), and of their mode of
occurrence in the different tanks of Ceylon.{ The blue-green vegetation
forms either (a) a dense felt or tufted growth or adhesive film on submerged
rocks, tree-stumps, etc.; or (0) floating masses, the latter in particular often
attaining a very prominent development. The more important and charac-
teristic floating forms belong to the genera Oscillaria, Lyngbya, Tolypothria,
Scytonema, Hapalosiphon, and fivularia. A member of the second genus
(LZ. majuscula (Dillw.) Harv.) is exceedingly common in many of the
tanks (eg., Nalandewewa, Tissawewa, Balankulam, Madawachyawewa,
Habaranewewa, etc.), forming large dark-green floating masses of densely
interwoven coarse filaments. At Nalande and Anuradhapoora it completely
dominates the vegetation of all the waters fed by the respective tanks; at
the latter place many of the smaller tanks threaten to become completely
silted up by the growth of this Alga and by the mud which collects amid
the tangle of its filaments. Other (smaller) species of Lyngbya also occur in
the tanks (eg., in Villamkulam, Andankulam), but play no very important
part (see, however, the next page). |

Another very important constituent of the algal vegetation of the tanks
are species of Rivularia (Gleotrichia). They absolutely dominate the algal flora
in Basawakkulam (Gleotrichia Rabenhorstw, Bornet), and also play a great part

* West and West, “Observations on the Conjugate,” ‘Annals of Botany,’ vol. 12,
No. LXV, 1898, p. 36.

t+ § Annals of Botany, April, 1907, p. 243 et seq.’

t When the water of the tanks falls in the dry season, a number of pools are usually
left behind, which, of course, again constitute a part of the tank in the wet season. The

vegetation of these pools is taken into account as a constituent part of that of the tank in
the following remarks, wherever it is deemed necessary.

1907. | Fresh-water Algal Flora of Ceylon. 227

in Lake Kantelai (Gleotrichia natans (Hedw.), Rabenh.). These two tanks, it
is interesting to notice, are both characterised by a very rich growth of water-
weeds, so that their waters certainly contain a very considerable percentage
of organic substance (cf. above, p. 223, footnote); further, they are both practi-
cally stagnant. Whether these factors are directly related to the abundant
occurrence of Aiwularia in these tanks must for the present remain an open
question. Whereas in Basawakkulam the Glwotrichia is the only common
Alga, this cannot be said of Lake Kantelai, where another blue-green form
(Lyngbya crugineo-cerulea (Kiitz), Gom.) is exceedingly common, and shares
the dominance of the former. The Lyngbya (like the Gleotrichia) is attached
to the segments of the abundant water-weeds, covering them with its thin
flocculent films for considerable stretches: in places this growth is so profuse
that it forms thick yellowish-green masses immediately below the surface of
the water. Exactly the same species occurs in abundance attached to the
submerged aquatics (Myriophyllum and Chara) in the open waters of Lake
Mineri (together with small quantities of Glwotrichia), but here it forms very
delicate films, tending to break up on being touched. In both Mineri and
Kantelai, however, the films do not consist of pure Lyngbya, but give shelter
to a considerable number of unicellular forms (various blue-green species ;
Diatoms, notably Epithemia and Pinnularia; Desmids, e¢g., species of
Cosmarium), the whole constituting a very characteristic assemblage of algal
species: The Diatoms and Desmids occurring in these films are in part quite
identical for the two tanks, and the films are thus a good example of the
“consortia” discussed below.* It is only open to surmise as to whether
there is anything more than a kind of “ space-parasitism ” in the case of these
composite films,f but there seem possibilities of more important (nutritive ?)
interrelations which we are not yet in a position to understand.

Oscillaria, though rarely of great importance in the floating flora of the
tanks, was a prominent feature in Mahakekirawa; here O. tenuis, Agardh.
var. natans (Kiitz),»Gom. occurred as small floating discs (2 to 6 inches in
diameter, and often almost perfectly round) on the surface of the water. The
filaments are arranged in a radiate manner in these discs, which also include
a considerable quantity of yellowish mud, serving to increase their compact-
ness. No doubt the discs originate on the mud at the bottom of the tank, and

* See p. 247 and cf. Fritsch, “ Problems in Aquatic Biology,” etc., ‘New Phytologist,’
vol. 5, 1906, p. 157.

+t Inasmuch as Lakes Mineri and Kantelai are the only large tanks I have had the
opportunity of studying from a boat, I consider it very probable that. such Lyngbya-
films may occur in the open water of a number of the other tanks, especially as the films
are not at all commonly found near the banks, but attain their greatest development over
the deeper portions of the tank.

228 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

are ultimately raised to the surface by gas-bubble development (cf. below,
p. 240). Similar discs are sometimes to be observed in temperate regions, but
their development has, as far as I am aware, not been studied.

Scytonema (S. tolypotrichoides, Kiitz) was a dominant form in only two of
the tanks, viz., Madokotaikulam near Vavoniya and Karambewawewa, near
Anuradhapoora. In both cases it formed spherical zegagropilous masses floating
round the margin and consisting of large quantities of knotted, more or less
radially arranged filaments, frequently of a yellowish colour on the surface,
although deep blue-green in the interior of the spherical structure. The
tangle of filaments, no doubt, includes air, by virtue of which the masses,
which are often about double the size of a cherry, float. The mode of
development of these masses has not been studied, but is probably analogous
to that of the egagropilous Cladophoras,* depending on successive movement
of the growing structure into various positions by the currents in the water.

There is much greater specific diversity to be found in the attached
Cyanophyceous growth than among the floating forms, which, as the above
remarks will have shown, are referable to a small number of species ; the most
important attached forms belong to the genera Oscillaria, Lyngbya, and
Tolypothriz. In comparison to our parts, the practical absence of unicellular
species in the attached blue-green flora is noteworthy. The dense pilose tufted
growth found on submerged rocks (especially Lake Kantelai), tree-stumps,
etc., is usually not due to a single species, but is composite (both blue-green
and green with intermingled Diatoms, viz., Synedra, Navicula, Surcrelia).
There is probably no essential difference between the attached growth in the
majority of the tanks, and that found in many of the small pools (cf. below):
in both cases we are dealing with algal consortia.t

We will now turn our attention to the composition of the green algal
element in the flora of the tanks. It exhibits a number of characteristic
features (common in part to the algal flora of all the inland waters), which are,
on the whole, just as striking as the dominance of the Cyanophycee, viz. :—

(a) The practical absence or great scarcity of certain genera, such as
Cladophora, Rhizoclonium, Vaucheria, Ulothriz, and Conferva, and the conse-
quent scarcity of a number of epiphytic Diatoms so characteristic of the fresh-
water algal flora of our parts (¢.g., Cocconeis Placentula, Synedra splendens).

(b) The replacement of Cladophora by the genus Pithophora.

* Cf. especially Brand, “ Die Cladophora-AXgagropilen des Siisswassers,” ‘ Hedwigia,’
vol. 41, 1902, p. 34 e¢ seg.; and Wesenberg-Lund, “Sur les Agagropila Sautert du lac
de Sor,” ‘ Bull. Ac. R. d. Sci. et d. Lettres Danemark,’ 1903, No. 2, p. 167 e¢ seq.

+ A few further facts regarding the attached growth on rock-surfaces in the tanks are
mentioned in the section dealing with the algal flora of the rock-pools (p. 248).

1907.] Fresh-water Algal Flora of Ceylon. 229

(c) The important part played by the genus Spirogyra to the practical
exclusion of other filamentous Conjugates, and the special systematic
composition of this Sprrogyra-element.

(d) The usually marked absence of broad filamentous forms (excepting
Spirogyra).

The genus Cladophora and its ally Rhizocloniwm are exceedingly rare in
Ceylon,* and the explanation of their absence seems no very difficult one.
In our parts they generally frequent water which is well aerated, and in
larger pieces of water, for instance, are in great part confined to near the
banks, where there is plenty of motion owing to the wind.t The practical
absence of these two genera in the fresh-waters of Ceylon is probably due
to an insufficiency of dissolved oxygen. The thick coarse walls and broad
filaments of these genera are not well suited for a ready diffusion of
gases. The absence of Vaucheria also may be due to its broad filaments,
although (as far as the tanks are concerned) this genus is not a very
common constituent of lake-vegetation, even in our parts. Vaucheria,
however, has a decidedly different assimilatory process to that of most other
green Alge, and it is possible that this process does not go on readily
amid tropical conditions. This view may receive some measure of support
in the fact that the entire group of the Heterokontz (Confervales, Borzi),
which probably have a similar assimilatory process,f is very rare in the
lowlands of Ceylon.§ Ulotrichacez are certainly also§ rare in Ceylon, and
their scarcity is not easy to understand. Some species of Ulothriz are
known to favour aerated water (although this does not apply to all of them),
and possibly the small percentage of dissolved oxygen may have something
to do with their absence. We have practically no data regarding the
influence of the amount of dissolved oxygen on the different genera of
Algze, but it seems very likely that some of them may be far more susceptible

* The following remarks (to last paragraph on p. 7a) apply to the fresh-waters of the
‘Ceylon lowlands as a whole.

t Cf. Chodat, “ Algues vertes de la Suisse,” ‘ Pleurococcoides—Chroolépoides,’ Berne,
1902, p. 77; also p. 90.

{ Regarding this assimilatory process, see especially K. Bohlin, “Studier dfver nagra
slagten af Alggruppen Confervales,‘Borzi,” ‘Bih. K. Svenska Vet.-Ak. Handl.,” vol. 23,
Afd. III, No. 23, German Résumé, pp. 52—54. The chloroplasts contain a relatively
Jarge amount of xanthophyll, and the first product of assimilation appears to be a glucose,
which subsequently becomes; transformed into oil. The assimilation is thus decidedly
different to that of the remaining green Alge, and in the conditions of illumination,
temperature, etc., prevalent in the tropics, there may be some factor or factors unfavour-
able to the process of photosynthesis in this case.

§ Messrs. West and West (“ Fresh-water Algze of Ceylon,” ‘ Linn. Soc. Trans.,’ vol. 6,
1902, p. 124) have already commented on this subject.

230 Dr, F. E. Fritsch. On the Subaérial and [Jan. 2,

in this respect than we imagine, so that tropical conditions may even
exclude forms which favour stagnant water in our parts.

We will now briefly notice the occurrence of representatives of the
groups and genera just discussed in Ceylon waters. I have practically only
met with fresh-water species of Cladophora in two wells (at Matale and
Ambalangodda, see p. 250), ae, in well aerated water. hizocloniwm, a.
genus which is exceedingly common in our temperate waters, was scarcely
met with except in the estuaries and lagoons. On the other hand, the:
most important representative of the Cladophoraceze in Ceylon is quite
undoubtedly the genus Pithophora, which, although neither a very common
form, was met with sufficiently frequently to make it a sensible constituent.
of the algal flora. It was never found in the actual tanks except in those:
of small size, but generally occurred in the side-pools above mentioned
(p. 226, footnote). The species of Pithophora have, on the whole, thinner
cell walls* than either Cladophora or Rhizoclonium, so that they may be. in
a better position to withstand the increased difficulty of respiration in the
tropics. Pithophora may, however, in its spores also have a far better
adaptation to the vicissitudes of the tropics (desiccation) than either of the
two other genera;f this is a point which should not be very difficult to
settle experimentally. If we examine into the existing records of the
occurrence of species of Cladophora and Rhizocloniwm in the tropics, we find
that in most cases (where the habitat is at all adequately described !) these
forms were growing in flowing or otherwise well aerated water.~ The
remaining cases do not prove anything to the contrary until it has been
definitely ascertained that the water in which these forms were growing was
actually stagnant. I think it may be safely stated that both Cladophora
and hizocloniwm are rare forms in tropical fresh-waters, and that they
normally only appear in a well aerated habitat. They are replaced by the
genus Pithophora, which apparently flourishes quite well in stagnant waters.

I have met with no species of the genus Vaucheria in the lowlands of
Ceylon (terrestrial and aquatic species in the uplands! cf pp. 217, 254),

* See especially Wittrock, “On the Development and Systematic Arrangement of the
Pithophoraceex,” ‘Nov. Act. Reg. Soc. Sc. Upsala,’ Ser. 3, 1877, p. 8: “The vegetative:
cells . . . . havealla thin membrane of cellulose without layers. In Cladophorezx,
particularly the cells belonging to the lower part of the thallus have often a thick
membrane in distinct layers.” See also the plates illustrating the paper cited.

+ It is interesting in this connection to mention that Miss F. Rich, working jointly
with me on the biology and ecology of British fresh-water algal growth, has come to the:
conclusion that Cladophora deserts those ponds which are subject to drying up in
summer.

t The records of tropical Algee found in the existing literature will be considered more.
fully elsewhere (‘ Annals of Botany,’ vol. 21, No. LX XXII, April, 1907, p. 248 et seq.).

1907.] Fresh-water Algal Flora of Ceylon. 231

although one may regard my collections as fairly representative of the algal
flora. A consideration of the published papers further brings out the
startling fact that very few determinable fresh-water species of this genus
have been recorded from the tropics, and there are only few records of
terrestrial species (chiefly V. sessilis (Vauch.), DC.).* In the course of my
travels I came across many likely localities for the occurrence of Botrydium,
but I never found it; there are only three records from the tropics (from
Loanda in Africa, Brazil, and Ecuador). We may, therefore, further charac-
terise the algal flora of the tropics by the practical absence of fresh-water
species of Vaucheria, and the extreme scarcity of the terrestrial Siphones
( Vaucheria and Botrydiwm).

I must here refer to another feature which is intimately connected with the
rarity of Cladophora, Rhizoclomum, and Vaucherva, viz., the scarcity and different
composition of the epiphytic growth, which in our waters finds one of its
main substrata on the filaments of these genera. The characteristic epiphytic
Diatoms (Cocconeis, species of Gomphonema, Synedra splendens, etc.) are very
rare in the Ceylon fresh-waters, which may, in part, be the result of the
Jack of a suitable substratum. But even where I found Prthophora with a
well-marked epiphytic growth, it was not made up of the above-mentioned
species of Diatoms, but usually consisted largely of species of Achnanthes.

The entire group of the Confervales seems to be poorly represented
in the tropics; only three genera (Characwopsis, Conferva, and Ophiocytvum)
have been recorded and, except in the case of Ophiocytwwm, the records are
rather few in number.f In Ceylon I have only met with occasional filaments
of Conferva in fresh-water, although future more detailed examination of my
material may disclose small numbers of the unicellular forms. Whether
the peculiar type of assimilation in this group is really responsible for its
scarcity in the tropics can only be settled by a careful investigation of those
tropical localities in which members of this group appear.

The Conjugates are amongst the most important constituents of the green
element in the algal flora of the tanks, and it will be best to consider the
Conjugate vegetation of these latter separately from that of the other pieces
ot Ceylon fresh-water. The Conjugates are best developed in those tanks
which possess a rich growth of water-weeds; where larger aquatics are
wanting, conditions are not favourable for the growth of any floating green
Alga, which is bound to rise to the surface during the hot hours of the day,
and so be fully exposed to the strong sunlight. The development of the
filamentous Conjugate element in the tanks takes place almost entirely at the
expense of species of Spirogyra; Mougeotia is exceedingly rare (JZ. parvula, Hass.,

* Cf. ‘Annals of Botany,’ los. cit., p. 254. + Loe. cit., pp. 256, 257.

232 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

var. angusta, Hass. in Tissawewa), whilst I never met with a Zygnema in any of
the tanks. Looking at the species of Spirogyra found’ in the Ceylon fresh-
waters in their entirety, it is very noticeable that they all belong to the
section in which the transverse walls between the cells are simple and not.
infolded, and that the large majority have more than one or even two spiral
chromatophores in each cell. These features are, in the main, common to all
the Spirogyras of tropical waters, although they do not appear to have been
noted before. Of 38 species recorded from the tropics, only nine* have cells.
containing a single chloroplast, and only five have infolded transverse walls.
Infolding of the end-walls of the cells is commonly met with in many of the
narrower Spirogyras of our waters, and is generally regardedt as a mechanism
for the dissociation of the cells of the filament, but its exact function is not
perfectly understood, nor do we know why it occurs in one species and not
in another. Since the large majority of tropical Spzrogyras are broad forms,
and infolded walls are only found in the narrower species, we could in no case
expect to find many examples of the latter type; yet the interesting fact
remains that the few narrow forms nearly all have simple end-walls. It is just
as difficult to account for the prevalence of broad forms with numerous closely-
arranged chloroplasts in each cell. Except for the species of Pthophora, the
Spirogyras are the only Algee with broad filaments found at all commonly in
the tropics, all the remaining forms being narrow and rarely exceeding 15 w in
diameter. I am inclined to associate this with the fact that Spirogyra gets on
well in stagnant water under relatively unfavourable conditions of respiration
(perhaps owing to the thin cell-wall), so that forms with broad filaments are
as successful as the narrow ones (cf. next paragraph and also p. 221). Iam
unable to offer any explanation for the usual abundance of chloroplasts.

The most important forms in the attached green algal flora are species
of Gdogonium.t Floating masses of this genus are rather rare (only in
Madokotaikulam and Madakanawewa), although this may be a seasonal
phenomenon. The species of Cédogoniwm found in the waters of Ceylon
are, as a rule, narrow forms with filaments having a diameter of 9—12 p,
even 15 w« being rather rare; species with broad filaments are exceptionally

* Sixty-two records of Spirogyras, with a number of chloroplasts, as compared with
20 records of forms witha single chloroplast. The details will be given elsewhere
(Fritsch, in ‘ Annals of Botany,’ vol. 21, No. LX XXII, April, 1907, p. 260 et seq.).

+ Of. Oltmanns, loc. cit., vol. 1, 1904, pp. 57, 58. On the whole, my material shows very
few cases of breaking up of the filaments into their constituent cells, though the
conditions must frequently have been favourable for dissociation. This is probably due
to the absence of those species of Spircgyra (viz., those with infolded end-walls), which
most commonly exhibit this phenomena.

t The genus Bulbochete is of rare occurrence in the tanks, and was always only
observed in small quantity amongst other attached Alge.

1907.] Fresh-water Algal Flora of Ceylon. 233

rare. The numerous records of species of Gdogoniwm from the tropics show
a very considerable preponderance of narrow forms.* As already pointed
out above, the filamentous algal flora of the tropics is prevalently narrow,
Spirogyra and Pithophora being the only broad forms at all commonly met
with. This is no doubt due to the smaller percentage of dissolved oxygen
in the water, forms with narrow filaments being better suited to these con-
ditions than broader ones. The phenomenon finds its parallel in the extensive
subdivision of the foliage in many of the higher aquatics. Spirogyra and
Pithophora are probably able to exist as broader forms, owing to their thin
cell-walls admitting of more rapid diffusion of gases. A careful study of the
tropical habitats of broad species of Gdogoniwm will probably disclose some
special factor favouring their development. Possibly the rather lmited
occurrence of Pithophora may also be due to the necessity of some special
condition. The Sprrogyras, however, are too widely distributed to admit of
such an assumption.

The Cheetophoraceze do not constitute a very important element, but species
of Stigeocloniwm were observed in some amount in a few cases (4,.,
Balaluwewa, Habaranewewa), whilst Aphanochete is occasionally found as an
epiphyte on other filamentous Algze. Chaetophora was not seen in the tanks.
Coleocheete is an occasional epiphyte.

The lake at Nuwara Eliya, the only large piece of upland water studied,
could, unfortunately, only be subjected to a very cursory examination. There
is no doubt, however, that the flora is in no way dominated by the blue-
green element. At the time of my visit there was very little algal growth.

(ii) Alge of Roadside Ditches and Pools in the Lowlands (excluding rock-
pools, see Section iv).—Although the tanks are the most important pieces of
inland fresh-water in Ceylon, a considerable bulk of algal growth is also
found in the numerous small pools and roadside drains occurring all over the
island. The general character of this vegetation is in most respects similar
to that of the tanks, but there are certain noteworthy differences which
require a brief discussion. The above-mentioned pools (p. 226, footnote) which
are found round about the tanks in the dry season, but constitute part of them
when the water rises, occupy an intermediate position between isolated pools
and the actual tanks as far as their algal flora is concerned. They generally,
however, approach more nearly to the latter, since their flora is (to a great
extent at least) dependent on that of the tanks, and is consequently dominated
by rather different conditions than apply to the pools, etc., which form the
chief subject of discussion in the present section. Most of the forms found
in the pools around the tanks are also met with in the main waters of the

* Of. Fritsch, ‘Annals of Botany,’ April, 1907, p. 265 et seq.

234 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

latter, but from what I have seen I think it probable that certain species are
able to flourish better in small isolated pieces of water than in the large
expanse of the waters of the tanks, and consequently attain to a degree of
development in these pools during the dry season, which is impossible except
in such small collections of water. Further, algal spores carried by the wind
or other means of dispersal may produce an algal growth in the side-pools,
which would never be successful in the main tank.

The conditions to which the Alge of the pools and ditches are exposed are
the same as those above defined for the tanks, although most of them are
more emphasised.* Thus,except where shading is afforded by the surrounding
trees and bushes, the shallow character of these pieces of water means little
or no protection from the strong light, and in the absence of shade algal
growth is rarely found. The temperatures attained are very high, and there
is a very considerable daily range. The risk of desiccation is great, and
drying up or inundation can take place very rapidly. The water is in almost
all cases quite stagnant, heavy downpours of rain being the only source of
aeration. The conditions of life are on the whole more uniform than in the
tanks, the most essential variations lying in the degree of shading and the
frequency of desiccation, conditions which are to some extent mutually
dependent.

In the pieces of water under consideration the blue-green element, although
still well developed, does not play the same predominant part as in the
tanks. The Conjugates are here the dominant group, almost every pool or
ditch examined containing a certain and often very considerable amount of
Spirogyra,t whilst other Conjugate genera (especially Zygnema, more rarely
Mougeotia) are of frequent occurrence, though rarely present in great quantity.
The dominance of Spirogyra is very noticeable on comparing the vegetation
of these pieces of water with that of the segregated pools round about the
tanks, for in the latter Spirogyra only occupies the second place, as in the
actual waters of the tanks. It is not easy to understand why the Conjugates
play so important a part in the pools and ditches, whilst the Cyanophycee
are subsidiary to them. It seems very probable that it is not so much the
occurrence of special factors which favours the Conjugate element, but rather
the existence of one or more conditions unsuitable to the blue-green element,
so that it becomes weakly developed and gives room for the Spirogyra, etc.

* Some of the smaller shallow tanks come very near to the pieces of water under
consideration (cf. footnote on p. 225).

+ The species of Sprrogyra in the pools and ditches show the same general features as
those of the tanks (viz., simple end-walls and a prevalence of broad forms with many
spirals in each cell).

1907. | Fresh-water Algal Flora of Ceylon. 235

Thus the Spirogyras may be better suited to meet the emergencies of desiccation
than most of the aquatic Cyanophycee (which generally have a rather poorly
developed sheath as compared with the subaérial forms) owing to the ease
with which they form resting spores. Azygospore-formation is not uncommon
in my material, and although we are quite ignorant as to the rapidity with
which such azygospores are formed, it seems reasonable to conclude that it is
a much more rapid process than the sexual development of zygospores. In
case of approaching desiccation, which in the tropics will often take place
very rapidly,.there will be a sudden concentration of the water, and this may
well act as a check on ordinary conjugation and lead to the formation of
azygospores.* The prevalence of Spirogyra may, however, also be due to the
frequent strong shading+ of these pieces of water, or to the success of
Spirogyra in perfectly stagnant waters such as these, both conditions which
though, perhaps, not unfavourable to the blue-green element, may still enable
the Conjugates to flourish and crowd out the Cyanophycee. In comparison
with pools and ditches in temperate regions, however, the blue-green Alga
still play a relatively important part, whilst Conjugates are much more
strikingly abundant. Without there being much difference in generic
constitution the facies is quite another one.

In a number of the pools and ditches examined, Spirogyra was practically
the only form present, but in other cases it 1s accompanied by a subsidiary
vegetation composed of Hdogoniwm, Microspora, Zygnema, Mougeotia, Bulbocheete,
Ulothrix, Strgeoclonium, and various Cyanophyceet (Anabena, Lyngbya,
Oscillaria, Scytonema, etc.) ; in a few cases one or other of these forms (nearly
always Cidogonium) was dominant, while the Spirogyra takes a second place.

* Cf. Klebs, ‘ Die Bedingungen der Fortpflanzung bei einigen Algen und Pilzen,’ Jena,
1896, p. 246 et seg. In Spirogyra Webert a 4-per-cent. solution of cane-sugar induced the
formation of a large number of azygospores, whereas in S. inflata a 6-per-cent. solution
was necessary to interfere with ordinary conjugation and produce parthenogenesis ; in
this latter species a 4-per-cent. solution induces ordinary conjugation, so that a sudden
increase in concentration from 4 to 6 per cent. leads to parthenogenesis.

+ Messrs. West and West (“Observations on the Conjugate,” ‘Annals of Botany,
vol. 12, No. XLV, 1898, pp. 35, 36) point out that the Conjugates can stand prolonged
exposure to fairly strong light in their natural habitat, which is not quite in agreement
with the statement that the effect of such exposure is to induce the formation of
zygospores (cf. also Ewart, “The Action of Cold and of Sunlight upon Aquatic Plants,”
‘Annals of Botany,’ vol. 12, 1898, p. 379 et seg.). It would seem as though strong
light stimulated the reproductive functions which probably means cessation of the
vegetative activities. On the other hand, Conjugates appear to be rather less sensitive to
illumination than many of the other filamentous green genera, and it is possible that the
tropical members of this order are adapted to a fairly strong light.

{ Except for occasional floating forms, the Cyanophycee occur most commonly attached
to water-weeds and branches and twigs lying loosely in the water.

VOL. LXXIX.—B. ‘i

236 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

It is interesting that if the latter genus is entirely wanting Cyanophyceze
generally take the lead (cf. the remarks in the preceding paragraph).
Desmids are occasionally very abundant, whilst other unicellular and
colonial green forms (Protococcales, ete.) are rare in these lowland waters ;
colonial Cyanophyvee (¢.g., Merismopedia, Gomphospheria) are sometimes not
uncommon. In comparison with temperate regions the scarcity of Diatoms
is very noticeable, which is uot difficult of explanation when one recollects
that these forms have a preference for cold water, and even in our parts
generally attain their maximum during the winter months. Yet both
Diatoms and Desmids are a good deal commoner in the pools and ditches
than in the tanks.

The large majority of these pools and ditches of the lowlands are
characterised by a Sprrogyra-Zdogoniwm* vegetation, the latter genus, though
relatively rarely attaining abundant development, being almost invariably
present in some quantity. This vegetation acquires different aspects accord-
ing to the subsidiary forms present and is particularly well emphasised in
certain pools and ditches owing to the almost constant association of other
species with the Spirogyra and Gidogoniwm.

The pools and ditches thus characterised always exhibit a dense, brownish-
red granular deposit on the bottom, and the same deposit often also con-
stitutes a filmy investment to all the water-plants. This deposit consists of
ferric hydroxide, and I will consequently talk of these pieces of water as

ferruginous pools.t Such pools are very common in all the lowland regions
with a considerable rainfall (e.g., Negombo, Colombo, Hanwella, etc.), and are
not rare in the uplands around Nuwara Ehya. Owing to the uniform
character of the algal flora, these ferruginous pools may all be considered
together (z.¢., both those of the lowlands and uplands). In a dozen typical
cases examined from different parts of the island, neither Spirogyra nor
Cdogonium were ever found to be absent from the pools, although the
one or the other (very rarely both) was occasionally only present in small
quantity.. Of other filamentous forms, various species of ulbochete-
(including B. minuta, West and West, 5. sprrogranulata, West and West,
ete.) are rarely wanting, and may be regarded as a constituent of almost
equal importance to the two genera previously mentioned; they invariably
occur as epiphytes on larger water-weeds, and possibly their absence in
some few cases is the result of the lack of a suitable substratum. Ulothriz,

* The species of @dogonium found in these pieces of water are again prevalently
narrow (see p. 232).

+ Similar deposits are also found in the rice-fields, and these waters show the same
characteristic flora as that now to be described.

1907.] Fresh-water Algal Flora of Ceylon. 237

Microspora, and Zygnema are all casual forms in these pools, these being the
only habitats in which species of Ulothrix occur at all commonly in the
island. Of much greater importance for the characterisation of the ferru-
- ginous pools are certain Desmids (fig. 3) and Diatoms. Foremost among

Fie. 3.—A few of the more Characteristic and Typical Desmids of the Ferruginous Pools
(all magnified about 260 diameters). A, Pleurotenium T'rabecula (Ehrenb.), Nag.
B, Euastrum ansatum, Ralfs. C, Huastrum obesum, Joshua f. punctatum. D, Pleuro-
tenium annulatum, West and West. E, Clostercwm Venus, Kiitz forma. F, C. pseudo-
diane, Roy. G, C. setacewm, Ebrenb. H, Netrium digitus (Ehrenb.), Itzigs. and
Rothe. J, Hyalotheca dissiliens, Bréb. K, Gymnozyga moniliformis, Ehrenb.
L, Closterium abruptum, West. M, Pleurotenium nodosum, Lund. N, Triploceras
gracile, Bail.

the Desmids are species of Pleuroteniwm (P. Trabecula (Ehrenb.), Nag. ;

P. annulatum, West and West; P. trochiscum, West and West; P. nodosum,

Lund.; P. doliforme,, West and West, etc.); diverse forms of Z7riploceras

gracile, Bail.; species of Closteriwm (C. setacewm, Ehrenb.; C. abruptum,

West; C. Pseudodiane, Roy; C. Venus, Kitz, etc.; in the uplands, also:
mig

238 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

C. Leibleinii, Kiitz, and C. Jenneri, Ralfs); species of Huastrwm (E. ansatum,
Ralfs; #. obesum, Joshua; EH. dideltoides, West and West); and Netrium
digitus (Khrenb.), Itzigs. and Rothe; Cosmarium, Peniwm, Stawrastrum
(more frequent in the uplands!) and Spirotenia are of minor importance
and often scarcely represented. A number of filamentous Desmids*
(Gymnozyga monirformis, Ehrenb.; Hyalotheca dissiliens, Bréb. ; Micrasterias
foliacea, Bail. ;+ and more rarely species of Desmidiwm) are also characteristic
forms in these pools. Amongst the Diatoms, Pinnularia and various small
species of Mavicula play the most important part, although only of occasional
occurrence. Plewrosigma and Surirella seem to be characteristic in some
cases, whilst, where water-weeds are present, Synedra lunaris, Ehrenb.,
sometimes occurs as an epiphyte on them. The blue-green element is often
entirely wanting in these pools, and is never of any importance.

Before proceeding to discuss the few data available regarding the biology
of this flora, it will be well to briefly sum up its constitution as follows :—

(a) Dominant.—Species of Cdogoniwm and Spirogya, Pleurotenium
Trabecula, P. annulatum, Clostervum setaceum, C. pseudodiane, Triploceras
gracile, Netrium digitus.

(b) Subsidiary.Species of Bulbochete, Huastrum, Pleurotenium, Closterium,
Cosmarium, Pinnularia, Navicula.

(c) Occasional—The remaining forms mentioned above and a considerable
number of others.

Of all the forms enumerated, only Spirogya and Gédogoniwm are absolutely
constant, all the others being occasionally absent.[ Their absence may
be due to actual variations in this ferruginous flora, or to the pools
examined being in diverse phases, the latter, on the face of it, seeming
the more probable explanation. This brings us to consider the relation
between the algal flora and the characteristic granular deposit found in these
pieces of water. We are confronted by two possibilities. The flora may
have nothing to do with the deposit, or the latter may be due to the
activity of the Algz present. Such evidence as I have been able to obtain
points to the latter conclusion. Whenever there is a considerable growth of

* Filamentous Desmids are, however, only a marked feature of the lowland pools; they
are rarely present in those of the uplands (cf. p. 253). There is a very marked filamentous
tendency amongst these Desmids of the lowland pools ; this is exemplified by the presence
of a filamentous species of Micrasterias, a genus in which the individuals are usually single,
and by the frequent occurrence of short filaments of 7riploceras gracile and Pleurotenium
(e.g., P. annulatum).

+ This is the only species of Micrasterias I have as yet noticed in these pieces of water.

t Pleurotenium, Bulbochete, Closterium setacewm, Triploceras, and the species of
Navicula are most widely present.

-1907.] Fresh-water Algal Flora of Ceylon. 239

filamentous Algz in these pools, large amounts of the granular deposit form a
dense, reddish-brown flocculent investment all round the filaments (see, for
example, fig. 3, J), so that the latter are often completely obscured; a
similar coating is generally also found on any water-weeds present. It
is hardly possible to account for this investment in any other way than by
the assumption that it has originated round about the algal growth and
water-weeds, as a result of their metabolic activities. The water cf these
pools contains a small amount of dissolved ferric chloride,* and this is
probably transformed (oxydised) into an insoluble form by the vital
activities of the plants present. As the ferric hydroxide accumulates, it
sinks down to the bottom of the pool, and forms a granular layer there.t
The gradual increase in the amount of the deposit may produce less
favourable conditions (shading ?) for certain algal forms, which thereupon
disappear. In the diverse pools studied, many different cases suggestive of
the above theory were met with. Thus, in some of the pools, the granular
matter merely formed a flocculent covering to the contained vegetation, and
there was little or none on the bottom; in others, there was a quantity of
the reddish-brown deposit on the bottom, as well as enveloping the plant-
erowth present; whilst, in still others, there was only a dense granular
layer on the bottom, and practically no vegetation rising up into the water
above it. In the pools last mentioned the vegetation was generally scanty
and least satisfactory, from the point of view of uniformity with the others.
Such pools generally also contained a quantity of Leptothriz, and it seems as
though this genus finds favourable conditions as the deposit accumulates ;
possibly it is the direct cause of the disappearance of the main mass of algal
growth. In any case, the ultimate result of the accumulation of the red
deposit is the elimination of a considerable mass of the vegetation. What
this process leads to finally I am unable to say, but there are some indica-
tions of the initial state of affairs to be found in pools and ditches having
no trace of the characteristic deposit, but containing a flora very similar to
that above described. |

* T have to thank Professor E. C. C. Baly, of University College, London, for kindly
analysing this water.

t It is not impossible that iron-bacteria may have a hand in this process. They are
well known to occur in such waters, and by their activity to give rise to extensive deposits

of hydroxide of iron (cf. 8S. Winogradsky, “Ub. Eisenbacterien,” ‘ Bot. Zeit.,’ vol. 46, 1888,
pp. 261— 270). They would probably tend to collect about the surface of the members of
the green vegetation, owing to the evolution of oxygen going on there; so that the red
deposit would primarily appear round about the water-weeds and Alge present. I do
not, however, think that these deposits can be solely due to their agency, for in many
cases there were very few bacteria evident ; they seem to occur more commonly in later
stages.

240 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

Although such ferruginous pools are very characteristic of Ceylon, they
are not essentially tropical, and are found quite frequently in temperate
regions. I have observed a similar reddish-brown deposit accumulating
since several years in one of the small ditches on Sheen Common, Richmond
(Surrey), and very much the same flora occurs associated with it as in the
Ceylon pools. Spirogyra, Edogonium, Zygnema, Ulothrix, Conferva, Pleuro-
tenvum, Clostervum, Netrium, Pinnularia, Navicula, Surirella, and Synedra
lunaris are the more important forms I have observed in the course of
casual visits during the last three years. The species are apparently in
many cases not identical with those found in the tropical pools, but that
alters little in the astonishing similarity. During the period of observation,
the granular deposit has steadily increased in quantity, while the Alge have
slightly decreased in numbers. Latterly, blue-green forms are putting in an
appearance, which tallies with what I observed in Ceylon. The vegetation
of these ferruginous pools thus constitutes one of the most striking cases of
parallel between temperate and tropical algal formations that I have noticed,
and probably stands isolated in this respect.

There is one more phenomenon noticed mainly in the pieces of water under
discussion (also in the padi-fields), that may be briefly described. As above
mentioned, pools without a noticeable Conjugate element are generally
inhabited by Cyanophyceous growth,—often Oscillaria. A visit to such
a pool in the early morning shows the muddy bottom covered with a more
or less continuous film of the Alga, as is characteristic of the species of this
genus. The film consists of a thin layer of algal filaments with a certain
amount of mud between them. Soon after sunrise gas-bubbles make their
appearance studded all over the surface of the films, whilst at many points
the latter show signs of inflation indicating a similar development of gas-
bubbles on the lower side. Soon after midday floating patches of Oscillaria,
still with numerous adhering gas-bubbles, are found on the surface of the
water, and a little patience enables one to follow the detachment and rising
of such a patch to the surface. This state of affairs continues for the rest of
the day, but early on the next morning there are again no floating patches of
Oscillaria, which merely forms a film on the bottom. In the course of the
day the same process repeats itself. This phenomenon is observed in waters
which are shallow (and almost unshaded) and in which the midday tempera-
ture is very considerable (viz., 35° C.). The rise of patches of the films to the
surface is, of course, due to the expansion of the gas-bubbles formed during the
assimilation and respiration of the Alga, whilst the cooling of the water after
nightfall involves a contraction and perhaps partial solution of these bubbles
(and probably their consequent detachment), and this results in the sinking

J 1907. | Fresh-water Algal Flora of Ceylon. — 241

_ of the floating patches. The phenomenon is certainly no rare one in the case
of Algze capable of forming compact discs like Oscillaria (cf. also p. 227).
Analogous, though not absolutely similar, is the rising of tangles of filaments,
occasionally also to be seen in Oscillaria, though more frequent in green
filamentous genera such as Conjugates, Gdogoniwm, Cladophora, etc.* The
facts mentioned in this paragraph are also, of course, commonly to be noticed
in temperate regions, but I am not aware that a daily rise and fall of such
constancy ever occurs in our parts.

It seems probable that the Algze in this case are merely the passive objects
of a useless physical phenomenon, and that the daily rise and fall is of no
service to them. But it seems well to point out that the rising of the
oscillariate patch to the surface may be advantageous from the point of view
of respiration and of assimilation (the latter owing to the considerable
percentage of mud included in these oscillariate films). In any case the Alea
only avails itself of a physical process, which would take place whether it was
of service to it or not.

(iii) Alge of Marshes and Padi-fields (ue, of ground permanently or
periodically saturated with water which does not form definite pools).—There
is very little difference on the whole between the algal flora of the padi-fields
and that of the lowland pools and ditches, but the biological conditions are a
little different, and it is a desire to emphasise these that leads me to hold the
two distinct. The rice-fields are fully exposed to the strong sunlight, and the
only (very scanty) source of protection is the frequent muddiness of the water.
Desiccation is a regular periodic phenomenon, and periodicity would probably
be found to be better marked in this flora than in the algal vegetation of any
other Ceylon fresh-water. In their exposed habitat the rice-fields resemble
the tanks, whilst the desiccation risks are analogous to those in the small pools
and ditches. In correspondence with this the blue-green element is rather
more pronounced than in the latter, although the Conjugates still very often
have matters their own way. The latter are sometimes almost the only forms
present ina padi-field, but just as often there is practically nothing but
Cyanophyceous growth. Mdogonium is not so commonly met with as in the
pools and ditches, but Bulbochete is again quite a common form. The Desmid-
flora is often very rich, whilst in one or two cases Diatoms (¢g., Gomphonema
attached to water-weeds) were present in exceptional numbers. <A very

* Cf. Oltmanns, ‘Morphologie und Biologie der Algen,’ vol. 2, Allgemeiner Teil, Jena,
1905, p. 142; also H. Devaux, “Du Mécanisme des Kchanges gazeux chez les plantes
aquatiques submergées,” ‘Ann. Sci. Nat. Bot.,’ 7th ser., vol. 9, 1889, p. 144. In the case
of filamentous green Alge, etc., the bubbles are retained between the tangle of filaments
and, by their alternate expansion and contraction, cause the rising and falling of the
tangles.

242 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

characteristic member of the vegetation of the padi-fields is Plewrosigma
curvulum, Pritch., though rarely present in any great quantity; another form
frequently found in small numbers is Fragilaria. I did not meet with
Pithophora or representatives of the Ulotrichales and Confervales in any of
the padi-fields examined.*

The periodicity of the algal growth in the rice-fields would be an interesting
study, and would probably disclose interesting adaptations, which a brief
examination such as mine could not possibly reveal. It seems that the first
growth is almost always blue-green, and that the vreen element only appears
subsequently when the rice and occasional water-weeds (especially Characez)
afford a certain amount of protection against the strong light. Two fresh
rice-fields at Matale showed practically nothing but blue-green forms, whilst
a slightly more advanced field at Matara contained abundant Osezlaria inter-
mingled with occasional filaments of Spirogyra and Diatoms and Desmids in
some numbers. Old rice-fields often contained only very scanty Cyanophycee,
but in their place quite a rich Conjugate and Diatom flora. It almost seems
as though after the harvesting of the rice, and with approaching desiccation
the blue-green element again gains the upper hand, since dry padi-fields often
bear a filmy growth of some blue-green form on the stiff mud. I think the
sequence just sketched out is near the actual one, but there are certainly
occasional exceptions. |

I met with very few examples of marshy ground other than that afforded by
the padi-fields. Mr. Lewis kindly pointed out a fairly typical marsh to me
about two miles inland from Kalutara; the ground was saturated with water
and covered by a rich growth of mosses with Urticularia, Nepenthes, etc. The
algal flora was that characteristic of such habitats in temperate regions, viz.,
a very rich Conjugate flora with numerous diverse Desmids and a certain
number of Diatoms. Blue-green forms on the whole played a minor part.
A small extent of low-lying swampy ground about half-a-mile to the east of
Matara was covered by a rich growth of grasses, and was in most respects
unlike the marsh at Kalutara. The algal flora though rich in Zygnemacez
was exceedingly poor in Desmids, and Diatoms were common only as
epiphytes. There was a considerable growth of a narrow species of
Gdogowum at some points.t

(iv) Algal Vegetation of the Rock-pools—At diverse points (Nalande,
Dambulla, Kurunegala, Habarane) in the northern half of Ceylon one meets

* Tt may once more be pointed out that the specific constitution of the green algal
flora is exactly the same as in the pools, ditches, and tanks (cf. p. 228 e¢ seq).

+ One or two further (brackish) marshes will be considered in conjunction with the
estuarine flora in the second part of this paper.

1907.] Fresh-water Algal Flora of Ceylon. 243

with huge black masses of basalt, which, besides forming the substratum
of the local tanks, generally contain numerous small but deep pools, which
often harbour an abundant growth of Alge of a characteristic type, although
the neighbouring tanks are generally very poor in littoral algal vegetation
{often with a very rich Plankton, ¢g., Dambullawewa, Habaranewewa).*
The temperature of the water in these pools is often high (31°5 C. at
Dambulla at 4 p.M.; 28°5 C. at Hanwella at 8 a.M.; particularly high in
the shallow pools at Kurunegala), but the daily range is certainly not very
considerable, especially where the pools are deep, as at Dambulla and
Nalande.t The pools are exposed to the full light, though the black rock-
surface is probably a good absorber. The deeper pools, in particular, give
one the impression of receiving relatively little light beyond the surface
layers. The decided prevalence of Cyanophycee (especially in the shallower
pools), however, speaks for the shading being less perfect than in the case
of the pieces of water considered in Section (ii). The risk of desiccation
is small in the deep pools, and it seemed as though many of the shallower
ones owed their origin to springs. This would also mean good aeration, as
is indicated by the whole aspect of the flora in many of the rock-pools ;
species characteristic of stagnant water are in most cases absent. The
special conditions in these pools are thus: rocky substratum, high
temperature of no considerable daily range (especially pronounced in the
deeper pools), full exposure to light (except in case of deep pools), and
probably fairly good aeration.

The algal vegetation in the rock-pools either takes the form of a close
pilose or fluffy covering on the rocky sides, or of a floating mass. More
than half the number (18) of typical pools examined contained a blue-green
flora with very few other forms; this is what we should expect under the
dominant conditions. The chief representatives of the Cyanophycez are
attached species of Rivularia and Hypheothriz (see below) and floating forms

_ * Similar masses of basalt are found round about other tanks (e.g., Punchikekirawa,
and between Kelawewa and Balaluwewa), and are no doubt covered by their waters in
the wet season, so that the pools in them then become part of the tank. During the
dry period, however, the pools are isolated and show a certain amount of individuality,
though strongly dominated by the flora of the tank. They are considered in the present
section, however, owing to certain similarities to the true rock-pools, as also are some
pools observed in rock-quarries at Hanwella. The characteristic rock-pools (¢.e., those
which are not in connection with a tank) are distinguished in the above description as the
“typical” ones.

t It should be pointed out that the water of these pools is not only heated directly by
the rays of the sun, but indirectly through the medium of the surrounding rock. The
latter will retain the heat much more tenaciously, and this will serve to decrease the
daily range of temperature in the pools in the rock, especially in the deeper ones.

244 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

of Anabena (sometimes also Lyngbya, mainly L. majuscula). Narrow
species of Oscillaria are often present in minor numbers, but this genus
plays a very unimportant part here in comparison to the pieces of water
discussed in the previous sections. The absence of Tolypothrix is noteworthy.
The blue-green element, though abundant, thus shows a very monotonous
constitution, which is in striking contrast to the great diversity displayed
by the green flora. This is very well developed in all the rock-pools not
dominated by Cyanophycee, and also shows some uniformity in so far as it
consists almost only of Conjugates and Protococcales. But, for the existence
of forms belonging to these two groups, conditions must often be eminently
suitable, for such pools have furnished a diversity of forms, which only
finds its parallel in the upland pools. Diatoms (Fragilaria, Navicula) are
rare—in fact often as good as absent (none in 11 of the pools), but
Peridiniacee are often quite common amongst the members of the Conjugate
vegetation. Cdogonium, if present at all, generally plays a very minor part
amongst the Conjugates, although it attained considerable development in
a pool at Kurunegala and one at Hanwella.* The species of this genus
found in the rock-pools have generally rather broader filaments than the
species inhabiting the waters previously discussed, although none of the
forms could really be called broad. This feature may be connected with
better aeration. }

The abundant Conjugate element comprises certain species of Zygnemacez
and Desmidiacez, and although these forms have been shown to play a
part in the minor waters of the lowlands generally, we shall find that they
exhibit a markedly different aspect in the rock-pools. In the first place
Spirogyra is mostly only a subsidiary form, in many cases, indeed, of isolated
occurrence ; only in three of the 18 typical pools examined did it play an
at all important part, whilst in nine of them it was completely absent
(even in some cases in which the other Conjugates flourished). Two of the
three cases of abundant occurrence of Spirogyra are from the deep pools at
Dambulla, and this lends support to the view above suggested of the

* The relative scarcity of species of @dogonium in the rock-pools may have something to
do with the absence of a suitable substratum. The young plants tend to be epiphytes on
other filamentous Algz or on water-weeds; the only filamentous forms present are
Conjugates, and these are well known to apparently afford an unsuitable substratum for
epiphytes, whilst water-weeds are rarely present. In the pool at Hanwella the
Edogonium (0. caleareum ?) was attached to the rocky base, but this is the only example
of the kind noticed. At Kurunegala there was a little of a water-weed growing in the
rock-pool, and this bore the @dogonium in this case ; in another rook-pool at the same
place many of the decomposing plant-portions forming a sediment at its bottom bore a
fair number of 2- or 3-celled young plants of the genus in question.

1907. | Fresh-water Algal Flora of Ceylon. 245

efficacy of the dark rock-surface as a light-absorber and the consequent
shaded character of the water in these deeper pools. The third case is from
a pool at Kekirawa shaded by the neighbouring jungle. Of equal importance
to Spirogyra are species of Zygnema which generally take its place; from
no other waters in the island have I seen as much Zygnema as in the rock-
pools. Mougeotia is completely absent, but in one case a considerable amount
of Gonatonema occurred. In other cases filamentous Desmids (fig. 4, F and
M) play a great part (species of Desmidiwm, eg., D. Aptogonium, Bréb.,

Fie. 4.—Illustration of the Types of Desmids characteristic of the Ceylon Rock-pools.
The figures are intended to show the prevalent type, and in no way aim at giving an
exhaustive representation of the great diversity of form presented by the Desmids of
these pieces of water. <A, Micrasterias apiculata (Ehrenb.), Menegh. var. (x 250).
B, Cosmarium decoratum, West and West (x250). C, Micrasterias zeylanica, n. sp.
(X 250). D, Staurastrum bifidum (Ehrenb.), Bréb., side view ( x 330 about). E, Ditto,
end-view ( x 330 about). F, Desmidiwm quadratum, Nordst. (330 about). G, Huastrum
zeylanicum, n. sp. (X560 about). _H, Cosmariwm (cf. punctulatum, Bréb.) (x 330
about). J, Staurastrum aristiferum, Ralfs? (x 330 about). K, Cosmariwm pseudo-
pachydermum, Nordst. (X250). L, Cosmariwm Lundellii, Delp. forma (x 250).
M, Spherozosma secedens, De Bary (x 560 about).

D. quadratum, Nordst., D. didymum, Corda, and Spherozosma, eg., S. secedens,
De Bary). Desmidiwum is particularly characteristic of these pieces of water,
while other filamentous Desmids (¢9., Hyalotheca and Gymnozyga) which are
common enough in the ordinary lowland pools, are almost or completely

246 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

wanting. The unicellular Desmids show similar characteristic features; thus
species of Clostervwm are either entirely absent or at least rare, and when
present they are mostly not those species which are found in the other low-
land pools; Huastrwm is also much rarer than in the latter, but Z. dideltoides,
West and West and £. zeylanicum, n. sp.* (fig. 4, G) were rather abundant
in one case. On the other hand, species of Micrasterias, which are rare
in the ordinary pools, play a great part in these pieces of water (e.,
M. apiculata, Menegh., M. zeylanica, n. sp.,t fig. 4, C, ete.), whilst species of
Staurastrum (eg., S. bifidum, Bréb., S. aristiferum, Ralfs?) and Cosmarium
(C. decoratum, West and West, C. pseudopachydermum, Nordst., C. Lundellii,
Delp., C. punctulatum, Bréb.?) are strikingly abundant in contrast to their
scarcity in the clayey pools. Plewroteniwm (P. Trabecula) is sometimes to be
found in some quantity. Looking at the Desmid-flora of the rock-pools in its
entirety, one is struck by the complexity of form of the diverse species in
contrast to the relatively simple forms assumed by the Desmids of the ordinary
lowland pools. This feature is illustrated both by the unicellular and
filamentous species (cf. figs. 3 and 4), and is certainly very noteworthy
(possibly due to aeration of the water?) As already mentioned, numerous

* Diagnosis.—Euastrum zeylanicum, n. sp. :—Cells small, about one and a-half times
longer than broad, very deeply constricted. Apical incision first slightly widened and
then narrowed to more or less of a point. Semi-cells pyramidate, with pointed basal
angles ; apex convexo-truncate, the margin on either side of the apical incision being
straight or slightly curved. Lateral margins with two incisions, of which the one next
the apex is deeper than the other ; upper portion of lateral margins (7.e., part between the
apex and the first lateral incision) retuse ; portions of lateral margins between first and
second incisions and between second incision and median sinus denticulate. Median sinus
very narrow and closed, slightly dilated at its inner end. Cell-wall smooth. Zygospore
not seen.

Length 45—50 p, breadth 30—35 p, breadth of apex 16—18 p, breadth of isthmus, 8 p.

L. zeylanicum has some points of resemblance to species like Z. radiatum, Turn., and
E. serratum, Josh., but the differences are perfectly obvious.

t+ Diagnosis.—Micrasterias zeylanica, n. sp.:—Cells small, length generally slightly
exceeding the breadth, rather deeply constricted ; sinus open, triangular acuminate with
a pointed inner termination ; semi-cells three-lobed, lobes separated by incisions, which
penetrate as deeply as the sinus, and are inclined towards the latter; ends of terminal
lobe bluntly pointed ; lateral lobes with two blunt processes, separated by a narrow and
rather shallow, rounded depression, the process next to the sinus being horizontal, the
other bent down towards the sinus; polar lobe widely spreading, slightly drawn out at
each extremity into a horizontal or deflected process ; apex flatly convex. Outline very
faintly granular. Cell-wall smooth or minutely punctate. Zygospore not seen.

Length 60—65 p, breadth 55—60 p, breadth of polar lobe 50—55 p, breadth of isthmus,
15—18 p.

Micrasterias zeylanica belongs to that group of species which includes J. oscitans, Ralfs,
and WV. incisa, Ralfs, but is more complex than either of these. In this respect it comes
nearer to M. truncata (Corda), Bréb., which is, however, much more abundantly incised.

-1907.] Fresh-water Algal Flora of Ceylon. 247

members of the Protococcales (Scenedesmus, Pediastrum, Celastrum,
Ehaphidium, Dimorphococcus, etc.) sometimes accompany the Conjugate
element in striking numbers, and in one or two cases Peridiniacez are quite
common. The exceptional abundance of the flora in these rock-pools can
only be referred to the high temperature of the water (perhaps also to the
favourable conditions of aeration ?).*

Before concluding the description of the algal vegetation in the rock-pools,
I wish to add a few remarks on the biology of the attached growth found on
the rocky sides of these pools, and we may extend the discussion to the algal
covering on submerged rock-surfaces in general. This attached growth is
either an adhesive tangle or is composed of velvety tufts of pile. The former
is often very compact, and consists either of a single species or more commonly
of a number of closely interwoven forms. The basis in the latter case is
generally blue-green, although green forms (Gdogonium, Mougeotia, Microspora,
Ulothriz, Stigeoclonium) often make up a large part of the mass, whilst
unicellular forms (Desmids, Diatoms, Protococcales) are frequently inter-
mingled. Such adhesive tangles are, no doubt, composed of certain definite
associations of species mutually dependent one upon another in certain
respects, and extensive investigation would probably show that similar (in
some cases perhaps identical) collections of species occur in different localities
under the same influencing conditions. The advantage of such composite
growth may lie in its forming a source of protection against the ravages of
aquatic animals, but there may even be a more intimate relation involving
the metabolic activities of the organisms concerned. For such associations of
species I propose the term “ consortiwm,’t one example of which has already
been noticed in the ZLyngbya-films found in Lakes Kantelai and Mineri
(p. 227); Pithophora, with its associated epiphytes, may be mentioned as a
second.{ Consortia are, of course, to be found in every piece of water, and I
hope at some later date to deal with those occurring in our waters.

* It may be suggested that the great specific diversity and relative individual
complexity presented by the algal flora in these pools (and especially in the deeper ones)
may be, to some extent, due to an absence of the external conditions influencing algal
growth in other tropical pieces of water. Especially in deep rock-pools desiccation
probably never takes place; the temperature is not subject to much daily variation ; the
aeration is probably fairly adequate ; and the light conditions are not extreme. These
characters of the habitat are probably favourable to the growth of a large number of
different forms, whose vegetation goes on undisturbedly. A considerable amount of
variation is thus possible in these waters, and I should not be surprised if a relatively
large number of new forms will be found in them as a result of a more extensive
investigation.

+ Consortium (Latin) = fellowship, participation.

t Cf. Fritsch, “Problems in Aquatic Biology, ete.,” ‘New Phytol.’ vol. 5, No. 7, 1906,

248 Dr. F. E. Fritsch. On the Subaérial and [Jan. 2,

The pilose growth (fig. 5), so often found coating rocky surfaces in the
tropical fresh-waters, is almost entirely blue-green, and presents features
analogous to those noticed above in connection with the st.baérial flora. The
pile consist of a single or of several different species, and in the latter case
we are again dealing with a consortium in which the interrelation could be
determined to some extent. In one of the rock-pools at Dambulla the whole
submerged surface of the rock was thickly covered with such pile. When
some of these were carefully scraped off, a dark dot was seen with the naked
eye at the base of each of them. Microscopic examination showed that this
dot was due to little radiating colonies of Aivularia (fig. 5, B and D), whilst
the remainder of the pila was constituted by a species of Hypheothrix, the
bundles of filaments of the latter arising from in between the threads of the
basal Rivularia. The branched bundles of the Hypheothriz are held together
to form the tuft by a very narrow species of Oscilaria ramifying in all
directions between the bundles of AHypheothria-filaments. In the outer
portion of each pila there was a considerable amount of mud and decomposing
algal substance, which afforded shelter to a number of green unicellular
forms (e.g., Scenedesmus, Tetraedron). The consortium here thus consists of a
Rivularia, a Hypheothriz, and an Oscillaria with a few unicellular forms. It
seems as though the Aivularza prepares the ground for the Hypheothria which
follows. A very similar pilose covering was found on the rocks in a small
pond near the tank at Habarane, but the pile in this case were considerably
longer. The basal Aivularia showed up very distinctly and, with a lens, its
filaments could be seen radiating out in all directions (fig. 5, A). The species
of Hypheothri« differed from the one observed at Dambulla, the filaments
forming thick unbranched bundles, each constituting a single pila; asa
result, there was no binding Oscidlaria in this case. The slight differences,
however, do not detract from the interest of the very considerable analogy.
Rivularia plays a very important part in all the rock-pools, and in some of
them was found growing on every conceivable object (including loose
sediment).

As above mentioned (p. 228) the rocks round the edge of Lake Kantelai were
covered by a thick pilose blue-green growth. This, again, consists of a species
of Hypheothrix, the bundles of which are bound together by a delicate
Oscillaria, but there is no basal Rwularia in this case. At the base of many
of the pilze a dark dot is indeed to be seen (fig. 5, C), but microscopic examina-

p. 157. Every piece of water contains forms, which are in part at least dependent one on
the other, be it from the point of view of substratum, of shading, of transference to a
suitable position in the water, or of protection from animal attacks : and all such associa-
tions of species occur together as a result of necessity.

1907.] Fresh-water Algal Flora of Ceylon.

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249.

Fig. 5.—IIllustration of Colonisation of Submerged Rock-surfaces. A (x 83), Single tuft
from pilose growth on submerged rocks of a small pond near the tank at Habarane ;
growth of Rivularia at base of Hypheothrix. B (x 134), Single tuft from pilose growth
on sides of rock-pools, Dambulla. D ( X35), the same enlarged. C (Xnearly 8),
Tuft from pilose growth on submerged rocks in Lake Kantelai ; the dark mass at the

base is due to air. Full descriptions of all the figures in the text.

250 Dr. F. E. Fritsch. On the Subaérial and ‘‘{Jan. 2,

tion shows this to be due to a collection of air at these points. Pressure on
the cover-glass breaks up this air into numerous small bubbles, which speedily
become dispelled into the surrounding water, and after this process the black
dot at the base of the pila has vanished. Perhaps we have here an air-bubble
which was originally clinging to the rock-surface and formed the base for a
growth of Hypheothriz, since some algal forms are occasionally to be found
attached to the surface-film of air-bubbles in the water.*

In concluding the discussion of this subject, I should like to point out that.
blue-green Algze in particular are noted for the frequent occurrence of dense
collections of very diverse forms, which have been one of the most fruitful
sources of the establishment of cases of so-called polymorphism. Had the
observers who dealt with this subject attacked the physiological problems
involved in these close associations of individuals, far more good would have
been done than in the proclamation of cases of polymorphism on superficial
data. For such collections of blue-green Alge are certainly nothing more
than highly interesting consortia.

(v) Alge of Wells and Springs (we, aerated but standing water).—I was
only able to examine three examples of the algal growth in wells, but they
have proved very instructive. The material is derived from three different
localities (Nalande, Matale, and Ambalangodda), so it may be looked upon as.
fairly typical for the island. The fundamental point about these pieces of
water is their good aeration. The water is generally at some considerable
depth below the top of the well and is consequently not exposed to a very
strong light, nor is its temperature probably in any way as high as in the
more exposed waters we have previously discussed. The substratum is stone
or wood work. The prevailing conditions are thus very marked and uniform
and produce a very typical algal flora.

The predominant forms are species of Pithophora, which were present in all
three cases and in two of them absolutely dominated the algal flora. In two
of the three wells at least there was also a certain amount of other
Cladophoraceze (Cladophora, Rhizoclonum). Since the material was in all cases
sterile and often covered with epiphytes, the discrimination of Cladophora and
Pithophora was not easy; the large mass of the Cladophoracez present,
however, showed the vegetative characters described by Wittrock} as
characteristic of Pithophora. In two of the wells there was a certain amount
of Spirogyra presenting the usual features, whilst blue-green forms (Lyngbya,
Oscillaria) are always present, sometimes in quantity. WM@dogoniwm was only

* Cf. also Fritsch, .““The Structure and Development of the Young Plants in

(Edogonium,” ‘ Annals of Botany,’ vol. 16, No. XLIII, 1902, p. 473.
+ Cf. Wittrock, in ‘Nov. Act. Reg. Soc. Sc. Upsala,’ Ser. 3, 1877, p. 4.

1907. ] Fresh-water Algal Flora of Ceylon. 251

observed in the well at Matale, where it occurred as an epiphyte on the
Pithophora. |

The only other forms of any importance are Diatoms, which were present
in enormous numbers in two of the wells; in the third (at Ambalangodda)
_there were practically none, which is due, perhaps, to the main mass of algal
growth found here being above water-level and consequently exposed to a
higher temperature than in the other two cases. In the wells at Nalande
and Matale the Diatoms occurred mainly as epiphytes on the Pithophora ; in
the former case there were epiphytic Gomphonemas and Synedras and loose
floating tangles of Fragilaria (with intermingled blue-green forms and
Synedra), whilst in the latter, except for a few Synedras, all the Diatoms were
epiphytic (Achnanthes, Gomphonema, and Synedra). Nowhere else in the
lowland fresh-waters was such a striking abundance of Diatoms observed, and
I think this feature is here rather a result of the colder water than of the
good conditions of aeration. Sragilaria is, however, a genus which prospers
quite well in warm water (cf below), and the conditions influencing its
presence or absence are very unclear.

The vegetation of these wells is thus characterised by a Cladophoraceous
and Diatomaceous element and shows a very uniform composition. The
conditions are, of course, exceptionally favourable for Cladophoracee, for not
only is the aeration adequate, but the lower temperature of the water involves
a greater power of dissolving the necessary gases. The wells are just the
kind of habitat in which we should expect to find Cladophoracee flourishing
in the tropics (¢f. p. 230), if anywhere. And their predominance in such
waters furnishes, to my thinking, a striking ‘confirmation of the causes
suggested above for the scarcity of this order in other tropical fresh-
waters.

A few words may be added on the vegetation of the hot springs at Kannia,
near Trincomalie, although they will be dealt with more fully elsewhere. In
hot springs we have aerated water at a high temperature, and this is the
marked distinction between them and the wells. The springs at Kannia are
roughly of two temperatures, viz., 37° C., and 40° to 41° C., and these two
different temperatures appear to call forth an entirely distinct algal vegetation
in the respective springs. Those at the higher temperature have the stone
sides above and below water-level covered with a continuous tangled or filmy
growth of blue-green Algee (mainly Zyngbya and Scytonema), whilst the less
hot springs (37° C.) have a brown, fluffy covering of Fragilaria on their sides.
This difference is very well marked, especially as regards the Fragilaria,
which scarcely appears at all in the hotter springs. Since there is no other
obvious factor than the difference of temperature to explain this diversity, we

VOL. LXXIX.—B. U

202 Dr. F. E. Fritsch. On the Subaérial and — [Jan. 2,

must look upon the higher temperature (only 4° C. above the other, however)
as being the cause of the absence of Fragilaria in the hotter springs.

(vi) Algal Vegetation in the Small Pools of the Uplands.—The vegetation of
the numerous small pools occurring round about Nuwara Eliya (especially
on the so-called Moon Plains and along the route from Nuwara Eliya to
Hakgalla) formed a ready means of study and furnished interesting materials
for a comparison with the lowland pools. The differences are just what we
should expect to find in view of the different conditions. As Mr. Nock kindly
informed me, these small upland pools dry up for about a month during the
dry period. Their water is generally almost quite clear, but stagnant, and the
midday temperature (October 29) in four cases was found to be 19°5C. The
bottom and sides in some cases seemed to consist of a white sandy loam, but in
other cases they were clayey. Some of the pools are of considerable depth,
others, again, shallow. Except for the slight shade afforded by the growth of
neighbouring grasses, etc., the only protection from the strong light is found
in the frequent occurrence of water-weeds, but some of the pools are quite
destitute of such growth. The important conditions are the fairly low
temperature, the clear water, and the probably rather intense illumination.

In very many of the pools the algal vegetation takes the form of a thin,
almost transparent filmy growth covering the abundant water-weeds, and
occasionally floating on the surface of the water. These films are so delicate
that they break up as soon as one attempts to remove the weeds bearing them
from the water, and the only way to collect them is to push the plants into the
collecting-tube under water. This character of the films is due to their being
composed almost entirely of unicellular forms, of which Desmids and a limited
number of Protococcales are the most important. Algal growth of this kind
is common enough in temperate regions,* and it is interesting to find it
developed here, in this pseudo-temperate part of Ceylon; as far as I have
seen, such films are not present in the lowland waters. A considerable part
of the films is composed of mud and decaying organic particles, and this
matter and the algal cells are no doubt held together to form a coherent whole
by the considerable masses of mucilagef excreted by the Algz forming the
films. This mucilage is, however, very delicate, in most cases not being
obvious without the help of stains, and this accounts for the very deli-
cate and frangible nature of the films. Although films of this type have
frequently been described, their origin has, as far as I am aware, not yet been

* Cf. Messrs. West and West, “A Monograph of the British Desmidiacez,” vol. 1, Ray
Society, 1904, p. 16.

+ See especially B. Schréder, “ Untersuchungen iiber Gallertbildungen der Algen,”
‘Verh. Nat.-Med. Ver.,’ Heidelberg N. F., vol. 7, 1902, pp. 1389—196, Plates VI and VII.

are
ee
[mers
ie

1907.| = Fresh-water Algal Flora of Ceylon. 253

adequately explained. It may be noticed in this connection that the Desmids
most involved are capable of slow movement with the help of the excreted
mucilage,* and they probably tend to collect in considerable numbers on the
surface of the water-weeds, attracted, perhaps, by the oxygen liberated during
their assimilation. The prolific division of the Desmids thus established will
soon lead to an accumulation of numerous individuals, whilst their mucilage-
investments will flow together and form a whole. The frequent inclusion of.
Protococcales and of muddy particles in these films is not difficult to under-
stand on this assumption, nor the occasional presence of Diatoms. Whether
these composite films can be regarded as a true consortium must be left to
further investigation.

Turning our attention now to the specific constitution of these films, we
have, in the first place, to notice that the blue-green element is practically
unrepresented ; isolated filaments of Oscillaria are sometimes present, and
in one case the films included a number of colonies of Chroococcus. As a
rule, however, Cyanophycee are sought for in vain, and from this point
of view alone these upland pools acquire an aspect rather distinct from
those of the lowlands. The Desmids are the most important forms (species
of Cosmarium, Euastrum, Closterium, Micrasterias, Peniwm, Xanthidium,
Pleurotenium, etc.), but filamentous species of this group are exceptionally
rare (only Spondylosiwm). This latter point is not difficult to understand
in the case of the films, for here the absence of filamentous Desmids is
probably due to the immobile character of these forms which, on the above.
assumption of the origin of the films, would be excluded from participation
in their formation. A more puzzling feature, however, is the great scarcity
of filamentous Desmids generally in the upland waters examined (cf. also.
p. 238, footnote). This is a second striking point of difference between the
upland and lowland pools, for in the latter, where the conditions are
favourable, filamentous Desmids are quite a prominent feature. It may.
be that the diversity is due to the different amounts of dissolved
oxygen in the water in the two cases. We have only the analogy of
the colonial Protococcales to go by, but in some of these (Scenedesmus,
Celastrum) Sennf found that colonies were formed when there was a
scarcity of oxygen, whereas an abundance of the latter led to the formation
of isolated cells. Possibly, then, the conditions prevalent in the low-
land pools are such as to favour the existence of filamentous Desmids,
while in the uplands the reverse is the case. The lowland pools certainly
afford striking examples of the filamentous tendency amongst the Desmids
_ * B. Schroder, loc. cit., pp. 157—159, Plate VII, figs. 3, 4, and 10a.

t “Ueber einige koloniebildende einzellige Algen,” ‘Bot. Zeitung,’ vol. 57, 1899, p. 97.

U 2

254 On the Subaérial and Fresh-water Algal Flora of Ceylon.

(eg., the occurrence of Micrasterias foliacea, one of the few forms of this
genus that has assumed the filamentous condition). It is, of course, quite
possible that there are other more obscure causes for the great scarcity of
filamentous Desmids in the upland pools. With reference to the above
suggestion, it may be pointed out that filamentous Desmids are, on the
whole, also a rather rare feature in our waters (except Hyalotheca dissiliens
(Sm.), Bréb., and Gymnozyga moniliformis, Ehrenb.), which may be due to the
higher percentage of dissolved oxygen.

Of the unicellular Desmids, Cosmariwm, Euastrum, and Closterium play
the most important part in the composition of the films, one of these three
being generally the dominant form. In some cases one or other of these
three genera is almost alone represented, and we obtain a practically pure
culture of a number of species of the respective genus. In other cases,
however, the films show a more varied aspect, containing most of the Desmid
genera above mentioned, though many of them only occur in subordinate
amount. Some of the films are composed of Desmids only ; in others various
Protococcales (Raphidium, Crucigenia, Colastrum, Hremosphera, Scenedesmus,
etc.) play a more or less important part. No doubt the presence or absence
of these latter forms depends on certain conditions—possibly on the quantity
of organic substance dissolved in the water.* Diatoms occasionally occur
in some quantity, but are often almost completely wanting.

The algal vegetation in the small pools of the uplands does not, however,
always take the form of these films. In some cases one finds a granular or
flocculose covering on the bottom, whose composition is quite identical to that
of the films. A mass of filamentous green Alge floating on the surface
(Spirogyra, Mougeotia, Ulothriz, frequently associated with Desmids) is not
very common. In a few cases various blue-green Algz are found, mostly
as a fluffy coating to weeds, etc. I was unable to determine whether they
are only developed in particularly exposed localities. Lastly, I may mention
that I met with a species of Vawcheria in the water of a small pool in the
Hakgalla Botanic Gardens. This is the first record of a fresh-water species
of Vaucheria from Ceylon.

Meagre as the facts thus are that I was able to derive from my brief
study of the vegetation of these upland pools, they yet point to a marked
diversity as compared with the lowlands and a distinct similarity to the
algal flora of temperate regions. These facts point to the temperature of
the water (and indirectly the lower percentage of dissolved oxygen) as one
of the chief factors influencing tropical algal vegetation.

* Cf. Schmidle, “Aus der Chlorophyceen-Flora der Torfstiche zu Virnheim,” ‘ Flora,’
vol. 78, 1894, p. 64.

255

Expermments made to determine the Conditions under which
“ Specific” Bacteria dered from Sewage may be present in
the Awr of Ventilating Prpes, Drains, Inspection Chambers
and Sewers.

By Masor W. H. Horrocks, R.A.M.C.

(Communicated by Colonel David Bruce, C.B., F.R.S., R.A.M.C. Received
January 11,—Read February 7, 1907.)

Most sanitarians at the present time believe that when sewage is in a
putrefactive condition and gas bubbles rising through it are bursting at the
surface, bacteria may be carried into the air of drains and sewers. It is also
considered possible that when sewage has dried on the surfaces of pipes,
bacteria may be separated as dried particles and carried some distance by
currents of air passing through the pipes.

The following experiments were designed to ascertain whether there are
any scientific facts on which to base these beliefs, and may be arranged in
three groups.

Grour 1.—Lxperiments to determine whether Specific Bacteria are ejected into
the Air by the bursting of Bubbles at the surface of Sewage.

In the first series of experiments, sewage obtained from a main sewer in
Gibraltar was inoculated with a rich emulsion of B. prodigiosus and then
poured into a deep glass jar so as to form a layer at the bottom about
2 inches deep. At a height of 4 inches from the surface of the sewage two
Petri dishes containing nutrose-agar were fastened, the medium facing
upwards, to wire tripods, which were then firmly wedged in the bottom of the
dish. A glass cover was then put on the dish and the fluid gently shaken by
a horizontal movement, until a layer of bubbles formed on the surface of the
sewage. This procedure was followed at intervals for three days, the plates
were then taken out and incubated at 22° C. After seven days’ incubation no
signs of the B. prodigiosus appeared. Only one colony composed of cocci
derived from the air was seen. This experiment was repeated again and
again, but invariably with negative results.

The sewage was next inoculated with a rich emulsion of B. typhosus, and
litmus-lactose-nutrose-agar plates were fastened to the tripods. The dish was
shaken as before, the plates were then removed and incubated at 37° C. No
signs of B. typhosus or B. coli were observed after incubation for one week.

256 Maj. Horrocks. Conditions under which “ Specific” [Jan. 11,

Old sewage, smelling strongly of sulphuretted hydrogen gas, was then placed
in the dish and the experiments were repeated as before. It was thought that
gas bubbles would form more readily in old than in fresh sewage ; this proved
to be the case, but the special ees added to the sewage never appeared
in the plates.

Soapy water from a lavatory basin was then inoculated with B. prodigiosus
and freely shaken in a glass bottle until it was permeated with bubbles; the
fluid was then transferred to the glass dish, the plates fastened to tripods were
rapidly placed 7 situ, and the cover put on. Twenty-four hours elapsed before
all the bubbles dispersed, the plates were then taken out and incubated as
before, but no signs of the B. prodzgiosus appeared.

In the above experiments there were no air currents circulating above
the sewage, and the bacteria could only be ejected by the bursting of bubbles.
The results appear to show that independently of air currents, bacteria will
not be ejected to a height of 4 inches by the bursting of infected bubbles.

The next series of experiments were made with the apparatus shown in
fig. 1. Plates of nutrose-agar were suspended by means of wire cages in the
vertical pipe, the uppermost plate
being 9 feet above the water in the
trap. Soapy water, inoculated with an
emulsion of B. prodigiosus, was shaken
up in a glass stoppered bottle until
the whole fluid was permeated with
bubbles, the contents of the bottle
were then poured into the trap until
it was filled. Under these conditions
currents of air passing up the vertical
pipe were able to carry bacteria
separated from the fluid by the burst-
ing of bubbles. The plates were
removed at the end of two hours and
incubated at 22°C. The result was
that colonies of Lb. prodigiosus ap-
peared in every plate at the end of
72 hours.

The last experiment of this series was made with a catch-pit on the storm-
water system of the town. Complaints having been made of foul odours

arising from the pit, the hinged cover was thrown back, and it was then seen
that bubbles were rising through the water retained in the pit as a result of
fermentation processes going on in the mud at the bottom. The pit contained

1907.] Bacteria may be present in Ventilating Pupes, ete. 257

180 gallons of surface water at the time. An emulsion of B. prodigiosus was
poured into the water and plates of nutrose-agar were then suspended as
shown in fig. 2. Twenty-four hours later the plates were removed and

incubated at 22° C. for"two days, when numerous colonies of B. prodigiosus
were found in all the plates. Control plates of nutrose-agar, exposed to the
air outside the pit, did not show any signs of the special organism used in the
experiment. ow |

_ These results show that bubbles rising through stagnant water may eject
bacteria, which will be carried away by currents of air passing over the.
surface of the fluid. - ,

eae 2.—Lxperiments to determine whether Bacteria dried on the surfaces of
Pipes are likely to be separated and carried by Currents of Air passing.
through the System.

The apparatus shown in fig. 3 was employed. Three 2-ft. lengths of
6-inch piping were laid on the ground and a rich emulsion of B. prodigiosus

258 Maj. Horrocks. Conditions under which “Specific” [Jan. 11,

was poured inside each of them. The pipes were then rolled backwards and
forwards until the fluid was uniformly diffused over the inner surface of each
pipe. Twenty-four hours later the pipes, being perfectly dry, were fitted
together with clay joints so as to form the vertical pipe of fig. 3. Nutrose-
agar plates were then suspended in the pipe and sewage was allowed to flow
through the horizontal piping at a rate not exceeding 3 feet per second for
20 minutes. The plates were then removed and incubated at 22° C.

At the end of 72 hours, all the plates were found studded with colonies of
B. prodigiosus. The experiment was repeated several times, and on each
occasion the same result was obtained. As the sewage passing through the
horizontal pipes did not contain Bb. prodigiosus, and control plates exposed to
the air were also free from this organism, it is fair to assume that the currents
of air produced by the passage of sewage through the horizontal pipes carried
up dried particles of B. prodigiosus detached from the walls of the vertical pipe.

Group 3.—LHxperiments to determine whether Specific Bacteria are ejected
into the Air of Drains, Sewers, etc., from Sewage flowing under Normal

Conditions.

In the first series of experiments, the trap of a 6-inch disconnecting trap
was filled with sewage, and two lengths of 6-inch drain piping, having a
junction bend turned upwards, fixed at one end, were fitted horizontally into
the house side of the trap. Similar lengths of drain piping, but with the
junction bend turned downwards, were also fitted to the sewer side of the trap.
The vertical portion above the trap was lengthened by the addition of three
2-ft. lengths of 6-inch drain piping. The apparatus is shown in fig. 4.

1907.] Bacteria may be present in Ventilating Pipes, ete. 259

Three litmus-lactose-nutrose-agar plates were next suspended with the
media facing upwards in the vertical pipe by means of wire cages, the
uppermost plate being about 6 feet 9 inches above the sewage in the trap.
Three gallons of sewage, taken from a main sewer in Gibraltar, were then
inoculated with a rich emulsion of the B. typhosus, and poured down the bend
on the house side of the trap. The sewage passed through the apparatus at a
rate not exceeding 3 feet per second, and was received in a bucket placed
under the bend on the sewer side of the trap. The inoculated sewage was
passed through the trap in the same manner on two successive days for about
half an hour. The plates were then removed and incubated at 37° C. for
24 hours, when numerous transparent blue colonies resembling those of the
Bb. typhosus were seen in each plate. Several of the colonies were tested with
anti-typhoid serum (horse) diluted 1—100 and, agglutination occurring at
once, the colonies were planted out on agar slopes. The growths thus
obtained were examined as to morphology and Gram staining, and then
planted out in the usual media. The following results were obtained :—

Medium. Result of incubation at 37° C.

Glucose-peptone .......scescsesesoscsccrscvece Acid, no gas.

Lactose-peptone ......ccsccsccccsssscsccccsces Unchanged.

PUATOSe-PCPtON’ ..ss.ccccsrseerocscoceeserces Acid, no gas.

Cane-sugar Peptone ......cceccscecsccccceseees Unchanged.

PAE ATE = POPLONE -..cscovecsassasessegeseasssscoses Unchanged.

UNPEMETAILT COs aca cesetskesssanvsseccencscsoeaees Unchanged.

POMMUIS-TTEKE 5. coc scetecslsevwevesedsacesveasseed Very faint acidity, no clotting.

IPG PtONG-WALED ., ci vessertcscessiasdccaceescece No nitroso-indol reaction.

BO eerste ce eich cvs omnicl’\Sepeenctsabanbaaccceses Colourless growth.

Proskauer and Capaldi, No. 1 .........06 No growth.

Bee BAU EM Ors Scene dcsceauteses ves nsosesedeNecwsscs Thin transparent growth, medium not
liquefied.

INAEEAGOSDEOED, -cis.0ccs0ss.ssetssccansoucveseses Reduced to NO,.

MMOrpholOgy, Ctl. ...0..0sasceccessesecacessesee Small motile bacillus.

SETA SUALDING ovisosceveesdoesacscesicosecs atnde Decolorised.

The bacilli were agglutinated by an anti-typhoid horse serum diluted
1—500.

A portion of the agar growth derived from one colony was then emulsified
in water and injected subcutaneously into a guinea-pig. As a control, a
similar emulsion of the B. typhosus used to inoculate sewage was injected into
a second guinea-pig of approximately the same weight. At the end of three
weeks the sera of both guinea-pigs agglutinated, in a dilution of 1—100, the
stock culture of B. typhosus.

It is plain that in this experiment the B. typhosus was ejected from the
sewage to a height of 6 feet 9 inches.

260 Maj. Horrocks. Conditions under which “Specific” [ Jan, 11,

Further experiments on the same lines were then carried out with the
Bb. prodigiosus, an agar growth emulsified in water being added to the sewage.
The same apparatus was used as in the previous experiments, the vertical pipe
being gradually lengthened by the addition of 2-ft. lengths of 6-inch piping.
The Bb. prodigiosus was recovered from the plates pusbendras 8 feet 9 inches
and 11 feet 9 inches above the sewage in the trap.

It was thought that possibly the resistance produced by the passage of the
sewage through the trap might have caused the projection of the special
bacteria employed into the air contained in the vertical pipe. i

Accordingly, in the next series of experiments, the trap was removed and
the apparatus fitted up as shown in fig. 3. Plates of nutrose-agar were
suspended in the vertical pipe, and the sewage mixed with B. prodigiosus was
made to flow at a rate not exceeding 3 feet per second, through the horizontal
pipes, which were’ never more than half filled with the sewage. The special
organism was again recovered from plates Buspendel titeet 9 ame above
the sewage in the trap. : :

As in all the above experiments, a very rich emulsion of the special
organism, ee as would x never be found under Ronanel, conditions, was added
1 c.c. of the Sileiom representing one-ninth of the cee on an agar slope
after 48 hours’ incubation at 22° C.,-to-ineeulate-the sewage. The same
results were obtained as when the rich emulsion was ‘used.’ )

It is evident from these experiments that special bacteria can be ejected
from flowing sewage independently of the resistance. offered to the flow by the
disconnecting trap. But as the plates were left in the vertical pipe for more
than 24 hours, it is possible that the colonies in the plates might have been
derived from particles dried on the surfaces of the pipes. In order to
exclude this source of the bacteria, the experiments were repeated again, but
the plates were withdrawn immediately ‘the’ sewage ‘had ‘ceased to flow, each
experiment only occupying 20 minutes. As before, colonies of B. prodigiosus
were found in all the plates, showing that they must have been produced
independently of dried particles carried by currents of air. As there were
very few bubbles visible to the naked eye in the flowing sewage, it is not
very probable that all the microbes found in the plates were ejected into
the air by the bursting of bubbles. I think it is possible that many of the
colonies were caused. by the ejection of minute infected droplets from the
flowing sewage. Gross splashing is out of the question, as the sewage was
flowing at a comparatively slow rate, and plates were found infected at a
height of 11 feet 9 inches above the surface of the fluid.

The next series of experiments were undertaken to test the value of the

1907.| Bacteria may be present in Ventilating Pipes, ete. 261

disconnecting trap as a means of protecting a house drainage system from
specific bacteria present in the air of. the sewer into which the house drain
discharges. | |

The apparatus employed is shown in fig. 5. It was a combination of the
arrangements used in the experiments already described. The straight run
of piping, with the vertical pipe attached to it, represents the sewer and an
attached ventilating pipe; joining the sewer is the house drain, the air in
which is separated from that in the sewer by the usual disconnecting trap.
The vertical pipe above the trap represents the air inlet or outlet, as the case
may be, of the house drainage system. Plates of nutrose-agar were suspended
in both vertical pipes... Sewage inoculated with B. prodigiosus was then
allowed to flow at a rate not exceeding 3 feet per second through the pipes
representing the sewer. The flow of sewage was continued for half an hour

Fia. 6.

on two successive days; the plates were then withdrawn and incubated at
22° ©.’ Colonies of B. prodigiosus were found in the plates placed in the
ventilating pipe of the sewer, but none were present in the plates placed in
the ventilating pipe above the disconnecting trap.

The experiment was repeated again, but during the flow of the infected
sewage through the sewer the disconnecting trap of the house system was
repeatedly flushed with 3 gallons of sewage. It-was thought that during the
flushing of the ‘trap, microbes in the sewer air might possibly enter the house
drainage system. This never occurred, the plates on the house side of the
disconnecting trap never showed any colonies of B. prodigiosus.

The vertical trap was then removed and the apparatus arranged as shown
in fig. 6.

Inoculated sewage was made to flow through the sewer as before, and
every five minutes a 3-gallon flush was passed through the house drain.
-Under these conditions colonies of B. prodigiosus appeared in the ventilating
pipe of the house system as well as in that of the sewer. These results show

262 Maj. Horrocks. Conditions under which “Specific” [Jan, 11,.

that a disconnecting trap prevents microbes present in the sewer air from
passing into the house drainage system.

The next series of experiments were made with actual drainage systems.

In the first experiment a newly laid system of a house in the town was:
tested by suspending plates in the junction and disconnecting chambers. A
plate was also fastened in front of the air inlet of the system, the air inlet.
being provided with a mica valve. An emulsion of B. prodigiosus was placed
in the pan of the w.c. of the house, which was then flushed out. The plates
were removed 24 hours later and incubated, with the result that colonies of
B. prodigiosus were found in every plate. “ Air” plates exposed showed no
signs of the special organism used in the experiment. 3

The second experiment was made at the military hospital, plates being
suspended in the top of a 6-inch ventilating pipe connected with a W.c. in
the basement. The plates were 50 feet above the ground level. A rich
emulsion of B. prodigiosus was placed in the pan of the W.c., and the contents.
were then flushed in the usual manner. At the end of 24 hours the plates.
were removed and incubated for 48 hours, when all the plates were found
studded with colonies of the special organism.

The third experiment was made in connection with the drainage at the
east end of the Centre Block of the military hospital. At the north-east.
corner of the block there is a ventilating soil pipe, 6 inches in diameter,,

West True North
at

Europa sewer

East

receiving the contents of a row of w.c.’s placed in an annexe on the first floor
of the hospital. The soil pipe is connected by a 6-inch drain to a discon-
necting chamber (B), which is 10 feet deep, and discharges its contents by a
short drain into a 9-inch sewer, running along the Europa Road. At the

1907. | Bacteria may be present in Ventilating Pipes, etc. 263

south-east corner of the block there is a similar disconnecting chamber (C),
which receives the drainage from a W.c. in the basement and is also connected
to the Europa sewer. There are six inspection chambers, with ventilating —
eovers at the road level, and a 6-inch ventilating pipe on the Europa sewer,
between the Centre Block and the Europa Pass Barracks, a distance of some
200 yards (see Plan of hospital drainage). Plates were suspended in the top
of the hospital soil pipe, at the north-east corner, some 30 feet above the
junctions of the W.c.’s, in the disconnecting chambers (A), (B), and (C), in all
the inspection chambers connected with the Europa sewer and in the top of
the ventilating pipe of the sewer. A rich emulsion of B. prodiyiosus was then
placed in each of the pans of the w.c.’s, which were then flushed. Twenty-
four hours later the plates were removed and incubated at 22°C. After
48 hours, all the plates, except those in disconnecting chamber (A), were
found studded with colonies of the special organism employed. Plates
exposed on the ground close to the Centre Block for 24 hours were also
incubated, but no signs of the B. prodigiosus appeared in them.

Judging by the results obtained with the experimental installations, it was
expected that the special organism would be found in the hospital soil pipe,
disconnecting chamber (B), and in the chambers and ventilating pipe of the
sewer. But as the disconnecting chamber (C) at the south-east corner of
the Centre Block is not in any way connected with the w.c.’s receiving the
emulsion of the B. prodigiosus, and is separated from the Europa sewer by a
_ modern disconnecting trap, the special organism should not have appeared in
this chamber. The experiment was repeated again three times, and, in
addition to the places already mentioned, plates were also suspended in the
disconnecting chambers (1) and (E) connected to the east side of the South
Block. The plates were left i sitw for 24 hours, and then incubated as
before. Colonies of B. prodigiosus appeared in the same places as in the
previous experiment, but none were found in the chambers (D) and (E).

It was then evident that there must be some means by which the discon-
necting chamber (C) was placed in communication with the Europa sewer.
A careful examination of the bottom of the chamber was made, and it was
found that there was no plug in the cleaning arm of the disconnecting trap:
consequently, the chamber was in direct communication with the air in the
sewer. Plugs were found firmly fixed in the cleaning arms of the discon-
necting traps in the chambers (D) and (E), and prevented micro-organisms in
the sewer air from entering the chambers.

The cleaning arm in the chamber (C) was then firmly plugged and the
experiment repeated. The result was that colonies of B. prodigiosus appeared
in the plates placed in the hospital soil pipe and in the chamber (B), but none

264 Maj. Horrocks. Conditions under which “Specific” [Jan. 11,

appeared in the plates suspended in chamber (C), proving conclusively that.
the patent cleaning arm had been the channel through which the B. prodigiosus
entered this chamber in the previous experiments.

As a control of the above experiments, plates were next placed in the
inspection chambers connected with the west end of Centre and South Blocks.
The contents of these chambers are removed by a separate drain, which
passes along the west of the hospital into the disconnecting chamber (F),
close to the south gate of the hospital. No colonies of B. prodigiosus were
found in any of the chambers, although the plates were treated precisely as
in the previous experiments. A fortnight later the experiment was repeated,
but on this occasion the pan of a W.c..connected with the drain on the west
side of the hospital was inoculated with B. prodigiosus, and the w.c.’s on the
east side were left in their natural condition. The result was that colonies
of the special organism appeared in the plates placed on the west of the
hospital, but none were found on the east side.

In all the above experiments the plates were left in the chambers and soil
pipes for 24 hours, consequently the 5. prodigiosus might have resulted from
dried particles of the growth separated from the walls of the pipes and
chambers and carried upwards by air currents, as well as from the bursting of
bubbles and ejection of droplets from the flowing sewage. In order to
determine whether the separation of dried particles was the source from which
the bacteria were obtained, the experiments were repeated, but on these
occasions the plates were withdrawn as soon as the w.c.’s had been flushed,
each experiment only taking 20 minutes. When incubated the plates
showed practically as many colonies of the BL. prodiqiosus as in the previous
experiments, showing that special bacteria may appear in the air of the
chambers and pipes independently of the separation of dried particles. -

It might be objected that in the experiments just related the bacteria were
suspended in water and had no organic matter adhering to them such as would
be the case under natural conditions. When fecal material is flushed down
w.c.’s and carried through a drainage system, it is probable that the B. colc and
the B. typhosus in the case of typhoid stools, will have an organic envelope
which may materially affect the results.

In order to ascertain whether the objection raised had any force, plates of
litmus-lactose-nutrose-agar were suspended in the ventilating pipe of the
Centre Block for 24 hours and then incubated. All colonies having a reddish
tint were fished and carefully studied, the result being that typical members
of the coli group were readily isolated. Plates were also suspended in large
inspection chambers connected with one of the main sewers of the town, about
10 feet above the flowing sewage. The plates were removed at the end of

1907.] Bacteria may be present in Ventilating Pipes, ete. 265

4 hours and incubated, when typical members of the coli group were again
readily isolated. As a final test, the possibility of the B. typhosus being
ejected from typhoid stools was ascertained by using the apparatus shown in
fig. 7. The trap was filled with sewage, and litmus-lactose-nutrose-agar plates,
the media facing upwards, were. suspended in the vertical pipe, which was
afterwards covered with a glass plate. A typical stool, obtained from a case
of enteric fever under treatment in the military hospital, was then mixed
with 2 gallons of water placed in the can connected by indiarubber piping,
1‘5 inches in diameter, with the house side of the trap. The taps were next
opened, and-the contents of the can having passed slowly through the trap,

were collected in the can connected to the P outgo of the trap. The taps
were then turned off and, the cans having been changed, the infected sewage
was again passed through the trap. This procedure was followed until the
sewage had passed 12 times through the trap. The plates were then removed
and incubated. Next day several transparent, blue colonies were observed

these were fished and planted on agar slopes. The growths resulting were
tested with an anti-typhoid horse serum, and one was found to be completely
agglutinated by the serum diluted 1—500. The growth was then submitted
to the usual tests, which showed that under the conditions mentioned a true
B. typhosus had been carried up the vertical pipe.

Two days later the experiment was repeated with another stool from the
same patient, with the result that three colonies of 5. typhosus were isolated,
one being in a plate 2 feet above the trap and the other two in a plate
3 feet 6 inches above the trap.

The experiments were so conducted that no splashing could possibly occur,
and on looking through the glass plate on the top of the vertical pipe, when
sewage was flowing through the trap, a few bubbles were seen bursting at the
surface of the fluid. The pipes and trap employed were quite new, and had
not been used in any of the previous experiments.

266 “Specific” Bacteria in Ventilating Pipes, ete.

These results show that bacteria existing in sewage under natural condi-
tions can be ejected into the air in the same manner as the naked bacteria
used in the experiments already recorded.

Conclusions.

The experiments show that :—

(1) Specific bacteria present in sewage may be ejected into the air of
ventilation pipes, Inspection chambers, drains, and sewers by (a) the bursting
of bubbles at the surface of the sewage, (0) the separation of dried particles
from the walls of pipes, chambers, and sewers, and probably by (c) the
ejection of minute droplets from flowing sewage.

(2) A disconnecting trap undoubtedly prevents the passage of bacteria,
present in the air of a sewer, into the house drainage system.

(3) An air inlet, even when provided with a mica valve, may be a source
of danger when it is placed at or about the ground level:

267

On the Pathology of the Dropsy produced by Obstruction of the
Superior and Inferior Vene Cave and the Portal Vein.—
Preluminary Commumcation.

By CHARLES Botton, M.D., Assistant Physician to University College
Hospital.

(Communicated by Professor Sidney Martin, F.R.S. Received December 19,
1906,—Read January 17, 1907.)
(From the Pathological Laboratory, University College.)

In August, 1903, I published a paper in the ‘Journal of Pathology’ (1),
in which I demonstrated a method of experimentally producing uncom-
pensated heart disease in an animal, which was compatible with life. This
method consisted in diminishing the size of the pericardial sac by stitches,
so that the diastolic filling of the heart was impeded. The main symptoms
of this condition were dropsy and diminution in the amount of urine
excreted.

As the immediate result of this interference with the action of the heart,
there occurred a rise of pressure throughout the whole systemic venous
system extending as far back as the capillaries, and a fall of the mean
arterial blood-pressure. Further, I found that the pressure in all the veins
fell to the normal limit again within the space of about one hour, and that
subsequently, when dropsy was being produced, the venous pressure in all
parts of the body was normal, and the arterial pressure had almost recovered
itself.

I wish to draw special attention to the fact that the venous pressure was
at first raised not only in the great veins near the heart, but also in the head,
limbs, and intestines, that it very rapidly reached the normal limit again,
and that the dropsy was produced whilst the venous pressure was normal,
the arterial pressure very little if at all lowered, and the capillary pressure
therefore not raised. |

My reason for so doing is because Starling’s(2) theory of hydremic plethora is
founded upon the assumption that the venous pressure primarily falls below
the normal in the limbs and intestines, although it rises in the great veins
near the heart. The low venous pressure in the limbs and _ intestines,
according to Starling, initiates the absorption of lymph, which continues
until the capillary pressure in the limbs as well as in the trunk is raised
above normal by the hydremic plethora so produced. Dropsy is then brought
about by the raised capillary pressure. He also considers that there must be -
an alteration in the capillary wall, and he lays great stress upon this point.

VOL, LXXIX.—B. | %

268 Dr. C. Bolton. Dropsy produced by [Dec. 19,

In order to examine the immediate effects upon the circulation of the
blood, Starling produced his uncompensated condition of the circulation by
introducing oil into the pericardium, which brings about precisely the same
immediate results as any other condition interfering with the diastolic filling
of the heart. He, however, registered the venous pressure in the limb by
means of a plethysmograph instead of by the direct introduction of a cannula
into a vein of the foot. The result was that he obtained a contraction of the
limb, which he interpreted as indicating a fall of venous pressure. I have
already pointed out in my paper that this contraction was an indication of
the vaso-motor constriction, which occurs as the result of asphyxia of the
vaso-motor centre. If the venous pressure be taken by means of a cannula
inserted into a vein of the foot, it is quite easy to demonstrate at once a rise
of venous pressure in that part, whether the pericardium be filled with oil
(see Ref. 1, experiment) or constricted by means of stitches (Ref. 1), or whether
the inferior vena cava be obstructed (see Ref. 1, experiment). I, moreover,
showed that it was impossible to keep up the high venous pressure by the.
injection of salt solution so as to produce hydremic plethora. This is owing
‘to the ready distensibility of the veins and the increased activity of the
excretory glands.

The results of my experiments were, therefore, antagonistic to Starling’s
theory of hydremic plethora, and I maintained that passive cedema was
primarily due to malnutrition of the capillary wall, raised capillary pressure
playing no actual part in its production.

- The present investigation was undertaken from three points of view :-—
(1) To confirm my previous results by employing a different method. (2) To .
discover a more easy method of obstructing the venous flow into the heart,
‘and one which admitted of different degrees of obstruction, which could be
accurately measured. (3) To bring the results of general venous obstruction
into line with those of local venous obstruction, in order to discover the
common principle underlying both. .

The subject will be treated of under the following headings :—

1. Method.

9. Effects of obstruction of the superior vena cava :—
(a) Upon the production of dropsy.
(b) Upon the circulation of the blood.
3. Effects of obstruction of the inferior vena cava :—

(a) Upon the production of dropsy.
(b) Upon the circulation of the blood..

~-1906.] Obstruction of the Vene Cave and the Portal Vein. 269

4, Effects of obstruction of the portal vein :—

(a) Upon the production of dropsy.
(6) Upon the circulation of the blood.

Te Effects of obstruction of the superior, together with the inferior, vena.
cava :— |
ao (a) Upon the production of dropsy.
(6) Upon the circulation of the blood.

6. Production of hydremic plethora after obstruction of the inferior vena.
cava.

7. Conclusions.
1. METHOD.

The animals used were cats. The anesthetic in all cases was ether, and
morphia was administered in addition in those experiments relating to the
effects upon the blood-pressures.

‘In order to obtain access to the superior and inferior vene cave, the chest:
was opened by an incision an inch or more long and parallel to the ribs in the
third right intercostal space in the case of the superior, and in the seventh
right space in the case of the inferior vena cava. The ribs were drawn apart:
by weighted retractors and the lung held aside by a small copper spatula.

If the vein were to be completely obstructed, it was ligatured with silk ; if
_incompletely obstructed, a piece of Jacques’ rubber catheter of the appropriate:
diameter and about } inch in length was placed round the vein and tied on.

In those experiments, which were done in order to determine, by a gradually
increasing constriction, at what point the venous or arterial pressure was first
altered, a fine wire was passed round the vein and the free ends of this wire
pushed through a piece of glass tubing so as to form a snare. The loop so
formed was gradually tightened until an effect was observed upon the blood-
pressure ; the vein was then cut through and the wire loop removed and its.
_ diameter measured. |

In all cases during the operation, artificial respiration was maintained
through a tracheotomy tube if the animal were to be killed at the end of the
experiment; but if it were to be allowed to live, a piece of glass tubing was.
introduced into the larynx through the mouth and artificial respiration
maintained in this way, the air being blown through a bottle containing ether. —
Strict antiseptic precautions were observed and a post-mortem bacteriological
examination made in most of the cases.

No difficulty with regard to pneumothorax was experienced after the chest
was stitched up, and the animals began to breathe naturally at once.

x 2

270 Dr. C. Bolton. Dropsy produced by [ Dec. 19,

The arterial blood-pressure was registered by means of a mercurial
manometer, the cannula being placed in either the carotid or femoral artery,
and a float, which traced the pressure curve on a kymograph.

The venous pressures were taken in the inferior vena cava, the cannula
being introduced into the external iliac vein ; at the lower end of the femoral
vein where it divides into two branches at the ankle, the cannula being inserted
into one of the branches and pushed on until its point was flush with the
opening into the femoral vein; in the portal vein, the cannula being intro-
duced into a branch of the splenic vein, the spleen being then placed back in
the abdomen; at the upper end of the external jugular vein, the cannula
being inserted into the posterior auricular vein and pushed down until its
point was flush with the opening into the jugular vein. The venous pressure
was registered on the kymograph by means of an air tambour connected with
the top of the venous manometer by means of rubber tubing. This simply
demonstrates whether a rise or fall of venous pressure occurs: it is no
indication of the exact amount of such rise or fall. The diameter of the
inferior vena cava is 5 mm., that of the superior vena cava practically the
same or a trifle smaller, and that of the portal vein 4 mm.

2. EFFECTS OF OBSTRUCTION OF THE SUPERIOR VENA CAVA.
(a) Upon the Production of Dropsy.

The vein was obstructed both above and below the entrance of the azygos
vein (7.¢., between the entrances of the internal mammary and azygos veins,
and between the azygos vein and the right auricle).

Obstruction below the Azygos Vein.

Complete Occlusion.—In each case death resulted either on the day following
the operation or five or six days later. The post-mortem findings were the
same in ail cases. There was marked cedema of the mediastinum, extending
up into the neck and down along the inferior vena cava to the diaphragm,
the whole of the cellular tissue being distended with clear serous fluid. <A
slight amount of clear fluid was found in the pericardium, and bilateral
hydrothorax was present. In one case 20 c.c. serous fluid was found in each
pleural cavity; sometimes this fluid is blood stained. The cedema of the
mediastinum may extend through the diaphragm, and out as far as the axille.
There is no free fluid in the abdomen. Céidema at the seat of the wound
occurs in the subcutaneous and muscular tissues, but this is not continuous
with the mediastinal cedema and is dependent upon mechanical laceration of

1906.] Obstruction of the Vene Cave and the Portal Vein. 271

the tissues. The urine is. diminished in amount, the animal refuses food, and
wastes.

Obstruction above the Azygos Vein.

Complete Occlusion—I have been able to show that an animal may com-
pletely survive this operation. The animal lived for three months, and at
the post-mortem examination the vein was absolutely occluded and not a trace
of cedema to be seen. The anastomoses were established by the comes nervi
phrenici, the internal mammary, and the azygos veins chiefly; the veins of
the diaphragrn were also distended. |

Before these anastomoses are established, however, cedema is regularly
produced, and apparently may directly lead to the death of the animal, or
absolutely disappear as in the above case. This can be proved by killing the
animal on the day after the operation or at a later date. (C&dema of the
mediastinum, slight hydropericardium, and bilateral hydrothorax are found.
It seems to be the exception for all the fluid to be absorbed. One animal
died 18 days after the operation in an attack of dyspnoea, and 145 c.c. slightly
blood-stained fluid was found in the right pleural cavity and 125 c.c. in the
left ; cedema of the mediastinum was also present. I have never found any
free fluid in the peritoneal cavity. The urine is diminished in amount.

Incomplete Occlusion. Diameter of Tube, 14 mm.—tThe result of constriction
to this size is absolutely the same as that of complete ligature. As a fact,
the tube practically obliterates the vein, since water cannot be made to flow
through it post mortem. Atany rate, if not absolutely obliterated, thrombosis
no doubt completes the obstruction.

Diameter of Tube,2 mm. (Cidema of the mediastinum and hydrothorax
are produced in this case also. Fluid flows slowly through the constriction
post mortem.

Diameter of Tube, 3 mm.—Cidema of the mediastinum is produced with
this constriction also, but only the merest trace of fluid in the pleural cavity
is found. Fluid flows quite easily through the constriction post morten. —

Diameter of Tube, 4 mm. No cedema is produced by this amount of con-
striction of the superior vena cava. There is slight hemorrhage and cedema
in the subcutaneous tissues at the seat of the wound, but this, as in all the
other cases, is the direct result of the injury to the tissues.

The wall of the vein is so very thin that its diameter is practically
the same as that of the enclosing tube in the case of 3- and 4-mm.
constrictions. The general statement may therefore be made that when the
diameter of the superior vena cava is constricted to three-fifths of its normal
size, oedema of the mediastinum and hydrothorax begin to appear, and that

DAG Dr. C. Bolton. Dropsy produced by [ Dec. 19,

it is possible, but not usual, for an animal to absorb the fluid and completely
recover when the superior vena cava is absolutely obliterated. This state-
ment applies to constrictions which are suddenly produced.

(b) Upon the Circulation of the Blood.
Arterial Pressure.

Complete ligature of the superior vena cava produces only a very slight
effect upon the arterial blood-pressure curve. The output of the heart
is slightly diminished, and there may be a very trifling fall in the pressure,
but usually there is none. On the following day, when the animal has
developed dropsy, the blood-pressure is of precisely the same height as it was wm
the normal animal. The fact that there is no immediate fall in the blood-
pressure on complete ligature of the superior vena cava was, of course, well
known in the days of Cohnheim (8).

Constriction of the superior vena cava similarly brings about no alteration
in the height of the blood-pressure tracing.

Venous Pressure.

Lixternal JSugular Vein.—A gradually increasing constriction of the superior
vena cava produces no change whatever upon the pressure at the upper end
of the external jugular vein, until the former vein is narrowed to a diameter
of 3or 24mm. At this point the pressure in the jugular vein rises to a
height varying from 20 to 40 mm. MgSQ, solution above what it was before
the constriction was commenced.

Complete ligature causes a rise of pressure at the upper end of the external
jugular vein of about 130 mm. MgSO, solution above the normal. This
high pressure is not maintained, for, whether the superior vena cava has been
completely ligatured or merely constricted, the pressure in the jugular vein
gradually falls, and within the space of about an hour has reached the normal
limit once more. On the following day, when the animal has developed dropsy,
the venous pressure in the external jugular vein rs still normal.

The following experiment shows the effects of obstruction of the superior
vena cava upon the pressure at the upper end of the external jugular
veln :—

1906.| Obstruction of the Vene Cave and the Portal Vein. 273

Oct. 10,1906. Cat; weight, 2280 grammes. Morphia, + grain, Ether and
artificial respiration.

Pressures. | Femoral artery. External jugular vein.

“Before obstruction .2.......<:... 60 mm. Hg 100 mm. HgSO, solution
Obstruction to 2 mm............. ~ 60 oe 111 a a
ratte later 0.0.6. ec. sees es see — 112 . -
- i OR eRe LES, ks re ee te e

SOO e ee nee ee 66 mm. Hg 113 . -
ai eds obstruction f eiSeotoitas 54 ie 205 = 5
& minutes later. ./....0.s.....0c080: — 221 ~ Bf
ae re eee -— 206 5 i
rit; ida dents eee mee — 185 A ”
Oy os he ar a — 174 FA ‘
ar ene ree EOE — 170 é
ee aT ra AR ee By en a 60 mm. Hg 130 .
ee yan Sean ae Ca a
Stal | % PAA Ris fats ee Se x — 105 » is

|

Inferior Vena Cava.—On complete ligature of the superior vena cava
there is a very trifling and temporary fall of about 10 or 15 mm. ae
solution in the pressure in the inferior vena cava.

Portal Vein.—Complete occlusion of the superior vena cava or constriction
of its diameter to 2 mm. causes a very transitory fall of pressure of igen
5 mm. MgSO, solution in the portal vein.

The only significance of this slight fall of pressure in the portal vein and
inferior vena cava is that it indicates the abstraction of blood from these
venous territories and its retention in that of the superior vena cava.

It is quite apparent, therefore, that the smallest degree of constriction of
the superior vena cava, which is necessary to produce any effect upon the
venous pressure, will give rise to dropsy. |

f

3. EFFECTS OF OBSTRUCTION OF THE INFERIOR VENA CAVA.
(a) Upon the Production of Dropsy.

In all cases the vein was obstructed just below the heart, between it and
the diaphragm.

Complete Occlusion.sComplete ligature of the inferior vena cava above the
diaphragm leads to death in a few hours. There is regularly found 15 cc.
or more blood-stained fluid in the peritoneal cavity. No oedema is found
anywhere else. This is exactly the result obtained by Richard Lower (4),
more than 200 years ago, by ligature of the inferior vena cava in the chest,
although it is usually misquoted in the text books, which state that he
obtained cedema of the hind legs. Antiseptics were not recognised in
his day and, so far as I am aware, he left the lung collapsed. In fact, it was

274 Dr. C. Bolton. Dropsy produced by [ Dec. 19,

precisely this collapse of the lung which induced Ranvier (5) to ligature the
vein in the abdomen, thinking that if dropsy were to be produced by venous
obstruction he should obtain it by this means; however, in this case the
anastomoses are too readily established, and Ranvier fell into error in
thinking he had disproved Lower’s results.

Incomplete Occlusion. Diameter of Tube, 14 mm—tThis practically amounts
to total occlusion of the vein, and death results with about 20 cc. fluid im
the peritoneal cavity. .

TIameter of Tubes, 2 and 24 mm.—Constrictions to both these diameters
may lead to death in a few hours, with 15 to 20 c.c. blood-stained fluid in the
peritoneal cavity ; on the other hand, the animal may survive the operation
and develop ascites, but for how long I have not determined.

Diameter of Tube, 3 mm.—The animal survives, but if killed 24 hours after
the operation, 25 to 55 c.c. clear straw-coloured fluid will be found in the
peritoneal cavity, and possibly a few drops of fluid in the pleure. One
animal lived four months, and at the autopsy showed no dropsy at all.
Anastomoses were well established in the abdominal wall, carrying the blood
to the superior vena cava. I have found ascites present 13 days after the
operation in one case.

The fluid, therefore, is absorbed when the anastomoses are established.
The urine is diminished in amount when dropsy is being produced.

Dnameter of Tube, 4 mm.—A constriction to this amount does not lead to
ascites.

The same general statement which was made with regard to constriction
of the superior vena cava may be made in the case of the inferior vena cava.
When the diameter of this vein is constricted to three-fifths of its normal
size, ascites appears, and when the anastomoses are completely established
this fluid disappears. If the vein is constricted below two-fifths of its
diameter, or if it is completely ligatured, death will result in a few hours, and
some ascitic fluid will be found in the peritoneal cavity.

This statement, of course, applies to suddenly produced constrictions.

(b) Upon the Circulation of the Blood.
Arterial Pressure.

A gradually increasing constriction of the inferior vena cava in the chest
is perfectly compensated for untila diameter of 3 mm. is reached, when the
arterial blood-pressure falls about 20 mm. Hg. Constrictions greater than
this produce proportionately greater depressions in the blood-pressure curve,
and complete ligature of the inferior vena cava causes the arterial pressure to
fall to 30 or 40 mm. Hg above zero.

1906.] Obstruction of the Vene Cave and the Portal Vein. 275:

It has for many years been known that ligature of either the inferior vena
cava or the portal vein causes a great fall in the arterial pressure owing to-
accumulation of blood in the splanchnic area (3).

On the following day, when ascites is present, the arterial blood-pressure is
usually below what it was before the operation, if the animal survives.

Venous Pressure.

Inferior Cava.—A gradually increasing constriction of the inferior vena.
cava in the chest produces no rise of pressure at the lower end of that vein
until its diameter has been reduced to 3 mm., when the pressure rises about
20 to 30 mm. MgSO, solution.

Constriction to a diameter of 2 mm. produces a rise of about 50 mm.
MgSO, solution, and complete ligature a rise of 100 mm. or more.
Whatever be the degree of constriction, however, the pressure in the infervor
vena cava gradually falls and after an hour or longer has reached the normal.

Moreover, on the following day, when the animal has developed ascites, the
pressure in the inferior vena cava still remains normal.

The following experiment shows the relative effects of constriction of the
inferior vena cava in the chest to a diameter of 24 mm., and of complete
ligature. The arterial pressure was taken in the carotid artery and the venous
pressure in the external iliac vein.

Sept. 22,1906. Cat; weight, 2600 grammes. Morphia, + grain. Ether and

U

artificial respiration.

Pressures, Carotid artery. | External iliac vein.
Before obstruction ............... iv 90 mam. Hig 95 mm. MgSO, solution
Obstruction to 2} mm. ......... 66 x Pans ie x
8 minutes later .................. 82 3 | 1380 a ys
Vein completely occluded ...... 30 - | 210 a |
Semimutes Water 1.206.000 sas 30 | 215 |

| | |

Veins of Foot—Complete ligature of the inferior vena cava in the chest
produces a rise of pressure in the veins of the foot of 60 mm. MgSO,
solution or more. Constriction to a diameter of 2 mm. may cause a rise of
50 mm. MgSOQ, solution in the veins of the foot. This is a more gradual
rise than that in the inferior vena cava, because it is produced in veins
farther removed from the seat of obstruction.

The pressure in these veins falls to the normal limit again in about one hour
after the occlusion has been produced.

_ The following experiment shows the effects of obstruction of the inferior
vena cava in the chest upon the venous pressure in the foot.

276 Dr. C. Bolton.
‘Oct. 24,1906. Cat; weight, 3200 grammes.
artificial respiration.
| Pressures. Carotid artery. |
| Before obstruction ................ 110 mm. Hg
| After complete obstruction ... 46 _
| Obstruction removed ............/ 104 i
| 2 MAIMUbEsmMaAbere see ae css cso 8s 110 Be
|. Obstruction to 2 mm............. 64 os
| Sop maimutesmlaveryneticc: once sess —
cea s is eure a RO EA Bo 96 mm. Hg
epee <6 Wel Ee eee ee
ae ok Ge einen gm ek —
cute eees SA Na RR —
fi RE 9 ete eRe aa
ec ane Rao bes 9 4 Rae ee
yee Sa ae A
IOI wah, iy She hse teen amen 70 mm. Hg
We 55 Shee aes eee a
ss irate go oie EAM ieee nee 56 mm. Hg
MBF, =f, 5 RE eae is
on Ss oe ee ee ee ae
| Obstruction removed ............ 74 mm. Hg
opminutes later: acc ccs-serescer 80 FS
The

Dropsy produced by

Morphia, 4 grain.

[ Dec. 19,

Ether and

View of foot.

142 mm. MgSO, solution

204s
1200
1e5niy oe
160,
195 9
ior iy
103!) cae
TSBer UTS
184
183, ;,
185) se
oe
#65 ae
GA
158 a
Nee
eee
een
1

following experiment demonstrates the fact that the pressure in the

veins of the foot rises when the pressure in the pericardium is increased by
the introduction of oil into that sac. |

Nov. 24,1906. Cat; weight, 3050 grammes.
artificial respiration.

Morphia, + grain.

Ether and

Pressures.

Carotid artery. |

Ce

Before introduction of oil

During injection, 3 minute intervals

Injection stopped eee Sr One
Oil allowed to run out

Cr

CO ee

84 mm. Hg

48 mm. Hg

84. ‘

Vein of foot.

|

(|...w she lew
Bi ees
4 P4652 i
1 148)
16208 te
Gil a a8) ewer
TATE
| 135, ba
J 135 95
132
omatasen :
I ene SO, 22
135 a
143 3
145 ee
iO
Cass) es
| | 135 9
i 136s
prem ae
[Wigs ae
lel els ae

120 mm. MgSo, solution

uo 1906.| Obstruction of the Vene Cave and the Portal Vein. 277

Portal Vein—Whether the inferior vena cava be completely occluded or
constricted to 2 mm. in diameter, the pressure in the portal vein rises, in the
former case about 90 mm., in the latter 50 to 60 mm. MgSO, solution. The
pressure falls to the normal limit again in about the same time as it does in
the case of the inferior vena cava. On the following day, when ascites is
present, the pressure in the portal vein rs still normal.

The following experiment shows the effects of obstruction of the inferior
vena cava in the chest upon the pressure in the splenic vein.

Oct. 1, 1906. Cat; weight, 2535 grammes. Morphia, + grain. Ether and
| artificial respiration.

| i}
Pressures. Carotid artery. Splenic vein.
a WS Be eee a
Before obstruction ............... | 120mm.Hg | 113 mm. MgSO, solution |
After complete obstruction...... 60 Ay | 200 * 5
BS minutes later... .......0-.doseeess 44 4 | 175 . 9
Bans EV ie eee = LSS ie. a: 3 |
i ee ek tered, ae ses A a |
| Obstruction removed ............ 80 mm. Hg ae ye 3 |
PeMMMAUGC LATCH 6. vajsentarcs eons v0 — | 90 ‘9 Of
pei. LAGAN), se reel am Hae if
aes Ris ante cie: tute oe Sictarets acaia’ 90 mm. Hg 113 . 5
Complete obstruction, 2nd time 42 a 170 Fe mn
2 UMTIUtES later. icicisscecseccoesess — LET 8 5
2 ial Rp i aon ss isae NS R
eat Lh aad eee oe riehise < ‘
Obstruction removed ............ 88mm. Hg | 85 3 "
2 minutes later... ...1.sadssse0s ees 86 Me | 120 Mi $5
‘Cee i ithe eal ae a ue, Wal ten url. i

Lxternal Sugular Vein—When the inferior vena cava is obstructed to
2mm., the pressure at the upper end of the external jugular vein falls to
the trifling extent of about 10 or 15 mm. MgSO,solution. This fall indicates
an abstraction of blood from the territory of the superior vena cava, on
account of the accumulation of blood in the portal area.

The following experiment shows that on obstructing the inferior vena
cava in the chest the pressure in the external jugular vein falls :—

Oct. 18, 1906. Cat; weight, 2750 grammes. Morphia, } grain. Ether and
artificial respiration.

|
Pressures. | Carotid artery. External jugular vein.
| 3 SEE Eaieeiaieain
Before obstruction ............... | 74 mm. Hg | 56 mm. MgSO, solution
Directly after complete ob-| 32 5 25 » ”
struction

Obstruction removed ............ 70 A 56 3g ”

278 Dr. C. Bolton. Dropsy produced by [ Dec. 19,

The smallest constriction, therefore, of the inferior vena cava, which is:
necessary to produce any effect upon the blood-pressures, will give rise to
ascites.

4. EFFECTS OF OBSTRUCTION OF THE PORTAL VEIN.
(a) Upon the Production of Dropsy.

Complete Occlusion.—Ligature of the portal vein at the spot where it enters:
the liver brings about death in a variable number of hours. There is
regularly found about 6 or 10 cc. slightly blood-stained fluid in the peritoneal
cavity and small hemorrhages in the mesentery may be seen. Since ligature
of the superior mesenteric vein causes hemorrhagic infarction of the intestine,
whilst ligature of the portal vein does not, the latter fact is to be explained
by the circumstance that the veins from the stomach and spleen are included
in the ligature, and, therefore, a certain amount of venous anastomosis can
become established and the spleen itself can act as a reservoir, but the
anastomoses are not sufficient to prevent the occurrence of dropsy or the
great fall of arterial blood-pressure which brings about the death of the
animal.

Incomplete Occlusion. Diameter of Tube, 14 mm.—Constriction of the portal:
vein down to 14 mm. diameter may or may not cause the death of the
animal,

If the animal dies, about the same amount of free ascitic fluid is found in
the peritoneal cavity as when the portal vein is completely ligatured. If the
animal lives and is killed on the following day, 20 c.c. fluid, free from blood,
may be found in the peritoneal cavity.

This is precisely what would be expected, since the animal lives longer than
when the vein is completely occluded, the obstruction not being so great.

Diameter of Tube, 5 mm.—Constriction to a diameter of 3 mm. likewise
produces ascites in small amount, about 10 c.c. perfectly clear fluid being
found in the peritoneum on the day after the operation. This fluid, however,
may be absorbed, because in an animal whose portal vein had been constricted
to this diameter seven weeks previously no ascites was found. The fluid may
also be absorbed when the portal vein is constricted to 2 mm. in diameter.

Diameter of Tube, 4 mm.—In most large animals the diameter of the
portal vein is just over 4 mm. in size, and a slight but definite constriction is
produced by a tube of 4 mm. in diameter. Ascites, however, is not produced
by such a procedure, the abdominal cavity being perfectly normal.

Speaking broadly, therefore, constriction of the portal vein to about
three-fourths of its normal diameter will give rise to dropsy of the

1906.| Obstruction of the Vene Cave and the Portal Vein. 279

peritoneum. Complete ligature will only produce a small amount of dropsy
of the peritoneum and small hemorrhages into the mesentery.

It is probable that constriction of the inferior vena cava must be carried to
a rather greater relative extent than that of the portal vein to produce dropsy,
because the anastomoses are more readily established in the case of the former
than in that of the latter.

(b) Upon the Circulation of the Blood.

The effects of obstruction of the portal vein, as is well known, are a marked
fall of arterial blood -pressure(3) and a rise of venous pressure in the
splanchnic territory. .

After complete ligature of the portal vein I have found that the venous
pressure in the territory of this vein, although it rises enormously behind the
ligature, falls to the normal limit agavn, as in the case of the inferior and
superior vene cave, and the small amount of free fluid found in the
peritoneum is formed whilst the portal pressure is normal. Small hemor-
rhages may be produced whilst the portal pressure is raised, but the dropsy
is produced after the venous pressure has reached its normal limit.

After constriction of the portal vein to 2 mm. diameter, dropsy of the
peritoneum is produced within 24 hours, but this may subsequently disappear
and complete compensation be established, the arterial and venous pressures
being normal.

5. EFFECTS OF OBSTRUCTION OF THE SUPERIOR, TOGETHER WITH THE
INFERIOR, VENA ‘CAVA.

(a) Upon the Production of Dropsy.

The superior vena cava was constricted, and after an interval of 14 or
20 days the inferior cava was constricted, or, vice versd, the superior vena cava
was constricted in an animal whose inferior vena cava had been constricted
14 or 20 days previously. In some cases the azygos vein was ligatured at the
same time as the superior vena cava was constricted.

The post-mortem findings, both the superior and inferior cave being con-
stricted, are exactly the same as I described in my former paper as resulting
from constriction of the pericardium, in other words, the condition is exactly
comparable to that resulting from an uncompensated heart lesion.

Dropsical fluid is found in each pleural cavity, in the peritoneal cavity,
and in the mediastinum.

If the inferior cava be constricted to a diameter of 4 mm., the superior to
that of 3 mm., and the azygos vein be ligatured, the animal may survive, and
a month later no fluid whatever be found post mortem.

280 Dr. ©. Bolton: Dropsy produced by [ Dec. 19,

Dropsy of the pleura and peritoneum is regularly found if the inferior cava
be constricted to a diameter of 3 mm., the superior to that of 3 mm., and the
azygos vein be ligatured.

This is a more easy method of producing uncompensated heart disease in
an animal than is constriction of the pericardium, because the exact amount
of constriction is known at the time of operation, and it does not depend
upon the judgment of the operator, so that there is less fear of the animal
dying or of the operation being unsuccessful...

(b) Upon the Circulation of the Blood.

In these experiments the inferior vena cava was constricted to a diameter
of 3 mm., and the superior vena cava was completely ligatured.

The same results are obtained whether the azygos be ligatured or not.

The effects upon the arterial and venous pressures are exactly the same
as those produced when the pericardium is constricted, with the exception
that the rise of venous pressure is most marked in the external jugular vein,
because the superior vena cava is completely obstructed.

The arterial pressure falls considerably,and the venous pressure in all parts
of the body rises. The venous pressure falls to the normal level again in
about the same time as it does when either vein is constricted separately, and
dropsy is produced whilst the venous pressure is normal, }

The effects of obstruction of both superior and inferior ven cave are,
therefore, precisely the same as those produced by an uncompensated heart

lesion.

6. PRODUCTION OF HYDRZ=MIC PLETHORA AFTER OBSTRUCTION OF THE
INFERIOR VENA CAVA.

If the inferior vena cava be constricted, and if, after the pressure in the
external iliac vein has fallen to its normal level subsequently, salt solution
be very slowly injected into the external jugular vein in quantities of 5 c.c.
at a time, a large amount may be introduced without raising the venous
pressure behind the constriction or the arterial pressure. If during the
injection the venous pressure rises 10 mm. MgSO, solution (less than a
pressure of 1 mm. Hg), it quickly falls again after the injection. In this way,
during one and a-half hours I have injected 130 c.c., and still the pressures,
arterial and venous, remained normal]. Absorption of lymph occurs much more
slowly than this, and the composition of lymph is different from that of salt
solution. It is therefore inconceivable to me how an animal by absorption
can raise its capillary pressure by the production of hydremic plethora. In
the case in point 10 c.c. ascitic fluid was found in the peritoneal cavity at the

al al iO ah Ore y

1906.] Obstruction of the Vene Cave and the Portal Vein. 281

end of the injections, and this fluid was produced with a normal capillary
pressure. pO | :

~~ Tf such a procedure be carried out in the case of a normal animal, no ascites.

is found after intravenous injection of 130 c.c. fluid.

7, CONCLUSIONS.

-- Of whatever nature the impediment’ be which opposes the free flow of
blood into the heart and diminishes thereby the diastolic blood quantum,
it will bring about precisely the same effects upon the circulation of the
blood. There will be a general rise of systemic pressure affecting all the
veins of the body and extending back as far as the capillaries, and a fall
of the mean arterial blood-pressure. This effect is due to accumulation
of blood in the veins. The venous pressure, however, soon falls to its
normal level again, if the animal lives; and the arterial pressure is raised
‘more or less completely to ‘its former level owing to vaso-motor constriction..
-Dropsy is produced after several hours; whilst the capillary pressure is
normal or even below normal.

The reason why the venous pressure returns to the normal level is because:
the veins passively distend and likewise the capillaries, which normally are:
not completely filled. The veins which distend are those of the thorax
and abdomen, because the superior and inferior cave can be seen distended
and the liver enlarged; the vene:cave are less supported than those in
other parts of the body, they are situated within the suction action of the:
thorax, the bed of the blood stream is here smaller than in the more
peripheral parts of the body, and muscular tension empties the veins in
the latter situation towards the heart. In the erect posture, when gravity
is of the greatest importance, the blood will tend to collect in the most.
dependent parts.

The dropsy is produced in this situation where the veins and capillaries.
are distended and where the blood flows with a diminished velocity, and it
probably depends entirely upon an altered condition of the vessel wall, and not.
upon an altered condition of the nutrition of the tissues themselves, as.
maintained by Lazarus-Barlow (6), although, of course, these experiments do. .
not disprove Barlow’s theory. .

This alteration is probably of a pathological nature and not Sachi as would.
lead to an increase of the normal function of the capillary endothelium (7)..
Raised capillary pressure plays no part in the production of this edema, and
the arterial pressure may be normal or less than normal, it matters not which..

It might be argued that. during the first hour the raised pressure which
occurs would damage the vessel: wall, but in those animals which recover:

282 Dr. C. Bolton. Dropsy produced by [Dec. 19,

and develop dropsy, and in which the constriction is not very complete,
the raised pressure is relatively insignificant and not to be compared with
that occurring during muscular movement. The earliest dropsy, therefore,
in uncompensated heart disease is strictly local in origin, and the capillaries
of the district affected become, practically speaking, an excretory organ.
In the remaining parts of the body absorption must occur pari passw with
this increased output of fluid, in order to keep up the normal amount of
blood in the body, but in the part affected absorption by the veins must be
retarded.

As the disease becomes more extensive the area of production of the
-dropsy must extend. |

When the superior or inferior vena cava is obstructed alone, the same
iitial rise of pressure occurs behind the obstruction as far back as the
capillaries, and it soon falls to the normal level again, owing to distension
of the particular veins in question and also, to some extent, on account of
the anastomotic channels, which after many days may effect complete
compensation if the constriction is not too complete. The dropsy is
produced in exactly the same way as described above, when al/ the venous
channels opening into the heart are obstructed, and it disappears in those
cases in which complete compensation is subsequently effected by the
anastomoses.

Absolutely the same remarks apply to obstruction of the portal vein.

Are there any conditions in which the dropsy of venous stagnation 1s
produced when the capillary pressure rs high ?

In answer to this question it may be said that the necessary conditions
are: Firstly, that the venous territory in which the obstruction is situated
be not too large in proportion to the rest of the vascular territories of the
body, so that sufficient blood:can be spared to maintain. a high venous
pressure, and, secondly, that the anastomoses be not too free. Under these
circumstances the dropsy of venous stagnation will occur with a high
capillary pressure, but the actual cause of the cedema is, however, an
alteration in the capillary wall, the high pressure being merely a concomitant
circumstance, or at the most a contributing factor. A parallel instance is
“geen in the case of hemorrhagic infarction. The escape of blood from the
vessels in hemorrhagic infarction may occur when the capillary pressure
is high, as in venous obstruction, but it may also occur when the capillary
pressure is low, as in arterial obstruction.

The dropsy of local venous stagnation is, therefore, produced in exactly
the same manner as that resulting from general venous stagnation, the
common factor underlying both being an altered condition of the vessel wall.

1906.] Obstruction of the Vene Cave and the Portal Vein. 288

In the former case the capillary pressure may be high, but not so in the
latter case. To maintain the volume of the blood normal in both these
conditions, absorption must occur in the unaffected portions of the vascula
system, but this absorption is not so great as it would be if the excretion
of urine were not diminished. The dropsy of general venous stagnation
is essentially local in origin, and the locality increases in extent as the
disease advances. |

Experiments are now being undertaken to estimate the lymph flow in the
various pathological conditions considered above.

REFERENCES.

1. Bolton, “ The Experimental Production of uncompensated Heart Disease, with
Especial Reference to the Pathology of Dropsy,” ‘Journ. of Path. and Bact.,
August, 1903, p. 67.

2. Starling, “Some Points in the Pathology of Heart Disease” (Arris and Gale Lectures),
‘Lancet,’ London, 1897, vol. 1, pp. 569—723. ‘‘ Physiological Factors involved
in the Causation of Dropsy” (Arris and Gale Lectures), ‘ Lancet,’ London, 1896,
vol. 1, pp. 1267—1407. ‘Practitioner,’ Nov. and Dec., 1905, pp. 633—786 (quoted
by Bainbridge). ‘ Lancet,’ Sept. 29, 1906 (quoted by H. Starling).

3. Cohnheim, “Vorlesungen iiber allg. Pathologie,” Berlin, 1882, Aufl. 2, vol. 1

_ (“Lectures on General Pathology,” ‘New Syd. Soc. Trans.,’ London, 1889, vol. 1,
p. 167).

4, Lower, “ Tractatus de Corde, item de Motu et Colore Sanguinis,” Editio sexta,
1728, p. 127,

5. Ranvier, ‘Compt. rend. Soc. de Biol.,’ Paris, 1869, vol. 59, p. 1326.

Lazarus-Barlow, “‘didema,” ‘ Phil. Trans.,’ B, 1894, vol. 185, p. 779.

7. Hamburger, “ Stauungshydrops und _ Resorption,” ‘ Virchow’s Archiv,’ 1895,
vol. 141, p. 398.

>

VOL. LXXIX.—B, ¥

284

The Influence of Increased Barometric Pressure on Man. No. 3.
—The Possibility of Oxygen Bubbles being set free in the Body.

Py LEONARD HILL, F.R.S., and M. GREENWoopD, Jun., M.R.C.S., Research
Scholar of the British Medical Association.

(Received January 26,—Read February 7, 1907.)

The avidity of the tissues for oxygen is such that this gas has never been
regarded as a factor in the production of decompression symptoms with
respect to the formation of bubbles.

Bert’s (1) analyses of the gas set free in the hearts of dogs rapidly decom-
pressed from 10 atmospheres gave—

Per cent. CO, Percent. O2. " Per centN;,

(i) ey 15:20 O90 82:8
(Zyrtec eace ag 0 84:1
(3) Reta 20°8 a trace 79:2

v. Schrotter and his colleagues (2) found a higher percentage of oxygen in

similar analyses—
Per cent. CO,. Per cent. O),. Per cent N,.
(1) 4 dogs yielded ...... 4:71 15°31 79:98
(2) 1 cha OO en 12°45 718 80°37

Leonard Hill and C. Ham (3) killed rats by rapid decompression from
+10 atmospheres, or more, and, cutting up their bodies under a funnel filled
with acidulated water, obtained the following results :—

Per cent. CO,. Per cent. O,. Per cent. N,.
Wee tee 10-7 21 87-2
(2). eerste sh oe 16°0 4:0 80°0

Hill and Macleod (4) submitted frogs and toads to 20 atmospheres of
oxygen (Brin’s oxygen, containing 93—95 per cent. O2) for five minutes, and
rapidly decompressed them. They found that the animals were convulsed
and enormously distended with gas. In the case of mice submitted to
10 atmospheres for five minutes and rapidly decompressed, no such distension
was observed, but the animals went into tetanic convulsions. These convul-
sions could be excited by a touch, the mice usually recovering and appearing
normal on the following day. Mice killed in the convulsive state showed,
here and there, a few bubbles in the vessels, some being found, on microscopic
examination, in the central nervous system, distending the capillary

The Influence of Increased Barometric Pressure on Man. 285

sized vessels and compressing the nerve-cells. The convulsions were due
to oxygen poisoning, and the bubbles, being formed of oxygen, were
eventually absorbed by the tissues. These bubbles supplied the nerve-cells
with oxygen until the circulation became re-established, so that no lasting
paralysis resulted, as occurs with nitrogen bubbles. It seemed, therefore,
probable that, if an animal be kept in oxygen until symptoms of poisoning
are well marked and there is danger of death, then the tissues will be unable
to combine with the oxygen liberated on sudden decompression, and embolism
will result. We decided to settle this question by analyses in the peritoneal
cavity, blood-vessels, and subcutaneous tissues.

The technic employed was that described in the paper of Hill and Ham (3),
the anima] being cut open under a funnel filled with water and inverted over
a basin of water. Before being passed under the funnel, the gas is carefully
washed out of the fur, and pains are taken to avoid opening the digestive
tract or lungs. The results obtained on frogs were as follows :—

(1) Frog exposed to +20 atmos. O2 during 1 hr. 10 mins. Rapid decom-
pression. Composition of gas obtained from the body —

a isi 80 per cent.
Ng ssies: 20

9

(2) Large Dutch frog. +16 to 20 atmos. O2 during 35 mins. Rapid
decompression.

COpcLane: 2°2 per cent.
{0 oc peee 75°6 Ay
Ne aire ais Dae, 4

(3) Frog. +20 atmos, O2 during 33 mins. Rapid decompression. Dead
on removal,

CO oes cas 2°5 per cent.
{o, eeccce 62:0 ”
No aisle of eck 35°d Be

The saturation time in frogs differs so much from that of the Mammalia
that no very direct conclusions could be drawn from these results, and we
therefore extended our observations to rats, the following experiments being
performed :— i

(1) Three rats were exposed to +70 lbs. Oz for one hour. Two were
convulsed in 35 minutes, and at the end of the experiment they were all
_ Moribund. One, however, still breathed after decompression, which took
3% seconds. Analysis of the peritoneal gas yielded :—

ss sits 54°5 per cent.
IN Hay, 4 45°5 es

286 Messrs. L. Hill and M. Greenwood, Jun. [Jan. 26,

(2) Three rats were exposed to +75 lbs. O2 for three-quarters of an hour,
Decompression time, 4 seconds. All three were paretic. The peritoneal gas
of one contained—

COs a. 2°2 per cent.
10. slele oe 28°3 s
Nia. Siete 69°5 5

(3) Three rats exposed for one hour to +75 lbs. O2. Decompression in
3 seconds. ‘Two rats were dead, the third moribund. Peritoneal gas—

COp Er 6°7 per cent.
{o, pies oiehe 26°7
Ne Sooua 66°6 3

(A sample of the chamber air contained only 75 per cent. Os, showing that it
had not been completely washed out at the commencement of the experiment,
since the gas in the cylinder used in this experiment contained 87°3 per
cent. Oz.) | |

(4) Experiment, 12.12.06—

11.55 A.M.—Six rats and six mice were placed in the pressure chamber,

which was washed out with oxygen and then raised to +75 lbs. Oz.

12.0.—Iwo of the mice convulsed.

12.5.—One rat exhibits a twitching of limbs, all the mice affected.

12.20.—All the rats affected, one convulsed.

12.35.—Decompression in 4 seconds. Several rats immediately thrown

into convulsions. | |

On opening the chamber, four mice were found to be dead, two others
affected, one of them dying in a few minutes. Of the rats, one was dead,
two convulsed, three moderately affected. Of these five, three died, one
was killed, and the fifth recovered. A sample was collected. One mouse
(dead) yielded a good deal of gas, one rat (dead) very little, and one rat (killed
for analysis) a considerable amount. |

The analysis gave—

hee VOOR) Goad: 67°9 per cent.
NGG cree eeaeee 32:1 oe

These experiments are not sufficiently numerous to permit of very exact
statements being made, but they seem to show that free oxygen is liberated
during decompression if the animal exhibit symptoms of oxygen poisoning.

The onset of convulsions immediately after decompression, mentioned in
the above account, is very suggestive, and may well have been due to the
sudden liberation of gas bubbles acting on the already poisoned tissues.

-1907.] Influence of Increased Barometric Pressure on Man. 287

The breathing of pure oxygen has been suggested by v. Schrotter (5) as a
means of washing the dissolved nitrogen out of the body before decompression.
He suggested that deep-sea divers might take down a small cylinder, wash
themselves out for five minutes, and then ascend rapidly without risk.

The experiments of Durig (6) show that, on breathing almost pure oxygen,
the body nitrogen is washed out in about five minutes, as shown by the
quantity of nitrogen in the expired air sinking in that time to a constant level.
L. Hill and C. Ham (7) have pointed out the extreme danger of breathing
oxygen for even five minutes at high pressures, and v. Schroétter (8) himself
has come to the conclusion that the use of oxygen should be confined to
an inhalation immediately after decompression.

The gas set free in the blood on rapid decompression contains 79—87 per
cent. No Of the alveolar air, nitrogen forms 79 per cent., so that the
difference of tension promoting an escape of bubbles is very small or nil
(5—0 per cent.). .
- If pure oxygen be breathed, the difference of tension will become 79—87 _
per cent., and this will greatly favour the escape of nitrogen bubbles from
the pulmonary vessels (Zuntz). There is little advantage in breathing
oxygen during the course of a rapid decompression, because the exchange is
between the tissues saturated with nitrogen and the blood. The amount
carried away by the blood depends on the respective tensions of nitrogen in
the tissues and the blood.

Oxygen inhalation cannot affect this, except in so far as it clears the
arterial blood of nitrogen in the lungs. If the pressure falls rapidly the
tissues will still be saturated—say, at 5 atmospheres—and hold 5 per cent.
of nitrogen, while the blood can only carry, say, 3 per cent., the pressure
having fallen to 3 atmospheres. Oxygen can have little or no effect in
arresting this decreasing power of the blood to carry nitrogen (v. Schrotter).

The only safe method is slow decompression at a uniform rate when the
body has been exposed long enough to become saturated.

REFERENCES.

(1) Bert, ‘La Pression Barométrique,’ p. 955.

(2) Heller, Mager, and Schritter, ‘ Luftdruckerkrankungen,’ etc., p. 800.

(3) Hill and Han, “ Phys. Soc. Proc.,” ‘Journ. Physiol.,’ vol. 33.

(4) Hill and Macleod, ‘ Journ. Hygiene,’ vol. 3, pp. 401, etc.

(5) v. Schrotter, ‘Der Sauerstoff in-der Prophylaxe, etc., der Luftdruckerkrankungen,
etc.,’ p. 71 ete.

(6) v. Schrétter, op. cit., p. 49.

(7) Hill and Ham, “ Phys. Soc. Proc.,” ‘Journ. Physiol.,’ vol. 33.

(8) v. Schrétter, ‘Zur Prophylaxe der sogennanten Taucherlahmung’ (Congresso
Internazionale per le Malattie del Lavoro).

.288

The Inheritance of Flower Colour in Antirrhinum majus.
cel Miss M. WHELDALE, Assistant Lecturer in Botany and late Bathurst
Student, Newnham College, Cambridge.

(Communicated by W. Bateson, F.R.S. Received February 12,—Read
February 21, 1907.)

[PLATE 4. ]

TABLE OF CONTENTS.

PAGE

Vint rodUction.....i.0..sseocaeueabenadan tes clsaeeeMneen GoMeMeRnCe Cre cena ace meee 288

Preliminary statement of resullts'...ic.i.ccisaocoedeednscabancacedactesenes 288
Details of various matings :—

iW hite x yellow crimson striped:a..c1...ccceesemonereneesecnmerenees 292

5 x crimson ‘delela® a. .7...0s.<00s.cumecotennetenun cence sence 295

99 M CLEMSON! oa. eaten ease seks Seememe eee Cena ase ewe boten 296

99 KP OMOW ieee sasosshbers Hitt at baceetecee Meee me eee eats aceite 296

Crimson delila x yellow crimson-striped ............secscscesseeees 297

i X CLIMSON: I ewsrennesss uwostcanc ah aueseeen meee ere 298

Ss KV OlOW “se isariale cote cinicise Uestocetes Perea en el ecemnanes 299

Crimson x yellow Crimson-Strioed ois. ssc vdduesteesccecteoeseeisal 299

. MX Vellows ches kveceueeehe oie-c+ ok Seem cece ee macece meme cme 299

Rose'< other ty Pes: vasscvoxcc. sett on nes aun emetic ce tnc uae eerceee 299

Experiments of De Vries on Antirrhinum............ccocccscssoscssees 300

Introduction.

These experiments were begun in June, 1903. The plants used were raised
from seed obtained from Messrs. Sutton. The course of inheritance in regard
to flower colour was the main object of the enquiry.

Preliminary Statement of Results.

There are as regards colour five easily-distinguishable types of Antirrhnum
flowers, namely :— |
1. White—Lips and tube pure white (PI. 4, fig. 1).

2. Yellow—Lips brimstone-yellow; tube ivory. The ivory tube is distinctly
recognisable from the pure white tube of the albino (fig. 2).

3. Ivory—Lips and tube ivory. Ivory colour is due to a faint tingeing
with yellow, and this type may be regarded as a very pale yellow, but
the pigment is, perhaps, distinct from that which gives the yellow
colour to Type 2 (fig. 3).

4, Crimson—Lips crimson ; tube magenta (fig. 4).

5. Magenta—Lips and tube magenta (fig. 5).

Lioy. Soc. Proé.B, Vou.79, PL.A.

yer
ade
oo
GINO er Marte srsreeee OE :
go a 5
paved 3
i
. 2
Le
a Fil
4 Z
?
pens 3 é
2 7
& 3
ee g $

Trap Lith? Leiden.:
1 White. 2.¥ellow. 3 lvory. 4. Crimson. &. Magenta.
6. Crimson della. 7. Magenta delila.

The Inheritance of Flower Colour in Antirrhinum majus. 289

The colours of all these types are sap-colours, not plastid-colours.

_ Forms of the crimson and magenta types occur in which the tube is ivory,
while the lips are magenta or crimson respectively (figs. 7 and 6). The term
“delila” was used by Professor De Vries* for such forms and has been
adopted in this paper.

_ Yellow and magenta colours are produced by the corresponding sap-colours
in the cells. Crimson colour in the lips is due to the simultaneous presence
of magenta and yellow saps in the cells. That such is the case is easily seen
in a microscopical examination of the tissue of the lips of a crimson flower.
Groups of cells containing pure yellow or pure magenta saps are found among
crimson cells in which the colour is undoubtedly due to a mixture.

With the exception of the albino type, the flowers of all types used have,
irrespective of the colour of lips and tube, a constant orange-yellow palate ;
smaller patches similar in colour generally appear at the base of the tube on
either side. This summer Mr. Bateson has shown me white types, some of
which had a little orange-yellow colour on the palate, while others had in
addition the yellow patches on the tube. Matings will be made with these
types, but all whites employed and described in the experimental work have
been entirely without colour.

The inheritance of the corolla colours can be represented by the following
factors, which have been found to follow the Mendelian inheritance :—

Y.—A factor representing yellowt colour in the lips associated with ivory
tube-colour.

I.—A factor representing ivory colour in the lips.

Des ie magenta _,, #

T.— r s) » 1n the tube.

Small letters, y, 1, etc., denote the absence of these factors.
From experimental evidence it appears that :—

1. All zygotes, from which Y is absent, are white, though they may
contain any of the factors I, L, and T.

‘2. The factor T is not manifested unless L is also present in the zygote ;
that is, no magenta colour appears in the tube unless magenta colour
be also present in the lips.

3. All zygotes containing Y are coloured. The actual colour may be
modified and determined by the presence of one or more of the
remaining factors. A zygote containing Y only or Y and T is yellow.

* “Mutationstheorie,’ Lief. IV, pp. 194—206.
+ This, of course, does not refer to palate colour, which may be regarded as a separate
character.

290 Miss M. Wheldale. The Inheritance of — [Feb. 12,

4, Ivory is “ dominant ”* over yellow ; a zygote containing Y and I or Y, I,
and T, is ivory.

5. Since. magenta superposed upon yellow gives crimson, a zygote
containing Y, L, and T is crimson, Y and L only, crimson delila.

6. Magenta superposed upon ivory gives, since the latter is very pale,
magenta. A zygote containing Y, I, L, and T is magenta, Y, I, and L
only, magenta delila.

The five types described are readily distinguishable and quite discontinuous.
Throughout the experimental work, all individuals met with have been at
once assigned, without any ditticulty, to one of the above classes. Magenta
colour, however, appears in at least three distinct shades or degrees of
concentration, which are probably determined by definite factors, though
evidence on this point is not yet complete. Upon a yellow ground, these
shades of magenta give the corresponding shades of crimson. In many cases
deep magentas have been bred from the lighter magentas, so we must conclude
that lighter forms are dominant to darker in the same sense that the pale
form, ivory, is dominant to yellow. Similar phenomena have been found to
occur in Sweet Peas and Stocks.t

The concentration of sap-colour in the tube and lips is generally approxi-
mately the same; the lips are somewhat darker in appearance, but this
difference depends probably on texture and not on difference in depth of
pigmentation. The tube may also be darker in shade than the lips, and this
is the case in one or two types not yet investigated.

The magenta sap-colour sometimes appears in irregular fleckings or
stripings, thus giving striped forms. If the striping occurs in the magenta
type, the ivory ground-colour shows where the magenta sap fails and the
flower may be described as ivory flecked or striped with magenta. When the
magenta sap fails in the crimson type, the yellow ground-colour appears and
the flower would be described as yellow flecked or striped with crimson. No
flecking or striping of ivory on yellow has ever been observed.

In several cases, where striped and non-striped forms have been crossed,
striping has disappeared in Fj, and has not appeared again either in F» or F3.
In another case of striped x non-striped (a paler magenta), the deeper striping
persisted in F; on the paler magenta ground ; a striped Fy has been given

* “T)ominant ” is here used to denote that the lighter colours overlie or suppress the
darker, as ivory, for example, conceals yellow ; and not in its strict sense of expressing the
relationship between a pair of allelomorphic characters: ivory and yellow are not, of
course, allelomorphic to each other.

+ ‘Third Report to the Evolution Committee, W. Bateson, E. R. Saunders, and
R. C. Punnett, p. 4.

1907.] Flower Colour in Antirrhinum majus. 291

also in the mating of two striped forms. In the two latter cases the results
are not. yet complete. |

The original wild Antirrhinum appears to be of the magenta type.
“ Reversion ” to this old form, which so often happens when crimson, ivory, or
yellow types are crossed with white, is explained by the introduction into the
zygote of the factors I, L (and T) by the white parent. The introduction of
the factor I alone will bring about “ reversion” when L is already present in
the zygote. .

The experimental evidence is not yet sufficient to determine whether the
phenomena in Antirrhinum are fundamentally similar to those observed for
Sweet Peas and Stocks. In the two latter cases,* production of colour depends
on the simultaneous presence in the zygote of two factors C and R; the
absence of both or either of these factors from the zygote renders it white. The
colour formed by the meeting of the two factors is red.

To treat the case of Antirrhinum on parallel lines, we might suppose the
production of yellow, the base-colour, so to speak, to be due to the presence
in the zygote, not of one factor only, but of two factors. Any zygote con-
taining both would be yellow; one only or neither would be white. If such
is the case, whites should be found, which, when crossed together, give yellow.
Whether this is the case or not cannot be decided yet, since the results of
crosses between whites are not known.

All phenomena so far observed in Antirrhinum could be equally well repre-
sented on the two-factor theory, provided we assume that the whites, originally
mated with coloured types, contained one of the factors which constitute
colour; this is quite possible, since so few white individuals (only three)
were employed in the matings. |

The question, then, as to whether colour in Antirrhinum depends on the
meeting of two complementary factors or not, must remain open for the
present. If two factors be necessary, albinos should be found, which, when
mated, give colour (here yellow), as in the cases of Sweet Peas and Stocks; if,
on the other hand, only one factor is necessary, then no two albinos when
mated will ever produce colour, and the case will be analogous rather to the
phenomena observed in animals.

In the Sweet Peas and Stocks, moreover, the colour of the zygote may be
further determined by the presence of a third blue factor B, which changes
the red colour to purple ; but B has no effect unless C and R are also present,
and thus may be carried by an albino without producing a sensible effect.

(Juite comparable to this third factor in Peas and Stocks are the factors I,

* ‘Third Report.

292 Miss M. Wheldale. Zhe Inheritance of — [Feb. 12,

L, and Tin Antirrhinum, none of which produce a sensible effect unless yellow
is also present. Any of these may be carried by an albino.

The factor I modifies yellow, giving ivory. The factor L, superposed upon
ivory, gives magenta ; with yellow it blends, giving crimson ; finally, the third
factor T is not manifested unless L in addition is present.

Details of Various Matings.
WHITE X OTHER TYPES.

White x Yellow Crimson-striped :—

The type yellow crimson-striped is, as explained above, really crimson, in
which the magenta sap occurs in irregular stripings and fleckings, so that the
lips appear yellow striped with crimson and the tube ivory striped with
magenta; a corresponding delila form exists, having an ivory tube.

White has always been found to breed true, but the selfings of the other
parent unfortunately failed, so that its constitution can only be deduced from
the results of crossing.

F, consisted of magentas and magenta delilas (Table I, p. 303).

F;—The cross-breds appeared to be of four kinds, giving respectively, on
selfing :— 7

Case 1. ase 2. Case 3. Case 4.
Magenta x self. | Magenta x self. | Magenta delila x self. | Magenta delila x self.
Magenta ......... 107 229 = =
. deliia 42 70 59 47
@rimsontsn.sc 2 42 74 = =
1 delila 12 19 18 5
ANG OR Ye 5 cles oo hstotec se 67 = 19 —
Yellow . 24 — 7 “=
WV MNIG Os ee tespeee.-2< 80 97 34 | 1)
Motalsi..... 374 | 489 | 137 ff
|
If we assume the original parents to be :—
VV LI AL ieaitiet er ccueece reece White
WY te eee eee Yellow crimson-striped,
then the cross-breds would be of four kinds :—
Mey i ILL tee ee ee ce Magenta (Case 1)
We Db be eee eee ee i (Case 2)
Vey Diet ee ee , delila (Case 3)

Voy Ti: tt, te. keen ee - » (Case 4),

1907.]

whole forms and delilas occurring in equal proportions.

Flower Colour m Antirrhinum majus.

here due to the introduction of I by the white parent.
From Case 1, on selfing, we should expect to obtain theoretically as the

result of the combination of four pairs of characters :—

293

“ Reversion ” is

Ratios Numbers Numbers

ar calculated. observed.
VILT ...|. Magenta ......... Sl or 118°38 107
a i eer ae » delila 27 39 44 42
Gn es re DV OMY ooic as tuusis vw cans 36 52 °59 67
eS Diss: CrimsOMl.......0006 27 39 °44 42
VG) eres DOUY cia veal esgaces — — aan
(ges a BW OULOW: iis c ic kate 12 17°53 24,
a See Crimson delila ... 9 13°15 12
MN Macias den WiellOw: icdsaacds vee — — —
Be lde ssh alte WSS? Ade dacsiae 64, 93 °50 80
Totals ..::.. 256 873 ‘98 374

From Case 2 we should obtain theoretically :—

Ratios Numbers Numbers

Bees calculated. observed.
YLT Lb Magenta ......... 27 or 206°30 229
‘Vfl Ul Were pere a delila 9 68 76 70
6 Nal a Crimsonc...e-:.dse 9 68 °76 74,
BV Toiviaresislenee delila. 3 22 92 19
a ar ee Wihite fie. 16 122 °25 97
Potala... 64 488 :99 489

\
From Case 3 :—

Rat; | Numbers Numbers

a utOg. | calculated. observed.
2 GE ries Magenta delila ... Zi Or 57 °80 59
GD: i aciaiias GORY, 22 5.\cisidgntne nae 9 19 °26 19
\008] (ee Crimson delila 9 19 :26 18
Mice uae WiehOW sind saciccedes 3 6°42 7
a wetietselsmareisrs WiGe) as de becese 16 34°25 34
Totals 034. 64, 136 99 v7

294 Miss M. Wheldale. The Inheritance of
And from Case 4:—

Rees Numbers Numbers
: calculated. observed.

YOT wee Magenta delila.. oor 39 -94 47

VOT ee Crimson seo, 3 ° 13°31 5

Sa elsfewlslrettawste WWihtte: ato: osecks.. « 4 76 S75) 19

Totals ...... 16 71-00 |

[Feb. 12,

The close agreement between the theoretical and experimental numbers
justifies the assumptions made in regard to the constitution of the original

parents.

Diagram I represents the composition of F, in Case 1 when we consider

xx
SSS

SERA

y1T y 1K ylt ylt yl1T ylT
yLT yLt yLT yLlt y1T WelliG

ad
ual

Neretatitetes x

S mS 5

‘ IK ro Bes RS Ha) sy DY Yj

SPOR Sh NI

SLT PYLE PALO PADO PATE Pann Part OP be”

k Lei ee Eek : uk EK a |
4

ROR OY RORSIRRRELLEY

RIO RAI rene iV Vi iz

ee Oe mh

SKK POPE KES ig YY We, ys

KECK, VSI GLI Mee

Ritece hoe erienes THER eohe Kye”
RXL 64 ee

\ AS i SIKH KKK KEPSHKPOD

Relate tatateniten Sr PSII OSES SO

RT ey WC VW 2 eZ

‘ KALKK aS We ty We,

SS SH LRP VACIINGBEILE

yLT | yLT | yt

y LT yLt y LT

Diagram I.—Scheme of Distribution of three of the Colour-factors in F, from

white x yellow crimson-striped.

Cross-hatched squares = crimson-flowered plants.
Single-hatched = yellow

Half plain — crimson delila flowered plants.
Plain = white-flowered plants.

oP)
99
99

—1907.] Flower Colour mm Antirrhinum majus. 295

three factors only ; the introduction of a fourth factor would give magenta
and ivory individuals. set
_ A similar diagram constructed for four factors shows us that, as regards the
composition of the Fs; they give on selfing, there are 16 kinds of magentas
in F, Among 13 magenta plants taken at random in F, and selfed, 8 out of
the 16 kinds were found. (For Case 1, Table II gives F; resulting from all
the Fy: individuals selfed.) The remaining 8 would doubtless occur among a
larger selection of Fy. individuals. -

Of magenta delilas, there are theoretically 8 kinds; 6 plants taken at
random gave 2.

Of crimsons, theoretically 8; 5 plants gave 3.
Of crimson delilas, 4; 3 plants gave 2.

Of ivories, 4; 6 plants gave 3. -

And of yellows, 2; 3 plants gave both.

A corresponding diagram can easily be constructed for Case 2.

From such a diagram one learns that the F, magentas are theoretically of
8 kinds; 11 plants taken at random gave 7 out of the 8. (Table III gives
F for Case 2.)

Of magenta delilas, 4; 4 plants gave 4.

Of crimsons, 4; 6 plants gave 4.

Of crimson delilas, 2; only 1 plant was taken.
Two white plants gave whites only.

Sunilarly, from a diagram constructed for Case 3, we find there are 8 possible
magenta delilas; 5 plants gave 5. (Table IV gives Fs; for Case 3.)

Of crimson delilas, 4; 3 plants gave 2.
Of ivories, 4; 1 plant only was taken.
Of yellows, 2; 1 plant only was taken.

Finally, from a diagram for Case 4, we find there are 4 possible magenta
delilas; 6 plants gave 2. (Table V gives Fs; for Case 4.) One white plant
gave whites only. No other F, plants were taken.

It is clear also from Diagram I that there are 8 possible albinos. Various
yellows this year have been crossed with a number of F, whites as a means of
testing for the characters which may be borne by an albino.

White x Crimson Delila :—

Both these types were found to be breeding true.
F, consisted of magenta delilas only. (Table I.)

296 Miss M. Wheldale. The Inheritance of — [Feb. 12,

F',—Two of the cross-breds were selfed, the combined result giving :—

Number.
Macemuarde lila vane ser eee 89
Crimson. too) Le 34
AVIANGO', kone. Sta aen pee ewes 41
LOG ccc nace 164
If we assume the original parents to be :—
yo WB Vp re is sande White
OG Gar Fi Dein ae rere en eeaee Crimson delila,
the cross-breds would be of one kind only :—
A yl tts ee cokers eee Magenta delila

“ Reversion ” is again due to the introduction of I by the white plant. —
From selfing the cross-bred we should obtain theoretically :—

Ratios Numbers Numbers

: calculated. observed.
VOD eu Magenta delila a 9 vor 92 °25 89
eG Bel Dh derma Crimson’ 5, 2: 3 30 °75 34
a ences WV GE. haces veca'ves 4 41 :00 41
Totals \.2.5.. | 16 164 00 | 164

There is a close agreement between the theoretical and experimental
numbers.

White x Crimson :-—

The crimson parent was hybrid as regards the characters Land T. Fy, as
would be expected, consisted of magentas and magenta delilas. (Table I.)
Except as regards striping, this is a case similar to white x yellow crimson-
striped, and the same series of types were obtained in F2 as in the latter case.
As yet, sufficient numbers have not been grown to make the details complete.

White x Yellow :—

Both parents were breeding true.
F, consisted of magenta delilas only. (Table I.)
If we represent the parents by :—

Voy ll LL tt sea ee Gee eeeneen White
WY tll th toccen koe a ae ones Yellow,

= 1907 | Flower Colour in Antirrhinum majus. =

_ then the cross-breds would be of one kind only :—
Le Magenta delila

Fs, has not been obtained yet.

CRIMSON DELILA X OTHER TYPES.

Crimson Delila x Yellow Crimson-striped :-—

The crimson delila parent was breeding true. The other parent was the
same individual used in the cross white x yellow crimson-striped.

F consisted of crimsons and crimson delilas. (Table I.)
_ F2—The cross-breds appeared to be of four kinds, giving respectively, on

selfing :—
Case 1. se Case 3. Case 4.
Crimson x self. | Crimson x self. | Crimson delila x self. | Crimson delila x self.
Crimson..........+. 56 35 = Ba
ue delila ... 24 17 20 23
Yellow ..... teeeees 32 — 8 ==
Totals’ <..... | 112 | 52 | 28 | 23

If we assume the original parents to be :—

NOG TM LAT iby cracidtotcohyieriainteim essai Crimson delila
Yes i ee Gee Re os gi stonsiofttalanaiarsloforore Crimson,
then the cross-breds would be represented by :—
Pasa an LD) Ue 2 rarstads ate sichasinsee Crimson (Case 1)
NA a EM Bb pertaeys ota tno anlge' bs (Case 2)
MOVE UG 3555 connxcorssesesees = delila (Case 3)
SONA SOS ACR easy Cy ae eee 3 » (Case 4),

whole forms and delilas occurring in equal proportions.
From Case 1, on selfing, we should obtain theoretically :—

F Numbers Numbers

Ratios, calculated. observed.
2 id ah Crimson.,........... 9 or 63 :00 56
YC achs.ce - delila ... 3 21 ‘00 24
i ig GR ena Yellow ..5 200.0000: 4, 28 :00 32
Yi : = — ==

Totals ...... | 16 112 °00- | 112

298 Miss M. Wheldale. The Inheritance of [Feb. 12,

From Case 2 :—

Raine Numbers Numbers
z calculated. observed.
Lf Res Gael pen Crimson: accc esc sce 3 or 39 ‘00 35
Nees s delila... 1 13 -00 ivf
MO GaIS fee aes | 4, 52 00 52
From Case 3 :—
: Numbers Numbers
| Ratios. calculated. | observed.
1h Coane ee Crimson delila .. 3 or 21 :00 20
Wicca acecke Yellow. Ge aeccc i 7-00 8
Motels! wacese | 4 28 :00 | 28

And from Case 4 we should get crimson delilas only, which agrees with the:
experimental result.

In Case 1 there are theoretically 4 kinds of F2 crimsons; 10 plants taken.
at random gave 3 kinds. (Table VI gives F3 for Case 1.)

Of crimson delilas there are two kinds; both were given by 4 plants.

Yellows are of 1 kind only; 8 yellows gave yellows only.

Crimson Delila x Crimson :—

The crimson delila parent was breeding true. The crimson parent was.
shown to be hybrid as regards T, giving, on selfing, 3 crimsons to 1 crimson.
delila. .

F, consisted of crimsons and crimson delilas. (Table I.)

F.—Three crimson delila cross-breds gave crimson delilas only. F, has not.
yet been obtained from the crimsons.

If we assume the parents to be :—

YY? ar De ING a ete Crimson

VY Littl 22 ee eee 2 delila,
the cross-breds would be :-—

Via Li Ge. eee Crimson

YOY Ce it acca e rn ree 2 ' hs delila,

whole forms and delilas in equal proportions, and the latter would give:
crimson delilas only on selfing, which agrees with the experimental results. ©

1907. |] Flower Colour in Antirrhinum majus. 299

Crimson Delila x Yellow :—

Both parents were breeding true.

F; gives the expected result, namely, all crimson delilas. (Table I.)
F has not yet been obtained.

CRIMSON X OTHER TYPES.

Crimson x Yellow Crimson-striped :—

The crimson parent was breeding true. The other parent was the plant
used in previous crosses.

F, consisted of crimsons only. (Table I.)

F.—Only one cross-bred gave seed and the offspring were crimsons and
yellows. The numbers are too small to be of value.

If we assume the original parents to be :—

VON eso gl SF Gand es keen Pane weenie ee Crimson

SON GEtIRN Dl (Ey | ease i een Yellow crimson-striped,
the cross-breds would be :—

SN Cem NS ee Crimson

OS Gata) 1d i Sa Oe RE ;

VGN SIT eS on do aya Nodes 53

“VES yen (Fe Le oe a x

All therefore are crimsons, and of these, one, on selfing, namely, YY ii Ll TT,
would give crimsons and yellows.

Crimson x Yellow :—
_ Fy gave the expected result, namely, all crimsons. (Table I.)

F2 has not yet been obtained.

ROSE X OTHER TYPES.

The type rose has ivory lips tinged with magenta and a pale magenta tube.
The individuals used in crossing were found to breed true. Crosses were
made between this and most of the other types, but sufficient numbers have
not been obtained in F, to give a reliable indication of the results. A type,
where “ rose,” 2.¢., a tingeing with magenta, is superposed upon a yellow ground
(the case where the factor I is missing), appeared in Fe, together with delila
forms of both types; this would lead to the supposition that “rose ” may be
represented by a Mendelian factor. |

An interesting point has arisen in connection with the cross rose x a deep
crimson delila. The cross-bred is a deep magenta, in which the tube takes
the same shade as the lips. This fact suggests, as pointed out elsewhere
(p. 301), that there may be a coupling between the shade of tube- and lip-

VOL. LXXIX.—B. Z

300 Miss M. Wheldale. Zhe Inheritance of — [Feb. 12,

colour ; magenta colour introduced into the tube takes the same concentration
as that in the lips.

Experiments of De Vries on Antirrhinum.

In the ‘ Mutationstheorie,’ Lief. IV, pp. 194—206, Professor De Vries gives
an account of experiments in crossing certain varieties of Antirrhinum majus.
It is stated that yellow colour was not taken into consideration and experl-
ments were confined to the white and red varieties. The following is a
description of the types mentioned :—

E40) Kel See a Tube and lips red, the lips deeper
Fleischfarbig... Tube and lips pale red

IDELIUE ee neni Tube pale or white, lips fairly deep red

WViCISB Ct ci scka White, often with a distinct very pale red tinge

Professor De Vries regards red as being made up of fleischfarbig F and
delila D, two dominant characters, and white as producing two characters, w
and w’, recessive to F and D respectively.

A red Antirrhinum was crossed with white, giving an Fy like the red
parent. From the F, cross-breds on selfing, four types were obtained :—the
original red, delila, flesh-colour, and the original white, roughly in the
proportions 9:3:3:1. This result would be represented :—

Red x white

FD x ww’
|

‘
FDww’ Red cross-bred

The cross-bred x self would give :-—

| Ratios. Per cent.
ig B AE Re ROG ee sce eaten: 9 or 56
| a ee a Flesh-colour...... 3 19
| DAaar oeee Delile 92. ake: 3 19
LA White. ase. cesne ss 1 6
Potalicc: 16 100

1907.) | = Flower Colour in Antirrhinum majus. 301

The reds in F2 are of four kinds and would on self-fertilisation give in Fs
the following results :—

1 FD —. FD red
2 FDw’ —~ FD (red) + Fw’ (flesh-colour)
2 FDw —- FD (red) + Dw (delila)
4 FDww’ —- the whole series again

The delilas are of two kinds :—

1 Dw —- Dw (delila)
2 Dww’ —~- Dw (delila) + ww’ (white)

The flesh-colours also :—

1 Fw’ —. Fw’ (flesh-colour)
2 Fww’ —- Fw (flesh-colour)+ ww’ (white)

Professor De Vries showed that a delila and a flesh-colour in F. gave, on
selfing, an F3 consisting of delilas only and flesh-colours only respectively.
Other F2 delilas gave delilas and whites and other F. flesh-colours gave flesh-
colours and whites. Further, among the F, reds, some gave reds and flesh-
colours in the proportion 3:1, while others gave reds and delilas in the same
proportion.

Professor De Vries has kindly informed me that his types correspond to the
following types mentioned in this paper, namely :—

Roth to Magenta
Delila x - delila
Fleischfarbig ,, Rose

Weiss > Lvory

» (tinged) ,, Rose delila

Rose xa delila form has been found to give a deep magenta whole form
(p. 299) and the result is consistent with the supposition that there is an inter-
dependency between the shade of colour in lips and tube; the magenta tube-
colour, introduced by rose, takes, in the cross-bred, the same shade as the
lip-colour. This result corroborates Professor De Vries’ suggestion that roth
consists of fleschfarbig and delila.*

Translated into the types used in this paper, the simplest expression for

* [In ‘Camb. Phil. Soc. Proc.,’ vol. 12, 1902, p. 50, I suggested an interpretation of
De Vries’ results with Antirrhinum, which then seemed to me preferable to that put
forward by him. Further evidence as to this and other cases of the resolution of
compound characters has shown that my suggestion was incorrect (see also ‘ Rep. Evol.
Com.,’ III, p. 12).—W. Barzson. |

Z 2

302 Miss M. Wheldale. The Inheritance of [Feb. 12,

Professor De Vries’ parent plants would be (Rl =rose lps, Rt = rose
tube) :—

YY FRY REEL Re Rt. 3... Magenta (RI Rt L = magenta)
Yor ilellsctarg to Ivory

and the cross-breds :—
NEE IVE laslrell GMM sires idyeeaanne Magenta

would give, on selfing :—

Batis: Numbers Numbers
; calculated. | observed.
per cent. per cent.
Rl Rt ....| Magenta, .......5.... 36 or 56 56
aie Nos 5 delila...... 12 19 24
RG oo ROG ...t0 32s ssi aston 9 14 15
HIRE £1). iee Magenta .........65. —_ — _
UL easibion tice Rose delila ......... 3
NG Rtv ston cat Magenta delila...... — } 7 11 5
dae rer: TWOrysre jstieocescsec: 4

The ivories and rose delilas are classed together as De Vries’ whites and
tinged whites. The numbers observed are the mean of those given for the
three experiments ; moreover, the various kinds of roth and fleischfarbig would
be found among the above F» forms.

In conclusion, I wish most sincerely to thank Miss Saunders and Mr. Bateson
for the advice and help they have kindly given me, both as regards many
practical details and in the writing of this paper.

I am also indebted to Mr. Lynch, of the Botanic Gardens, Cambridge, for
allowing me space under cover every year for the germination of my
seedlings.

1907.]

Flower Colour mv Antirrhinum majus. 303

Table I.—F, from Matings between various Types.

Matings.

White x yellow crimson-striped ...
Pe MCHIMNBON Aelia ..2...6/c.0cnens

» crimson eae aeastnnee

PE EV CLOW avsinn psinncp sooceedsiece
Crimson delila x yellow crimson-
Stripedien sree.

re X CFIMEOND «2.000000

,, MVE LOW 4 cai crcidteiows
Crimson x yellow crimson-striped...
PEAY CLOW Svnccc occ caencnces
OBE WYMELE: Csioe:n'Fs wba secivnmsie ict aves ee
PRPC ETIMISOM sais 50.020cecceecen cosmes

a ) x Crimson. Gelila.......00...c00.0

» x yellow crimson-striped ......

PM OW CLOW: ccsiede co seslsrnincecndtsdenne

MM TVOLY | ponte, cusice ese osernnes seas

Magenta.

Ns:

Colours in F).

ae is Crimson.
19*
33
4,
21
36
8
36
23

Crimson R
delila. Oey
35
10
22
20

20 (paler)

|

* The small total here may be due to the fact that a large number of F, individuals were

destroyed by frost.

Table II1.—Fs3 from Case 1 in the Mating White x Yellow Crimson-striped.

Colours in F3.

Ivory. | Yellow. | White.

—_—_—_- —---er—vwmnn>- oo OS _- SOU Cees See eee _e ee

Number of Colour of

) F, 2

individuals.) individuals. M

agenta.

3 Magenta +
1 ate Me scarcr +
3 Py ss cit +
1 7A ale en ee +
2 pe Le eee Ce +
1 ey ee | eee +
1 one OE ee +
1 Mae me vaisiasces +
5 Magenta delila as
1 bP) bP)
2 Crimson......... |
i oat a
2 OR aren
2 Crimson delila
1 ” ”
2 ORs Fes tices «
2 LAF | tle
2 ”
2 Yellow? A.iiese.
1

Magenta Gara Crimson
delila. ‘| delila.
+ + +
+ + =
goc + vee
+ oes sive
cee + slere
Gre eee e0e
+ + +
+ di +
+ oe ds
‘A + +

+ si:
+ ae
a +

+

+3

tans

Pete:

++

+ +

+

+ +

+

oe +

a +

+ +

ie -

+ ~

+ +

+

+

Beet cgh lace

+

via +
ai

|

304 The Inheritance of Flower Colour in Antirrhinum majus.

Table III.—F3 from Case 2 in the Mating White x Yellow Crimson-striped.

Colours in F3.

Number of Colour of
individuals.| individuals. Magenta : Crimson d
Magenta acta. Crimson. ABE White.

2 Magenta ........ + + + + ate
2 Pe mate: + + + +
3 ay rere + + 5
1 PST Aa hae + +
1 se ee ee: + + +
1 ca Oa ee + +
eRe ate weccceed +
1 Magenta delila se + + a
1 ” ” + +
1 5 % + | bide +
1 ” ” ot
i Grimson: aac me | + + +
1 Hee ne + Pi +
2 4 ee | - +
EIR on iceyy’ . dateeneneene | +
J Orson delila ee + +
2 White: . oe | +

Table IV.—F from Case 3 in the Mating White x Yellow Crimson-striped.

Colours in Fs.

Number of Colour of VEE
F, F, ‘
indivi .| indivi : M. rimson ,
individuals individuals pie Bea Ivory. Vallone White.
1 Magenta delila + ss oF 4p +
1 13 + + + +
1 * + + ae ae
1 mS + dae =5
1 3 + +
2 Crimson delila oe + Ate +
1 is + en +
D4 MLVOFY « .teedeSee ve ‘fe + + +
1 + +

Table V.—Fs from Case 4 in the Mating White x Yellow Crimson-striped.

Colours in F;.

Number of Colour of

_ 42 _,, Be

individuals, | individuals. Magenta delila. | Crimson delila.| White.
2 | oe delila + LE
4, | + eh +
1 | NWI RR DS esters So +

Longitudinal Symmetry in Phanerogamia. 305

Table VI—F3 from Case 1 in the Mating Crimson Delila x Yellow Crimson-

striped.
Number of Colour of Colours in Fy.
2 2
individuals. | individuals. Crimson. Crimson delila.| Yellow.
5 Crimson ......... + + +
3 Oy) le esanaeagor = ae +
2 fake teeceedts graiat +
2 » delila Les + ze
2 ” ? +
8 Molllow?,cacecee Sh +

Longitudinal Symmetry in Phanerogamia.
By Percy Groom, M.A., D.Se., F.LS.

(Communicated by Dr. D. H. Scott, F.R.S. Received January 24,—Read
February 21, 1907.)
(Abstract.)

The first object of this communication is to put forward a simple graphic
method of recording measurements concerning the longitudinal symmetry of
plants, and to test its efficiency as a means of solving morphological problems.
The method is as follows:—Measurements of the successive internodes of a
stem are made, and are recorded on squared paper as successive ordinates ;
the resultant curve is termed the internode curve. The longitudinal distances
apart of other members are dealt with in the same manner, and ranged into
other curves. |

The second object of the paper is to describe the general and particular
results obtained by use of the method.

In a typical herb, the internode curve of the main axis is a regular and
characteristic ascending-descending one (see Curve 1 in fig. 1), while those of

ANA AY

Fie. I.

306 Dr. P. Groom. [Jan. 24,

the successive branches, commencing at the base of the plants and ascending
the main stem, gradually change from the ascending-descending curve to a
purely descending one (see Curves 2—6 in fig. 1), or tend to do so. It is this
gradual change in the internode curve of the successive branches that explains
the two maxima in the internode lengths (and possibly in the rate of growth)
met with in branches.

By reference to plants of different habit, and growing under different
climates or in different habitats (including water), it is proved that these
internode curves are characteristic of the species, and inherent though lable
to disturbance by external conditions.

Alternate-leaved Chenopodiacec, both in the main axis and in the branches,
display a remarkable periodic zig-zag in the internode curve (see the curve
printed plain in fig. 2). By connecting the alternate ordinates, this can be
analysed into two consistent and often regular sub-curves (see the dotted
curves in fig. 2); one of these sub-curves is the “internode sub-curve,”’ and

ig./2:

the other the “ displacement sub-curve.” By comparison of the curves of the
main axis and branches of Chenopodiacee and typical herbs, also between
those of alternate-leaved and opposite-leaved Chenopodiacez, as well as by
other means, it is shown that the displacement sub-curve represents the
distances up which one leaf at each successive node has been relatively
displaced from a primitively opposite arrangement. Both sub-curves of the
main axis assume the ascending-descending form, but in the successively
higher branches the internode sub-curve changes from the ascending-descend-
ing form to a purely descending one, while the displacement sub-curve tends
to do likewise, or does so, at a later stage. This inherent peculiarity of the
chenopodiaceous internodes sheds light upon the affinity of the family to the
Caryophyllaceze, whose opposite leaves do not develop simultaneously; and
there are indications that at least several, if not all, of the families comprising
the Centrosperme are opposite-leaved in reality, or, like the Chenopodiacee,
in design.

To test this theory of displacement, reference was made to the admitted

1907.] Longitudinal Symmetry in Phanerogamia. 307

case of displacements of branches above their leaf-axils in Boraginacee. In
_ this connection, the curve was constructed by recording as successive ordinates
the heights of the successive branches above their subtending leaves. The
resultant displacement curve of the branches is of startling regularity, and
conforms in type with the displacement sub-curve of the leaves on the
chenopodiaceous main stem.

One significant point is that these old-established fixed (hereditary) dis-
placements in Chenopodiacee and Boraginacez follow the same rule in respect
to their distribution and relative dimensions as that formulated by De Vries
mm respect to anomalies and monstrositres.

The Boraginacee are shown to be probably opposite-leaved in design, though
alternate-leaved by displacement, and are thus brought into closer connection
with the Labiatee.

In the advanced case of Solanum Dulcamara, the graphic method showed
for the first time that the familiar displacements of leaves high up the axis
are anticipated lower down by similar ones.

Investigations of slighter or more fluctuating leaf-displacements in Lysimachia
vulgaris, Veronica virginiana, and Cinothera, showed that where the phyllo-
taxis is sometimes cyclic, and at other times acyclic, on the same axis,
individual, or species, the internode curve becomes irregular when any of the
leaves are alternate or sub-opposite. This irregular curve could be analysed
into consistent sub-curves when the displacements were slight. But in some
instances of more considerable displacements I failed to accomplish this
analysis, partly because the number of leaves originally associated with a
node is apt to vary at different ‘points of the same axis. The irregularity of
the curve is due to two causes: (1) the method of measurement rigidly
adopted, namely, from one leaf to the next one, may obviously lead to a
merely seeming diminution in length of an internode; (2) but, beyond this,
the displacement of a leaf causes a true (relative) abbreviation of the inter-
node up which it takes place. The irregularity of the internode curve in
these cases, and in others investigated, is of import in indicating that the
construction of an internode curve of merely one specimen (living or fossil)
may suffice to show that the phyllotaxis in the family varies from acyclic to
cyclic, just as similar treatment of one axis of a herb (living or fossil) may
show at once whether the axis be a main one or a branch and, if the latter, it
may give some information as to its insertion on the mother axis.

Incidentally, observations were made on the double-leaves of Lysimachia
vulgaris and Rhinanthus, and on the increase and decrease in the number of
leaves at a node in these plants. The morphological significance of the
observed facts is discussed. Of particular interest and novelty was one case

308 Longitudinal Symmetry on Phaneroganna.

in Lhinanthus, in which the opposite phyllotaxis changed to alternate by a
process which some botanists would describe as one of the concrescence of the |
two opposite leaves.

The observations just mentioned, as well as some regarding displacements,
more or less clearly indicated than when phyllotaxis is cyclic in design,
alternate internodes, nodes, and associated displacements are more closely
correlated than are successive ones. This may imply that morphogenous
impulses travel along orthostichies, but it at least sheds light upon a number
of varied morphological problems.

The theory of the construction of the phanerogamous stem is discussed, and
stress is laid upon the fact that the longitudinal displacement of leaves
demonstrates that internodes may be of different phylogenetic age and
lineage, and are, therefore, not serially homologous segments. The interpreta-
tion of these displacements adopted in this paper would also imply that the
stem is partly constituted of tissue originally appertaining to leaves, so that
“stem ” and “leaf” are not sharply distinguished members.

The graphic method was also tested by reference to other morphological
problems. It does not afford the means of distinguishing between a sympode
and a monopode in the cases of the sympodes of Petunia and Myosotis, though
the curves harmonise with the accepted view. But more decisive results were
obtained with the tendrilled axis of Ampelopsis hederacea ; here the complex,
periodic, yet apparently irregular, internode curve could be analysed only into
three consistent sub-curves (by Joining every third ordinate); and this exactly
corresponds to the interpretation of structure adopted by Eichler and others,
while it directly conflicts with rival theories.

‘sa 309

The Estimation of Chloroform in the Blood of Anesthetised

Ammals.

By G. A. Buckmaster, Assistant Professor of Physiology, University
College, University of London, and J. A. GarpNeErR, Lecturer on
Physiological Chemistry, University of London.

(Communicated by Dr. A. D. Waller, F.R.S. Received February 1,—Read
February 21, 1907.)

(From the Physiological Laboratory of the University of London.)

In a former paper* we expressed doubt whether the methods employed by
Dr. Maurice Nicloux and other French observers for accurately determining
the percentage of chloroform in the blood of anesthetised animals were
capable of yielding such satisfactory results as those obtained by Nicloux
when known quantities of chloroform were added to blood in witro;
on the ground that the mixing of blood and chloroform in vitro is an
essentially different process from that which takes place during the
physiological process of inhalation, since it has been shown that, during
anesthesia, chloroform associates itself particularly with the red corpuscles.

In order to test the validity of our criticism, and to enable the results
obtained by the French observers to be correlated with our own, and also to
gain an idea as to how far the method employed by Nicloux is capable of
giving reliable results in the case of tissues.to which our method may not be
readily applicable, we have madea series of parallel estimations by the two
methods on the blood of anzesthetised animals.

In our experiments with the method of Nicloux, we followed closely the
procedure which he has detailedt, and we take this opportunity to express
our indebtedness to Dr. Nicloux for his kindness in supplying us with the
distillation-apparatus which he uses, and in giving us a demonstration of his
method. As Nicloux points out,t the chief difficulty in his method consists
in determining exactly the end-point of the reaction which occurs in
titrating with silver nitrate, using potassium chromate as an indicator.
While taking every care to hit the end-point as closely as possible, we
uniformly slightly over-stepped rather than under-stepped the mark. In
carrying out the method, we found it necessary to use caustic soda made

* “Roy. Soc. Proc.,’ B, vol. 78, p. 418, 1906.

t “Dosage de petites quantités de chloroforme,” ‘Extraits du Bulletin de la Société
Chimique de Paris,’ 3rd series, vol. 33, p. 321, 1906.

{ Lbid.

310 Messrs. Buckmaster and Gardner. Estimation of [Feb. 1,

from sodium instead of caustic potash as recommended by Nicloux, as we
were unable in this country to obtain potash absolutely free from chloride.

I. Analyses of Chloroform by the Carvus and by the Nicloux Method (Dumas
Reaction).

The chloroform used in these experiments was kindly distilled for us by
Dr. J. Wade of Guy’s Hospital. It had a boiling-point of 61:14°—61:15° C.
at 760 mm. and a specific gravity 1:5088 15°/15°. The results of the analyses
are given in Table I and Table II.

Table I (Carius’ determinations).

Amount of Percentage of paige: f Percentage of
chloroform taken. chlorine. P chines chloroform found.
gramme.
O-L177 89-172 89 *102 100 :07
0 °2721 89 "139 89 °102 100 °03

0 ‘2683 89 084: 89 °102 99-97

Table II (Nicloux’ determinations).

| Theoretical | Percentage of

Amount Percentage of
of chloroform hlori ia q,| Percentage of chloroform
taken. Sav eae chlorine. found.
gramme.
I. Made with a solution 0 :0184 89 -262 89 °102 100 °18
of 0°92 per 1000 c.c.
of alcohol |
II. Made with a solution 0 -03815 | 88 -041 89 °102 98 °8
of 1 °575 per 1000 c.c.
of alcohol |
III. Made with a solution 0 0385 87 -935 89 -102 98 -69
of 1 -6764 per 1000 c.c.
of alcohol | |
IV. Made with a solution| 0 -06785 86 -66 | 89-102 97 :26
of 3 °3556 per 1000 c.c. |
of aleohol |
V. Made with a solution 0 ‘018735 90 °54 89 102 101 °6 |
of 9 °3675 per 1000 c.c. |
of alcohol |
VI. Do. do. 0 04684 87 °378 | 89 -102 98 -06

From the second table it will be seen that the average percentage of
chlorine found by the Nicloux method from the sample of chloroform 1is
88°3, a figure which is 0°8 per cent. below the theoretical value. The figures

1907.| Chloroform in the Blood of Anesthetised Animals. 311

are comparable with those given in the following table taken from Nicloux’
‘paper. |

| Poids de chloroforme.

Quantité
de chloroforme

Mis. Trouvé. retrouve.

millegrammes. | millegrammes. par cent.
5°0 100-0
I. Faite avec une solution 10 9° 98-0
alcoolique de chloroforme 20 19 *4 97 -0
4 10 grammes par litre 50 48 °6 97 -0
100 96 :0. 96:0
II. Faite avec une solution a a 2 ae :

alcoolique de chloroforme i. :

a 2 grammes par litre 20 19 °7 98 °5
& p 40 39 °8 98 °5

II. Determination of Chloroform in the Arterial Blood of Ancsthetised
Animals by our Method and that of Nicloux (Dumas’ Reaction).

The experiments were conducted on cats. The animals were anesthetised
by ether, and the necessary operations for introducing cannule into the
trachea and carotid artery were performed. The tracheal cannula was fitted
to a Chauveau’s valve and chloroform administered by a Woulff’s bottle.
Samples of blood were taken before the administration of chloroform, and at
some selected period in the course of the anesthesia a measured quantity of
blood (20 to 30 cc.) was withdrawn, and this was at once divided up into
various proportions for analysis by the two methods. For the purpose of the
Nicloux estimations some samples were allowed to clot before estimation,*
some were treated with sodium oxalate to prevent clotting, others with
hirudin, and others were defibrinated by shaking with mercury. In the case

of blood withdrawn at the asphyxial point it is, in the case of cats, very

difficult to weigh any sample before clotting takes place, since this occurs
with great rapidity.

1. Experiments in which the Blood was allowed to clot before treatment with
acidified Alcohol.

Varying weights of blood were taken on which to carry out duplicate
experiments. During distillation, the clotted blood was found to become
detached as a disc of leathery consistence, about the size and thickness of

* Dr. Nicloux has kindly informed us, by letter, of a detail that we did not gather from
his paper, viz., that in his observations the blood is withdrawn with a syringe and
immediately mixed with alcohol so that it does not clot en masse.

312 Messrs. Buckmaster and Gardner. Estimation of [Feb. 1,

a four-shilling piece, which persisted to the end of the process. The
formation of this leather-like mass was only partially prevented by breaking
up the clot with a rod before distillation. The results of these experiments
are given in the following table.

Table III.
Number of Weight of : Chloroform Chloroform
experiment. blood taken. State of ansesthesia. by Carius. by Nicloux.
grammes.
5 954 0 053
1 7 925 } Asphyxis c2cfhhas. estcutecr en ; — 0 *0324,
7 °6908 — 0 0354
5 1381 0 °0575
2 | 9-735 ; Near asphyxia ............ceees. — 0 0269
5 °4656 == 0 ‘0404,
8 { 7 0302 Between vanishing of reflexes { — 0 -0333
9 5823 and asphyxia — 0 0236

2. Experiment in which Blood was defibrinated by shaking with Mercury before
the addition of Alcohol.

Only one determination was made by this method, since it might be
objected that some chloroform was lost in the shaking process.

Table IV.
Number of Weight of State of thesi Chloroform
experiment. blood taken. adem iauasi mame by Nicloux.
grammes.
4 9 -4017 After 55 minutes’ inhalation of 2 per { 0-021
5 9302 cent. chloroform 0 025

1

3. In this series of experiments a comparison was made, using oxalated
and clotted blood.

1907.] Chloroform in the Blood of Anesthetised Animals. 318
Table V.

, ai Chloroform | Chloroform
Number of | Weight of | Condition ‘
experiment. blood. of the blood. State of ansesthesia. b

Carius. Nicloux.

5 :0748 — Between disappearance of| 0 -°0298
5 reflexes and asphyxia
7°02 Oxalated ...... — — 0 0237
5 *5657 Who eames — — 0 0244.
5 *2192 —_ After 47 minutes with| 0°034
} 3°7 per cent. chloro-
6 3 616 — form 0 0358
6 *883 Oxalated ...... — — 0 -0285
8 °2588 PO oer: ~ _ 0 0256
10 ‘97 Clotted......... — — 0-017
4, °6723 ~— After 40 minutes’ inha-| 00256
lation of 2 per cent.
7 chloroform
8°2447 | Oxalated ...... — — 0 022
6 °3256 La arene? — —- 0 0248
4, °5388 — After 35 minutes’ inha-| 0°016
lation of 2 per cent.
8 chloroform
70821 | Oxalated ...... — — 0 0235
7 °7917 Seva. teens — — 0 -0206
4 °1422 _— After half an hour inha-}| 0 °0447
9 { lation of 3 per cent.
chloroform
7°9383 | Oxalated ...... — — 0 0338
4, *2004 — After a prolonged anes-} 0°0485
thesia. Chloroform,
given intermittently,
about 3 per cent.
10 6°6885 | Clotted and — — 0 -0333
clot broken
up with
glass rod
59814 | Oxalated ...... — — 0-031
42491 _ — 0 0354
5 °2041 — — 0 -0339
11 11 6862 | Clotted......... — — 0 0236
l 5 °7553 rc nue ene — — 0-03
9°4907 | Oxalated ...... — — 0 027

4. In the last series of experiments the cat was treated with hirudin in
such proportion as to render the blood incoagulable for some minutes. We
find that this is an excellent substance for checking the tendency to clotting,
which is go liable to occur in cats at the moment of asphyxia.

314 Chloroform in the Blood of Anesthetised Ammals.

Table VI.
Number of Weight of Chloroform by Chloroform by
experiment. blood. Carius. ‘ Nicloux.
grammes.
4,579 0 0319
12 4 °1432 0 °0327
14 °9269 = 0 0334
13 ‘879 | = 0 -0359
Conclusions.

1. The method of chloroform determination, based on Dumas’ reaction,
though not possessing a very high degree of precision as an exact chemical
method, as is apparent from a comparison of Tables I and II, is sufficiently
accurate for most practical purposes and, as used by Dr. Nicloux, is a simple
and rapid method, and one capable of giving satisfactory results for the
estimation of small quantities of chloroform in air and in simple solution in
such liquids as urine, etc.

2. In the case of the blood of anesthetised animals, if the blood for
analysis is allowed to clot before admixture with acid alcohol, the results by
Nicloux’ method are uniformly too low. An inspection of Tables III and V
will show that results concordant amongst themselves are obtained when
sensibly equal quantities of blood are taken, but, as might be expected, the
figures decrease the larger the amount of blood used. In cases where smaller
amounts of blood are taken and, consequently, the amounts of chloroform
dealt with are very small, the results are often near the truth; but this, we
think, is largely due to the inherent difficulty in the method as regards the
determination of the end-point.

3. In cases where the clotting of blood was prevented by oxalate, the
results more nearly approach those given by the methods we have previously
adopted, but still are somewhat lower.

If we take the results given in Table V, the average percentage of
chloroform indicated by our method is 0°0319, and by that of Nicloux in the
case of oxalated blood is 0°0259. In our method we are satisfied that the
average Maximum error is not more than 5 per cent., generally considerably
less. We find the difference between the results given by our method and
that of Nicloux’ method averages 19 per cent.

4, In the single experiment quoted with hirudin, the figures by the two
methods are remarkably concordant.

Cyanogenesis in Plants. 315

5. On account of the variable and low results obtained with clotted
blood, it may be inferred that the method of Nicloux may give low results
when applied to organs or tissues which do not disintegrate when boiled
with acid alcohol. We take this opportunity of stating that the expenses of
the foregoing work were defrayed out of a grant made by the Royal Society.

Cyanogenesis in Plants. Part VI.—On Phaseolunatin and the
Associated Enzymes in Flax, Cassava, and the “ Luma Bean.”

By Professor WynpHam R. Dunstan, M.A., LL.D., F.RS., T. A. Henry,
D.Sc. (Lond.), and 8. J. M. Auxp, Ph.D. (Wiirzburg).

(Received February 27,—Read February 28, 1907.)

In a previous paper of this series* it has been shown that the production
of prussic acid by the seeds or beans (“ Lima beans”) of Phaseolus lunatus is
due to the interaction of a cyanogenetic glucoside, phaseolunatin, with an
enzyme, both these substances being proved to exist in the seeds.

Phaseolunatin was proved to have the composition and constitution of
a dextrose ether of acetonecyanohydrin, but it was not then obtained in
sufficient quantity to ascertain precisely the structure of the dextrose residue
in the glucoside. Recently, however,f large supplies of “Java beans” (the
beans produced by Phaseolus lunatus grown in Java) have been imported into
this country, and we are indebted to Dr. Bernard Dyer for a small consign-
ment of these beans, which has constituted the raw material from which
the considerable quantities of phaseolunatin required in the course of the
present investigation have been prepared.

Since the publication of our previous paper, it has been asserted by Kohn-
Abrest{ that these “Java beans” contain not one, but several cyanogenetic
glucosides, and that none of these yield acetone on hydrolysis by hot dilute
mineral acids or by the glucosidolytic enzymes present in the beans.

We have considered it necessary, therefore, to examine carefully the
glucosidic product obtained from “Java beans” by the process originally
used by us§ in the investigation of the beans of Phaseolus lunatus obtained
from Mauritius and we have been unable to detect the presence of any other
cyanogenetic glucoside in Java beans except phaseolunatin, identical in all

* Dunstan and Henry, ‘ Roy. Soc. Proc.,’ 1903, vol. 72, p. 285.

+ ‘Bulletin of the Imperial Institute,’ 1905, vol. 3, p. 373, and 1906, vol. 4, p. 329.
t ‘Comptes rendus,’ 1906, vol. 143, p. 182.

§ Dunstan and Henry, loc. cit.

VOL: LXXIxX,—B. 2)

316 Prof, Dunstan and Drs. Henry and Auld. [Feb. 27,

respects with that first isolated by us from the practically wild Phaseolus
lunatus of Mauritius.* We have found no difficulty in confirming our
previous observation that this glucoside yields acetone on hydrolysis, and is,
in fact, a dextrose ether of acetonecyanohydrin.

We have shown recently} that the linamarin first isolated by Jorissen and
Hairs from flax (linseed) is identical with phaseolunatin, and that the latter
also occurs in the cassava of tropical countries. This identification of
linamarin with phaseolunatin makes it possible to discuss the divergent
views, which have been held by various investigators regarding the nature of
the enzyme, which occurs in association with phaseolunatin in flax and in
the beans of Phaseolus lunatus.

Jorissen, in his first paper{ dealing with the production of prussic acid by
flax, stated that the seed contained a substance on which emulsin acted in the
same way as on amygdalin. Ina later paper, Jorissen and Hairs§ recorded
the isolation of linamarin (phaseolunatin) from flax and stated that it was
decomposed by the enzyme associated with it in flax seed, but not by the
emulsin of almonds. Subsequently linamarin was re-examined by Jouck,]|
who stated that 1% was decomposed by emulsin. In the third paper of the
present series,{] dealing with the isolation of phaseolunatin, the identity of
which with linamarin had not then been established, it was stated that this
glucoside was decomposed by emulsin, and that as the range of activity of
the enzyme, occurring with phaseolunatin in the beans of Phaseolus lunatus,
appeared to be identical with that of the emulsin of almonds, it might be
assumed provisionally that these beans contained emulsin.

This statement was based on the result of a single experiment, in which
prussic acid was undoubtedly liberated when a commercial emulsin
preparation was added to an aqueous solution of the glucoside. This
experiment has, however, been repeated frequently, using several different
commercial emulsin preparations as well as emulsin prepared by ourselves
from sweet almonds, and in no case have we been able to observe again the
formation of prussic acid within a reasonable time, though in every case
prussic acid was liberated on the further addition of the mixture of enzymes
prepared from the beans of Phaseolus luwnatus or from flax or cassava.

* M. Kohn-Abrest’s results are dealt with more fully in a recent paper (Dunstan and
Henry, ‘ Annales de Chimie et de Physique,’ January, 1907).

+ Dunstan, Henry, and Auld, ‘ Roy. Soc. Proc.,’ 1906, vol. 78, p. 145.

+ Jorissen, ‘ Bull. Acad. Roy. Belg.,’ 1884 (iii), vol. 6, p. 718.

§ Jorissen and Hairs, ‘Bull. Acad. Roy. Belg.’ (iii), vol. 21, p. 529.

|| Jouck, ‘ Beitrage zur Kenntnis der Blausdure abspaltenden Glycoside,’ Strassburg,
1902.

4 Dunstan and Henry, ‘ Roy. Soc. Proc.,’ 1903, vol. 72, p. 285.

1907. | Cyanogenesis in Plants. 317

The statement that phaseolunatin is decomposed by the emulsin of
almonds must therefore be withdrawn. It is probable that in the cases
(recorded by Jorissen,* Jouck,t} and in the third paper of this series{) in
which the liberation of prussic acid from phaseolunatin has been attributed
to the action of emulsin the decomposition was in reality due to some
secondary cause and not to the direct agency of emulsin.

There is, therefore, the fundamental difference between the emulsin of
almonds and the enzymes occurring in Phaseolus lunatus beans, flax, and
cassava, that whilst emulsin decomposes amygdalin and salicin and is
without action on phaseolunatin, the enzymes of Phaseolus lunatus, flax, and
cassava decompose all three of these glucosides. The question at once arises
as to whether the enzyme associated with phaseolunatin in these three plants
is merely an enzyme of the emulsin type having a greater range of activity,
or is a mixture of emulsin with a second enzyme, and that the decomposition
of phaseolunatin is due to the action of the latter. The first of these
hypotheses seems at first sight the more plausible, since it is difficult to admit
that the same mixture of two enzymes should occur in the three plants
already mentioned, especially since none of them contains, so far as has been
ascertained, any glucoside or complex sugar which could be attacked by
emulsin. The further evidence we have obtained, however, leaves no doubt
that, accepting present theories of enzyme action, these three plants do
contain two enzymes, one of the emulsin type and the other of the maltase
type.§ It is probable that to the action of the “ maltase” the decomposition
of phaseolunatin is due.

- Fischer|| has shown that thé glucosidolytic enzymes so far systematically
examined are divisible into two classes, the one capable of decomposing the
a-alkyl ethers of the hexoses and the other the stereo-isomeric @-alkyl ethers
of these sugars. The first class of enzymes may be regarded as typified by
the maltase of yeast and the second by the emulsin of almonds.

It is obviously possible from Fischer’s generalisation to classify an

* Jorissen, loc. cit.

+ Jouck, loc. cit.

t Dunstan and Henry, loc. cit.

§ Since the present investigation was concluded, some results have been published by
Marino and Fiorentino (‘ Gaz. Chim. Ital.,’ 1906, vol. 36, p. 395), which seem to indicate
that the maltase of malt is capable of decomposing both a- and #-glucosides ; thus,
according to these authors, it effects the decomposition both of maltose and salicin. They
reject the obvious and simple explanation of these anomalous results, viz., that the maltase
they used contained emulsin, on what appear to be insufficient grounds. If their conclu-
sions were established, our present views respecting the nature and mode of action of the
group of glucosidolytic enzymes would require to be considerably modified.

|| ‘Zeit. Physiol. Chem.,’ 1898, vol. 26, p. 75.

Dp:

318 Prof. Dunstan and Drs. Henry and Auld. [| Feb. 27,

unknown glucosidolytic enzyme by ascertaining whether it is active towards
the a-alkyl ethers of the hexoses or towards the stereo-isomeric (-ethers.
EK. F. Armstrong* has extended Fischer’s work in this direction by showing
that when the e-alkyl ethers of the hexoses are decomposed hydrolytically
by enzymes of the maltase type the sugars immediately liberated are the
a-forms of the hexoses, and that, similarly, when the stereo-isomeric §-ethers
are decomposed by enzymes, they furnish the @-forms of the hexoses.

In both cases the forms of the hexoses first berated in the solution
change, gradually if left to themselves, or immediately if a trace of alkali be
added to the liquid, producing the equilibrium mixtures of the «- and @-forms
of the hexoses, thus exhibiting the phenomenon of mutarotation which
Lowryf first explained in this way.

EK. F. Armstrong} has extended these results to a number of the naturally
occurring sugars and also to several of the synthetic glucosides, and has
succeeded in showing that by the action of invertase on sucrose and raffinose
the a-form of dextrose is produced and that emulsin decomposes @-methyl
glucoside, liberating the @-form of dextrose.

Action of Yeast on Phaseolunatin.

Since it had proved impossible to decompose phaseolunatin by emulsin,
which is an enzyme of the §-class, it seemed advisable to ascertain if 1t could
be decomposed by yeast-maltase. The latter was prepared by mixing yeast,
previously dried at 30° C., with sand and grinding the mixture with a small
quantity of water in a mortar. The paste so obtained was set aside with
more water for 24 hours and maintained at a temperature of 35° C., 1 per
cent. of toluene being added as an antiseptic.

The aqueous extract so obtained was filtered through a porous tube of
baked clay, and by this means a pale straw-coloured liquid was obtained free
from yeast cells. This liquid readily decomposed «-methyl glucoside and
phaseolunatin. The results of several of the experiments made are given in
the following table :—

* ‘Chem. Soc. Journ.,’ 1903, vol. 83, p. 1305.
+ ‘Chem. Soc. Journ.,’ 1899, vol. 75, p. 213.
t Loe. cit.

1907. | Cyanogenesis in Plants. 319

Amount of

maltase Time Eamets OF

Name of glucoside used. preparation | of action. | | eee
c.c. hours. per cent.
A. a-Methyl glucoside, 0'2 gramme 20 25 59 -0*
-Phaseolunatin, 0°3 gramme ...... 20 25 30 ‘St
B. Phaseolunatin, 0°3 gramme ...... 30 20 22 -5t
0°3 gramme ...... 30 30 30 ‘OF
a Methyl glucoside, 0°2 gramme 10 20 26 °0*

* Determined by estimating the dextrose produced.
+ e. Ks 3 prussic acid produced.

The action was found to proceed more readily, and could be more easily
followed when, as suggested by Fischer,* dried yeast was substituted for the
extract. Thus, in one particular case, using yeast dried at 30° C., 85 per
cent. of the phaseolunatin was decomposed in 24 hours.

These results indicate that phaseolunatin is an a-glucoside, since it is
decomposed by yeast-maltase. Attempts were therefore made to ascertain
by experiment whether a-dextrose is produced initially when the glucoside is
hydrolysed by an enzyme.

Nature of the Dextrose Residue in Phaseolunatin.

A preparation of the enzymes contained in the beans of Phaseolus lunatus
was made by extracting the beans with cold water, exposing the extract in
a vacuous desiccator over sticks of potassium hydroxide to remove the
hydrocyanic acid formed and then pouring the aqueous extract into excess of
alcohol. The precipitate so obtained was dried on a porous tile at the
‘atmospheric temperature. :

The characterisation of the sugar produced by the decomposition of
phaseolunatin was carried out as follows. To a solution of 3 grammes of the
glucoside in 20 cc. of water, 0°5 gramme of the enzyme preparation was
added and the mixture kept at 40° C. in a stoppered bottle.

For each observation a few cubic centimetres of the liquid were withdrawn,
mixed with a small definite quantity of alumina cream and immediately
filtered through a closely felted pad of asbestos. The optical rotation of the
clear filtrate was then determined before and ajter the addition of a drop of
ammonia solution, the latter being used as indicated by Lowryt to establish
equilibrium. The same polarimeter tube was used throughout the

experiments.
* ‘ Berichte,’ 1894, vol. 27, p. 1479.
t+ ‘Chem. Soc. Journ.,’ 1899, vol. 75, p. 213.

320 Prof. Dunstan and Drs. Henry and Auld. _ [Feb. 27,

Series 1—Temperature, 39° C.

Rotation after

FS Fac Initial rotation. addition pene
5 of ammonia. ’

hours. ° / (e) / fe} 4

(0) —115 —l 15* 0 O

1 —0O 46 —1 11 —O 25

2 —O 35 —0O 58 —0O 23

3 —0O 34 —O 57 —O 23

20 —0O 56 —0O 56 0 O

* The non-occurrence of any immediate change in rotation indicates that the optical rotation
of phaseolunatin is not affected by the addition of the minute quantity of alkali necessary in
carrying out these experiments.

In this series of experiments 2°85 grammes of phaseolunatin were dissolved
in 20 c.c. of water, and to the solution 0°5 gramme of the enzyme preparation

was added.
Series 2.—Temperature, 40° C.

Rotation after

; Ate Initial rotation. addition Ae eeeee
: of ammonia. :
hours. Shy) Be ey
0:0 —l O —l1 O 0 O
0°5 —O 44 —0O 59 —0O 15
1°5 —0O 38 —O 55 —0 17
3 °0 —0O 29 —0O 50 —O 21
20 °0 —0O 50 —0O 50 0 O

The various portions of the liquid resulting from these two series of
experiments were mixed and boiled with dilute sulphuric acid to ensure
complete decomposition of the glucoside. The acid liquid was then
neutralised with barium carbonate, evaporated to dryness on the water bath,
and the residue extracted with strong alcohol. This extract was then
decolourised with animal charcoal, and the contained sugar precipitated by
adding excess of ether. The sticky precipitate was dissolved in diluted
alcohol, from which on standing the sugar separated in the form of minute
colourless plates, melting at 85°, which is the melting point of dextrose. It
had, moreover, the same specific rotation as dextrose. It was shown in the
previous paper* that the sugar produced by the hydrolysis of phaseolunatin
is dextro-rotatory, and yields a phenylglucosazone, melting at 208°. The sugar
produced by the hydrolysis of phaseolunatin is therefore dextrose.

* Dunstan and Henry, ‘ Roy. Soc. Proc.,’ 1903, vol. 72, p. 285.

1907. | Cyanogenesis in Plants. 321

Further, the tabulated results given above show that an increase in the
levo-rotatory power of a solution of phaseolunatin, partially hydrolysed by
the natural enzyme, is produced by the addition of ammonia. This can only
mean that a highly dextro-rotatory substance initially produced by the action
of the enzyme is transformed by the ammonia into one of lower dextro-
rotatory power.

It will be seen that, taking the following data into account,

epectic rotation of phasecolumatin ..............cecceeccsessusesverese —27°4
6 - EMO ENOGE) Memo yee CAME waleeier cunt Saws rene +105°
. . f= Ue MOSER ice cane cetera keen i ceciacoinieneenes -+ 22°
. * the equilibrium mixture of «+ 8-dextrose +52°5

these results can only be explained on the assumption that the sugar initially
produced by the decomposition of phaseolunatin is «-dextrose, and that the
glucoside itself is the a-dextrose ether of acetonecyanohydrin. Phaseolunatin
appears to be the only naturally occurring «-glucoside so far known, all the
other glucosides so far examined yielding the 8-forms of the sugars on
complete hydrolysis by enzymes.

Nature of the Glucosidolytie Enzymes present in Phaseolus lunatus.

Since a-dextrose is produced from phaseolunatin by the enzyme preparation
obtained as already described from the beans of Phaseolus lunatus, it must
be assumed that this preparation contains an a-enzyme and, since the
elucoside is also decomposed by the maltase of yeast, it would appear that this
a-enzyme may be identical with yeast maltase, or is at any rate of the same
type.

The only other possible view is that the e-enzyme of Phaseolus lunatus may -
be identical with the invertase of yeast, but Fischer has shown that the
latter does not decompose a-methyl glucoside, and we have found that yeast
washings, containing invertase, have no action on phaseolunatin. In these
two respects, therefore, invertase differs from the a-enzyme of Phaseolus
lunatus.

The identity of the «enzyme of Phaseolus lunatus with yeast maltase
cannot, however, be definitely established, since, although the range of
activity of the two enzymes appears to be similar, yeast maltase decomposes
a-methyl glucoside and maltose more rapidly than does the a-enzyme of
Phaseolus lunatus, and the latter hydrolyses phaseolunatin more quickly than
yeast maltase does.

Since the enzyme preparation obtained from Phaseolus lunatus decomposes

322 Cyanogenesis in Plants.

amygdalin and salicin, and in this respect resembles the emulsin of almonds,
it must also contain a @-enzyme identical with or similar to emulsin. The
only other possible hypothesis, viz., that Phaseolus lunatus contains a single
elucosidolytic enzyme capable of decomposing both «- and #-glucosides,
seems incompatible with our present views of the relationships of enzymes
and glucosides.

A mixture of the same two enzymes must also occur in cassava and in
flax* since, as we have shown previously, enzyme preparations obtained from
these two sources behave in precisely the same manner as the enzyme
preparation obtained from Phaseolus lunatus. It is also worthy of mention in
this connection that yeast is capable of decomposing phaseolunatin, amygdalin,
and salicin, and on this and other grounds has been assumed to contain an
emulsin-like enzyme as well as maltase.t

* Dunstan, Henry, and Auld, ‘ Roy. Soc. Proc.,’ 1905, vol. 78, p. 145.
+ Henry and Auld, ‘ Roy. Soc. Proc.,’ 1905, vol. 76, p. 568.

323

Capillary Electrometer Records of the Electrical Changes during
the Natural Beat of the Frogs Heart. (Prelimnary
Communication. )

By Francis Gotcu, M.A., D.Sc., FLR.S.

(Received March 5,—Read March 14, 1907.)

It was shown, in 1880, by Burdon-Sanderson and Page, that when the
ventricle of the excised frog’s heart is connected with a capillary electrometer
by two contacts, one lying on the base and the other on the apex, then every
contraction of the heart was associated with electromotive changes of the
following type. The electrometer meniscus was displaced in a given direc-
tion, the displacement then suddenly returned and, after a certain time, a
second final displacement occurred in the opposite direction, which returned
slowly ; the recorded displacements are therefore of the type shown in fig. 1
of the annexed diagram. Most of these earlier experiments were carried out
on the excised heart rendered motionless by a suitable ligature around the
sino-auricular junction, and excited to activity by an induced current which was
applied to the tissue, near one of the electrometer contacts. The observed effects
were demonstrated to be due to the algebraic sum of an active process under one
contact, of the propagation of this process to the tissue under the other contact,
and of the occurrence of a similar active process at this further point. The
two phases, such as occur in fig. 1, were further shown to be indicative of the
time relations of these active processes. The electromotive change during the
active process causes this tissue to become relatively negative as compared
with the inactive tissue. This negativity commences under the proximal
contact, nearest to the seat of excitation; its development is, however, cut
short by a similar change occurring under the distal contact situated further
off from the seat of excitation, thus giving the first electrometer displacement
the character of a single spike. Whilst both changes are in full progress
there is no electrical difference between the contacts, and thus an iso-electrical
interval occurs; finally, the change at the proximal contact, having commenced
first, ends first, and the distal change outlasting the proximal one, a terminal
displacement of opposite sign is produced. All these well-known results have
been supposed, with little experimental warrant, to occur in the beating
frog’s heart in situ, and a discrepancy thus exists between the assumed
electromotive phenomena of the beating frog’s heart and the electrical changes
actually observed in the beating heart of mammals and of man. Numerous
- records have been obtained in man and mammals by making use of Waller’s

324 Dr. F. Gotch. Records of Electrical Changes [Mar. 5,

discovery that the electrometer, when appropriately connected with the
surface of the body, shows the electromotive changes which accompany each
beat of the heart im setu. These have been supplemented by observations
upon the exposed beating heart (Bayliss, Starling, etc.). The latest records
of this type in the case of man are those which Einthoven has obtained by
the use of his delicate string galvanometer. All these records differ
fundamentally from the records obtained by Burdon-Sanderson with the
excised heart of both the frog and the tortoise, for, although the effects are
diphasic, the second phase in the mammalian heart is of the same sign as the
first (as in fig: 2), instead of being of opposite sign. It has thus been
suggested that there is a want of parallelism between the activities of the
mammalian heart and those of the heart in the frog and the tortoise.

The present experiments show that the changes observed when the frog’s
heart remains in sitw and supplied by blood are precisely similar to those
known to occur in the mammalian heart, they also bring out a further new
point ot no little interest, since, under certain conditions, a return wave of
activity proceeding from the apex to the base is clearly demonstrable; this
wave succeeds that which proceeds from the base to the apex. It is this
return wave which is largely responsible for the extensive terminal phase of
the whole electromotive effect in the beating heart.

The observations now carried out have been made under a considerable
variety of conditions, only a few of which can be at present referred to. The
recording instrument employed was the extremely sensitive electrometer
used by myself for the investigation of nerve. In order to indicate its
sensitiveness, I may state that with one contact on a locally injured surface
the full change under the other uninjured contact caused a very extensive
displacement of the mercury meniscus which, when magnified by the lens
used for its projection upon the recording photographic plate, displayed itself
as a movement of from 150 to 180 mm.

The heart of the pithed frog was carefully exposed and left covered by
pericardium; two contacts were then placed, one across the auriculo-
ventricular groove, the other upon the ventricle apex. Hach natural beat
of the heart, thus left im situ, was associated with electromotive changes
which caused electrometer movements of the character shown in fig. 2. In
the right hand of this curve is the commencement of a heart beat, the rise of
the curve indicating the relative negativity of the tissue under the contact
upon the groove; the point (a) is the commencement of the auricular con-
traction, the point (0) the commencement of the ventricular contraction 0’46
later. In the left part of fig. 2 the whole of the main ventricular change is
displayed ; it begins at the point (6) and commences to subside at the point

1907.] durng the Natural Beat of the Frog’s Heart. 325

(7). The successive events comprise an initial rise at (0) (negativity of the
base), then a sudden fall at (c), occurring 0’’04 later; this gradually ceases,
and after some little time a final or terminal rise (negativity of the base) recom-
mences at (¢). Such a record indicates that the base activity, commencing at
(6) and beginning to subside at (/), is maintained longer than the apex
activity which commences at (c) and begins to subside at (e); the duration of
the base activity in this case was 1’24, that of the apex activity was 1°7-08.
The curve is in all essentials similar in character to that already referred to as
obtained from the mammalian heart in situ, both the first, or initial, phase and
the second, or terminal, phase having the same sign. Similar records of less
extent were obtained when the contacts were appropriately placed upon the
external body wal] of the frog without exposing the heart, whilst similar
ones of larger extent were obtained when the pericardium was opened and
the contacts placed upon the actual cardiac surface. If the heart is excised,
then the records gradually change in character until ones of the character
shown in fig. 1 may be obtained; these correspond in type with those described
by Burdon-Sanderson as obtained from the immobile excised heart aroused
by an artificial stimulus. Itis, however, certain that the diphasic effect with
phases of opposite sign, such as that shown in fig. 1, is not characteristic of
the frog’s heart when examined in sitw, supplied with blood and beating
naturally.

An interesting modification in the type of the records occurs if the
ventricle is distended with blood; such distension is readily produced by
slight pressure upon the vessels leading from the truncus arteriosus. In
order to obtain the modification, the contact upon the groove is now so
placed as to comprise that portion of the ventricle base which lies near the
exit of the aorta. The modification is shown in fig. 3, which gives one full
ventricular change. At the point (0) the base negativity due to activity of
this part commences, the rise is cut short, as before, at the point (c), 0°05
later, this being due to the propagation of the active state from the base to the
apex and the commencement of the apex change; the apex change is, how-
ever, itself.now cut short at the point (d) by a third change occurring about
0’-05 later, and this last change is of the same type as.the initial base one,
v.€., indicates a second development of base negativity. On the subsidence
of the apex effect, which begins at the point (e), these two more prolonged
base effects, being in the same direction, are summated and produce the con-
spicuous terminal phase which culminates at the point (/). There is no
doubt that the period from (0) to (c) indicates the time taken by the wave
of activity in its propagation from the base to the apex, in this instance
(temp. 12° C.) at a rate of from 130 to 140 mm. in 1 second; it follows that

326 Dr. F. Gotch. Records of Electrical Changes [| Mar. 5,

the period from (c) to (d) may be interpreted as the time taken by the
propagation of the active state back again from the apex to that part of the
base from which the aorta springs, in this instance also at a rate of from
130 to 140 mm. in 1 second.

Control experiments, with a eontact upon the aortic bulb, showed that this
second reversal was not due to the development of electromotive changes in
the aortic wall or bulb, these being too feeble to affect the recording instrument.
Moreover, although curves showing double reversals of the type shown in
fig. 5 are obtained when the heart begins to get distended with blood, if, by
further aortic compression, this distension becomes considerable, then the
records tend to return to the type indicated in fig. 1, the ventricle base effect
near the aorta being now almost imperceptible; on removing the distension
and letting the blood out of the swollen heart by cutting through the bulb,
curves of the type shown in fig. 3, with the double reversal of the first phase,
are again obtained. It thus appears that adequate blood supply through
intracardiac pressure brings into prominence the activity of that part of the
ventricle which leads up to the aorta, and that this favourable influence,
although interfered with by considerable distension, continues after the blood
has left the beart; hence the new phenomena of double reversal are not
directly caused by intraventricular blood pressure, but are indirectly asso-
ciated with a previous adequate blood supply of the muscular tissue forming
the ventricular wall. A number of experiments carried out with different
positions of contacts and under a variety of conditions (local slight injury,
local warming, etc.) have convinced me that the cause of the double reversal
is the circumstance that, during the ventricular contraction, a wave of activity
starts in that part of the base which is not immediately related to the aortic
exit, and that this, having been propagated to the apex, then returns from
the apex to the aortic part of the base.

Only one class of such experiments need be referred. to in this preliminary
communication. If contacts are placed on each lateral margin of the base
near the groove, so that one hes upon the left side remote from the aorta, the
other upon the right side near the spring of the aorta, then each natural
beat of the heart is associated with electromotive effects of the type shown
in fig. 4; these become more and more pronounced under the special con-
ditions which favour the appearance of the double reversal. The curve given
in fig. 4 shows the auricular beat commencing at the point (a); this is
followed after an interval of 0’°45 by the base effect which begins at the
point (6). Now,since the rise of the curve indicates relative negativity of the
tissue under the contact on the left margin of the groove, which is remote
from the aortic exit, it is clear that the change activity must commence here

1907. | during the Natural Beat of the Frog's Heart. 327

Time 2%" ai

PTT PMN eat INS CLAY A PT EY Nea ears aly yey
‘

b ei ee

DESCRIPTION OF FIGURES.

The curves given in the above figures are outlines of four photographic records of the
displacement of the capillary electrometer meniscus. On each figure a rise in the curve
indicates relative negativity of the tissue under a contact upon the auriculo-ventricular
groove ; all the curves are to be read from left to right.

Fie. 1.—Excised frog’s heart, contacts upon auriculo-ventricular groove and upon the
ventricle apex; natural beats; (a) auricular activity, (6) commencement of
ventricular activity at base of ventricle, (ce) commencement of ventricular
activity at apex of ventricle, (7) summit of final ventricular phase.

Fie. 2.—Frog’s heart ¢n situ, contacts as in fig. 1; (a) commencement of auricular
activity, (6) commencement of ventricular activity at base, (c) commencement of
ventricular activity at apex, (f/f) summit of final phase.

Fie. 3.—Frog’s heart in situ, but during or after distension with blood, contacts on
ventricle base near spring of aorta and on ventricle apex ; (6) commencement of
general base activity, (c) commencement of ventricular activity at apex, (d) com-
mencement of base activity localised to neighbourhood of aorta, (f/f) summit of
final ventricular phase.

Fic. 4.—Frog’s heart in situ, contacts on each side of base near groove, one on left side
remote from aorta, the other on right side close to spring of aorta; (a) com-
mencement of auricular activity, (6) commencement of activity under contact on
left side of base remote from aorta, (c) commencement of activity on right side
of base near aorta.

before a similar change commences under the other contact, that is, in the
tissue near the aortic exit. The rise is, however, cut short at the point (c)

328 Klectrical Changes during Natural Beat of Frog's Heart.

by the commencement of such a change under the aortic base contact; this
occurs in a number of measured records after a delay of from 0’06 to
0’-08, we. a longer interval than can be accounted for by the propagation
time from base to apex. Moreover, the base change which commences first
at (b), subsides first at (¢), that commencing later at (c) subsiding later at (7),
and thus causing a second or terminal phase of opposite sign to the first, or
initial, one. Many records of this type have been obtained, and in every
instance the first phase is of the same character; hence in the natural beat
the whole base does not pass simultaneously into the active state with its
associated relative negativity, the manifestation of such activity in the part
near the exit of the aorta being always delayed.

The development of the frog’s heart from a primitive S-shaped tube, and
the well-known fact that a contraction of the wall of the aortic bulb occurs
as the last stage of the whole cardiac cycle, are in accordance with the
observations now described. These show that the frog’s heart, when fully
developed, continues to display signs of its ontogeny, and that even when
these are structurally imperceptible, they can be revealed by appropriate
physiological methods.

[The experiments here described were all carried out upon winter specimens
of Rana temporaria during the months of January, February, and March. |

329

The Fermentation of Glucosides by Bacteria of the Typhord-colr
Group and the Acquisition of New Fermenting Powers by
Bacillus dysenterize and other Micro-orgamsms. Prelim-
nary Communication.

By F. W. Twort, M.R.C.S., L.R.C.P.

(Communicated by Leonard Hill, M.B., F.R.S. Received March 7,—
Read April 18, 1907.)

(From the Bacteriological Laboratory, London Hospital, E.)

For a number of years bacteriologists have largely relied upon certain
fermentation reactions of sugars and alcohols for the differentiation of
various micro-organisms which may otherwise be almost indistinguishable.
This applies more especially to the case of bacteria occurring in the so-called
“typhoid-coli group,” although recently this method of differentiation has
also been extended by Mervyn Gordon (1), Andrewes, Horder (2), and others,
to the group of micro-organisms described under the general name of
Streptococcus. As fresh fermentible substances have been introduced as
tests, the number of different species occurring in such micro-organismal
groups has become very large. Mervyn Gordon, in his studies on Strepto-
coccus and Staphylococcus, introduced tests on certain sugars and upon such
glucosides as coniferin and salicin, and he considered that these reactions
were of help in differentiating different Streptococci from each other.
MacConkey (3) has also published extended observations on the capability of
certain lactose fermenting fecal bacteria to attack sugars like saccharose and
dulcite, and from the reactions obtained he was led to subdivide these
lactose fermenters into four groups as follows :—

(1) A group represented by Bacillus acidr lactici, Hueppe, capable of
fermenting lactose, but without action on dulcite or saccharose.

(2) A group, the typical representative of which is Bacterium colt,
Escherich. In this case lactose and dulcite are fermented, but not
saccharose.

(3) A group of the types Bacillus neapolitanus, Emmerich, and Bacillus
pneumome, Friedlander, containing organisms which ferment
lactose, saccharose, and dulcite.

(4) A group containing micro-organisms capable of fermenting lactose
and saccharose, but not dulcite. In this group were placed
Bacillus lactis aerogenes, Escherich, Bacillus ‘capsulatus, Pfeiffer,
and Bacillus cloace, Jordan.

330 Mr. F. W. Twort. Mermentation of Glucosides by [Mar. 7,

On utilising these sugar tests, MacConkey was able to differentiate such
closely allied bacteria as B. coli, Escherich, B. acidi lactici, Hueppe, B. lactis
aerogenes, Escherich, and 5. pneumome, Friedlander.

It was with a view to obtaining other fermentation tests which might be
of service in separating organisms still remaining undifferentiated that the
following experiments with glucosides were undertaken.

In carrying out these tests, an ordinary peptone water medium containing
2 per cent. of the glucoside to be tested was used. Such a medium was
distributed in a number of test tubes, each of which contained a small
inverted glass tube sealed at the upper end after the method recommended
by Durham. In one series of experiments litmus solution was purposely
left out of the medium to see whether any colour change might be observable
in the glucoside under examination. In another series 5 per cent. of
Kahlbaum’s litmus solution was added, the medium being otherwise identical
with the above. All the glucosides used were obtained from Merck.

In the case of insoluble glucosides an amount was added to the peptone
water which would represent a 2-per-cent. solution.

All those media containing glucosides which by their colour obscured the
reaction of the litmus solution were repeatedly tested on litmus paper
during the growth of the organism.

Forty-nine glucosides were tested with the following 18 species of bacteria
belonging to the typhoid-coli group :—

B. fecalis alcaligenes, Petruschky.

B. dysenterie, Kruse.

. Flexner (Pseudo-dysentery IV of Firth).

B. typhosus, Eherth-Gaffky.

B. paratyphosus, Brion and Kayser.

i" Schottmiller.

B. enteritidis, Gaertner.

B. paracoli, Widal.

B. colt, Escherich.

B. pneumonie, Friedlander (MacConkey strain).

(laboratory strain).
(Kral strain).

2) 9

9 2)

B. neapolitanus, Emmerich.
B. pyogenes fetidus, Passet.
B. acidi lactict, Hueppe.

B. lactis aerogenes, Escherich,
B. capsulatus, Pfeiffer.

B. cloace, Jordan.

| 1907.] Bacteria of the Typhoid-coli Group, ete. 331

At least six tubes of each glucoside medium were examined, the period of

observation extending over four weeks.
Of the 49 glucosides examined, the following showed no reaction which

could be ascribed to fermentation :—

Ononin. Jalapin. Convallarin.
Ericolin. Scammonin. Quabin.
Digitalein. Colocynthin. Scopolin.
Helleborein. Plumierid. Frangulie acid.
Cyclamin. Absynthin. Smilacin.
Apiin. Quercitrin. Condurangin.
Hederaglucoside. Hesperidin. Convolvulin.
Tannin.

The glucoside plumierid gave a dark green coloration with B. capsulatus
and to a less extent with 5b. lactis aerogenes, B. cloace, and B. pneumonie, but
no real fermentation was observed.

The remaining 27 glucosides showing some degree of fermentation with
One or more of the test micro-organisms were as follows :—

Euonymin green. Periplocin. Populi.
Euonymin brown. Cathartinic acid. Camellin.
Iridin. Amyedalin. Globularin.
Senegin. Sapotoxin. Cerberid.
Coniferin. Saponin. Baptisin.
Arbutin. Bryonin. Coronillin.
Salicin. Convallamarin. Gratiolin.
Syringin. Digitalin. Adonidin.
Quillajinic acid. Strophanthin. Phloridzin.

In addition to the 18 test bacteria mentioned above, experiments were also
carried out in the glucoside media with 26 other members of the typhoid-
coli group. The latter cultures were isolated in the London Hospital from
cases mostly of cystitis and colitis.

The results obtained with the whole 44 bacteria are set forth in the
accompanying table, the type cultures are distinguished by an asterisk.

The sign + indicates a positive reaction, the sign — indicates that there
was no change in the glucoside under observation. Vacant spaces indicate
that no tests were made, the quantities of glucoside available being
insufficient. “A” is used to denote acid formation ; “G” gas formation.

In the table it can be seen that the bacteria have been subdivided into
seven sub-groups according to their fermentative action on glucose, lactose,
dulcite, and saccharose. |

VOL. LXXIX.—B. 2B

332 Mr. F. W. Twort. Fermentation of Glucosides by [Mar. 7,

The last four of these sub-groups correspond to the four groups of
MacConkey.

On looking at the table it is apparent—

1. That there is comparatively little difference between the fermenting
capabilities of many of the lactose fermenters. |

2. That two micro-organisms of a sub-group may differ in their ability to
ferment glucosides, whereas micro-organisms in different sub-groups may
present the same fermentative changes when tested on glucosides.

It will also be noted that the chief fermentations occur in the four sub-
groups of lactose fermenters, but whereas the members of the true coli
group give practically identical results when tested on glucosides, the results
with members of the Friedlander group show greater variations, the chief of
which is the inability of B. neopolitanus and Bacillus No. 41 to ferment
salicin, arbutin, and syringin, which glucosides are easily fermented by
£. friedlénder. It must also be pointed out that the fermentation of many
glucosides is neither marked nor constant.

Taking the individual glucosides in the table and reading from left to
right, the following are the chief general characteristics of the fermenta-
tions :—

Brown euonymin. Gas production very variable, often marked. Acid
production seldom present, never marked.

Green euonymin. Gas and acid formation never very marked.

Tridin and senegin. Acid and gas extremely variable, often marked.

Coniferin, arbutin, Usually show very distinct fermentation, both acid and

salicin, syringin. gas being frequently very marked, occasionally slight.
Quillajinic acid. Gas sometimes marked, acid always slight.
Populin. Gas never excessive, acid production very variable.

Camellin, globu->
larin, cerberid, | Gas production always slight, acid never very pro-
periplocin, ca- nounced.
thartinic acid.
Amygdalin. Gas never formed, acid production variable and some-
times marked, especially with members of Capsulatus
and Friedlander sub-groups.

Sapotoxin. Gas production always slight, acid variable, but most
pronounced, with Capsulatus and Friedlander sub-
groups.
Saponin. Gas very rarely present, acid usually slight.

Bryonin and con- Acid and gas never very pronounced and very incon-

vallamarin: stant.

aud

as 7
- »
_
=
\
{

:

(To face p. 32.)

Twort Table showing the Action of Bacteria, belonging to the Typhoid-coli Group, on Various Glucosides.
| es) ae eS can he ey eee z | | |
= = i | | i k “a
| = | reall | z | Ps | 5 2 | ai | Ci | 2 oI é
Ee ao : | =} | g a a a | —E = a . : Ss 2
SE | 88 | 2 io We Sil |e £ a it ne 4 EL ake Nee wilh ee 5 : = E : 5 é re | 3
ae | 2 | £ 3 F & £3 | ¢ z EP) et | oes el et El 4 | 4 2 3 & B E=| s 3 é é z
| Ca meee eos ae = lod | eB lee oa | es] |} es |e) | 2 | a | 6 | 8 | soe ies |e eee ees
No. || Sianies a ie A | Oe = a | ee | & z 2 z 8 |< Po EW ee il s | © £ z z e | 8 2 go | os 3 z
| a !a 4 | a | Ss | 4 a a | ce | oS eo | oe | ty Ss | 4 a | a | w is} Aa Z is} 6 | « 4 a 3 4 A | a
| Pana = I | a Ts, ae | eee nas lpae ; | = Th Mipeon ee Aer | = Palle | | aie PM (aww ss lies:
| AG | AG/AG AG | A @)/A G | AG|AG/)AG/AG/AG | AG@jiA G/A @G | AG AG@j|,)AG,AG | PAN (GH |} AN 1G) || AS 1G |) ASG: | AG|AG/|AG A@ | AG ]A @G@ | A @ | A Gj/|A G |
| | | \ | | |
| | | | eee | “(5 a
Te Bacitius) fescalts |aloalizenerPetrusohky, (Conrady strain) —)— ||) — = | — —)— —}— —)— —j— —)— —)— —j— — jj) — j— —4— —)-— |= =/= = l= Si] 242 22 Sis 2. 2 2a Se }- -J- - - - - -|- -)
2 Bacillus dysenterie, Kruse (Kral strain). = =) => j=) + H= + HJ] - =] - - - | S = SS SS] HS] ] SS) Sie SoS Sse Sle al= = | eh rae et | (ese inet Pee Pe a ee a) Se ae I
3* Bacillus dysenteriae, Flexner (Firth strain) =el= =)|/2 = =|] S42 Si] jal] S eb Sie sale ele =e S222 Sse SSeS Sle SS] Sl] ala ae al = SS SS aS aS aa Soe
4° Bacillus typhosus, Bberth (Danish strain) = = = Pt He SHS a] Ha] aH] SS She ae Hla aa SS }- —-|/- -j- — | = Hb eS KJ er Kf er Oa a Oa eS es a te Ha f Re a tt HK FP Ha Ss a = J = =
| | | |
5* Bacillus paratyphosus, Schottmiller (Kral strain) Niet reece) fe cra) | a Bc Oe | ha SL Pe a Ne sfle ole ele slag | j= fe Hoe Kt = Kf KH Hf te +] =] =) + +] =H
6* | Bacillus enteritidis, Gaertner (Kral strain) ... a a aE | | — dtr | - -j- - = =—- =- -J|-—- —-J +t ¢)/- -—- + €/- = Sub.
7* Bacillus paratyphosus, Brion and Kayser (Kral strain) Soy Se a i ee | a ee te | ub-group IT
8* Bacillus paracoli, Widal (Kral strain) HS b= ee ab ee SoS Sle Sl= Sie ela alo Sa SS SNS SS SS So SiS oi IS ;- - - = |S oil aor tant lbh + +o —- —-) + ¢+)/- =
| | | |
9 Bacillus from urine—cystitis .... - - = =) + = = = | —- -{- -J=- -,;- -) + -J= #+]4+ - SS) Se SS FS eS SS oS SS SS |] Ses aS. Shee Su ee eee
10 Bacillus from urine—cystitis .... —- +/=] —/4 =|-— = = sis sie Sle Sle ele S58 = - = -]- -f- -'- -1- -);- -J- -|- -} f= - + -| - -|- - + - = = =) + + Sub-group Il
11* — Bacillus acidi lactic’, Hueppe (MacConkey strain) ......... St Rte a te Tee Ne er |r Te Vest eee mess | Senet || ee |e eee Se SSeesieesitrs eee os Slo =F
12 Bacillus from stool—colitis — + + =!|4 ¢°4+ = + +) + + | + + + +/+ -J|- +)- + SSS ae SSS SS ia i SS SHS Sle Bae ee] Slo =
13 Bacillus from stool—eolitis . Sal Seaecian a | ae he MS Ne ee ey ee ea a ee - =) = =) + -f- =) = -)- -)- -J- -,;- -J- -}- = = -}- -J/- -J+ +]4+ + = -}]- -)
14 Bacillus from urine—cystitis. =P De Sere ere rent fof lop ae Ge a eS SSO =o lS 3/5 Si] Sy Sie SS Sie Sle ee sia aie Silo Sle sills elle 412 <]= = Sub-group TV
15 — Bacillus from urine—eystitis Se a mh a aR eal fey ll th te fa a a I a = =—- = =-J/= —-/- -/}|=- =j=- -}=- =]/- -|=- = L= =| Sy Ee Peel 2S. Sia eee |e
16 Bacillus from stool—colitis — + + +i t+ + + + | Se ae ge ge te ge bya ei ae Woatte ay t+ —- = -}+ -|- -)- -] + a (cu el era -|- -|}+4 =-,- -|+ -—- -— -—]+ +) + t+ - = = =p)
| | | |
17* Bacillus coli, Escherich (MucConkey IBGreilnl)) eeswee sce eneaee es et at Tecmo cE Mtan ee cteiict sect ete elle ee Wc ge I ee eS eS | fe S| a ee er ee fom e Bo £) = =))
18 Bacillus from stool—colitis 5 Spee ae | A aE ER A terme aee | Chere semen epee Te a py ee eee ny gm Re |e ee ee ines eo ae Sa Sy a pa a SS a ee
19 Bacillus from stool—colitis = +) =I =) + + tpl bit +) 4 4/4 =) = +] — + Stsalo Sis Sito Sls Soo sllis Spe eis ce S05 Sse sla elle e)a ¢% a ala =
20 Bacillus from stool—colitis — +) + +) 4+ +/+ + igh obj fie ela em He oe Sl fo esle oslo sls Siac sls sis slo slo a seselao slo sre alle Bie 41a elo =
21 Baciilus from stool—colitis Be See ee a ere | oe he | he re fis | re >a i/o = sie asyjSe Bla Ge Ste ai] Sila ala = Selo Sele ea sear fla a gc elie =
22 Bacillus from stool—colitis ee sss eh | ta) rs + + | a fs Pgh ak | ae |e a fio 4 es ey ce. ||| a Sh ee See Sl See oS es a eS Sala aye Ge Seale e/42 4), “& |] = =
23 Bacillus from urine—cysti fey as 5 ||) boo ae I a | | ear re rh ae em ||| Re fae | eae ns Se SS es Ss lle ree | _ _ (Sub-group V
24 Bacillus from urine—eystitis . a ae | Se ed erty entire ates ce Be re Ree Fn (es ra | ey | ene (ie Re) (ene) Bee wr | eg ee. | Sea || oe ee, + hae ee ee |
25 Bacillus from urine—eysti Sea oo se etree cal Ga || eee | ieee re ae Bae eka a ee || | ies | ee ee ioe = ea = =) eee pane Vrms | Naesn ee a PS
26 Bacillus from stool—colitis Seje S]a aye = GeO) alee | retire Puente, Ere, MnP Armen he etter TE ao ek |v, at | oe met pe Ske = || See eee | tee e @ ey = Fal
27 Bacillus from urine—eystitis . t+ +) tt] +i + 4 Jt t+] + +14 4+ + +] 4+ —] + 4+ — +/| [ieee SSE ie SS 1 el Se | ER eh | Pea | ae EE = es | Sat = oe iH i Wes z fa % as
28 Bacillus from urine—eystiti: — +]/4 —]# +) 4 + Hote itp ae le ge Sl) ek Sao Ho Ok pe | eee | Peery pe Ne FN Ee al | = | 2 aS Se Si 3 Ge
| | | | | | | | B
29* Bacillus pne Friedli = | | | H | | | | | | |
20 | ee ee ae eae Conkey strain). a fe z zi M i Bi ze Got x weap Sel] Ge sel te ae |e elle ee i eth eles el ee SE eS | ee Weems =o) Sheed Sills WES caliae ee ise se Hh)
STR ae ee y wp ap ge |) ae ab ap Shoot See ele ee aa Mesa t+ —|+ + + —-]| + =! — +) - =] 4+ + &o=f— —-|— += —]t +/+ FY) 4] +]
wu sean he Al at een ees ee ae SP | Se Sp a de ap i ae ther eee SR) a er SS ae = || 4 = =! SsiySs Slo Sve = | | + |
Bacillus from stool—colit oh, ct eee | ie op fe rN ht | ae fae Sell oP SEAN ie ah ¥ a Fel a oat
= ed qe ue a) ap Salle ela ae eee ce | atts arg | |e TEE ee ee re ee eR a a a mse ae tte Se ee a mae an lige Seip se a
Bacillus from urine—eystitis de |i on Re ine fe Toy) Se ea rh fh a oer rte re Ge ra (ear | | | |
Bacillus pneumonia, Friedlander (Inboratory strain) Serra ee | tale aia ely ale ole a |= colle se id Pe ast ci ary yee aa Iara he ae leat | as SS SS SS See Ea
_ Bacillus from sputum. ” + +) 4 —|+ +) 4 + the af: pane ea sali Sie e SWS SS Sle 2/2 21S S/S Sl] S) 2 2) SS ee ee ea ee a ee eee a
Bacillus from stool—colitis Se. oe || 2 es bo eee ea me at iia ewe le 2p lS am ee ipes ep eS SS Se es = |e pee ee aeeeies a ei eee
Bacillus pneumonia, Friedliinder (Krl strain) Sen ere reat ee SE ee SS eS ES Ey She sige ceed ee [ree ng | ie aed Cnet | Ran a | SaaS SS) SES See ee
Bacillus from stool—colitis ..... re eile at ie ae si fan Wee | eee chap" ree | Serle So ew ee | tena pct — fast SSS |= Sites eee | ee
Bacillus pyogenes factidus, Passet ( key strain). Peewee) a el ae Syl Bye | Base | SEV lo Eee Po aca Bo al ae emit Ea j= —-|- =|+ -|= -Pt +] 4+ +] + FF] 4 4]
Bacillus neapolitanus, Emmerich (MuucConkey strain) SMe ce Gea eh She ol Shige lS Sys moe abs =] SS SS SS i a | a Pe ee
Bacillus from stool—colitis .....0.......2..000020. 3 Rear a leermmrreermey Lo SS es er ee Pie es SS Fle SS SS = |S OS] fe. =H] = = = | = ae = SS PS SS hae ar ae se ae ae {jeans oer
| | | aii, ca eal | ea et ee [ee a se [SSS aa se ae she
42® Bacillus capsulatus, Pfeiffer (MacConkey strain) .. f= ile als ae] | | | |
, oom + ae cre = = | | |
43* — Bacillus lactis aerogenes, Escherich (MucConkey strain)... — + + + | 4 4 + x + + iH z i x is x i % z x is So eames |) ee ys t+ -[+ +/+ -]+ +/- +]- + + be Pip SP Sk Se ee ee
44* Bacillus clouce, Jordan (MacConkey strain) amare ee Ne le Se ENS SiS Et Fes SPS FPP Sle cece ee pS Se a eee nN a a oS ML
\: | | | | | ie eae (ee A is PN | ee ie ieee | eee em |e es et mts 8)
_ a =_— = — Se ee | | | | | i |

(4149) Pe = Seat | ie |

1907. | Bacteria of the Typhoid-coli Group, ete. 333

‘Digitalin and stro- Gas always small, acid production often well marked.
phanthin.

Coronillin, gratio-
hn, mpi, Aci and gas usually slight and inconstant.
adonidin.

Phloridzin. No gas production, acid formation slight.

Colour changes are observed in the case of certain glucosides, the most
striking being the following.

Media containing coniferin often turn yellow or orange even although no
apparent fermentation has taken place. Arbutin-containing media
frequently turn brown when the cultures are old and alkaline. This is quite
independent of any sign of fermentation.

Syringin media turn to a bright pink colour when fermented. In some
instances, however, the colour is pale.

Amygdalin media turn lemon yellow in old alkaline cultures, showing no
fermentative change.

Phloridzin media inoculated with Sb. capsulatis, B. cloace, B. pneumonia,
B. lactis aerogenes, and Bacilli Nos. 30, 31, 32, 33, 38 and 41, show a bright
brick red colour.

Plumierid becomes dark green in old alkaline cultures of B. lactis aerogenes,
and the saine may occur to a less extent with other micro-organisms.

The results quoted above seem to show that there is no sharp line of
demarcation between the various sub-groups of lactose fermenting micro-
organisms. It is true that nearly all the bacteria tested show slight
individual characters, but they are so closely allied to each other that
attempts to group them according to their lactose, dulcite, and saccharose
fermenting powers must be regarded as artificial. |

It seems, therefore, probable that the separate micro-organisms in the
various sub-groups are not to be regarded as distinct species, but as varieties
or hybrids of one or more species. If this be so, one might expect them to
be constantly varying, losing old characters and gaining new ones according
' to the conditions under which they are grown, and it was with the object of
testing this hypothesis that further series of experiments were undertaken.
In the first place attempts were made to hybridise such organisms as JS. cola
and B. typhosus, but owing to technical difficulties no conclusive results were
obtained.

Attempts were then made to produce variations in the pure cultures of the
18 test types of micro-organisms, the method employed being to grow
each micro-organism for a succession of generations in a fluid medium
containing a sugar which it had failed to ferment, the object being to see

2B 2

3834 Mr. F. W. Twort. Fermentation of Glucosides by [| Mar. 7,

how far it could acquire a new fermenting property. After these experi-
ments were practically finished, my attention was directed to a paper by
Oscar Klotz (4), who had used the same method on one particular microbe of
the coli group.

Klotz, however, only attempted to make his microbe regain characters
which it had lost. In this he was successful after daily sub-cultures were
made for about four generations, but it must be noted that his organism
regained its fermentative powers even when grown on ordinary agar,
although in this instance it was slower. The case with which Klotz was
dealing was really one in which certain physiological functions were lost
after the bacterium had passed through the body of an animal, these
functions being regained in sub-cultures a vitro.

In the experiments which I have carried out the micro-organisms chosen
had been grown in the laboratory for some years, their origin and properties
being thoroughly well known. Many of them were obtained directly from
Dr. MacConkey.

The media used in the experiments contained a sugar which the particular
microbe is ordinarily unable to ferment. The sugar was added to ordinary
salt peptone water to the extent of 2 per cent. s

To ensure that the cultures were pure, they were first plated out, sub-
cultures being obtained from single colonies. These sub-cultures were then
passed through the usual sugar tests, being grown for four weeks at 37° C.
Each culture so obtained and proved to be pure was transferred to a medium
containing the sugar which at first it had failed to ferment. In such
a medium it was allowed to grow for 14 days, when sub-cultures were again
made to the same sugar medium and so on for successive generations.

All the sub-cultures were incubated for four weeks, the experiments being
prolonged over months. The reason why the cultures were incubated for
14 days was to try to induce the microbe to attack the sugar after it had
used up the other nutritive material in the medium. If division was still
going on, it was assumed that any variety with a tendency to ferment the
particular sugar would survive and multiply, since it could utilise the latter ~
which the other members of the culture could not. In this way it might be
expected that in successive generations a culture of increasing fermentative
power would be obtained.

Such indeed proved to be the case; for several microbes were ultimately
able to attack sugars which they, in accordance with universal bacteriological
experience, at first failed to do. This change of function was slow, in most
cases many successive generations being required before the new property
was fully developed. Thus all the members of the para-typhoid sub-group

1907. | Bacteria of the Typhoid-colt Group, ete. 335

were ultimately able to ferment saccharose. B. typhosus also acquired the
property of fermenting dulcite and lactose.

The dysentery bacilli of Kruse and Flexner were ultimately able to
ferment saccharose within 24 hours. B. acidi lactic, after several generations,
fermented saccharose, and thus fell into the same sub-group as B. lactis
aerogenes. B. dysenteriae Kruse was also induced to ferment lactose, and so,
fermenting glucose, saccharose, and lactose, came to be placed in the same
sub-group with B. lactis aerogenes, and in this respect may be regarded as
having fallen from the top of the typhoid-coli group to the bottom. Similar
changes in the case of other micro-organisms ultimately led to the belief that
division into distinct sub-groups separated by fermentation tests is impossible.
The fermentation reactions are characterised by acid reactions, but are rarely
accompanied by the production of gas. Special experiments were carried
out with a typhoid bacillus which had acquired the power to ferment dulcite.
When such a culture was plated out on agar, sub-cultures from single colonies
retained the dulcite disruptive powers, although they were still capable of
being agglutinated by a typhoid immune serum, thus proving that the
fermentation was not due to any contaminating microbe. On inoculating
the dulcite fermenting typhoid culture into a guinea-pig, sub-cultures were
obtained showing the same reactions and these reactions were also maintained,
even when the microbe was grown for several generations on ordinary peptone
agar.

Conclusions.

1.,A large number of glucosides may be fermented by many members of
the typhoid-coli group of bacteria. The fermentations vary with the micro-
organism tested, and the variations are as marked inside each sub-group
of bacteria as between adjacent sub-groups.

2. The sugar-fermenting powers of an organism may be artificially changed
by growing the said organism for a succession of generations in media
containing a sugar which at the commencement of the experiment it was
unable to ferment. ;

By this means a pathogenic organism may be altered until it gives
fermentative reactions characteristic of a non-pathogenic member of its
group. It is possible, indeed, that pathogenic organisms in the typhoid-coli
eroup may so alter their characters that they become unrecognisable when
growing for some time outside the body in soil, water, ete.

If this is so, it might partly account for the difficulty experienced in
isolating B. typhosus from these situations.

It also seems possible that a non-pathogenic organism may lose its

336 Fermentation of Glucosides by Bacteria of Typhoid-coh, ete.

fermenting powers and become pathogenic should it find a suitable medium
such as the alimentary canal, and regain its old characters when outside the
body. This is, however, only a suggestion which at present is in no way
proved.

In view of the results obtained with the typhoid-coli group of organisms,
it seems quite possible that other organisms may show similar changes, and
that the fermentation tests worked out by Mervyn Gordon for the Strepto-
cocci may also be inconstant if the same means of experimentation are
employed.

In conclusion, I have to thank the Royal Society for the Government
grants which enabled me to purchase the glucosides used in this research,
and also Dr. MacConkey for many of the typical cultures used.

REFERENCES.

1. Mervyn Gordon, Thirty-third Annual Report of the Local Government Board,
1903—04, Supplement.

2. Andrewes and Horder, ‘ Lancet,’ 1906, September 15, 22, and 29.

3. MacConkey, ‘ Journal of Hygiene,’ 1905, vol. 5, p. 333.

4. Klotz, ‘Journal of Infectious Diseases,’ 1906, Suppl. No. 2, February.

337

On Reciprocal Innervation of Antagomstic Muscles.—

Tenth Note.
By C. S. SHERRINGTON, D.Sc., F.R.S.

(Received March 9,—Read April 18, 1907.)
(Physiology Laboratory, University of Liverpool.)

I. In previous notes* on this subject the examples of reciprocal innervation
taken from the limb muscles have usually been from muscles acting at the
knee. An advantage offered there is that the vastz and crureus, extensors
of the knee, act directly on that one joint alone. A muscle acting on two
joints is often an extensor of one of the joints and a flexor of the other.
When such a muscle is thrown into contraction by reflex action, there may be
a dilemma as to whether it is as an extensor or as a flexor that it is acting,
and the answer will depend partly on the mode of fixation of the joints at the
time. A double-joint muscle of this kind is gastrocnemius; it extends the
ankle and flexes the knee. It is, therefore, of interest to examine how this
muscle behaves when its antagonist, tibialis anticus, a flexor of the ankle, is
thrown into contraction by reflex action.

Stimulation of any among various points of skin, or of the proximal
end of various afferent nerves of the leg, excites reflex contraction of tibialis
anticus. A nerve often used for the purpose in my observations has been the
cutaneous branch of the peroneal nerve about a centimetre above the
ankle (cat). In the reflex which then occurs in the limb, the following points
can be made out. The ankle is flexed, and with the flexion of ankle occurs
also flexion at knee and hip. If the anterior crural, obturator, hamstring,
and popliteal nerve trunks, the insertions of iliopsoas, and psoas parvus, and
the origin of pectineus muscles be severed, active flexion of knee and hip
are excluded from the reflex. The active flexion at ankle still persists. On
inspecting the tendons of the pretibial flexors of the ankle, that of tibialis
anticus is seen to be drawn upward at each reflex, and with it also those of
extensor longus digitorum and peroneus longus, but in my experience these
latter are retracted less. If, in addition to the nerves and muscles. mentioned
above as severed, the peroneal nerve itself have all its muscular branches
severed, but the branch from the popliteal nerve to the gastrocnemius be
spared, and that muscle be exposed to inspection, usually no change in that
muscle appears.

* ‘Roy. Soc. Proc.,’ vols. 52, 66, 76, 77.

338 Prof. C. 8. Sherrington. On Reciprocal [ Mar. 9,

But if the conditions of experiment provide a pre-existent reflex contraction
in the gastrocnemius, relaxation of it is seen to occur. A pre-existent reflex
contraction of the gastrocnemius can be provided in several ways. I have
for the purpose generally employed stimulation of the central end of the
external plantar nerve of the opposite leg, ¢eg., right. A light weight is
attached to the left foot in such a way that the gastrocnemius acts against
the weight in maintaining extension at the ankle. On stimulating the
proximal end of the above-mentioned cutaneous branch of the left peroneal
nerve, the ankle then flexes (fig. 1). The gastrocnemius being the only
muscle of the leg whose innervation remains, this flexion must indicate
reflex relaxation of that muscle. The requisite precurrent reflex contraction
of the gastrocnemius may also be obtained by inducing decerebrate rigidity.

Fic. 1.—Myograph tracing from isolated right gastrocnemius (spinal cat). Upward
movement of the recording lever registered contraction of the muscle, downward
movement registered relaxation. The upper of the two signals below the myograph
line marked the moment and duration of mechanical stimulation applied to central
end of a cutaneous branch of the musculo-cutaneous division of left peroneal nerve.
This stimulation caused reflex contraction of right gastrocnemius. The lower signal
marked the times of application of weak faradism to central end of the similar
cutaneous nerve of the right limb. In the right limb all other branches of the
peroneal nerve, and all the branches of the popliteal except that to the gastrocnemius,
had been severed, as well as obturator, anterior crural, and hamstring nerves, and the
insertions of iliopsoas and psoas parvus, and the origins of adductor and pectineus
muscles. The inhibitory relaxation of gastrocnemius is seen to be followed on
cessation of the stimulation by marked after-rebound to contraction, especially after
the second and third repetitions of the inhibitory stimulus. Time is marked above
in fifths of seconds.

One of the features of that rigidity is tonic extension of the ankle joint.
Under this condition the reflex which produces contraction of the pretibial
flexors of the ankle produces also, simultaneously, a relaxation of the
gastrocnemlus.

1907. | Innervation of Antagonstic Muscles. 339

This reflex relaxation of gastrocnemius resembles that produced in the
extensor muscles of the knee by stimulation of the proximal end of a
cutaneous branch of the crural] nerve. If, instead of the cutaneous branch of
the peroneal nerve, the branch of the peroneal to the tibialis anticus muscle
is severed, and then stimulated proximal to the section, other conditions
remaining as above, the gastrocnemius similarly relaxes. In this case,
therefore, stimulation of afferent nerve-fibres from the flexor muscle of the
ankle evokes reflex relaxation of the extensor muscle of that joint. The
case resembles, therefore, the reflex relaxation of the extensor of the knee
evoked by stimulating the afferent fibres of the nerve of the flexor muscle of
the knee.*

Let the conditions of experiment remain still as above except that the
nerve of extensor longus digitorum is left unsevered, and let the tendons of
that muscle at the ankle be severed to prevent their acting on the ankle
joint. The extensor longus digitorwm is seen to contract at the time when the
gastrocnemius relaxes in the reflex excited from the central end of the nerve
of tibialis anticus. And, conversely, stimulation of the central end of the
nerve of extensor longus digitorwm causes reflex contraction of tibialis anticus,
together with concurrent relaxation of gastrocnemius.

II. In these instances, as in others previously reported,f stimulation of
afferent nerve-fibres issuing from a muscle excites reflex contraction in that
muscle and in muscles synergic with it, and, concurrently, causes reflex
relaxation of the antagonistic muscles. Such instances are furnished by the
nerves of biceps cruris (posterior part), biceps brachu, gracilis, semi-tendinosus,
tibialis anticus, extensor longus digitorum, peroneus longus, etc. But it was
shownt{ that if the nerve of the vasto-crureus muscle in the thigh be carefully
split and one of its divisions be then severed, the central end of that division,
when stimulated, provokes reflex relaxation of the vasto-crureus muscle
itself, with concurrent reflex excitation of the antagonists of that muscle.
Here the reflex exhibits, as in the other cases, reciprocal innervation of
antagonistic muscles, but the case differs from the others in that that muscle
whose nerve is stimulated is itself the field for relaxation, while, conversely,
its antagonists, instead of being inhibited, contract. I find a parallel case to
this is given by the nerve of gastrocnemius.

If all the nerves of the limb be severed except that of the outer head of
gastrocnemius, and if then the central end of the nerve of the inner head of
that muscle be stimulated, reflex relaxation of the outer part of the gastroc-

* © Roy. Soc. Proc.,’ vol. 52.

t+ ‘Roy. Soc. Proc.,’ vol. 52.
t ‘Roy. Soc. Proc.,’ vol. 77.

340 Prof. C. 8. Sherrington. On Reciprocal [ Mar. 9,

nemius occurs. If the nerves of tibialis anticus and extensor longus digitorum
have not been severed, though their tendons at the ankle have been cut in
order to prevent their mechanical action at that joint, reflex contraction of
tibialis anticus, and, to a less extent, In my experience, of extensor longus
digitorum, can, by inspection, easily be seen to accompany the reflex relaxation
of the unparalysed part of the gastrocnemius. The inhibition can be demon-
strated well by producing from the plantar nerve of the opposite leg a reflex
contraction of the gastrocnemius just prior to exciting the reflex inhibition
from its own nerve. The inhibition can also be demonstrated under
decerebrate rigidity. The experiment can be varied by severing the nerve of
the outer instead of the inner head of the gastrocnemius and keeping that of
the inner head intact for revealing the effect on the muscle. The result is
similar in the two cases. The nerve of soleus exhibits a similar reflex; so
also in the fore limb do the nerves of anconeus, supraspinatus, and parts of
deltoid and triceps brachii respectively. These likewise, while causing
inhibitory relaxation of their own muscles, cause also reflex contraction in
their antagonistic muscles, which flex respectively the elbow and the shoulder
joints.

III. Among afferent nerves proceeding from the muscles of the limbs, there
are, therefore, as regards their reflex effects upon the musculature, two groups.
Both of these evoke reciprocal innervation in antagonistic pairs of muscles;
but one group evokes a reflex which excites that muscle from which the afferent
nerve itself proceeds and concurrently inhibits the antagonistic muscles, while
the other group evokes a reflex which relaxes the muscle from which the
afferent nerve proceeds and concurrently excites the antagonistic muscles.

In both cases the reflex inhibition is not confined to one single muscle, but
involves a number of muscles, just as the reflex contraction is not confined to
one single muscle, but involves a number. Thus, with the inhibition of
gastrocnemius, there can be seen to be associated, in one and the same reflex,
reflex relaxation of soleus, crwreus, vastus lateralis, and vastus medialis.
Similarly, with the reflex contraction of tzbialis anticus there can be seen to
occur concurrent contraction, more or less pronounced, in extensor longus
digitorum, peroneus longus, biceps cruris (posterior part), sem-tendinosus,
sartorius, tensor fascice femoris, gracilis, psoas, iliacus, and psoas parvus. It is
obvious, therefore, that the reflex inhibition is distributed over a group of
synergic muscles, and that, similarly, the reflex contraction is distributed
over another group of synergic muscles. The former are extensors of the
ankle, knee, and hip; the latter are flexors of the ankle, knee, and hip.

In view of the synergic relation apparent in the muscles over which the reflex
relaxation is distributed, it might be expected that stimulation of the afferent

1907.] Innervation of Antagonstic Muscles. 341

nerve of a muscle whose own nerve inhibits (reflexly) that muscle itself would
also inhibit the other muscles whose nerves reflexly inhibit their own muscles.
Experiment shows this to be actually the case. Thus the afferent nerve of
vasto-crureus inhibits reflexly the gastrocnemius; the nerve of gastrocnemius
inhibits reflexly the vasto-crureus ; the nerve of soleus inhibits vasto-crureus,
the nerve of vasto-crwreus inhibits the soleus, and so on, so far as I have
tested.

Sunilarly, stimulation of the afferent nerve proceeding from a muscle whose
nerve throws its own muscle reflexly into contraction might be expected to
excite reflex contraction in other muscles whose afferent. nerves excite reflex
contraction in their own muscles. Experiment finds this to be actually the
case. Thus the nerve of semz-tendinosus excites, reflexly, contraction of the
tibialis anticus; the nerve of tzbialis anticus excites reflex contraction in the
semi-tendinosus; the nerve of extensor longus digitorum excites reflex con-
traction in the tibialis anticus; the nerve of tibialis anticus excites reflex
contraction in the extensor longus digitorum; the nerve of psoas excites
contraction in the semi-tendinosus; the nerve of semi-tendinosus excites
contraction in the psoas; the nerve of psoas excites contraction in the tibialis
anticus ; the nerve of tibialis anticus excites contraction in the psoas, and so
on, so far as my experiments go.

IV. The reflexes elicitable from some of the limb nerves other than the
above have also been observed in the experiments (cat). Among these
additional nerves have been the following: the first dorsal digital, external
plantar, internal plantar, the branch from the anterior tibial trunk to the
foot (taken close above the ankle), the cutaneous branch of the musculo-
cutaneous division of the peroneal nerve, the external saphenous nerve on
the calf, the cutaneous branches of the sciatic given off above the popliteal
space, the branch of the obturator to pectineus, the branch to gracilis, the
branch to the adductor, the whole obturator nerve at the obturator foramen,
the external cutaneous and the internal saphenous at various points along its
course. These evoke reflex contraction in psoas, semi-tendinosus, biceps cruris
(posterior part), tibialis anticus, extensor longus digitorum, peroneus longus—
the reflex contraction in these two last is relatively slight, in my experience—
gracilis, sartorvus, tensor fascie femoris, gluteus maximus (anterior part),
gluteus minimus, pectineus. The muscles, therefore, in which the reflex
contraction has been observed to take place may be broadly described as those
producing flexion of the limb, with some abduction and inward rotation.
Consonantly with this, the reflex movement observed to result from
stimulation of each of the afferent nerves, mentioned in the list at the outset
of this section, has been flexion at knee, hip, and ankle, the flexion tending

342 Prof. C. 8. Sherrington. On Reciprocal [ Mar. 9,

to be more marked at one or another of those joints according to
circumstances, of which one has seemed to be the particular nerve chosen for
excitation. These afferent nerves have also provoked reflex relaxation of vasto-
crureus, adductores, quadratus femoris, semi-membranosus, gastrocnemius, and
soleus. These reflex results have throughout the experiments been perfectly
constant, in the sense that a nerve which provoked reflex contraction of
a certain muscle never yielded reflex inhibition of that muscle, either in
the same experiment or in another. The modes of stimulation of the nerves
were, in each case, faradisation and mechanical compression separately
employed for each nerve.

Of these afferent nerves examined, some are cutaneous, some deep,
eg., issue from muscles, and some contain fibres from both cutaneous and
deep sources. The reflex result of each was the same, in so far that each
provoked contraction of the flexor muscles and reflex relaxation of the
extensor muscles. I have shown previously* that the reflex movement
provoked in the limb by faradisation of skin points in all parts of the
surface, below the knee at least, is likewise flexion at knee, hip, and ankle.
Putting these results together, it seems, therefore, that whereas in some
reflexes, ¢.g., the scratch reflex and the pinna reflex, the receptive field of the
reflex 1s entirely cutaneous, in the flexion reflex of the lmb the receptive
field is both cutaneous and deep. For reasons adduced elsewhere,t the deep
receptors of the body, ¢g., receptive end-organs in muscles, tendons, joints,
bones, etc., can be conveniently classed together as proprioceptive, the
immediate agent of stimulation in their case being usually not an object of
the environment, but a part of the body itself. On the other hand, the
receptors of the outer surface of the organism, ¢.g., the skin, receive their
stimulation usually more directly from the external environment, and have
been termed exteroceptivet The fields of reception of such reflexes as the
scratch reflex and the pinna reflex are, therefore, purely exteroceptive; but
the field of reception of the flexion reflex of the hind limb is both extero-
ceptive and proprioceptive. The same appears to be true, so far as my
experiments go, of the flexion reflex of the fore limb, and also for the
crossed extension reflex of the hind limb and the crossed extension reflex of
the fore hmb. ‘The field of reception of each of these reflexes appears to be
both exteroceptive and proprioceptive.

* “Journ. of Physiology,’ vol. 34, p. 28; ‘Integrative Action of the Nervous System,’
London and New York, p. 127.

+ ‘Integrative Action of the Nervous System,’ pp. 130, 336, etc.; ‘Ergebnisse d.
Physiologie,’ p. 824, 1905,

t ‘Integrative Action of the Nervous System,’ loc. cit.; ‘Ergebnisse d. Physiologie,
loc. cit.

1907.] Innervation of Antagonistic Muscles. 343

In the scratch reflex, the reflex, though from each point of skin in the
receptive field a scratch reflex is produced, varies in certain characters as the
locus of stimulation in the receptive field is. shifted. The scratch reflex,
considered as a generic type (the type reflex)* is, therefore, not really one
reflex but a group of allied reflexest differing somewhat one from another, yet
presenting certain similar characters in common. The same is true of the
flexion reflex of the hmb. Although the movement is always flexion of the
limb, the flexion tends to differ somewhat according as this or that skin-
point, or this or that afferent nerve branch, is the seat of stimulation.
These differences are more easily apparent In my experience under weak
stimulation than under strong. The flexion reflex as a generic whole (the
type reflex) is, therefore, not strictly one reflex, but is a group of allied
reflexes, closely similar in general character, yet possessing individual
differences one from another.

V. The crossed reflex excited in a hind limb by stimulation applied to
the fellow limb of the opposite side exhibits, in my experience, a regularity
almost as marked as does the uncrossed flexion reflex. The distribution of the
effect seems mainly; if not entirely, to muscles affected by the uncrossed
reflex. The crossed reflex has been elicited in my experiments by stimulation
of the following afferent nerves: The plantar nerves, the anterior tibial
close above the ankle, cutaneous branch of the musculo-cutaneous division
of the peroneal, external saphenous, external cutaneous, internal saphenous
at various heights up the limb, the nerves of tibialis anticus, extensor longus
digitorum, peroneus longus, p. brevis, p. quartidigitt, vasto-crureus, gracilis,
pectineus, adductor, and the larger nerve-trunks, such as obturator, peroneal,
popliteal, and hamstring nerve. In the case of these nerves, the reflex
effect observed in the crossed hind limb has been contraction of crureus,
vastus lateralis, vastus medialis, adductores, semi-membranosus, gastrocnemius,
and soleus. The resulting movement has been observed to be extension at
knee, ankle, and hip. This movement is observed not only in the cat and
dog but in the rabbit ; though in the rabbit, in the ordinary progression of
that animal, the hind limbs flex and extend synchronously usually instead of
alternately as in cat and dog.

When psoas, gracilis, semi-tendinosus, biceps eruris (posterior part), tensor
fascie femoris, and extensor longus digitorwm are examined in the crossed
reflex, they are, in my experience, not seen to contract. One might imagine
that the reflex exerted no influence on them. But of this one cannot be

* “Tntegrative Action of the Nervous System,’ p.127; ‘Ergebnisse d. Physiologie,’ Joc.
cit., p. 822.
+ ‘Brit. Assoc. Reports,’ 1904. Address to Sect. I.

344 Prof. C. 8. Sherrington. _On Reciprocal [ Mar. 9,

sure until they have been thoroughly examined under conditions favourable
for the detection of reflex inhibition. It seems not unlikely that they are
inhibited in the crossed reflex.

It would seem therefore that, as said above, the distribution of the reflex
effect is in the crossed reflex mainly, if not entirely, to the same muscles as
those affected by the uncrossed reflex, but that the incidence of excitation
and inhibition upon the motor neurones of those muscles is converse in the
two reflexes. Those muscles which undergo reflex relaxation in the idiolateral
reflex exhibit reflex contraction in the crossed reflex. In giving the above
detailed lists of muscles observed to contract or relax in thése reflexes, it is
not intended to assert that the reflex throws into or out of contraction no
other muscles of the hmb. All that is meant is that in the muscles cited the
reflex effect mentioned has been regularly observed.

VI. It seems noteworthy that those muscles, the afferent nerves issuing
from which evoke reflex relaxation of those muscles themselves, possess
certain physiological characters in common. They are all extensors, extend-
ing respectively the knee, the ankle, and the elbow. ‘They are all muscles
which, after decerebration of the animal (rabbit, cat, dog, monkey), markedly
exhibit decerebrate rigidity. They all are muscles which, in the standing
posture and in progression, counteract, by tonic contraction, the effect of
gravitation and prevent the limbs yielding under the weight of the body.
Two of them certainly yield “jerk-phenomena,”’ e.g., “knee-jerk” and
“elbow-jerk.” Another among them yields the allied phenomenon, ankle-
clonus. They are all muscles for which the toxic agents, strychnine and
tetanus-toxin, show marked predilection. They seem, therefore, in these
respects to belong to a homogeneous functional group.

A question naturally arising in regard to these muscles is, whether in
the afferent nerves issuing from them there exist no fibres at all which can
provoke in them reflex contraction. In my experiments on their nerves the
stimulus applied to the nerve trunk has been faradism or mechanical com-
pression. It must be remembered that when stimulated in this way the
plantar nerves likewise always evoke reflex inhibition in these muscles in
the idiolateral hind limb. Yet there is a reflex—the “extensor thrust ” *—
obtainable from the planta (dog), which causes, not relaxation, but contraction
of these very muscles; and this reflex ceases when the plantar nerves are
severed. There appear, therefore, to be in the plantar nerves some afferent
fibres which can excite reflex contraction of the extensor muscles of the limb,
although when the whole of either plantar nerve trunk is excited by faradi-
sation or by mechanical compression, the reflex produced is,in my experience,

* “Roy. Soc. Proc.,’ vol. 66, p. 66; also ‘Journ. of Physiology,’ vol. 30, p. 39.

7 1907.] Innervation of Antagonistic Muscles. 345

never contraction of these muscles, but is inhibitory relaxation. Similarly,
therefore, with the afferent nerves issuing from these muscles, it may be that
they contain some fibres which, if stimulated by themselves apart from the
other fibres in the nerves, would evoke reflex contraction of the muscles,
although the whole collection of nerve-fibres in the nerve-branch causes reflex
inhibition of the muscles when the mode of stimulation is faradisation or
mechanical compression of the nerve. This possibility is emphasised by
other facts. These may be briefly stated as follows :—

1. The tonic rigidity of vasto-crwreus in decerebrate rigidity appears to be
reflex, and the afferent channel of the reflex seems to lie in the nerve issuing
from vasto-crureus itself. The tonic rigidity of this muscle is maintained when
all the nerves of both hind limbs have been severed, excepting only the
nerve of the vasto-crureus itself. The rigidity of the muscle immediately
ceases on severance of the spinal afferent roots through which pass the
afferent fibres of the vasto-crureus nerve itself. Similarly, also, with the
tonic contraction of gastrocnemius under decerebrate rigidity, and with that
of soleus. The tonic contraction of these muscles ceases only on severance
of the afferent fibres issuing from those muscles themselves.

2. When all the nerves of both hind limbs have been severed, excepting
only the nerve of vasto-crureus, squeezing the lower end of the shaft of the
femur, exposed from the outer side, after exsection of the middle portion of
the length of the shaft, evokes contraction of the vasto-crureus, and this
contraction becomes inelicitable on severing the afferent spinal roots of the
limb. Similarly, when the popliteal nerve remains as well as the nerve
of vasto-crureus, contraction of vasto-crureus is elicited by squeezing the
shaft of the tibia, but the reaction ceases on severing the afferent spinal
roots of the limb. Again, under decerebrate rigidity, when all the nerves of
both hind limbs are severed, excepting only the nerves of gastrocnemius and
soleus of one side, light tapping on the shaft of the tibia excites increase of
the tonic contraction of gastrocnemius and soleus; and this persists after the
shaft of the femur has been cut through, but ceases on severance of the
afferent spinal roots of the limb.

3. If, under decerebrate rigidity, the animal being in the inverted posture,
the hind limb is lightly lifted by the foot into a position of full extension at
knee and the foot is then released so that the limb below the knee drops.
into flexion, the free fall is seen to be suddenly checked much before full
flexion is reached. The impression given is that of a contraction reflexly
_ evoked in the extensors of the knee. The reaction occurs when all nerves of
both hind limbs have been severed, excepting only the nerve of the vasto-
crureus under observation. It ceases when the afferent spinal roots of the

346 Prof. C. 8. Sherrington. On Reciprocal [ Mar. 9,

hmb are severed. <A similar thing is seen with adductor minor, and with
quadratus femoris under appropriate conditions for their tension. Similarly,
under decerebrate rigidity, the tonic contraction of the vasto-crureus is
markedly increased by a few passive extensions and flexions of the knee;
and this holds good when the only nerve unsevered in the two hind limbs is
that of the vasto-crureus in question. Similarly, the tonic contraction of
the gastrocnemius-soleus can be markedly increased by a few passive
extensions and flexions of the ankle, and this holds when all nerves have
been severed except those issuing from these muscles themselves; the effect
ceases when the spinal afferent roots have been cut.

4. On lifting a dog in which spinal transection above the lumbar region
has been performed, and the period of shock passed over, it is extremely
frequent for strong reflex extension of both hind limbs to occur. Both
limbs are simultaneously extended at knee and ankle. This does not occur
if, when the animal is lifted, the hind limbs are prevented from dropping
under their own weight. Passive support of the limbs, under the thighs
for instance, precludes the reflex.

5. It was stated above that faradisation or mechanical compression of
the nerve of the vasto-crureus provokes with regularity an inhibitory
relaxation of that muscle and of soleus and gastrocnemius. Occasionally,
however, though, in my experience, quite rarely, if the nerves of the limb
be all of them severed, excepting only that of the vasto-crureus itself, and
that nerve be split into two divisions, central stimulation of one division
may provoke slight extension at the knee if the initial posture of the knee
at the time be full flexion.* But the same stimulation provokes inhibitory
relaxation of the extensor if the initial posture of the knee at the time
be fair extension. This result seems to resemble some reflex results recorded
by v. Uexkiill in Invertebrata.t

6. Finally, it may be recalled that various observers regard “ knee-jerk ”
and “ankle-clonus ” as reflex reactions, in spite of the admitted brevity of
the latent time of the former. The knee-jerk is obtainable in the spinal
animal when all nerves of the two hind limbs have been severed, excepting
only the nerve of the vasto-crureus muscle in which the “jerk” is being
examined.t It then ceases at once on severing the afferent spinal roots,
through which pass the afferent fibres of the nerve of that muscle. Again,
under decerebrate rigidity, ankle-clonus is obtainable (cat) when all the
nerves of both hind limbs have been severed, excepting only the nerve of

* Cf. ‘Roy. Soc. Proc.,’ vol. 66, p. 66 ; and vol. 76, B, p. 291.

+ ‘Zeitschrift f. Biologie,’ vol. 44; ‘ Ergebnisse d. Physiologie,’ 1904, p. 1.
t ‘Roy. Soc. Proc.,’ vol. 52, p. 557.

1907. |] Innervation of Antagonistic Muscles. 347

gastrocnemius-soleus. The reaction then ceases on severing the afferent
spinal roots, through which pass the afferent fibres from the nerves of those
muscles.

7. It seems possible that the diphasic result described in the following
section (Section VII) may likewise point to the admixture, in the afferent
nerves stimulated, of fibres provocative of opposite reflex effects (reflex
relaxation, reflex contraction) in the same muscle.

VII. When the isolated vasto-crureus muscle with its nerve intact, or,
similarly, the isolated gastrocnemius, soleus, swpraspinatus or triceps brachii are
under examination for the reflex effect upon them of excitation of the afferent
nerves of their own limb, the inhibitory relaxation provoked is, on cessation
of the stimulus, very frequently followed at once by contraction (fig. 1).
This I have noted before,* but in systematically working through the
afferent nerves of the hmb it is so usual a reaction as to demand further
mention here. It occurs with mechanical as well as with electric stimula-
tion. It occurs in reflexes excited from skin as well as in those excited
from isolated nerves, both cutaneous and deep. It occurs in the fore-
limb as well as in the hind-limb. It seems of central origin.

Similarly, the inhibitory relaxation of the hamstring muscles, ¢., left,
provoked by stimulation of the central end of a nerve, eg., vasto-crureus
nerve or plantar nerve, of the crossed hind lmb (right), is sometimes
followed, on cessation of the stimulus, by an immediate rebound to
contraction in those muscles. This may be well seen after the nerve of
the corresponding extensors (left) of knee has been severed. A similar
result can be obtained from an afferent nerve, ¢g., median, of the crossed
forearm (right). The stimulation of the median, giving inhibitory relaxation
of the hamstring muscles (left), is followed, on cessation of the stimulus, by
an immediate rebound to contraction. Again, under decerebrate rigidity,
when all the nerves of the hind limb (left) have been severed, excepting
only the nerve of the vasto-crureus, the reflex contraction of the vasto-crureus
excited by stimulation of a nerve, eg., musculo-cutaneous, of the crossed
(right) forearm, is often followed, immediately on cessation of the stimulus,
by a relaxation of that muscle far more complete than that which existed
in it immediately prior to the reflex contraction. The phase of reflex
contraction seems here to be immediately followed by a phase of reflex
relaxation ; just as, conversely, in other cases the phase of reflex relaxation is
immediately followed by a phase of reflex contraction.

The after-contraction following the inhibition seems best obtained when the
stimulus is not very strong, but appears more marked after moderate than
* * Roy. Soc. Proc.,’ vol. 77, B, p. 486.

VOL. LXXIX.—B. 2¢

348 Prof. C. 8. Sherrington. On Reciprocal [Mar. 9,

after very weak stimuli. It seems favoured by shortness of duration of the
stimulus, eg., a duration of half a second or a second seems more favourable
than one of 4 seconds. It is seen under decerebrate rigidity as well as after
spinal transection. The after-contraction in vasto-crwreus or gastrocnemius
often does not extend the knee or ankle to as great a degree as existed under
the decerebrate rigidity, immediately before the reflex inhibition—the first
effect of the stimulus—set in. Sometimes, however, the after-contraction
actually exceeds the degree of contraction existent prior to the reflex
inhibition (fig. 1). The after-contraction never, in my experience, sets in
during the actual application of the stimulus, but follows as a rebound after
its cessation. The rebound is evidently reflex, for it often involves the
extensors of hip, knee, and ankle together.

The inference was drawn above that the reaction is central in its
mechanism. That is also borne out by the fact that it is met with under
direct stimulation of the cross-section of the spinal cord itself. Thus I have
met it when faradising, by the unipolar method, the lateral column of the
spinal cord at the 10th thoracic level in the dog. The 7th cervical segment
had been exsected seven months previously. From three points in the left
ventrolateral column flexion of left hip and knee was obtained as a conjoined
movement, together with extension of the right knee. There followed
regularly, on cessation of the stimulus, a marked after-extension of left hip
and knee. This after-extension was immediately cut short on stimulating
the previous point again. But the after-extension was frequently increased
by stimulating the left dorsal column. The face of the spinal section
stimulated was that distal to the transection made at the time to expose the
cross area of the cord for direct excitation.

[Addendum.—In Section VI above, it was pointed out that the muscles
reflexly inhibited in the instances of reciprocal innervation given possess
certain physiological characters in common. Among these characters, one
that was mentioned was their counteracting gravity by tonic contraction in
the habitual posture of the body when erect, ¢.g.,in standing. An instance
of reflex reciprocal innervation is, however, furnished by the hind limb (cat),
in which there occurs inhibitory relaxation of muscles which in some
respects can hardly be supposed to have the function of counteracting
gravity in the ordinary erect posture, eg., standing. This instance is well
exemplified when the afferent nerve chosen for exciting the reflex is either of
the plantar nerves or the musculo-cutaneous branch of the peroneal below
the knee. Stimulation of any of these nerves excites reflex contraction of
the flexors of hip, knee, and ankle, and reflex inhibition of the extensors of

1907. | Innervation of Antagonistic Muscles. 349

hip, knee, and ankle. But the stimulation excites, in addition, gluteus
mintmus and the anterior part of gluteus maximus. These two muscles
produce abduction and internal rotation of the thigh. Together with the
reflex contraction thus excited in these two muscles, the stimulation provokes
reflex inhibitory relaxation of adductor minor, adductor major, and quadratus
femoris. Of these three muscles, the two first are adductors of the thigh,
the two last are external rotators of the thigh; all three are extensors of the
thieh. In this reflex, therefore, not only are extensors relaxed when flexors
contract, but adductors are relaxed when abductors contract, and external
rotators are relaxed when internal rotators contract. It would seem,
therefore, in this case that, as in the case of reciprocal imnervation of
antagonistic lateral muscles of the eyeball, the reciprocal inhibition of the
muscles occurs even where the muscles are not by their tonus engaged in
counteracting gravity.— April 16, 1907.]

iT A 3 ; Pe 7 f > ‘ ye
: é 4a x : ; rhe nlbebanee
a ‘ i : , we
: i : ' {
“1 Fai § ;
‘ 7 j i
e
as
(
=~
ae ‘
ia) ¥

1907. | Innervation of Antagomstic Muscles. 349 |

hip, knee, and ankle. But the stimulation excites, in addition, gluteus
minimus and the anterior part of gluteus maximus. These two muscles
produce abduction and internal rotation of the thigh. Together with the
reflex contraction thus excited in these two muscles, the stimulation provokes
reflex inhibitory relaxation of adductor minor, adductor major, and guadratus
femoris. Of these three muscles, the two first are adductors of the thigh,
the two last are external rotators of the thigh; all three are extensors of the
thigh. In this reflex, therefore, not only are extensors relaxed when flexors
contract, but adductors are relaxed when abductors contract, and external
rotators are relaxed when internal rotators contract. It would seem,
therefore, in this case that, as in the case of reciprocal innervation of
antagonistic lateral muscles of the eyeball, the reciprocal inhibition of the
muscles occurs even where the muscles are not by their tonus engaged in
counteracting gravity.—April 16, 1907.]

bo
0

VOL, LXXIX.—B,

390

Studies on Enzyme Action. IX.—The Enzymes of Yeast :
Amygdalase.

By kh. J. CALDWELL, B.Sc. (Lond.), Leathersellers’ Company’s Research
Fellow, Chemical Department, City and Guilds of London Institute, and
5. 12 COURTAUED, Be.

(Communicated by Professor Armstrong, F.R.S. Received May 9,—
Read May 23, 1907.)

[International Catalogue of Scientific Literature.

Author’s title slip :—D. Q. R.

Subject slips :—
D 1850 Amyegdalin, REE of, by amygdalase.
D 8014
Q 1240
R 1910 Yeast, new enzyme in (amygdalase). |

| Amygdalase, a new enzyme in yeast.

The number of the ‘ Gazzetta Chimica Italiana’ published on February 12,
1907, contains a communication received by the editor on December 17, 1906,
“Su le azioni idrolitiche diverse prodotto da un solo enzima,” by L. Marino
and G. Sericano,* in which the conclusion is arrived at that the resolution of
amygdalin by an extract of yeast into glucose and amygdonitrileglucoside
is conditioned not by the enzyme maltase but by invertase.

We have long been aware that maltase does not effect the hydrolysis of
amygdalin and to this extent can confirm Marino and Sericano’s observations ;
but the conclusion that the cyanophoric glucoside is hydrolysed by invertase
is not in accordance with our observations, which show that a specific enzyme,
not hitherto recognised as a constituent of yeast, is the active agent in
separating the glucose: it will be convenient to speak of this enzyme
as amygdalase.

Amyedalin is one of the few natural glucosides which give two molecules
of glucose when hydrolysed, the other product being benzylidene cyanhydrol,
which—in dilute solution—decomposes spontaneously into benzaldehyde and
hydrogen cyanide. There is reason to suppose that the two molecules of
glucose are attached to one another—that is to say, that amygdalin is derived
from a biose.t

It was shown by Emil Fischer, in 1894,f that an extract of dried Frohberg

* Vol. 37, pp. 45—51.

+ Evidence has already been adduced in favour of the view that this biose is not
maltose (Caldwell and Courtauld, ‘Trans. Chem. Soc.,’ 1907, vol. 91, p. 670).

t ‘Deut. chem. Ges. Ber.’, 1894, vol 27, p. 2989.

Studies on Enzyme Action. 351

yeast (a bottom yeast) is capable of resolving the biose section of the
molecule alone, a true glucoside and glucose being formed :—

CgH;CH(CN)O-Cj2H21019 + H2O > CgHs;CH(CN )O-CgHi105 + CgHi20¢.

In his earlier paper, Fischer speaks of the action as due to “ invertin,” but
afterwards attributed the hydrolysis to the enzyme present in yeast which
hydrolyses maltose.

Fischer’s investigation was carried out at a time when little was known of
the specific character of the enzymes. In the light of later work—especially
that of E. F. Armstrong—it was improbable that maltase was the agent,
emulsin (although highly active towards amygdalin) being without action on
maltose and methyl-a-glucoside, which are both easily resolved by maltase.
Our experiments were instituted to clear up this discrepancy:

To prepare amygdonitrileglucoside, Fischer digested a mixture of amygdalin
with yeast-extract at 35°. On repeating the experiment in accordance with
his directions, using a dried English brewer’s yeast, we were surprised to find
that the action took place far less rapidly than Fischer’s description had led
us to suppose it would ; on testing the efficiency of the yeast-extract with
maltose it was found to be almost inactive: judging from the relative
stability of amygdalin and maltose in presence of acids, it was to be expected
that the maltose would have been more rapidly hydrolysed than the
amygdalin.

On considering all the circumstances, it appeared that we had worked at
a higher temperature (35°) than that at which maltase exerts its maximum
activity according to E. F. Armstrong’s observations. The question arose
whether the inactivity of the extract were not a consequence of overheating :
comparative experiments were made, therefore, in which extracts prepared at
different temperatures were digested, each at several temperatures, with
solutions containing equivalent quantities of amygdalin, maltose and
methyl-a-glucoside. These led to the discovery that the action on amygdalin
and also on methyl-a-glucoside persisted after heating the solutions to such
a temperature that the action on maltose was at an end—consequently,
that maltase was not the active agent. Similar experiments at higher
temperatures afforded evidence that the enzyme which hydrolysed amygdalin
could be destroyed without depriving the solution of its power of hydrolysing
cane-sugar. : 3

The proof was thus obtained of the existence of a specific enzyme capable
of effecting the separation of glucose irom amygdalin. Whether or no this
enzyme also hydrolyses methyl-«-glucoside or whether besides maltase two
different enzymes are present in the extract, one of which attacks amygdalin,

2 D2

352 Messrs. R. J. Caldwell and 8. L. Courtauld. [May 9,

the other methyl-«-glucoside, cannot be decided at present: the discovery of
an a-glucase different from maltase, however, is of considerable interest as an
indication of the existence of separate «-glucosidoclasts.

It may be pointed out that the disproportion observed by E. F. Armstrong*
in the relative rates at which maltose and methyl-«-glucoside are hydrolysed
by maltase and acids respectively was perhaps in a measure due to the fact
that the “ maltase ” contained the two enzymes which are capable of hydro-
lysing the glucoside, but only one capable of acting on the maltose.

Method followed—The experiments were carried out in small closed
flasks, which were heated ina Hearson incubator; excepting pipettes and
funnels, all the vessels used were of hard Jena glass. The top yeast used was
a pale ale yeast, for which we were indebted to Mr. Julian Baker. The
freshly pressed yeast was first well washed with water saturated with
toluene and without loss of time thoroughly drained on a vacuum filter ; it
was then rapidly dried on bibulous paper, the drying being completed by
exposure over sulphuric acid im vacuo. Professor Emil Fischer had the
kindness, at Dr. E. F. Armstrong’s request, to obtain for us a quantity of
dried bottom yeast (S. Cerevisiw, Race 12, Berlin), which was ostensibly
similar to that which he had used.

To prepare the extract, one part of dried yeast was added to 20 parts
of water and a little toluene; the mixture was then digested at the required
temperature, being shaken at intervals; it was finally clarified by filtration.
Usually, the liquor was in contact with the yeast during 4 to 6 hours, experi-
ment having shown that nothing was gained by extending the period.

Solutions of the hydrolytes were prepared containing four-tenths of a
gramme-molecular proportion per litre. Equal volumes of the solution (20 ce.c.)
and of the yeast extract, heated to the temperature at which hydrolysis was
to be effected, were quickly mixed in a flask; a sample of 10 c.c. was at once
withdrawn and, the enzyme having been destroyed by the addition of a single
drop of concentrated sulphuric acid (or in the case of cane-sugar, a solution
of caustic soda), 10 c.c. of a 10-per-cent. solution of sodic acetate was run in;
the liquid was then heated to 80° to precipitate the dissolved protein as far as
possible. Finally, the filtered solution was examined polarimetrically in a
20-mm. tube at 25°. Meanwhile, the main bulk of the mixture had been
placed in the incubator, where it was kept at the required temperature; at
intervals, usually after 24,48 and 72 hours from the.commencement of an
experiment, 10-c.c. samples were withdrawn, and treated as before described.

The amount of the substance hydrolysed was estimated by dividing the
observed change in rotatory power by that calculated to correspond to

* ‘Roy. Soc. Proc.,’ 1904, vol. 74, p. 198. ,

1907. | | Studies on Enzyme Action. 353

complete hydrolysis; as the total change exceeded 200’ in every case, the
error in the polarimetric reading being not more than about 1’, the values
found may be regarded as accurate to within about } per cent.

Table I.—Action of Extracts of Top Yeast (S. Cerevisiw) on Solutions of
Equimolecular Concentration at 25° and 35°.

Amount hydrolysed in
G1 d d Yeast Hydrolysis
maces extracted at | effected at P
24 hours. 48 hours. 72 hours.
per cent. per cent. per cent.
25° 25° 15 °*4 23 °3 29 °6
Perec dalin 35 25 15 °4 21-2 28 °3
oe eat 25 35 18 °3 32° 50°8
35 35 18-7 25 °4 31°1
25 25 “as 93 ‘8 100 ‘0
iaTkose 30 25 21°8 26 °9 30 °3
WALES COCTGCOCOCCOCL? 25 35 65 5 yh) 6 69 ‘9
35 35 Lis 15 °9
25 25 (HEHE 100 °O
; 35 25 45 °3 60 5
Methyl-a-glucoside 95 35 9-8 86 ‘8
35 35 42 °9 44, °3

Table II.—Action of Extracts of Top Yeast prepared at Temperatures
between 0° and 55° on Solutions of Equimolecular Concentrations.

Amount hydrolysed in

Glucoside used. Yeast extracted at eel lanl
24 hours. | 48 hours. | 72 hours.
| per cent. | per cent. | per cent.
c 0° 25° 4.°8 13 °4 17 °4
15 15 11 °4 16 °4 24 °4,
15 25 16 °9 24 °5 38 ‘0
Amygdalin ......... < | 15° (extract heated 25 15 °1 21°0
at 45°—1 hour)
| 45 25 16 ‘1 26-7 36 ‘0
ie 55 25 racemised | racemised | racemised
0 25 541 69 °6 759
15 15 57 °9 68 -O
15 25 70 °0 78 °6 80 °4:
MaIEOSC 4.......0cesises + | 15° (extract heated 25 3°9 29 °7 32 °3
at 45°—1 hour)
| AS 25 1°8 2-7
L 55 25 0-0 0-0 0-0
( 0) 25 49 -O 65 °6 749
| 15 15 50 °6 64:°9
Methyl-a-glucoside + | 15° (extract heated 25 39 *4, 50 6 63 *9
L at 45°—1 hour)
55 25 0°0 0°0 0°0

354 Messrs. R. J. Caldwell and 8. L. Courtauld. [May 9,

Maltase not Effectwe in Hydrolysing Amygdalin.—The results recorded in
Table I show that amygdalin is more readily hydrolysed at 35° than at 25°,
at whichever temperature the extract be prepared. In the case of maltose,
the higher temperature is prejudicial, whether prevailing during the extraction
or the hydrolysis.

Results are recorded in Table II of experiments in which the yeast was
extracted both at lower (0° and 15°) and at higher (45° and 55°) temperatures
than those before mentioned. In the first place, it is obvious that 15° is a
less effective temperature than 25° not only in the case of amyedalin but
also in that of maltose, a smaller percentage being hydrolysed at the lower
temperature. But the extract prepared at 15° was the most active, 0° being
apparently an altogether unfavourable temperature. The results obtained
with an extract prepared at 45° show that the activity towards maltose
is almost non-existent whilst unimpaired towards amygdalin. Extracts
prepared at 15° and then heated to 45° show considerably reduced activity
towards maltose. Extracts prepared at 55° were destitute of activity towards
the three glucosides.

The experiments made with bottom yeast (Table III) gave results which
showed that this yeast was far less active towards amygdalin than the top

Table III.—Action of Extracts of Bottom Yeast (S. Cerevisiw, Race 12,

Berlin).
Amount hydrolysed in
Glucoside used. Yeast extracted at are ies
effected at
24 hours. | 48 hours. | 72 hours.
per cent. | per cent. | per cent.

( 25° 35° 4°6 4° 1
| 35 25 6-2 6-2 75
3 , 25 25 6°2 46 8°3
Ay CCA LIT; o.a oe ; | 15 25 3-3 Aee 5:8
15° (extract heated 25 4°6 3°3 4°6

at 50°—1 hour)
a 25 35 67 ‘8 83 °6 86 ‘9
| 35 25 52 2 68 “1 74-0
Malt 25 25 58) O0/ 74°3 81 °2
J a ose CO GDiOIL OD ULC CEO 15 25 53 ‘l 68 3 TA, 6
15° (extract heated 25 15 °5 21°8 26 ‘0

L at 50°—1 hour)

ig 25 35 61-9 76°3

| 35 25 40 ‘6 55 ‘8 65 °5
. 25 25 59 ‘O (ae 82 °7
Methyl] a-glucoside < | 15 25 43 °3 66 “2 71-9
| 15° (extract heated 25 22:5 34 °1 35 °3

L at 50°—1 hour)

1907. | Studies on Enzyme Action. 355

yeast, although the two extracts were almost equally active in hydrolysing
maltose. The maltase in the bottom yeast appears to have been somewhat
more resistant, a considerable proportion surviving in the extract which had
been heated to 50°. The extract prepared from this yeast at 25° was more
active than that prepared at a higher or lower temperature, 15° being the
most favourable temperature in the case of the English top yeast ; moreover,
the hydrolysis of maltose was best effected by this yeast at 35° instead of
at 25°.

Results similar to those recorded in Table III were obtained with another
sample of yeast reported to be a bottom yeast grown in maize wort.

In view of the high temperature at which it exerts its maximum fermen-
tative power, it appeared desirable to examine the remarkable yeast
S. Thermantitonum described by Grove Johnson. Mr. Johnson was good
enough to give us some quantity of the yeast: unfortunately, the extract pre-
pared from it was so highly coloured that satisfactory measurements could not
be made with it. The impression was gained that it resembled that prepared
from the bottom yeast and that possibly amygdalin was not attacked by it.

Results are recorded in Tables [IV and V which finally led to the conclusion
that maltase can be entirely destroyed by heating, whilst the extract retains
its activity towards amygdalin almost unaltered. Owing to the greater
stability of the maltase in the case of the bottom yeast, it was necessary to
heat the extract during a longer period (three hours instead of one) in order
to render it inactive towards maltose.

Table [V.—Action of Extract of Top Yeast prepared at 15°. -

Amount hydrolised at 25°.

Glucoside used. Treatment of extract.
24 hours. 48 hours. 72 hours.
per cent. per cent. per cent.
(Wmihcatedl “aie. clave vastieh es 13 °9 PAN GP 28 °3
Amygdalin ............ Heated at 45° (2 hours) ... ies 25 °4 31°7
el 502 (il hour) ss... 18 °3 25 °8 34 ‘6
Winkewted eo. lace ccce ose basse 65 °8 76 °4 1
Heated at 45° (1 hour)...... 16 ‘1 22-7 23 °3
WEGIGOS6® os.ceeeeccbess. i ASO nati) o. 1:5 1°8 1°8
a oO2\@ hour) t.., 0-0 0-0 0:0
(Umitieabed yy 00s. secsiccekee ose 60 ‘9 (Alot 751
Methyl-a-glucoside Heated at 45° (2 hours) ... 16 °5 26 ‘9 32 °5
2) 60° (1 hour) ....| 15:0 23-7 29 °3

356 Messrs. R. J. Caldwell and 8. L. Courtauld. [May 9,

Table V.—Action of Extracts of Bottom Yeast prepared at 25°.

Amount hydrolysed at 25°.
Glucoside used. pice Treatment of extract.
extraction.
24 hours. | 48 hours. | 72 hours.
hours. per cent. | per cent. | per cent.
20 Whhested nena: a3 15 °4
Wes odalin 4 siihy, ase eeevauemaemne 4 °6 8 °8 10 *4
Nee ee 4, Heated at 35° (3 hours) 3°8 feel 8°8
4 {ORS Orme te 2°9 5-0 5-0
20 Unheated) i ches cca 7BL@ 82 ‘1
Mbaltcse 4 MRR er ede seers 59 1 100 78 ‘8
ne 4 Heated at 35° (3 hours) 591 73 4 81°5
4 s vi 0-0 1°4 1:2
20 Unheatedt. cesses 76 °6 88 °8
; 4 iat See es 55 °8 7-1 80 ‘3
pee ie euucoside 4, Heated at 35°(3 hours)| 50 °2 67 °9 78°7
4 ptaee 25 Ou tek 2-1 1°7 3°6

Non-identity of Invertase and “ Amygdalase.”—The observations (recorded
in Table IT) that amygdalin was not attacked by an extract which had been
prepared at 55° not only led us to think that the hydrolysis of amygdalin
by yeast was not conditioned by maltase, but also that invertase was not the
active cause of change—inasmuch as it is well known that invertase is far
from being destroyed at this temperature. Experiments which are recorded
in Tables VI and VII were made to test this point directly. The results
show that the assumption made by Marino and Sericano before referred to
cannot be accepted. An extract which has been heated at 60° and is entirely
without action on amyegdalin and methyl-«-glucoside still preserves much of
its activity towards cane-sugar. |

Marino and Sericano removed the maltase from the mixture of enzymes
derived from yeast by subjecting the extract to diffusion ; they overlooked
the possibility that the less diffusible fraction—which they proved to be
free from maltase—contained any other enzyme than invertase.

It may be mentioned as a point of interest that bottom yeast, although
equally rich in maltase, appears to contain a far smaller proportion of both
invertase and amygdalase than top yeast. At the end of 24 hours only
2°7 per cent. of amygdalin and 91:5 of cane-sugar had been hydrolysed by an
extract of bottom yeast as compared with 10°5 per cent. of amygdalin hydro-
lysed by an extract prepared under like conditions from top yeast, which also
hydrolysed cane-sugar completely in the course of a much shorter period than
24 hours. The extreme activity of top yeast towards cane-sugar is clearly
indicated by the results given in Table VII.

| a Studies on Enzyme Action. 357

Table VI.—Action of an Extract of Top Yeast prepared at 25° before and
after Heating at Higher Temperatures.

|

Amount hydrolysed at 25°.
Glucoside used. Treatment of extract.
24 hours. 48 hours. 120 hours.
per cent. per cent. per cent.
Winhexted? ..c<ncrssvece ses 10°5 te9
Amygdalin ............... Heated at 50° (1 hour) 48 17 ‘9
A GOP 0:0 0:0 racemised
Wuheated ssc... <inve.we 100 °O0 100 -O
CAMEBULAP ...... 0000000 Heated at 50° (1 hour) 100 0 100 -0
a GOOF os 54 °2 86 ‘8 100 ‘0
Uniheated) 256 cise i ccscetves 40 °2 51 °4
Methyl-a-glucoside ... Heated at 50° (1 hour) Lf 32
% GO"; 0:0 0-0 0°0

Table VII.—Action of an Extract of Top Yeast prepared at 25° before and
after Heating at 60° during 1 hour.

Amount hydrolysed at 25° in

Glucoside used. Treatment of extract.
52 hours.
per cent.

Wal abOce drats ct. cktees ett hoe. 27 5
2 000/2(0 6) 1) See ae arene Floated attO0 visit acaccnctewatct ee 0°0
fe 60° and acidified* ... 0:0
Unheated, diluted to 1 in 50 ... 96 °6
WAMC-SUZAL .....0.001 00 oseaseone Hea teat abi OO? snacaee hetctcnerencstiwne 100 0
Heated at 60° and acidified ...... 100 °0

Methyl-a-glucoside ......... | Heated at 60° and acidified ...... 0°0

* The extract which had been heated at 60° became sufficiently alkaline to convert amygdalin
into isoamygdalin after a long period of digestion (Table VI). N/1000 acetic acid does not
condition any measurable amount of hydrolysis. The expression “acidified” in the table is to be
interpreted as meaning that acetic acid was added in the proportion of 0°001 gramme-molecule per
litre of extract.

Hydrolysis of Methyl-a-Glucoside-—It will be observed on reference to the
tables, that methyl-a-glucoside is always more or less attacked so long as
amygdalin is acted upon, but that a very marked diminution in activity
towards this glucoside attends the disappearance of the maltase (Tables IV
and V). The inference that the methyl-«-glucoside is attacked by the two
enzymes maltase and “amygdalase” cannot well be disputed—it is clearly
necessary to assume that either an «-glucase exists together with amygdalase,

358 Messrs. R. J. Caldwell and 8. L. Courtauld. [May 9,

or that one and the same enzyme (amygdalase) is concerned in the hydro-
lysis of both amygdalin and methyl-a-glucoside.*

It is noteworthy that after the maltase is destroyed there is a coincident
variation in the amount of amygdalin and methyl-«-glucoside which undergo
hydrolysis in the case of different yeasts (Tables IV and V).

Moreover, a yeast extract in which the maltase has been destroyed
produces an effect on a mixture of methyl-«-glucoside and amygdalin markedly
smaller than the sum of its effects on the two compounds taken separately
(Table VIII). As there is competition for the enzyme when two substances
are hydrolysed together (cf Henri),} this result would also seem to show that
a single enzyme is active.

It has been stated by Ter Meulen{ that only glucose retards the hydro-
lysis of amygdalin by emulsin and that maltose is without effect. Apparently
maltose is without effect on the hydrolysis of amygdalin by amygdalase
(Table VIIT); and in this respect galactose and lactose are also inert, whilst
glucose has a marked inhibitory effect.

It is impossible at present to identify amygdalase with any known enzyme.
The enzyme is present in varying amounts in the different yeasts we have
examined, and appears to be equally well extracted from the dried material
at all temperatures from 15° to 45°. Whereas a low temperature of extrac-
tion, 0°, affords a very small proportion of active amygdalase (Table IIT),
the quantity is increased by heating the extract for a short time at 45°; a
similar effect is produced by heating an extract prepared at 15° (Table IV):
that is to say, the amygdalase is originally dissolved as part of a more
complex protein or zymogen molecule, which is hydrolysed at the higher
temperatures. Proof of its stability is afforded by the fact that it is con-
tained in invertase prepared by O’Sullivan and Thompson’s method, which
involves the autolysis of the yeast and precipitation of the enzyme by
alcohol—a process which entirely destroys maltase.

* Marino and Sericano speak of using 20 kilogrammes of yeast. After this had been
washed and dried, it was extracted with water; the precipitate produced by adding
alcohol to the extract was purified by dialysis. Eventually they obtained only 2°5 grammes
of dried substance. They state definitely that their invertase was prepared by continuing
the dialysis until methyl-a-glucoside was no longer atfected. However, they give no
details of the experiments which proved this to be the case and it is noteworthy that
amygdalin appears to have been but slowly hydrolysed by their material, even when this
was used in large proportion.

t+ ‘C. R. Soc. Biol.,’ 1903, vol. 55, p. 868.

t ‘Rec. Trav. Chim.,’ 1905, vol. 24, p. 444.

1907. | Studies on Enzyme Action. 359

Table VIII.—Action of Amygdalase from Top Yeast on Amygdalin in
presence of Maltose, Methyl-«-Glucoside, Glucose and Galactose at 25°.
The extract was prepared at 15° and subsequently heated at 50° during
two hours to kill the maltase.

Rotatory power change at 25°.
Substance hydrolysed.

24 hours. 48 hours. | 72 hours.

PSMA DIONE 2505 ieroeviceniccasvescetueteste ses —0° 37’ —0° 58’ —1° 10’
BeMR RC MALOMO Mae tie uiiideclitaeny Gee ceededeneven ces +0° 1’ +0° 0’ +0r 0}
Silas esses cre edse. —0° 36’ —0° 58’ —1° 10

Amygdalin and maltose together ............... —0° 39’ —1° 0’ —1° 14’
Retardation ......... OF ae: Or 2 ait: 4/

EPPO AIOUC .06ss0vecereteecdscdeeessevacvedece —O0° 37’ —O° 58’ —1° 10’
Methyl-a-glucoside alone .............cssecevenes +0° 25’ +0° 42’ +0° 54/
UM, Foe cb aeeondie sa —0° 12’ —0° 16’ —0O° 16’

Amygdalin and methyl-a-glucoside together —0° 18’ —0° 28’ —0° 40’
Retardation .. ...... iy) aba? 6’ 0° 12’ 0° 24)

Amount hydrolysed at 25°.

per cent. per cent.
amy eclaliM ALOME .. 2. ..casdccdeessdienstieanserenases 17 °5 26 °2
Amygdalin and glucose together ............... 10 °4 241

Amyedalin and galactose together ............ 167 28 °3

360

Studies on Enzyme Action. X.*—The Nature of Enzymes.

By Henry E. ArmsrroneG, F.R.S., and E. FRANKLAND ARMSTRONG.

(Received May 9,—Read May 23, 1907.)

[International Catalogue of Scientific Interature.

Author’s title slip :—D. Q.

Subject slips :—
D 1820 Cane sugar, hydrolysis by invertase.
D 8012

Q Soap + Enzymes, nature of (invertase, maltase). ]

The study of the action of enzymes has now reached a stage at which it may
be well, in view of their importance as vital agents, to consider what is estab-
lished and to call attention to some of the principal issues which remain to be
elucidated. :

Specific Activity of Maltase and Hmulsin.—As the investigation is extended,
the evidence becomes more and more convincing that the action which an
enzyme exercises is specific; in other words, that it is hmited to compounds
of a particular type—to a greater extent, indeed, than is recognised in
earlier communications of this series, which in some few particulars need
modification.

In No. IIIa table is given (p. 520) in which it is indicated (on the authority
of Emil Fischer) that maltase hydrolyses «-galactosides as well as «-glucosides
and that galactose has a slight retarding effect. At the time when the experi-
ments to which the table relates were carried out, no opportunity had been
found of verifying Fischer’s statement. Subsequently, when experiments
were made with methyl-e-galactoside, extracts from both top and bottom
yeast proved to be entirely without action. It was then ascertained that the
summarised account of Fischer’s work which had been consultedft contained
an incorrect statement and that the results we had obtained were in harmony
with those recorded by Fischer in his original papers.t

The slight retardation of the action of maltase by galactose did not appear
surprising so long as it was supposed that galactosides were hydrolysed by
the enzymes; when, however, this was found not to be the case, it became

* Former Communications.—I, ‘Chem. Soc. Trans., 1903, vol. 88, p. 1305 ; II, ‘ Roy. Soc.
Proc.,’ 1904, vol. 73, p. 500 ; ITI, zbed., p. 516; IV, zbed., p. 526; V, zbed., 1904, vol. 74,
p. 188°; VI., cbed., p. 195 ; VII, zbid., 1905, B, vol. 76, p. 592; VIII, zbid., p. 600 ; also
ibid., 1904, vol. 73, p. 5387 ; 1906, B, vol. 78, p. 376.

+ ‘Zeit. Phys. Chem.,’ 1898, vol. 26, pp. 66 and 69.

t ‘Ber.,’ 1894, vol. 27, p. 2987, and 1895, vol. 28, p. 1480.

Studies on Enzyme Action. 361

necessary to re-examine the action: ultimately, the retarding effect of
galactose was traced to the presence of an alkaline impurity.*

In No. V, in which the action of emulsin on milk sugar is discussed,
attention is called to the evidence in favour of the view that emulsin is
capable of acting on §-galactosides (7.¢., milk sugar) as well as on @-gluco-
sides ; also to Bourquelot and Hérissey’s contention that probably lactase is
present in ordinary emulsin. Experiments to be described in a later com-
munication, made specially with the object of clearing up this question, leave
no doubt that the activity of emulsin as ordinarily prepared is due, as
Bourquelot and Heérissey and also Pottevin have contended, to the presence
of a distinct enzyme capable of affecting the @-galactoside, on which emulsin
proper has no action.

To state our opinion in brief, maltase is capable of hydrolysing a-glucosides
alone, whilst emulsin hydrolyses @-glucosides.

Correlation of Enzyme with Hydrolyte.—In view of these considerations, the
suggestions should be canvelled which were put forward in the third com-
municationt with reference to the extent to which configuration may be
modified without nullifying the action of an enzyme.

Attention was directed in this communication to the evidence of a close
correlation in configuration between enzyme and hydrolyte afforded by the
controlling influence on the rate of change of a glucoside which the hexose
derived from it exerts; welcome confirmation of this conclusion is afforded
by Ter Meulen’s observations.t

A further series of experiments has been made with specially purified
materials which enable us to confirm and extend the earlier observations ;
it 1s unnecessary to describe these in detail, as the method followed has
already been described. The results are summarised in the following table :—

|
Enzyme. | Hydrolyte. Inhibitants. Destitute of inhibiting power.
Maltase ...... Maltose......... Glucose, methyl-8-| Galactose, fructose, mannose, sorbose,
glucoside arabinose, mannitol, dulcitol
Invertase ...... Cane sugar ...... Glucose, fructose... Galactose, mannose, mannitol, dulcitol
Lactase ......... Milk sugar ...... Galactose, methyl- | Fructose, mannose, arabinose, xylose,
a-galactoside mannitol

The evidence that enzyme and hydrolyte must be in complete correlation
appears to be little short of absolute in the case of the sucroclasts.
* Cf. Caldwell, ‘Roy. Soc. Proc.,’ 1906, vol. 78, p. 283.

t Vol. 73, p. 525.
t ‘Rec. Trav. Chim.,’ 1905, vol. 24, p. 444.

362 Prof. H. EK. Armstrong and Dr. E. F. Armstrong. [May 9,

The Hydrolysis of Cane Sugar.—The evidence as to the manner in which
the hydrolysis of cane sugar is affected by the products of change is
conflicting. O’Sullivan and Tompson stated, in 1890,* that the products of
inversion are without influence on the rate of change. In 1902, however,
Henri assertedt that invert sugar retards the hydrolysis and ascribed the
inhibiting effect almost entirely to the fructose.t

In the same year, Adrian Brown} also showed that invert sugar had
a marked effect, whilst milk sugar had but little influence in retarding
inversion.

Barendrecht has since published two communications|| dealing with the
influence of a variety of substances. His results are of a most contradictory
character and scarcely admit of rational interpretation; it is difficult to
regard his radiation hypothesis seriously.

Apparently, the difficulties which attend experiments on the subject are
not sufficiently appreciated. Invertase being extraordinarily active, it is
necessary to work with highly dilute solutions of the enzyme: minute
quantities of impurity therefore exercise an important influence. Unless
hard glass vessels are used, it is impossible to obtain consistent results.
A very large number of experiments have been made at various times
(chiefly in 1904—05) to test the influence of glucose, fructose, galactose,
mannose, dulcitol and mannitol: in many cases the results were conflicting,
so that the problem cannot be regarded as solved: the general impression
has been gained, however, that glucose and fructose alone retard the action,
probably to an equal extent.

Conclusions similar to ours have been arrived at by Ford with reference to
the action of diastase on starch,{1 and he has used amino-acids in order
to neutralise impurities. From this point of view, we had used glycine and

* ‘Chem. Soc. Trans.,’ vol]. 57, p. 927.

t ‘Compt. Rend.,’ 1902, p. 917.

{ This statement was incorporated in the table given in No. III.

§ ‘Chem. Soc. Trans.,’ vol. 81, p. 382.

|| ‘Zeit. Phys. Chem.,’ 1904, vol. 49, p. 456 ; 1906, vol. 54, p. 367.

I ‘Soc. Chem. Ind.,’ 1904, vol. 23, No. 8; ‘Chem. Soc. Trans.,’ 1904, vol. 85, p. 980.
Ford expresses the opinion that the extraordinary results obtained with starch by various
workers are due to a lack of recognition of the important influence of traces of impurity
on the course of the change. The results of recent French workers justify this view.
According to Ford, the action of diastase attains'its maximum in neutral solution and is
not augmented by asparagine unless there has been a previous restriction ; this is true
also of the various salts which are said to accelerate the action.

The accelerating influence of traces of acid on the activity of invertase (cf. O'Sullivan
and Tompson) is. probably a consequence of the neutralisation of alkalinity. We are

inclined to think that the effects produced by salts are also of a secondary character and
that they do not influence the action of invertase in any specific manner.

1907. | Studies on Enzyme Action. | 363

asparagine in experiments with maltase prior to the appearance of Ford’s
communication: finding them to be practically without influence, we felt
safe in using them with invertase. As a rule, the activity of invertase is
much increased by amino-acids; the experiments on which we rely were all
made in presence of glycine.

Nature of Enzymes.—Granting that both glucose and fructose inhibit the
action of invertase on cane sugar, it would seem to follow that this enzyme
is so constituted that it can adapt itself to both sections of the biose.
Unfortunately, no satisfactory expression of the structure of cane sugar has
been arrived at hitherto, so that we are unable to formulate the changes
which the biose undergoes on hydrolysis in a definite manner. It is an
altogether peculiar substance, its extraordinary instability in presence of
acids being most remarkable in comparison with that of other bioses* from
which it does not differ markedly in most respects.

Invertase, the enzyme correlative with cane sugar, is equally remarkable
among enzymes on account of its extreme activity.

Apparently, cane sugar is a derivative of a-glucose, to judge from
O’Sullivan and: Tompson’s observations;+ nevertheless it is not attacked by
maltase and as this acts on all the simple a-glucosides, it cannot well belong |
to their class. Moreover, taking into account Pottevin’s statementt that
methyl-fructoside is not hydrolysed by extracts of S. octosporus, Mucor mucedo
and Mucor alternans, which hydrolyse cane sugar and maltose, it must be
supposed, in like manner, that cane sugar is not a simple tructoside.

These conclusions are in entire harmony with that derived from the study
of inhibiting agents, so that there is little room for doubt that during hydro-
lysis the enzyme extends its influence over the whole of the cane sugar
molecule—being intimately correlated in all its parts with the carbohydrate
in the manner postulated in No. ILI§ of these communications.

The question arises whether this is true of other biases.|| The experi-
mental material is not yet sufficient for the discussion of the problem,
but there are already indications which make it probable—at all events, in
the case of milk sugar—that what is true of cane sugar and its correlative
enzyme applies generally.

It is commonly assumed that maltose is an e«-glucoside, because maltase,
which hydrolyses it, also acts on simple glucosides such as methyl-

* Of, IV, p. 530.

t CL ELF. pemeirene ‘Chem. Soc. Trans.,’ 19038, vol. 88, p. 1308.

{ ‘An. Inst. Past.,’ 3903, vol. 17, p. 43.

§ Pp. 522—524.

|| To avoid periphrasis, it appears desirable to term those enzymes which act on simple
glucosides monases and those which attack the biose sugars b7ases.

364 Prof. H. E. Armstrong and Dr. E. F. Armstrong. [May 9,

a-glucoside. But independent proof of its character is afforded by the fact
that the primary product of the hydrolysis of maltose is a-glucose (1.1307).
Maltose and apparently lactose also appear to differ from cane sugar in that
they are hydrolysed by enzymes which also act on the corresponding simple
elucosides (monosides).

The existence of this difference affords further proof that the configuration
of cane sugar and of its correlative enzyme are peculiar and altogether
different from that of other bioses and biases. To account for the wider
activity of a biase such as maltase, it is only necessary to suppose that not only
are the simple glucosides of the same general type as the correlative bioses,
but that this resemblance also extends to the biases and that it is sufficient
in some cases if the attachment of the biase be secured over one section of
the hydrolyte. Thus, representing the glucose radicle by G and other radicles
such as are present in simple glucosides by h, EY being the hypothetical radicle
of the enzyme correlative with the radicle G, the following diagram may be
used, the horizontal line representing the section of the molecule over which
attachment of enzyme to hydrolyte takes place—

GLO G

Es . Kg

Gr Or k
Ey . Kg

Formation of Enzymes.—It is conceivable that the enzymes themselves are
subject to hydrolysis and simplification—in other words that a biase may give
rise to a monase. The existence of monases in admixture with biases is,
therefore, to be expected. Attention has already been called in the previous
communication to evidence indicative of the presence in extracts of dried
yeast of an enzyme (a-glucase) capable of hydrolysing methyl-«-glucoside, but
without action on maltose; this discovery may well prove to be significant
from the point of view now advanced.

There can be little doubt that the sucroclastic enzymes are products of
hydrolytic changes conditioned by enzymes (mainly by proteoclasts, in all
probability); the very different results obtained with extracts prepared under
different conditions (at different temperatures, etc.) are scarcely to be
accounted for in any other manner. And it is to be supposed that several
enzymes may arise in this manner, all of one type, yet all capable of
hydrolysing a particular hydrolyte and differing only in activity, much in the
same way that the various simple glucosides represented by the general
formula R’.0.G. (such as methyl-, ethyl-, phenyl-glucoside, salicin, etc.) differ
in stability towards hydrolytic agents.

YS Studies on Enzyme Action. 365

In the case of enzymes other than those which affect carbohydrates, the
range of activity would appear, however, often to be greater than is
ever manifest in the case of enzymes of the sucroclastic class. Thus lipase
hydrolyses not only fats but also numerous ethereal salts; and many of the
polypeptides prepared by Emil Fischer are hydrolysed by trypsin, which
clearly exercises a wide range of activity.

As the only radicles common to the ethereal salts and to the polypeptides
respectively are in the one case the group—CO.O—and in the other the group

>N.C.CO—present in the amino-acids represented generally by the formula

NH:2.CHR.CO.OH, it is to be supposed that these simple groups are the
active centres and that the specific influence of the enzyme is exercised at
these centres. The variations observed in the activity of an enzyme towards
a variety of hydrolytes are, from this point of view, to be ascribed mainly to
differences in the stability of the hydrolytes; a single key, as it were, is
provided which fits a variety of locks almost equally well; but as the locks are
constructed with springs varying in stiffness, the effort which must be exerted
to open the locks varies from lock to lock. But the interesting problem
to be solved in this connection is whether, in the case of compounds
such as the proteins, several junctions may not be resolved practically
simultaneously by “compound” keys, as it were. :

Bertrand’s remarkable observations* on the limiting oxidising power of
Bactervum xylinum have shown that configuration is of consequence even in
the case of oxydases. It is conceivable that the oxidation of compounds such
as the higher fatty acids is a regulated process consequent on the attachment
of an oxidase to the terminal carboxyl group, the which oxidase serves to
bridge over the interval between this group and some more or less distant
CHg group and to locate oxygen against it.

* © Ann. Chim. Phys.,’ 1904, ser. 8, vol. 3, pp. 181—288.

bo
&

VOL, LXXIX.—-B,

366

The Solubility of Air in Fats, and rts Relation to Carsson Disease.
By H. M. Vernon, M.D., Fellow of Magdalen College, Oxford.

(Communicated by Dr. J. S. Haldane, F.R.S. Received April 1,—Read
May 30, 1907.)

(From the Physiological Laboratory, Oxford.)

As is now well known, the very varied symptoms produced by rapid
decompression from high atmospheric pressures, and popularly known as
“caisson disease” or “diver’s palsy,” are due to liberation of bubbles of
gas—chiefly nitrogen—in the blood and tissues. In the course of a recently
published investigation, I found that airis much more soluble in certain oils
than in water. Dr. J. S. Haldane pointed out to me the interest of this fact
in connection with the causation of caisson disease, and at his suggestion I
have repeated and extended my observations.

The fats experimented with were olive oil, cod liver oil and lard. In the
case of the first two, the solubility was measured at 15° and at 37° C.,
whilst for lard it was gdetermined at 45° C. In the observations made at
room temperature, the oil was shaken violently with air in a bottle for several
minutes, and was allowed to stand for 1 to 14 hours till all the air bubbles
had risen to the surface. It was then weighed, and about 40 to 50 grammes
of it were sucked up into the vacuous flask of a Geissler’s mercury pump.
This flask contained 70 to 100 cc. of 0°5 per cent. sulphuric acid which had
previously been well boiled for an hour so as to get rid of all traces of air.
The mixture of oil and water was now boiled for half an hour, the oil breaking
up into a very fine emulsion and giving up practically all of its gas in the first
few minutes. This gas was pumped off and analysed with Haldane’s* gas
analysis apparatus. The oil was boiled with dilute acid instead of water, so
as to obtain the whole of the carbon dioxide present, both combined and in
solution. In determining the solubility at 37°, the oil, previously saturated
at room temperature, was warmed to about 38° to 39°, and was shaken
vigorously with air for about two minutes. At the end of this time its
temperature had fallen to about 36°5. It was warmed up a second time and
the shaking repeated, and was then kept in a water bath at 37° for about
half an hour in the case of the cod liver oil, and an hour in the case of the
olive oil, these being the times required for all the bubbles of air to rise to
the surface. The gaseous content of a weighed amount of the oil was then
determined as before. The results obtained are given in the tables. They

* Haldane, ‘Journ. Physiol.,’ vol. 22, p. 465, 1898.

Solubility of Air in Fats, and its Relation to Caisson Disease. 367

represent the volumes of gas, reduced to 0° and 760 mm., contained in 100 c.c,
of the oil at the temperature recorded, when saturated with air at a pressure
of 760 mm. To calculate these values, it was assumed that the specific
gravity of olive oil at 15° is 0°917, and at 37°, 0:902.* The specific gravity
of cod liver oil, compared with that of water at 15°, was found by direct
experiment to be 0-928 at 15°, and 0:914 at 37°.

|

100 e.c. of olive oil contain—

At 15° C. Mean. Atisn Cy Mean.

c.c. | C.c. c.c. C.c. C.c C.c. C.c. C.c.

Oxygen ROR Seiccavee 2°20 2°23 2 °42 2°28 || 2°33 2 °36 2°30 2°33
DatrOPen ......... 5°23 5 °30 5 40 5 *26 5 a9 5°23 fs Ls 5°19
Eos cic ene ses 0°19 0 °24 0°16 0°20 Os 0°13 0:18 0:16

100 c.e. of cod liver oil contain—

| At 15° ©; | Mean. Atco CC, Mean.

c.c. c.c. C.c. c.c. C.c. c.c. C.c. C.c.

Oxygen Bre cridlors 2°34 2°31 2°22 2-29 2-21 2 22 2°22 Ve} yp
Mitrogen ...:..... 4°95 5°15 5:07 5:06 5°05 5°10 5°08 5°08
0)» Een eee 0°19 0°25 0:19 0°21 0°18 0°24 0°20 0°21

Considering only the mean values, we see that in the case of each oil the
solubility at 37° is within the limits of experimental error the same as its
solubility at 15° C. A similar constancy was found by Bunsen for the
solubility of oxygen in alcohol at temperatures from 0° to 24°, though
Timofjewt has since found that the solubility does slightly diminish with the
temperature. In any case the diminution is small (6°8 per cent. diminution
for oxygen and 5:2 per cent. diminution for nitrogen between 0° and 24°) and
if it exists at all in the case of these oils, it must be smaller still. A simple
proof of the slight effect of temperature on solubility is obtained by saturating
the oil at room temperature, and heating it gradually in a water bath. Nota
trace of gas is observed to come off if the temperature be kept at 97°, but at
100° it bubbles off in very small quantities.

The observations on lard were carried out at one temperature only. Lard
melts at 36° to 48°, and solidifies at 27° to 30°, but if melted and shaken with

* See Lewkowitsch, “Chemical Analysis of Oils, Fats and Waxes,” London, 1898,
p. 404.
t Timofjew, ‘ Zeit. Phys. Chem.,’ vol. 6, p. 151.

2 We

368 Dr. H. M. Vernon. Solubility of Air in [Apr. 1,

air at 37° it is too viscid to allow the air bubbles to rise to the surface in a
reasonable time. Hence the solubility was determined at 45°. The lard was
heated to about 48°, and then shaken violently for a minute with air, its
temperature at the end of this time having fallen slightly below 45°. The
process was repeated, and after keeping the lard in a water bath at 45° for
about 35 minutes the gases were boiled off in the usual way. They came off
quite as readily as from the olive and cod liver oils. The data obtained are
given in the table, they being calculated on the supposition that the specific
gravity of lard at 45°, as compared with water at 15°, is 0°895.* If the mean
values be compared with the mean values for the other fats, it will be seen
that the solubility of oxygen is practically constant throughout. That of
nitrogen varies slightly, it being 5°23 cc. for olive oil (an average of the
values at 15° and at 37°), 5-11 cc. for lard, and 5°07 c.c. for cod liver oil.
Still these differences are almost within the limits of experimental error. In
the case of the lard, it is possible that the two minutes’ shaking with air was not
quite sufficient for complete saturation, but more thorough shaking was not
possible, as it brought down a slight precipitate which prevented the rapid
clearing of the air bubbles.

100 e.c. of lard at 45° C. contain— Mean.
C.c. C.c. C.c. c.c.
Oxygen fi) acces 2°23 2°40 2°35 2°33
NatEOgeM Been. se 5°05 5 ‘09 5°18 5°11
CO. cee 0°12 0°12 0°15 0°13

In that fats of such very different composition as the three experimented
with dissolve practically the same amounts of oxygen and nitrogen, we are
fully justified in assuming that human fat would behave similarly. From the
physical and chemical constants collected in the accompanying table,f we see
that the melting point of human fat les between those of cod liver oil and
lard, whilst its iodine value is 61°5 per cent., or about the same as that of
lard. In that cod liver oil has an iodine value of about 145 per cent., and

| Cod liver oil. | Olive oil. Lard. Human fat.
Solidifying point 0° to —10° —6° (?) 27° to 30° 15°
Melting point ... — = 36° to 48° 17°5°
Specific gravity 0°928 at 15°/15° 0°917 at 15°/15° | 0°895 at 45°/15° | 0°903 at 25°/25°
Todine value...... 123 to 168 per cent. |79 to 88 per cent.|50 to 70 per cent.| 61°5 per cent.

* Lewkowitsch, oc. cit., p. 564.
t+ Lewkowitsch, loc. cit., pp. 454, 477, 562 and 564.

1907. ] Fats, and its Relation to Caisson Disease. 369

olive oil one of about 83 per cent.,it follows that the proportion of unsaturated
acids present in a fat has little or no influence upon its solvent powers for
oxygen and nitrogen.

From the absorption coefficients for oxygen and nitrogen given by Bohr
and Bock* it is calculated that 100 cc. of water saturated with air at
15° absorb 0°733 c.c. of oxygen and 1°411 c.c. of nitrogen. Taking a mean
of all the values obtained with cod liver oil and olive oil at 15°, the solubility
of oxygen is found to be 3:1 times greater than in water, and of nitrogen
3°7 times greater. At 37° Bohr and Bock’s values show that 100 cc. of
water absorb 0°507 c.c. of oxygen, and 0975 cc. of nitrogen. Taking
a mnean of all the values obtained with cod liver oil and olive oil at 37°, and
with lard at 45° (for doubtless the solubility at this temperature is practically
the same as at 37°), the solubility: of oxygen is found to be 4°5 times greater
than in water, and that of nitrogen 5°3 times greater. It has been found
by Bohr,+ that blood plasma dissolves 2°5 per cent. less nitrogen than an
equal volume of water, whilst blood dissolves slightly less still; hence we
may conclude that at body temperature the fat of mammals dissolves at least
Jive times as much mtrogen as water or as blood and blood plasma, |
_ The solubility of oxygen in fats, though apparently less than that of
nitrogen, may in reality be as great. It is well known that oils are able to
absorb and combine with oxygen, and the rate of this absorption is by no
means inconsiderable. For instance, a sample of olive oil was saturated with
air at 15°, and was kept for 23 hours at 18° in a stoppered bottle filled to
the brim. It then yielded only 0°49 c.c. of oxygen per 100 c.c., whilst some
of the sample from which the air was boiled off an hour after saturation
yielded 2°40 cc. Owing to its richness in unsaturated acids, cod liver oil
absorbs oxygen more quickly still, and a sample saturated with air at 15°,
and kept 23 hours at 18° in a stoppered bottle, gave only 0°05 c.c. of oxygen
per 100c.c. Undoubtedly, therefore, there must have been some absorption of
dissolved oxygen during the half to one and a-half hours the air-saturated
oils were kept before analysis.

It might be thought that the solubilities determined by the above described
method are invalidated, or liable to be invalidated, by leakage of air into the
vacuum pump. This is not the case if due precautions are taken. The
pump must be in such good order that no appreciable leakage occurs when it
is left vacuous for several days, and it must be kept vacuous for a week or
more before use, for if air be left in it a small amount attaches itself to the
glass walls of the apparatus, and only escapes slowly when the pump is

* Bohr and Bock, ‘ Wied. Ann.,’ vol. 14, p. 318, 1891.
t Bohr, ‘ Nagel’s Handb. d. Physiol.’ vol. 1, p. 62, 1905.

370 Dr. H. M. Vernon. Solubility of Air in [Apr. 1,

made vacuous again. Air likewise attaches itself to the walls of the boiling
flask, but it is got rid of by the preliminary boiling of the dilute acid previous
to the introduction of the oil. In all analyses of gases, whether in oil, water,
or salt solutions, such a preliminary boiling was invariably adopted, and by
taking these precautions many hundreds of analyses have been made without
invalidation of results by leakage of air. I have elsewhere* recorded nearly
a hundred analyses of the gases in sea water, and the mean nitrogen values
then obtained are almost identical with the nitrogen absorption values
determined by Dittmar.

The bearing of the above recorded results upon many phases of caisson
disease is probably a direct one. The commonest symptoms observed in
caissoniers consist of joint and muscular pains, and in paralysis, the latter
being the result of injury to the spinal cord. Thus in autopsies of caissoniers,
Bert observed softening of some inches of the spinal cord in the dorsal region.
v. Leyden, in a case of death 15 days after decompression, observed signs of
myelitis, and small irregular fissures in the mid-dorsal cord, probably
produced by the escape of gas. Autopsies of divers have shown necrobiosis
and hemorrhages in the spinal cord,t and distention of the blood vessels by
air bubbles.t

Chemical analysis shows that spinal cord and peripheral nerves contain
nearly 20 per cent. of fat and fat-like substances. Adipose tissue contains
about 83 per cent. of fat, and yellow bone marrow (as distinct from red
marrow, which is poor in fat) contains no less than 96 per cent. of fat
(Gorup-Besanez). If these tissues were saturated with nitrogen at, for
instance, four atmospheres of atmospheric pressure, 100 c.c. of them would
contain, instead of the 0°975 x4 = 3°90 cc. of dissolved nitrogen present in
an equal volume of water, about 7 c.c.in the case of the spinal cord and
nerve, and 20c.c. in the case of yellow bone marrow. On _ sudden
decompression, therefore, the volume of nitrogen bubbling off in a gaseous
form might be two to six times as much as that from the non-fatty fluids and
tissues of the body.

It has been found that the severity of the symptoms developed by
caissoniers is influenced greatly by the time of exposure to increased pressure.
In sinking the foundations of a bridge at St. Louis, a pressure reaching at
a maximum to 50 lbs. above normal was used, and a large proportion of the
workers were affected. As the depth of the caissons increased, the shifts
were shortened from four hours to one hour, with the result that the serious

* Vernon, ‘ Journ. Physiol.,’ vol. 19, p. 68, 1895.
+ Cited by Hill and Macleod, ‘ Journ. Hygiene,’ vol. 3, pp. 408 and 409, 1903.
t See Hill’s “‘ Recent Advances in Physiology,” p. 250, 1906.

1907. | Fats, and its Relation to Caisson Disease. 371

accidents were reduced to ni.* Presumably, therefore, the body tissues
were not so fully saturated with nitrogen in the shorter as in the longer
period. On the other hand, Hill and Greenwoodf found that on exposing
themselves to a pressure of 30 to 45 lbs., their urine became saturated with
the theoretical amount of nitrogen in 10 to 15 minutes. This quicker
saturation must be due to the blood-flow through the kidney being much
more rapid than that through the spinal cord, and very much more rapid
than that through adipose tissues. Even if it were as rapid through
fat-containing tissues as through the kidney, the fats, in virtue of their

greater solvent power, would take longer to become fully saturated than the
fluids of non-fatty tissues.

Conclusion.

At body temperature, fats dissolve more than five times as much nitrogen
as an equal volume of water or blood plasma.

The special tendency of the fat-containing tissues (such as subcutaneous
tissues, spinal cord and peripheral nerves) of caisson workers and divers to

suffer injury from the liberation of gas bubbles after rapid decompression 1s
dependent on this great solubility.

* See Paul Bert’s “ La Pression Barométrique,” Paris, 1878, p. 404.
t+ Hill and Greenwood, ‘ Roy. Soc. Proc.,’ B, vol. 79, p. 21, 1907.

372

On Mitosis in Proliferating Epithelium.
By J. O. WAKELIN Barratt, M.D., D.Sc. Lond.

(Communicated by Professor C. S. Sherrington, F.R.S. Received April 4,—
Read May 30, 1907.)

(From the Cytological Laboratories, University of Liverpool.)

The object of the present investigation is to determine the character of the
mitosis occurring in cutaneous epithelium which has been artificially stimu-
lated to proliferate freely. In the epithelial proliferation of carcinoma, in
addition to somatic mitosis, a synaptic mitosis is also met with.* In
attempting to decide upon the significance to be attached to this latter
phenomenon it is necessary to ascertain whether reduction mitosis is met
with exclusively in cancerous proliferation of the surface epithelium, or
whether it is also encountered in non-cancerous proliferation. If it is present
in the latter case, then synaptic mitosis cannot be regarded as exclusively
related to epithelioma, or, for that matter, to sexual reproductive tissue; if,
however, only somatic mitoses occur in non-cancerous epithelial proliferation,
then the possibility is present that the altered biological characters
presented by the epithelial cells of malignant growths may be in some way
directly connected with the reduction taking place in the number of
chromosomes.

The abundant epithelial proliferation occurring in many cutaneous diseases,
é.g., psoriasis, lichen planus, and dry forms of eczema, is unsuitable for this
investigation, since the amount of material available from such sources is too
small and the supply too inconstant to fulfil the requirements of research.
Recourse was therefore had to epithelium, which had been made to proliferate
freely by the use of scharlach R,* dissolved in olive oil, the injection of which
beneath the skin of the rabbit’s ear causes extreme and, it may, irregular
hypertrophy of the Malpighian layer.t It was further found of advantage in
obtaining an abundance of division figures to implant pieces of such
proliferating epithelium under the skin of the rabbit. Using the material
obtained in these two ways the present investigation was carried out. The
effect of scharlach R, it may be observed, is to cause marked increase of the
prickle layer, both of the surface epithelium and of the hair follicles. This
increase is at first fairly regular, but later, when considerable in degree,

* Farmer, Moore, and Walker, ‘ Roy. Soc. Proc.,’ B, vol. 77, p. 226.
+ Scharlach R is azo-orthotoluol-azo-8-naphthol : C,H,N = NC,H,N = NC,,H,OH.
+ B. Fischer, ‘ Miinch. Med. Wochenschr.,’ 1906, 53. Jahrg., S. 2041.

Mitosis in Prohferating Epithelium. 373.

becomes irregular, and ingrowth of epithelium occurs, comparable to, though
considerably less extensive than, that occurring in squamous epithelioma. If
the proliferation be set up in scar tissue, the resemblance to the latter
condition is striking. With the disappearance of the scharlach R the
process comes to an end and involution occurs, though the skin appendages
may be more or less destroyed. The character of the epithelial proliferation
is, therefore, intermediate between that of such affections as psoriasis and
lichen planus, in which epithelial hypertrophy without downgrowth takes
place, and epithelioma, in which downgrowth of epithelium is the character-
istic feature, and is the means by which neighbouring tissues are invaded and
extension of the growth occurs.

When marked proliferation of the epithelium of the rabbit’s ear has been
brought about under the conditions above described, sections hardened in
Flemming’s solution and. stained by Heidenhain’s iron alum hematoxylin
method show numerous mitoses, amounting occasionally to as many as five
mitoses to every hundred cells encountered. The mitotic cells are of large
size, being 10m to 16m in diameter. In most cases the individual
chromosomes can be recognised to be more or less curved rods of varying
length, too closely-packed to be counted. In a relatively very small number
of the mitotic figures, however, it is possible to enumerate the chromosomes,
and in this way to determine the forms of mitosis present.

The form most commonly met with appears to be the ordinary somatic
type shown in fig. 1,* which presents no essential points of difference
from the corresponding type in the testis of the rabbit, taken as a standard,

aN
F oS
\

Fie. 1. —Normal Somatic Mitosis in Proliferating Epithelium of Rabbit’s Ear.

* The mitoses exhibited in this and the succeeding figures, all of which have the same
magnification (cp. scale in fig. 5), were drawn to scale in plan and elevation, and were then
modelled. The illustrations were prepared from the models. The use of models was
adopted in order to permit a simultaneous view of all the chromosomes present, in their
natural relation, instead of the succession of views at different levels afforded by the
microscope.

374 Dr. J. O. W. Barratt. [Apr. 4,

shown in fig. 2. In this form the number of the individual chromosomes
which could be distinguished ranged between 28 and 32, while in the testis,
taken for comparison, the number of chromosomes which could be recognised

Y
YP

KK

Fic. 2.— Normal Somatic Mitosis in Testis of Rabbit.

lay between 28 and 36. In the somatic mitoses of the ear of the rabbit,
after injection of scharlach R, the chromosomes appear larger and coarser
than in the testis, but are otherwise of the same type.

In addition to the above, another type of mitosis, shown in fig. 3, is
met with less frequently than in the former. Here the chromosomes, instead
of being curved rods, mostly assume the form of large spheroidal or
ellipsoidal masses, taking the stain chiefly in their peripheral layer and to
a less extent centrally; some of the chromosomes, however, form short
rods, straight or curved, occasionally swollen at the extremities ; again, some
of the chromosomes form long rods (two such are shown in fig. 3, one thick,
one thin). All these chromosomes are arranged in pairs, with or without
connecting threads of chromatin, in the same manner as the so-called
gemini of the first maiotic division of the testis (fig. 4), with the exception
of the two last mentioned, which are evidently equivalent to pairs, though

1907. | Mitosis in Proliferating Epithelium. 375

no division or constriction can be recognised. It will be seen that the
chromosomes in figs. 3 and 4 are on the equator of the spindle. In fig. 3

a
YSo @ VEC

ted

Fie. 3.— Reduction Mitosis in Proliferating Epithelium of Rabbit’s Ear.

the number of pairs is 14; in other mitotic forms of the same type it was
found to vary from 14 to 18. A glance at the heterotype mitosis from the
testis of the rabbit (fig. 4), in which the number of gemini or pairs of
chromosomes which could be counted was found to vary from 14 to 18,
shows the essential morphological identity of the synaptic form occurring
in the proliferating epithelium of the rabbit’s ear (fig. 3) with the first
maiotic mitosis in the testis of the same animal (fig. 4). In both forms the

Ao a
O \\
Sa
—————————————————————————
sig / : S
Fig. 4.—-Heterotype Mitosis in Fig. 5.—Post-synaptic Mitosis in

Testis of Rabbit. Proliferating Epithelium of Rabbit’s Ear.

376 Dr. J. O. W. Barratt. [Apr. 4,

chromosomes are for the most part large spheroidal or ellipsoidal masses,
minor differences being recognisable in the smaller gemini.

In addition to the above type, another less frequent form of mitosis was
encountered. This is exhibited in the dyaster shown in fig. 5. Here the
chromosomes, which form elongated narrow rods, curved or bent, are also
reduced in number, varying from 16 to 18 in each aster. This form may
represent a post-synaptic division. If this is so, it is impossible to determine
whether it is the first or a subsequent post-synaptic division, since there
is no means of identifying its particular generation.

Spireme forms are fairly common. In these the chromatin granules are
not unfrequently abundant, but well-defined double beading of the spireme
thread has not so far been observed. It may be noted, however, that in the
testis of the rabbit double beading could not be clearly exhibited. Late
prophases, in which different permanent forms of the chromosomes com-
posing the gemini, similar to those described by Moore and Arnold,* were
recognisable, have not been met with, nor have masses of archoplasm or
archoplasmic vesicles been up to the present encountered in proliferating
epithelium of the rabbit’s ear.

No suggestion can be offered, from the observations made in the course of
this investigation, as to the significance of the reduction division which takes
place. Nor has it been possible to ascertain the further history of the
reduced cells, and, in particular, to determine whether division with the
reduced number of chromosomes may be repeated for more than one
generation.

It may be observed that the number of chromosomes counted has not
been constant, but has varied from 28 to 36 in somatic mitoses, and from
14 to 18 in the reduction mitoses. The difficulty of accurately ascertaining
the number present under the conditions obtaining in stained sections
viewed under high magnification may in part account for the variation met
with, particularly in the former case. It is, however, not at all clear
that this explanation is completely satisfactory; on the contrary, the
constancy with which variation is met with suggests, particularly when
reduction occurs, and enumeration of chromosomes becomes in consequence
much less difficult, that the number is not absolutely fixed, but exhibits
a certain degree of variation.

Considerable variation was exhibited in different experiments in respect of
the number of division figures encountered. As a rule, the number was
greatest when the epithelial proliferation was most marked. An extreme
degree of mitosis was observed at the end of a week in some of the implanted

* ‘Roy. Soc. Proc.,’ B, 1906, vol. 77, p. 563 ; also Moore and Embleton, zbid., p. 555.

1907. | Mitosis in Proliferating Epithelium. 377

portions of skin. The reduction mitoses were met with in those cases in
which proliferation was considerable, but owing to the very large number of
division figures which are not sufficiently well defined to enable their
character to be determined, it is not possible to give statistics of the relative
frequency of the somatic and reduction mitoses occurring at different periods
in the proliferating epithelium.

Summary.

The preceding observations may be briefly summarised as follows :—

1. In epithelial proliferation brought about by scharlach R, both normal
somatic and reduced mitoses occur. This statement applies to epithelium
proliferating 7m situ, and also to the same implanted under the skin.

2. In the reduction mitoses the number of chromosomes which dbuld be
counted varied from 14 to 18. In the somatic form the number counted
varied from 28 to 36.

3. Reduction mitoses could be recognised less frequently than somatic
mitoses.

4. Post-reduction mitoses were met with.

5. The character of the mitoses occurring was not definitely altered by
implantation under the skin.

es ee =<

378

Increase in the Complement-content of Fresh Blood-serum.

By J. HENDERSON-SMITH, M.B., Philip Walker Student of Pathology in the
University of Oxford.

(Communicated by Professor Gotch, F.R.S. Received March 26,—Read
May 9, 1907.)

Very soon after coagulation has occurred in blood withdrawn from the
vessels, serum begins to be expressed. The amount, at first small, increases
rapidly, and in a few hours, at room temperature, contraction of the clot is
practically complete. Some observations made in the course of other work
suggested that the composition of the serum first yielded differs from that
of the serum expressed later, and a special investigation of the point led
to the results recorded in this paper. The serum given off from the clot
at varying intervals of time after bleeding was tested for its content in
hemolytic complement. This body was selected for examination, not only
because it can conveniently be studied in the test-tube, but also because
much greater quantitative accuracy of result can be obtained with it than
with, for example, a bactericidal complement. In most of the experiments,
the serum of rabbits was tested against the red blood-corpuscles of the ox,
and the necessary immune-body was derived from the serum of rabbits
immunised by repeated injections of these corpuscles. In some instances
other combinations were used, as is stated later, but in every case the red
cells used for injection and for examination were fresh, and had been freed
from serum by being washed thrice in many times their bulk of isotomic
salt solution. The experiments fall into two groups, according as the serum
examined was from a normal animal or from an animal that had been
previously immunised.

1. Normal Serum.

The method adopted was as follows :—A rabbit was treated with injections
of ox red blood-corpuscles, and when its serum had reached a sufficiently
high titre, its blood was withdrawn and the serum obtained. This was then
heated for half an hour at 56° C. to destroy the contained complement, and
served as a stock of “immune-body ” (by which name I shall refer to it in
the rest of this paper). The neck of a normal rabbit having been shaved
and sterilised, the large vessels of the neck were cut, and the issuing blood
was caught in a sterile flask. As soon as coagulation had occurred, the
clot was loosened from the sides of the flask by a sterile glass-rod. At
varying intervals of time, samples of the separating serum were taken and

Increase in the Complement-content of Fresh Blood-serum. 379

rapidly centrifuged to free them from cells, and the supernatant fluid was
then tested. Each sample was examined separately as soon as it was
obtained, instead of waiting for the last sample and examining them all
together. The latter method was used as a control in a few cases, but the
interval between the taking of the first and last samples extended over
several hours, during which time spontaneous deterioration or other change
of the complement might easily occur in the earlier samples. Such a
change does, in fact, occur, as will be shown later, and it was necessary to
avoid this source of error.

The examination of a sample was made in the following way :—A series
of test-tubes was prepared, and into each was measured the same amount
of immune-body (the necessary dose having been previously ascertained).
Diminishing amounts of the sample of serum were then added to the test-
tubes, the first tube receiving a large dose, the next a slightly smaller, the
third a still smaller, and so on. The volume of fluid in all the tubes was
then made the same by adding salt solution, and finally 4 c.c. of a 1-per-cent.
suspension in salt solution of ox corpuscles were added to every tube.
Control-tubes having been prepared, all tubes were shaken and placed in an
Ostwald water-bath at 37° C. After two hours the tubes were withdrawn
and of the series the lowest tube, in which total hemolysis had occurred

y)

2

was noted. The dose of serum in this tube contained just enough comple-
ment to produce total hemolysis in that time. The same procedure was
carried out with every sample, the amounts of immune-body and red cells
being kept constant throughout.

given below.

An example of such an experiment is

Lxample 1—Dose of immune-body = 0:04 ¢.c.; 4 ¢.c. 1-per-cent. suspen-

sion ox red blood-corpuscles. Total = complete hemolysis. Time, two hours

au ol.
Time in hours between bleeding of rabbit and withdrawal of sample.
i | 4 1 2. 34 5 vs
: total total total total total total total
; bP] 3) 3”) g 23 3) bP) pe)
; bb) bP) bp) 3’) bP) 9 3)
! ; not total ” ” ” ” ” oe)
: less not total ks 9 35 5 .
: a less not total . % 45 Fy
y Lay et less not total 5 pe a
: ee oe sa less not total Py not total
: she — By ay less not total less
. wate oy as Mee Fret less

380 Mr. J. Henderson-Smith. Increase in the [Mar. 26,

No lysis was produced by 0°35 c.c. of the 5-hours sample without immune-
body; nor by 0:04 c.c. of the immune-body without the normal serum.

In this case, then, 0°3 cc. of the serum, withdrawn 20 minutes after
bleeding, was required to produce total lysis; but with the successive samples
the amount required steadily decreased, until of the serum which had remained
five hours in contact with the clot 0°08 c.c. was sufficient. The amount of
complement contained in the later sample, or at least its activity, must be
more than three times as great as that of the complement in the earliest
sample. It increased steadily for five hours, and then began to diminish
(decreasing afterwards till it eventually disappeared).

This example is typical. In all the experiments (seven) done with normal
rabbit’s serum and ox corpuscles, a similar increase in the complement has been
observed. The amount of the increase varies, but as a rule it is to double,
sometimes to three times, the quantity found in the first sample. The time
during which increase occurs also varies. The longest time observed was
14 hours; but this is exceptional, and in most cases the rise is complete in
five to seven hours. The same phenomenon occurs when, instead of ox
corpuscles and the appropriate immune-body, horse corpuscles are used with
immune-body from rabbits treated with injections of horse corpuscles. When
normal guinea-pig serum is used instead of rabbit serum to supply the
complement for either ox or horse corpuscles (with appropriate immune-body
from rabbit), the same increase to about double the amount is found. The
duration of the increase, however, is shorter, extending to two to four hours.
(In one of six experiments with guinea-pig serum the rise was very slight, and
complete in one and a-half hours.) —

It may be suggested that this increase in the complementing-power is only
apparently a change in the serum, and that the change really occurs in the
red corpuscles used as the reagent. In the interval between the examination
of the first and last samples the red cells may become more sensitive, and so
give a false appearance of increased power in the serum. This, however,
cannot be the explanation of the facts observed. The red corpuscles were
kept in the cold room (about 3° C.) till immediately before use, and were
always fresh. Under these conditions they do not become perceptibly more
sensitive within so short a time as seven to eight hours, when tested with
other lytic bodies, such as bacterial hemolysins and chemical reagents.
Further, the results, described later, obtained with immune sera prove that
no such change as is here suggested occurs in the corpuscles used.

It was of interest to determine whether this change in the complement-
content of serum occurs even when the serum is removed from the clot, or
takes place only when it remains in contact with the clot. This was

1907. | Complement-content of Fresh Blood-serum. —- 381

investigated in a number of the normal serum experiments. A portion
of the first sample was set aside in the cold after centrifuging, and examined
some hours later, at the same time as a sample which had remained on the
clot during the same period. The results show that a rise does occur in
the serum, even when separated from the clot. The amount of this rise is,
however, very small, only a small fraction of the increase which has taken
place in the serum left on the clot. /g., in the example given above, the value
of the 20 minutes sample rose in five hours, when off the clot, from 0°3 c.c. only
to 0-2 c.c., as against the rise to 0°08 c.c. of the serum left on the clot during
that time. It is not easy to suggest an explanation of this phenomenon. We
might suppose that complement exists in serum or plasma partially in an
inactive or zymogen phase, which is slowly converted into the active body.
Or again, on the view that complement is derived from leucocytes, we may
suggest that even after prolonged centrifugalisation, fragments of white cells
remain suspended in the serum and gradually disintegrate. Such explana-
tions, however, have as yet no real experimental basis, and we know too little
of the nature and source of complement to make speculations profitable.

In the normal sera examined, then, hemolytic complement increases by
100 to 200 per cent. during the first few hours after the drawing of blood, the
duration of the increase extending in the case of the rabbit over a period of
five to seven hours, in the case of the guinea-pig over a period of two to
four hours. It is probable that this variation is not peculiar to the hemolytic
complements. Ainley Walker(1) found the bactericidal action of serum to
increase during the first few hours after the blood was shed, and attributed
this to an increase in the amount of complement. According to Macdonald (2),
whose results have been confirmed by Craw, the opsonic power of serum
undergoes a similar increase.

J. H. Craw has pointed out that all these bodies are relatively unstable,
while the hemolytic immune-body, which does not vary, is comparatively
stable; but whether this fact is of significance can only be determined when
further evidence has accumulated on the subject. The results obtained in
the experiments recorded here have no immediate bearing on Metchnikoff’s
view that alexine does not exist as such in circulating plasma. Sufficient
serum for examination does not separate until 15 to 20 minutes have elapsed
from the time of bleeding. In the serum. then obtained the amount of
complement, though much less than is found later, is still always considerable,
and there was nothing to indicate that at the moment of bleeding the blood
contained no active complement.

Example 2.—Dose of immune-body: 0: 025. Horse red idod:cawpuneladt
-4 cc. of 1-per-cent. suspension; two hours at 37°. Quantity of normal
VOL. LXXIX.—B. 2F

382 Mr. J. Henderson-Smith. Increase in the [Mar. 26,

rabbit serum necessary for total hemolysis was, of 1 hour sample, 0°6 c.c.
24 hours sample, 0°5 c.c.; 5 hours sample, 0°3 cc.; 7} hours sample,
0325; 9 hours sample, 0°35 c.c.; 12 hours sample, 0:4 c.c. Hence a rise to
double the amount in 5 hours. In the control tubes, 0°5 cc. of the
1 hour sample gave no lysis, but the same dose of the 5 hours sample gave a
trace.

Example 3.—Dose of immune-body, 0:04 e.c. Ox corpuscles: 4 c.c. 1-per-
cent. suspension; 2 hours at 37°. Quantity of normal guinea-pig serum
necessary for total hemolysis, of $ hour sample, 0°05 c.c.; of 1 hour sample,
0:025 cc. ; of 4 hours sample 0:03 c.c. ‘Viet

2. Immune Serum.

The immune sera examined were those of animals, mostly rabbits, injected
with blood-corpuscles, usually of the ox. In some cases the serum came
from rabbits, which had been injected with horse corpuscles, and in one case
from a goat injected with ox corpuscles. The results in all the cases were
concordant. Two factors in such sera require to be examined: the immune-
body and the complement.

The immune-body is easily tested, since it does not appreciably, if at all,
deteriorate spontaneously. The immunised animal was bled as before, and
samples of serum withdrawn in the usual way. Each sample, after centrifuging,
was at once heated 30 minutes at 56° to destroy the complement, and, when
the last was ready, all were examined together. The method was similar to
that described in the last section. With a constant quantity of corpuscles
and a constant quantity of serum from a normal animal as complement, the
minimum dose of each sample necessary to produce total lysis was
determined. This was found to be the same for all the samples; and in all
the experiments this proved to be the case. The immune-body, therefore, .
does not vary, but is present in its full amount from the first.

Example .4.—Dose of normal rabbit serum as complement, 0°15 cc.;
4 c.c. 1-per-cent. suspension ox corpuscles; 2 hours at 37° C. Immune
serum from rabbit injected with ox corpuscles. The dose required of this.
serum after heating was for the 1 hour, 2 hours, 4 hours, 6 hours, 8 hours,
and 12 hours samples the same, viz., 0°04 cc. The dose of the 10 hours

sample was 0:045 c.c., which is probably an experimental error.

'The investigation of the complement in immune serum is more difficult. |
We have no means of destroying immune-body, and leaving complement in
the serum, and Khrlich’s (3) method of absorption of the immune-body in
the cold is not suited to quantitative estimations of this kind, and has been

1907.] | Complement-content of Fresh Blood-serum. 383

shown by Mainwaring (4) to be open to serious objections. On the other
hand, we cannot simply ignore the immune-body contained in the serum.
By taking varying doses of the serum we should be taking not only varying
quantities of complement, but also varying quantities of immune-body ; and
Morgenroth and Sachs (5) have shown that the amount of complement
necessary for hemolysis depends in some measure on the amount of immune-
body present. Now, in the process of immunising, the animal responds by
a great increase of immune-body in its serum, but there is no corresponding
increase of complement. Of the serum of a highly immunised animal,
therefore, it is possible to find a dose which contains enough immune-body to
cause total laking of the chosen quantity of corpuscles, but: does not contain
enough complement. Complete hemolysis, therefore, does not occur with
that dose, unless we make good the lack of complement by adding some from
another source. By making use of this fact, a method was devised of
estimating the complement in immune serum. An immunised animal was
bled and samples of serum were withdrawn from the clot in the usual way,
each sample being tested at once after centrifuging. Such a dose as has been
just described, containing enough immune-body but not enough complement,
was taken, and the same dose was taken for all the samples. Since, as we
have just seen, the immune-body does not vary in the different samples, we
are thus taking always the same amount of immune-body. The deficiency
in complement was supplied by adding the serum of a normal rabbit, and the
quantity of this serum which .had to be added was carefully determined for
each sample. The greater the quantity of normal serum required, the less
complement must the dose of immune serum contain. The amount of
normal serum that would be required, if the dose of immune serum
contained no complement, is readily found by first heating the latter and so
destroying its contained complement. The difference between this amount
and the amount we had to add when the sample was unheated gives us
- a measure of the value of the complement contained in the unheated dose,
this value being expressed in terms of the normal serum. We can thus
determine the amount of complement contained in each sample, and so
ascertain the extent of the variation, if any.

Hxample 5.—Four cubic centimetres of 1-per-cent. suspension of ox
corpuscles; 2 hours at 37° C. Immune serum from rabbit, injected
with ox corpuscles; of this serum, when heated, 0°02 c.c. required. the
addition of 0°25 cc. normal rabbit serum (¢.¢e., complement) to produce total
hemolysis, and this quantity of the normal serum by itself produced no
lysis. !

2 2

384 Mr. J. Henderson-Smith. Increase in the [Mar. 26,

Cc. C.¢.

To 0°02 unheated immune serum, ¢ hr. after bleeding, must be added 0-225 normal serum.
99 99 1 99 99 0°2 9
5 9 2 9 9 0°225 9
99 99 32 99 99 0°25 9

” ” i 5 Wien , 0°25 ”
0°02 c.c. unheated serum by itself, no perceptible lysis.
Since 0:25 ¢.c. normal serum was required, when the immune serum had
been heated, there was contained in the dose of the unheated immune
serum—

Cel prec C.c.

Of the } hr. sample complement equivalent to 0°25—0:225 = 0°025 of the normal serum.
99 1 99 99 99 0:05 99 3)
99 2 99 9 99 0°025 9 99
» oF FP S is No appreciable amount.

9 5 2? 9 99 9 39

The amount of complement increased to double the amount by the end of
the first hour, but had decreased again after two hours, and by four hours had
become inappreciable.

Example 6—Immune serum from rabbit injected with ox corpuscles; of
this when heated 0°05 c.c. required 0°13 c.c. normal rabbit serum. To 0:05 of
the immune serum unheated—

Ge;
From the 4 hr. sample niust be added 0°1 normal serum.

9 1 99 P) 0°085 39
29 1$ ? 99 0°08 99
99 3 99 99 Ol 99
PP) 5 9 79 01 bP)
Cie,
Therefore the dose of the 4 hr. serum contained the equivalent of 0°03 normal serum.
” } 9 1 ” ” 0:045 ”
” ” 1s ” 0 0°05 ”
” ” 3 ” ” 0:03 ”
oD) ” 5 ” 9 003 ”

The complement increased by about 66 per cent. in 13 hours.

By this method we can demonstrate the occurrence of change in the
quantity of complement contained in the immune serum, and can determine
the time during which such change is occurring. It is true that we
supplement an “immune” complement.by a “normal” complement; and it,
is probable that partial complements do not exist in the same proportions in
the two sera. It is even possible that they are not identical, even when
derived from animals of the same species, although there is no evidence of
this, and such evidence as we have is against this view. Even if it were so,
however, the fact would not invalidate the method, in so far as it indicates.
the occurrence of change and the duration of that change. The amount of

1907.] Complement-content of Fresh Blood-serum. 385

change which it shows to occur is, however, more open to question. It is
assumed that the immune-body undergoes no alteration in the process of
heating, and this, probable as it may be, has not been proved. Further, the
existence of different proportions of partial complements in the two sera
would affect the quantitative accuracy of the results obtained. In any case,
great stress should not be laid on particular figures, since the experimental
error is quite appreciable, though remarkable regularity and accuracy are
obtainable with hemolytic sera. While we may rely, then, on the method
showing us whether any change occurs, and the duration of such change if
present, the amount of change indicated is probably only an approximate
measure of what really occurs.

In example No. 5 given above, the amount of complement had risen by
the end of one hour to double the amount first observed. As a rule, the
increase is rather less. In some of the experiments (10 were made with
immune sera) no increase at all was found, the complement diminishing
steadily from the first observation. Further, in all the experiments where
any rise took place it was complete in one and a-half to two hours after the
blood was drawn.

There is a difference, therefore, between normal and immune serum in this
respect. In normal serum we found the complement to increase by 100
to 200 per cent. and the duration of the increase to be from five to
seven hours. In immune serum the full amount of complement is often
present from the first, and where an increase does occur, it is complete in
a short time and is not so great as the increase in normal serum. By the
process of immunising, therefore, complement, or the source of complement,
has been altered in such a way that it is developed in the serum with
greater readiness than is the case with normal blood. The repeated
injections of red cells may have induced repeated new formations of
complement, with the result that the leucocytes or other cells have been
“ educated,’ in Metchnikoft’s sense, to secrete complement, or its precursor,
more quickly than where such stimuli have not been given. But here, again,
explanation becomes pure hypothesis.

On the suggestion of Professor Muir, an experiment was made to see
whether the change produced by the process of immunising affected all the
hemolytic complements of the animal immunised or only those specially
concerned in the laking of the red cells used for the immunisation. A rabbit
immunised against ox corpuscles was bled, and samples of serum withdrawn
in the usual way. Two series of experiments were then carried out. In
the first, ox corpuscles and the appropriate immune-body were used, and the
variation of the complement determined by the method for immune serum.

386 Increase in the Complement-content of Fresh Blood-serum.

The rise was complete in one and a-half hours, and amounted to under
70 per cent. In the second series, guinea-pig corpuscles were used along
with immune-body derived from a rabbit immunised to these cells. Here
the samples were treated as if they were samples of a normal serum, and the
minimal dose giving total hemolysis with constant amounts of immune-body
and corpuscles was determined for each. The rise of complement was not
complete after three hours, and amounted to more than 100 per cent., i.e., it
followed the rule of variation of a normal serum. The change produced
by immunising with ox corpuscles in this case, therefore, did not affect all
the complements laking guinea-pig corpuscles, but was confined to the ox-cell
complements. As only one such experiment was made, it is impossible to
say whether this is a general rule or not, or in what degree the complements,
which are able to take part in lysis of either ox or guinea-pig corpuscles,
are affected.
Summary.

1. The serum first separating from shed blood may differ in composition
from that separating later.

2. With normal blood of rabbit or guinea-pig, the amount of hemolytic
complement contained in the first serum to separate is comparatively small,
and progressively increases in serum separating later for a period of 5 to 7
hours in the case of rabbit, 3 to 4 hours in the case of guinea-pig, to reach
a maximum 2 to 3 times as large as the amount first observed.

3. Even after removal from the clot, the amount of complement may
increase in serum which separated soon after the blood was drawn, but this
increase is slight.

4, This increased activity of serum is not peculiar to the hemolytic
complements.

5. With the blood of immunised animals, immune-body does not increase
in this manner.

6. With the blood of immunised animals, the hemolytic complements may
increase in amount, but this increase is not always present, and when it does
occur is less, and reaches its maximum more quickly than is the case with
normal blood.

7. The alteration thus produced by the process of immunising probably
affects only the hemolytic complements related to the immunising agent, and
not other hemolytic complements which may be present in the serum.

A portion of this work was done at Queensbury Lodge, Aldenham, and
I desire to express my thanks to the Directors of the Lister Institute of
Preventive Medicine, and also to Dr. Dean for the facilities afforded me

The Correlation of the Ovarian and Uterine Functions. 387 |

there. I am glad also to have this opportunity of thanking Professor Ritchie,
of Oxford, for the interest he has taken in my work and for suggestions made
in the course of it.

REFERENCES.

1. Ainley Walker, “On some Factors in es Action,” ‘Journal of Hygiene,
vol. 3, p. 52, Jan., 1903.

2. Macdonald, G. G., “On Immunity in Bena Infections,” in ‘Studies in
Pathology,’ etic Bulloch, Aberdeen, 1906.

3. Ehrlich and Morgenroth, ‘ Berlin Klin. Wochenschr., 1899, No. 1.

4. Mainwaring, ‘Journal of Infectious Diseases,’ vol. 2, pp. 446 and 460.

5. Morgenroth and Sachs, ‘ Berlin Klin. Wochenschr.,’ 1902, No. 5.

The Correlation of the Ovarian and Uterine Functions.
By E. 8. CaRMICcHAEL, M.B., F.R.C.S.E., and F. H. A. MarsHatt, M.A., D.Sc.

(Communicated by Professor E. A. Schafer, F.R.S. Received April 10,—
Read May 30, 1907.)

(From the Physiological Laboratory, University of Edinburgh.)

A considerable body of evidence has been adduced in support of the
hypothesis that the uterus is dependent upon ovarian influence for the
maintenance of its structure and the discharge of its functions, and that the
removal of this influence at any time during reproductive life causes the
uterus to atrophy, besides producing a profound effect upon the entire
metabolism of the organism. Some writers, on the other hand (eg.,
Blair Bell), contend that it is the uterus which is of prime importance,
and observations have recently been cited pointing to the conclusion that
this organ exercises a control over the growth and activity of the ovaries.
Others, again (eg., Bond), adopt the view that the ovaries and uterus are to
a very large extent inter-dependent upon each other.

The clinical evidence brought forward by surgeons after removal of the.
uterus and ovaries in the human subject has supported to a great extent the
experimental evidence of the existence of such an inter-relation. The modern
tendency to conserve these organs as far as possible during surgical inter-
ference has been brought about largely by the belief in the dependence
of the one organ upon the other. Important work bearing upon this subject
has been published recently by Zweifel (1899), Mandl and Birger (1904),
Bond (1906), and many other surgeons.

It is the purpose of this paper to record a series of experiments dealing |

388 Mr. E. S. Carmichael and Dr. F. H. A. Marshall. [Apr. 10,

with these questions, and to state the conclusions which we have reached
regarding them. |
Influence of the Ovary upon the Uterus.

A brief véswmé of the experimental evidence adduced upon the existence
and nature of the ovarian influence has been recently given in a paper
by Marshall and Jolly (1907), who cite observations showing that the uterus
undergoes undoubted degenerative changes after the removal of the ovaries.
Further evidence, which is in accord with this conclusion, has been inde-
pendently reached by Carmichael (1907). For accounts of the literature
reference may be made to these papers.

Since, however, certain of the conclusions arrived at are still matters
of controversy,* it has been thought worth while to. place on record the
results of further experiments upon rabbits, dealing, firstly, with the effects
of ovariotomy in full-grown adults at different periods after the operation,
and secondly, with the effects of the same operation in young animals.

It has been suggested that the changes occurring in the uterus may be due
to an interference with the blood supply to that organ, in the removal of the
ovary. The uterus in the rabbit, as in the human subject, has an independent
vessel of supply derived from one of the pelvic vessels, and it is inconceivable
that any operation undertaken with care for the removal of the ovaries,
situated as they are high up in the abdominal cavity, could interfere with
this vessel. Care was also taken in our experiments to avoid injuring the
anastomotic branch of the ovarian artery, which passes along the Fallopian
tube to join the uterine.

For this purpose in several of the experiments a lateral incision was made
over the ovary on each side, so as to obtain better access and interfere with
the mesovarium as little as possible by traction, a condition which is
unavoidable in removal of the ovaries by a mesial incision. Thus all dangers
of fallacy from vascular interference were avoided.

A mesial incision is often unsatisfactory for the removal of ovaries, in
that the upper pole of the ovary may be left behind in dividing its superior
attachment, considerable traction being required to expose the organs freely.

In the following experiments fully grown rabbits were used :—

Expervment 1—The ovaries were removed by a mesial incision, and the
animal killed after an interval of three and a-half months. Sections showed —
that the uterine stroma had undergone considerable fibrosis and was reduced
in amount. The glands had almost entirely disappeared. The superficial
epithelium was, however, fairly normal, but degenerated in places, some

* See the latter part of the present paper, where recent theories upon the nature and
importance of the uterine influence are discussed.

1907.] Correlation of the Ovarian and Uterine Functions. 389

of the cells being vacuolated. The muscular wall did not show much
degeneration, but was somewhat thinner than usual. Superficially the
uterus, whose size was carefully noted at the time of the operation, was
perceptibly smaller.

Experiment 2.—The ovaries were removed by a mesial incision, and the
animal killed three and a-half months subsequently, as in the preceding
experiment. The glands and epithelium did not show the same degree of
degeneration as in the previous case. Some fibrosis was, however, clearly
discernable throughout the stroma. The muscles appeared to be not well
developed, but otherwise were fairly normal.

Experiment 3.—The ovaries were removed from a pregnant rabbit by two
lateral incisions. Two days afterwards the rabbit aborted. After five and
a-half months the rabbit was killed. The uterus appeared very pale and
atrophic looking. Mammary tissue was almost non-existent. Adipose
tissue was super-abundant. Sections through the uterus showed that the
mucosa was very thin and also fibrotic, the nuclei of the stroma being few
and far between, excepting in the immediate neighbourhood of the
epithelium, just below which they were much more numerous. The glands
were represented by mere traces. The epithelial cells were somewhat
degenerated, not taking on the stain so well as normally. There were very
few blood-vessels.

Haperiment 4.—The ovaries of a rabbit were extirpated by means of two
lateral incisions a week after parturition. The animal was kept alive for
five and a-half months, during which time it received 22 injections of
commercial ovarian extract, made to determine whether this would have
the effect of arresting the uterine degeneration. The extract was injected
intra-peritoneally, one injection on an average being made every week.
It was found, however, that whereas sometimes the injection appeared to
result on the following day in a congestion of the mucous membrane of the
external generative organs, the atrophy of the uterus was in no way
prevented. Sections showed a very thin fibrotic mucosa, and a thin
muscular coat. As in the preceding case, the glands had almost disappeared,
and many of the epithelial cells were vacuolated and obviously degenerate.
In the cervix the changes were not so pronounced, the epithelial cells being
nearly normal in appearance.

Expervment 5.—The ovaries were removed by two lateral incisions. The
rabbit was killed six and a-half months subsequently. Very pronounced
changes had taken place. The uterine stroma was extremely fibrotic, and
contained no glands. The epithelium was much attenuated, the nuclei
having failed to take up the stain to the usual extent. A few very small

390 Mr. E. S. Carmichael and Dr. F. H. A. Marshall. [Apr. 10,

capillaries, however, could be still seen in the stroma. The muscle fibres
were much broken up. The cervix had not undergone the same amount of
degeneration, but some of the epithelial cells appeared vacuolated.

In the following two experiments very young immature rabbits were
used :—

Experiment 1.—The ovaries were removed on May 29. The rabbit
weighed 660 grammes. On August 8 it weighed 1640 grammes. On
March 20 the rabbit was killed after first being weighed. The weight was
3450 grammes, the animal being fully grown and in perfect health. The
uterus was found to be absolutely infantile, being no bigger than a rat’s.
Sections showed that the muscular wall was almost totally undeveloped.
The mucosa was somewhat thicker, but fibrous throughout. The lining
epithelium was, however, present.

Experiment 2.—The ovaries were removed from a very small rabbit on
June 5. The animal weighed 740 grammes. On August 8 it weighed
1450 grammes. On March 20 it was killed after being first weighed. The
weight at that time was 2080 grammes. The rabbit was quite fully grown
and very fat. The uterus, as in the preceding experiment, was very small,
being not so large as an adult uterus after the removal of the ovaries some
months previously, as shown by our first series of observations.

These experiments show that the removal of the ovaries at an early
period of life, while arresting the development of the other generative
organs, has no effect on the growth and general health. The weights, as
given above, compare favourably with those of unoperated rabbits and with
those of animals in which the uterus had been removed (see below).

Influence of the Uterus upon the Ovaries.

Although the bulk of evidence brought forward clinically seems to show
that the uterine functions fall into abeyance after removal of the ovaries,
a considerable number of cases have been reported in which menstruation
has persisted after odphorectomy. Pozzi (1905) has recently recorded.
several such cases, but the uterus in the majority contained a uterine fibroid
or other pathological condition.

The explanation of such cases has always been a difficult one, but it has
probably led to the ideas now held by some surgeons that the uterus is
capable of performing its functions independently of the ovaries, and even
that the functions of the ovaries themselves depend largely on the presence
of the uterus and its secretions.

Zweifel and Abel, in a communication to the German Gynecological
Congress (1899) in tracing the after-histories of cases of hysterectomy, found

1907.] Correlation of the Ovarian and Uterine Functions. 391

that when the body of the uterus was removed, entire atrophy of. the
ovaries always followed, so that menopausal symptoms set in similar to
those occurring after odphorectomy. In three cases, however, in which a
portion of the uterine mucous membrane was conserved, . menstruation
persisted and menopausal symptoms were absent.

Doran (1905), after following up the after-history of 60 cases of sub- total
hysterectomy, is inclined to support Abel and Zweifel,in that the uterus
should be removed above the cervix, in order to prevent sudden artificial
menopause, while he also cites two cases where menstruation persisted
after removal of the body of the uterus and both ovaries, the cervix being
left behind. 7 7

Mandl and Biirger (1904) in an exhaustive monograph on the slathiots of
hysterectomy with and without the ovaries hold that in those cases in which
the ovaries are left behind there is a gradual cessation of ovarian function due
to atrophy or degeneration of the ovaries.

Holzbach (1906), however, states that, as a rule, the ovaries Htdlo not
degenerate after hysterectomy, and that where such degeneration does occur
it is probably due to the interference with the nervous connections consequent
upon the operation of removal.

Blair Bell (1906) holds that the theory of the internal secretion of the
ovary is inconclusive, and that the clinical facts go to show that menstruation
is due to a uterine secretion, and the menopause due to its absence, while he
supposes ovulation itself to depend on the circulation of this secretion, which
he calls “uterin.” He mentions that there have been many cases in the
_ Royal Infirmary at Liverpool in which ordinary menstruation continued after
the removal of the ovaries, and makes further statements pointing to the
conclusion that the ovaries invariably atrophy after the removal of the uterus.

Bond (1906), on the other hand, believes that the ovarian secretion 18.
influenced by a saline secretion from the endometrium, the two, however,
acting antagonistically to one another, so that prevention of the uterine
secretion by hysterectomy favours hypertrophy of the ovary. Bond’s view,
therefore, is diametrically opposite to that of Blair Bell.

Bond records two experiments on the results of hysterectomy. In one of
these the entire uterus of a rabbit was removed and the animal killed after
five months. Both ovaries were found to be normal. In the other experiment
the left uterine cornu only was extirpated and the rabbit killed after five
months. The ovaries in this case also were in no way atrophied. Bond
expresses the belief that the prevention by previous hysterectomy of the
secretion of the saline fluid by the endometrium of the ancestrous uterus,
favours the overgrowth of luteal tissue in the ovary.

392 Mr. E.8. Carmichael and Dr. F. H. A. Marshall. [Apr. 10,

Stress has also been laid upon the well-known fact that whereas the
corpora lutea of the ovary continue to undergo development for a considerable
period of time if pregnancy supervene after ovulation, this hypertrophy soon
ceases in the absence of pregnancy. Bond records an experiment on a rabbit
in which the ovary after being grafted to an abnormal position contained a
somewhat aberrant “corpus luteum of pregnancy” in association with a
gravid uterus. Such observations are regarded by him as affording evidence
of an internal uterine secretion (differing from that elaborated by the
‘ancestrous uterus), acting on the ovaries and so inducing a growth of luteal
tissue. It must be remembered, however, that the occurrence of pregnancy
‘exercises a profound effect over the whole maternal organism, and not merely
npon the condition of the ovaries.

Certain other authors have adopted views which imply a dependence of the
ovaries upon some function of the uterus. Thus, Loewenthal (1884) appears
to have held that ovulation occurs as a result of a stimulus in some way set
up by the uterine changes of the procestrum. Moreover, Heape (1900) has
expressed the belief that cestrus (which depends normally upon the presence
of the ovaries) “is possible only after the active changes due to procestrum
have taken place in the uterus,” but this statement clearly applies to normal
animals.

Excepting for the two experiments of Bond, which have just been referred
to, no experimental work appears to have been published upon the effects of
hysterectomy on the ovaries. The following experiments, therefore, notwith-
standing the fact that the results reached were negative, are not without
amportance.

Expervment 1.—On April 8 the uterus was removed entire from a very
young rabbit which weighed 420 grammes. On August 8 the weight was
1640 erammes. The rabbit was killed on November 11 after being weighed.
The weight was then 2060 grammes, the animal being apparently fully
grown. The ovaries were well developed, having increased greatly in size
since the operation. On histological examination they were found to be
normal, containing numerous growing Graafian follicles, and showing no signs
whatever of degeneration.

Hzperiment 2.—On April 8, the uterus of a very young rabbit was
removed, excepting the cervix. Weight, 390 grammes. On August 8 the
weight was 1640 grammes. On November 11 the rabbit was killed.
Weight, 1940 grammes. The ovaries were normal in every way.

EHxperument 3.—On May 2 the uterus was removed entire from a young
rabbit. Weight, 650 grammes. On August 8 the weight was 1660 grammes.
The rabbit was put with the buck at intervals and noticed to copulate. On

1907. | Correlation of the Ovarian and Uterine Functions. 393:

January 20 it was killed when quite full grown and in perfect condition.
Weight, 3020 grammes. The ovaries were extremely well developed and
contained numerous discharged follicles.

Experiments 4 and 5.-In each experiment the uterus was removed from
a very young rabbit, excepting the cervix, and the rabbit killed after several.
months (in the one case after eight months and in the other after 10: months).
(Estrus was noticed in both cases after the animals had reached maturity.
The ovaries in both cases were normal, those of one animal containing large
follicles, and those of the other (which had been with the buck) recently
ruptured follicles.

Experiment 6.—The uterus was removed entire from a very young rabbit
on May 17. Weight, 470 grammes. In August the animal was observed to
be ill and on August 28 was killed. The ovaries had undergone development,
since the operation and were normal in histological appearance. ,

Experiment 7.—The uterus and Fallopian tubes were completely removed
from a young rabbit on December 9. The rabbit was killed on April 3, when:
the ovaries were found to be normal and to contain many follicles.

In four experiments on rats the uterus was removed entire in the month
of July. Two of the rats were killed in January, when the ovaries showed
no indications of degeneration. The other two were killed in April, the.
ovaries being large in size and containing many protruding follicles.

On the other hand, marked uterine degeneration has been recorded in rats.
after the removal of the ovaries for shorter intervals of time (Marshall and
Jolly, 1907).

Further, in the case of a ferret in which the uterus was removed, the
histological appearance of the ovaries was normal after three months, when
the animal unfortunately died. The interval which had elapsed, however,
was probably too short to admit of the deduction of definite conclusions.

It may be concluded as a result of these experiments that the growth and
development of the ovaries are in no way dependent upon the presence of
the uterus. Such a conclusion is no doubt in opposition to some of the
clinical evidence, but it is one which on phylogenetic grounds might be
expected, since the uterus is an organ which came into existence compara-
tively recently in the course of vertebrate evolution, whereas the ovary is
common to all Metazoa.

It has been shown, further, that cestrus and coitus may occur after the.
complete removal of the uterus. Whether under such a condition the
other generative organs undergo any of the changes which normally
characterise the procestrum was not determined, since such changes are in.
any case difficult to detect in rabbits.

394 The Correlation of the Ovarian and Uterine Functions.

It was likewise impossible to determine definitely whether there was any
increase of ovarian activity consequent upon hysterectomy (such as one
would infer if Bond’s theory of an antagonistic uterine secretion were correct),
but we failed to discover any clear indication of its existence.

Summary of Conclusions.

The results obtained by these experiments lead us to summarise our
conclusions as follows :—

1. The removal of the ovaries in young animals (rodents) prevents the
development of the uterus and Fallopian tubes. These remain in an infantile
condition. The subsequent growth and general nutrition of the animals seem
to be unaffected.

2. The removal of the ovaries in adult animals (rodents) leads to fibrous
degeneration of the uterus and Fallopian tubes (most marked in the mucous
membrane). The animals’ subsequent health and nutrition remain good. |

These observations, for the most part, support the evidence obtained
clinically in the human subject after surgical operation.

3. The removal of the uterus in a young animal has no influence in
preventing the further development of the ovaries. These are capable of
ovulating and forming corpora lutea after adult life has been reached.

4, The removal of the uterus in an adult animal does not give rise to any
degenerative change in the ovaries, if the vascular connections of the latter
remain intact.

These latter observations do not support the contentions of those surgeons
who advocate sub-total hysterectomy, believing that the functional activity
of the ovary is in some way dependent on the presence of the uterus.

The expenses of this investigation were defrayed by grants from the
Carnegie Trust for the Universities of Scotland.

LITERATURE.

Blair Bell, ‘ Liverpool Medico-Chirurgical Journal,’ July, 1906.

Bond, ‘ Brit. Med. Journ.,’ July 21, 1906.

Carmichael, ‘ Journ. Obstet. and Gynec.,’ March, 1907.

Doran, ‘ Lancet,’ November 4, 1905.

Heape, ‘Quart. Journ. Micr. Sci.,’ vol. 44, November, 1990.

Holzbach, ‘ Arch. f. Gynak.,’ vol. 80, 1906.

Loewenthal, ‘ Arch. f. Gynak.,’ vol. 24, 1884. .

Mandl and Biirger, ‘Die Biologische Bedeutung der Eiersticke nach Entfernung der
Gebarmutter,’ Leipzig, 1904.

Marshall and Jolly, ‘ Roy. Soc. Edin. Trans.,’ vol. 45, 1907, and ‘ Edinburgh Med. Journ.,’
March, 1907.

Pozzi, ‘ Annales de Gynéc. et d’Obstet.,’ December, 1905.

Zweifel, ‘ Zentralb. f. Gynak.,’ No. 21, 1899.

Magoo ”

The Ascent of Water in Trees : (Second Paper).

By A. J. Ewart, D.Sc. Lond., Professor of Botany, University of
Melbourne.

(Communicated by Francis Darwin, For. Sec. R.S. Received April 18,—
Read May 9, 1907.)

(Abstract.)

' The physical conditions in the vessels interpose no obstacle to the exertion

of a pumping action on their contents controlled by the medullary ray cells. —

There are many theoretical ways in which surface tension action could be
brought into play in this manner, but no practical proof of the existence of
any such action in the wood vessels has as yet been obtained. Similarly,
the bleeding of roots and stems affords no absolute proof of the existence of
a vital pumping action in them, since all the observed cases can be explained
as the result of osmotic action coupled with a reabsorption of the osmotic
materials from the ascending stream, and their surface adsorption by the walls
of the vessels. Instances are given in which an apparent pumping action
proved to be of this character.

Nevertheless, the experiments detailed or quoted in the above paper all

tend to show that the continuous ascent of water is only possible in living

wood, and that the power of conduction is rapidly lost on death, without any
mechanical blocking of the vessels being necessarily responsible for the
change. Hence we are forced to conclude that the living cells in tall trees
continually restore the conditions for the ascent of water wherever these are
affected by the excessive emptying of the vessels, and decrease the resistance
to flow, as far as possible, by maintaining continuous water columns in parts
at least of the wood. So long as these are present ab initio,a pumping
action only becomes necessary in trees over 20 to 50 metres in height, but
suspended columns cannot be maintained for any length of time in the vessels
of tall trees without the aid of the living cells of the wood.

The energy required to pump water upwards in the tallest trees represents
only a small fraction of that produced by the daily photosynthetic assimi-
lation, and it is the feeble character and diffuseness of the pumping action
which renders it so difficult to demonstrate practically.

‘Contrary to Strasburger’s statement, no vessels appear to run as open
channels from end to end of any tree, the longest vessel observed being
564 cm. as measured by the length of stem in which it ran (Wistaria). In
this plant, however, and others also, owing to the irregular course followed by

396 The Ascent of Water in Trees.

the vessels, these are always longer, and sometimes considerably longer, than
the piece of stem in which they occur.

Experiments on the suction and exudation of trees at different levels, and
upon the influence of the entry of air and water under pressure, showed that
no continuous suspended water columns, or high internal tensions, existed in
the conducting elements of the trees experimented on (Maple and Poplar)
during active transpiration, or, indeed, at any period of the year.

The same was shown by direct measurements of the pressure in intact
vessels of Wistaria during active transpiration. This fact, coupled with the
high total resistance to flow, shows that this resistance is overcome locally
from point to point, and not by any enormous tension from above or pressure
from below, neither of which exists, nor could be maintained to a sufficient
extent to carry on the elevation of water in a tall tree. A high tension from
above leads to rapid blocking with air; a high pressure from below leads to
great loss by lateral exudation from the vessels.

The apparent differences of osmotic pressure previously observed between
leaves at the base and apex of a tall tree are not evidence of the existence of
any greater osmotic suction at the higher level, but are the result of the
different ages and sizes of the cells, and of the different conditions to which
they have been exposed. Equally great differences may exist between leaf-
cells at the same level.

The surface adsorption of dissolved solids in the vessels plays a very
important part in their function as translocatory channels, causing a delay
in the ascent of dissolved solids, such as sugar and salts, and an accumulation
of them along the outer walls of the vessels. The latter facilitates their
outward diffusion, but at the same time renders the transference of
small quantities of material between widely removed organs difficult or
impossible.

The tallest trees in Australia do not appreciably exceed 300 feet in height,
so that the values previously given for the maximal total resistance to the
upward flow of sap in actively transpiring trees must be reduced to between
30 and 50 atmospheres.

In a Maple tree whose younger wood had been killed by formalin the
transpiration current turned to the older, partially blocked, but still living
wood, and none of Strasburger’s experiments show definitely that efficient.
conduction is possible in the dead wood of tall trees. The ascent of water
is, therefore, a vital problem, in so far as it depends upon conditions which
hitherto can only be maintained in living wood.

397 -

The Role of the Various Elements in the Development and
| Regeneration of Bone.

By Sir WILLIAM MaceweEn, FBS.

(Received and Read January 24, 1907.)
(A bstract.)

The present inquiry has been undertaken with the view of obtaining
data, chiefly: by direct experiment, as to the rdle which the various elements
play in the development and reproduction of bone. This communication
deals with a part of the subject, under two heads :—A, the potentiality of
the periosteum as a factor in the production of bone, and B, the regeneration
of bone from proliferation of osseous tissue. The following is a brief réswmé
of the paper :—

A. POTENTIALITY OF PERIOSTEUM AS A FACTOR IN REPRODUCTION OF BONE.

(1) To test this, a complete cylinder, constituting a portion of the shaft of
a long bone, was removed while the periosteum was preserved intact. This
showed, ten weeks afterwards, an osseous defect, constituting a gap in the
continuity of the shaft.

(2) Periosteum free from osseous plaques was removed and transplanted.
This was not followed by reproduction of bone, but by absorption of the
periosteum.

(3) Duhamel’s silver ring experiments are discussed and the correctness of
the deductions drawn therefrom are questioned.

In order to test whether the bone cells or the periosteum produces the
bone which covers the silver rings, three experiments, each differing from the
other, were performed, in which silver rings were placed on bone deprived of
its periosteum, with the result that in each case the rings became covered
with bone.

B. THE REGENERATION OF BONE, FROM PROLIFERATION OF OSSEOUS TISSUE.

The periosteum is shown to be a limiting membrane controlling the
osteoblasts, as illustrated in fractures, when the periosteum is intact and
when it is torn. The production of callus is not inherently greater in the
lower animals than in man—the amount of callus in both depends on the
limitation of the periosteum and the amount of movement. (4) Direct
experiment, showing that a long bone deprived of its periosteum continues
to grow, (5) so also do the flat bones of the skull. (6) Bone may be made

VOL, LXXIX.—B, 26

398 Development and Regeneration of Bone.

to grow in the midst of lacerated muscles by the mechanical distribution of
osteoblasts. Observation. Riders Bone. Suggestion as to the production of
myositis ossificans.

(7) Can shavings of nude bone grow on being placed between muscles in
a gap in the continuity of the shaft? Experiment and result seven weeks
after show that not only do they grow, but that they also proliferate to a
very marked extent. : |

Is there any direct evidence to show that transplanted living bone actually
grows and proliferates instead of forming, like blood clot, a passive framework
for the granulation tissue to penetrate, which framework will then become
absorbed? There is, as may be illustrated (8) in an instance of bone growm
in sponge filled with granulation tissue.

(9) To test the osteogenic power of bone cells constituting the shaft of
a long bone, they were grown inside of a glass tube. Result. |

(10) Intra-human transplantation of bone. Result 28 years after. Data
are obtained from this experiment as to the growth of the humerus.
from the proximal and distal epiphyseal cartilages respectively. Also as to
interstitial osseous increase—evidence of the increase in length of the
diaphysis from the epiphyseal cartilage toward which the nutrient,
vessel runs.

psa at
=

;

: ;

399

An Experimental Enquiry into the Nature of the Substance
im Serum which influences Phagocytosis.

(Second Communication. )
By GrorGEe Desay, M.A., C.M., M.B., Chief Bacteriologist, Lister Institute. .

(Communicated by Dr. C. J. Martin, F.R.S. Received April 23,—Read
May 30, 1907.)

In a previous comiunication it was shown that in certain immune sera,
é.g.,1n anti-staphylococcus, in anti-dysentery, and in anti-typhoid serum, there
are present thermostable substances which, by their action, prepare the
homologous organisms for phagocytosis. The thermostability in the case of
these immune sera could be demonstrated by the use of Wright and Douglas’
method, whereas in the case of normal serum another method was, as a rule,
necessary.. In the case of the serum of the horse, however, it was pointed
out that even by Wright and Douglas’ method the increase of phagocytosis,
observed by using normal heated serum, was, as compared with salt solution,
generally considerable.

In view of certain experimental data and having regard to Ehrlich’s theory,
the conclusion was arrived at that the thermostable substances of normal and
immune sera were probably identical.

It was further shown that, when submitted toa temperature of 60° C., both
immune and normal serum suffered a rapid and sudden diminution in opsonic
power. This change occurs in the first few minutes of heating, after which
further loss appears to take place very slowly.

The results obtained suggested that the sensitising action of the serum
might be due to the combined action of two substances, the one thermostable,
the other thermolabile ; the one of the nature of the “ substance sensibilisatrice,”
the other of the nature of an alexin.

The present paper is an account of certain experiments carried out with the
view of elucidating this point:*

* In the course of writing this paper, a communication by Neufeld and Hiine has reached
me, in which, in a criticism of my interpretation of these curves, they appear to have
overlooked the fact that I had indicated the possibility that the sudden fall may be due to
a destruction of complement. As at the time of the publication of my previous com-
munication I had no experimental data to support that view, I preferred to leave the
matter for further investigation. What I said was:

“Whether free complement may take part in the preparation of the microbe is difficult

to determine. From the experiments detailed in this paper it is certain that it is not a
necessary participant in this action. At the same time it is not improbable that the

2 @ 2

400 Mr. G. Dean. Enquiry into the Nature of the [Apr. 23,

THE INFLUENCE OF THE STRENGTH OF THE EMULSION UPON THE NUMBER
OF ORGANISMS TAKEN UP BY THE LEUCOCYTES.

In Table I are shown the results obtained in two experiments carried out
in the usual way by mixing one volume of serum, one volume of washed
blood corpuscles and one volume of bacterial emulsion, putting the pipettes
containing the mixtures in the incubator at 35°5 C. for 15 minutes,
after which the preparations for microscopical examination were made.
The number of phagocytosed bacteria were subsequently counted, and the
average number per leucocyte taken as the phagocytic index. As a rule, the
number of organisms in 50 to 80 leucocytes were counted, and in many
of the experiments the mean of two or three such counts by independent
observers was taken. The emulsion of Staphylococcus pyogenes awreus was
employed in different concentrations, full strength, half strength, quarter
strength, one-eighth strength, and one-sixteenth strength.

Table I.
Average number of cocci phagocytosed in the
Emulsion of cocci case of —
concentration.
Thick emulsion. Thin emulsion.
1 86-0 | 16°5
4 §2°5 | 9°6
4 17 °4 | 6 °4
1 7°9 | 2°9
as 3 °0 1°3

The number of organisms taken up is, within the error of experiment,
directly proportional to the concentration of the bacterial emulsion
employed.

INFLUENCE OF DILUTION OF THE SERUM UPON THE NUMBER OF ORGANISMS
TAKEN UP BY THE LEUCOCYTES.

When in similar experiments different concentrations of the same normal
serum are employed, no direct proportionality can be demonstrated to exist
between the serum concentration and the number of microbes phagocytosed.
For example, the number of ingested organisms may be as great, or even greater,
with the half, quarter, or even the one-eighth concentration, as with the full
immune body, when aided by complement, may act more powerfully, and that the sudden

fall in the “ opsonic” power of both normal and immune serum, on heating, is due to the
destruction of the complement. I may revert to this subject on another occasion.”

1907.] Substance in Serum which influences Phagocytosis. ‘401

strength serum. Thisappears ina table in Wright and Douglas’ (1903) paper,
but no remark is made on the significance of the phenomenon. —

The normal serum of several species of animal tested behaved in a Tous
manner in respect to dilution, as appears in Table IT.

Table I1.—Effect of Dilution of Normal Serum of Different Species. The
Numbers indicate the Average Number of Micro-organisms taken up
‘per Leucocyte. |

| “49
Human Horse’s Pig’s panel oak d | Human :
Deut st ee naeti e 2. «ss serum and serum and | serum and aa serum and |
leucocytes. | leucocytes. | leucocytes. lesanezton leucocytes. »
‘Ar: . Staphylo- Staphylo- Staphylo- Staphylo- Tubercle
PECTO-OTEAMISM «..... { coccus. coccus. coccus. coccus. bacillus.
Concentration of serum—

1 9°7 23 °7 12 °7 16 *4 8 °3

3 9°6 28 °5 15 °8 170 12 °2

a 10-0 14 °6 14.°9 12 °6 9°5

2 8 2 13 ‘0 12°3 9-2 5°3

a5 8°5 11°9 5 °2 3 °0 4°8

ae 6 *4 6°8 6:7 2°4 | 2:5

ee 2°3 = = = | 1°6

In addition to the experiments with staphylococcus, an experiment with
the tubercle bacillus was also made, which shows that this relation is not
limited to one micro-organism. Five dilution experiments on normal human
serum, the diluent being normal salt solution, and the organism being the
Staphylococcus pyogenes aureus, have been made.

The mean figures of these experiments for the pure serum, and the successive
dilutions down to 1 in 32, if expressed in terms of unity are,

# YEG) 4:02; O71; 0°53, 0:35,
and the corresponding concentrations of serum,
I, O05, 0:25, 0125, 0:0625, 0:03125.

I am indebted to Dr. J. C. G. Ledingham for pointing out to me that after
the second number there exists an approximate constant ratio between the
second series and the squares of the first, as shown below :

(1:02)? = 42 (0°25),
(0°71)? = 4:03 (0°125),
(0:53)? = 44 (0:0625),
(0°35)? = 3:9 (003125),

and that if the points (0:25, 1:02), (0°125, 0°71), (0:0625, 0°53), (003125, 0'35)

402 Mr. G. Dean. Enquiry into the Nature of the [Apr. 23,

are plotted, they are found to satisfy almost exactly the parabola whose
equation is y? = Kz, where K = 41 (mean of 4:1, 4:03, 4-4, 3°9). The points
(05, 1:16) and (1:1) obviously do not he on the parabola.

The curve shown in the figure has been drawn from the mean of the figures

for each dilution.

JET AS eee
ZL Ss
| So
ee a ee.

ee De.
HERRERA

orale
fT eE ee
ZERRRERSEUES
fief Tec | [i
pp

' Concentration 4
of Serum. 32 16 s ze

<. Phagocytic index.
ae Pek sts ;

Ni-

~ Tt will be seen that with the half concentration of serum there is a slight
rise in the curve. This occurs so consistently that it can hardly be due to
experimental error. From the quarter concentration onwards a fall is seen
in the curve.

It is interesting to note that this square-root proportionality is analogous
to what was found by Schutz (1885), Volhard (1901) and others working
with albumin-pepsin and fat steapsine, etc. If these data are sufficiently
accurate to be relied upon, it means that the phagocytosis is proportional to
the square root of the dilution of serum for these concentrations, but that
another factor is probably at work to account for the upper part of the
curve. |

The importance of this question of dilution in relation to the estimation of
the amount of sensitising substances in the serum in normal, pathological,
and immune conditions is obvious.

1907.] Substance in Serum which influences Phagocytosis. 408

A striking instance may be given here. One specimen of an anti-
staphylococcic horse serum, when diluted 32 times with saline, was found to
have lost only 25 per cent. of its sensitising power, whereas the corresponding
dilution of a normal serum of the same age had lost 90 per cent.

This subject in its various aspects will be dealt with more fully upon
a future occasion.*f .

THE INFLUENCE OF THE ADDITION OF FRESH SERUM UPON THE OPSONIC
POWER OF HEATED IMMUNE SERUM.

As was pointed out in my previous communication, thermostable immune
substance is capable of sensitising the microbes for phagocytosis in the
absence of free complement. Metchnikoff’s view, that in such cases the
complement may be suppled by the leucocytes, is a possible interpretation,
but will not be dealt with in the present paper. I propose to discuss here
the question as to whether the heated immune substance in the presence of

* Some misunderstanding (Wright and Reid, ‘ Roy. Soc. Proc.,’ 1906) has arisen in
regard to my remarks (‘ Roy. Soc. Proc.,’ vol. 76, p. 506) on the limitations of Wright and
Douglas’ technique. I would again point out that their method may fail to demonstrate
sensitising substances where these are present in considerable amount, and may show no
difference between two fluids, notwithstanding that one contains a large amount of
sensitising substance, the other only a small amount, or none.

To illustrate this, 1 may quote some experiments which were carried out with the view of
determining the limits of sensitising action in an immune staphylococcic serum. Dilutions
of 1 in 100 of this immune serum, and 1 in 100 normal serum, when tested by the ordinary
technique of Wright and Douglas, showed no difference in their action, which -was
practically ned. On the other hand, if one mixed a volume of these dilutions with an
equal volume of a coccal emulsion, so that the ultimate dilution was 1 in 200, and placed
the mixtures at 35°°5C. for one hour, and, after re-emulsifying both, carried out the
ordinary technique, a very marked difference could be determined. The results obtained
with the same sera, at an interval of some months, by three different observers, who
endeavoured to adhere to standard conditions as to emulsions, etc., were as follows :—

Phagocytic index of

Immune
Normal serum. staphylococcic
serum.
oe 24, 21°0
Pe ee 3°3 18 °7
(C) Geen ee 1°9 23 °7

+ After I had made a communication on the effect of dilution at the Pathological Society
of Great Britain and Ireland, a paper by Simon, Lamar, and Bispham, which deals with
the importance of this subject of dilution, came into my hands.

404 Mr. G. Dean. Enquiry into the Nature of the [ Apr. 23,

fresh serum produces a greater effect than results from the two substances
acting independently ; in other words, is the action similar to, or identical
with, the action of a bacteriolytic amboceptor and complement? At least
two methods may be employed in the study of this problem: one, the direct
method, of estimating the activity of the bodies when acting separately and
in combination ; the other, indirect, by employing something which will throw
out of action one of the constituents of a combination, leaving the other
unaffected.
THE Direct METHOD.

Laperiments with Staphylococer.

The ordinary technique was employed. In this experiment 1 volume of
leucocytes, 1 volume of coccal emulsion and 1 volume of the serum were

employed. The result was as follows :-—

| Average cocci
per leucocyte.

Normal serum of rabbit, fresh, Livolame finns. oc ssececsacccccien neon sort ueeeeaeee seems 11°9
Immune serum of rabbit, heated to 60° C. for 20 mins., 1 volume ............... 71
33 °O

1 part immune heated and 4 parts fresh normal serum, 1 volume ...............

The calculated value of the mixture at the highest estimation does not
exceed 19, whereas the experimental value is 33. Some other factor must
therefore be at work: in this experiment probably a complementing of the
immune heated serum by the fresh serum.

Another experiment of the same type may be recorded in which a slightly
different technique was employed.

Average cocci |
per leucocyte.

Normal rabbit serum, heated, 1 volume, +

Immune staphylococcus rabbit serum, heated, 1 volume, +
Normal salt solution, 1 volume, +

Leucocytes, 1 volume, +

Cocci, 1 volume.

—~-—_~
JS)
©

Normal rabbit serum, heated, 1 volume, +
Fresh normal rabbit serum, 1 volume, +
Normal salt solution, 1 volume, +
Leucocytes, 1 volume, +

Cocci, 1 volume.

or)
No)

Normal rabbit serum, heated, 1 volume,* +

Immune staphylococcus rabbit serum, heated, 1 volume, +
Normal rabbit serum, fresh, 1 volume, +

Leucocytes, 1 volume, +

Cocci, 1 volume.

15:1

VY SS SS

* This was added to control a factor in another experiment.

1907.] Substance in Serum which influences Phagocytosis. 405

Here the calculated value does not exceed 10, weit the experimental.

value was 15.

Experiments with the Dysentery Bacillus of Shiga.

In the case of such organisms as the Dysentery bacillus, Typhoid bacillus,
and Cholera vibrio, a difficulty presents itself owing to the occurrence of
bacteriolysis. If the full strength of certain fresh normal sera is employed,
an accurate estimation of the phagocytosis by this method of investigation
may be impossible on account of the fact that many of the bacilli disappear
altogether or are visible only as shadow forms both inside and outside the-
leucocytes. These phenomena are still more pronounced when one mixes.
with such a fresh normal serum a heated immune serum of any potency..
The intracorpuscular and extracorpuscular bacteriolysis in such am experi-
ment may be extreme. By a process of dilution of both the normal and the-
immune serum it was found possible to overcome this difficulty. For
example, after a number of orientating experiments with dysentery,
cholera, etc., it was found that if a dilution of 1/100 of a particular sample
of heated anti-dysenteric serum was employed with a dilution of 1/20 or
1/30 of fresh serum, marked phagocytosis occurred with slight intracellular
but no obvious extracellular bacteriolysis. When 1 volume of these:
dilutions is mixed with 1 volume of leucocytes and 1 volume of bacterial
emulsion, the ultimate dilutions reached are 1/60 or 1/90 of the fresh normal
serum and 1/300 of the heated immune serum.

The procedure carried out in such an experiment is as follows. Tubes and.
preparations are made in the usual manner from the following combinations :—

Average number of
bacilli per leucocyte.

A. Heated anti-dysentery serum (dilution 1:100), 1 volume, +
Normal salt solution, 1 volume, +
Leucocytes, 1 volume, +
Dysentery bacillus (Shiga), 1 volume.

4°3

B. Fresh normal serum (dilution 1:30), 1 volume + |
Normal salt solution, 1 volume, +
Leucocytes, 1 volume, +
Dysentery bacilli (Shiga), 1 volume. |
C. Heated anti-dysentery serum (dilution 1:100), 1 volume, +
Fresh normal serum (dilution 1:30), 1 volume, + 16°1
Leucocytes, 1 volume, +
Dysentery bacilli, 1 volume.

In Table III are shown the results of four such experiments with anti-
dysentery serum and fresh normal sera of different ages, with ae mean.
values obtained.

A06 Mr. G. Dean. Enquiry into the Nature of the [Apr. 23,

Table IfI.—Average Number of Bacilli Ingested per Leucocyte.

| ; | : | C:
Experiment , Normal serum fresh. | Anti-dysentery serum heated to—

number. Dil. 1: 30, ultimate | 60° C. for 20 minutes. _ A mixed with B. |
| dil. 1: 90. | Dil. 1: 100, ultimate dil. 1 : 300. |
| |
(1) gis 1°0 7°5 |
(2) 0°5 1 5-9 |
(3) 5°6 4°3 16°1 |
(4) 255 1°3 9-0 |
otal anton. 10°3 78 38 °5 |

Mean of averages | 2°5 | 18) 9°6

Ii the effect produced by the combined action of the normal fresh serum
and the immune heated serum were merely additive, the calculated value
should be 4:2, whereas the experimental value is 9°6. Taking into con-
‘sideration the results obtained in the experiments upon the influence of
dilution, we must assume that the mixture containing the higher serum
concentrations should have given a lower count even than 4:2, whereas, by
‘experiment, a count of 9°6 is obtained. Another factor, therefore, must be at
work in the production of this result, in all probability the complementary of
the immune serum by the fresh normal serum.

Hxperiments with the Typhoid Bacillus and the normal Sera of the Guimea-pig
and of the Rabbit.

Certain normal sera have been very thoroughly investigated for the
‘presence of amboceptor and complement in connection with bacteriolysis. It
seemed of interest to examine some of these for sensitising and complementing
action from the standpoint of phagocytosis. |

If a like relation is proved to exist, it will be at least an indication that the
substances which prepare the microbes on the one hand for extracellular
bacteriolysis, and on the other hand for phagocytosis, are identical, and that
free complement, while an essential factor only in the former process, yet
‘plays in the latter also an important, if secondary, 7éle.

The sera of the rabbit and guinea-pig, which have been investigated by
Wechsberg (1902) and others for amboceptor and complement action in
relation to the bacteriolysis of the typhoid bacillus, offer excellent material
for a comparative study of the two processes.

The following experiment, which is a repetition and confirmation of an
‘experiment of Wechsberg, shows how the matter stands for bacteriolysis in
the case of the guinea-pig and of the rabbit.

1907.] Substance in Serum which influences Phagocytosis. 407

Experiment.—A 24-hours’ agar culture of a typhoid bacillus, recently
isolated from the blood of a patient, was emulsified in bouillon and diluted.
Into each of a series of small test-tubes there was introduced a quantity
corresponding to 1/1000 of the whole bacterial mass. Serum was added in
varying amount, and the volume in each tube made the same by the addition
of normal salt solution. A small volume of broth was also added to each
tube.

_ These tubes were then placed in the incubator for three hours, after which
a fixed volume was taken from each and plated.
At the same time the various serum constituents were tested for

sterility.
Table IV
Normal rabbit’s serum :
Fresh unheated ° of. Number of colonies
Culture. guinea-pig’s serum. heated to 60° for : on agar plate.
20 minutes. |
c.c C.c. .
(@ 0°5 @) 109,998
| 0°25 O uncountable
i of anager | 0:1 ©) uncountable
ae a iene of F 0°5 10 4,365
“OP 0°25 10 21,604
O°l 1°0 uncountable
li 0:0 1°0 uncountable

This experiment shows that inactivated rabbit’s serum is capable of being
reactivated by normal guinea-pig’s serum, so that the combination of the two
is capable of producing a marked bacteriolytic effect in quantities which,
acting apart, have little or no such action. Comparative tests with fresh
rabbit’s serum and fresh guinea-pig’s serum showed that the latter had much
less bacteriolytic action than the former, and the above experiment proves
that this difference is due to a deficiency of amboceptor.

Employing the opsonin technique, a number of tests was performed with
the object of ascertaining how these two sera influenced the bacilli in
regard to phagocytosis.

| ‘Experiment. Phagocytic index.
1 Guinea-pig’s serum, heated to 60° for 20 minutes .......... & 0:2

) 2 Rabbit’s serum, heated to 60° for 20 minutes ..,..........0606 0°4
3 CAUMMCHAPIC a seLMM, Lrosh ...... 0c. cccescssccsenseserscugusecsesseus 5°7

l 4 PPA Oe SEMI, TPES) oe cck scenes gegen seceagenssoressponges» eas 105

| 5 Rabbit’s serum (60° for 20 minutes) + guinea-pig’s serum, 15 ‘6

fresh

408 Mr. G. Dean. Enquiry into the Nature of the [Apr. 23,

All were brought to the same volume.

A repetition of Experiments 3, 4, and 5, with a thinner emulsion of the
typhoid bacillus, gave the relation 0°9, 2:0, and 3°6.

These experiments show, in the case of the sera of the rabbit and of the
guinea-pig in relation to the typhoid bacillus, a marked parallelism between
the extra-corpuscular bacteriolytic function and the sensitising or opsonising
function by means of which the organisms are prepared for phagocytosis,
and that a complementary action takes place in both cases.

The Indirect Method.

These experiments were based on the observations made by Ehrlich,
Morgenroth, Bordet, Pfeiffer, and others that the injection of the serum of
one species into another gives rise to the elaboration in the blood of the latter
of anti-bodies, which may act either on the amboceptor or on the complement.
Anti-complement formation is a common phenomenon, Whereas anti-
amboceptor is of rare occurrence. The mechanism of the action of such sera
has been the subject of much discussion which cannot be touched on here.
In the present paper the term “ anti-complement serum ” will be used without.
prejudice, the possibility that such a serum may act on other bodies besides.
complement not being overlooked. In relation to the present subject, it.
seemed of interest and importance to ascertain the effect produced by an
anti-compleinent serum in experiments of the type of these with which we
are dealing. Whilst these experiments were in progress, Muir (1906) pub-
lished results obtained by a similar method with which, in the main, the
present series are in agreement.

Technique Employed.

The anti-complement serum was prepared by injecting a rabbit on several
occasions with human or horse serum which was previously heated to
58° to 60° C. for half an hour. The anti-complement serum, after separation,
was itself heated to 60° C. for 20 minutes. Varying proportions of this
serum were then added to the homologous fresh serum, or to the mixture of
which it was a constituent part. The tubes containing these were incubated
for two hours at 35°°5 C. In this way a precipitin reaction was produced.
The supernatant fiuid removed from the precipitum was employed in the
experiment. By doing a series of eight or 12 such tests, the most effective
combinations of the serum were ascertained. In some of the experiments
there was no such preliminary treatment of the fresh serum by the anti-
serum, this being simply made one of the constituent parts of the mixture of

1907.] Substance in Serum which influences Phagocytosis. 409

leucocytes, bacilli, etc., and allowed to act during 15 or 20 minutes. The
further carrying out of the experiments was as follows :—

(A) Normal rabbit serum, heated to 60° C. for 20 minutes, 10 volumes, +
Fresh normal horse serum, 5 volumes.
(At 35°°5 C. for two hours.)

(B) Anti-complement (horse) serum of rabbit, heated to 60° C. for
20 minutes, 10 volumes, +

Fresh normal horse serum, 5 volumes.
- (At 35%5 C. for two hours.)

({C) Normal rabbit serum, heated to 60° C. for 20 minutes, 10 volumes, +
Normal horse serum, heated to 60° C. for 20 minutes, 5 volumes.
(At 35°°5 C. for two hours.)

{D) Anti-complement (horse) serum of rabbit, heated to 60° C. for
20 minutes, 10 volumes, +
Normal horse serum, heated to 60° C. for 20 minutes, 5 volumes.
(At 35°'5 C. for two hours.)

The supernatant fluid from each of the above mixtures A, B, C, and D, was
subsequently mixed with an equal volume of human leucocytes and staphylo-
coccic emulsion and kept at 37° C. for 15 minutes.

When horse serum had been injected into the rabbit to obtain the anti-
complement serum, it was found impossible to use horse leucocytes in the
experiment, on account of the rapid agglutination of the red cells and
entanglement of the leucocytes. In this case, therefore, human leucocytes
were used, and by so doing the possible danger of a certain amount of
specific leuco-toxic action was avoided.

The results of seven experiments are detailed in Table V (p. 410).

It is seen that a great loss in the sensitising power has resulted in
Experiment 2, where the anti-complement serum was used. The higher
count of Experiment 4, as compared with Experiment 8, is probably largely
due to the fact that the heated anti-complement serum had a_ higher
sensitising power than the normal rabbit serum employed, the ratio being
about 2:1. This last observation applies to all the corresponding figures in
the table, which summarises the results of seven such experiments, with
& varying proportion of anti-complement and normal sera versus fresh horse
serum. On comparing the second and fourth columns of that table, it will be
seen that fresh normal serum, after having been acted on by the anti-
complement serum, gives almost the same phagocytic average as is

410

Mr. G. Dean. Enquiry into the Nature of the [Apr. 28,

abtained by heated horse serum under the same conditions, ze, a loss has
occurred comparable to that produced by heating to 60° C. for 20 minutes.

Table V.—Results of Experiments with Anti-complement (Horse) Serum
obtained from Rabbit, together with Control Results.

Fresh normal serum of horse,

Serum of horse heated (60° for 20 mins.),
+

+

ee eee c. D,

Normal rabbit’s | sualn Goulet Normal serum of Anti-complement

serum of rabbit. ae serum of rabbit.

| serum. Heated to Heated to 60° C rabbit. Heated to Heated to 60° C
60° C. for 20 mins. for 20 mine: 60° C. for 20 mins. for 30 ana.
1 17 °5 15 41 5°5
2 10°7 3°5 1:2 3 °4
3 8°3 2°1 2°6 0°8
4 8 ‘4 a Hil DK OI 3 °2
5 6°3 0°6 1°3 0 °2
6 Zc 4, *4, 1°4 3 °0
7 6°0 2°5 1°0 Leg
Mobal inc sec 64 °3 21 °7 12 °7 17 °2
ane Be 3‘ 1:8 2°4
experiments

The serum of the horse, after having been heated for 20 minutes to 60° C.,
still shows some sensitising power, even when tested by Wright and
Douglas’ method.
where a large number of leucocytes had been counted, show that heated
normal horse serum, compared with normal salt solution, gave average counts
in the ratio of about 4: 1.

Several comparative tests by my assistants and myself,

Action of Anti-Complement Serum in Complemented Miatures.

The following experiments with staphylococci may be reported in full
as the type of such experiments. The usual opsonic technique was
employed, the constituent parts of the mixture introduced into the tubes
being as follows (see table, p. 411). |

A comparison of 1 and 4 shows that, by the action of anti-complement.
serum, the phagocytic average has been reduced from 15°1 to 2°4.

Similar experiments with the dysentery immune serum of the horse com-
plemented with normal human serum showed, as the result of the action of
the anti-complement serum, a reduction in the phagocytic index in one
experiment from 7°5 to 1:3, in another from 5°9 to 1:7. In this case human
anti-complement serum and horse leucocytes were employed.

1

1907:| Substance in Serum which influences Phagocytosis. 411

Phagocytic
| index.

1. Normal rabbit’s serum, heated to 60° C. for 20 minutes, 1 volume, +
Immune rabbit’s staphylococcus serum, heated to 60° C. for 20 minutes,
1 volume, +
Normal human serum, fresh (dilution 1:10), 1 volume, +
Leucocytes, 1 volume, +
Cocci, 1 volume

bt
Or
=

2. Normal rabbit’s serum, heated to 60° C. for 20 minutes, 1 volume, +
Normal salt solution, 1 volume, +
Normal human serum, fresh (dilution 1:10), 1 volume, +
Leucocytes, 1 volume, +
Cocci, 1 volume.

6°9

3. Normal rabbit’s serum, heated to 60° C. for 20 minutes, 1 volume, +
Immune rabbit’s staphylococcus serum, 1 volume, +
Normal salt solution, 1 volume, +
Leucocytes, 1 volume, +
Cocci, 1 volume.

3 °0

4, Anti-complement rabbit’s serum, heated to 60° C. for 20 minutes, 1 volume, +.
Immune rabbit’s staphylococcus serum, heated to 60° C. for 20 minutes,
1 volume, +
Normal human serum, fresh (dilution 1:10), 1 volume, +
Leucocytes, 1 volume, +
Cocci, 1 volume.

2°4

5. Anti-complement rabbit’s serum, heated to 60° C. for 20 minutes, 1 volume, +)
Normal salt solution, 1 volume, +

\ Normal human serum, fresh (dilution 1:10), 1 volume, +

-Leucocytes, 1 volume, +

Cocci, 1 volume.

if

6. Anti-complement rabbit’s serum, heated to 60°C. for 20 minutes, 1 volume, +
Immune rabbit’s staphylococcus serum, heated to 60° C. for 20 minutes,
1 volume, +
Normal salt solution, 1 volume, +
Leucocytes, 1 volume, +
Cocci, 1 volume.

3°7

a ee ee oe a ee

Summary.

1. Dilution of fresh unheated serum is not accompanied, so far as the higher
concentrations are concerned, by a fall in the sensitising power for certain
organisms (staphylococcus, tubercle bacillus). The diminution in this was.
found, as a rule, to begin at the quarter concentration.

2. So far as the present experiments go, the points corresponding to the:
1/4th, 1/8th, 1/16th, and 1/32nd dilutions of normal human serum lie on
a parabola whose equation is y? = 4X, «e, for these dilutions the phago-
cytosis is proportional to the square root of the serum concentration.

3. The phagocytic index obtained by mixing appropriate dilutions of
a heated immune serum with a normal fresh serum is greater than results
from the two substances acting separately.

412 Nature of Substance in Serum which influences Phagocytosis.

4. In the case of certain normal sera (guinea-pig and rabbit) previously
investigated for amboceptor and complement in relation to the extra-
corpuscular bacteriolysis of the typhoid bacillus (Wechsberg), an exact
parallelism can be demonstrated to exist between that function and opsonisa-
tion. The normal amboceptor can be complemented by fresh serum in
regard to both functions.

5. An “anti-complement ” serum, when mixed with a fresh normal serum
alone or in a mixture containing heated immune serum, throws out of action
the thermolabile substance, whereas it does not appear to influence the
thermostable substance.

REFERENCES.

“Muir, R., and Martin, W. B. M. (1906), “ On the Combining Properties of Opsonins of
Nowual Serum,” ‘ Brit. Med. Journ.,’ December 2, 1906.

Neufeld, F., and Hiine (1907), “ Watcmechanece iiber baktericide Immunitat und
ena coce tenet ‘ Arbeiten a. d. Kaiserlichen Gesund.,’ vol. 25, Part 1.

‘Schiitz (1885), ‘Zeitschr. f. Physiol. Chemie,’ von Hoppe: Seyler, “Eine Methode zur
Bestimmung der relativen Pepsinmenge,” vol. 9, p. 577.

‘Simon, C. E., Lamar, R. V., and Bispham, W. N. (1906), * A Contribution to the Study
of the Opsonins,” ‘ Journal of Experimental Medicine,’ vol. 8, p. 651.

‘Volhard (1901), ‘Zeitschr. f. Klin. Medizin,’ “Ueber das fettspaltende Ferment des
Magens,” vol. 42, p. 414, vol. 43, p. 397.
“Wechsberg, F. (1902), “ Zur Lehre der natiirlichen Immunitaét und iiber baktericide
Heilsera,” ‘ Zeitschr. f. Hygiene und Infections-Krankheiten,’ vol. 39, p. 171.
‘Wright, A. E., and Douglas, 8. R. (1903), “ An Experimental Investigation of the réle of
Blood Fluids in connection with Phagocytosis,” ‘ Roy. Soc. Proc.,’ vol. 73, p. 357.

‘Wright, A. E., and Reid, T. (1906), “On Spontaneous Phagocytosis and on the Phago-
cytosis which is obtained with the Heated Serum of Patients who have responded
to Tubercular Infection, or, as the case may be, to the Inoculation of a Tubercle
Vaccine,” ‘ Roy. Soc. Proc.,’ B, vol. 77, p. 211.

413.

CroonrtaAN LECTURE ——On Globulins.
By W. B. Harpy, M.A., F.RS. |

(Being the Croonian Lecture delivered May 25, 1905.—MS. Received
April 9, 1907.)

Globulins are a class of proteids which occur in both animal and vegetable
tissues. They are peculiar in the complexity of their relations to electrolytes.
Insoluble in water, they are soluble in low concentrations of acids, alkalies,
or neutral salts. In presence of acids the globulin is electro-positive, in
presence of alkalies it is electro-negative, in presence of neutral salts it is
electrically neutral. Electrically active globulin (7.e., dissolved by acids or
alkalies) is precipitated by minute amounts of neutral salts; also, no matter
what its electrical state may be, or how dissolved, globulins are precipitated
by neutral salts near the saturation point of the latter. The problem I
propose to consider is their diversified relation to electrolytes.
~ Connected with this problem is another, namely, the relation of solutions
of globulins to colloidal solution. Do they form hydrosols at all, and, if so,
to what extent? Krafft urged the colloidal nature of soap solutions, because,
within certain limits of temperature and concentration, they gelatinise, and
have the vapour pressure of pure water. Kahlenberg and Schreiner,
however, regard soap solutions as being crystalloid in character, because
over the whole range of concentration the soap is a good electrolyte—it
ionises and undergoes hydrolytic splitting, like other salts of a weak acid and
strong base. Smits, again, by measurement of the vapour pressure over a
wide range of concentration; is convinced that above a critical concentration
the soap passes wholly into the colloidal state.

Exactly the same points arise in connection with proteid. Waymouth
Reid has shown that the proteids in a solution exert no measurable influence
upon the vapour pressure. On the other hand, solutions of globulins are
relatively good conductors over the whole range of concentration through
which it is possible to follow them. The dilution curve shows no break
indicating a general change of state, even when concentration is pushed to
the point where fluidity almost vanishes.

Again, colloids, as a class, are chemically inert. But globulins react
actively with acids or alkalies to neutralise them. Globulin solutions, even
at extreme concentration, form syrups and not true gels, except, perhaps,
under special circumstances. Clearly, therefore, it is pertinent to ask
whether globulins form colloidal solutions. aie |

VOL, LXXIX.—B. 2H

414 7 Mr. W. B. Hardy. [Apr. 9,

The globulin used throughout this research is that which is precipitated
from ox serum by dilution and slight acidification.

Solution by Acids or Alkalies. Electro-positive or electro-negative globulin.
When acid or alkali is gradually added to a suspension of globulin in water,
the opaque suspension gradually changes to a clear transparent fluid.
Between the opaque suspension, from which the globulin settles on standing,
to the stage of limpid transparency there is no break. The most minute
addition of acid or alkali (in the absence of salts) converts the suspension
into a non-settling “ solution ” of low grade, which is opaque white from the
large size of the particles of globulin dispersed throughout it. Further
addition of acid or alkali raises the grade of solution. The globulin particles
become smaller and smaller until complete transparency is reached.

The process can be reversed by dialysis, save that, at the lowest grade
attainable, no precipitate settles in the absence of disturbing factors.

With the first addition of acid or alkali, and at the last stage of dialysis,
the globulin is electrically active, so that it moves in an electric field, the
direction of movement being that which one would expect if it combined
with the acid or the alkali to form a salt, and the specific velocity is quite,
or nearly, at its maximum value. A rise in the grade of solution is, on the
whole, associated with a fall in the specific velocity, never in my experience
with a rise in this value. The electric charge on the surface of the globulin
particles therefore appears to reach maximal density when the solutions are
still of exceedingly low grade. :

These facts can best be explained by assuming that true salt formation
occurs. This agrees with the view of all previous workers on the
interaction of proteids with acids or alkalies (Sjoqvist, Bugarsky and
Liebermann, and Cohnheim). Since globulins combine either with acids or
bases, they have both an acid and a basic function—they are amphoteric
electrolytes.

The globulin salts ionise in solution; therefore, in an electric field the
entire mass of proteid moves. They also hydrolyse, but the hydrolysis
offers special features resembling those which Jordis has pointed out in the
case of sodium silicate, and Chevreul in the case of soaps. In both of these
cases hyper-acid salts are formed, while hyper-acid salts are formed on
dialysis of a solution of globulin by alkali, and hyper-basic salts on dialysis
of a solution of globulin by acid. |

By dialysis the degree of hyper-acidity or hyper-basicity can be raised,
but, as Jordis finds in the case of silica, with continuously increasing
difficulty. With the rise in the degree of hyper-acidity or hyper-basicity,
the “grade” of solution diminishes, but, in the case of globulins, precipitation

1907. ] On Globulins. A415

- does not occur. Electrically active solutions of globulin cannot be precipi-
tated by dialysis, nor at any stage do they cease to be electrically active.

If G be used to denote globulin, the equation of hydrolysis or dialysis
would be:
| «GHS+y HOH = (GHOH),(GHS),-,+y HS,

or «GB+y HOH = (GH), (GB),-,+yBOH.

In dialysis, the ratio z/y varies continuously. It is as indeterminate as
the ratio between the combining salts has been found to be by Bédlander,
Abegg, Sherrill, and others in the double salts of mercury and silver.

Clearly a relation of this kind agrees with van Bemmelen’s definition of
absorption compounds as chemical combination with variable composition.

Removal of water from a solution of acid or alkali globulin does not
produce precipitation, and the dried gummy residue reabsorbs water and
passes slowly again into a state of solution. Acid and alkali globulin,
therefore, form, with water, solutions which have the feature characteristic
of colloidal solutions, in that there are no saturation points.

In order to compare the solvent power of different acids or alkalies, it is
necessary to fix upon some arbitrary point, such as the point of minimal
opalescence. By the use of a system of controls and proper illumination
this point furnishes very concordant values.

Measured in this way, it appears that for strong and medium acids:
solvent power 1s measured by the number of gramme molecules present.
not by the number of gramme equivalents :

HCl = H.SO, = HsPQ,.

Very weak acids have a lower solvent power: HCl =5HA =

These relations are explained by the very weak basic function of globulin.
Salts with weak acids are much hydrolysed, and to reach the same grade
of solution an excess of acid is needed in order to lower the degree of
hydrolysis. |

With alkalies, the weak alkali NH,OH dissolves as well as the strong
alkalies, owing to the fact that globulin acts as an acid of considerable
strength.

The acid and basic functions were measured by the well-known methods—
the catalysis of cane sugar and of methyl acetate—and the acid function
found to be much the greater.

The molecular relation noticed above recalls the salts of amido acids:

NH: NH-,HCl1, NH: NH2H2SO,
COOH COOH ” COOH COOH °
2H 2

R +HCl=R R +H.80, = R<

416 Mr. W. B. Hardy. [ Apr. 9,

Data derived from measurements of electric conductivity, and from the
behaviour with indicators, support the view that true salts are formed, and
that the globulin acts much more strongly as an acid than as a base.

Direct Measurement of the Specific Velocity of Globulin “ Ions.’—This was
carried out by the boundary method.

As the basic function of a globulin is weaker than its acid function, the
salts GHS (globulin + acid) will be hydrolysed more than the salts GB
(globulin + base). Therefore, in the equation of hydrolysis given above, y/a
will be larger for the former than the latter. Similarly, comparing salts with
HA and with HCl, y/z will be greater for the former.

Now with increase in the value of y/«# the size of the proteid particles
increases also. By dialysis, the size can be increased until the particles
diffract white heht. The growth in the case of solutions of sodium silicate
can be traced by the appearance of molecular states incapable of passing
through a parchment membrane. In a similar way, by continuous addition
of AgCl to a solution of NaCl, molecular states are produced large enough
to diffract white lght. .

In each of these cases these large molecules are ionic, that is to say, they
take part in the electric transport. And their specific velocity is exceptionally
high; that of (AgCl), Cl, being 57 x 107° at 18°, whereas Ag at infinite
dilution is only 58.

“Jons” of this order of magnitude have the properties of matter in mass.
Each is defined by a surface and moves under the influence of a surface
contact difference of potential. With their appearance the fluid ceases
to be homogeneous. It has internal surfaces. I propose to call such ions
colloidal ions, or pseudoions. Their specific velocity is high, and, within
wide limits, is independent of their size, and is controlled by the laws of
electrical endosmose. |

These conclusions are borne out by the boundary measurements and by the
electrical conductivity of colloidal solutions.

For instance: theoretically the proportion of pseudoions in the following
solutions of globulin should be :—

In HA > in HCl, > in NaOH,
and the specific velocities at 18° were found to be 23x107°, 115 x10™°,
76x10-* By Ostwald’s law of the relation of specific ionic velocity to the
number of atoms in an ion, the value 23x 107~°is an utterly impossible one
for an ordinary ion of the magnitude of the proteid molecule.

Solution by Neuwiral Salts—Globulins are dissolved by neutral salts owing
to the formation of molecular compounds (G.BS). These compounds are
readily decomposed by water with liberation of insoluble globulin

1907. | | — On Globulins. — . A17

(G.BS+HOH = G.HOH+BS). Therefore they are stable only in presence
of a large excess of salt. Hence the solvent power of salts is from 200 to 500
times less than that of acids or alkalies. Hence, also, the presence of the
globulin lowers the electric conductivity of the salt to only a small extent:
(2 to 6 per cent.).

A double salt of the form AB.CD. ionises according to AB.CD = AB.C+ Dy

I have never succeeded in detecting any trace of such ionisation in the
ease of globulin and neutral salts; possibly owing to the extreme instability
of the ions. The proteid does not move in an electric hele and it seems to
take no part in the electric transport.

Owing to the insolubility of the dissociation products, the globulin can be
precipitated from its solution with neutral salts by simple dilution. No
degree of dilution will precipitate globulin from solution in dilute acid
or alkah.

The compounds of globulin and alkalies (GB) are more readily dissolved
by neutral salts than is simple globulin. Compounds of globulins and acids
are insoluble by neutral salts, being decomposed with liberation of the acid.

If on the analogy of an amido acid a globulin combines owing to the
presence of the NH, and the COOH group, then it forms salts with alkalies
by replacement of hydrogen, and with acids by the change of the trivalent
nitrogen of the amido group to the pentavalent form.

Ho He
| (1
ee eee NOT ROO os 14 BOR
COBY, Qu 6
C<oOn C<ONa
is H
UN . H.
He ii eG
Ke on ‘
C<OH

Where, then, does the neutral salt link on ?

There are two possible places—the unsatisfied valencies of the nitrogen, or
the unsatisfied valencies of the upper O of the COOH group. According to
the oxonium theory, O has a maximum valency O=, but O= is much less
stable than N=; therefore, one would expect the linkage to be by the amide
group— .

He He
{| | Na
ue aR WN ail: =e OL
A 41,0

418 Mr. W. B. Hardy. (Apr. 9,

And this would account for the fact that the acid can be turned out of the
combination with globulin by an excess of neutral salt, but the alkali
cannot be.

It is easy to prove by the boundary method that the globulin, when
dissolved by salts, takes no part in the electric transport. Dissociation of the
salt-globulin compound by the water may be regarded as being practically
completely suppressed by the excess salt present. This being the case, the
diminution of the electric conductivity of the salt by the globulin may be
used as a measure of the fraction of the salt actually combined with it.

In the following table a few values are given. Comparison is made
between solutions containing the same amount of salt per litre; but in the
one case the solution is saturated with globulin, in the other case it is a
simple solution of the salt in water. No correction is made for the diminu-
tion of the molecular conductivity of the salt owing to replacement of a
portion of the water by globulin, since it would amount to less than 0:01 per
cent. No. III is of the nature of a control, to prove that electrolytic
impurities adherent to the purified globulin may be neglected. The salt
solution was made up with a dialysate in equilibrium with the suspension of
_ globulin.

Serum centrifuged, diluted to 10 vols., globulin precipitated by HA. The precipitate

suspended in a large volume of distilled water and collected by the centrifuge, and
re-suspended in water freed from gases by boiling.

Specific conductivity of the water employed, 6 x 1076 rec. ohm, 18°.

J. Ox globulin suspended in water. 100 c.c. = 4:08 grammes dry globulin. Tem-
perature, 18° :—

Specific Specific
conductivity, conductivity, :
salt globulin, salt water, K salt globulin
Salt. Normality. xallOz x 10°. K salt water
KS Ol wear on cos 01716 1445 1539 0 ‘938
pie Weeseee ces 0 °1679 1411 1506 0 937
NBO esc neces 0°27 1850 1950 0 949

Specific resistance of the globulin suspension 3°1 x 10~°.

II. Sheep globulin. 100 cc. = 4:4 grammes :—

Nal tos. . .te. 2145 2280 0 ‘938
Syl Sowers 2150 2278 0 943

The measurements in II were duplicated in order to test the degree of accuracy of the measure-
ments of volume.
III. Ox globulin dialysed until in equilibrium with water saturated with toluol :—
MgSQ, ...... 0:11 517 543* 0-95

In this case the salt solution (*) was made up with the dialysate, not with water.

[ Mote——In a recent paper* it is stated that globulin in solution does not alter the

* Mellanby, ‘Journ. of Physiology,’ vol. 33, p. 354, 1905.

=
= P
“y
%

1907. ] On Globulins. 419

conductivity of the salt used to dissolve it. The conductivity of the salt “is the same as
that of a similar solution in pure water.” This statement is directly contrary to my own
observations. It leads me to add a few words as to the precautions which are necessary
in dealing with proteid solutions.

Electrodes coated in the ordinary way with platinum black are quite useless. The
proteid in their immediate neighbourhood is changed very rapidly and very drastically.
An instance selected at random will show the magnitude of the effect.

18°. Globulin dissolved by dilute NaOH. 3°3 per cent. globulin. Readings at
intervals of two minutes approximately :—

2560 ohms.
DGAD Ss. 5,
2537,
2530 __,,
2522,
2538,

The rise in the last observation followed upon slight shaking of the cell to wash the
changed portion of the solution away from the electrodes. Platinum grey electrodes,
prepared as Whetham directs, give no trouble. Readings with globulin solutions of all
kinds remain quite constant after being for 48 hours and longer in contact with them.

Even with platinum grey electrodes, however, it is necessary so to adjust the cell that
the observed resistance does not fall under about 500 ohms. With a lower resistance, and
therefore a larger current, the readings become unsteady and too low. |

On the assumption that the whole of the drop in electric conductivity is
due to association of the salt with the globulin, the above figures show that,
at the particular concentration and temperature, 1 gramme of dry globulin
combines with 33 x107° gramme equivalent of NaCl and 26 x 10~° gramme
equivalent of K2SQO,, while to dissolve 1 gramme of dry globulin 10 x 107°
equivalent of alkali, or 18 to 36 equivalents of acid are needed. The
figures for salt and acid are of the same order of magnitude, and this is what
might be expected if salt and acid combine in the same way with the proteid.

APPENDIX.

(Added December, 1906.)

Viscosity of Solutions of Globulins-—The measurements of this value, very
briefly touched upon in the lecture, have been amplified. They show the
following interesting features :—

In dilute solutions of globulins the viscosity, as measured by the rate of
flow through a capillary tube, is of the same order of magnitude for each of
the three types—acid, alkali, and salt globulin.

With increasing concentration the viscosity increases, but the increase is
much greater for alkaline globulin than for acid globulin, and for acid globulin
than for salt globulin. The difference is very striking; thus, at a concentra-

42.0 Mr. W. B. Hardy. [Apr. 9-

tion of 7:59 grammes globulin per litre, the transpiration times are in the:
ratio—

Wiatenginackinl.acstn. bon 1
MgSO, globulin......... 4°66
HCl eceenene 155
NaOH 5s , Pawaetonn 67:9

In solutions of salt globulin, the globulin particles do not carry electricity ;
“jonic” proteid is completely absent, therefore the higher viscosity of acid
and alkaline globulin solutions is connected with the presence of “ionic”
proteid, that is, of very large molecules carrying a charge.

Since globulin acts much more strongly as an acid than as a base, there
will be in similar solutions of HCl globulin and NaOH globulin a greater
concentration of “ionic” proteid in the latter. Here, again, increased concen-
tration of “ionic” proteid goes with increased viscosity.

These conclusions agree with the observations of Reyher* on the viscosity
of solutions of soap, and of Sackiirt on solutions of the proteid caseinogen..
It would, however, be rash to fix upon the simple proteid or soap ion as the
agent. The effect of dilution, and of varying proportions of acid or alkali.
make it possible that the high viscosity is due to the presence of the
molecular complexes, which carry a surface charge, and which I have called
“eolloidal” ions, or pseudoions. Thus, further addition of small quantities of
ammonia, beyond the amount necessary to dissolve the globulin to a trans--
parent solution, decreases the viscosity. Ammonia is a very weak base, which,
in solution, has a specific molecular conductivity much less than that of. its.
compound with globulin. One cannot, therefore, refer the diminished viscosity
to diminished ionisation of the ammonia-globulin compound, but the optical
properties of the solution show that slight excess of ammonia does diminish
the size of the particles of the globulin. It raises the “grade” of the
solution.

A comparison of the different types of solution of globulin raises, in a
special way, the question of the meaning to be attached to the phrase
“colloidal solution.” Mixtures of very diverse character have been classed
as colloidal, some because they form jellies, others because they are physically
heterogeneous, others because the osmotic pressure is exceedingly low, others,.
again, because the electric conductivity is abnormally low. Of these, perhaps,
the only constant attribute is physical heterogeneity. Solutions which
present any of the other distinctive features of the colloidal state always.
contain particles which are large enough to scatter ght.

* € Zeits. f. Physik. Chem.,’ vol. 2, p. 7438, 1888.
+ Ibid., vol. 41, p. 672, 1903.

1907.] ~ On Globulins. 421

Amongst the attributes of colloidal solutions is the possession of an
abnormally high viscosity. Judged in this respect, the salt globulin solutions.
are only slightly colloidal as compared with the solutions of alkali or acid
globulin. They differ also from acid and alkali globulin in the relatively
greater definiteness of the relations between the components. Dilution of a
solution of salt globulin, for instance, brings about a sharp separation of solid
and liquid. No such clean saturation points are found with the other two
systems. The definiteness of the relations makes it possible to compare the
system—neutral salt, water, globulin with other similar three-component
systems. er # “ts

Data sufficient for the purpose can be obtained from the measurement of
the solubility of globulins in aqueous salt solutions made by Osborne and
Harris,* and by Mellanby.t The curves which these authors give fail to.
show the true relations, since only two co-ordinates are used, namely for the
salt and the globulin; the third component, water, is neglected. For the
following curves, trilinear co-ordinates are used in the manner recommended
by Stokes. © 2! 3
_ Fig. 1 shows curves plotted to scale from values derived from Osborne and.

Ss

Fie. 1.

* © American Journal of Physiology,’ vol. 14, 1905.
it Loe. cit.

A422 Mr. W. B. Hardy. [Apr. 9,

Harris.* S is pure salt, W pure water, G pure globulin (edestin). The
‘points on the curves, fixed by actual experiment, are shown in the usual
way by dots or crosses. A’B’C is the curve for Na2SO,., ABC the curve for
NaC]. The broken line extending to just beyond B is the probable form
-of the curve for H,SO,. The diagram is drawn as an isotherm for NaCl,
water, edestin, the areas for the other salts being left out. The context is
not very clear on the point, but the temperature of observation seems to
‘have been 20°. .

Each curve clearly consists of two distinct parts, AB, which is. in
‘equilibrium with solid salt, and BC in equilibrium with globulin crystals.
The whole surface, therefore, consists of certain areas:—ABC, all points
within which represent a homogeneous state, namely, a fluid solution of
‘globulin, salt, and water; ASB, which is an area of heterogeneous states.
Each point in it represents a mixture which cannot exist as a uniform
state, but divides into two phases, one a fluid solution on the curve AB, the
-other a solid which is pure salt. WBG, again, is an area of mixture which
cannot form a uniform state, but which divides into a fluid solution on CB,
-and into globulin crystals. SBG, each point within which represents a
mixture which divides into a fluid having the composition of B and solid
salt and solid globulin.

In fig. 2, portions of three isotherms for NaCl, water, serum globulin are

Aq. 99 8 | Glob

1907. | — 3 On Globulins. 423

plotted to scale, the data being derived from Mellanby’s paper. They cover
only a very small fraction of the whole curve, ABC, but they are sufficient to
show, (1) that the curve encloses an area which is placed on the line WS, as
in the preceding case. This follows from the form of the curve, and from the
fact that if sufficient salt be added, a point is reached on the line WS where
salt solution is in equilibrium with solid globulin. (2) That the area
enclosed increases with a rise of temperature.

This last fact, together with some observations of my own, enable us to
approximate roughly to an isotherm for edestin at, say, 30°. When edestin
is present in sufficient quantity, on warming a further amount passes into
Solution, so that we have a series of solutions in equilibrium with crystals.
Beyond a certain temperature the crystals fuse, and we then have two
fluid layers in equilibrium, the lower layer (as I have found by actual
analysis) containing all three components.

Fig. 3 is a diagram of an isotherm at a higher temperature; it shows the
area ABC enlarged, and it shows a new area, ROP, due to the appearance of

Fic. 3.

‘a second series of solutions (the melted crystals). Of the shape of this area
we know nothing. The curve ABC now contains three parts, AD in
equilibrium with 8, DE with G, and EF with the solutions which lie on OR.
Fig. 1 does not represent the conditions for blood globulin at any tem-
perature, since, in presence of water alone, a solid solution of water in
globulin is formed. The tie lines from the curve BC, therefore, instead
of focussing at G, end on a curve of unknown slope, which starts from about

424 _ Mr. W. B. Hardy. [Apr. 9,

the middle of the line WG. In other words, the isotherms of blood globulins,
over the region where we have any information to guide us, are of the form;
shown in fig. 4.

Fre. 4.

This 1s as far as the known facts carry us. It now remains to compare
the system with a similar one. Such a one is found in water, sodium
chloride, and succinic nitrile, which has been studied by Schreinemakers.*
In both there is a pair of immiscible bodies, salt, and globulin, or salt and
succinic nitrile, each of which is partially miscible with the third substance,
water. Fig. 4, A and B are two isotherms reproduced from Schreinemakers’
paper. It is obvious that, in all essential features, they resemble the:
isotherms for globulin-salt-water already given.

It is clear, from this comparison and from the low viscosity, that slouaine
salt-water is a system showing few abnormal features. It manifests decisive
points of equilibrium, which resemble those in a comparable and purely
crystalloid system. The other two globulin systems are as decisively
abnormal, especially in the high viscosity, and the absence of definite:
transition points, and it is significant that, regarded from the purely physical
standpoint, the essential difference should be the presence in the latter of
large molecular aggregates, each carrying an electric double layer.

The presence of internal electrified surfaces adequately accounts for the:
high degree of inertia which obscures the transition points. What is the:
source of the electrification? In all cases in which it can be traced, the
charge is due to molecular interaction of the type classed as chemical, and of
that particular chemical type associated with the decomposition of neutral
electricity, to which the name “ionisation” has been given. Burton?+ clearly
shows that metals form hydrosols because they react with the solvent to form
hydrides or hydroxides. Acid globulin, alkaline globulin, and soaps are salts.

* ‘Zeits. f. Physik. Chem.,’ vol. 23, p. 417, 1897.
t ‘Phil. Mag.,’ April, 1906.

1907.] On Globulins. | A425

which form colloidal solutions because one of the radicles is of large size, is
‘almost or quite immiscible with the solvent, and therefore readily forms
molecular complexes which have the electric sign proper to the radicle when
‘dissociated from its fellow in the salt molecule.

Each colloid particle undoubtedly carries on its surface a charge, since,
without exception, they move in a uniform electric field. But in the system
formed by each particle there cannot be any free electricity, any resultant
charge. If there were, the system would, by Earnshaw’s theorem, be
unstable, and settling would occur the more rapidly the greater the charge.
Experiment shows the opposite to be the case. The charge on each particle,
therefore, must be bound by an equal and opposite charge on the liquid face
opposed to it. The condition of double electric layers is therefore realised.

' It has been shown by Waymouth Reid* that in a solution of proteid no
osmotic pressure can be traced to the proteid, and I find that when a
solution of globulin is dialysed against water until equilibrium is reached,
the dialysee and dialysate have exactly the same electric conductivity. These
facts, added to others already stated, afford overwhelming evidence that in
these solutions the chief portion of the proteid is, in effect, removed from
solution by being gathered into masses of more than molecular dimensions
which are separated from the rest of the solvent by a surface. The masses
of probably hydrated proteid thus form an internal phase. In colloidal
salts such as soaps, solutions of globulin in dilute acid or alka, solutions of
caseinogen or of acid or alkali albumen, in which the internal phase is
bounded by double electric layers formed by ionic interaction with the
external phase, the stability of the system with reference to forces such
‘as gravity will depend, in part, upon the potential difference between the two
faces round each particle. The electric double layers will contribute to
stability, simce any movement of a particle through the fluid will develop
free electricity in quantity proportional to the potential difference between
the layers.+

Salts in small amount diminish the stability of these colloidal systems
and bring about concentration ; and salts, as has been shown experimentally
by Wiedeman, Perrin,t Burton,§ and others, diminish the potential difference
in the condenser system round each particle.

The contribution which the electrification of the internal surfaces makes
to mechanical stability is seen in the high viscosity of these solutions as

* ‘Journ. of Physiology,’ vol. 31, p. 438, 1904.

t+ Helmholtz, ‘Wied. Ann.,’ vol. 7, p. 337, 1879.

f ‘Journ. de Chim. Physique,’ vol. 2, p. 61, 1904 ; vol. 3, p. 50, 1905.
§ ‘Phil. Mag.,’ November, 1906.

426 On Globulins.

compared with solutions of globulin by salts which resemble them in
containing large molecules, but differ in the total absence of electric layers
from these molecules.

Solutions of globulins in salts introduce us to an entirely different aspect
of colloidal solution. Here, again, the proteid contributes nothing to the
osmotic pressure,* and, therefore, must be withdrawn from true molecular
solution; but as all ionic interaction is suppressed by the large excess of
salt present, there are no double electric layers formed, and in their absence
viscosity is low and stable equilibrium conditions are rapidly reached.

The origin of the mechanical stability of this system, the nature of the
influence which maintains the even dispersion of the proteid throughout
the fluid, is difficult to explain, since any factor to which we can appeal
seems also to involve the exertion of a finite osmotic pressure by the proteid.

If the distribution of the energy be considered, we arrive at the
following :—Let the state of thorough mixing of the components be
established, and let a movement under the influence of any mechanical force
take place, so that the proteid becomes more concentrated in some region.
In this region, owing to the concentration of the colloidal salt, the degree of
hydrolysis and of ionisation will be diminished, and the available energy
thereby increased.

The equation of hydrolysis which Arrhenius} gives shows that hydrolysis
and ionisation diminish faster than concentration increases; therefore, with
unequal distribution of the proteid the gain in available energy (E—7d)
will be greater in the regions of higher concentration than the loss in the
regions of diminished concentration. The system as a whole, therefore,
would gain in available energy, and the change from uniform distribution to
unequal distribution could not take place. unless work were done on the
system.

* Waymouth Reid, loc. cit.
t ‘ Zeits. f. Physik. Chem.,’ vol. 5, p. 16, 1890.

427

Fatty Degeneration of the Blood.
By 8. G. SHarrock and L. S. DuDGEon.

(Communicated by Professor J. Rose Bradford, F.R.S. Received March 18,—
Read April 18, 1907.)

Erroneous as histological research has proved Hunter’s view of the
organisation of effused blood to be, yet Hunter was the first to discern the
biological truth that the blood is alive—alive in as strict a sense as is any
other component part of the living body. |

To view the blood as a tissue is a natural extension of the same conception,
a tissue, that is, of mobile elements, and one in which the plasma repre-
sents a fluid, in place of a solid, intercellular material. Yet even in regard
to this quality we may discover gradations between the opposite. extremes
like those presented by blood and cartilage, in such intermediate forms as
mucous tissue, where the intercellular substance may be equally well called
semi-fluid or semi-solid.

If there is a paradox in such an application of words, it is not so much
that the incongruity marks an error of conception as that terms used in one
stage of science become inadequate to compass biological truth, which as
it grows under observation defies the artificial limitations which language
would impose upon it.

In a similar manner has the common conception of a crystal been
dissipated by the sheer force of observation. For the researches of Lehmann
and of Schenck* have made it clear that certain fluids may retain the
characteristic crystalline feature of containing doubly refractive bodies.
The conception of a fiuid crystal is not less paradoxical than that of a
fluid tissue.

One peculiarity in the constitution of the blood whereby it differs from
other tissues is, of course, the fact that although its cells, as in the more
stable forms, undergo division, both mitotic and amitotic, and have in this
sense a life of their own, yet the primary sources of its elements are the
blood-forming organs—the bone-marrow, the hemolymph and lymphatic
glands, or other lymphatic tissue, and the spleen.

In considering diseases of the blood, therefore, there is a double question
involved: there are firstly the changes in the blood arising from disease of
the blood-forming organs, and, secondly, the abnormal changes of the formed
blood, arising from lesions proper to the blood itself.

* ‘Fliissige Kristalle,’ Leipzig, 1905 ; cited by J. C. Adami, F.R.S., and L. Aschoff,
Roy. Soc. Proc.,’ B, vol. 78, 1906.
VOL. LXXIX.—B. peer

428 Messrs. 8. G. Shattock and L. 8. Dudgeon. [Mar. 18,

Conceiving of the blood, then, as a complex tissue, and subject like any
other to parasitic and bacterial infection, or partaking in the morbid changes
of many other specific diseases, it might be imagined that it would likewise
be subject to degenerations, and more particularly to the chief and most
widespread of all of these, viz., fatty.

The blood-degeneration which has been as yet most studied (other than
the so-called polychromatophilic of the red cells) is that usually regarded
as glycogenic, and consists in the abnormal, 2.¢., excessive, appearance of
glycogen, chiefly in the finely granular polymorphonuclear leucocytes, although
the granules occur also in the eosinophile or coarsely granular polymorpho-
nuclear cells, and to a lesser degree in the lymphocytes.

To this form of degeneration (which is known also as iodinophilic or
iodophilic, since the grains and granules take a brown colour after treatment
with iodine solution) we will refer again in connection with certain
ambiguous appearances met with by ourselves in blood-films treated with
Scharlach solution.

If we except pathological effusions, such as those of pleurisy and peritonitis
and purulent exudations, the process of fatty degeneration has hitherto
been looked for and studied only in the solid tissues. Yet it will be
apparent on theoretical grounds that in cases where fatty degeneration of
the last-named tissues is due to a deficient oxygen-carrying power of the
blood, or to a toxic condition of it, the state of the blood might react upon
itself so as to induce similar changes in its own cells.

The chief causes of fatty degeneration in general, excluding those of a
local character which produce only a local change and have no interest in
connection with the present subject, are profound anzmias and certain forms
of toxemia, whether bacterial or of inorganic causation, as especially
illustrated in phosphorus poisoning. )

In the first the fatty degeneration of the various organs results from the
reduction in the oxygen-carrying power of the blood. In the second, it
results directly from the injurious action of the toxin upon the cells of the
tissue.

Our inquiry, therefore, is: Does the blood itself suffer similarly in these
two groups of morbid conditions ?

(1) Do the leucocytes (leaving the red cells out of consideration) suffer
from a defective oxygen-carrying power of the blood itself ?
(2) Do the leucocytes suffer from the direct action of toxins circulating
in the blood in profound toxzemia ?
In the blood, as in other tissues, the distinction must be drawn between
fatty infiltration and fatty degeneration.

1907. | Fatty Degeneration of the Blood. | 429

Without entering into the chemical question as to the source of the fat
in fatty degeneration, how far, 2.¢., it is due to a transformation of the proteid
of the cell (as once held), and how far to the accumulation of fat
reaching the cell from without and appearing in it because not utilised, the
cardinal difference between the two processes remains, even though the
source of the fat should be the same in both.

In the one case the fat accumulates or is stored in a cell of which the
cytoplasm is wninjured; in the other it appears for the reason that the latter
is damaged and rendered incapable of utilising the fat which reaches it.

With the fat that is not free, but combined, in the cell, neither term
deals; the presence of this is not recognisable, either by its microphysical
or microchemical properties.

Technrque.

At the outset it 1s obviously essential to exclude the use of fat solvents,
like absolute alcohol and ether, as fixing reagents, and essential also that
the blood should not be dried. One preliminary method we tried was the
use of blood clot, but it proved less satisfactory than that of making films.

Microscopic sections are readily cut from cylinders of blood that has been
received into, and allowed to clot in, narrow tubes, the clot being afterwards
fixed in salt-formol followed by 80-per-cent. alcohol. Such sections can
be stained for fat like those of other tissues. The method, again, of using
washed corpuscles (1.¢., corpuscles washed in salt solution after having been
separated by the centrifuge from blood in which clotting has been prevented
by the addition of citrate of sodium) offers no special advantages, and after
making a trial of it we did not adopt it.

The method finally chosen was that of making films on slides and cover
glasses. The film from first to last must be kept moist, and treated, in
short, throughout as a section of any tissue which is to be studied for the
presence of fat. The reason for this will be obvious, for any fat in the
leucocytes would, in the dried film, tend to transude, and become lost by
diffusion in the surrounding area.

The film having been made on the slide in the usual manner, by drawing
a second slide in front of the blood, the slide is immediately placed with the
film downwards in a specially devised chamber of formol vapour.

The chamber we use is a deep, oblong glass with a ground edge, and is
itself hardly larger in sectional area than an ordinary microscopic slide.
This is packed nearly to the top with cotton wool thoroughly soaked with
formol (40 per cent.), and on the top of the wet wool is laid a grating of
perforated zinc, near either end of which is soldered a cross strip of zinc

212

430 Messrs. 8. G. Shattock and L. 8S. Dudgeon. [Mar. 18,

wire; the latter prevents the film from touching the grating, through
which the vapour freely passes to fix the film. The chamber is covered
with a piece of overlapping plate glass, after vaseline has been painted over
the edge.

Before the slide is prepared, cover-glass films can be made in the usual
manner, and placed with the blood upwards upon the perforated zinc. The
glass lid being immediately replaced, the slide film is prepared and introduced
with the film downwards over the cover glasses, from off which it is kept by
means of the cross pieces of zinc wire on which it rests.

After not less than 15 minutes (though the films may be left in the vapour
for 24 hours or longer, if convenient) the slide is removed and at once placed
in the vertical position in a glass of Scharlach for 24 or 48 hours. The
cover glasses are placed separately in hollowed glass blocks of the dye,
accurately covered to prevent evaporation. ,

After a certain amount of experience we adopted, as a check, the practice of
treating one of the cover glasses after removal from the formol, and before its

Fic. 1—A diagram showing the glass formol chamber and zinc grating used to fix the
blood films. The glass lid is not represented. Below the perforated zinc the space
is filled with cotton wool soaked in formol solution, 40 per cent.

transference to the Scharlach, with absolute alcohol, followed by ether and
again by absolute alcohol; the object of this was to remove any fat or fat-like
bodies from the cells of the film. At first,in making the cover-glass films, we
adopted the plan of adding salt solution to the blood on the cover, in order to
prevent any possibility of the film drying; the film itself was made on each
cover glass separately by means of a platinum loop. This precaution was
found to be unnecessary, and we therefore discarded it.

The use of alcoholic solution of Scharlach as a fat stain requires much care,
for the dye readily precipitates. The solution we employed was made by
saturating 75 per cent. absolute alcohol in the cold, and subsequent filtration
through paper first wetted with alcohol. In the case of a slide, the latter was

~ ——

1907. ] Fatty Degeneration of the Blood. 431

placed straight from the formol chamber into the Scharlach solution, and after
24 or 48 hours withdrawn, washed for a few seconds in 75-per-cent. alcohol,
then in distilled water, and after this stained in a bath of hemalum for
three minutes; this was followed by washing in distilled water, and then in
tap water, the film being finally protected with a long cover glass after the
application of Farrant’s medium. The cover glasses were prepared in a similar
manner.

Our observations were commenced in April, 1906, and on May 29, 1906, at
the meeting of the Pathological Society of London, held on that date,* we
made a preliminary statement to the effect that we had discovered the
existence of fatty degeneration in the blood in certain morbid conditions. We
are not aware that any similar observation has been published before this
announcement. The only work of a similar kind of which we know, indeed,
is that carried out by Professor Antonio Cesaris-Demel, at the Institute of
Pathological Anatomy of the University of Pisa. Of this a preliminary note
_ was published very shortly after our own, viz., in June, 1906, having been
communicated to the Royal Academy of Medicine of Turin at its sitting of
June 8,1906.+ The title of the note is, “On Degenerative Changes of the
Leucocytes of the Blood, studied by the Method of Coloration in the Fresh
State.” The author gives no detailed account and no figures, although he
states that the stain used by him was Sudan III, one closely allied to that
used by ourselves.

Our examination of blood films extends altogether to 79 cases and comprises
various morbid conditions. Nevertheless, it is in comparatively few that we
have satisfied ourselves of the presence of fat in the leucocytes, 7.¢., of points
or droplets presenting the same physical characters and coloration with
Scharlach as those seen, eg., in the typical form of fatty degeneration of the
cardiac muscle or renal epithelium.

Inst of Diseases in which the Blood was examined for Fatty Degeneration.

Pernicious anemia. Myelemia.

Toxemia of pregnancy. Influenza.

Subphrenic abscess. Exophthalmic goitre.
Chlorosis. Acute pneumonia.
Diphtheria. Chronic Bright’s disease.
Appendicitis. Purpura.
Lymphadenoma. Pericarditis (rheumatic).
Acute meningitis. Anemia (secondary).

* ‘Brit. Med. Journ.,’ June 2, 1906.
t ‘Giornale della R. Accademia di Medecina di Torino,’ Giugno-Luglio, 1906.

432 Messrs. 8. G. Shattock and L. 8. Dudgeon. [Mar. 18,

Post nephric abscess. Septic nephritis.
Anemia of undeterminal origin. §Myxcedema.

Pleurisy and pericarditis. Hepatic suppuration.
Septic bronchopneumonia. Ulcerative endocarditis.
Carcinoma of pylorus. Rickets.

Sub-acute rheumatism, probably | Acute cerebro-spinal meningitis.
gonorrheeal. Diabetes and lipemia.

In the case of some of these diseases more than one example was studied.

List of Diseases in which Fat was found in the Leucocytes.

Chlorosis. Toxeemia of pregnancy.
Chronic Bright’s disease and Acute pneumonia.
anemia of chlorotic type. Purpura.
Carcinoma of pylorus and Diabetes and lipeemia.
aneemia of chlorotic type. Myelemia.
Influenza. Lymphadenoma.
Pleurisy and pericarditis. Acute cerebro-spinal meningitis.

If these cases are analysed it will be found that most of them come in the
category of toxic diseases, some of an acute kind, others of a chronic. Of the
three cases of Myeleemia in which fatty degeneration of the finely granular
leucocytes was found, one was acute, two were chronic.

The degree of fever present in these toxzemic diseases varied according as
the condition was one of intoxication or infection. In one of the most
pronounced examples of fatty change, viz., the toxemia of pregnancy, the
pyrexia was very slight.

In the toxic series may be placed :—

Toxeemia of pregnancy. Influenza.

Acute pneumonia. Pleurisy and pericarditis.

Myeleemia. Lymphadenoma.

Purpura. Chronic Bright’s disease (associated

Acute cerebro-spinal meningitis. with some degree of chlorotic
anemia).

In regard to this group of cases we believe that the fatty change present in
the finely granular polymorphonuclear leucocytes results from the direct action
of the toxic substances present in the blood itself; that is to say, that the
change is one of proper fatty degeneration as distinguished from the ingestion
or storage of fat by a healthy cell.

1907.] 3 Fatty Degeneration of the Blood. 433

The fineness of the fatty points, their varying number in different cells in
the same film, and the fact that they occur in abundance in cases where the
condition of the patient excludes the possibility of so large an amount of fat
being merely ingested from the blood by the circulating leucocytes, all point
to this conclusion. Thus in the three cases of acute pneumonia the blood was
taken, in one, on the sixth day, two hours after the crisis; in another, on the
fifth or sixth day, before the crisis; and in the third, five hours after the
crisis. Furthermore, the fact that in such an acute disease fatty degeneration
actually does occur in the renal epithelium and cardiac muscle proves the
ability of the toxins to effect such damage also upon the circulating
leucocytes.

That the degeneration of the leucocytes has occurred in the blood and not
in the bone-marrow may be held as almost certain, since had so marked a
change taken place whilst the cells were still in the marrow, it is unlikely
that they would have entered the vessels ; they would have remained 7m sztw.
Upon the actual condition of the bone-marrow we have, with one exception,
no observations. |

When, as in myelemia, the fat occurs likewise in myelocytes in the blood,
we should think, similarly, that the change occurred in the cells after they
had reached the blood-stream.

The leucocytes most degenerated probably die, and might by their
disintegration furnish a certain amount of free fat in the plasma.

Not that this is the sole source of the free fat in the blood which was
present in some of the cases, for its amount is too great for such an explanation.
Most of the free fat must be regarded as derived from the intestine, or as
transferred from the common connective tissue to the blood plasma for the
use of the more important organs. The fat in some of the leucocytes under
these circumstances is doubtless ingested, but the fact that in some of the
cases where the fat in the leucocytes was well pronounced no free fat was
present in the plasma shows that this cannot be advanced as the sole
explanation.

The presence of fat in some of the myelocytes of the blood film in
myelemia, again, cannot be due to ingestion, since these cells have no
phagocytic powers. It might have been thought, a priori, that the presence
of fat in the finely granular polymorphonuclear leucocytes would be a sign
of grave significance as indicating not only a high degree of toxemia but an
interference with the function of cells, the phagocytic integrity of which is of
such importance in the cure of a disease hke pneumonia. For the leucocytes
so altered must have lost for the time their physiological capacities. They
would be much in the same condition as the pus cells in an acute abscess,

434 Messrs. 8. G. Shattock and L. 8. Dudgeon. [Mar. 18,

rendered fatty by the toxic action of pyogenic micro-organisms. Indeed,
a study of cover-glass films made from the pus of an acute abscess and
treated precisely as those of blood reveals the presence of scarlet points and
droplets within a large number of the finely granular polymorphonuclear
leucocytes, which are exactly matched by those in the leucocytes of certain of
the blood films described. In some of the pus cells (as in the blood) the
red points are quite minute (as they were in the case of influenzal pleurisy) ;
in others larger (as in one of the cases of acute pneumonia); or still more
so, as they were in the case of pyloric carcinoma associated with chlorotic
anemia.

It is noteworthy, too, that in the pus cells so affected the nuclei may
exhibit no fragmentation. Nor have we ever seen nuclear fragmentation in
the fatty leucocytes in blood films.

This prognostic conjecture, nevertheless, is not borne out by observation.

Thus in the three cases of acute pneumonia in which a certain number of
the polymorphonuclear leucocytes contained fat, recovery ensued. In one
case the blood was examined on the sixth day, two hours after the crisis; in
another on the fifth or sixth day, before the crisis; and in another five
hours after the crisis.

The actual number of leucocytes showing the fatty change in these three
cases, however, was not excessive, and although the phagocytic power of such
cells would, doubtless, be diminished or destroyed, the great excess of
leucocytes present would compensate for such a loss. Within two or three
days after the crisis the leucocytosis has disappeared ; the leucocyte count
is practically normal. .

This rapid disappearance of the leucocytic excess from the blood is largely
due to the vast immigration of leucocytes into the infected organs, the lung
more especially ; the surplus yet remaining would disappear, as having no
further purpose to serve. For all observation goes to show that the
rapid amelioration following the crisis is due to the activity of the finely
granular leucocytes.

MacDonald* has shown that the opsonic index attains its maximum at the
crisis. And Rosenow+t concludes from his work that pneumonia serum has
no pneumococcidal effect ; that the opsonin during and shortly after crisis (in
cases that recover) seems to rise above the normal, and that the pneumo-
coccidal action of pneumonic blood is the result of the combined action
of serum and leucocytes—phagocytosis and intraphagocytic digestion. It
must not be forgotten, moreover, as a general consideration, that the

* © Path. Soc. Trans.,’ London, vol. 57, p. 45, 1906.
+ ‘Journal of Infectious Diseases,’ vol. 3, June 30, 1906, Chicago.

1907.] Fatty Degeneration of the Blood. 435

degenerating cells in the blood may recover, like those, eg., of the kidney in
toxic diseases, in which the damage done to the renal epithelium is shown by
the temporary presence of albumen and casts in the urine.

Tn all the cases we have examined the fatty changes were confined to the
finely granular polymorphonuclear leucocytes, that is, to the cells which
take the active part in phagocytosis ; the most active cells, as being the more
highly differentiated, are those most affected by the toxin.

We have never seen any such change in the lymphocytes—a class of cell
devoid of phagocytic power.

The inactivity of the lymphocytes is a striking comment upon the fanciful
views once held, that the lymphatic tissue, whether of the tonsils, lymphatic
glands, or intestinal track, constituted a highly important line of defence
against microbial invasion. As a matter of observation it is in the solitary
and Peyerian glands of the intestine that tubercular infection takes place ;
and once a tubercular précess has commenced in a member of a lymphatic
chain it spreads from gland to gland until the whole are involved.

Anemia.

In this group of diseases the following of the cases in which fat was found
in the leucocytes may be placed :—

(1) Chlorosis.

(2) Carcinoma of the pylorus, associated with anemia of the chlorotic
type.

In chlorosis the fatty changes occurring in the finely granular poly-
morphonuclear leucocytes are attributable to the deficient oxygen-carrying
power of the blood. No direct observations upon the state of the organs
appear to have been made in this disease, although it is commonly assumed
that they are affected with fatty degeneration ; the dilatation of the heart
observed clinically being especially so explained. Observations upon the
blood-forming organs are equally wanting.

In chlorosis the most marked change in the blood is the excess of water—
hydremic plethora. The number of red cells in a given volume Is
diminished; the absolute number is increased; and similarly, whilst the
percentage of hemoglobin may be reduced to 40 or even to 30, the total
hemoglobin content remains normal, and the oxygen-capacity undiminished.

As the reduction in the oxygen-carrying power affects the tissues supplied
by the blood, so it may affect the nutrition of the leucocytes in the blood
itself.

In the particular case of chlorosis in which fat occurred in the leucocytes
the blood was taken at 2 o’clock p.m., the patient having had no food since

436 Messrs. 8. G. Shattock and L. 8. Dudgeon. [Mar. 18,

breakfast. The plasma, as studied in the blood films, contained free fat, the
presence of the latter, under the circumstances mentioned, being possibly due
to its less ready manipulation in consequence of the excessive bulk of water
in the blood.

The amount of fat in the finely granular leucocytes in this case is very
striking; every cell held a certain number of fine scarlet points or
droplets, and many are loaded. How much of the fat in the leucocytes is to
be ascribed to ingestion, and how much to degeneration, it is impossible
to say.

As in the case where cedema follows venous obstruction the transudation
occurs only after the stasis has le@ to damage of the endothelium, so in
a somewhat similar way, in the case of anemia, we should regard the
deficient oxygenation as involving injury to the leucocytes; the intra-
cellular fat appears in consequence. |

In the case of pyloric carcinoma there was an as§ociated chlorotic anemia
arising from the ill-nourished condition of the body in general, including the
blood-forming organs.

The fat droplets in the leucocytes, of which only a certain number were
affected, are of conspicuous size, the change being a far advanced one. No
free fat was present in the plasma.

And finally, in the case of diabetes and lipamia, in which typhoid infection
terminated life, the amount of free fat in the plasma makes it clear that
much of the fat in the leucocytes must be ascribed to phagocytic ingestion,
but how much to this, and how much to the toxic condition of the blood,
cannot be stated. The peritoneal fluid removed after death was full of fat
droplets. The bone-marrow showed a highly abnormal number of large
endothelial cells holding an abundance of multiple fat droplets. The gall-
bladder held no bile, and its contents were so white that they were at first
thought to be purulent ; microscopic examination revealed the presence of
free fat and shed columnar cells.

It is interesting to note, in passing, that the bases of these cells were loaded
with fat, which had evidently been ingested by them whilst in satu, as 1t 1s by
those of the intestine. The absorptive capacity of the epithelium of the gall-
bladder has been shown, also, by Aschoff.

The Presence of Granules in the Leucocytes, which Stain of a Deep Brown
Colour.—Scharlach Granulation.

From the foregoing list of diseases in which fat was found in the finely

granular polymorphonuclear leucocytes, some, in which a fatty degeneration
might have been particularly anticipated, are conspicuous by their absence.

1907. | Fatty Degeneration of the Blood. 437

_ Two such would certainly be diphtheria and pernicious anemia. In both
these diseases, however, we have found the leucocytes laden with fine
granules, which, though deeply stained, do not exhibit the transparency and
proper red colour of ordinary fat.

We may first give a list of the cases in which these deep brown granules
occurred; the phenomenon itself we may call, for convenience, Scharlach

granulation :— :
Subphrenic abscess. Diphtheria (6 cases).
Exophthalmie goitre. Appendicitis.
Pneumonia and empyema. Pericarditis (rheumatic).
Pernicious anemia. Acute meningitis.

In discussing the nature of these granules, various possibilities require
consideration. That the granulation is not a mere precipitate of Scharlach
within the cytoplasm of cells that have been imperfectly fixed 1s shown by
the fact that the diphtheria blood films (in which, amongst others, it occurred)
were fixed in formol vapour for over 24 hours.

It is noteworthy that the normal granules of the eosinophile leucocytes
exhibit a somewhat similar coloration, though of a less intense degree.
Against the identity of the granulation with the substance composing the
eosinophile grains, however, must be placed the circumstance that the latter
are equally well coloured in both pathological and normal blood films after

the films have been treated with absolute alcohol; the fine granules under

discussion, on the contrary, are not demonstrable by means of Scharlach if the
films are first treated with absolute alcohol.

The degree of Scharlach granulation, moreover, present in some cases
(every finely granular leucocyte being laden) excludes, we think, the explana-
tion that the granules in question are eosinophile, which have been discharged
from coarsely granular cells, disintegrated, and subsequently ingested by the
finely granular leucocytes. |

In this connection it may be further mentioned that no eosinophilia has
been present in any of the cases as a possible source of such an excessive
amount of Scharlach-staining substance.

The Scharlach granulation is not due to the coloration of the proper
granules of the cell ; for in some of the leucocytes comparatively few granules
occur, and in these cells the proper granules are recognisable, though
unstained.

The granulation is not unlike the glycogenic, as displayed in the leucocytes
after treatment of blood films with iodine solution. In most cases the iodine
reaction takes the form of a diffuse brown coloration of the cytoplasm of the

438 Messrs. 8. G. Shattock and L. 8. Dudgeon. [Mar. 18,

finely granular polymorphonuclear leucocytes, but it may take that of a fine
stippling. A still further likeness is to be found in the occasional presence
of similar iodophilic granules in the plasma; for fine extra-cellular granules
may occur also in the Scharlach preparations.

Although the iodophilic reaction may be seen in normal blood, in pro-
nounced degree it occurs only under pathological conditions, and observers
are agreed in viewing it as evidence of cell degeneration, brought about in
most cases by a toxic cause. |

We exclude this explanation, however, for the reason that the Scharlach
granulation is not to be observed in control cover-glass films if treated with
absolute alcohol before being transferred to the dye. The glycogenic reaction,
on the contrary, is as readily obtained after this treatment as without it.*

We have, moreover, found that glycogen is uncoloured by Scharlach. If
fat-free glycogen (from the rabbit’s liver) is dissolved in distilled water and
precipitated by alcohol, the wet sediment, when treated with Scharlach for
48 hours and examined microscopically, is seen to consist of fine spherical
granules which are quite untouched by the dye. The same sediment stains
of a mahogany brown when submitted (after drying on an albuminised cover
glass) to the action of iodine vapour for 20 minutes, and afterwards mounted
in Ehrlich’s iodine medium.

The absence of the Scharlach granulation when the blood films have first
been treated with absolute alcohol excludes the possibility of the granules
being blood pigment which has been ingested by the leucocytes.

We have found the Scharlach granulation in some of the finely granular
polymorphonuclear leucocytes in cover-glass films made from the splenic pulp
in a case of acute peritonitis, the films having been fixed in formol and treated
precisely like those of the blood. In this observation, also, the exclusion of
blood pigment is brought out by the fact that the deep brown granules are
not to be seen in the films stained by Leishman’s method.

Does the Scharlach granulation represent a substance in which fat is not
free, but combined? Is it allied to lecithin or myelin, both of which are
known to occur in many kinds of cell? Lecithin stained from egg-yolk is not
coloured with Scharlach.t

In sections of an enlarged prostate which we have examined, although the
presence of fat was demonstrated by Scharlach treatment in some of the
epithelial cells, the free granules in the acini (generally held to be lecithin)
were quite uncoloured.

By the same want of colourability, after treatment with formol, myelin

* J. Barnicot, ‘Journal of Pathology,’ vol. 11, p. 304.
+ T. R. Elliott and I. Tuckett, ‘Journal of Physiology,’ vol. 34, p. 350.

1907. | Fatty Degeneration of the Blood. 439

may be excluded. In medullated nerve fixed in formol and afterwards
teased and immersed in Scharlach solution for 24 hours, the myelin of the
individual fibres exhibits practically no coloration; it is equally unaffected
by osmic acid, if the nerve is first fixed in formol.

As regards the coloration of human fats, that is, of their fatty acids which
are the proper colourable substances, pure oleic acid is stained of the typical
red colour with 75-per-cent. alcoholic solution of Scharlach.

From human liver Leathes has extracted another fatty acid, the colour-
ability of which we have been able to test upon cover-glass smears. The
smear, after being treated with formol vapour, stained with Scharlach, and
finally washed in distilled water and mounted in Farrant’s medium, is trans-
parent, and of an orange or orange-brown colour; it presents nothing
suggestive of the Scharlach granulation.

From human heart-muscle Leathes has also extracted a fatty en other
than oleic, and not identical with that referred to from the liver.

The fact, however, that in fatty degeneration of this muscle, as studied in
diphtheria, the fat that appears is from the first stained of a brilliant red by
Scharlach shows that, whatever fat besides olein may be present, it is not the
substance constituting the Scharlach granulation.

There is, to conclude, the possibility that the granulation is due to a soap,
the colloidal nature of which prevents its diffusion into the substance of the
cell.

Various fermentative capabilities have been ascribed to the leucocytes.
They are certainly proteolytic ; and both amylolytic and lipolytic powers have
been attributed to them.

It is conceivable that the fat droplets, on their first appearance in the
leucocytes, might be split, with the resulting formation of an intracellular
soap arising from the combination of fatty acid with calcium, sodium, or
potassium.

Using pure oleate of calcium (the only soap of the three not soluble in
water), we find that cover-glass smears, after being treated as the blood
films, fail to exhibit the dark brown colour and opacity of the Scharlach
granulation ; the particles take a pale brown or orange tint.

For the present, therefore, we are forced to leave the nature of the
Scharlach granulation undetermined. Nevertheless, its appearance in
diseases in which a fatty degeneration of the leucocytes might have been
anticipated, and the fact that in some cases of acute pneumonia the
proper Scharlach reaction of the points in the cells is obtained, whilst in
others it is the brown opaque Scharlach granulation, makes us conclude that
the granulation is indicative of a degeneration; and seeing that it is not

440 Prof. E. W. MacBride. Some Points in the [May 6,

obtainable after the treatment of the blood films with absolute alcohol, we
conclude that it is allied to that known as fatty.

In conclusion, we desire to express our thanks to Dr. G. Buckmaster, and
to Dr. J. B. Leathes, for criticism and help in regard to certain of the points
raised by the investigation.

Some Points vw the Development of Ophiothrix fragilis.

By E. W. MacBripg, D.Sc., F.R.S., Strathcona Professor of Zoology in McGill
University, Montreal.

(Received May 6,—Read June 13, 1907.)

The subject of which the present communication is a brief preliminary
account has occupied my attention during the last five years, and a full
account will be published shortly. Meanwhile, a brief outline of the main
results is given here.

Ophiothri« fragilis is one of the commonest species of British Ophiuroidea.
Especially in the neighbourhood of Plymouth, where the material for this
research was obtained, this species is found in immense swarms, so that, on
certain kinds of bottom, hundreds of individuals are brought up with each
haul of the dredge. During the months of June and July it spawns, and on
some occasions the Plankton captured above the beds of gravelly sand on
which it lives is full of its larvee in ail stages of development.

A large quantity of this Plankton, captured during July, 1899, formed the
principal part of the material, but this was supplemented by artificial
fertilisation carried out in the laboratory of the Marine Biological Association
in the years 1898, 1899, and 1905.

The eggs are small (about 0:1 mm. in diameter) and opaque owing to the
possession of a reddish-brown yolk. The development was observed to be
completed in 28 days, but the rate of development depends on the amount of
food supply in the water, and it is probable that in the sea the eggs
develop more quickly than under the most favourable circumstances in the
laboratory.

Only on one occasion (June, 1898) was it found possible to rear the larve
through their development until metamorphosis was completed; this was
due to the presence of an unusual amount of Phytoplankton in the water

1907. : Development of Ophiothrix fragilis. 44]

on that occasion, but on other occasions an abundant supply of the early
stages of development was obtained, so that the gap in the series of larve
captured in the Plankton was completely filled.

If artificial fertilisation is carried out in the usual way, viz., by dissecting
out the ovaries and shaking them over a beaker of clean sea-water in order to
let the-eggs fall out, and then shaking in the water a piece of testis extracted
from a ripe male, the development takes place in the manner which I have
already described to the American Society of Zoologists (1), and which is
illustrated in figs. la, 1b, and le. The egg segments so as to give rise to
a solid mass of cells or morula.

Id. Te. i
Fias. la, b,c. Three stages in the abnormal development of Ophiothrix fragilis.
Mes., mesenchyme. Ce., ccelomic rudiment.
Fics. ld, e, f. Three stages in the normal development of Ophiothrix fragilis.

When the embryo escapes from the egg-membranes and commences to lead
a free-swimming existence it has an ellipsoidal form, and consists of an outer
mantle of columnar ciliated cells and of an inner mass of rounded cells. It
very much resembles the “planula” larva of a Ceelenterate. Subsequently an
invagination takes place at one pole of the egg, and the archenteron is formed.
But the invaginated cells do not form a single layer. A multiplication of
cells takes place at the vegetable pole before the invagination takes place,
and when this occurs it takes place in such a manner as to give rise to a sac
with a tongue of cells projecting into the lumen. A similar development in
the case of Ophiura brevis has been described by Grave (2).

The larva soon assumes a triangular form (fig. 1c), one angle being the

442 Prof. E. W. MacBride. Some Points in the [May 6,

posterior end of the animal, whilst the other two are the rudiments of the
postero-lateral ciliated arms, which in this species attain an extraordinary
length.

The interior cells of the morula are now seen to be the skeleton-forming
cells of the caleareous spicules which support these arms. The ccelom arises
as a vesicle from the apex of the archenteron, and shortly afterwards the
stomodzeum makes its appearance in the centre of the anterior surface. The
tongue of cells projecting into the archenteron is gradually absorbed.

Until 1905 no doubt existed in my mind that the course of development
above described was the normal one; but in that year, in addition to the
ordinary methods of artificial fertilisation, the device was tried of placing
males and females together in half-gallon jars filled with pure sea-water and
allowing them to spawn naturally. A vigorous culture was obtained in this
way, and the development differed in some important particulars from the
type described above. |

Segmentation results not in the formation of a morula, but of a thick-
walled blastula (fig. 1d), one side of which becomes specially thickened and
buds off mesenchyme cells. The embryo now escapes from the egg-membrane
and becomes a larva. Soon at the pole from which the mesenchyme was
produced a regular invagination takes place, with no trace of the tongue
of cells described above.

Mesenchyme cells continue to be budded off from the apex of the growing
archenteron. At the same time a conspicuous feature makes its appearance
at the animal pole of the larva. This is a crest of vacuolated cells (fig. 1e),
which has a triangular shape, and seems to be an apparatus for the floating
of the larva, which lasts until the ciliated arms are developed.

The ccelom next makes its appearance as a thin-walled sac, greatly
resembling in shape the ccelomic rudiment in the larva of Asterina gibbosa
(fig. 1f),and at the same time the rudiments of the postero-lateral arms appear.
The vacuolated crest diminishes slowly in size; when it first appears its
height is as great as the entire length of the larva, but by the time the
stomodeum appears it has disappeared. Previously to this the cclomic
rudiment has become separated from the gut as a single vesicle. This then
divides into right and left halves, and the stomodzum joins the archenteron,
which now shows beginning of the constrictions which mark it off into
cesophagus, stomach, and intestine.

Pausing for a moment to reflect on the meaning of the two types of develop-
ment described above, it is clear that the difference between them cannot be
due to the fact that in the one case the genital organ was dissected out, and
in the other case that the animal was allowed to spawn. In some cases larve

1907.] Development of Ophiothrix fragilis. 443

with a high vacuolated crest were obtained from artificial fertilisation. It
appears to be clear that we must assume that eggs are capable of fertilisation
before they are quite ripe, and that the fact that eggs can be fertilised is no proof
that they are fully ripe or that the resulting development is normal. This
conclusion has, I think, a somewhat important bearing on the experimental
studies for which the eggs of: Echinodermata have furnished the material.
Notably the statements which some authors have made about obtaining
ripe eggs from sea-urchins like Strongylocentrotus all the year round must be
received with great caution.

When engaged in the study of the development of Lchinus esculentus,
I noticed that if one attempted to fertilise the eggs of half-grown specimens,
larve with imperfectly developed arms resulted, which only lived a few days.

The fact, however, that the abnormal development of Ophiothria fragilis has
a considerable resemblance to the normal development of Ophiwra brevis is of
interest, as showing how far reaching in its influence on the subsequent
development is the condition of the egg at the moment of fertilisation, and
the idea is suggested that here, perhaps, is to be found the origin of variations.

We left the normal development at the moment when the ccelom had been
completely separated from the gut and had been divided into right and left _
halves. The ciliated band had been formed, and its first two outgrowths, the
postero-lateral arms, had appeared. The stomodzeum had joined the gut.

The madreporic pore is now formed by the union of an ectodermic ingrowth
with a short outgrowth from the left celom. The left ccelom is now distin-
guishable from the right by its more distinct lumen—an indication of the
fact that it gives rise to the so-called ampulla of the stone-canal. I have
searched in vain for any trace of a right pore-canal such as has been found —
by Grave in the case of some ophiurid larve. From both cceloms, muscle
fibres are developed which surround the cesophagus of the larva and endow
it with the power of peristaltic motion. The other larval arms then appear,
first the antero-lateral pair at the sides of the mouth and then the post-oral
pair at the sides of the anus. The ccelom now undergoes transverse division
on both sides into anterior and posterior cceloms, the division on the left side
being completed before that on the right (fig. 2). The posterior ccelom on
both sides is at first a solid nodule of cells which later acquires a lumen.
Next, the last pair of ciliated arms, the postero-dorsal, appear, and then the
anterior ccelom on both sides again prepares for division. As before, a
nodule of cells buds out from the posterior end, but this time the division
is not completed. On both sides the posterior portion, which will form the
middle division of the ccelom, is not completely separated from the anterior
division. On both sides it becomes hollow. On the left side it buds out

VOL, LXXIX.—-B. 2K

444 Prof. E. W. MacBride. Some Points in the [ May 6,

five finger-like prolongations (fig. 4), thus proclaiming itself to be the rudiment
of the hydroccele, or water-vascular system of the adult. Sections demonstrate
that its persisting connection with the anterior ccelom which constitutes the
stone-canal, as in the case of Asterina gibbosa, is situated between lobes 1
and 2.

Fig. 2.—Diagram of larva, showing ccelom divided on left side and dividing on right side.

Fie. 3.—Diagram of larva, showing formation of rudiments of hydrocceles from the
posterior ends of the anterior coelomic sacs.

Fie. 4.—Diagram of larva, showing first stages of metamorphosis. Notice migration of
pore towards the right. Jl.a.c., left anterior ccelom; J.p.c., left posterior
ceelom ; U.hy., left hydroccele ; l’.p'.c’., right horn of left posterior ccelom ;
7.a.c., right anterior celom; 7Ay., right hydroccele ; 7.p.c., right posterior
coelom ; .c., pore-canal.

The metamorphosis of the larva into the adult form now commences, This
is initiated by a preponderant growth of the left side. The hydroccele enlarges,
its lobes grow longer, and its anterior end commences to extend dorsally to the
stomodzeum (fig. 4). Soon the left antero-lateral arm is involved and is pushed
over to the right (fig. 4). This is the first sign of the metamorphosis visible
to the naked eye. As metamorphosis proceeds, this left arm comes to lie close
to its fellow of the right side, and the first lobe of the hydroccele becomes
completely dorsal to the cesophagus. The left posterior ccelom sends a
horn over to the right side beneath the cesophagus, and the posterior end
of the hydroccele extends over to the right, parallel to this horn, eventually
meeting the anterior end and converting the hydrocele into the water-
vascular ring.

The movement of the anterior end of the hydroccele carries with it, as
already indicated, all the neighbouring parts of the animal, so that the pore-
canal and left anterior ccelom are carried over to the right and thus come into
close proximity with the right hydroccele, which occasionally, as is shown in
fig. 4, 7.dy., undergoes a development similar to that of the left side, presenting
five lobes. Often it presents two lobes, but normally it is a thin-walled
vesicle without lobes.

(a r
: y
.

1907. | Development of Ophiothrix fragilis. 445

In Asteroidea and KEchinoidea, the right hydroccele is from the beginning
close to the dorsal pore, and on this account Masterman (3) and Goto (4) have
called it the central ecelom and denied its homology with the left hydroccele.
In Ophiothrix fragilis there can be no question on the subject, for the right
hydroceele is, from the beginning, on the right side of the larva, and its dorsal
position in the other groups may be explained by supposing that it is right
with regard to the adult position of the mouth. As the adult mouth
is on the left side of the larva, right with respect to this mouth will be
nearly mid-dorsal with respect to the larval mouth. But in Ophiuroidea,
where the larval mouth persists as the adult mouth, the right hydroceele is on
the right of it from the beginning, and the pore, originally situated on the
left side, moves round so as to assume a mid-dorsal position.
Little remains to be added in this preliminary account. The arm rudiments
are outgrowths of the left posterior ccelom and, counting from the dorsal end,
‘arm rudiment No. 1 comes to support hydroccele lobe No. 2, as in Asterina
gubbosa, whilst arm rudiment No. 5 supports hydroccele lobe No. 1. As in
Echinus esculentus, inter-radial ridges alternating with the lobes of the
hydroccele grow out and extend outgrowths to the right and left, so as to
roof over what would be the ambulacral grooves and thus form the epineural
canals. The perihzemal canals also arise from hollow inter-radial outgrowths
of the left posterior ccelom, except that the first arises, as in Asterina gibbosa,
from the axial sinus. The arms of the larva gradually disappear, except
the two postero-lateral, which are only cast off when the larva has sunk
to the bottom and metamorphosis is complete.

LIST OF PAPERS CITED.

1. MacBride, E.W. “Some Points in the Development of Ophiothrix fragilis,” ‘ Proc.
American Soc. of Zoologists,’ Philadelphia, 1903.

2. Grave, Caswell. “ Ophiura brevispina,” ‘Mem. from Biol. Lab. Johns Hopkins,’ 45.

3. Masterman, A. “The Early Development of Cribrella oculata, with Remarks on
Echinoderm Development,” ‘Trans. Roy. Soc. Edin.,’ vol. 40, Part 2.

4, Goto, S. “The Metamorphosis of Asterias pallida, with Special Reference to the Fate
of the Body Cavities,’ ‘Contributions from the Zool. Lab. of Mus. of Comp.
Zool. at Harvard,’ No. 88.

2 KZ

446

Croontan Lecrure.—On the Structural Constituents of the
Nucleus, and ther Relation to the Organisation of the
Indinidual.

By J. BRETLAND FARMER, D.Sc., F.R.S.

(Received May 13,—Lecture delivered April 25, 1907.)

The latter half of the nineteenth century witnessed three events of
supreme importance to biology. The first of these consisted in that
reasoned theory of the mode of origin of new species with which the name
of Charles Darwin will always remain associated. The second lay in the
discovery, made by Strasburger in 1875, that the nucleus is not only
a permanent organ of the cell, but that certain definite constituents of it are
transmitted in unbroken sequence from one cell generation to another.
Thirdly, Oscar Hertwig, also in 1875, showed that fertilisation consists not
only in the union of male and female cells, but that the union of the two
nuclei forms an essential part of the process.

At the present time, when evolutionary problems are being attacked at
their very roots by the experimental study of variation, results are being
accumulated which are capable of being dealt with from a cytological
standpoint. Much is to be expected from a joining together of the forces
engaged on what are really only different aspects of the same problem.
What we really want to know is the nature and mode of working of the
machinery which is responsible for the appearance of the characters
manifested, as well as inherited, by the organism. We also are concerned
with the nature of those inner changes which find their outward expression
in what we designate as variation.

Whilst I do not of course pretend that all these questions can be
satisfactorily answered at present, so many new facts which bear upon them
have come to light within recent times that I venture to indicate some of
what I conceive to be the more important results that have been obtained.

Every animal and plant either consists of a single cell, or it is made up of
a cell-colony the members of which have had a common origin, inasmuch as
they have all sprung from one mother-cell, the egg. And it is in the cells
that the properties of an individual must ultimately be sought.

The more closely the cell is studied, the more irresistibly are we compelled
to admit the supreme importance of the nucleus in directing and controlling
its metabolic activities. Experiments have abundantly proved that if a
nucleated protoplasmic mass be divided by artificial means, so that one

On the Structural Constituents of the Nucleus, etc. 447

portion contains, while the other lacks, the nucleus, the former alone is able
to continue to discharge the ordinary vital functions, and to manifest those
chemical and physical properties which necessarily underlie them. The
enucleated fragment, on the other hand, although it may persist for some
time, nevertheless loses, more or less rapidly, all those special faculties which
should enable it to maintain a separate existence. Synthetic and secretory
activities disappear, and death sooner or later supervenes.

Seeing, then, that the nucleus plays so fundamental a part in maintaining
the due sequence of chemical change in the cell, the question naturally
arises as to whether all those properties of the individual which find their
expression in form, and in other externally recognisable characters, may not
be attributed to the interaction of the nucleus with the cytoplasm external
to it; whether they are not merely the inevitable result and outcome of the
series of material changes that succeed each other within the confines of
the constituent cells. I believe the answer must be in the affirmative. <At
the present time, however, such a general statement has but little value
beyond expressing the general direction in which the many lines of investiga:
tions appear to be advancing. We are not likely, within the near future, to
be in a position to trace all the stages which intervene between the importing
of raw material and the turning out of the finished fabric. Modern research
clearly indicates that the proximate problems are more intricate than they
were formerly supposed to be. Nevertheless, I venture to think that the
very complexity of the regulatory processes emphasises, rather than
diminishes, the probability that a comprehensive physical explanation of
organisation and inheritance will one day fall within our reach. But the
final synthesis can only be made possible when we have understood the
details, when the more immediate problems have been clearly stated and
successfully solved.

Some of these problems are already in process of solution; thus numerous
investigators during the last 30 years have done much to clear up the
essential features of fertilisation, although it cannot be said that as yet there
exists complete agreement on all points. For while it has been generally
conceded since Hertwig’s discovery, already alluded to, that the nuclear, and
not the cytoplasmic, association constitutes the significant part of the process,
there are still some who hold that the fusion of the cytoplasm is of direct
and material importance in the transmission of the hereditary properties of
the individual.

As this is a matter of great weight, an examination of the facts yielded by
a study of fertilisation is demanded. It is true that the male gamete carries
with it into the egg a small amount of cytoplasm. On penetrating the egg,

448 Prof. J. B. Farmer. On the | May 13,

the nucleus escapes from its cytoplasmic investment which then disintegrates
and mixes with that of the egg, whilst the two nuclei approximate and often
fuse. Although the amount of cytoplasm thus introduced is exceedingly
minute, as compared with that of the egg, probably no one at the ‘present
time would regard this circumstance as necessarily implying a correspondingly
small degree of pe unless other evidence pointed also to the same
conclusion.

Boveri has shown that when an enucleated fragment of the egg of one
species of echinoderm is fertilised by the sperm of another species, or even
genus, the resulting larva resembles the male parent. These statements
have been sometimes challenged, but the explanation of the discrepant
results obtained by different observers probably les in the fact that the eggs
themselves may be readily stimulated to develop parthenogenetically.
Enucleated fragments of eggs already in this condition, even in the earliest
stages, will have already received a developmental stimulus of a material
nature from their own («e., the female) nucleus. This must inevitably
produce an effect on the final result. The recent discovery* that if eggs which
are known to be in the early stages of parthenogenetic development are
fertilised by the sperm of another species, the offspring actually do show
a markedly increased resemblance to the maternal parent, is of the highest
theoretical interest in connection with the mode of interaction between the
nucleus and the cytoplasm.

The entrance of more than one sperm, and the fusion of Shei nuclei with
that of the egg, is commonly followed by great disturbance in the course of
development of the embryo, if, indeed, it is able to proceed at all. It is
remarkable how very seldom polyspermy occurs in healthy cultures. I have
myself examined many thousands of the eggs of Fucus, and I have only seen
two or three examples of the entrance of more than one sperm into each egg.
In some cases, however, polyspermy can. be readily induced by anesthetising,
or in other ways interfering with the normal conditions of the egg; and the
abnormal results that ensue serve to emphasise the correlation existing between
nuclear constitution, and cell- and tissue-development.

The rarity of polyspermy, at any rate amongst the majority of plants, points
to the existence of some means of actively preventing its occurrence in
ordinary eggs. It is often stated that a cell wall is instantly excreted over
the surface of the egg on fertilisation, and that in this way the entrance of
additional sperms is prohibited. Such an explanation, if, indeed, it ever
suffices, will not cover every case. In some eggs, as, for example, those of
the Fucacez, the membrane is not formed till an appreciable time has elapsed

* Curt Herbst, “Vererbungsstudien, IV,” ‘ Arch. f. Entw.-Mech. d. Organismen,’ vol. 22.

1907. | Structural Constituents of the Nucleus, etc. 449

after fertilisation. Hence the reason for the exclusion of the extra sperms
must be sought in another direction. It seems probable that the cause of
exclusion is connected with a sudden chemical change immediately resulting
from the entrance of the first sperm into the cytoplasm of the egg. I shall

show that in one case, at any rate, there is direct evidence of such a change

in the reaction of the egg, and it may be remarked that it is quite intelligible
that such a reaction should be affected or even arrested by anzesthetics, or other
substances that interfere with the proper metabolism of the cell.

In the first place, it is certain that sperms are attracted to the egg by
specific substances which are excreted from it and diffuse into the surrounding
medium. Different sperms are affected by different substances, and what will
attract those of one plant may be entirely without influence on those of
another. For example, if two vaccine tubes, filled with very weak solutions
of sugar and malic acid respectively, be placed in a vessel containing a mixture
of the actively motile sperms of a moss and of. a fern, those of the former will
crowd into the tube containing the cane sugar, those of the fern into that
filled with the solution of malic acid.

Since, then, the swarming of the sperms towards the egg is effected as
a response to a definite chemiotactic stimulus, it is inherently probable that
a correspondingly negative or repellant stimulus provides the egg with the
means securing it against the entrance of more than one sperm. We have
been able to show that this is the case in several of the Fucacee. If the
large eggs of Halidrys be allowed to extrude into sea-water together with the
sperms, the latter will, very shortly, be found to congregate on the surface of
the egg. Each sperm possesses two cilia, with one of which it holds on to the
egg, whilst with the other it lashes the water, endeavouring to force an
entrance through the limiting layer of the protoplasm. So active are the
movements that the relatively huge egg is seen to be rocked about in the
‘water. In a short time a sudden change takes place in the egg. It swells,
and then in a moment it becomes rugose or prickly first at one spot, and
thence rapidly over its whole surface. At the same instant, all the sperms
that were moving over its periphery rapidly leave it, and those that are
unable to detach themselves sufficiently quickly are seen to become
immediately paralysed and to die. Soon afterwards the egg shrinks in size
and becomes quite smooth again. Later on a membrane is excreted over its
surface, proving definitely that fertilisation has occurred.

In this instance, then, the case for the emission of a repellant, and even
lethal, substance from the surface of the egg as the result of fertilisation is
quite clear, and it perhaps diffuses from the fine mucilage-like threads that
are shot out from the rugose periphery. Since making these observations on

450 Prof. J. B. Farmer. On the [May 13,

Halidrys, I have been able, though not so easily, to detect the repellant effect
in some other Fucacez. Still more recently, in an investigation on ferns
involving the details of fertilisation, we have observed that after one
sperm had entered the egg, the others close to its surface showed signs of
greater disorganisation than those which were further removed from it. In
this instance also, then, it seems clear that the entrance of a sperm produces )
a chemical change in the egg which is reflected over its whole surface, although
we are not as yet in a position to make any definite statement as to the
precise nature of the substance concerned,

It is another question whether the zhange is produced directly in the
cytoplasm, or whether it emanates in the first place from the nucleus.
Although certain facts seem at first sight to favour the latter view, I think
the former is the more correct one. In many gymnosperms the two sperms
which are formed in the pollen tube are both extruded into the egg. But
only one of them is functional; the other soon disintegrates in the cyto-
plasm, and does not approach the egg-nucleus. But it must be borne in
mind that the sperms are rather passively shot into the egg from the pollen-
tube, than individually attracted into it. In Cycads, however, where the
sperms are actively motile, only one enters the egg as a rule, and when more
do succeed in penetrating it, they always perish at once. It may be
suggested that as the egg of these plants is of enormous size as compared
with that of the Fucacee, the change which is produced instantaneously
in the latter may take longer to complete in the case of the gymnosperms.
In any event it docs take place, and the destruction of the additional sperms
occurs in time to prevent them getting near the nucleus of the egg.
Further evidence in favour of the cytoplasmic origin of the repellant
substance is again afforded by Halidrys. It often happens that the eggs, as
they are being shed, are broken into two pieces, of which one is destitute of
a nucleus. Such fragments, however, as Williams has shown, commonly:
attract the sperms, and on the entrance of one of them the same repellant
influence on the rest is as evident as in the case of normal eggs. Wilson,
also, found conclusive evidence to show that when enucleated fragments
of echinoderm-eggs are fertilised, only a single sperm is concerned in the
process. |

These observations seem to me to prove that the essential act of fertilisa-
tion consists not only in the union of two, and not more than two, nuclei,
but also in the retention by them of a definite structwral basis. For we have
seen that the mere addition of nuclear material cannot be regarded as the
important act, inasmuch as any extra sperms entering the egg are without
influence on development, prejudicial or otherwise, provided that, as in the

1907.] Structural Constituents of the Nucleus, ete. 451

gymnosperms, they are destroyed so that their structural individuality is lost.
That the egg-cytoplasm is essential as building-material is, of course, not
disputed, and in so far as it consists of different substances, these will have
different prospective values in the final result. In the case of many animal
eggs, the cytoplasm shows physical differences in its various regions, and there
is reason to think that this diversity in material is, at any rate sometimes,
connected with differences in the structures finally to be developed. If, then,
the ege be mutilated, so that a definite kind of raw material is abstracted
from it, an imperfect embryo might be looked for, unless the removed raw
material could be regenerated. The nucleus, as architect, would otherwise
only be able to build a body correspondingly defective in certain parts. But
such considerations as these do not weaken the position of the nucleus as the
agent responsible for the particular direction and control of development
which is followed by a cell, or, collectively, by a group of cells. In short,
just as we have the strongest grounds for associating secretory and other
metabolic activities of the cell with the nucleus as the prime mover, 80, also,
various lines of evidence indicate the same body, or rather certain con-
stituents of it, as guiding the course of those chemical transformations
in the cell and in the organism which make themselves apparent in
characters.such as form, colour, and the like, which can be appreciated by our
senses.

I have indicated the possibility that certain constituents of the nucleus,
rather than this body as a whole, may be charged with the control and
direction of cellular development. It remains therefore to consider the
evidence on which such a view, which in some form or another has often been
put forward, is based. Darwin, Weismann, De Vries, and others, have each
suggested the existence of particles which are responsible for the characters
of the individual. And I may at once say that, apart from some such material
units, |! am unable to understand how the large mass of facts of which we are
now in possession can be explained.

Within recent times our knowledge respecting the results of breeding, and
also respecting the details of cellular organisation, has been greatly widened,
and I believe that the evidence thus drawn from two entirely different
sources irresistibly points to the same conclusion, and that the existence of
discrete units which the breeder, on purely theoretical grounds, has been
compelled to assume, is entirely borne out by the observed presence of
certain structures which exist in vast numbers in the nucleus. These
structures enter into the composition of those remarkable bodies, the chromo-
somes, which form such striking objects in the nucleus at the periods of
division. Of course, I need not suy that the particles in question are not to,

452 Prof. J. B. Farmer. On the [May 13,

be regarded as the immediate characters themselves; they may, perhaps,
be roughly compared with that class of bodies known as ferments, setting up
in the extra-nuclear cytoplasm those changes which, in their sum-total,
constitute development, and producing those chemical re-arrangements of
which form, colour, etc., are the visible expression.

It will be convenient, in the first place, to examine the results obtained
from experiments in cross-breeding, a field in which Bateson and others have
achieved so great a measure of success. We can afterwards try to relate these
results with the salient facts of nuclear structure which have been elucidated
within recent times.

If two pure parents which differ in, say, one character from each other be
chosen and mated, the offspring almost always behaves in a definite and
uniform manner with regard to the character in question. Often it appears
as if they all inherited the character of one parent only, that of the other
having been entirely lost. But on allowing the hybrids to interbreed, it soon
becomes plain that the alternative character was only latent, and not lost at
all. It reappears in a fixed percentage of the offspring, usually to the extent
of 25 per cent. When these individuals, thus exhibiting the recessive
character, are mated, they continue to breed true, and the alternative
dominant character, which replaces the recessive one in 75 per cent. of the
individuals of that generation, never reappears amongst the recessive progeny
so long as they interbreed only with one another.

As regards the individuals which make up 75 per cent. of the second
(the £2) generation, it is found that whilst all of them show the dominant
character exclusively, only one-third continues to breed true, whilst two-thirds
of them, when mated together, prove to be hybrids; and in the next generation
these continue to throw out one-quarter pure dominants, one-quarter pure
recessives, and one-half hybrids. This is a remarkable result, and although it
is not always directly or obviously obtained, it occurs in so large a number of
instances as to prove sufficiently that the characters in question do behave as
independent units. It is not surprising that there should be apparent excep-
tions to the rule; some of them have already been shown to be due to an
incomplete analysis of the real characters themselves, so that composite features
have been mistaken for units. But, at any rate, in the presence of so large a
number of cases which do conform to the Mendelian rule, they may be safely
left on one side so far as our present purpose is concerned, which is to find
an explanation of the numerous positive instances that are everywhere
recognised.

It is possible to make certain statements, which almost partake of the
nature of inevitable conclusions, from a consideration of the mode of

1907. | Structural Constituents of the Nucleus, ete. 453

behaviour of those alternative characters so well termed allelomorphs by
Bateson. In the first place, it is difficult to escape from the inference that

the unit characters must be due to material primordia which are ultimately

responsible for their appearance, and that these several primordia have a
separate and persistent individuality of their own. The union of the
primordia at fertilisation, as judged by. the results, rather resembles a
mechanical mixture than the formation of a chemical compound.

Inasmuch as two sexual nuclei unite at fertilisation, each one carrying with

it characters of that parent from which it originated, it is clear that each
sexual cell or gamete must possess, in its nucleus, half the whole lot of the
primordia that will be contained in a fertilised egg. Furthermore, the
manner in which the characters are distributed amongst the members of any
one generation is such that no sexual cell can contain more than a single
individual of any given pair of allelomorphs which are present in the body
of the organism producing the sexual cells. This circumstance is expressed
by the term “ Purity of gametes,” and it implies that, as regards any particular
character, the primordium contributed by one parent only will be present in
a gamete, the other primordium or allelomorph having been distributed to
another gamete. When two gametes unite having identical primordia of any
character, the resulting offspring must be pure bred as regards that character,
and their progeny, if interbred, will keep true. But this is only so for each
separate character ; the different characters behave quite independently of one
another. Thus, if we take Aa as representing one pair of allelomorphs, and
Bd as representing another distinct pair, no gamete can contain A together
with a, or B together with 6, but it can contain either member of each pair,
1.¢., AB, Ab, aB, or ab. When two of these gametes unite, it can easily be
seen that the characters will be combined as I have already stated.
_ The assumption as to the purity of gametes, which is in accordance with
the facts it attempts to explain, is in harmony with cytological observations,
and therefore may be accepted as representing more than a mere working
hypothesis. :

The clear results of statistical investigations of this kind seem to me to
completely dispose of a view of fertilisation, otherwise quite possible, as being
of the nature of a chemical union of the fusing nuclei. The facts conclusively
point to the inference that there is only a mechanical mixture of the
structural units contained in each of the sexual nuclei that take part in the
act of fertilisation; the units retain their own identity, and they are again —
sorted out, though in different combinations, in connection with the differen-
tiation of the sexual cells for the next generation.

We may now turn to consider the structure of the nucleus, and enquire

454 Prof. J. B. Farmer. On the | [May 13,

4
into the nature of the evidence for the existence of permanent structures
within it which will agree with what. seems to be demanded by the facts of
heredity as elucidated by experiments on breeding.

The resting nucleus is externally hmited by a membrane, within which is
contained a gelatinous, more or less reticulated, framework, the linin; and
this is bathed in a more fluid medium, the paralinin. There are also one or
more spherical bodies, the nucleoli, which form storehouses of materials that
become separated and used up during times of nuclear activity. As long as
the nucleus is in the resting condition, it betrays little of that intricate
structure which it exhibits at periods of its division. Much of the complexity
apparent at those times must be attributed to temporary physical changes
proceeding in the colloidal masses which are then undergoing rapid change of
state ; but underlying these there is a permanent structural configuration
which is constantly preserved and is a determining factor in controlling the
course of those cellular changes on which the organisation of the individual
ultimately depends.

When a nucleus is about to enter on division, the linin becomes impreg-
nated with a substance termed chromatin, belonging to the nuclein class of
compounds. The chromatin, or its precursors, is mainly to be found in the
nucleoli as long as the nucleus is at rest; it is only during the period of
division that it becomes aggregated in the linin. But the chromatin is not
evenly distributed through the linin. It appears as very regularly arranged
discontinuous swellings in the thread. These small swellings, formed of
chromatin and linin, have been called chromomeres. The linin, made up of
alternate discs of chromatin- and non-chromatin-containing segments, now
either forms a convoluted filament, which finally breaks up into a definite
number of segments, the chromosomes ; or else these bodies are visible at the
first onset of division as discontinuous areas of linin mapped out by the
accumulation of chromatin in groups of chromomeres. The chromosomes are
commonly stated to be constant in number for a given organism, but this is
certainly not universally true, especially in the vegetative, or somatic cells.
On the other hand, in the reproductive cells the number is on the whole very
uniform, and even peculiarities of form distinguishing certain chromosomes
may continually recur.

The chromosomes then take up definite positions at the equator of the
nucleus, and each chromomere in the thread has already split into two exactly
similar halves. This carries with it the longitudinal fission of each chromo-
some as a whole, and the two halves then move apart and are finally
distributed, one to each of the two daughter nuclei which are thus formed.

The chromosomes are the only parts of the original nucleus of which it

1907. ] Structural Constituents of the Nucleus, ete. 455

can be certainly affirmed that they are equally shared between the two

daughter-nuclei. Seeing, then, that the net result of the process is to

accurately divide the chromosomes (and consequently the chromomeres) by
longitudinal fission, it would seem to be immaterial whether the actual
numbers of these bodies remained constant or not, at any rate in the somatic
divisions. The uniformity so commonly observed, especially when coupled
with the occasional departure from it, probably indicates that the number
typically present is to be taken as an expression of the organising function of
the cell as a whole, rather than as evidence of the independent nature of the
chromosomes themselves.* And this point of view is intelligible when we
reflect that they are themselves only organised bundles of chromomeres.

At fertilisation, two nuclei unite, and their chromosomes consequently
mingle. The immediate descendants of the cell thus produced are all
similarly provided with double the number of chromosomes contained in the
nuclei of the sexual cells before fusion occurred. It is, of course, clear that,
in any organism, a limit to such duplication must soon be reached. It is
found, as a matter of fact, that each act of fertilisation, with its concomitant
doubling of the chromosomes, is associated with a correlative process of
reduction to one-half. To this process the name of Mevosist has been given.
The way in which meiosis is effected is surprisingly uniform in both animals
and plants, and its physiological importance may be gathered from the fact
that, whilst it occurs in every organism that reproduces itself sexually, it is
not bound up with morphological periods in the life history. It antedates
all morphological differentiation, and is only directly related to sexuality, of
the continuance of which it is both a consequence and a condition.

The great theoretical interest attaching to the phenomenon of meiosis lies
in the convincing proof which it affords of the existence of persistent
structural units which are directly responsible for the appearance of the
various characters manifested by the developing organism.

It was formerly held by many investigators that the significance of
fertilisation consisted merely in doubling the amount of chromosome-
substance, and that reduction merely paved the way for this addition of bulk.
The mass was supposed to act as a whole, rather than to be made up of
separate and independent units. All the results of recent investigations have

* Much has been made of the fact that the chromosomes of successive cell generations
can sometimes be almost certainly identified with those that went into rest during the
previous telophase. But it should be remembered that such may easily happen,
especially when divisions succeed each other rapidly, for the chromosome substratum
chiefly consists of colloidal substance, which would only slowly lose its individuality under

quiescent conditions.
t Farmer and Moore, ‘ Quart. Journ. Micr. Sci.,’ vol. 48.

456 Prof. J. B. Farmer. On the | May 13,

tended to upset this view, and to prove that just as the body itself is made
up of individual cells, each an entity in itself, so also, and even more emphati-
cally, the permanent elements of the nucleus are structurally distinct and
physiologically diverse.

Reduction or meiosis is no mere halving of the bulk of the chromosome-
substance; its significance is to be discerned rather in the sorting out and
distribution between two daughter cells of the structural entities—the
primordia of characters—which are handed on from one generation to
another.

We may now follow, as briefly as possible, the way in which the chromo-
somes behave during the meiotic divisions, and contrast the process with that
which obtains during an ordinary or somatic fission.

In the first place it is observed that as the nucleus advances towards
division, and the chromosomes are able to be identified within it, their number
is only half of that characteristic of the nuclei of the preceding cell
generations that have arisen from the fertilised egg. A careful study of the
facts has shown that this reduction is as yet only a false one, and that the real
process is far more complicated. For each apparent chromosome is in reality
made up of two chromosomes which have become temporarily united together.
This union is, for a time, very intimate, and we have good grounds for saying
that it is not mere chance which determines which two particular chromo-
somes shall unite to form a pair. For example, it may happen that two
chromosomes in a premeiotic nucleus are different from the rest. Sometimes
there are several dissimilar pairs. At meiosis, the pairing always takes place
in such a way that like joins with like, to form the pseuclo-chromosomes, as
these bodies have been well called.

During the earlier stages, each of the paired chromosomes divides longitudinally, as in
an ordinary somatic division, but the two halves do not yet become separated from one
another. When the time comes for the splitting asunder of the two daughter-chromo-
somes to form the two nuclei, we find that the paired individuals now disunite and travel
apart. When this has taken place, and the two daughter nuclei have been formed, the
longitudinal fission, to which I have already referred, finally takes effect, and the two
nuclei at once proceed to divide again. Each chromosome splits along the line of
cleavage already marked out during the previous division. The process essentially
resembles that of a somatic fission, but with this important difference, vz., that the
reduced number of chromosomes is retained. And this feature continues to mark all the
nuclei of the post-meiotic cells, until the number is again doubled at the next act of
fertilisation.

The net result of the meiotic phase is to effect the reduction in the number
of chromosomes, and hence of the chromomeres, by the device of distributing
half of the entire duplicated stock to one, and half to the other, nucleus,
I would remark, in passing, that this fact points to the conclusion that the

1907. ] Structural Constituents of the Nucleus, etc. 457

real importance of the chromosomes lies more in their structure as bundles of
chromomeres than in their material composition alone.

I have referred to the peculiar circumstance that simclar chromosomes pair
to form the pseudo-chromosomes. It is, of course, evident that one of each

of them will pass into the daughter-cells at meiosis, and hence ultimately into

the nuclei of the gametes, which are the direct descendants of these cells.
But it is also evident that when the gametes unite at fertilisation, not only
will the chromosomes be doubled in number, but that the peculiar ones when
present at all will again occur in pairs. The special interest of this lies in the
indication that the chromosomes which pair at meiosis are derived from the
male and female parents respectively.* This being so, meiosis is seen to be
invested with profound significance in relation to the sorting out of the
structural elements originally contributed by the parents, and the distribution
of entire sets of them, but in different combinations, into the sexual cells.
Thus the facts of meiosis are seen to fall completely into line with the
conclusions drawn from experiments on breeding as far as the numerical
distribution of characters is concerned.

That this pairing of the chromosomes is a real fact, and not a mere fanciful
interpretation put on a complex series of phenomena, is abundantly proved
by the work of the last few years. I will give two examples in illustration.
It is well known that hybrids are frequently sterile, and this is often found to
be associated with the failure on the part of the organism to accomplish the
reduction of the chromosomes satisfactorily at the meiotic phase. This may
happen if the chromosomes contributed by the two parents are unequal in
number, and Rosenberg showed that it occurs in the hybrid between two of
our common Sundews. Drosera rotundifolia has 10, and D. longifolia has
20 chromosomes in the sexual nuclei. When these two species are crossed,
the somatic cells of the hybrid will, of course, exhibit 30 (10+ 20) chromo-
somes in their nuclei. Hence, at meiosis, it might be expected that there
would be 15 pseudo-chromosomes produced. Instead of this, the number is
seen to be 20. But, on examination, the 20 prove to be made up of 10 large
and 10 small ones. What has happened is that the 20 contributed by the one
parent, as well as the 10 furnished by the other, have retained their autonomy,
but the 10 from D. rotundifolia have united with 10 of the 20 belonging to
D. longifolia. The small ones represent the remaining 10, for which there
were no mates. The statement that at meiosis the pseudo-chromosomes are

* T have purposely avoided discussing in detail the peculiar heterochromosomes that
have been observed by Wilson and others in insects, as we are not at present in possession
of sufficient evidence to fully estimate their possible significance.

458 Prof. J. B. Farmer. On the | May 13,

composed of individuals derived from the male and female parents respec-
tively could hardly receive stronger support.

The second example I will take from a hybrid fern which is at present
under investigation in my laboratory. The plant in question, Polypodiwm
Schneideri, is the result of a cross between P. aurewm and P. vulgare var.
elegantissumum. The nuclei of the former contain 35 chromosomes after meiosis,
whilst P. elegantissimum has about 97 or 98. The chromosomes of the hybrid,
at meiosis, instead of exhibiting a mean between the two parents, 2.¢. 66, has
a much larger number. There is a little variation in the actual numbers,
but the average les between 90 and 100. That is to say, the quantity
characteristic of the elegantissimum parent is retained. Besides this, we find
at meiosis a large number of small, unpaired, chromosomes; these obviously
represent the surplus over and above those furnished by the awreum parent.
At division they betray their real nature by passing in their entirety to one
of the daughter-nuclei, when these are formed. |

Facts such as those I have recounted seem to emphasise the permanent
individuality of the chromosomes, and indeed they have often been appealed
to in this sense. The case is further strengthened by the not unfrequent
occurrence in cells, before they have undergone meiosis, of one or more pairs
of chromosomes that are different from the others, a circumstance to which I
have already alluded.

Moreover, the mode of sorting out, and the general behaviour of the
chromosomes throughout the cell-generations of the organism, are at first
sight in striking accord with the results of statistical investigations on
heredity, and they are looked upon by many people as representing the
primordia of the characters which we have already considered.

But I believe there is a fatal objection to this point of view, and one which
conclusively proves that we cannot regard the chromosome itself as the
structural unit responsible for the characters of the organism. The objection
is based on their relatively small numbers, and it was urged, though in
a somewhat different connection, by Weismann, many years ago. I will
illustrate my meaning by one example, and I wish to express my thanks to
my friend, Mr. A. D. Darbishire, for kindly collecting the facts of the case.
In the two pure races of Pisum sativum and FP. arvense, there are no less
than 18 pairs of characters respectively which in the hybrids behave as
allelomorphs. Since, however, there are only seven chromosomes in the
nuclei of the gametes, it is evident that the 18 allelomorphs could not all
occur independently of each other, but that, if the chromosome be adopted
as the unit, the allelomorphs would appear in at most seven groups, and
further, that the members of each group would always appear in association.

1907. | Structural Constituents of the Nucleus, ete. 459

Now such a correlation does sometimes occur in respect of a few characters,
but it becomes relatively insignificant when the total number of independent
pairs of allelomorphs is borne in mind. And if this causes difficulty in
dealing with relatively few characters, it obviously gathers in force when the
vast numbers of features which distinguish an organism are remembered.

_ But if for the chromosomes we substitute the chromomeres as representing
the discrete units which are responsible for the production of the hereditary
characters, the last objection loses its force. The chromomeres, consisting of
special chromatin-containing linin bodies, are present in great quantities; so
numerous that, as far as I am aware, they have never been counted in
a single chromosome. They fulfil the condition of being distinct entities ;
and also, at meiosis, the divergent chromosomes contain, or rather are made
up of, chromosomes arranged in two opposing groups, and derived from
different parents.

At the same time, I am aware that there are certain difficulties introduced
by this suggested substitution of the chromomeres for the chromosomes,
though I think these are more apparent than real.

The cardinal facts which we have to take into account are: first, the large
number of the allelomorphs and, secondly, their mutual independence. These
two circumstances, taken together, are irreconcilable with a view that would
regard the allelomorphs as invariably assigned to particular chromosomes. But
if we attach to the chromomeres, or to any other still smaller particles, the
properties of separate character-producing substances, the widely entertained
view as to the real structural persistence of the chromosomes themselves will
require some modification. or in order to give that complete independence
observed to exist between most of the allelomorphs, it is clear that any given
chromosome must be correspondingly indifferent as to which chromomeres
enter into its composition.* This, however, amounts to a surrender of the
doctrine that there exists any permanent structural arrangement in an
individual chromosome lasting from one generation to another.

They might, perhaps, be compared with the hands that are successively
dealt out from a pack of cards: each new hand, in respect of the number of
cards, may resemble, but is not really identical with, those of the preceding
deals. So, too, the chromosomes which reappear at each division would be
similar to, but not necessarily the same as, those of the preceding division.
The material particles of which they are built up are shuffled in the intervals

* In the heterotype pseudochromosomes, each member must, however, be composed of
homologous primordia contributed by the male and female parent respectively. It may
well be that this is the significance of synapsis, which forms so characteristic a feature of
the heterotype mitosis.

VOL, LXXIX.—B. AME

— 460 | Prof. J. B. Farmer. On the [May 13,

elapsing between one division and another. This might well happen without
any change of form; just as in some crystals, for example, certain atoms
or groups of atoms may be replaced without altering the crystalline form. In
the organic world, we know that the precise form of a particular organ may
be produced, although the cells of which it is made up may not be those which
should have entered into its composition. I need only refer to the experi-
ments proving that a substitution of blastomeres may take place in the early
stages of development of the frog’s egg without affecting the subsequent
differentiation of the tissues. Again, I might refer to the replacement of the
crystalline lens, when extirpated from a salamander’s eye, by a new lens
formed not from ectodermal, but from mesodermal, cells. And, as a matter of
fact, the abundant anastomoses which the linin exhibits during the earliest
stages of nuclear division seem to provide just that mechanical condition for
distribution which theory requires. This anastomosis has always been a
difficulty in the face of assumptions of chromosomal permanence. ;

The chromosomes, then, would represent similarly organised groups of
chromomeres, but they would not necessarily represent permanent or per-
sistent structures in the sense that each one is to be looked on as being
invariably composed of the same chromomeres. ‘Their constancy in form
and number would be the expression of organisation within the cell, and not
of an unchanging aggregation of the same constituents.

The relation between the chromosomes and the origin of them from the
male or female parent respectively of course remains unaffected; what
evidence we possess points to the conclusion that the two sets of chromosomes,
and consequently their chromomeres, remain distinct in all the cell-generations
up to meiosis.* In a number of animals and plants they reappear in two
distinct groups at every nuclear division during the earlier stages of ontogeny ;
and Blackman has shown, for the Uredinee, that the nuclear approximation
which corresponds to fertilisation is not really consummated until many cell-
generations afterwards; not, indeed, until the meiotic phase comes on. In
these plants, then, the chromosomal isolation is carried to an extreme, for the
sexual nuclei do not unite during the somatic phase to form a single nucleus ;
they co-exist side by side as separate bodies, although they periodically
divide in a synchronous fashion.

In suggesting the chromomeres as the agents which are responsible for the
production of the characters, I am aware that I may be laying myself open
to the charge of merely retreating from a position never very strong, and now

* Even if it should be found that this distinctness is definitely lost in the premeiotic
nuclei, it would not weaken the strong evidence in favour of the shuffling of the
primordia, and their re-arrangement in groups of homologous pairs, at meiosis.

1907. | Structural Constetuents of the Nucleus, ete. 461

beginning to prove untenable, to another that will perhaps turn out to be no
‘less vulnerable. But I think we are bound to take into account the structures
we can actually see, and the evolutions of which we can definitely follow,
before we take refuge in other and merely hypothetical entities. At any rate,
the plain facts of heredity, on the one hand, seem to postulate the existence
of large numbers of real units in the cell in order to account for the observed
phenomena, and, on the other hand, a study of the cell shows that such
structures are really present, although they may be crowded together into
larger or smaller distinct packets. And this will continue to be true,
whether the chromomeres, or still smaller particles, are adopted as the units.

This view as to the variable nature of the chromosomes seems to me to-
render intelligible certain other facts which have often proved puzzling, and
have become especially so during some recent investigations on the cytology
of ferns. . In studying the nuclei of a series of varieties that have sprung, and
are still originating, from some of our common species, ¢g., the Male Fern,
one cannot fail to be struck by the somewhat unexpected occurrence of
chromosome-numbers which show a great, but quite irregular, deviation from
that characteristic of the type.

Sometimes the new numbers are in excess, sometimes they fall below that
which one would have anticipated. Thus in two varieties of the Male Fern
known as Polydactyla, the numbers in the prothallial cells are 66 and 90
respectively. The two ferns are remarkably alike in external features, as is
indicated by their common name; but they are known to have originated as
distinct plants. In spite of their similarity, they show many points of
important difference when the various stages of their life-histories are
compared. They are both apogamous, and both replace the normal act
of fertilisation by the union of nuclei derived from adjacent vegetative cells,
in spite of the fact that they produce perfectly active sperms. One of them,
however, entirely lacks the female organ—the archegonium—whilst in the
other, though present, it is quite functionless. Along with these peculiar
features there is associated the change in the number of the chromosomes
already referred to. Now, it is important to notice that the differences do
not form multiples either of each other or of the number (72) of the typical
fern from which they are known to have originated. And when other
varieties are brought into the comparison, all grades of deviation are shown.
It would thus appear, since there is no corresponding diversity in the sizes
of the individual chromosomes, as though the variation must have arisen
through a re-arrangement of the whole substance of which they are made up;
in other words, the chromomeres have become thrown into new combinations
quite apart from any further modifications they may have undergone. The

2L 2

462 Prof. J. B. Farmer. On the [May 13,

consideration of such an example as this serves to illustrate the truth of the
general statement that the chromosomes, like other organs, are subject to
change, both as to size and number. Such a difference is not uncommon
between closely allied species; and in several of the ferns to which I have
referred, the number is not even constant in the same individual. It would
seem, then, that the bodies in question form rather a frail support for the
heavy weight of speculation that has been piled upon them.

Much of the importance which has been attached to the chromosomes in
connection with organisation is connected with the fact that in the plants
the process of meiosis is not directly followed, as it is in animals, by the
differentiation of sexual cells. In most plants the meiotic phase forms the
starting point of a definite stage in the life-history of the organism, and all
the cells are distinguished by the possession of the half, or the post-meiotic,
number of chromosomes. Thus in the fern, the plant as we know it,
possesses the double set. Meiosis is associated with the formation of the
spores, and when these germinate they give rise to the sexual plant, the
prothallium. All the cell-nuclei of this structure continue to show the
reduced or post-metotic number of chromosomes as the consequence of meiosis.
It seemed natural, then, to regard this change of nuclear structure as
connected in some causal way with that alternation of a sexual and a sexless
generation which is so striking a feature in the vegetable kingdom, especially
in the higher classes, of which the ferns may be taken as examples.

The investigations on apogamy, and especially on apospory, have shown this
view to be untenable. Indeed, it has been known for some years that eggs of
species of Echinoderms when stimulated to develop parthenogenetically
proceed to segment on perfectly normal lines in so far as they can develop at
all. Their nuclei retain the post-meiotic number of chromosomes throughout
the short life of the early larval stages, that is up to the death of the
embryonic animals.

We have been able to show, in the case of ferns, that in apospory there
may be a direct transition from the fern plant to the prothallium, accompanied
by the total suppression of the meiotic phase which normally marks the
passage from the one generation to the other. The nuclear character of such
an aposporous prothallium exactly resembles that of the fern plant from
which it sprang, instead of only having half the chromosomes. The chief
difference of importance which the prothallium itself exhibits, as contrasted
with an ordinary one, lies in its inability to produce fertilised eggs. The
sexual organs may be fully formed, and the sperms are frequently found to
be vigorously motile. Fertilisation, however, never occurs, and the fern
either arises directly from the unfertilised egg, or from the tissues of the

1907. | Structural Constituents of the Nucleus, ete. 463

prothallium. In such a case, then, the entire life-history is passed through
without any reduction of the chromosomes; the egg, like the other cells of
the prothallium, already contains the full complement, which normally is not
supplied until fertilisation. There is, therefore,no reason to be surprised at
the omission of fertilisation in these cases.

It is sometimes possible to induce prothallia which are normal as regards
their nuclear contents, 2.¢., are reduced, to produce embryos apogamously.
But in all these instances, excepting one, we have found that the normal
sexual fusion, which is here prevented from taking place, is replaced by a
fusion of nuclei from adjacent vegetative cells. In this way the normal
doubling of the chromosomes is secured. This circumstance has been used as
an argument to prove that at any rate the double set is necessary for the
production of the sporophyte generation.

We have encountered one instance, however, in which there seems no room
for doubt that the fern plant sprang from a prothallium of post-mevotie
structure, without any preparatory nuclear fusion. The plant in question*
appeared in a close fernery, and, from what we know of its history, it must
have started as a spore. Presumably, therefore, it had passed through
meiosis, and this is borne out by the small number of chromosomes present
in its nuclei. From the prothallium, fern plants were produced apogamously,
and they are all characterised by the possession of the reduced, 1.e., the post-
meiotic, number of chromosomes.

It thus becomes evident that, given a complete set of normal chromosomes,
whether in single or in duplicate, the entire life-history may be passed
through. The peculiar fusions between the nuclei of adjacent prothallial
cells, together with the extreme rarity of the formation of the fern plant
without the double set, may perhaps be taken to indicate some normal
relation as existing between the duplicate number and the sporophyte, but it
clearly cannot be of any fundamental importance.

The fact that both stages of the life-history can be gone through in the
absence of that nuclear change which ordinarily corresponds to each one of
them is a proof that the organisation of the species, whatever may be the
phases through which it passes, can be completely effected eather with the
single, or with the double, set of chromosomes; but the meaning attached to
sexuality, as importing into the organism the means of producing variation,
becomes even more clearly defined than before.

I desire, in conclusion, to obviate a possible misapprehension which,
perhaps, might arise from the way in which I have spoken of the chromo-
meres as the agents responsible for the appearance of the characters

* Lastrea pseudo-mas cristata apospora, see ‘ Annals of Botany,’ vol. 21, p. 180.

464 On the Structural Constituents of the Nucleus, ete.

manifested during ontogeny. Of course, in calling them primordia, I only
mean to imply that they are the agents that determine the particular
sequence of chemical changes which shall occur in the unstable cytoplasm. I
do not mean that they are themselves the characters in parvo. The very
fact that, as agents, they may persist in a dormant state, and thus fail to
excite the appearance of the characters appropriate to them, as may happen
in the concurrent presence of dominant and recessive allelomorphs, suffi-
ciently emphasises this. For as long as the course of metabolism can be
completely impelled by one of the two allelomorphs, the influence of the
other will remain latent. We may, perhaps, find an analogy in the behaviour
of Penicillvum, which, although it is provided with a powerful battery of
ferments, does not indiscriminately attack all the nutrient materials of the
substratum when plentifully provided with suitable carbohydrates.

Such an example serves also to illustrate another aspect of the problem,
and one that perhaps often meets with less attention than it deserves. For
admitting that there are good grounds for assigning the production of
hereditary characters to the action of discrete bodies, we are still without an
explanation of the obvious facts of functional correlation and adaptation. The
environment, in the widest sense of the term, often determines which of the
many potential characters of a cell shall actually develop.

We know something as to the manner in which this may be achieved in
certain instances. For example, nutritional, as well as other and more
obscure, stimuli were found by Klebs to determine the particular one out of
several alternative courses of development which was followed in the case of
certain alge. And again, the Hormones of Starling may be quoted as
types of substances, apparently of relatively simple chemical structure,
which serve as excitors of specific cellular activities in the complex animal
body.

The conceptions of primordia which are responsible for the appearance of
hereditary characters on the one hand, and also of specific exciting substances
on the other, seem to be both necessary, and the one to supplement the
other.

Every organism and every cell has larger potentialities than are ever
realised in any single ontogeny. The hereditary mechanism imposes the
limits within which development can take place ; but within those limits other
conditions may determine the path actually followed.

4695

On Certain Phenomena of Inactivation and of Inhibition exhibited
by Precipitin Antosera.

By D. A. Wetsu, M.A., B.Sc., M.D., Professor of Pathology, and H. G. CHAPMAN,
M.D., B.S., Demonstrator of Physiology in the University of Sydney.

(Communicated by Dr. C. J. Martin, F.R.S. Received April 30,—Read
June 13, 1907.)

(From the Physiological and Pathological Laboratories of the University of Sydney.)

General Phenomena of Inactivation and of Inhibition exhibited by Heated
Antisera.*

When a precipitin antiserum, prepared with blood serum or with egg white,
is heated to 75° C. for 10 or 30 minutes, it loses its power of yielding a
precipitate with the homologous protein (inactivation), and it acquires the
power, when added in sufficient proportion, of preventing the formation of the
precipitate that would otherwise appear when the homologous protein interacts
with unheated antiserum (inhibition). An identical heat exposure does not
‘produce the same degree of inhibitory power in different homologous antisera,
the inhibitory capacity being estimated by the amount of unheated antiserum
required to elicit a precipitate.

In the majority of the antisera tested, inhibition was complete, that is, no
precipitation occurred, when (a) 0:1 c.c. heated antiserum, (0) 0-1 c.c. unheated
antiserum, and (c) 0:001 cc. or 0:005 cc. homologous protein were allowed
simultaneously to interact, or when (a) was mixed with either (0) or (¢) for
24 hours before the other ingredient was added. Inhibition was always
complete when the amount of the heated antiserum was three times that of
the unheated antiserum, and always incomplete when these proportions were

* Our conclusions are based on more than 1000 separate interactions, involving
20 different antisera, about 50 different combinations of heated and unheated antiser
_ having been tested. The antisera were prepared in the rabbit by intraperitoneal
injections of blood serum or of egg white. The general procedure was the same as that
indicated in a previous communication (‘Roy. Soc. Proc.,’ B, vol. 78, p. 298, 1906), and
most of the interacting substances were employed in a fresh undried condition, To
obviate coagulation, the antisera were diluted with two or four volumes of water before
being heated, but this water is not included in the amounts of heated antiserum to which
reference is made in the text. Thus 0°1 c.c. heated antiserum indicates 0°1 ¢.c. antiserum
heated together with two or four volumes of water. We ascertained that the additional
water did not noticeably affect the interaction.

466 Prof. D. A. Welsh and Dr. H. G. Chapman. [Apr. 30,

reversed. Incomplete inhibition was manifested by delay in appearance or
by diminution in amount of the precipitate, or by both phenomena.

When complete inhibition was established, it did not cease with lapse of
time, nor was it overcome by any excess of homologous protein, but it could
be overcome (that is a precipitate would appear) on a suitable addition of
unheated antiserum, or on a smaller addition of unheated antiserum together
with homologous protein.

When, as occasionally happened, 0-1 c.c. heated antiserum was inadequate
to prevent precipitation in a mixture of 0:001 c.c. or 0:005 e.c. homologous
protein and 01 cc. unheated antiserum simultaneously added, it was
invariably also inadequate when allowed to stand in contact with the unheated
antiserum for 24 hours before homologous protein was added, but it was
frequently adequate to prevent precipitation when allowed to remain in
contact with the homologous protein 24 hours before the unheated antiserum
was added. In view of the fact that no excess of homologous protein could
overcome any inhibition, it is futile to conclude from such evidence that the -
heated antiserum antagonises either the homologous protein or the unheated
antiserum more than the other. As a matter of fact, its influence is probably
exerted not so much on either of the interacting substances as on the product
of their interaction (v. infra).

Analogous results have been obtained by other observers, notably by
Miiller,* in respect of lactosera, and by Eisenberg.t

Relation between Inactivation and Inhibition.

On the hypothesis that precipitins constitute, on Ehrlich’s scheme,
receptors of the second order, equipped with a relatively unstable ergophorous
and a relatively stable haptophorous affinity, inactivation is produced by
destruction of the former, while inhibition is due to retention of the latter
alone. Any factor that would abolish the ergophorous affinity would, zpso
facto, render the precipitin inactive and inhibitory, so long as the haptophorous
affinity was not thereby damaged. Precipitin would thus become transformed
into precipitoid. In other words, inactivation and inhibition would be merely
different aspects of the same phenomenon, and this is the accepted relation-
ship between them (Kisenberg, Miller, and others).

We have evidence, however, that inactivation and inhibition are separable
phenomena, that inactivation does not, in the above circumstances, necessarily
imply inhibition, so that the relation between them does not admit of so

* Miller, ‘ Archiv f. Hygien.,’ vol. 44, p. 126, 1902.
+ Eisenberg, ‘ Centralb. f. Bakt.’ (Originale), vol. 31, p. 773, 1902.

1907. | Phenomena exhibited by Precipitin Antisera. 467

simplea statement. Thus a precipitin antiserum (No. 38) prepared with hen-

egg white, when heated to 70° C. for 30 minutes, did not become noticeably
inactive or inhibitory. When heated to 72° C. for 10 minutes, it was rendered
almost completely inactive, but not noticeably inhibitory. Exposure to 72° C.
for 30 minutes produced complete inactivation without, however, any
appreciable inhibition. Further exposure to 75° C. of a portion of the
antiserum already heated to 72° C. for 30 minutes yielded a solution still
wholly inactive, but now strongly inhibitory. We have repeated these
observations with concordant results.

It is noteworthy that the conversion of antiserum from the active to the
inactive condition appears to be a temperature change rather than a time
effect, and that inactivation occurs rapidly at the temperature at which we
succeeded in obtaining, in an acidulated solution of a homologous antiserum, a
heat coagulum not found in natural rabbit serum.*

_A second means of effecting a separation of the phenomena of inactivation
and of inhibition was accidentally revealed. Another homologous antiserum
(No. 11), prepared in the rabbit by injections of hen-egg white, had been
stored in a dry condition. At the expiry of 11 months it was found to be
still readily soluble in salt solution, but to have become wholly inactive, no
trace of precipitate appearing on interaction with homologous protein. When
0:1 ec. of this unheated inactive antiserum solutiont was allowed to interact
with an equal volume (0:1 c.c.) of active homologous antiserum (Nos. 23 and 25
were separately employed) and with homologous protein, no appreciable
inhibition could be detected. When, however, a triple volume (0°3 c.c.) of the
inactive antiserum solution interacted with a single volume (0°1 c.c.) of the
active antiserum in otherwise similar circumstances, the full precipitate was
in every instance obtained, but in three out of six tubes its appearance was -
delayed for about 24 hours. When it is borne in mind that three volumes
(0°3 c.c.) of any antiserum heated to 75° C. was invariably found to completely
inhibit precipitation when one volume (0°1 c.c.) of a different but homologous
antiserum interacted with homologous protein, it must be admitted that any
inhibition effected by the unheated inactive antiserum (No. 11) was uncertain
and exceedingly slight. It is, indeed, possible that the occasional delay in
the appearance of the full precipitate may have been due to some slight non-
specific inhibition exerted by increased serum concentration.

When, however, the inactive antiserum (No. 11) was heated to 75° C. for
30 minutes, it developed a pronounced inhibitory capacity—so much so that

* Welsh and Chapman, ‘ Roy. Soc. Proc.,’ B, vol. 78, p. 309, 1906.
t+ O-l cc. of the inactive antiserum solution contained 0°01 gramme dried antiserum
(No. 11) and was, therefore, roughly equivalent to 0°1 c.c. undried antiserum.

468 Prof. D. A. Welsh and Dr. H. G. Chapman. [Apr. 30,

0:1 cc. heated antiserum mixed with homologous protein for 48 hours was
able to prevent precipitation when 01 c.c. active antiserum (No. 23 or
No. 25) was subsequently added. The inhibition was constant, but
incomplete, when 01 c.c. heated antiserum was mixed with 0°1 cc. active
antiserum (No. 25) and homologous protein was simultaneously or
subsequently added. The inhibition was complete when 0-2 c.c. heated
antiserum interacted with 0:1 cc. active antiserum and homologous protein,
and was independent of the order in which the substances were added.

Both by heating to 72° C. and by spontaneous degeneration, therefore, 1t
was possible to obtain antisera which were wholly inactive and yet not at all
or only doubtfully inhibitory, and thus to effect a separation of the
phenomena of inactivation and of inhibition. In both instances, also, heating
to 75° C. so altered the constitution of the inactive, non-inhibitory antisera,
that they came to acquire powerful inhibitory properties.

If inactivation and inhibition were inseparable phenomena, it sere be
supposed that inactivation was merely a particular manifestation of
inhibition, that precipitin was not necessarily altered by heating to 75° C.,
but that an independent inhibitory substance was developed at that
temperature. Our observations render this supposition untenable.

The mere separation of the phenomena of inactivation and of inhibition
might be reconciled with the commonly accepted statement of Ehrlich’s
theory of the constitution of precipitins by the twofold assumption of
a diminution, followed by an increase, in the combining affinities of the
haptophores. It might, for example, be assumed that an immediate effect of
the weakening or destruction of the ergophorous affinities, resulting in
inactivation, would be some weakening of the haptophorous affinities also,
whereby molecules of the nature of epi-precipitoid, or precipiton, would be
produced. On the analogy of epi-toxoids, or toxons, such epi-precipitoids,
or precipitons, having feebler combining affinities than the unaltered
precipitin molecules, would be readily evicted by them, and so might give
rise to little or no noticeable inhibition of the precipitate. It would be
necessary also to assume that heating to 75° C. restored or enhanced the
potency of the haptophores, so that epi-precipitoids were transformed into
syn-precipitoids or even into pro-precipitoids, having combining affinities
respectively equal to or greater than those of unaltered precipitins. We do
not see how the presence of epi-precipitoid in the inactive, non-inhibitory
antisera described by us can be either proved or disproved, unless the
occasional doubtful inhibition above noted is to be taken as an indication of
its presence. |

Other facts, however, render this explanation inadequate. It has been

1907. | Phenomena exhibited by Precipitin Antisera. 469

shown that, after the interaction of 0:001 <.c. or 0-005 c.c. homologous protein
and 0-1 cc. antiserum, the addition of further quantities of antiserum
produces a fresh deposit proportional to each added mass of precipitin.*
This is strongly suggestive that, after each interaction, unoccupied binding
affinities of the homologous protein are available. Further, if inhibition is
produced by the binding of the thermostable haptophorous groups of the
heated antiserum to receptors of the protein molecule, it is remarkable that
the addition of considerable masses of homologous protein fails to provide
receptors for the precipitin groups of the unheated antiserum and to bring
to an end the inhibition.

All that we can say is that inactivation and inhibition are separable
phenomena, commonly appearing together because of the proximity of their
respective temperatures of origin, and depending on substances whose
interactions do not admit of. so simple an expression as that afforded by the
usual statement of Ehrlich’s theory.

Specificity of the Inhibition exercised by Heated Antisera: Phenomena of
“ Crossed” Inhibitron.

The non-appearance of a precipitate in an otherwise effective precipitin
interaction is not always due to the same conjunction of chemical and
physical factors, and of these factors some exert an inhibitory influence that
is altogether non-specific. But the inhibitory power of heated antiserum
is markedly specific, probably not less specific than the reaction of the
corresponding unheated antiserum. The nature of this specificity is best
illustrated by a concrete example. Thus, a heated hen-egg antiserum will
prevent the formation of a precipitum between hen-egg albumin and any
hen-egg antiserum, but not between ostrich-egg albumin and ostrich-egg
antiserum, nor will it have any effect on non-avian precipitin interactions.
The phenomena of “crossed” inhibition are revealed by the fact that heated
hen-egg antiserum will inhibit precipitation between hen-egg albumin and
ostrich-egg antiserum and between ostrich-egg albumin and any hen-egg
antiserum. Moreover, when ostrich-egg proteins were substituted for the
corresponding hen-egeg proteins in each of the above experiments, identical
results were obtained. These results may be summarised in the following

Table I :—

* Welsh and Chapman, ‘ Roy. Soc. Proc.,’ B, vol. 78, p. 305, 1906.

470 Prof. D. A. Welsh and Dr. H.G. Chapman. [Apr. 30,

Table LI.
Homologous protein, | Active antiserum, | Heated antiserum, = ee

Ne: 0-005 c.0. O11 c.c. Ol ce. Prebipienee

1 | Hen-egg white Hen-egg Hen-egg Inhibited

2 Ostrich-egg white Ostrich-egg son Unaffected

3 | Horse serum Horse 55 5

4 | Hen-egg white Ostrich-egg 5 Inhibited

5 Ostrich-egg white Hen-egg “ 5

6 3 % Ostrich-egg Ostrich-egg .

7 | Hen-egg white Hen-egg i Unaffected

8 | Horse serum Horse es i

9 | Ostrich-egg white Hen-egg white 5 Inhibited
10 | Hen-egg white Ostrich-egg di 9

The Nature of inhabition as indicated by the Specific Solubility of Precipitates
in Heated Homologous Antisera. |

Miller* was the first and, so far as we are aware, the only observer
to point out that a precipitate resulting from a precipitin interaction was
soluble in the heated antiserum, but his observation had reference only to
lactosera. The possible significance of this phenomenon led us to under-
take a more extended series of tests,in order to determine how far this
property was possessed by other heated antisera, and how far the capacity
for dissolving precipitate might be identified with the capacity for
inhibition.

Our experiments gave the following results :—(1) The precipitum formed
between hen-egg albumin and hen-egg antiserum is soluble in any hen-egg
antiserum heated to 75° C., but not in ostrich-egg antiserum, nor in horse
antiserum, similarly heated; (2) the precipitum formed between ostrich-
egg albumin and ostrich-egg antiserum is soluble in ostrich-egg antiserum
heated to 75° C., but not in hen-egg antiserum, nor in horse antiserum,
similarly heated; (3) the precipitum formed between horse serum and
horse antiserum is soluble in any horse antiserum heated to 75° C., but not
in hen-egg or ostrich-egg antiserum similarly heated; (4) such precipita
are insoluble in water, in salt solution, in natural rabbit serum, and in
natural rabbit serum heated to 75° C., all of which are likewise non-
inhibitory; (5) the amounts of heated antisera used in dissolving the
precipitates were the same as those used in producing inhibition (from
0-1 to 0°3 c.c.), and where the lesser quantity was unable to inhibit, it was
also found to be unable to dissolve the precipitate. When a precipitate
was soluble in heated antiserum, it rarely happened that the whole of the

* Miiller, doc. cit., p. 175.

1907. | Phenomena exhibited by Precipitin Antisera. 471

deposit was taken up into solution, but by far the greater portion was so
taken up, only a small trace being left in marked contrast to the original
deposit, and in marked contrast also to the full deposit which reappeared
when a precipitate was shaken up with any heated antiserum other than
that strictly homologous. It seems legitimate to draw the conclusion that
the capacity for dissolving precipitate is a specific property of inhibitory
antisera.

This conclusion is reinforced by the results of testing the solubilities of
the precipitates obtained in “crossed ” interactions, as indicated in Table II,
and by comparison of these results with the “ crossed ” inhibitions shown in
- Table I.

Table IT.
. . Precipitate ae
N Homologous protein, | Active antiserum, ne serum, 0°1 c.c., Result
ne 0°005 c.c. O'l c.c. ee added to to precipitat
24 hours Hee | hk he a
precipitate.
mm.
1 | Hen-egg white Hen-egg 2°0 Hen-egg Dissolved
2 | Ostrich-egg white Ostrich-egg 3°0 3 Unaffected
3 | Horse serum Horse 1°5 . #
4} Hen-egg white Ostrich-egg 0°5 ‘ Dissolved
5 | Ostrich-egg white Hen-egg 0°5 ap Zz
6 ss 5 Ostrich-egg 3 °O Ostrich-egg .
7 | Hen-egg white Hen-egg 2°0 3 Unaffected
8 | Horse serum Horse 1°5 i F
9 | Ostrich-ege white Hen-egg 0°5 S Dissolved
10 | Hen-egg white | Ostrich-egg | 0°5 % Ls

These results suggest that the precipitum given by hen-egg antiserum
and ostrich or any egg albumin, other than hen-egg albumin, may be
regarded as similar to that produced by ostrich-egg antiserum and any egg
albumin other than ostrich-egg albumin. We may assume this precipitum
to result from the general avian character, or component, of the proteins
used in the immunisation, while the greatly increased precipitum produced
by hen-egg antiserum and hen-egg albumin, or by ostrich-egg antiserum
and ostrich-egg albumin, we may assume to be due to the specific hen or
ostrich character, or component, of the protein used for injection. A heated-
egg antiserum would thus inhibit and dissolve the general avian precipitum
plus its own strictly homologous precipitum, but would neither inhibit nor
dissolve the non-homologous component of any precipitum, however closely
related. This principle is probably of general application, and might be of
value in the practical differentiation of closely related proteins.

The phenomena above described emphasise the close relationship that

472 Phenomena exhibited by Precipitin Antisera.

exists between the capacity for solution and the capacity for inhibition
of precipitate, but we have still more striking evidence to offer. For
example, an antiserum rendered inactive, but non-inhibitory, by heating to
72° C., has no power of dissolving precipitate, but when the antiserum is
rendered inhibitory, by heating to 75° C., it coincidently acquires the power
of solution. Antiserum (No. 11), rendered inactive, but non-inhibitory, by
long-continued desiccation, did not dissolve precipitate, but when heated
to 75° C., and thereby made capable of inhibiting, it also became capable
of dissolving precipitate. When antisera are mixed with a sufficiency of
homologous protein, their precipitable content may be entirely thrown out
of solution and the antisera rendered inactive. Such inactivated antisera,
when heated, do not gain the power of preventing precipitation (a corre-
sponding result was obtained by Miiller in respect of lactosera), and neither
do they gain the power of dissolving precipita.

Further, an antiserum rendered inhibitory and solvent, by heating to
75° C., has almost completely lost both capacities after heating to 85° C.
for 10 minutes, and has completely lost both by exposure to 85° C. for
30 minutes, or to 100° C. for five minutes. Such destruction of inhibitory
and solvent capacity does not reactivate the antiserum, nor does the addition
of heated or unheated natural rabbit serum restore its inhibitory or solvent
capacity.

Notwithstanding many experiments of the above nature, we found it
impossible to dissociate the capacity for inhibition from the capacity for
solution of precipitate. Both appear and disappear together, and it would
seem not unreasonable to draw the further conclusion that an antiserum
acquires the power of preventing precipitation in virtue of its acquiring the
power of dissolving precipita; in other words, that an essential part of the
mechanism of specific inhibition is the development of a no less specific
capacity for solution of precipitate.

Conclusions.

1. Inactivation and inhibition are separable phenomena, the former
appearing without the latter in hen-egg antisera heated to 72° C., or exposed
to long-continued desiccation, the latter not becoming appreciable until the
antisera are heated to 75° C.

2. Inhibition and solution of precipitate by heated antisera are specific
inseparable phenomena.

3. The specific capacity for dissolving precipitate acquired by heated
antisera is probably an essential part of the mechanism of specific inhibition.

New Paleozoic Lycopod with a Seed-like Structure. 4738

4. Certain phenomena of precipitin reactions, including precipitation,
inactivation, inhibition, and solution of precipitates, appear to be incompatible
with the commonly accepted statement of Ehrlich’s theory of the substances
concerned in precipitin interactions.

Miadesmia membranacea, Bertrand; a New Paleozoic Lycopod
| with a Seed-like Structure.

By M. Benson, D.Se., F.LS.

(Communicated by Dr. D. H. Scort, F.R.S. Received April 26,—Read
June 13, 1907.)

(Abstract.)

The vegetative organs of this interesting new type were discovered by
Bertrand in 1894. He found them in sections of a calcite nodule from the
Gannister beds of Hough Hill, England. <A large quantity of new material
has become available, and now not only are more details known as to the
vegetative organs, but a fairly complete knowledge of the reproductive organs
is possible. ,

Miadesmia was exceedingly minute, its stem slender and without any trace
of skeletal tissue. It is the first. Paleeozoic Lycopod of herbaceous character
known structurally. The megasporophylls which were identified by
Dr. D. H. Scott, F.R.S., in 1901, show a more advanced type of seed habit
than has hitherto been met with in Cryptogams. The megasporange gives
rise to but one thin-walled spore, which in development and structure
resembles an embryo sac and germinates im situ. An integument surrounds
the sporange, leaving but a small orifice as micropyle. This is surrounded by
numerous long processes of the integument, which formed a collecting and
incubating apparatus for the microspores. There is no trace of an envelope
about the microsporange. The carpellary leaf was shed at maturity, and
resembles a winged seed.

Disregarding the structural modifications of the megasporophyll, the
nearest affinity of Miadesmia among forms so far known seems to be with
the non-specialised species of Selaginella, such as Selaginella selaginoides, but
the foliage leaves show the archaic leaf-base comparable with that of
Lepidodendree.

474

On the Identification of Chitin by tts Physical Constants.
By Iczrna B. J. Souzas, Fellow of Newnham College, Cambridge.

(Communicated by Professor W. J. SoLLAS, F.R.S. Received May 27,—
Read June 13, 1907.)

The determination of the distribution of chitin in the animal kingdom is
hampered by the absence of any test or positive means of identifying it.
Gamgee, in his ‘Text-book of Physiological Chemistry, gives a list of
structures of invertebrate animals in which chitin has been described. But
when those cases are eliminated in which the identification has been based
solely on the negative character of insolubility in caustic alkalis or weak
acid, the revised list, as it appears, for instance, in von Fiirth’s ‘ Vergleichende
chemische Physiologie der niederen Tiere, is greatly curtailed. It is true
that chitin yields a characteristic decomposition product, the amido-
derivative of sugar known as chitosamin, in definite proportions, but the
amount of material available is not always sufficient to allow of the prepara-
tion of this product. In cases, however, where we have other reasons for
suspecting the presence of chitin, the reducing action of the chitosamin
resulting from the treatment of the original substance with sulphuric acid is
a valuable confirmatory test.

Under these circumstances it seemed that it might be worth while to make
a determination of the specific gravity of chitin by the well-known method
of a diffusion column. The specific gravity of any substance as thus
determined stands in real and intimate relation to its chemical constitution,
for the mass dealt with is always small and can therefore be rendered
homogeneous by various cleansing processes. It is readily permeated by the
suspending fluid and the absence of any other sources of error may be
ensured without difficulty. |

In addition to the determination ge the specific gravity, that of ae
refractive index should be made, for two substances may possess the same
specific gravity, but not the same chemical composition, though when we have
other reasons for suspecting their identity this is unlikely.

The refractive index of any substance has been shown by Gladstone and
others to be connected with the specific gravity by a relation which, whether
expressed as a or more correctly as oo is constant for that sub-
stance and is independent of the temperature. Therefore the demonstration

On the Identification of Chitin by its Physical Constants. 475

of common values for the specific gravity and the refractive index of two
substances affords strong evidence of their identity.*

Unfortunately, I do not possess a reading microscope, so that I have taken
my readings direct from a scale at the back of the experimental tube. But
I have appreciated the advantage of the use of glass beads: when these are
made and suitable fluids chosen the process need occupy but little time.

The fluids I have used are chloroform, of which the specific gravity is
roughly 1:48 at 21° C., and “absolute” alcohol of specific gravity 0-791 at
21° C. The specific gravity of chitin from the integuments of Astacus
having been found roughly to lie somewhere between 1°39 and 1:41, mixtures
of chloroform and alcohol of specific gravity 1°36 and 1°44 were made with
the help of a Westphal’s balance, and these mixtures formed the two fluids of
the column. In using light fluids it is not possible to float glass beads, such
as are used for dealing with minerals in heavy fluid columns, and which are
described in ‘Nature’ :+ it is necessary in the present case to blow the beads
dumbbell-shaped, but with both ends hollow. Hence it is possible to obtain
_ beads which float with their long axis horizontal and this is an advantage, for
the nearer the axis of the bead approaches to the horizontal position the less
is likely to be the error which arises in estimating its centre of gravity.

Chitin from various sources was prepared in the usual way by soaking: in
10-per-cent. hydrochloric acid, boiling in 5-per-cent. caustic potasht for many
hours with frequent changes of the liquid, washing in water, very dilute
hydrochloric acid, and extracting in alcohol and ether. In some cases this
treatment did not render the chitin quite colourless: the last traces of
pigment were removed by Mayer’s method of bleaching, in which chlorine is
the active agent.

The specific gravity of chitin from integuments of Astacus cleaned in this
way was found to range between 1°400 and 1404. The chitin of a cater-
pillar, Bombyx cynthia, was slightly heavier, 1:404 to 1:408, of the cockroach
wing 1°402, of the pupa case of the buff-tip moth 1404: of Oniscus and
spiders, again, the value agreed very nearly with that of Astacus. Two
species of Myriapoda gave the value 1°397 to 1°399; one of these was the
common English Lithobius, the other a large millepede which I picked up in
the Karroo, South Africa, but have not identified.

* This method was originally published in 1885 by Professor Sollas in the ‘Roy. Soc.
Dublin Proc.,’ new ser., vol. 4, p. 378, and subsequently in ‘ Nature,’ 1891, vol. 43, p. 404,
and ‘Quart. Journ. Geol. Soc.,’ vol. 58, p. 163, 1902.

t+ Loe. cit.

{ The use of potash of this strength, which is weaker than that employed by Kruken-

berg, was recommended to me by Dr. F. Gowland Hopkins, to whom I am indebted for
much kindly interest in this investigation.

Vor. LAXIx.—B, 2M

476 Miss I. B. J. Sollas. On the Identification of [May 27,

The chitin which had the lowest specific gravity of any I have measured
was that of Limulus and of Scorpio. This had a value 1:393 when cleaned as
above described. If, however, stronger potash is used, chitin from Astacus and
from Limulus approach each other in a diffusion column and give a zone at a
level corresponding to a specitic gravity 1:°398. This figure seems to represent
the value of the specific gravity of pure chitin, for, as stated below, it is
that of chitin precipitated from its solution in strong acid.

The strength of potash which gives a common value for the specific
gravity of chitin from both Limulus and Astacus is 40 per cent. It must be
used for a short time only after the usual long boiling in 5 or 10 per cent., as the
prolonged action of strong potash causes chitin to become soluble in weak acid.

Neither boiling in 50-per-cent. hydrochloric acid nor soaking in strong
hydrofluoric acid alters the specific gravity of chitin.

Young chitin from a crayfish which had recently undergone ecdysis was
heavier than the older skin; it formed a zone at a level corresponding to
a specific gravity of 1:410, with outlying pieces down to 1-420. Unfor-
tunately, I have not obtained the specific gravity of the cast skin. It proved
to be more readily soluble in weak acid after boiling potash than the younger.
chitin and, not foreseeing this, I inadvertently dissolved it before I had taken
measurements.

The refractive indices for red light of the chitin from various sources
which I have measured he between the limits 1°550 and 1°557. The
measurement was made by Becke’s method.* That from Astacus and
Limulus integuments lies between 1°554 and 1557. The fluids used in these.
determinations were aniseed oil and mixtures of aniseed and fennel oils
calculated to have the refractive indices 1:550 and 1°557.

As stated above, chitin was precipitated from its solution in strong
hydrochloric acid, in order to determine which of the obtained values, ranging
between the extremes 1°393 and 1°408, represents the specific gravity of
pure chitin.

The solution was performed in a refrigerator, carapaces of Astacus and
Limulus being used. At the end of a fortnight no difference in the bulk
of the solid was noticeable on inspection. The acid was then poured off
and diluted in the refrigerator with 10 times its volume of water cooled
till a thin film of ice formed on the surface. A copious milky precipitate
was formed. Fresh acid was poured over the remaining pieces of chitin,
and slight precipitates obtained at intervals of two days from this freshly

* “Ueber die Bestimmbarkeit der Gesteinsgemengtheile auf Grund ihres Licht-
brechungsvermégens,” ‘Sitzungsber. der K. Akad. der Wissensch.,’ Vienna, vol. 102,
p. 358, 1893.

1907.] : Chiten by its Physical Constants. 477

added acid. The solution in. hydrochloric acid, when kept at a temperature
of less than 0° C.,is thus seen to be not unstable. The precipitate, which
was flocculent, was washed by decantation until an acid reaction was no
longer obtained; it was then boiled in alcohol and ether. Placed in a
specific gravity column, it formed a broad zone, extending from 1°380 to
1:390. Some of the precipitate was then dried at a low temperature
(40° to 60° C.). It formed a thin film on the evaporating dish, which was
scraped off with a scalpel, and then formed a white powder. This powder
had a specific gravity 1:398, and this, I conclude, is the specific gravity of
chitin. If dried too rapidly at a higher temperature than 60°, the precipitate
flakes off the dish and its specific gravity is no longer uniform.

The substances of unknown chemical composition which I have examined
by this method are Lumbricus bristles, the organic portion of the molluscan
radula, the pupal skin, of Pieris, and of some other Lepidoptera stated by
Griffiths to be “pupin.” I had hoped to deal also with the skeletal
substance of the branchial bars of Amphioxus, which are stated to be
insoluble in alkalis, but I could obtain no residue after boiling the pharynx,
even in weak caustic potash. .An account of the specific gravity of the
organic portion of the shell of Sepia, in which chitin has been found by
Krukenberg, is also given below. Lumbricus chetz were isolated in the
following manner:—The worm was slit up along the mid-dorsal line and
the alimentary canal and its contents removed. The remaining body wall:
was then washed in a current of water to remove any matters which might
have escaped in the process of removal of the gut. The contents of the
gut are troublesome unless removed at once. The worms thus far cleaned
are now treated with 10-per-cent. hydrochloric acid, and then boiled in
potash as usual in the preparation of chitin. If after prolonged treatment
with potash any matter other than chetz remains undissolved, this is most
readily got rid of at a later stage of the cleansing process (after extraction
in alcohol) by separation in a diffusion column, say of chloroform and alcohol.
The bristles will float on the chloroform, the insoluble dirt (presumably
sand, undigested wood fibres, and cellulose, set free in removing the
alimentary canal) sinks to the bottom. The bristles can then be removed
with a pipette and extracted in ether. In this way a considerable quantity
of cheetz can be collected with very little trouble. They form a neat zone
in a diffusion column at a level corresponding to a specific gravity 1:392.
They are doubly refracting. I have measured one index only, it lay between
1557 and 1°550. I conclude, therefore, that the chetze of Lumbricus
contain chitin. This is in agreement with the results obtained by Goodrich.*

* ‘Quart. Journ. Micr. Sci.,’ 1897, vol. 39, “ Notes on Oligocheetes.”
2M 2

478 Miss I. B. J. Sollas. On the Identification of [May 27,

From their solubilities and their behaviour with Millon’s reagent and with
the xanthoproteic test, Goodrich considered that the bristles of Oligocheta
are evidently chitin or some nearly allied substance. But more recently
Schepotieff,* after a lengthy research, concludes: “ Diese Reaktionen zeigen,
dass die Borsten jedenfalls nicht aus einer einheitlichen Substanz bestehen,
sondern aus mindestens zwei. Die eine derselben zeigt die Reaktionen der
Eiweisskorper. Die andere kann schwerlich Chitin sein, wie der Mangel
der Zuckerbildung bei Behandlung mit Schwefelsiure ergiebt.” With
regard to the last difficulty, the absence of sugar formation after treatment
with sulphuric acid, I am unable to find that it exists, for I obtained copious
sugar reduction with Trommer’s test. Schepotieff (p. 672) dissolved the
bristles in 89-per-cent. sulphuric acid, kept them for 25 hours at a tempera-
ture of 40° to 50° C., diluted the solution with 10 times its bulk of water,
and kept the diluted solution at 100° C. for some hours. He then
neutralised with barium carbonate, and tested the solution with copper
sulphate and sodium hydrate. It was suggested to me by my friend Miss
Durham that the use of barium carbonate in place of the usual caustic
potash for neutralisation may have led to Schepotieff’s difficulty, as the
precipitate, consequent on neutralisation with barium carbonate, would carry
down the sugar with it. At any rate, sugar is certainly present after
treatment with sulphuric acid, and there is no reason why one of the two
constituents should not be chitin. That more than one substance should be
present in the bristles is not surprising. I have not met with any chitinous
skeleton which did not give the proteid reaction to which Schepotieff alludes
before it was cleaned.

Pupa cases of Pieris brassice and P. napi, cleaned in the same way as
chitin, had a specific gravity of 1-400 and refractive index between 1°554
and 1557. Griffitht has examined these integuments chemically, and has
stated that they consist of a new animal substance which he calls “ pupin.”
He dissolved the skins (after they had been cleaned with potash, water,
alcohol, and ether) in hydrochloric acid, and precipitated from this solution
a substance to which he assigned the formula Cy,HeN20;5, and found that
it split when boiled with strong mineral acids into leucin and carbon
dioxide. Von Fiirth remarks that the proportions of C, H, and O point to
an albuminoid, but the N-content is strikingly small. He suggests that a
closer investigation would be worth while, as he supposes that the products
of splitting, besides leucin and carbon dioxide, must have been overlooked.

* ‘Zeitschr. Wiss. Zool.,’ vol. 74, 1903, p. 674.

t+ ‘Comptes Rendus,’ IT, vol. 105, pp. 320—32]1, and ‘ Bull. Acad. Roy. Belg.,’ (3), vol. 24,
1892, p. 592. ,

1907. | Chitin by wts Physical Constants. 479

I can only state that the substance has, like the integument of other
Lepidopteran pups, the physical properties of chitin, and that I have
obtained evidence of the presence of sugar in it after treatment with
90-per-cent. sulphuric acid at a temperature of 40° C. for 24 hours. The
sugar was detected by Trommer’s test, as in the other cases mentioned.*
The molluscan radula I have studied from this point of view by this method
chiefly in the case of the periwinkle, Litiorina littorea, the limpet, Patella
vulgata, and the whelk, Buccinwm undatum. The radula of Patella, as I have
shown elsewhere,t contains silica and iron in considerable quantities.
To cleanse it we must, therefore, treat it for some time (24 hours) with
strong hydrofluoric acid. A control experiment showed that the specific
gravity of chitin from the integument of Astacus was unaltered by this
treatment. After washing out the hydrofluoric acid the usual method of
preparing chitin was used. The radule still retain a faint yellow colour
after this treatment ; they were placed in chlorine water, when they became
colourless and somewhat transparent. They were cut into short lengths,
and these were found to form a neat zone in the diffusion column at a level
corresponding to a specific gravity 1405 to 1-407. Littorina radule gave
a closely similar result. In the case of Littorina I used the confirmatory
test, and obtained reduction of copper salts by Trommer’s method.

Buccinum radule differ from the others which were measured in that
they form a broad irregular zone in a diffusion column. The extreme tips
of the radule, taken from the radular sac, however, give a uniform specific
gravity of 1404. The remainder of the radule form a band extending from
1-400 to 1:411, or even lower. It is noticeable that the pieces in this band
for the most part hang with their greater length vertical, and the oldest
end of each length is lowermost. It, therefore, seems possible that some
second resistent substance, in addition to chitin, is present in this case in
increasing quantities as we pass forwards from the radular sac. If this
is the correct interpretation of the facts, it is interesting in connection with
the occurrence of free sulphuric acid in the salivary glands of Buccinum, and
it is not surprising that an organ used to bore holes in shells with the
aid of sulphuric acid should need to be specially resistent. For some time
I fancied that the irregularities in the specific gravity of this radula might
be due to small particles of sand caught between the bases of the teeth and
the basal membrane. But the specific gravity of the teeth, when isolated

'* Since this was written, von Fiirth and Russo have shown by chemical methods that
the integuments of Lepidopteran pup consist of ordinary chitin, ‘Beitr. z. chem.
Physiol. u. Pathol.,’ vol. 8, 1906.

+ ‘Quart. Journ. Micr. Sci.,’ 1907, vol. 5), pp. 115—136.

480 Miss I. B. J. Sollas. On the Identification of [May 27,

from the basal membrane, shows that this cannot be so. The same radule
of which I had measured the specific gravity were treated for a moment
with strong hydrochloric acid, with the result that the basal membrane was
instantly destroyed and the teeth freed; after washing and reboiling in
alcohol they were found to have specific gravities of even wider range than
that of the intact radula, the heaviest of them being 1-430. By soaking the
radula of Buccinum for several weeks in very strong potash (above
40 per cent.) the teeth can be isolated, the basal membrane being destroyed.
This fact, discovered by chance, aroused some doubt as to whether the basal
membrane were really chitin, and a repetition of the experiment was made
in which the skeletons of two segments of Astacus were subjected to the
action of the same strength of potash. The two segments became isolated
from one another, the abdominal feet (pleopoda) were also isolated from the
segments, the soft connecting membranes being destroyed. And yet these mem-
branes have the same specific gravity and the same refractive index as chitin,
- and no one has ever suggested that they are anything else. These membranes’
differ in staining reactions from the chitinous pieces which they connect in just
the same way as the basal membrane of Buccinum differs from the teeth.

A third method of isolating these teeth is to soak the radula in hydro-
fluoric acid for 24 hours previous to the usual cleansing process. The teeth
will then be liberated when the radula is boiled in 5-per-cent. potash
solution. Teeth thus prepared gave the least range that I have found in this
material ; they formed in all cases a dense zone at the level 1:°393; in one
instance they were confined to this zone, but in two other columns a certain
number of teeth were scattered down to 1:411, and a very few, perhaps five
or six, hung at various depths below this. Astacus integuments subjected
to precisely the same treatment had a specific gravity of 1406; and, as
happened when strong potash was used, the connecting membranes were
destroyed. Some of the heavier Buccinum teeth were removed with a
pipette from the column; in one or two of them a single large granule,
apparently a foreign body, was visible: it appeared to have undergone partial
solution. Since, in the case of Murex, I have seen obvious sand grains
firmly embedded in the teeth of the radula, it would seem probable that the
exceptionally heavy teeth are to be accounted for by accidents of this kind.
The minor variations in specific gravity may perhaps be due to some second
organic substance accumulated in the thick body of the tooth remote from the
margin, and thus not affecting the measurement of the refractive index, which

les constantly between 1°550 and 1:554. The fact that the heavier teeth hang
in the column with their fangs upwards is in accordance with this view.

The refractive indices of the organic portion of the Molluscan radula,

1907. ] Chitin by tts Physical Constants. 481

which I have determined in Littorina, Patella, and Buccinum, lie between
1550 and 1°554.

The shell of Sepia has been shown by chemists to contain chitin, and the
percentage yield of chitosamin has been measured by them. After decalci-
fication with 10-per-cent. hydrochloric acid and boiling in 5-per-cent. potash
it readily breaks up, the thick outer wall of the shell with the rostrum and
lateral expansions becoming freed from the mass of delicate septa. This outer
layer quickly becomes transparent and colourless in parts, the rostrum and
thickened region in its neighbourhood being yellowish. The septa remain
opaque, they adhere to one another by a maze of low sinuous ridges, the
capillary spaces between which readily become filled with air, unless special
care is taken to avoid this. The specific gravity of the outer layer is not
a constant quantity, but ranges from 1°385 to 1393. Alternate boiling in
various strengths of potash higher than 5 per cent. and in 50-per-cent.
hydrochloric acid do not reduce this range. The septa, on the other hand,
form a zone in the diffusion column at the level of chitin. The refractive
index of the inner lamine lies between 1°550 and 1°554; that of the outer
is variable, lying sometimes between 1°550 and 1:544, sometimes between
1550 and 1:554. The variable specific gravity of the outer layer of the
Sepia shell points to the presence of more than one substance. As the
change in specific gravity goes hand in hand with a corresponding change in
refractive index, the suggestion naturally arises that there may be a series of
chitins, of which a lighter and a heavier member are present in the Sepia shell.

Rosenheim* has given some reason to suppose that the fossil carapace of
Pterygotus osiliensis contains chitin. It would be interesting to apply the
method described in this paper in confirmation of his results, and to extend
it to the study of other organic substances.

Summary.

The determination of the physical constants of chitin forms a useful
method of identifying it. The specific gravity of chitin from various sources
approximates to the value 1:398, a number which represents the specific
gravity of chitin precipitated from its solution in strong acid. The HOE
index lies between the limits 1:550 and 1°557.

The bristles of Lumbricus, the pupal skin of Pieris and other Lepidoptera,
the radula of Mollusca and the shell of Sepia, when freed from mineral
matter and easily soluble organic substances, have specific gravities and
refractive indices which lie between the same limits as those of chitin from
varicus sources.

* “Roy. Soc. Proc.,’ B, vol. 76, 1905, pp. 398—400.

482

The Inhibitory Action upon Subsequent Phagocytosis, exerted on
Actwe Normal Serum by Inactive Normal Serum through
which Bacilli have been passed.

By J. C.G. Lepincuam, M.A., M.B. (Aberdeen), Assistant Bacteriologist, Lister
Institute.

(Communicated by Dr. C. J. Martin, F.R.S. Received April 30,—Read
June 13, 1907.)

Considerable difference of opinion still prevails regarding the nature of the
opsonic substances present in normal serum. Wright, Bulloch and Atkin, etc.,
uphold the view that the opsonin of normal serum is a simple thermolabile
body. Muir, on the other hand, regards the opsonin as a body which behaves
like complement, while Dean holds that it is essentially thermostable and in
all probability co-operates in its action with a thermolabile complement. The
demonstration of anti-bodies by complement-deviation experiments (Bordet,
Gengou, Pfeiffer and Friedberger, etc.) has recently proved fruitful in
‘connection with the bacteriolysins, hemolysins, precipitins of immune sera,
and the following experiments were designed to test whether, by a similar
method applied to phagocytosis, the presence in normal serum of opsonic
amboceptors could be demonstrated :—

EXPERIMENT I.

Normal human serum was heated for 30 mins. at 60° C. (denoted “A”). A very thick
emulsion of tubercle bacilli in 1 : 1000 salt solution was added in equal volumes to “ A ”
and kept in contact therewith for 1 hr. 30 mins. at 37° C. The mixture was then centri-
fugalised (7000 revolutions per minute) for 1 hr., and the supernatant fluid pipetted off
(denoted “B”).

Equal volumes of “B” and fresh normal human serum were now mixed and kept in
contact at 37° C. for 1 hr. (final fluid denoted “'T”).

The steps employed in the preparation of the control fiuid were essentially similar to
the above. Thus, heated normal serum was added in equal volume to salt solution
(instead of to tubercle emulsion) and the mixture kept in contact for 1 hr. 30 mins. at
37° C. Equal volumes of this mixture and fresh normal serum were now taken, and
retained in contact for 1 hr. at 37° (control fluid so obtained denoted ‘C”).

The opsonic contents of “T” and “‘C” were now compared in the usual way, fresh
tubercle and staphylococcal emulsions being employed. The result was as follows :—

Bacilli per leucocyte.
Test fluid ‘'T”+leucocytes+ B. tuberculosis ........000+ 05
Control fluid “ C” +leucocytes+ B. tuberculosis......... 2°3

Cocci per leucocyte.

Test fluid “'T”+leucocytes+staphylococci ............ 1°4
Control fluid “ C” +leucocytes+staphylococci ......... 3°57

Inhibitory Action upon Phagocytosis, ete. 483

The difference between “T” and “C” was sufficiently striking, but it seemed desirable
to re-test the two fluids with stronger emulsions of B. tuberculosis and staphylococcus.
Accordingly, after standing in the cold room over-night at a temperature of 2° C., they
were again compared, with the following result :—

Bacilli per leucocyte.

Test fluid “T ”+leucocytes+ B. tuberculosis ........005. 0°8
Control fluid ‘*C ”+leucocytes+ B. tuberculosis......... 4°5

Cocci per leucocyte.
Test fluid “'T”+ leucocytes + staphylococcus ............ 3°9
Control fluid “‘C”+leucocytes+staphylococcus ...... 24°5

Thus the ratio of “C” to “T” in the case of tubercle rose after 16 hrs. from 4°6 : 1 to
5°6 : 1, and in the case of staphylococcus from 2°5 : 1 to 62 : 1.

EXPERIMENT IT.

Fluids “T” and “C” were prepared in the way described above and their opsonic
contents compared. Result :—
Bacilli per leucocyte.

PeSt HUNG ecaeeec selvdhecne 2°6
Control Hurd °° CO? 2.0 ev ecaend 6:0
Cocci per leucocyte.
MESG ULI el, Say ea eee 4°2
Control Hurd §°C? ec cccckccewse 172

After standing over-night in the cold room, they were re-tested with fresh emulsions of
bacilli and cocci. Result :—
Bacilli per leucocyte.

Pest thud, sic. <a eatioo te 1:0
Control fluid “©? oo s..00055-15. fa |

Cocci per leucocyte.
Mest tudo eect cenweeecclevsss 2°3
Control, Muitd.“C.” . diasslt.ccseon ss 90

The ratio of “C” to “T” thus rose after 16 hrs. from 2°3:1 to 7°7:1 in the case of
B. tuberculosis, while in the case of staphylococcus it remained practically the same.

Experiment III.

In this experiment the heated serum and the salt solution were emulsified directly with
B. tuberculosis in an agate mortar. Result :—
Bacilli per leucocyte.

MERE MUI OV cco sie edes See's 2
Control fur C2 ccs cccesses 5°0

Cocci per leucocyte.
Mest hud “Tok cticesese sees 54
Comol fluid Coico lice tess 159

EXPERIMENT LV.

Instead of B. tuberculosis, staphylococci were brought in contact with heated normal
serum. The further steps in the preparation of “T” and “C” remained the same.
Result :—

484 Inhibitory Action wpon Phagocytosis, ete.

Cocci per leucocyte.
Bests Gund ee eee tcnou- secre 1Os7,
Control thud aCe sere-scn.ceeeer 15°7
(Opsonic content with regard to B. tuberculosis omitted.)

The difference between “C” and “T” is thus relatively small. A similar experiment
was made, in which staphylococci, which had been heated at 60° C. for 1 hr. 30 mins., were
passed through normal heated serum. Result :—

_ Cocci per leucocyte.

Control Haid Ce ee ee 18

So far, then, as these latter experiments are concerned, it does not appear
that much, if any, inhibitory action on fresh normal serum is exerted by heated
serum through which staphylococci have been passed. In view of the con-
sistent results obtained with tubercle bacilli in the first series of experiments,
it was important to determine whether saline solutions, through which bacilli
have been passed, exhibited a similar inhibitory action on fresh normal
serum.

A saline solution (1: 1000) was digested at 37° with tubercle bacilli for
1 hr. 30 mins. and then centrifugalised. The extract so obtained was added
in equal volume to fresh normal serum and kept at 37° C. for 1 hr.
A control fluid was made by digesting the normal serum with salt solution
(1: 1000). The contents in tubercular opsonin of these two fluids were then
compared, with the following result :—

Bacilli per leucocyte.
Pesbehiidl), coe cevacieecsssaetese 54

Combtrow iid eects see ed
Three similar experiments yielded the following figures :—
Bacilli per leucocyte. Cocci per leucocyte.
Testinid 3. ee 6°7 \ 5 \ 11°9
Control fuid 27-25... 1064 6 11°9

Extracts of tubercle bacilli in salt solution have, therefore, little, if any,
inhibitory action on the tubercular and staphylococcal opsonins of fresh
normal serum.

Conclusions.

Heated normal serum, through which tubercle bacilli have been passed,
exerts a marked inhibitory action on the tubercular and staphylococcal
opsonins of fresh normal serum. Such an action points to the presence in
normal serum of opsonic amboceptors, the combination of which with the
free receptors thrown off from the tubercle bacilli deviates the complement
from the fresh normal serum, and thus inhibits phagocytosis. Further
experiments of this nature are In progress.

485

Contributions to our Knowledge of the Poison Plants of Western.
Australia.

' By E. A. MANN, Government Analyst, and W. H. Incz, Ph.D., F.1.C.

(Communicated by Professor J. Norman Collie, F.R.S.)
(Abstract. )*

The State of Western Australia has for many years had an unenviable
reputation by reason of the large numbers of indigenous plants which, by
their poisonous properties, have caused heavy losses amongst cattle and other
varieties of stock. At the present time these losses are estimated by the
Agricultural Department at.about £15,000 to £20,000 per annum.

Mr. Jas. Drummond, in 1842, was the first to make a comprehensive
report on these plants. Since then other references may be found from time
to time. In July, 1895, the West Australian Government Bureau of
Agriculture proposed to initiate a full enquiry into the properties of these
plants, and placed the matter in the hands of Mr. 8. S. Dougall (Municipal
Chemist).

Mr. Dougall’s report is to be found in the ‘ Journal of the Department of
Agriculture of Western Australia, December, 1900. He reports that every
effort to detect the presence of an alkaloid in the “ York Road” and “ Box ”
poison plants had been unsuccessful.

In 1899 the Western Australian Department of Agriculture sent specimens
of these plants, together with the “Heart Leaf,’ to Professor Balfour, at
Edinburgh, by whom they were forwarded to Professor Stockman, of the
University of Glasgow, for chemical examination. Professor Stockman
attributed the toxic principle to a substance belonging to the saponin series.+

In 1902, samples of several of these plants were again sent to England by
the Department of Agriculture, and submitted to Professor Dunstan, F.R.S.,
of the Imperial Institute, for examination. A report was received (1905) on
one only of the plants (the “ Narrow Leaf”), in which Professor Dunstan
stated that, by extraction with alcohol, he obtained “a yellow crystalline
substance, probably identical with, or closely related to, quercitrin, the —
glucoside found in the bark of Quercus tinctoria.”

As far as the present authors are concerned, the chemical investigation was

* The full paper was communicated in two parts :—

Part I, dealing with Cygnine, was received February 14, 1906, and read February 22,

1906.
Part II, dealing with Lobine, was received November 5, 1906, and read December 13,

1906.
+ ‘Journal of the Department of Agriculture, Western Australia,’ December, 1900.

486 Mr. Mann and Dr. Ince. [Feb. 14—Noy. 5,

begun by one of them in 1897, and continued intermittently for some years ;
it was not till 1905, however, that a systematic examination of these plants
was commenced in the Government Laboratory.

Isolation of the Alkaloid from the “ York Road” Poison Plant, Gastrolobium
calycinum. Cygnine, CigH29N20s.

One pound of the powdered plant was extracted with 76-per-cent. alcohol,
rendered slightly acid with acetic acid. The bright green percolate was
evaporated at 50° C., and the residue treated with 1-per-cent. sulphuric acid,
filtered and the filtrate cleared with lead acetate, the excess of lead being
removed by means of sulphuretted hydrogen. The cleared solution was
neutralised and precipitated with tannic acid, the precipitate was collected
and mixed with freshly prepared lead hydrate. This mixture was then care-
fully dried below 50° C. and extracted with hot 70-per-cent. alcohol. The
alcoholic solution was evaporated and made acid with hydrochloric acid.
From the solution a crop of colourless crystals separated. These were
purified by recrystallisation. On injection into a guinea-pig, however, they
exhibited no toxic action. ;

In the next experiment 10 lb. of the powdered plant were extracted with
alcohol and treated as before, but with this difference, that in none of the
operations was the temperature allowed to rise above 30° C.

The result was an indefinite crystalline mixture that under the micro-
scope consisted of needle-shaped and cubical crystals. These were separated
by careful solution in alcohol and evaporation in vacuo. (The cubical crystals
are only slightly soluble in alcohol.) By this means a hydrochloride that
crystallised in fine acicular crystals was obtained. That it possessed toxic
properties was proved by the fact that one-tenth of a grain killed a guinea-
pig in seven minutes. The cubical crystals, mentioned above, were often met
with in later investigations; they are colourless, very soluble in water, but
only slightly so in alcohol. They contain no nitrogen or chlorine and possess
faintly acid properties. The mean of two analyses gave :—C = 687 and
H= 81. Calculated for C,2Hig03, C = 69:2, H = 7-7. It seems probable
that this substance is a decomposition product of the alkaloid, for whenever,
during the extraction of the plant, a high temperature (100° C.) had been
used for evaporation, the cubical crystals either preponderated or were the
only crystalline substance obtained. Moreover, on warming the acicular
crystals with water, for the purpose of recrystallisation, the result was
always the same, an increasing crop of the cubical crystals and a non-
crystallisable brown substance.

The acicular crystals were the hydrochloride of a nitrogenous substance,

1906. | On the Poison Plants of Western Australia. 487

and they must be kept dry, otherwise they slowly decompose; moreover, in
their preparation from the plant, it is absolutely necessary that in none of the
operations should the temperature be raised above 30° C.

The free alkaloid is still more unstable, and, when set free from the hydro-
chloride by means of ammonia, is uncrystallisable and decomposes readily.

Hydrochloride of the Alkaloid—tThe acicular crystals, when obtained pure,
were analysed. The mean of seven analyses gave the following results :-—
C= 626, H=66, N=79, Cl=10°5. Calculated for CigHe2N203HCl,
e029, 2H = 6:3, N = 77, Cl = 98.

The salt-is not very soluble in water, 100 cc. dissolving 1°932 grammes at

5° C. The aqueous solution gives the usual alkaloidal reactions.

From its chemical composition, behaviour, and physiological effect, this
substance seems to be new to science and has been named “ Cygnine.”*

The gold salt was prepared by allowing a solution of the hydrochloride in
alcohol, together with chloride of gold, to evaporate in vacuo. It crystallises
in yellow needles, m. p. 220° C., with decomposition.

On analysis it yielded the following results:—mean of six analyses,
0, Hs, N=45, Cl=210, Au= 29-7. Calculated for
CigHaN2O3sHAuCh, C = 34:3, H = 3°5, N = 4:2, Cl = 21:3, Au = 29'5,

The gold salt is fairly stable in a dry atmosphere, but eee decom-
poses under the influence of light and moisture.

Cygnic Acid, CyoH1904—This acid was obtained from the lead precipitate
obtained during the clearing of the alcoholic extracts. The precipitate was
suspended in water, and treated with excess of sulphuretted hydrogen, and
filtered. The solution was treated with barium hydrate, the resulting brown
precipitate washed with alcohol, and decomposed with ammonium sulphate,
filtered, and the filtrate evaporated on a water-bath. The residual crystalline
mass was dissolved in a small quantity of water, reprecipitated as a crystalline
powder by alcohol. This redissolved in water and, evaporated in vacuo, gave
large colourless needles, which decomposed on heating. On analysis they
gave :—-mean of three analyses, C = 52°70, H= 75, N=12°5. Calculated
for CipHgOu4(NH,4)2, C = 52°6, H = 7:0, N = 12:3.

From the ammonium salt the barium salt was prepared; on analysis it
gave Ba = 41:0, Cy)Hs0,Ba = 41°6 of Ba. Attempts to prepare the pure
acid from the barium salt in dilute sulphuric acid only gave a colourless
amorphous residue. The acid is precipitated by lead, barium and silver
salts. The silver salt easily reduces on heating, forming a mirror.

Gastrolobic Acid, C;Hi)0;.H,0.—It was noticed that a flocculent yellow

* Western Australia was originally known as the Swan River Settlement, and a black
swan is still the emblem of the State.

488 Mr. Mann and Dr. Ince. _‘[ Feb. 14-—Nov. 5,

precipitate was formed during that part of the process of clearing the
alcoholic percolates from the powdered plant, which was partly effected by
blowing air through the liquid to get rid of excess of sulphuretted hydrogen.
This precipitate, after repeated recrystallisation from water and alcohol, and
treating with decolourising agents, still remained yellow. It is soluble in
alkalies with a deep yellow colour. The silver salt is a faint cream-white
precipitate. The free substance contains no nitrogen of sulphur. It is a
very weak acid, for the ammonium salt (red-yellow tufts of needles) decom-
poses in the air, losing ammonia and reverting to the original acid. The
sodium salt is more stable (red-yellow tufts of needles). The acid decomposes
without melting on heating. The mean of three analyses, C = 43:5, H = 6:3,
H,O = 10-7. Calculated for C;H;,0;H20, C = 43:7, H = 6:3, HO = 94

The sodium salt gave Na = 19°6. Calculated for C;HipOgNae, Na = 19°4.

Gastrolobin, a vegetable gelatinoid—tIn clearing the syrupy percolate from
the sulphuric acid extraction, it was noticed that, after the addition of lead
acetate, the solution became quite hmpid. To isolate the mucilaginous
principles, the lead precipitate was washed, suspended in water, and decom-
posed with sulphuretted hydrogen. The filtrate had regained its viscous
character. Absolute alcohol precipitated a flocculent substance, which was
soluble in water. The substance does not reduce Fehling’s solution and does
not contain nitrogen or sulphur. The aqueous solution is precipitated by
tannic acid, lead acetate, dilute sulphuric acid, and sodium carbonate. The
substance was purified by solution in water and reprecipitation with alcohol
(five times). An analysis was made: C = 44:2, H = 6:0; CsHi00; requires
C = 444, H = 6:2. The substance was oxidised with nitric acid, no indica-
tions of saccharic, oxalic, or tartaric acids were given, but it yielded the
pyrrol test for mucic acid. The substance probably belongs to that ill-defined
family of vegetable carbohydrate gelatinoids, of which agar-agar is the best
known member. The body is apparently identical with a similar substance
described by Professor Stockman in his report.

Cygnose, CeHi204.—After the removal of the gastrolobin by means of lead
acetate, the limpid solution gave tests with phenylhydrazine and Fehling’s
solution that pointed to the presence of a sugar in the solution. It was
found quite impossible to obtain a pure sugar from the solution. The
osazone was therefore prepared in the ordinary way. It can be crystallised
from alcohol in the form of slender yellow needles, m. p. 179° C. The
mean of four analyses gave C = 60:0, H = 63, N=195°7. Calculated for
‘CgH1904 N2H.CeHs)2, C = 60°3, H = 6:1, N = 156.

The sugar itself seems to be optically inactive both before and after
inversion. It is not fermented by yeast.

1906. | On the Poison Plants of Western Australia. 489

In view of the presence of saponin, quercitrin, or allied bodies in plants
of this group, reported by Professors Stockman and Dunstan, special search
was instituted for these two bodies, but with negative results. »

Toxic Action of Cygnine.—During the course of the research, the poisonous
nature of the products obtained was from time to time established. With
cygnine hydrochloride the following tests were made, with the assistance of
Doctor Blackburne, Government Bacteriologist.

One-tenth of a grain of the hydrochloride dissolved in a few minims of
water, and injected subcutaneously into a guinea-pig weighing 568 grammes,
killed the animal in 7 minutes. One-twentieth of a grain killed. another
guinea-pig (348 grammes) in 22 minutes. Confirmatory evidence was
also obtained by an experiment on a sheep. Seven grains of the salt in
aquegus solution was injected into a sheep weighing 50 to 60 lbs. The first
spasm was manifested 5 minutes after injection ; in 6 minutes the animal fell
to the ground in a rigid condition, and was subjected to renewed spasms up
to 12 minutes after injection, when death ensued.

The following is an extract from Dr. Blackburne’s notes on the first
experiment :—

“After remaining quiet for 2 or 3 minutes it became rigid, with head
stretched forward, then returned to normal, but in a minute or so rapid
convulsions came on. The teeth could be heard to shut with a snap, the
body became rigid, but there was no obvious opisthotonos. These spasms
occurred three or four times in 7 minutes; the animal was quite dead.
A post-mortem examination was immediately made. There was marked
venous engorgement throughout the parenchymatous organs and the lungs,
and all the large veins were much dilated. There was no local reaction
at the point of injection. The heart was greatly overfilled, every chamber
being much distended with dark blood. One hour and a-half afterwards, the
heart (still im situ) responded to stimuli. Death was due evidently to
asphyxia, probably secondary to convulsions. It would appear from this
that the poison belongs to that group of which the principal one is
strychnine.”

Isolation of the Alkaloid from the “ Box” Poison Plant, Oxylobium parviflorum.
Lobine, Coz3HaN30x.

Experience having been gained in the examination of the “ York Road”
plant, the same kind of treatment was adopted in this case. The temperature
throughout the whole investigation was kept as low as possible, never being
allowed to rise above 35° C.

By this means a crystalline hydrochloride was obtained. This salt

490 On the Poison Plants of Western Australia.

crystallised in needles, was very hygroscopic, and decomposed when its
aqueous solution was heated above 35° C., a substance crystallising in cubes
being formed ; in spite of its hygroscopic character, it is but sparingly soluble
in water (less than 1 per cent.). Its toxic action was shown by injection of a
guinea-pig: one-tenth of a grain killed in five minutes. The symptoms were
similar to those caused by cygnine.

The alkaloid itself was only obtained in a semi-crystalline condition by
very careful spontaneous evaporation of the alcoholic solution in the dark ;
as a rule, it remained as an amorphous substance resembling isinglass. It
decomposed on heating. An analysis gave the following results (the mean of
four analyses) :—C = 67:1,H =7°7.,N = 10:2. Calculated for Co3H3:N30,,
Ci 16015, 6 —) 7-3) N02:

The gold salt was prepared as an amorphous yellow precipitate ; it decom-
poses on exposure to light and moist air, and melts at about 100°C. It is
freely soluble in alcohol, but sparingly so in water. On analysis, the mean
of five analyses gave C = 363, H= 44, N=55, Au= 261, Cl= 19%)
Calculated for Co3H3,N304, HAuChl, C = 36°7, H = 4:25, N = 56, Au = 26:1,
Cl= 18-9. This new alkaloid has been named “ Zobzine.”

In nearly all its characteristics it resembles cygnine, but it is more stable,
for the free alkaloid was isolated. Like cygnine, its hydrochloride, on heating,
yields a non-nitrogenous substance, crystallising in cubes, and a nitrogenous
uncrystallisable compound. These cubical crystals are fairly soluble in water,
very soluble in alcohol, but insoluble in ether. They melt without decom-
position at 107° C. They are non-nitrogenous and faintly acidic. The mean
of two analyses was C = 63°3, H = 8-4. Calculated for CyHi,03, C = 63°5,

H = 8'2.
Isolation of Malic Acid from “ Box” Poison Plant.

The lead precipitates obtained during the clearing operations in the
isolation of the alkaloid were treated with sulphuretted hydrogen. From
the filtrate an acid was obtained that gave a white amorphous precipitate
with silver salts; a white, cloudy precipitate, heavy on boiling with calcium
hydrate; and a precipitate with lead salts. The acid solution was neutralised
with ammonia, treated with alcohol to precipitate mucilaginous matter, and
evaporated to dryness. Deliquescent crystals resulted. These were then
heated on a water-bath with excess of lime water, when a crop of crystals
gradually deposited. The free acid prepared from this salt was very soluble
in water. The mean of two analyses gave C = 35°83, H= 46. Calculated
for C,H,O;, C = 35°83, H = 4:5. From this and the properties of the acid
it is probably malic acid. The calcium salt was also analysed, found

Observations on the Life-history of Leucocytes. 491

Ca = 20°9. Calculated for (C4H505)2Ca.H,0, C = 21:0. The free acid melts
at about 100° C.
Examination for Carbohydrates—Sugars seemed to be absent. <A small
quantity, however, of a mucilaginous carbohydrate similar to gastrolobin was
separated.

Owing to the reported presence of quercitrin and saponin bodies, by Dunstan
and Stockman respectively, special search was made for these bodies, but with
negative results.

Observations on the Infe-history of Leucocytes. Part I[.—On the
Origin of the Granules.

By C. E. WALKER, Assistant-Director of the Cancer Research Laboratories,
University of Liverpool.

{Communicated by Prof. C. 8. Sherrington, F.R.S. Received August 2, 1906,—
Read February 7, 1907.)

[PLATE 5.]

The granules that are so frequently found in leucocytes* generally seem
to lie scattered quite irregularly in the cytoplasm of the cell in which they
occur. It is possible, in the case of the leucocytes found in the spleen,
lymphatic glands, and the blood of mammalia, that there never is any ordered
arrangement of the granules. In the bone-marrow, however, where leucocytes
containing granules are often extremely numerous, a section of properly
preserved material will show that the granules in a large proportion of these
cells are arranged in a more or less definite manner. The granules in these
are, as a rule, oval in shape, and are seen to le in sequence close to each
other, so that a line drawn through their long axes would appear as a thread
or wire coiled up irregularly in the cytoplasm of the cell (fig. 1). Given
this line connecting them, the granules would exactly simulate beads threaded
on a wire bent into irregular curves, and put into a small spheroidal space.
There are many gradations in the regularity of this arrangement of the
granules. It varies from the mere suggestion of some of them having been
strung together to very definite order, and the joining of several end to end
(fig. 2). |

There are, again, other cells in which a large number of the granules are

* As in a previous communication (“ Observations on the Life-history of Leucocytes,”

“Roy. Soc. Proc.,’ B, vol. 78, p. 53), the term “leucocyte” is used in the widest possible
sense, and is intended to include all the wandering nucleated cells of the body.

VOL, LXXIX.—B. 2N

492 Mr. C, E. Walker. [Aug. 2

joined together, forming in places a thick, deeply-staining thread, the axis
of which is continuous with the axis of the strings of separate granules
(fig. 3). In some such cells the separate granules and the thick thread
appear to be about equal in amount and continuous with each other (fig. 4),
while in others there are but very few granules, and the thread pre-
ponderates. From these it is possible to pass, by almost insensible
gradations, to cells where there are no granules, but only a thick thread
coiled up around the nucleus (fig. 6). In some of these cells it is practically
impossible to distinguish the nucleus, so dense and darkly-staining is the
coiled thread (fig. 5). So misleading are these figures that for several months
{ took them for, and drew them as, the spireme stages in the prophase of
division in which the nuclear membrane had disappeared. It was only when
I found nuclei in a resting condition existing in the cells which also contained
this thread, that the present interpretation was arrived at. A careful
examination of the cells in this stage, however, will frequently, though not
always, demonstrate the presence of a resting nucleus. Where the nucleus
cannot be seen, the reason is generally very obvious: the thread is so deeply
stained with the basic stain, and is so closely coiled, that it entirely obscures
the similarly stained nucleus. Moreover, while the thread stains very deeply,
the nucleus, apparently, stains less readily than usual, thus adding to the
probability of its being overlooked.

From this stage it is possible to pass again by practically insensible
gradations to cells where the coiled-up thread occupies a space slightly
larger than (fig. 7), equal to, or smaller than the nucleus, until we arrive
at some where it seems to be about the same size in proportion to the
nucleus as is the archoplasm in the case of the spermatid. laiee

Though it has not been possible as yet to trace the origin of this thread
farther, it is strongly suggested that it arises in the archoplasm, which is
often seen to be connected with it (fig. 9).

During the whole of its existence the thread stains very deeply, and always
with a basic in preference to an acid stain. It seems to show best with
saffranin and basic fuchsin. When it has entirely broken up, the granules
formed from it still stain in the same manner; but as they begin to lose their
regular arrangement, so they seem to lose their affinity for the basic stain.
It appears, however, that this happens but slowly in all such cells, and
probably in many the granules never stain with the acid dyes. In bone-
marrow large areas are often encountered where over 30 per cent. of the
cells contain granules staining with a basic dye. In other parts of the body
the cells containing such granules are not present in such large proportions,
and we meet with more in which the granules take the acid dye.

1906.] Observations on the Life-history of Leucocytes. 493

The thread, which subsequently breaks up to form the granules, has only
been met with in the cells in the bone-marrow, and has not been observed in
leucocytes anywhere else in the body.

It would seem, then, that the granules arise from a_ threadwork
forming part of the archoplasm, and which is probably derived from it
originally. This thread grows and eventually breaks up to form the
granules. During the whole of its existence it stains with a basic stain, —
reaching the maximum in staining capacity shortly before it breaks up.
The staining capacity of the nucleus during this period seems to vary
inversely with that of the thread. There seems to be no appreciable
difference between the material forming the thread and the chromatin in an
actively dividing cell. There is, however, no evidence of its having been
derived from the nucleus.

The position, from a cytological point of view, is much obscured by the current
methods of dealing with leucocytes. It is extremely difficult to compare the
results obtained by the methods used by cytologists with what has been
observed in specimens that have been dried, heated, fixed with alcohol,
or otherwise treated in such a way as to materially alter the appearance and
inter-relationship of the parts of the cell. The methods commonly used,
however, have proved to be of the greatest value clinically, and with regard
to the staining reaction of the granules, which have so largely entered into
the current classification of leucocytes, there is nothing incompatible with
the observations here recorded.

It has been seen that the staining reaction in some, at any rate, of the
granular cells changes from basic to acid. It is highly probable that this
change may be hastened or retarded by influences external to the cell while
it is still in the body. This probability is increased by the fact that the
proportion of acid to basic staining granules is increased by so crude an
influence as additional heat and dryness after the cells have been removed
from the body.

The presence, therefore, of varying proportions of cells containing the
so-called acidophile and basiphile granules in different diseases is just what one
would expect, and is no argument against a common origin of both from the
thread here described as occurring in some of the cells in the bone-marrow.*

* The material used has been chiefly obtained from the guinea-pig and rat. The

_ same fixatives were used as described in “ Observations on the Life-history of Leucocytes ”
(‘ Roy. Soc. Proc.,’ 1906). Attention is also drawn to the remarks upon preservation in the
same paper, as smears or films of bone-marrow will not show the figures here described.
No matter how carefully a smear or film be prepared, there must always be a grave risk

that the cells will be partially dried, and the least approach to drying is fatal to the proper
preservation of the cells. | |

DauNinezs

Mr. C. E. Walker.

494 [Aug. 2,

I have to thank Professor Sherrington for his kind advice, which has been
of the greatest value to me, particularly when beginning my researches with
regard to leucocytes.

[Addendum, July, 1907.—I would take this opportunity of pointing out
the relationship between certain structures which arise from true archoplasms.
Among such structures are the archoplasmic vesicles found in the cells of the
testis, which develop into the cephalic cap of the spermatozoon; the similar
structures (Plimmer’s Bodies) which appear in some of the cells in malignant
erowths; and the granules in leucocytes. Whether these phenomena are
connected in some way with the Maiotic Phase is not clear, though something
of the kind would seem to be suggested.

Since the above communication was presented, my attention has been
redirected to acommunication by Professor Mathews, of Chicago University, on
“The Changes in Structure of the Pancreas Cell.”* I give the following

summary of the results obtained by Professor Mathews and myself.

Mathews.

(1) Certain spiral twists or coils
are found in the cytoplasm of the
pancreas cells at certain periods.
They probably arise from the nucleus.

(2) The threads, or some of them,
subsequently collect in a mass, form-
ing the “so-called nebenkerns of the
pancreas.”

(3) These spiral twists or threads
‘proceed to decompose.

(4) A stage apparently intervenes
between the disappearance of the
threads and the appearance of the
granules in any given portion of the
cytoplasm of the cell.

(5) It is probable that the “zymo-
gen granules and the cytomitoplasm
of the inner zone arise as the products
of the decomposition of the threads.”

Walker, |

An archoplasm, 2.¢., an area in the
cytoplasm showing a denser and more
homogeneous structure than the rest,
is found in certain leucocytes. This
archoplasm does not arise from the
nucleus.

A coiled up thread, arising in the
archoplasm, may be observed in some
leucocytes. The archoplasm in the
leucocyte is something entirely
different to the “nebenkern of the
pancreas cell.”

The coiled archoplasmic thread
proceeds to grow until it surrounds
and obscures the nucleus. So far
from any decomposition taking place,
there is never any cessation in the
growth of the thread until it has
invaded the greater part of the
cytoplasm.

The thread breaks up directly into
a number of short lengths, which
subsequently acquire an oval, and,
later, perhaps a spherical shape.

The granules found in the leuco-
cytes described arise by the direct
breaking up into definite segments of
the thread. In these cells there is no
cytomitoplasm, and the thread from
its first appearance never shows any
sign of decomposition.

* © Journal of Morphology,’ vol. 15, 1899, Supplement.

Roy. Soc. Proc., B. vol. 79, Plates:

Walker.

Fier 3:

Fie. 2.

ie.

Fic. 6.

FIG. 5:

4

Fic.

1906.] Observations on the Life-history of Leucocytes. 495

From the above it will be obvious that the second series cannot be
regarded as any kind of confirmation of the first, for the sufficient reason that
they possess nothing in common and deal with entirely different phenomena,
For the same reason the two series do not contradict each other. |

DESCRIPTION OF PLATE.

(Drawn with a 2-mm. Zeiss apochromatic, 1°40 aperture, specially constructed for a
10-inch tube, and a 27 compens. ocular.)

All the cells illustrated are from the bone-marrow of the guinea-pig.

Fig. 1.—Cell showing the granules arranged more or less in strings.

2.—The stringing of the granules is more marked, and some of them are joined end
to end.

3.—Still more of the granules are joined together, forming thick threads.

4,—An earlier stage where granules and thread are about equal in quantity,

5.—The thread before it has begun to break up. The nucleus is hidden.

6.—The same stage with the nucleus showing.

5 7.—An earlier stage. The nucleus here stains more faintly.

8.—A slightly different form of thread, which is not uncommon.

» 9.—A very early stage. The archoplasm is seen connected with the thread.

9

Observations on the Infe-history of Leucocytes.— Part III.
By C. E. WALKER, Assistant-Director, Cancer Research Laboratories,

University of Liverpool.

(Communicated by Professor J. B. Farmer, F.R.S. Received May 15,—Read
June 27, 1907.)

[PLATE 6.]

The phenomena here recorded were first observed among the leucocytes of
Axolotl, which had collected in a mass owing to the presence of a foreign
body or to the infliction of a slight wound.*

Some among the mass of leucocytes thus obtained were seen to be sending
out protrusions from their nuclei. Often where this occurred the action

* The methods used for obtaining leucocytes apart from the other cells of the body
were adapted from those of other observers who have investigated inflammatory
processes. Small celluloid tubes were introduced under the skin of the animal and
removed after being left in the body for varying periods of time. The tubes and their
contents were, after removal, dropped into a suitable fixative and sections made in the
usual manner. Better and more uniform results were, however, obtained by fixing
portions of the coagulated exudation from a small scratch upon the skin of the animal.

496 Mr. C. E. Walker. [May 15,

seemed to be mutual on the part of two contiguous leucocytes (fig.1). These
protrusions were quite different in character from the processes which are
sent out by the nuclei in polymorphonuclear leucocytes and myeloplaxes,
and from the appearances presented during the phenomenon of amitosis.

Shortly after the nuclear protrusions had reached the periphery of the
cytoplasm, which was always reached at adjacent points of the contiguous
cells, the ends of the protrusions joined.* After the joining of these two
processes, a ring of chromatin masses appeared at either end, a short distance
outside the points where the peripheries of the respective nuclei of the two
cells wquld have been if no protrusions had been formed (fig. 2). Strands
of chromatin extended from these masses just under the membrane of both
nuclei, and also along the tube which joined them. Other strands seemed to
pass along the tube between the two nuclei. After this apparatus had been
fully formed, the chromatin in one of the two nuclei began to disappear, and
the amount of chromatin in the other was frequently very markedly increased.
This process continued until one nucleus was completely denuded of its
chromatin, and the nuclear membrane collapsed (figs. 2 and 3). The
chromatin ring at the end of the tube contiguous to the collapsing nucleus
remained long after the other had disappeared, and sometimes the termination
of the tube and the ring appeared in a shape something like an acorn on a
stalk (fig. 3). While this process seemed to be quite constant in so far as
the disappearance of the chromatin from one of the nuclei was concerned, the
increase of the chromatin contained in the other nucleus was not always very
apparent. Frequently, however, there was no doubt that a very large increase
indeed had occurred.

The leucocytes of Axolotl upon which these observations were made were,
of course, in a more or less abnormal condition, but in view of the relatively
small size of mammalian cells when compared with those of Axolotl,+ it
appeared likely that what was very striking in the case of the large cell
might easily be missed in the case of the small. Some slides of normal
mammalhan tissues (bone-marrow, spleen, lymphatic gland, etc.) were
re-examined, and all the stages in the phenomenon described above, excepting

* It can but rarely happen that two contiguous cells will be in such a position in the
section that the mutual sending out of nuclear processes will be presented to the observer
in profile, as shown in fig. 1. Much more often either only one of the two cells wiil be
found in the same section, or the two will be super-imposed either directly or at an angle.
The phenomenon is therefore probably more common than would at first sight appear, as
anything but a lateral presentation of the two cells would be difficult, often impossible,
to interpret.

+t Compare figs. 1—3 with figs. 4—9, which give a fairly accurate idea of the relative
sizes of these cells. |

1907. ] Observations on the Life-history of Leucocytes. 497

the first, were found in the normal mammalian spleen (figs. 4—8). In
addition, an apparently later stage was found, where the collapsed nuclear
membrane had almost disappeared, leaving only a few shreds (fig. 9). Hitherto
this phenomenon has not been observed in any other mammalian tissue than
normal spleen. It is probable, had the series of observations upon what
happened among the leucocytes of Axolotl been carried further, that the later
stage observed in the mammalian spleen would have been found. One
‘would also expect to find the same phenomenon taking place among the
leucocytes gathered together in the mammalian body under certain abnormal
conditions.

There are some points of difference between the appearance of the figures
in Axolotl and those in mammalian spleen. The ring of chromatin masses
in the latter class of cells does not appear to be constant, or even frequent,
at that end of the tube nearest the nucleus which is destined to absorb the
other. Again, the evidence that the process is at first mutual, is questionable
in the case of mammalian leucocytes, as no undoubted cases (such as
shown in fig. 1) have hitherto been discovered. It may be, therefore, that
the process commences mutually between two cells, one eventually gaining
the upper hand in the case of Axolotl, while in mammals the process may
never be mutual at any stage; but one celi always plays an active part, the
other remaining passive. No other material points of difference have yet
been observed. - ,

In both Axolotl and mammalian spleen, however, there is an immediate
conclusion with regard to these observations that appears quite clear. The
nuclear contents, at any rate the chromatin, and probably the linin, of the
one cell are directly absorbed by the other. The probability that the linin
as weli as the chromatin is absorbed is very great. The chromatin masses
-forming the rings at either end of the tube, and the strands of chromatin
extending from these masses, are so sharply defined that it would seem that
something must be holding the chromatin together in a definite form. The
equally sharply defined strands of chromatin that pass through the rings
appear to be continuous with the chromatin and linin of the nucleus.

The process of absorption begins by the mutual throwing out of nuclear
protrusions of a peculiar form by two contiguous leucocytes in the case
of Axolotl, while in the mammalian spleen one leucocyte apparently sends
‘out a protrusion from its nucleus, which attaches itself to the nucleus of
another. In either case the obvious result:is: (1) one cell with a nucleus
containing a double complement of chromatin, and probably of linin, and
(2) a mass of protoplasm without a nucleus, which represents What was
originally the other cell.

498 | Mr. C. E. Walker. | May 15,

The mass of protoplasm which is left without a nucleus may be dismissed
in a few words. It closely resembles what has been described elsewhere as
the red blood corpuscle after it has lost its nucleus, and before it has become
differentiated to its concavo-convex shape.* Whether some red corpuscles
arise in this way must remain doubtful at present. There is, however,
nothing against this being the case in the observations referred to, as red
corpuscles seem to arise in the spleen, as well as in the bone-marrow.

The case of the leucocyte with a nucleus containing a double complement
of chromatin and linin, however, calls for a careful consideration.

In a previous communication upon the life-history of leucocytes, observa-
tions were recorded which seemed to show that some, at any rate, of the
leucocytes in the bone-marrow passed through the Meiotic Phase, and, after
the number of the chromosomes had thus been reduced to one-half the
normal somatic number, many post-meiotic generations occurred both in
the bone-marrow and in the lymphatic glands, in which the reduced number
of chromosomes was retained.t| A comparison was made between these
phenomena occurring among leucocytes and what happens in the case of
many plants. At the same time, it was suggested that, while the large
number of post-meiotic generations, and the fact that but few of the cells
of these generations were destined to conjugate with other reduced cells
in the case of plants, offered a parallel with what apparently happened
in the case of leucocytes, an important difference existed, in that there
was no evidence that any among the post-meiotic generations of leucocytes
ever reached a stage where they conjugated with other reduced cells.

The observations recorded in the present communication suggest that
this comparison may now be carried much further, not only between certain
phenomena exhibited by plants and leucocytes, but also between those
exhibited by plants and unicellular organisms on the one hand and
leucocytes on the other. This further comparison depends upon the inter-
pretation placed upon the absorption of the nucleus of one leucocyte
by another, and is only possible if this is regarded as a process of
fertilisation,

There is a considerable amount of evidence that these leucocytes in which
the fusion of nuclei takes place have passed through the Meiotic Phase. The
members of this particular class of leucocytes arefrequently found to be dividing
mitotically, very commonly exhibiting the reduced number of chromosomes.

* C, E. Walker, “On the Origin and Differentiation of the Red Blood Corpuscles in
Mammals,” ‘Trans. Path. Soc. London,’ vol. 58, Part I, 1907.

+ C. E. Walker, “Observations on the Life-history of Leucocytes,” ‘Roy. Soc. Proc.,’
B, vol. 78, 1906.

1907.] Observations on the Life-history of Leucocytes. 499

This class of leucocytes, which is described in more detail elsewhere,* forms
a large proportion of the cells in the spleen and the majority of those in the
lymphatic glands.

It is necessary, before considering the probability of fertilisation being a
correct interpretation, to dismiss the probability of the process being
simply the destruction of one cell by another. This is a very common
process, but seems always to take the form of engulphment of the whole or
part of the cell destined to destruction into the cytoplasm of another cell.
The usual sequence in the case of vertebrates is, that one leucocyte or other
kind of cell engulphs another bodily into its cytoplasm. The engulphed cell
is gradually disintegrated. The nucleus first shows signs of degeneration.
At a later stage the whole cell somewhat resembles an archoplasmic vesicle
or Plimmer’s body, and eventually it disappears altogether, both nucleus and
cytoplasm. The engulphed cell:is never taken into the nucleus, but remains
in the cytoplasm of the engulphing cell, and is there apparently digested.
This phenomenon may be seen taking place in many different tissues,
particularly where there is any disturbance from the normal condition. Itis so
totally unlike what has been here described as taking place among the leucocytes
in Axolotl and mammalian spleen, that it is unnecessary to go into further
details.=| The process of engulphment and destruction of one cell by another
is, in fact, morphologically a very simple and straightforward affair, and takes
place without the development of any special apparatus on the part of either.
On the other hand, the absorption of one leucocyte nucleus by another, as
here described, seems to involve the development of a special and complicated
apparatus, formed in such a manner that the chromatin and linin of one
nucleus may be transferred directly to the other, without any process of
digestion by the cytoplasm being possible.

It might possibly be suggested that certain prolongations of the nucleus
and cytoplasm which are very common among the leucocytes found in the
mammalian spleen might be confounded with the figures here described. A
careful examination shows that they are of quite a different nature, and so
dissimilar as to be hardly comparable.

The morphological evidence would therefore appear to be in favour of
fertilisation, in that the nuclear contents of two cells are apparently fused
without any disintegration of the contents of either nucleus taking place,

* C. E. Walker, “On the Origin and Differentiation of the Red Blood Corpuscles of
Mammals,” loc. cit. bid., ‘‘ Observations on the Life-history of Leucocytes,” loc. cit.

+ Figures illustrating the appearance of engulphed cells may be seen in the ‘ First
Report on the Cytological Investigation of Cancer,’ Moore and Walker, 1906, Cancer
Research Laboratories, University of Liverpool.

500 : Mr. C. E. Walker. [May 15,

and that the fused nuclear contents continue to exist in a common
cytoplasm.

If we accept the interpretation that the apparent fusion of the nuclear
contents of two leucocytes is to be regarded as the fertilisation of the
one by the other, a remarkably complete, although complex series of
phenomena is suggested; a series of phenomena which, however surprising
it may appear at first, is found on careful examination to be analogous in
many respects, even in detail, to several equally complicated series of
phenomena that have been generally acknowledged to occur.

It is necessary here to describe briefly those phenomena in plants with
which it is proposed to compare those occurring among leucocytes.

A certain group of cells belonging to the soma becomes differentiated and
passes out of somatic co-ordination. Some of these, the tapetal cells, without
passing through the Meiotic Phase, exhibit changes which render them
different in function to the cells of the soma. They are destined to
disintegration in a comparatively short time, quite independently of what
happens in the soma or among the resi of the cells of this differentiated group.
They serve a nutritive purpose. Other cells of the group pass through the
Meiotic Phase. Of these, only a few of the individuals produced by many
subsequent generations are destined to reach the stage where conjugation
may take place with other cells in the same condition. The rest produce
a comparatively unlimited number of post-meiotic generations, and carry
out the function of nourishing and supporting those cells destined to
conjugate.

Apparently a number of the cells belonging to the early generations of
leucocytes do not pass through the Meiotic Phase, but though they are
apparently out of co-ordination with the soma, they serve nutritive and other
purposes, some among them developing granules.*

Other leucocytes, apparently belonging to later generations, pass through
the Meiotic Phase. Of this group the greater number appear to be
converted into red corpuscles, thus serving a nutritive purpose.t Others of
this group, if the fusion of nuclear contents be regarded as fertilisation,
conjugate with other reduced cells.

It may be that when, during the process of evolution, leucocytes or their
immediate precursors were developed and passed out of co-ordination with
the parent soma, these cells reverted to the characters of a remote protozoan

* C. E. Walker, “Observations on the Life-history of Leucocytes, Part II,”
supra.

t+ C. E. Walker, “On the Origin and Differentiation of the Red Blood Corpuscles in
Mammals,” loc. cit. igs

1907.] Observations on the Life-history of Leucocytes. 501

ancestor. This phylogenetic reversion of certain cells produced by the soma
may have become constant, and new characters may have been developed
from this fresh point of departure. This would account for the phenomena
exhibited by the leucocytes in the vertebrates. It has been considered
probable that spermatozoa are also examples of phylogenetic reversion to the
characters of protozoan ancestors.

The possible conclusions to be derived from these observations may be
briefly stated as follows: Certain cells, probably only existing in the bone-
marrow in the case of the adult mammal, give rise to a group of cells which
are, or will immediately pass, out of somatic co-ordination. Some of these
are destined to comparatively rapid disintegration, probably including the
nutrition of other cells among their functions. Others pass through the
Meiotic Phase, and continue to divide, producing an apparently unlimited
number of post-meiotic generations of cells possessing half the somatic
number of chromosomes. The great majority of these are converted into
red corpuscles, serving a nutritive purpose. The remainder are fertilised in
the manner described in the observations here recorded, and, as is the case in
many unicellular animals, after fertilisation divide a number of times, thus
producing fresh generations of leucocytes, which may or may not sub-
sequently pass through the Meiotic Phase again.

This would naturally lead to the further conclusion that the leucocytes
passing out of co-ordination with the soma live as parasites upon the parent
organism, and in themselves possess a complete life cycle.

These observations would appear to have some bearing upon what has been described
as occurring in malignant growths. It has been stated that during the early stages of
malignant growths, leucocytes, entering into the cytoplasm of some of the tissue cells,
proceed to divide mitotically, synchronously with the nucleus of the cell that they have
invaded, the chromosomes of the leucocyte and the tissue cell becoming mixed and being
distributed between the daughter nuclei resulting from the mitosis.* This bastard form
of fertilisation seems to suggest some properties in the leucocytes different to those
possessed by any other cells in the body, excepting perhaps the sexual cells. This
suggestion is strengthened by the observations here recorded.

A fusion between the cells of certain graftable tumours in mice has been described.t
These tumours were said to be malignant, though some doubt has since been thrown upon
this statement. A re-examination of the figures iJlustrating this alleged fusion of cancer
cells of mice suggests very strongly that some of them may be fusions of leucocytes such

* Farmer, Moore, and Walker, “On the Behaviour of lLeucocytes in Malignant
Growths,” ‘Trans. Path. Soc. Loudon,’ vol. 56, Part III, 1905. Jbid., “On the Cytology
of Malignant Growths,” ‘ Roy. Soc. Proc.,’ B, vol. 77, 1906. Moore and Walker, ‘ First
Report on the Cytological Investigation of Cancer,’ University of Liverpool, Cancer
Research Laboratories, 1906.

+ Bashford and Murray, ‘ Roy. Soc. Proc.,’ 1904.

502 Observations on the Life-history of Leucocytes.

as are here described, and that the observers were mistaken in supposing that the cells in
question were cancer cells. That a large congregation of leucocytes does occur in the
case of Jensen’s graftable tumour renders this extremely probable, for we have seen that
among the leucocytes brought together by the introduction of a foreign body in the case
of Axolotl, some of them conjugated. Moreover, nothing in the nature of a conjugation
between cancer cells has been recorded by any other observers, although the alleged
fusion has been diligently sought for during several years.

DESCRIPTION OF PLATE.

Fie. 1.—Two leucocytes in Axolotl in which the nuclei are sending out protrusions.

Fic. 2.—The two nuclear protrusions have joined, rings of chromatin masses have
appeared at either end of the tube thus formed, and the chromatin is disappear-
ing from the lower of the two nuclei.

Fig. 3.—The nuclear membrane of the cell on the right is collapsing. The ring of
chromatin masses has disappeared at the end of the tube nearest the nucleus
that has absorbed the chromatin of the other.

Fic. 4.—Two leucocytes from the spleen of the guinea-pig, showing the nuclear protrusion
from one attaching itself to the nucleus of the other.

Fic. 5.—A slightly later stage.

Fias. 6, 7, and 8.—Stages showing the absorption of the contents of one nucleus by the
other.

Fic, 9.—Showing the complete collapse of the nuclear membrane of one leucocyte, with
only a few shreds of the membrane remaining.

Walker. Roy. Soc. Proc., B. vol. 79, Plate 6.

oe

903

The Time taken in passing the Synapse vn the Spinal Cord of
the Frog.

By FLorENcE BucHAanan, D.Sc., Fellow of University College, London.

(Communicated by Professor Sherrington, LL.D., F.R.S. Received
March I, 1907.)

(Abstract.)*

The time taken by an impulse to cross a single synapse has been estimated
by the measurement and comparison of cord-delays in different reflexes of
the decerebrate frog. The cord-delay was determined by measuring, (1) the
interval of time which elapsed between the moment of application of a single
break induction shock to a nerve containing afferent (sensory) fibres and the
moment of appearance in a particular spot of a particular muscle of a
response, known to have no latency of its own (the electrical response), and
shown to have been produced by the excitation of the afferent fibres; by
then measuring and deducting from this time-interval, (2) that which elapsed
before a response occurred in the same spot of muscle when the same or a
similar induction shock was applied to the efferent (motor) fibres of the
nerve supplying it. From the remainder the time was deducted which would

be taken by an impulse travelling at the rate of 30 metres per second to

traverse the measured length of nerve between the two points of excitation.
The recording of the intervals was automatic, the movements of the
meniscus of the capillary electrometer, the two terminals of which were.
connected with the muscle, being photographed on plates moving at known
rates. The recording muscle chosen was the gastrocnemius, and the nerves
stimulated were the sciatics of the same and of the opposite side. The
experiments were all made on autumn or winter frogs.
It was found that a single induction shock was only capable of producing
a true reflex response in the gastrocnemius from a normal cord when applied
to the sciatic of the same side. The same stimulus applied to the opposite
sciatic only produced the response when the excitability of the cord had
been raised by some drug. The drugs used for the purpose were strychnine
and phenol. .
When the same-side sciatic was stimulated, the reflex response in the
muscle was quite distinct from the response produced directly by the excita-
* The paper will be published in full in the first number of the Physiological Part of

what was the ‘Journal of Anatomy and Physiology,’ to appear in January, 1908, edited
by Professor Schafer, F.R.S.

504 Time taken in passing Synapse in Spinal Cord of Frog.

tion of the motor fibres. The difference of time between the two responses
was hardly altered by the administration of strychnine so long as the dose
was not strong enough to produce spasms or to affect’ the general circulation.
It was, however, slightly shortened, the cord-delay varying in normal cords
between 0°012 and 0-022 second, and in strychnine cords between 0-009 and
0020 second. With stronger doses of strychnine the cord-delay was con-
siderably lengthened, probably as the result of impairment of the circulation.

When the sciatic of the opposite side was stimulated and the excitability
of the cord raised just sufficiently for a reflex effect at all to be produced
by the stimulus, the cord-delay was always about double what it was in the
same-side reflex. In the strychnine cord of preparations the circulation of
which was still unimpaired, the extra-cord-delay in the case of the crossed
reflex became very rapidly shorter as the action of the drug continued and
might even be reduced to one-fifth of its original value in a preparation in
which the total cord-delay in the same-side reflex was hardly altered during
the experiment. It was, however, never reduced to less than 0:004 second.
In preparations in which the reflex responses were spasms, or in which the
circulation was impaired, it also was always long, although not usually
quite so long as the total cord-delay in the same-side reflex. In such
preparations in both kinds of reflexes the central stimulus often failed to
produce its maximum effect on the muscle at the beginning of the response.

The effects of altering the temperature of the cord, and of altering the
strength of the artificial stimulus applied to the nerve, and of fatigue, on
cord-delay and on extra delay respectively in the two kinds of reflexes are
dealt with in the full paper.

It is suggested that the method of measuring cord-delay employed gives
the clue required for ascertaining the number of synapses involved in
succession in any particular reflex, and that there is normally one synapse
interposed in the conductive path of each individual fibre concerned in the
same-side reflex investigated, two in that of each fibre concerned in the
crossed reflex. It is pointed out that the numerical results obtained refer,
as a rule, rather to the modal time than to the shortest time in which a
synapse may be passed, and reasons are given for believing that the set of
synapses alone involved in the same-limb reflex, the primary synapses, are
wont to act much more synchronously than the set of secondary synapses
involved, in addition, in the crossed reflex, when the cord is normal. The
nature of the action of strychnine on the cord is also discussed.

The experiments were made in Oxford, in the autumn of 1906, with sé
aid of the Government Grant administered by the Royal Society.

a
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i

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7 =

905

A Preliminary Summary of the Results of the Experimental
| Treatment of Trypanosomiasis in Rats.

By H. G. Piimmer, F.LS., and J. D. THomson, M.B., C.M.
(Communicated by Sir Ray Lankester, K.C.B., F.R.S. Received July 20, 1907.)

The experiments of which the following note is an abstract have been
undertaken under the direction of the Tropical Diseases Committee of the
Royal Society.

The strains of trypanosomes which have been used in these experiments
are a Nagana from the original strain brought to England, and a Surra from
Professor Lingard in India. The Nagana strain kills rats in an average time
of 5°5 days, and the Surra strain in 69 days.

Chinolin Compounds.

The knowledge of the action of quinine in malaria suggested the trial of
various chinolin compounds. As is well known, quinine itself has no
influence in trypanosomiasis. Cyanin was the substance first tried, as this
in extremely dilute solutions (1—10,000) is very poisonous to trypanosomes
outside the body. A series of Nagana and Surra rats were given doses of
from } to 1 milligramme (which is a poisonous dose for small rats), but all of
these died about the same time as the controls, and in every case the
trypanosomes had increased in the usual manner, so that at death the blood
was as swarming with trypanosomes as if no treatment had been used.
Messrs. Bayer and Co., of Elberfeld, kindly placed at our disposal nine
chinolin and chinaldin compounds and six “cyanin farbstoffe,’ but none of
these produced any effect on the development or course of the diseases.

Dichlorobenzidine+ Acid HH.

This substance, which was first used by Nicolle in trypanosomiasis of mice,
was found, when used both on mice and rats, to lengthen the course of the
diseases, but in no case was the extension of the course of the disease very
marked. Nagana mice, after one dose of 1 c.c. of a 1-per-cent. solution, lived
from 8 to 11 days, and Surra mice from 10 to 16 days, one living as long as
29 days; in Nagana rats the disease was prolonged to an average time of
14 days, and in Surra to an average of 16 days. In all the animals treated
with this substance the spleen and liver were very much enlarged, and living
trypanosomes were found post mortem, showing that the substance had not
practically influenced the pathological processes. In no case was the
substance completely absorbed.

506 Messrs. H. G. Plimmer and J. D. Thomson. [July 20,

Trypanroth.

This was discovered and first used by Ehrlich. When used alone it
lengthened the course of the disease in both Nagana and Surra to about
14 days, but in all cases the spleen was found to be as much enlarged as in
the control, and living trypanosomes were invariably found post mortem.

Arsenious Acid.

This was used at first simply dissolved in water, and later, in order to try to
avoid the irritation and sloughing invariably produced, in a solution slightly
over-neutralised with carbonate of soda. When injected before the fourth
day of Nagana and Surra, arsenious acid caused the disappearance of the try-
panosomes from the blood, but they invariably recurred before death. It was
given in doses of 1 milligramme, and one rat lived as long as 26 days, two to
16 days, and one to 17 days, each of these rats having taken 6 milligrammes
altogether. A very considerable effect was produced on the vitality of the
trypanosomes, as a rat inoculated from the bone-marrow of one of the 16-day
rats did not show trypanosomes in the blood until the ninth day after
inoculation. Whether neutralised or not, sores were’ produced at the points
of injection. The spleen was found post mortem to be about as large as
that of the controls.

Atoxyl.

This substance, which was first used by Thomas and Breinl, has been
found by Ehrlich and Bertheim to be the sodium salt of para-amido-phenyl- —
arsenic acid, with the formula

NH,(C.H,)AsO.ONa.OH,3H20,

and Moore, Nierenstein, and Todd* have confirmed this view of its com-
position, which differs from that originally published, which stated it to be an
anilide of metarsenious acid.

This is the most important substance, so far discovered, in relation to the
treatment of trypanosomiasis. Im all cases of Nagana and Surra (with the
exception of the atoxyl-proof cases mentioned below), atoxyl causes the
entire disappearance of the trypanosomes from the blood, so that rats
inoculated with the blood when it was microscopically free from parasites
failed to take the disease; but the trypanosomes, in our experience, have
invariably recurred, and death was only delayed for a period varying with
the dose and with the time of commencement of the treatment. When the dose
was sufficient, eg., 1 to 15 c.c. of a 5-per-cent. solution in three to five doses

* “On the Treatment of Trypanosomiasis by Atoxyl .... followed by a Mercuric
Salt,” etc., ‘Biochemical Journal,’ vol. 2, Nos. 5 and 6.

1907.] Experimental Treatment of Trypanosomiasis in Rats. 507

(a dose being given every, or every other, day), even after as many as four,
recurrences the spleen was generally found post mortem to be very little or at
most moderately enlarged. A too large dose apparently produced wasting,
and a bad condition of the hair, and, we think, a quicker return of the
trypanosomes.

When atoxyl is given more continuously or too freely than is required, in
cases in which there have been many recurrences, and probably under some
other conditions of which we are ignorant, in a certain small proportion of
rats so treated a race of trypanosomes is produced which entirely resists
atoxyl, and continues to develop and multiply in spite of continued exhibition
of the drug. This strain, when inoculated into fresh rats, retains its resistance
toatoxyl. Ehrlich, who has produced such a strain in mice, calls them
“ atoxyl-fest,” and we have obtained this atoxyl-proof variety of trypano-
some in rats, both in Nagana and Surra, of which mention will be made

later.
1. Monophenylarsenie acid.

2. Nitrophenylarsenie acid.
3. Paratolylarsenie acid.

These three bodies were sent for experimental purposes to Professor
Cushny, F.R.8., by Professor Michaelis, who discovered them. They are of
interest in connection with the treatment of trypanosomiasis, since atoxyl is
an amido derivative of phenylarsenic acid, which was also discovered by
Michaelis. They are extremely poisonous substances, and were given in doses
of 1 c.c. of a solution of 1/400. |

1. Monophenylarsenic acid.—This, in common with other arsenic com-
pounds, notably affected the development of the trypanosomes. In doses of
1 e.c. of 1/400, repeated once or twice in untreated animals, it diminished
greatly the number of trypanosomes; and in three recurrent cases, after
initial treatment with atoxy], it caused the trypanosomes to disappear, but it
did not save any animal; and it is, as are all these three compounds:
poisonous in effective doses. | )

2. Nitrophenylarsenic acid—In untreated animals, and in two recurrent
cases after atoxyl, this caused a temporary disappearance of the trypanosomes ;
in a case of second recurrence after atoxyl, it produced no effect on the
trypanosomes. It is the most poisonous of the three.

3. Paratolylarsenic acid.—This caused a more effective disappearance of the
trypanosomes than the other two compounds, and it was better borne. One
Nagana rat, treated only with it from the beginning, lived for 25 days, having
had, however, three recurrences during this period. At each recurrence the
trypanosomes disappeared after two doses of 1 c.c. of 1/400. In recurrent

VOL. LXXIX.—B. 2 0

508 Messrs. H. G. Plimmer and J. D. Thomson. [July 20,

cases after atoxyl a temporary disappearance of the trypanosomes was also
effected.
Other Arsenic Compounds.

Arrhenal (di-methyl sodium arseniate) and sodium cacodylate were
also tried, but were found to have no effect whatever on the course of the
disease, nor upon the development of the trypanosomes either in first infections
or in recurrences.

Other Bodies,

Fluorescein was tried without any effect; and also sodium cinnamate, on
account of its reputed property of producing leucocytosis, which might have
been useful from the phagocytic standpoint. But it failed to have any effect
in these diseases.

Treatment with two or more Drugs.

The deduction from the above-mentioned experiments with single bodies
is that, at the present time, there is no substance known which alone will
cause a permanent disappearance of the trypanosomes from the affected
animal, 7.¢., effect a cure; so that experiments have been carried out to see
whether any combination of bodies would produce the desired effect. The
compounds of mercury seemed from their known properties to be the most
promising, and the results of experiments in this direction, lately published
by Drs. Moore, Nierenstein, and Todd, are most encouraging. We have not
used the form of mercury (liq. hydrarg. perchlor.) with which these good
results have been obtained; but we have used the lactate, sozoiodol, and
succinimide, and also Donovan’s Solution (liq. arsen. et hydrarg. iodid.). Of
these the succinimide appears to us to be the best, as it is unirritating, and
will apparently mix with atoxyl without change—at any rate it does not
interfere with the action of the atoxyl; the lactate is very irritating, even in
doses of one-third of a minim of the ordinary hypodermic solution, and it is
not so effective as the succinimide; the sozoiodol appears to come between
these two in point of efficiency. But in our experience there is a great
difference in the individual equation of rats with regard to dosage and
poisonous effects, which the consideration of relative weight does not help us
to gauge, so that there has been difficulty in ensuring the best method of
giving these drugs, and of determining their appropriate quantity. The
duration of the disease has been very greatly prolonged in most cases, and in
some we have confidence that a cure has been effected.

Of all the substances. tried, atoxyl has had by far the most favourable
action, and it is easily tolerated, and produces no irritation. But, as mentioned
before, it has no permanent effect; it causes the disappearance of the

1907.| Experrmentul Treatment of Trypanosoniasis in Rats. 509

trypanosomes from the blood, even when in very large numbers, but there
remain behind certain forms of the parasites which can resist it, and which
after an interval can reproduce the general infection with the indifferent forms.
These recurrences can take place, in our experience, up to as many as seven
times. The resistant forms appear to be located in the intermediate
periods in the bone-marrow, but there is some doubt about this yet, as in
some cases we have failed to produce an infection, even with a quantity of the
marrow. It is probable also that some of these resistant forms may be found
in the glands, and observations are being carried out on these points. But
when an infection is produced from the marrow of these atoxyl-treated rats,
the incubation period is very much prolonged, and it may be as long as
nine days before trypanosomes can be found in the blood of the inoculated
aninials. | |

_Atoxyl appears to act not by virtue of the arsenic in it alone, but its effects
are probably due to this in combination with some other of its constituents,
as its action is much more rapid and complete than that of any other of the
simpler arsenic compounds which we have tried.

The following Tables I and II give the results of treatment with atoxyl
and succinimide of mercury : this latter has been given either with the atoxyl
or immediately after it, the best method being, we think, to give two or three
doses of atoxyl and to give a dose of succinimide of mercury at the same time
as the second and third doses, with perhaps one after. Some of our rats have
had too much, and we are now giving less with better effect. The succinimide
has the great advantage of being unirritating to the tissues, and it will mix
with atoxyl without precipitation, and without, at any rate, interfering with the
action of the latter.

910

Messrs. H. G. Plimmer and J. D. Thomson. - [July 20,

Table L—Nagana Rats treated with Atoxyl and Succinimide of Mercury.
Average duration of untreated disease, 5°5 days.

Quantity | Quantity of
No. | Weight. | of 5 p.c¢. | succinimide | Recurrences. | Lived.
atoxyl. of mercury.
grammes. CC. milligrammes. | days.
1 175, ||" 2-4 1°5 | 2 23 | Had young on 20th day.
2 210 2°85 a Derg) | 1 - 80 | Did not die of disease.
3 250 Biol 2°5 iL _ 28 | Kidneys degenerated.
4 200 aie 2°75 | 0 — | Living 143 days.
5 175 2°0 0°75 | 1 27 | Did not die of disease.
6 150 1:8 1:25 | 0 — | Living 140 days.
7 200 18 1°25 0 — | Living 136 days.
8 150 2°75 DOr ae 6 79
9 175 2°6 1°25 0 22 | Succinimide given too long
after the atoxyl.

10 175 3°0 1425 1 — | Living 92 days.

Al 125 18 0°8 0 38

12 200 2 0°75 1 — | Living 85. days.

13 160 2°8 0°75 3 36

14 210 45 1°75 4 59

15 175 i653 0°5 0 — | Living 78 days.

16 190 1 “4 0°25 0 — | Liwing 75 days.

17 200 41 1°75 4 42 | Trypanosomes plentiful at
time of death, probably

. atoxyl-proof.

18 175 6°35 2°0 7 107 | This rat had 5 minims. of
Donovan’s solution on
the initial appearance of
trypanosomes, but, as it
had no effect, atoxyl,
and subsequently succin-
imide, were given.

19 160 4°1 0°75 3 61

20 150 3°7 0°5 3 44. |Given _ paratolylarsenic
acid after third recur-
rence, which killed the
rat.

21 150 Ong 0°75 0 — | Living 37 days.

1907.| Hxperimental Treatment of Trypanosomiasis in Rats. 511

Table I1.—Surra Rats treated with Atoxyl and Succinimide of Mercury.
Average duration of untreated disease, 6°9 days.

|
Quantity of |

|
Quantity |
No. | Weight. | of 5p.c. | succinimide Recurrences. Lived.
atoxyl. of mercury. | |
grammes. C.c. milligrammes. days.

} 175 1°5 2°25 0 43 | Did not die of disease.

2 175 15 2°25 0 68 | Did not die of disease.

3 150 1°5 1°25 0 44 | Did not die of disease.

4, 200" | ok 2°75 3 | 638

5 125 | 1° 1°5 0 | — | Living 137 days.

6 t25 |. 1:8 0°5 0 | 120 | Did not die of disease.

7 joo % | 18 0°75 1 24.

8 WW 2°45 1-0 1 23 | This quantity of succin-
| | imide produced an acute
| | nephritis in this rat, with

necrosis of the epithe-
lum of the convoluted
tubes, and many hemor-
rhages.

9 160 3°3 1°0 1 — | Living 91 days.

10 150 3°0 0°75 4, | 55
11 150 2 4 0°75 | 2 | 32
12 160 2°1 0°37 1 22 | Treatment suspended on
10th day. |
13 160 Tes) 0°25 2 32 | Did not die of disease.
14 145 2°1 0°75 0 — | Living 42 days.
15 120 1°9 0°5 O — | Living 39 days.
16 125 3 °O 1°0 2 | 34 | This rat had 2 c.c. iodipin
| on 38rd day.
17 200 41 1°75 4, |; 42
18 145 1°05 0°25 O | — | Living 42 days.
19 120 0°95 0°5 O | — | Living 39 days.
20 145 1°05 0°65 0 — — | Living 33 days. |
21 125 4}, 2-1 0°75 3 | 654 | This rat had at the out-
| | set 8 mgms. in all of
| arsenious acid, the atoxyl
| being given on the first
and subsequent recur-
| rences.

* 22 155 | 1°5 1:0 1 | 382 | This rat had also initial
| treatment with arsenious
| acid, of which it had

6 mgms. in all, the
atoxyl being given on

(See also No. 8, Table ITI.)

the first recurrence. The
rat did not die of the
disease, but from injury
during pregnancy.

Table III gives the results of treatment with atoxyl and mercury sozoiodol,
from which it will be seen that the results are not so good as those with
succinimide of mercury. Mercury sozoiodol will not mix with atoxyl, a
dense precipitate being formed when the solutions are mixed. The solution
used was the ordinary hypodermic solution (15 per cent.) prepared by
Martindale.

2 O22

ile Messrs. H. G. Plimmer and J. D. Thomson. — [July 20,

Table I]].—Nagana and Surra Rats treated with Atoxyl and Mercury Sozoiodol.
Average duration of untreated diseases 5°5 and 6:9 aeye respectively.

|
| | Quantity | Quantity Racie |
Disease.! No. | Weight. | of 5p.c. | of mercury Sia eee Lived.
| | atoxyl. sozoiodol. | |
| | |
grammes. Cc.) 7 ie sminims. } 4 days.
hye 2 200 1°35 1 Ir ee 24.
2 150 1 °4 if 1 26
NIGI Snel hy 145 18 1 0 — | Living 108 days.
4, 155 2°4 | 14 T+) 126
( 5 275) 18 14 0 Living 122 days.
| 6 LES O RE a Lets! 1 0 35 | Did not die of disease.
| | 7 815 5-7 13 4 59
| 8 150) 4). 16°58 12 a 81 After second recurrence
ies J | with mercury sozoiodol,
aa | succinimide of mercury
| | was substituted, of which
| | 0°75 mgm. was given in
| | all.
reg) 150 747 5 1 33 | After first recurrence, this
| | rat was given succinimide
| of mereury, of which it
| | | had 1 mgm. in all.
| |

Table IV gives the results of treatment with atoxyl and lactate of mercury,
which are less good than those with succinimide and soziodolate of mercury.
This drug has the disadvantage of being very irritating to the tissues, and
also of forming a precipitate when mixed with atoxyl. The solution used
was the ordinary hypodermic solution (25 per cent.) prepared by Martindale.

Table IV.—Nagana and Surra Rats treated with Atoxyl and Lactate of Mercury.
Average duration of untreated diseases, 5°5 and 6°9 days respectively.

| | Quantity | Quantity of | Bees | |
| Disease. | No. | Weight. _ of 5p.c. | lactate of | a ve | Lived.
| | atoxyl. | mercury. | ; | | |
| | | | | |
i | |
grammes. | c.€. minims. | days.
if 7st ae 85 if | Deghy ) ee
Shen 150) yl 2. 1 Teh 2
|) 3 POOR) elke 1 OP 238
| 4 175 3°65 | 1 1 31 | This rat had 0° mgm. of
| succinimide of mercury
| after first recurrence. |
bes ZOO Nel wedice 1 1 32 |
| [6 He i Bs 1 1 30
Ne Surrey villa 7 75s bate gs 1 0 21 | Did not die of disease.
| 8 Signe asa i 1 24.
9 125° 9 4 43:20 2 2 35 | This rat had 1 mgm. of suc-
| | cinimide of mercury after
| | first recurrence.

il

a

1907.| Laperimental Treatment of Trypanosomiasis in Rats. 513

Table V gives the results of a few experiments made with atoxyl and
Donovan’s solution (lig. arsenic et hydrarg. iodid.) which are somewhat

_ encouraging. During the last month a fresh and more extensive series of

experiments have been started. The poisonous effects of Donovan’s solution

show themselves in the intestines, which become intensely inflamed, whereas

~ the effects of too large doses of succinimide of mercury show themselves

principally in the kidney, in the form of an acute inflammation going on to

necroses with multiple hemorrhages.

Table V.—N agana and Surra Rats treated with Atoxyl and Donovan’s Solution.
Average duration of untreated diseases, 5°D and 6:9 days respectively.

Quantit Quantity of

Disease.| No. | Weight. | of 5 ne Teuawens re Lived.

| atoxyl. solution. ;
grammes. Cie minims. | days.

Nagana |. 1 130 1°8 5 2 38 | This rat had 2°5 c.c. 1/400
monophenylarsenic acid
after first recurrence; this
removed the trypanosomes
from the blood, but a
second recurrence took
place 11 days later.

(ae 175 3°7 lg a — | Living 67 days.
3 150 2-4 15 3 72
A 200 2°25 4, 1 28 | The 0°5 c.c. of Donovan’s
+0°5 c.e solution was given to test
dosage.* Next day the
Surra /4 rat became very ill, and
died two days after with
acute enteritis. No kidney
lesion.
5 190 2°25 5 1 23
ak, Gx: 300 1°0 8 0 — | Living 36 days.

* See paper by Drs. Moore, Nierenstein, and Todd, before referred to.

Table VI gives the results of a series of experiments made with atoxyl

and iodipin.

This latter is a solution, or mixture, of iodine in oil of sesame,

unirritating, and it can be given in large doses with safety.

made by Merck, who kindly placed some at our disposal, and it is stated to be
The results are.
sufficiently encouraging to suggest a further trial of this combination. The
oil takes a very long time to get absorbed; it becomes colourless from
absorption of the iodine, and appears to cause no irritation in the tissues.

514 Messrs. H. G. Plimmer and J. D. Thomson. [July 20,

Table VI.—Nagana and Surva Rats treated with Atoxyl and Iodipin. Average
duration of untreated disease, 55 and 6:9 days respectively.

|

Quantit Bn. ‘ | |
Disease.| No. | Weight. | of 5 p. = Saennly on Bec | Lived. |
oral iodipin. rences. | |
| 7S i oe
| grammes. c.c. ¢.c. days. |
pa 275 4,°9 7°5 2 35
2 150 5 *4 9°5 5 71
3 150 LES 6°0 0 26
Nagana|+ 4 195 1°5 2°0 0 112 | Did not die of disease.
| 5 175 Zana 6°0 2 23 | Did not die of disease.
6 275 4 °O 75 0 29 | Did not die of disease.
ews 175 45 7-0 3 A
| |
G28 175 ts: 40 0 57 | Had eight young towards
| | | end of treatment.
| 9 195 15 2-0 0 — | Living 132 days.
| | 10 195 1°5 2°0 O — | Living 132 days.
i lhe GL 143 6°6 12:0 2 44,
Surra.../< 12 250 41 4:0 1 29 Did not die of disease.
13 250 4°O 4 °O 2 24:
| | 14 215 4°5 6°5 0 — | Living 108 days.
| | 15 165 2°9 4°5 2 33
| | 16 150 3°3 6-0 3 31 | Did not die of disease.
| (S07. 150 1} 2°5 1 20 | Did not die of disease.

The Production of an Atoxyl-proof Race of Trypanosomes in Rats.

This phenomenon is of great biological and therapeutical interest, and
needs much further research yet, both for its explanation, and its bearing on
treatment.

In certain rats, after treatment with atoxyl, a number of recurrences may
take place—we have seen as many as seven; these recurrences at first yield
to treatment with atoxyl, but as the case progresses towards its end, the
recurrences occur at shorter and shorter intervals, until one comes to a time
when they can be no longer affected by atoxyl. The trypanosomes in this
case have. become atoxyl-proof. Ehrlich succeeded in producing an atoxyl-
proof strain of Nagana in mice, and we have succeeded in producing atoxyl-
proof strains of both Nagana and Surra in rats. One point of importance
biologically is that these atoxyl-proof trypanosomes do not lose their
resistance to atoxyl on transference into other rats, and we have carried this
strain on in Nagana through a series of eight successive rats, and in Surra
through a series of six rats without any loss of their atoxyl-proof properties.
When treated with atoxyl, even in large doses, multiplication goes on just as
rapidly as in untreated animals, and the rats die at the usual time. Our
cases have been given atoxyl to test them, and have been then treated with
various substances, as set forth in the table below. Of these substances,

1907.| Haperimental Treatment of Trypanosomiasis in Rats. 515

trypanroth and some form of mercury have given the best results. In
thinking of human trypanosomiasis in this connection, the danger of the pro-
duction of an atoxyl-proof strain will be at once apparent: so that: atoxyl
should be given to human beings with the greatest care and forethought.

The following Table VII summarises the few results we have obtained so
far. Rat No. 1 was inoculated from a Nagana rat which had become
uninfluenced by atoxyl after the third recurrence, before which large and
repeated doses had been given : and Rat No. 11 was inoculated from a Surra
rat in a similar condition.

A

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Expermmental Treatment of 1

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517

The Pressure of Bile Secretion and the Mechanism of Bile
Absorption in Obstruction of the Bile Duct.

By Percy T. Herring, M.D., and SUTHERLAND Simpson, M.D., D.Sc.

(Communicated by Professor Schafer, F.R.S. Received May 1,—
Read June 27, 1907.)

(From the Physiological Laboratory of the University of Edinburgh.)

In the course of a series of experiments which were made for the purpose
of determining the mode of entry of bile into the lymphatics of the liver
during obstruction of the common bile duct we had occasion to measure the
pressure of bile secretion in several animals. Our results were so different
from the figures generally given that we have ascertained the maximum
pressure of bile secretion in a number of dogs, cats, and rabbits, in two
guinea-pigs, and in two monkeys. The first part of the present paper deals
with the pressure of bile secretion; in the second part the mechanism and.
site of bile absorption are discussed.

We are indebted to Professor Schafer for help and advice in our work..
The expenses of the research have been defrayed by a grant from the Moray
Fund for the prosecution of research in the University of Edinburgh.

Part I.—THE PRESSURE OF BILE SECRETION.
Previous Observations.

Kstimations of bile-pressure were first recorded by Friedlander and
Barisch (5). These observers found that the maximum pressure attained by
the bile in guinea-pigs varies between 184 and 212 mm., measured in terms
of the height of a column of bile and water. Their method of procedure
consisted in ligaturing the common bile duct near the duodenum, and tying a
cannula into the fundus of the gall bladder which was then brought forward
into the abdominal wall. A vertical glass tube was connected to the cannula
by rubber tubing, the animal placed on its side and a millimetre scale affixed
to the vertical tube so that the zero mark was opposite the fundus of the
gall bladder. The bile mounts in the tube at first rapidly, then with
decreasing speed until a maximum is reached. The column of fluid moves
up and down with the respirations of the animal, and sinks again after a
maximum is attained.

Heidenhain (8) in 1868 published the results of a number of experiments
on bile secretion, and among them were several in which the bile-pressure

VOL. LXXIX.—B. 2P

518 Drs. P. T. Herring and 8. Simpson. [May 1,

was recorded. In a curarised dog the maximum pressure attained was
146 mm. bile. In a rabbit a pressure of from 163 to 168 mm. bile was
recorded, in another rabbit 115 mm. bile was the maximum, while in a third
rabbit a maximum pressure of 242 mm. bile was reached.

In Hermann’s ‘Handbuch der Physiologie,’ Heidenhain (9) gives the
secretion pressure of bile in five dogs as varying from 119 to 220 mm. of a
solution of sodium carbonate. A simultaneous measurement of blood-pressure
in a branch of the superior mesenteric vein in each of these animals gave
figures varying from 50 to 90 mm. of a solution of sodium carbonate. From
these figures and those of Friedlander and Barisch, Heidenhain concluded
that secretion of bile is not a mechanical effect of blood-pressure in the liver,
but a vital act, and that the pressure of secretion is produced by the activity
of the liver cells. Heidenhain showed, moreover, that the maximum pressure
reached in any case is not an absolute measurement of the force that the liver
cells can exert in secretion, but is rather the pressure at which bile secretion
is balanced exactly by bile absorption.

Most text-books of physiology give Heidenhain’s figures. Afanassiew (1)
is stated to have recorded the maximum pressure in dogs as 275 mm. bile.

Richet(15) gives the maximum pressure in cats as varying between
158 and 264 mm. bile. These figures appear to be taken from the results of
experiments by Kowalewsky (12), who finds the pressure varies from
12 to 20 mm. Hg. in curarised cats.

In 1905, Freese (3) measured the force of contraction of the gall bladder in
dogs. He introduced a cannula into the gall bladder through the common
bile duct and cystic duct, then tied a hgature round the common duct near
the duodenum. Stimulation of the splanchnic nerve caused contraction
of the gall bladder sufficient to support a column of Ringer’s solution 214°5 to
313 mm. high. The method does not appear to have excluded the secretion
pressure of bile. Ligature of the common bile duct near the duodenum must
have brought about a rapid rise of pressure in the bile passages which would
be transmitted to the interior of the gall bladder. It is possible that some
of the pressure changes in Freese’s experiments, ascribed by him to the
musculature of the gall bladder, may have been due to changes in the rate
of secretion and absorption of bile in the liver.

Methods employed in the Present Research.

The animals were anesthetised with chioroform. Tracheotomy was then
performed, and the anesthetic subsequently continued by a tracheal tube.
In some cases ether was employed after the chloroform. In all experiments
the animals were kept deeply under the anesthetic and killed by an overdose,

1907. | The Pressure of Bule Secretion, etc. 519

or by opening the inferior vena cava in the thorax. -After anesthesia was
complete, the animal was partially immersed in the supine position in a large
bath of 0-9 per cent. salt solution kept at a constant temperature of 37° C.
The abdomen was opened and the cystic duct clamped by a small strong clip
near the gall bladder. A silver cannula was inserted into the common bile duct
and tied in position there. A fine piece of rubber tubing was slipped over
the end of the cannula and attached to the lower end of a vertical piece of
glass tubing 1 mm: in internal diameter. A millimetre scale was attached
behind it, and so arranged that it and the glass tube could be raised or
lowered to adjust the zero mark. The rubber tubing had in it a T-piece, from
the third limb of which another piece of tubing led off to the side of the bath,
and was so arranged that when open the drops of bile fell on an electric
marker, by means of which the rate of secretion at zero pressure could
be recorded on a slowly moving drum. The abdomen was freely opened, so
that movements of the diaphragm affected the pressure as little as possible ;
in this way the respiratory spring of the column of bile was diminished and
rarely exceeded 1 or 2mm. The opening in the abdomen was covered with a
sheet of cotton wool, and the animal was then entirely immersed, with the
exception of the head and neck, in the warm salt solution. The vertical glass
tube was adjusted so that the zero mark was at the level of the hepatic duct
‘at the portal fissure. Part of the liver was thus above the level of the zero
mark, but the greater part of it was at a lower level. This we believe to be
as exact a method as that adopted by Friedlander and Barisch. It is certainly
advantageous to maintain the temperature of the animal by warm saline when
the abdomen is open, seeing that the experiment may be of some considerable
duration. :

In some animals we also determined the blood pressure in the splenic vein
as near the portal vein as possible. The pressure was recorded by means of
an apparatus similar to that described for recording the bile pressure. In this
case a T-tube led to a pressure bottle containing a saturated solution of sodium
carbonate, and the apparatus was filled and the pressure recorded in terms of
a column. of that solution. Before removing the clip from the vein after
insertion of the cannula, the fluid was allowed to fill the vertical tube to a
height approximating to the probable blood pressure ; the pressure bottle was
then shut off by a clip, and the clip on the splenic vein removed. The method
was found to give good results.

Occasionally we made intravenous injections of extract of duodenal mucosa
or of bile into a jugular vein. |

In beginning an observation we usually recorded the rate of flow of bile

secretion at zero pressure by the marking of drops in the way described on
pang 2 Na

520 Drs. P. T. Herring and S. Simpson. [May 1,

a slowly moving drum. After a time the tube leading to the dropper was
clamped, the bile began to rise in the vertical tube and the height reached
was recorded every five minutes, and in some cases every minute. Readings
were continued for some time after a maximum pressure had been registered.
We frequently allowed the bile to flow again at zero pressure, and after
a time recorded the rate of secretion again. In some animals several estima-
tions of the maximum pressure were made with intervals between of
secretion at zero pressure. In a few cases we took simultaneous records of
bile and pancreatic pressures; the latter we intend to deal with in a sub-
sequent paper.

Results Obtained.

The bile pressure was measured in 19 cats. One of these was unhealthy
and suffering from diarrhoea. The maximum bile pressure in this cat was
210 mm. bile, and was the lowest of the series. The lowest maximum of
the other 18 was 240 mm., and the highest pressure of any we recorded was
373 mm. The average maximum pressure for 19 cats was 3044 mm. bile.
The subjoined table gives the figures for each animal.

Cat. Sex. Weight. yy eae ie

grammes. mm.
A Male 3300 366
C , 2850 330
D Male (castrated) 2900 280:
E Female 2650 373
EF Male 2600 310.
G f 2950 335
H Female 2500 321
if Male 3200 336
J ‘ 2750 282
L Female 2430 289
M Male 3100 210
N Male (castrated) 3720 310
O Female 2500 325
P Male 3200 315
Q f 2850 319
R ff 3850 240
N) Female 2600 272
T i 2900 24.4,
U

29

2300 327

Measurements of bile pressure were made in eight dogs. The lowest
maximum recorded was 243 mm. bile; the highest was 342 mm., figures
considerably in excess of those given by Heidenhain. The lowness of the
readings obtained by Heidenhain in the dog may possibly be due to his
having employed curari. The average pressure in the eight dogs we

1907. ] The Pressure of Bile Secretion, ete. 521

experimented on was 300 mm. bile. The table presents the figures for each

animal.
| Due cae Weight Maximum{pressure
ae ; ; ; of bile.

l
| kilos. mm.
A Male 10 °5 328
B . 13-0 295
C Fe 12 °6 243
D Female 11°5 285
E Male 12 °2 342
F a 11°0 285
G4 3 9 25 304
H va 10 °5 Bly

Four ordinary rabbits gave us maximum bile pressures ranging from
181 to 204 mm. bile. The average pressure was 197 mm.

In two Belgian hares pressures of 225 mm. and 306 mm. bile respectively
were recorded. The latter animal was a large one, and the rate of bile
secretion was rapid.

The subjoined table gives the details.

Animal. Sex. Weight. SUS SHES | AUERSURE
of bile.

grammes. mm.

| Belgian hare A ............ Female 2800 225

} Ordinary rabbit B......... Male 2100 203
Ordinary rabbit C ......... Female 1900 181
Belgian hare D ............ Male 3300 306
Ordinary rabbit E......... Female 2700 200

| Ordinary rabbit F ......... i 2250 204:

|

|

We made two estimations in guinea-pigs, but found these animals difficult
to work with. A male guinea-pig, weighing 480 grammes, gave us a
maximum bile pressure of 122 mm.; a female, weighing 650 grammes, gave
a maximum of 154 mm. bile. These figures are less than those given by
Friedlander and Barisch. Both guinea-pigs took the anesthetic badly, and
died before many observations had been made. Our figures are for this
reason in all probability lower than the average.

In one monkey (Macacus rhesus) the maximum bile pressure was found
not to exceed 220 mm. bile. This animal was unhealthy, and had suffered
for some time from diarrhoea. In another monkey (Macacus rhesus) the bile
pressure rose te 321 mm. bile.

The highest bile. pressures were recorded in cats, 373 mm. bile in Cat D

522 Drs. P. T. Herring and 8. Simpson. [May 1,

being the greatest. The average maximum for cats, 3044 mm., in our series
corresponds closely with the average maximum for dogs, 300 mm. Rabbits
have a lower pressure, in round numbers about 200 mm. bile. In a healthy
monkey the maximum pressure was 321mm. Bile pressure in the monkey
probably averages about the same as that in the cat and dog.

In all our experiments the pressures recorded are measurements of the
pressure in the common bile duct in terms of a vertical column of bile.
The gall bladder was occluded in every case, so that its contractions could
have no influence on the pressure; that the occlusion was complete was
verified in every animal after death. The free opening of the abdomen
under warm normal saline prevented extraneous pressure on the liver by
abdominal and diaphragmatic movements ; consequently, little oscillation of
the column of bile ever occurred, and the readings may be accepted as
indicating a true secretion pressure.

Graphic representations of the rise of bile pressure and its time relations
are interesting. The accompanying chart gives several examples of pressure
curves. In it the figures on the left-hand side denote the pressure in
terms of millimetres of bile, while the figures below the chart represent time
in five-minute intervals.

As a general rule, the bile pressure rises rapidly at first, the rate
depending chiefly on the rate of bile secretion. After reaching a pressure
of 100 mm. or thereabouts, the rise of pressure begins to slow down.
Cat A, Dog E, and Rabbit D (see chart) gave typical pressure curves. In
these animals the bile pressure rose rapidly, but the amount of rise
diminished at every reading until a maximum was reached, after which the
pressure began to fall. The curve of pressure in Cat P represents another
type in which, after attaining a certain height, the pressure remains
stationary or even falls, but only temporarily, afterwards rising gradually,
but slowly. In this cat the maximum pressure, 315 mm., was attained
3 hours 55 minutes after commencement of the experiment. We had several
examples of this slow rise to a maximum. In the chart is also placed a
short record of the height of portal vein pressure of Cat P; the large drop
in bile pressure was due to interference with the portal circulation in
putting the cannula into the splenic vein; some of the sodium carbonate
solution also entered the circulation. A short record of portal vein pressure
in Dog Eis also given. A simultaneous reading of bile pressure and portal
veln pressure shows that the two coincide in their variations. Rise of
blood pressure is accompanied by a rise of bile pressure; fall of blood
pressure by a fall of bile pressure. Changes in blood pressure in the portal
vein at once show themselves in the rise or fall of bile pressure. The rapid

1907. | The Pressure of Bile Secretion, ete. 523°

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524 Drs. P. T. Herring and S. Simpson. [May 1,

variations thus brought about point to a mechanical explanation of their
occurrence. Heidenhain argued that bile absorption took place with greater
rapidity at a low blood pressure, while the rate of secretion by the liver
cells was also diminished. No doubt this is a potent factor in the long
run, yet the slight and rapid changes in pressure that occur must be
mechanical. Stimulation of the vagus nerve, for instance, causes a rapid and
- simultaneous fall of both bile pressure and portal vein pressure. In Dog OC,
with bile pressure 204 mm., and portal vein pressure 107 mm., stimulation
of left vagus by faradic current for five seconds caused an immediate fall of
bile pressure to 195 mm. and blood pressure to 95mm. One minute later
bile pressure had risen to 214 mm. and blood pressure to 115 mm.

Injection of extract of duodenal mucous membrane into a vein frequently
caused a rise of bile pressure, and was found to increase the rate of secretion
at zero pressure. It was not always successful, and often, while found to be
very active on pancreatic secretion, had no effect on bile secretion or
pressure.

The injection of bile into a vein was invariably attended by increased rate
of secretion. It usually brings about an increase of bile pressure ; but if the
latter is high at the time of injection very little effect is noticed.

The animals we experimented on were, with a few exceptions, well
nourished, and had been fed in the morning some hours before being
anesthetised. The rate of bile flow as determined before estimation of bile
pressure was found to be very variable. A rapid rate of secretion is not
essential to the production of a high pressure, but it influences the form of
the pressure curve. The size and weight of the animal does not appear to
influence bile pressure, provided the animals are of the same species.

We did not take many subsequent records of bile pressure in the same
animal. The rate of secretion of bile is usually diminished after the bile
duct has been obstructed for some time, but not invariably; we have
obtained records of bile flow in which the rate has been increased.
Subsequent records of bile pressure give as a rule lower readings than the
first, as was pointed out by Friedlander and Barisch. An exception may be
quoted. In dog D a maximum pressure of bile of 285 mm. was recorded.
After release of the bile from the bile duct a record of bile flow was taken,
and, half an hour later, secretion was more rapid than it had been prior to the
first experiment. A second record of bile pressure, taken an hour after the
first, reached a maximum of 305 mm. bile, an increase of 20 mm. pressure.
A lower second reading is not therefore essential, and with an increased rate
of secretion by the liver cells there may be, as in this case, a higher pressure

reached.

1907.] _ The Pressure of Bile Secretion, ete. 525

Part IJ.—ABSORPTION OF BILE FROM THE BILE PASSAGES IN THE LIVER.—
PREVIOUS OBSERVATIONS.

Ligature of the common bile duct in the living animal is followed sooner

or later by the presence of the constituents of bile in the blood. It was at

one time believed that the bile enters the blood directly in the liver.
Haller (6) stated that communications exist in the liver between the hepatic
veins and bile ducts, and in support of this adduced the fact that injection of
the bile ducts with fluid is followed by its escape into the veins.
Saunders (16), in 1795, published the results of experiments performed on
dogs. In a living dog he lgatured the common bile duct and killed the
animal two hours afterwards. He found the lymphatics of the liver
distended with bilious fluid and the thoracic duct filled with moderately
diluted bile. There was no appearance of jaundice as indicated by the
conjunctiva, and he stated that it was probable that bile was only just
beginning to enter the blood vessels.

Tiedemann and Gmelin(17), in 1827, recognised that most of the
observations of Saunders were correct. They found jaundice appeared not
earlier than the third day after ligature. Frerichs(4) noted a yellow tinge
of the conjunctiva first appearing between the 60th and 70th hour after
ligature. He challenged the statement of Saunders that bile is found in the
blood after two hours.

That the bile is absorbed by the lymphatics of the liver in obstructive
jaundice has received abundant confirmation from the works of
V. Fleischl (2), Heidenhain, Vaughan Harley (7), and many other observers.
Heidenhain determined experimentally the site of the escape of bile from the
bile channels into the lymphatics. He injected a solution of indigo-sulphate
of soda into the bile ducts and found that the skin, mucous membranes, and
urine were rapidly coloured blue. Examination of sections of the liver
showed him that there was no coloration of the interior of the lobules, the
blue was present in the interlobular bile ducts, but not in the bile capillaries.
Secretion of bile must have been going on while absorption was taking place.
Heidenhain concluded that the bile escapes from the interlobular bile ducts
into connective-tissue spaces and is carried away by the lymphatics.

Most observers, and especially Vaughan Harley, have insisted that in
obstructive jaundice the bile reaches the blood solely by the lymphatics.
Nauwerck (14), on the other hand, denies this and believes that the bile
enters the blood directly through fine channels in the liver cells.

. Mall (13) has recently stated that when the lymphatics of the liver are
injected from the bile duct there is always an extravasation in the centre of

526. Drs. P. T. Herring and S. Simpson. [May 1,

the “portal unit.” Extravasation does not take place from the bile
capillaries, but only from the duct as it communicates with the capillaries, as
well as from the larger bile ducts. Mall finds that the lymphatics of the
liver do not drain all portions of the liver, but only those parts formed by
the centres of the “portal units.” He states that there are no lymphatics in
the centres of the lobules, nor at the nodal points.

In a paper (11) published last summer we gave reasons for concluding that
there are no lymphatics in the liver lobules at all; they are confined to the
portal spaces, 7.e., to the centres of the “ portal units” of Mall.

The question of bile absorption resolves itself into that of the mode of
formation of lymph in the liver. If there are no lymphatics in the lobules
of the liver, and the production of liver lymph is to be regarded as purely
mechanical, its source must be confined to the portal spaces. In this case
the interlobular veins must be extraordinarily permeable to allow the
passage outwards into the lymphatics of so large an amount of concentrated
lymph. On the other hand, if the liver cells take part in its formation, the
lymph must be secreted at the periphery of the lobules, and if all the cells
in the lobule participate there must be something of the nature of a com-
munication from cell to cell throughout the lobule. Given such a mechanism,
the secretion of lymph may be regarded as a vital act on the part of the
liver cells. We hoped that we might by our experiments throw some light
upon this question.

Methods Employed.

After determining in the manner already indicated the maximum pressure
of bile secretion in an animal we allowed the obstructed bile to escape from
the bile passages. A piece of rubber tubing leading from a reservoir
containing carmine gelatine was then slipped over the cannula in the
common bile duct, and the bile passages were injected with carmine gelatine
at pressures varying in different animals. We made use of an apparatus by
means of which we could inject at pressures measured in terms of the height
in millimetres of a vertical column of water. The difference in pressure
between similar heights of a column of bile and a column of water being
practically negligible, the pressures may be regarded as sufficiently alike.
In most cases we employed a pressure of injection not exceeding the
maximum of bile pressure previously recorded in the animal. In some cases
the injection was continued and the bile duct ligatured before the animal was
killed. In others the injection was made post mortem, the animal being
killed by an overdose of chloroform, or by opening the inferior vena cava
above the diaphragm so as to avoid a rise of blood pressure in the liver.
Sometimes we used an injection pressure higher than the maximum bile

1907. | The Pressure of Bile Secretion, etc. 527

pressure, in other cases a lower pressure was employed. Injection was
continued for periods of time varying from one to six hours.

On completion of the experiment, the bile duct was ligatured and the liver
removed and placed in cold 10-per-cent. formalin. After a time it was cut
up, fixation completed in formalin, and pieces cut in paraffin. Sections were
lightly stained with hematoxylin and examined.

Results Obtained.

Injection of the bile duct at the maximum pressure of bile secretion was
in every case sufficient to fill the larger bile ducts with carmine gelatine.
Where the animal was living throughout the experiment the secretion of bile
continued, the lymph vessels in the portal fissure were distended with bile-
stained lymph, and the yellow colour could in some cases be seen extending
into the thoracic duct. In several experiments both in the cat and dog
carmine gelatine was found in the lymph trunks of the liver and in the
portal lymph glands. Whenever injection mass appeared in the lymph
trunks outside the liver the injection had been at a comparatively high
pressure or had been continued for some time. In Cat A, the maximum bile
pressure of which was 366 mm., the injection pressure was 410 mm. for
40 minutes, and was then raised to 520 mm. water for 30 minutes longer.
The animal was living throughout the time of injection, and carmine gelatine
was freely mixed with lymph both in the portal lymphatics and thoracic
duct. In Dog A, maximum bile pressure 328 mm., the injection was at
a pressure of 400 mm. water for three hours (dog living). In this case also
carimine gelatine was present in portal lymphatics and thoracic duct. Cat L,
maximum bile pressure 289 mm., was injected for six hours at a pressure of
120 mm. water. The animal was dead before injection began, but the
inferior vena cava had been ligatured above the diaphragm ; carmine gelatine
was found in the portal lymphatics on completion of the experiment.

We attempted to ascertain the time at which bile appears in the portal
lymph trunks after ligature of the bile duct. For this purpose we clamped
the cystic and common bile ducts in a cat, opened a lymph trunk near the
portal fissure and gathered the lymph every two minutes by pieces of
blotting paper which had previously been soaked in a solution of cane sugar
and dried. After drying the lymph-filled pieces we tested them with strong
sulphuric acid for bile acids. We were not satisfied with the reaction, but.
the lymph trunks issuing from the liver were strongly bile-stained one hour
after obstruction of the bile duct. Heidenhain found indigo-sulphate of
soda to appear in the lymph very soon after its injection into the bile ducts,
but in such an experiment there is no guarantee that the entry has not been

528 Drs, P. T. Herring and 8. Simpson. — [May 1,

made by rupture of the interlobular ducts. Heidenhain indeed explained its
occurrence on this supposition. It is difficult to show the presence of small
quantities of bile in the blood, but in many of our experiments the urine
contained bile acids. Dr. Cramer kindly tested the urine for us; Gmelin’s
test for bile pigment gave negative results, but bile acids were readily
demonstrable. Bile acids were found in the urine two hours after the
beginning of a record of bile pressure.

In animals which were kept alive during injection of the bile ducts at
a pressure lower than or not exceeding the maximum bile pressure attained,
sections of the liver show little injection. The larger bile ducts are filled
with carmine gelatine and the injection passes into the smaller interlobular
ducts. In the dog and cat very few of the bile capillaries are injected, a few
are filled at the periphery of the lobules, but the injection does not extend
more than one or two cells inwards. The adjoining liver cells are frequently
filled with a lighter coloured (diluted) carmine gelatine. These cells appear
to have been injected from the bile capillaries. The rabbit’s liver shows the
same appearance, but more of the bile capillaries are injected. Hering (10)
noted the filling of liver cells from the bile duct. He stated that Berlin
blue, when injected into the bile duct under so small a pressure that no
extravasation takes place, fills only the bile capillary network in the
periphery of the lobule. Higher pressure produces extravasation first into
the liver cells in the form of small round drops, later irregular large drops,
lastly, the whole liver cell appears blue. Further injection bursts the cell
wall, and the injection flows into masses or escapes into the blood capillaries.
We have noticed a similar appearance in sections of the livers examined.
Injection of the bile ducts at a pressure near to or exceeding slightly the
maximum bile pressure almost invariably causes extravasation into the blood
vessels, and this is especially liable to occur when the injection is carried
out in the dead animal. The extravasation appears to take place in the
manner described by Hering by a filling of the cells at the periphery of the
lobule and escape of the injection from them into the neighbouring sinusoids.
In livers injected after death this filling of liver cells and escape into blood
vessels renders the interpretation of the appearance produced a difficult one.
The injection undoubtedly gets into the blood vessels at the periphery of the
lobule. In an animal injected during life the appearance is somewhat
different. In Cat E, which was injected during life for 55 minutes only at
a pressure of 443 mm. water (maximum bile pressure, 373 mm.), the lymph
of the portal lymphatics and thoracic duct contained carmine at the end of
that period. Sections show extravasation of carmine gelatine into the cells
at the periphery of the lobules, but no injection of the blood vessels as

1907. | The Pressure of Bile Secretion, etc. 529

in the other livers. There has been escape into the blood vessels at the
periphery of some of the lobules, and the carmine gelatine has been taken up
by Kupffer’s cells, and appears in them in the form of small round drops.
Kupffer’s cells are greatly distended with drops near the periphery of the
lobule, and contain carmine gelatine in decreasing amounts towards the
central vein, in the neighbourhood of which they are generally free.

The most valuable injections are those in which the bile duct has been
injected at comparatively low pressures so as to avoid extravasation, and yet
fill the interlobular bile ducts. If the pressure is higher, rupture occurs as
stated, but there is also the appearance of injection mass having passed out
of the finer interlobular ducts into connective-tissue spaces. It is probable
that with a high pressure of injection most of the fluid passes out in this
manner and so reaches the lymphatics. Heidenhain was undoubtedly right
in assigning the site of rupture to the fine interlobular ducts leading from
the lobules. When a low pressure is employed there is no trace of such
rupture, although the ducts are filled, yet in this case bile is escaping from
the liver by the lymphatics. In the rabbit’s liver, where injection penetrates
along the bile capillaries further into the lobules, one often sees a number
of cells in a column suffused with diluted injection mass, as though the
material were passing through them in the direction of the periphery of the
lobule.

The readiness with which obstruction of the bile duct is recovered from,
the bile again following its natural course when the obstruction is removed,
and the observations of Heidenhain that the bile secreted after removal of
the obstruction contains the same relative proportions of solids to fluid, show
that no permanent damage is caused by the leakage, assuming it. to be
a leakage. One would hardly expect so complete a recovery were there
a mechanical rupture of the wall of the bile ducts. One must rather assume
that there is some physiological explanation of the absorption of bile. It is
possible that the walls of the interlobular ducts are capable of allowing bile
to pass out without any physical damage resulting, and that such passage
cannot occur when the internal pressure is reduced, thus acting as a kind of
safety valve allowing escape into the lymphatics when required. Carmine
gelatine, however, does not appear capable of passing out in this way;when
injected at pressures too low to produce extravasation, but high enough to
turn the flow of bile into the lymphatics.

Escape of bile into the lymphatics, if it occurs from the liver cells
directly, can only be from the cells at the peripheral part of the lobule.
Inside the lobule the only escape possible is by the natural channels, the
bile capillaries, or into the fine plasmatic channels which permeate the liver

530 Drs. P. T. Herring and S. Simpson. [May 1,

cells. It is remarkable that the protoplasm of the liver cell, containing as it
does the plasmatic channels and the origin of the bile capillaries, should be
able to sustain so great a difference of pressure without bile passing from
the bile capillaries into the blood sinusoids. That the liver cells should be
capable of injection in much the same manner both from the blood vessels
and from the bile ducts is a fact of extreme importance. The pressure
necessary in either case is a comparatively small one. In none of our
experiments on the injection of intracellular canals in the liver from the blood
vessels did we find any escape of injection material from the cells into the
bile passages. On the other hand, injection of the bile ducts in the dead
animal appears to be invariably attended with filling of a few of the cells
at the periphery of the lobule and escape into the blood vessels.

The intracellular plasmatic channels are in direct communication with the
blood vessels, but when injected from the latter it is often difficult to make
out in every cell the mode of entrance of the injection. It is probable that
the liver cell exerts a controlling influence on the amount of plasma which
passes in from the blood. We found it difficult to wash the injection mass
out of the channels by a subsequent perfusion through the blood vessels of
normal saline.

We suggested in our previous paper that the intracellular plasmatic
channels of the liver act as an intermediate system, linking the blood vessels
in the lobules to the lymphatics outside, and that the lymph passes from
cell to cell on its way to the periphery of the lobule. The absorption of
bile by the lymphatics in obstructive jaundice seems to us to favour this
view. The mechanism of bile absorption appears to be in the liver cell.
When the passage of bile is obstructed, the pressure in the bile ducts and
capillaries rapidly rises, secretion continues, but is diverted in the peri-
pheral cells of the lobules into the lymph stream; when obstruction is
removed, the bile again follows its normal course. The amount of pressure
which the liver cell has to sustain, and which is exerted on it when the bile
duct is occluded, is comparatively low, and does not materially affect its
vitality. An example of this is furnished by Dog D, already instanced, in
which a maximum bile pressure of 285 mm. bile was recorded in the first
experiment. The obstruction was then removed, and a record of the rate of
bile flow taken. Therate of secretion half an hour later was more rapid than
it had been prior to the experiment. A second record of bile pressure taken
an hour after the first gave a maximum pressure of 305 mm. bile. That sub-
sequent estimations of bile pressure are not as a rule as high as the first may
quite well be explained by a diminution of activity of the liver cell, due to
the length of time the animal is under the anesthetic.

1907. | The Pressure of Bile Secretion, ete. 531

_ The form of the curves of bile pressure shows that there is not much
absorption until the pressure has reached about 100 mm. bile. The rate of
rise of pressure then begins to diminish more rapidly in some cases than in
others. Absorption is at first slow, but gradually increases until its rate is as
rapid as the rate of secretion when the column of bile becomes stationary.

On the death of the animal, the bile pressure, if high, sinks rapidly at
first, but on approaching a pressure of about 100 mm. falls much more slowly
and becomes almost stationary. The same thing occurs even if death has
been caused by opening the inferior vena cava above the diaphragm. Blood
pressure in the liver rapidly falls to zero, but the bile pressure falls slowly.
The curve of fall of bile pressure after death is given on the chart.
In Cat O, the sudden fall of bile pressure near the end of the tracing is due
to death from an overdose of chloroform. When the pressure had fallen
to 149 mm. bile, the inferior vena cava was divided above the diaphragm ;
bile pressure fell very slowly, and six minutes later was 134 mm. In
Cat P, death was brought about by division of the inferior vena cava, and
the pressure of bile secretion, which was 276 mm. at the time, fell rapidly at
first, then at a decreasing rate. Ten minutes after the vein had been opened
the bile pressure was 108 mm., and falling very slowly. If the bile had been
escaping from ruptured bile ducts into the lymphatics or into the blood
vessels, it is improbable that this slowing of the fall of pressure would have
taken place.

The absorption of bile seems to us to be bound up with the formation of
lymph in the liver, and while we advance no definite proofs in favour of
Asher’s view, we are inclined to think that the phenomena observed in the
absorption of bile can best be explained on the hypothesis that lymph is a
product of the activity of the liver cells, and that in obstructive jaundice the
bile secreted leaves the liver cells with the lymph, instead of by its proper
channeis, the bile canaliculi.

Summary.

The maximum pressure reached by the bile in obstruction of the bile duct
exceeds the figures originally given by Heidenhain. In the dog, cat, and
monkey the average maximum pressure is about 300 mm., measured in terms
of a vertical column of bile. The highest pressure recorded was 373 mm.
bile inacat. In the ordinary rabbit the pressure reaches about 200 mm. bile,
but may exceed 300 mm. in the case of the Belgian hare.

After obstruction of the common bile duct and cystic duct, the pressure
in the bile passages rises rapidly at first, but slows as a maximum is reached.
Subsequent records of bile pressure after temporary release of the bile may
exceed the first maximum, but are usually lower. After death the bile

532 The Pressure of Bile Secretion, ete.

pressure falls rapidly until it has reached about 100 mm. bile, after which the
fall is comparatively slow, even when the blood pressure in the liver is at zero.

The height of bile pressure attained is, as Heidenhain showed, the pressure
at which as much bile is secreted every moment as is taken up from the
bile paths by re-absorption. The obstructed bile leaves the liver by the
portal lymphatics. Injection of the bile ducts with carmine gelatine at a
pressure not exceeding the maximum bile pressure is also followed by the
appearance of the injected material in the liver lymphatics. The examina-
tion of sections of livers thus injected reveals the presence of the injection
in the liver cells at the periphery of the lobules. Appearances suggest that,
the bile escapes from the liver cells through the intracellular plasmatic
channels described by Schafer, and that these channels are to be regarded
as an intermediate system linking the blood vessels of the liver with the
lymphatics at the periphery of the lobules.

LITERATURE REFERRED TO IN THE TEXT.

1. Afanassiew. Cited from Landois and Stirling, ‘Text-book of Physiology,’ vol. I,
p. 387 (1886).
2. Fleischl, v. ‘“ Von der Lymphe und den Lymphgefiissen der Leber,” ‘ Bericht iiber
d. Verh. d. Konigl. Sachs. Ges. d. Wiss., math.-phys. K1.,’ vol. 26, p. 42 (1874).
3. Freese. ‘The Force of Contraction of the Gall-bladder and the Course of its Motor.
and Inhibitory Fibres,” ‘Johns Hopkins Hosp. Bulletin,’ vol. 16, p. 235 (1905).
4. Frerichs. ‘ Klinik der Leberkrankheiten,’ vol. 1, p. 99 (1858).
5. Friedlander and Barisch. “ Zur Kenntniss der Gallenabsonderung,” ‘ Arch. f. Anat.
Physiol. u. wiss. Med.,’ p. 659 (1860).
6. Haller. ‘Elementa Physiologie,’ vol. 7, p. 81 (1776).
7. Harley, Vaughan. “ Leber und Galle wihrend dauernden Verschlusses von Gallen-
und Brust-Gang,” ‘ Arch. f. Anat. u. Physiol.,’ Physiol. Abt., p. 291 (1893).
8. Heidenhain. ‘Weitere Beobachtungen betreffend die Gallensecretion,” ‘ Studien
d. physiol. Instit. zu Breslau,’ vol. 4, p. 226 (1868).
9. Idem. “ Physiologie der Absonderungsvorginge,” ‘Handbuch der Physiologie, von.
Hermann,’ vol. 5, pp. 268-—278 (1883).
10. Hering, E. “ Ueber den Bau der Wirbelthierleber,” ‘Wiener Sitzungsber.,’ vol. 54,
Abt. 1 (1866).
11. Herring and Simpson. “ On the Relation of the Liver Cells to the Blood-vessels and
Lymphatics,” ‘ Roy. Soc. Proc.,’ B, vol. 78, p. 455 (1906).
12. Kowalewsky. “ Zur Lehre tiber die Mechanik der Gallenbewegung,” ‘ Pfliiger’s.
Archiv,’ vol. 8, p. 597 (1874).
13. Mall. “A Study of the Structural Unit of the Liver,” ‘Amer. Journ. of Anat.,”
vol. 5, No. 3, p. 298 (1906).
14. Nauwerck. “ Leberzellen und Gelbsucht,” ‘Miinchener Med. Wochenschr.,’ Jahrg.
44, p. 29 (1897).
15. Richet. ‘Dictionnaire de Physiologie,’ Paris, vol. 2, p. 155 (1897).
16. Saunders, W. ‘A Treatise on the Structure, Economy, and Diseases of the Liver,’
pp. 88—91 (1795).
17. Tiedemann and Gmelin. ‘Die Verdauung,’ vol. 2, p. 40 (1827).

533

Further Studies of Gastrotoxic Serum. (Progress Report.)

By CuHares Bouton, M.D., Research Scholar of the Grocers’ Company.

(Communicated by Professor 8S. Martin, F.R.S. Received June 10,—Read
June 27, 1907.)

(From the Pathological Department, University College Hospital Medical School.)
[Puate 7.]

In two (1 and 2) previous communications to the Royal Society I was able
to demonstrate the production of a gastrotoxic serum by the injection of the
mucous membrane of the stomach of one animal into another. In this way,
a heterogastrotoxin was formed by injecting the stomach cells of the guinea-
pig into the rabbit, and an isogastrotoxin by injecting the stomach cells
of the rabbit into the rabbit. I also showed that a human gastrotoxin
could likewise be produced by injecting human stomach cells into another
animal.

The heterogastrotoxin possesses poisonous properties for the tissues of the
variety of animal whose stomach has been used for injection, but the
isogastrotoxin does not. The latter, however, is toxic for the tissues of a
nearly allied animal.

The gastrotoxin, on injection into an animal for which it is poisonous,
produces necrotic patches in the mucous membrane of the stomach, which
develop into ulcers. In the test-tube the serum also produces effects upon
the tissues of the animal concerned. It was proved to be of a complex
nature, and to be capable of effecting the following reactions :—

1. Heemolysis of the red blood corpuscles.

2. Precipitation of the soluble proteids of the stomach cells, and also
of other proteids of the body.

3. Agglutination of the gastric protoplasmic granules.

4. A more or less definite hyaline transformation of the intact gastric
cells.

The present communication will deal with the four following points :—

1. The multiplicity of the precipitins.

2. The identity of the substances producing the precipitating and aggluti-
nating reactions.

3. The production of immunity to the gastrotoxin.

4, The réle which the gastric juice plays in the actual formation of the
necrotic patches and of the subsequent gastric ulceration.

VOL. LXXIX.—B, 2 Q

534 Dr. C. Bolton. | [June 10,

The gastrotoxic serum was in all cases obtained by injecting the washed
stomach cells of the guinea-pig into the rabbit, the blood serum of the rabbit
developing toxic properties for the guinea-pig’s tissues.

1. The Multiplicity of the Preciprtins.

My object was to ascertain whether the same precipitin or different precipi-
tins were concerned in the precipitation of the various proteids.

Method.—The proteid solutions examined were albuminous extracts of the
gastric, hepatic, and intestinal cells, and the blood serum of the guinea-pig.
The extracts were made by grinding up the cells to a pulp, shaking up the
latter with salt solution, and filtering through a Berkefeld filter. The proteid
solutions were all made of such a strength that a faint cloud was formed on
testing them with potassium ferrocyanide and acetic acid. It is necessary to
use the solutions of the same strengths, because the precipitate is soluble
in excess of the precipitable substance, and therefore if albuminous fluids of
different strengths are employed, erroneous results will follow.

If equal volumes of such a proteid solution and anti-serum be mixed
together and incubated for four hours, and if the precipitate which forms in
the solution be removed by centrifugalisation, the resulting solution, which
will consist of a two-fold dilution of anti-serum, will be found to be
incapable of precipitating any further the proteid solution. In this way one
can easily remove the precipitin for any particular proteid, and the solution
can then be tested with regard to its power of precipitating a different
proteid. In all cases, of course, a control must be prepared, so as to prove
that the precipitin has been actually removed. ,

In this way the precipitins for each of the four proteid solutions mentioned
above were removed, and the resulting serum diluted to different degrees and
tested as to its capacity of precipitating the three remaining proteids, with the
following results :—

Removal of the Precipitin for Stomach-cell Proteid—A solution of gastro-
toxin from which the precipitin for stomach-cell proteid has been removed
is thereby rendered quite incapable of precipitating the proteids of liver cells,
intestine cells, or blood serum.

Removal of the Precipitin for Lwer-cell Proteid—Removal of the precipitin
for liver-cell proteid slightly weakens the power of the gastrotoxin to preci-
pitate the proteids of stomach cells and blood serum, and it completely
deprives it of the power of precipitating intestine-cell proteid.

Removal of the Precipitin for Intestine-cell Proteid—Removal of this
precipitin renders the gastrotoxin incapable of precipitating liver-cell proteid,

1907. | Further Studies of Gastrotoxic Serum. 535

but only weakens its precipitating power for the proteids of stomach cells
and blood serum.
— Removal of the Precipitin for the Proteid of Blood Serum—If the
precipitin for the blood serum be removed, the gastrotoxin is still able to
precipitate all the three remaining solutions, but not quite to the same
degree as before. |

It appears, therefore, that the precipitins of gastrotoxic serum are multiple
and to a great extent specific, but that their actions overlap to some extent.
The gastrotoxic precipitins for liver-cell and intestine-cell proteids appear to
be more nearly allied than the others, if not identical. In confirmation of
the above statements I may say that if the blood serum of an immunised
rabbit be examined every day, it will be found that the first precipitins to
appear in the serum are those for the proteids of the stomach cells and
blood serum, and that the precipitins for the liver-cell and intestine-cell
proteids appear at a later date during the process of immunisation.

These results are in agreement with the statement of Nuttall (3) and others
to the effect that precipitins are to a large extent, but not absolutely, specitic.

2. The Identity of the Substances producing the Precipitating and Agglutinating
Reactions.

The precipitin for stomach-cell proteid was removed from a solution of
gastrotoxin in the way described above and the capacity of this solution to
agelutinate gastric-cell granules then tested. In order to prove that the
precipitin was removed from the solution, a control was in each case prepared,
in which stomach-cell proteid was added to the solution instead of gastric
granules.

In this way I have been able to show that in the absence of the precipitin
no agglutination of gastric granules occurs, in other words the gastrotoxin
does not contain a specific agglutinin for the gastric granules. When
unwashed granules are used for the performance of the agglutination test, the
gastrotoxin precipitates the albuminous fluid in which the granules float and
the latter are agglutinated by this precipitate. When washed granules are
used, the gastrotoxin no doubt produces some change of a coagulative nature
in the superficial layers of the granules and they are therefore agglutinated
as they sink to the bottom.

On the other hand there is no doubt that the gastrotoxic agglutinin for red
blood corpuscles is a specific substance, because if the gastrotoxic precipitins
be removed as described above, the resulting solution still agglutinates red
blood corpuscles in exactly the same strengths as untreated gastrotoxic serum
does. The serum is, of course, heated to 55° for half an hour before

2Q 2

536 Dr. C. Bolton. [June 10,

performing this test, in order to prevent hemolysis. My results disagree
with those of observers such as Deutsch (4) and others, who hold that specific
agglutinins are formed for the protoplasmic granules of cells, and who make
no mention of any precipitating action. Their position, according to my
experiments, is untenable.

3. The Production of Immunity to Gastrotoxin.

The effect of repeated injection of gastrotoxic serum is to produce
immunity to the gastrotoxin.

It seemed that a determination of the results obtained from the continued
administration of gastrotoxin would be interesting from two points of view :—

(1) With regard to whether chronic ulceration of the stomach could be
produced in this way, or whether the animal would become immune.

(2) If the animal became immune, whether it would develop protective
substances in its blood against one or all of the constituents of the gastrotoxic
serum. In this way light would probably be thrown upon the actual cause of
the ulceration, whether it was of hemorrhagic origin primarily, or due to a
direct action upon the gastric cells.

Method.—The method which I have found to be the most satisfactory is the
intraperitoneal injection of inactive (heated to 55° C.) gastrotoxic serum.

The injected animal was, of course, the guinea-pig, and the serum was
derived from rabbits immunised by injection of washed guinea-pig’s stomach
cells.

One injection of 5 cc. was given each week. In order to produce a
satisfactory degree of immunity, 10 or 12 injections are necessary.

In this way I have immunised 10 guinea-pigs. The immunity of these
animals was tested both by experiments 7 vwo and also in witro.

1. Examination in vivo: Active Immunity—Four or five injections of the
serum will not protect an animal against a lethal dose (10 cc.) of gastrotoxin,
which still produces necrotic patches in the stomach.

After an animal has, however, received 10 injections (50 c.c. serum in all)
a lethal dose (10 c.c.) of gastrotoxin fails to produce necrotic patches in the
stomach of that animal (fig. 1, Plate 7).

Passive Immunty.—The blood serum of an actively immune animal is
capable of conferring passive immunity upon another animal. .

In an experiment of this kind a lethal dose (9 cc.) of active gastrotoxic
serum was mixed im vitro with 6 cc. inactive protective serum and the
mixture injected into a guinea-pig weighing 360 grammes. No lesion was
produced in the stomach. The control animal was inoculated with 9 c.c.
active gastrotoxic serum and 6 c.c. inactive normal guinea-pig’s serum. This

1907. ] | Further Studies of Gastrotoxic Serum. 537

animal showed the usual patches of necrosis in its stomach. Normal guinea-
pig’s serum is itself able to protect an animal to a slight extent (fig. 2).

2. Examination in vitro.—The examination was conducted from two points
of view :— |

(a) To find out whether the tissues of an immune animal were as susceptible
to the gastrotoxin as were those of a normal animal.

(6) To ascertain what protective power the serum of the immune animal
possessed with regard to preventing the action of gastrotoxin i vitro.

(a) Action upon the Tissues of an Immune Animal: Blood Corpuscles.—The
gastrotoxic hemolysin dissolves the red corpuscles of an immune animal to
exactly the same degree as it does those of a normal guinea-pig.

Proteid Solutions.—The stomach proteid, intestine proteid, and even the
blood serum of the immune animal are all precipitated by the gastrotoxic
serum.

Stomach Cells.—The same action is seen upon the immune cells as upon the
normal cells, namely, more or less clearing of the cells.

In the case of several other poisons it has been proved that the tissues of
the immunised animal are still acted upon by the poison.

(b) Protectwe Power of the Serum in vitro.—The protective serum was, 1n
all cases, heated to put the complement out of action. |
Hemolysin—Hemolysis is completely prevented by the immune serum.

The immune serum obtained from an animal after four injections prevents
hemolysis to some extent. After eight injections it has more power. After
11 injections 0°5 cc. serum completely prevented any hemolysis occurring
in any of the tubes. (Hemolytic strength of the serum used -—0:25 ce.
serum completely dissolved 1 c.c. 5-per-cent. suspension of guinea-pig’s
corpuscles.)

Probably smaller amounts than this would suffice. ‘The experiments were
only made, however, to demonstrate the presence of an anti-hemolysin.

Precipitin.—I have not been able to show the presence of any anti-
precipitin. The serum of an immune animal, on being added to the gastro-
toxin in sufficient amount to dissolve any precipitate that forms, fails to
prevent the precipitation of stomach-cell proteid.

Action on the Gastric Cells——The. changes in the cells are not sufficiently
definite for any very accurate determinations to be made, but my impression is
that the cells are as much acted upon, whether the immune serum is present
or not. This point must, therefore, for the present be left an open question.

Anti-precipitins have been described, but my results fail to confirm such
observations.

Anti-hemolysins have, of course, been recognised for a long while.

538 | Dr. C. Bolton, _ [June 10,

4. The Réle which the Gastric Juice plays in the Production of Gastric
Ulceration.

I have been able to prove that the actual ulceration produced by
injection of the gastrotoxic serum is brought about by the action of the gastric
juice. |

Although it is considered at the present day that the gastric juice plays
an important part in the production of gastric ulcer, I have not been able to
find any reliable experimental evidence in support of such a supposition. Of
course, it. is clear that if the gastric cells are killed by any means, the gastric
juice will digest them, as it does any foreign proteid substance, but I refer
to self-digestion of the stomach, in which the gastric juice attacks a
gastric cell which shows no deviation from the normal on microscopic
examination.

The whole question of self-digestion of the stomach is extremely interesting
and important, but as yet very little is known about the subject.

Methodi—My method is to neutralise the gastric juice with sodium
bicarbonate solution before injection of the gastrotoxic serum.

In order to do this a soft rubber catheter is passed down the cesophagus of
a guinea-pig into its stomach, and about 14 e.c. of a 4-per-cent. sodium
bicarbonate solution introduced through a small funnel. The gastrotoxic serum
is then injected into the animal’s peritoneal cavity. A control pig is at the
same time injected with the same amount of the same serum. I have done
eight such experiments, and in ali the cases in which the gastric juice was
previously neutralised no necrosis of the stomach could be seen, but in all
the control animals the usual black patches were visible in the mucous
membrane of the stomach (fig. 3).

The stomach contents in the pigs which were previously treated with
alkali were found to be strongly alkaline, whilst the contents of those not
so treated were strongly acid. I am not yet prepared to state the weakest
strength of the soda solution which will prevent necrosis.

I have hitherto found no microscopic change in the stomach which precedes
digestion.

These experiments conclusively prove what I had previousiy shown in
another way, namely, that the initial lesion is not a hemorrhage, for in that
case neutralisation of the gastric juice would certainly not prevent a
hemorrhage occurring. It is also well known that precipitins cause no
lesion on injection, because any precipitate formed would be dissolved by the
excess of the animal’s fluids present. It appears to me that a functional
disturbance is set up in the gastric cells which renders them susceptible to

1907.] Further Studies of Gastrotoxic Serum. 539

the action of the gastric juice. What that disturbance is and whether or not it
bears any relationship to the slight hyaline change which I have described as
occurring 7 vitro, I am not prepared to state. I think, however, that it is a
different process, because the action is destroyed by heat, no visible change
is to be seen in the cells, and an antibody is formed against it.

If a solution of hydrochloric acid (0°5, 1, or 2 per cent.) be introduced into
the stomach previous to the injection of the gastrotoxic serum, the resulting
necrotic lesions are more pronounced than they are in the control animals
(fig. 4). A 2-per-cent. solution of hydrochloric acid alone produces no effect
whatever when introduced into the stomach of a guinea-pig.

I think, therefore, that hyperacidity of the gastric juice must play some
part in the formation of gastric ulcers; the essential factor, however, is some
alteration in the cell itself, and probably this alteration may be brought
about in a variety of ways.

| Conclusions.

1. A gastrotoxic serum contains many precipitins which are more or less
specific in their actions, but these actions overlap to some extent, and absolute
specificity is unknown.

2. There is no specific agglutinin for the gastric granules, the agglutination
being brought about by the precipitins. |

3. By repeated injection of the gastrotoxic serum, immunity is established.
The immune substance is present in the blood serum of the animal, its tissues
being still susceptible to the poison.

It seems, therefore, impossible to establish a chronic lesion by gradual
absorption of the gastrotoxin, if the immunising mechanism of the animal be
intact. Any lesion which is produced occurs within a few hours, as I have
shown. If a chronic lesion occurs as the result of the action of such a cyto-
toxin, the explanation is probably to be found in the perpetuation of an
acutely produced lesion by some cause or other. Whether this cause be
secondary bacterial infection or hyperacidity of the gastric juice remains to
be proved.

4, The actual necrosis and ulceration of the stomach is produced by the
gastric juice acting upon a cell which is functionally damaged. Hyperacidity
of the gastric juice increases the tendency to such ulceration.

REFERENCES.

1. Bolton, ‘ Roy. Soc. Proc.,’ vol. 74, p. 1385, 1904.

2. Bolton, ‘Roy. Soc. Proc.,’ B, vol. 77, p. 426, 1906.

3. Nuttall, “Blood Immunity and Relationship” (text-book).

4, Deutsch, “Sur le sérum antihépatique.” XIII* Cong. Internat. de Méd., Sect. de Bact.
et Parasitol., 1900, Paris, ‘Compt. Rend.,’ pp. 55—56.

540 Further Studies of Gastrotoaic Serum.

DESCRIPTION OF PLATE.
Fie. 1.—Active Immunity to Gastrotoxic Serum.

Upper stomach: This is the stomach of the control animal, which was injected with
10 c.c. gastrotoxic serum. It shows patches of necrosis of the mucous membrane. .

Lower stomach: This guinea-pig had been immunised with 10 injections of gastrotoxic
serum. A lethal dose (10 c.c.) of gastrotoxic serum was injected. No lesion has
resulted.

Fie. 2.—Passive Immunity.

Upper stomach: This animal received a mixture of 9 cc. gastrotoxic serum and 6 c.c.
protective serum. No lesion is to be seen.

Lower stomach: This animal received a mixture of 9 c.c. gastrotoxic serum and 6 c.c.
normal serum. Necrotic patches are to be seen in the stomach.

Fia. 3.—Neutralisation of the Gastric Juice by Sodium Bicarbonate.

Upper stomach: This is the stomach of the control animal, which was injected with
10 c.c. gastrotoxic serum. It shows necrosis of the mucous membrane.

Lower stomach: 14 ¢.c. sodium bicarbonate solution (4 per cent.) were introduced into
this stomach, and the animal was then injected with 10 c.c. gastrotoxic serum.
No lesion has resulted.

Fia. 4.—Hyperacidity of the Gastric Juice.

Upper stomach :| This is the stomach of the control animal, which was injected with
10 c.c. gastrotoxic serum. It shows some necrosis of the mucous membrane.
Lower stomach: 27 c.c. hydrochloric acid solution (1 per cent.) were introduced into this
stomach, and the animal was then injected with 10 c.c. gastrotoxic serum.
Extensive necrosis of the mucous membrane has resulted, and it is much more

marked than in the control animal.

Bolton. Roy. Soc. Proc., B. vol. 79, Plate. 7.

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On the ee between the Output of Uric Acid and the Rute of
Heat Production in the Body.

By E. P. Catucart, M.D., and J. B. LEATHES, M.B.

(Communicated by Dr. ©. J. Martin, F.R.S. Received June 22,—Read
June 27, 1907.)

(From the Lister Institute of Preventive Medicine.)

The rate at which uric acid is turned out of the body is very different at
different times of the day, even when the food contains no ready-made
purine derivatives. It is higher during the early hours of the day than at
any time, and it is considerably lower at night. The reason for this, as
was pointed out by one of us,* is not likely to be that the excretory
functions are depressed at night, since these functions, to Judge from the
total nitrogen of the urine, are more active during the first hours of sleep
than at any time in the 24 hours. And since, when the diet is confined
to bread, butter, and milk, the uric acid must be derived from the body
substance and not from the food, it seems probable that there is some
function of the body which is in abeyance during sleep and is, to a consider-
able degree, responsible for the output of uric acid; some function, that
is to say, which is effected by chemical reactions involving the production
of uric acid, and of nothing else that can be detected in the urine, unless it
be phosphoric acid, and possibly in some measure creatinine.

If it is possible to identify this function, the activity of which can, on
a suitable diet, be measured by the amount of uric acid excreted, it may
be possible to give a clearer account of the processes by which, at the onset
of fever, the ternperature of the body can be sent up independently of any
voluntary muscular activity ; for while the temperature is rising, the output
of uric acid may be four times as great as it otherwise would be.f Similarly,
in the study of other pathological conditions in which uric acid plays a part,
it must be of importance to be able to point to the kind of activity which
is accompanied by increased uric acid production.

The following series of observations, which we have worked out con-
jointly, was designed to show how the reaction of the body to loss of heat
on the one hand, and the heat produced by ordinary muscular exertions
on'the other, might affect the amount of uric acid in the urine. 1t was
the outcome of several earlier, as yet unpublished, experiments, which

* J. B. Leathes, ‘Journal of PUNO y), vol. 35, p. 125, 1906.
+ Loe. cit., p. 205.

542 Dr. E. P. Cathcart and Mr. J. B. Leathes. [June 22,

pointed, though less clearly, to the same conclusions as we think may be
drawn from this one.

A diet was taken by the subject of the experiment (EK. P. C.) which
consisted of 2 quarts of milk (2°28 L.), 12 ozs. of bread (360 grammes),
4 ozs. of cheese (120 grammes), and 2 ozs. of butter (60 grammes). This
was distributed equally over six identical meals taken every three hours
from 7 A.M. to 10 P.M.

The urine was collected immediately before each meal, and in each
sample the total nitrogen (Kjeldahl), the creatinine (Folin), the uric acid
(Folin), and the phosphoric acid (by titration with uranium acetate) was
estimated, and the hourly rate of output calculated for each period of the
day. The diet was taken from May 17 till June 4. On the first two days
the urine was collected for 24 hours and mixed before analysis.

On the eighth day of the diet, May 24, two and three-quarter hours were
spent stripped at a swimming bath. For the greater part of this time the. |
subject of the experfment stood about, and entered the water only from
time to time as the skin became dry, the object being to lose heat with as
little compensatory production of heat by voluntary exertion of the muscles
as possible. The rectal temperature was reduced in this way from 100°8,
at 2.50 P.M. immediately after undressing and after the 10 minutes’ walk to
the bath, to 97°"7 just before dressing again at 5.30. The reading at 4 P.M.
was 98°83, and at 5 p.m. 98°. The temperature of the water was 71°, and
that of the air 64°. At fairly frequent intervals spasmodic attacks of
shivering came on, but these were not so severe as in a “rigor.” Although
the body temperature fell 3° F., the greater part of this fall would have
occurred even with clothing, as the observations taken every three hours
throughout the whole experiment showed that a rectal temperature of 98°8,
or even slightly less, was normal for this time of day on abstaining from
muscular activity. The body temperature, therefore, fell barely a degree
beyond the normal range of variations, and the regulating function was not
seriously overtaxed. This was the result aimed at, as a previous experiment
had shown that a more severe and rapid removal of body heat introduced
disturbing factors and gave a different result.

The effect on the output of uric acid was to increase it more than 80 per
cent. in comparison with the mean of the four preceding days. The figures
observed are given in Column C of Table I appended. —

Three days later, May 27, the exposure to cold was repeated, only with
this difference that active muscular exertions were introduced. The subject
was stripped for two and three-quarter hours and kept his skin wet by
repeated immersion in, and sponging with, cold water, T.° 59 to 60. In

~

1907] On Uric Acid and Heat Production in the Body. 543

the intervals quick-running movements, maintained for five minutes at a
time, and dumb-bell exercises, sufficient to cause pronounced hyperpneea,
were carried out. In this way actual shivering was almost entirely avoided,
although the periods of immersion, longer than those on the 24th, and the
sponging with water 10° colder than that at the swimming bath, the
measures that is for removing body heat, were more drastic than on the
former occasion. The body temperature, which was 100°4 at 4.10 P.M.
when the treatment began, was still 100° at 5.30; it was lowest (98°°8) at
6.30, but was slightly raised again (99°1) at 6.50, when the treatment
ended.

The effect on the output of uric acid, shown in Column D of Table I, was
in the succeeding 24 hours to increase it, but only to about half the extent
to which it was increased by the cold without muscular exertions three days
before, and if the comparison be made with the output of the 24 hours
immediately preceding, 2¢, 312 milligrammes, the increase is less than
20 per cent., and during the three hours of the treatment the output was
remarkably low. |

Three days later, on May 30, the subject of the experiment, dressed in
winter clothing, with, in addition, a thick knitted jersey, spent the greater
part of two and three-quarter hours in severe muscular exertions. At
3.10 P.M., with the rectal temperature at 99°8,a sharp walk of 24 miles,
lasting half an hour, raised the temperature to 102°4. Then, after an
interval of a quarter of an hour for food, a heavy floor rubber was worked to and
fro for half an hour, at the end of which time the temperature was 101°8.
The walking was then repeated (T.° 102:1) and finally the work with the
rubber for half an hour more giving a temperature at 5.50 P.M. of 102°.

The output of uric acid fell from 311 milligrammes in the preceding
24 hours to 223 in those including and succeeding the exercise. The figures
are given in Column E of Tabie I.

Finally, four days later, on June 4, an attempt was made to repeat the
experimental conditions of the first exposure to cold on May 24 in the
swimming bath. It was not possible to do this exactly, because the air
temperature was 8° lower, 56° F., and at this temperature the loss of heat was
sufficient to cause shivering, which was fairly continuous for two hours. The
body temperature was 98°4 at 4 P.M. as compared with 98°°8 at the same
time on the earlier occasion, but at 5.30, just before dressing, it was a trifle
higher than the first time, 98° instead of 97°°7. Whether these differences
in the conditions account for the result shown in Column EK, which is of the
same character though not so pronounced as that in Column C, we are not
yet prepared tosay. ‘The output in the 24 hours preceding the final exposure

544 Dr. E. P. Cathcart and Mr. J. B. Leathes. * [June 22,

was 311 milligrammes, this was raised to 386 milligrammes. This is less
than the output on the former occasion, but, as on that occasion, the figures
for each of the periods in the 24 hours are above the average and particularly
that for the period ending at 7 P.M., which included the greater part of the
exposure to cold.

On May 19, the third day of the diet, the hark: weight was 70°5 kilo-
grammes; on June 4 it had risen to 73:2 kilogrammes.

The results of this experiment point to the inference that one of the factors
largely concerned with the production of uric acid in the body and its
appearance in the urine is the reaction of the body to loss of heat; and that a
part, it may be a large part, of the endogenous uric acid is, so to speak, the
chemical expression for the intensity of this reaction; further, that this
reaction involves some form of activity distinct from voluntary movements of
the skeletal muscles, since such voluntary movements have, on the contrary,
the effect of diminishing the output of uric acid, at any rate while they are
going on.

At the same time we are not prepared to say that the activity for which
the chemical expression is the output of uric acid, has no other physiological
expression than heat production in response to increased loss of heat. That,
indeed, would not at all represent our view. For it is at any rate clear, from
this experiment taken by itself, that the reaction set up by exposure to cold
may last on for a considerable time after the exposure. A glance at the
column in Table II, in which the output of uric acid for each 24 hours
throughout the experiment is shown, makes it obvious that the interval of
three days that elapsed between each abnormal phase of the experiment was
not sufficient to allow a complete return of the rate of output to its value in
the days before the first exposure.

‘The nature of the activity concerned in this reaction to cold, and certain
other points arising out of the data in Table II, will be entered into more
fully on another occasion.

1907.] On.Uric Acid and Heat Production in the Body. 545

Table I—Showing the Rate of Output of Uric Acid, in milligrammes per
hour, for the different periods of the day ending at the hours indicated
in the first column.

A. B. C. D. E. F,
Mean of — Exercise Second
exposure to | Cold + exer- 2 exposure to
Mean four normal . warmly ;
cold, without | cise, from cold, without
of all the | days before £ 4.10 to 6.50 - dressed, from ane
16 days. | exposure to SEN oe : 3 10'to REO!) se ee
| ld 2.50 to 5.30 P.M. oat 2.50 to 5.30
Te P.M. me P.M.
7 P.M. Saal 11°5 19 °5 6-2 3 °6* LiF
19° ,, 14°7 13-0 21°1 20 °4 6°9 15°5
7 AM. BOS | Oe DU pata 12 °4 9 °2 14°7
Lo. 18 ‘6 15 °3 28 °7 20°7 13 °8 21°6
1 P.M. 14 °5 12°0 20 °2 19 -2 11 °0 14°7
Bae. 13 ‘9 11°5 17 °4 19 6 11°3 14°9
“Total for Learo 0 259 0 475-0 369 “0 223 -0 386 ‘0

* This figure is almost certainly too high.

Table II.—Total Daily Output of the various Urinary Constituents estimated.

Date, ce he erayiie
24 hours Uric Total ane ;
ending acid. | nitrogen. Creatinine. P,O;. BG. eae en ee Remarks.
4 P.M, 6 p.m.| Noon.
mgms. | grammes.| grammes. | grammes. €:€. i i
May 20 ...... 335 16 °25 1°50 3°27 1260 49 47
cg ee 280 17 37 1°49 3 ‘69 1582 45 51
yy Sai keesen|) |) 225 18 -22 1-46 3°77 1544 52 53
ee eee 259 17 -40 1°51 3°74 1378 59 65
eee! 2F0 17°73 1-49 3°35 1314 67 65
be IONE and 475 18 ‘81 1 44, 4 04, 1083 64, 70 | 22 hours stripped at
. swimming bath at
beginning of this period
4) 20 ..wace| 338 18°74 1°51 3°83 1295 67 57
Pee ee Ble 17°57 1-48 3°64 « 1429 58 61
28 saeu|, SBD 17 -88 1°51 3°33 1295 66 57 | 22 hours’ exercise stripped
and cold
ee eee) BLS 17 -59 1-49 4°20 1616 51 54
Oras) vo BLL 17-07 1-49 3°93 1578 52 54
PN 0.| 22S 17 ‘98 1-57 3°65 1002 57 64 | 2% hours’ exercise heavily
dressed
| June 1......| 875 18 -62 1-56 4°19 984, 61 64
Pe eres.) BOS 18 -95 1°54 4°06 1266 59 58
aa x s.|, 209 19 -39 1-46 4°24, 1625 54 54
eaien..| 286 | 20-11 1°43 4°12 1608 56 — | 2$ hours stripped at
—_—|—__-— — aoe swimming bath
Mean of West f 2
16 days B21 : 18-10 1-49 3°89

546

Implantation of Actively Proliferating Epithelium.
By J. O. WAKELIN Barratt, M.D., D.Sc.

(Communicated by Professor Sherrington, F.R.S. Received July 20, 1907.)

(From the Cytological Laboratories of the University of Liverpool.)

The present investigation deals with the course of events following
subcutaneous implantation of actively proliferating adult epithelium. To
avoid the possibility of disturbing factors being introduced by the formation
of antibodies, the epithelium was implanted into the same animal or into an
animal of the same species.

The effect of implantation of normal adult epithelium has, more particu-
larly during the last two decades, been the subject of considerable study by
a large number of observers, whose investigations have shown that
epithelium may be successfully implanted and an epithelial cyst result, but
that the implanted epithelium shows a marked tendency to die out in its
new habitat.* .

In the present research, instead of using normal epithelium for implanta-
tion, rapidly proliferating epithelium was employed. It was anticipated
that additional information would thereby be afforded upon two important
points, namely, upon the immediate influence of implantation on the activity
of the epithelial cells, and (owing to the large bulk of epithelium available
for implantation) upon the influence exerted by the implanted epithelium
upon non-epithelial tissue elements. Both these anticipations have been
fulfilled. )

The use of rapidly proliferating epithelium has been rendered possible by
Fischer’s discovery of the action of scharlach R upon the epithelium of the
skin of the rabbit. The extent to which epithelial proliferation occurs may
be judged by a glance at fig. 2, which shows the appearance of a section of
the ear after the injection of the dye, fig. 1 showing, for comparison, a
normal section drawn to the same scale.t

* Cp. E. Korschelt, “ Regeneration und Implantation im Tierreich,” ‘ Verhandlungen
der Gesellschaft Deutscher Naturforscher und Aerzte, 1906, Erster Teil, S. 164.

+t B. Fischer, ‘‘ Die experimentelle Erzeugung atypischer Epithelwucherungen und die
Entstehung bésartiger Geschwiilste,” ‘ Miinch. Med. Wochenschr.,’ 1906, 53 Jahrg.,
S. 2041.

t The sections represented are stained by Heidenhain’s iron alum hematoxylin method,
except in fig. 1, where methylene blue eosin is used for staining. Figs. 1 and 2 are
magnified 33 diameters ; the remaining figures are magnified 800 diameters.

Implantation of Actively Proliferating Epithelium. 547

In the middle

Hair follicles, containing hairs, are present ; also a few sebaceous glands.
The thickness

some are contained in fat cells, some

Fig. 2.

Fat globules, stained black, are seen :
have come from the oil injected. The cartilage of the ear is shown in the section, and is not

The stratum corneum is also hypertrophied, and considerable increase in the nuclei of the
markedly thickened.

hypertrophy of the Malpighian layer of the surface epithelium and of the hair follicles.
corium is present.

of the ear has been increased about five-fold, the increase being chiefly due to irregular

the cartilage is seen.
Fie. 2.—Section of rabbit’s ear injected with scharlach R dissolved in olive oil.

Fig. 1.—Section of normal ear of rabbit, for comparison with the succeeding figure.

A series of 31 implantations of skin from the rabbit’s ear, after injection of
scharlach R dissolved in olive oil, was made, the site of implantation being
the skin of the back (Table I). The epithelium of the skin implanted was
very actively proliferating, being in the condition shown in fig. 2. The
thickness of the portion of skin implanted was about 1 mm. Before removal
of skin for implantation the rabbit’s ear was shaved, all loose horny
epidermis removed, and the surface washed with absolute alcohol, followed
in some experiments by the application of a 1 in 500 solution of mercuric
chloride. The latter did not, however, present any obvious advantage, nor

548 Dr. 3.0. W. Barratt: [July 20,

Table I—Experiments in which Portions of the Skin of the Ear of the
Rabbit, previously injected with Scharlach R so as to cause Epithelial
Proliferation, were implanted subcutaneously.

Condition of implanted skin at end of period of implantation.

No. of Duration of |
SIGHS SRE | Lucie TE NOU Condition of pena a Necrotic Scharlach R
epithelium. P epithelium. oil globules.

cells.

days.
1 3 In part living Absent Present Present
2 6 None living 3 Abundant ms:
3 6 In part living Present Present a”
4, 6 None living Absent Abundant ss
5 6 ” ” ” ”
6 7 In part living Present Present s
ob 7c ? +P 3) 29
| 8 Mi None living Absent Abundant is
9 ( ” ” ” ”
10 7 In part living Scanty Present :
11 Wi Ms Absent ie pa
12 if ” ” ” ”
13 8 5 Numerous a) vgs
14, 14 | Very little living Absent Scanty
15 14 ae Scanty A
16 14 5 ¥ Present
17 31 | a | Absent *
18 31 ” ” ”
19 31 None living
20 31 Very little living 5S
i 21 ie \ Scanty
22 71 None living
f 23 71 53 Very scanty
24 71 ie
25 80 Very little living) Scanty
| 26 80 ” ”
27 80 None living
28 80 been:
29 90 5
30 90 i
31 135 is

was it in any way inimical to the success of the implantation. The portions
of skin implanted were removed at the end of from three days to four and a
half months. In 17 out of the 31 implantations, living epithelium was
found on removal; in the remaining 14 experiments no living epithelium
was present on removal. 7

For comparison with the above a second series of seven implantations
of normal skin from the rabbit’s ear (cp. fig. 1) was carried out (Table II).
In five of these experiments living epithelium was found on examination at
the end of from seven days to four and a half months.

q

1907.] Implantation of Actively Proliferating Epithelium. 549

Table [1.—Control Experiments in which Healthy Living Skin of the Rabbit’s
Ear was implanted subcutaneously.

Condition of implanted skin at end of period of

| implantation.
No. of Duration of |
experiment. implantation.
Condition of Mitoses in Necrotic
epithelium. epithelium. epithelium.
days
1 Mi In part living Very scanty Present
2 7 None living a =
3 31 In part living Very scanty
4 31 ” 3
) 31 ”
6 31 None living —
7 135 Very little living Absent
|

In order to be in a position to interpret some of the appearances found in
the area of implantation, a third series of five implantations was made with
normal skin of the rabbit’s ear, previously kept for two months in absolute
alcohol. These experiments will be referred to later.

When actively proliferating epithelium was implanted it was found that,
during the first fortnight of implantation, such portions of the basal prickle
layer as remained living continued to divide rapidly, so that in the implanted
epithelium mitoses were about as numerous as before implantation, while, in
addition, loose horny epidermic scales were produced in abundance at the
free surface of the epithelium. The circumstance that cell division continued
for a time apparently undiminished in implanted actively proliferating
epithelium was quite unexpected, for although it is known that normal
epithelium preserved in a moist chamber may remain, for as much as a
fortnight after removal, living and capable of being grafted on to a healthy
granulating surface,* nevertheless such survival seems to be regarded, not
unnaturally, as a species of suspended animation, unattended with active
growth. Normal epithelium is, however, owing to its relatively slow growth
and its scanty amount, unsuitable for determining the effect of implantation
on cell activity, while the use of actively proliferating epithelium, on the
contrary, enables this effect to be readily ascertained. The proliferating
epithelium, after implantation, would appear to possess a restricted supply of
nourishment, for it is not possible to recognise capillaries penetrating into
the implanted skin, even at the end of the first week. The character of the

* K. Garrey, “'Transplantationen in der Chirurgie,” ‘ Verhandlungen der Gesellschaft
Deutscher Naturforscher und Aerzte,’ 1906, Erster Teil, 8. 205.

VOL. LXXIX.—B. 2k

550 Dr. J. O. W. Barratt. [July 20,

mitoses in the implanted epithelium is the same as that noted before
implantation, both somatic and reduction mitoses being seen, the former
predominating, as described and figured in a previous communication.*

|

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they are not so flattened as in fig. 3.

Wein Re) AGN
AAV Or0 Tse Weac\) :
HN He A tee Yel Ua NEE AS
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Fie. 4.—Section of skin of rabbit’s ear, in which a slight degree of epithelial proliferation has been produced

Fig. 3.—-Section of normal skin of rabbit’s ear, for compar

How long active proliferation continues in implanted skin cannot be
definitely determined from the experiments so far made, but it was found

* J. O. Wakelin Barratt, “Mitosis in Proliferating Epithelium,” ‘Roy. Soc. Proc.,’
B, vol. 79, 1907, p. 372.

1907.| Implantation of Actively Proliferating Epithelium. 551

that by the 31st day the epithelium was considerably reduced in amount, and
that, as in normal skin, division figures were rare. The epithelium still
surviving at this period resembled normal surface epithelium, consisting of
a flattened expansion of prickle cells, covered with epidermic scales and
lying upon a bed of fairly dense connective tissue. No hair follicles or
sebaceous glands were seen at this period. Subsequently the epithelium
surviving became very scanty in amount.

When the normal skin of the rabbit’s ear was used for implantation
(Table IT), after the lapse of about a month, the appearance of the portions
implanted was similar to that of the former series at the end of the same
period of time.

In implanted skin a variable amount of the prickle layer always died.
Polynuclear leucocytes appeared in large numbers about the dead portion,
which could be readily recognised by the shrivelled character of its nuclei,
which stained imperfectly and diffusely. Not unfrequently dead and living
_prickle cells lie close together, the latter showing numerous mitoses and
spireme figures. Subsequently the polynuclear cells were accompanied, and
later, for the most part, replaced by large mononuclear cells with abundant
cytoplasm (cp. figs. 5 and 7), before which the prickle cells gradually
disappeared, so that ultimately the place of the latter was occupied by large
mononuclear cells accompanied by multinucleated giant cells, the nuclei of
which lay chiefly in the centre of the cell (cp. figs. 6, 7, and 8). As in such
cases the horny epidermic scales still persisted unaltered, acting like inert
bodies, as did also the hair shafts (figs. 7 and 8), it might at first appear as if
these large mononucleated cells and giant cell masses were really epithelial
cells, which, owing to their changed environment, had undergone meta-
plasia, losing their prickles and their capacity for undergoing conversion into
keratin. That this is not so, however, was shown by the experiments of the
third series, in which the implantation of alcohol-hardened skin led to
precisely the same appearance of large mononuclear and giant cells, before
which the prickle layer disappeared (fig. 8), while the appearance of mono-
nuclear cells was preceded by that of polynuclear leucocytes, just as in the
first two series. This action of dead sterile epithelium in attracting poly-
nuclear leucocytes shows that the presence of the latter is not necessarily
due to contamination with bacteria. No evidence of the presence of bacteria
in the area of implantation could be obtained by staining methods, and the
appearance of the site of implantation was that of a purely aseptic process.
In this connection it may be observed that, when the normal skin of the
rabbit’s ear was used for implantation, the same appearance of polynuclear
leucocytes at first, and later of mononuclear and giant cells, occurred. Fig. 6

2. BR 2

Fig. 5.

<a Dr. J. O. W. Barratt. [July 20,

represents a section of a portion of the ear of a healthy rabbit implanted
sub cute for 31 days (Experiment 3, Table II). It must therefore be con-
cluded that the dead prickle layer exerts a chemiotactic action, which is at

The
In

Still lower are portions of two large

v4) 4 > oS

4.
~S
PO ;

v7

Fe Chae

well-defined collagen bundles are not present.

ER
AEE, 6 .

Below this are seen large mononucleated cells, together with

reve '
OLS :
Go}

Se

ill-defined spherical masses, among which darkly staining bodies, apparently degenerated nuclei, are found.
t cells.

Fie. 6.—Section of healthy skin of rabbit’s ear, implanted for 31 days under the skin of the back of the rabbit.

prickle cells are still living, and are normal in aspect, one division figure being seen. The nuclei in
’
contact with the epithelium in the upper left-hand corner necrotic material is seen, consisting of

Previous to implantation, epithelial proliferation had been brought about by means of scharlach R.
the corium are surrounded by abundant cytoplasm

two polynuclear cells, one of which shows basophile granules.

Above is the stratum corneum unaltered.

gian

t
Fie. 5.—Section of skin of rabbit’s ear, implanted for seven days under the skin of the back of the rabbit.

“, Be !
Sey. URS
oA AY Se

first acute, leading to the attraction of polynuclear leucocytes, and, later,
becoming less intense, affects mononuclear cells. The source of the latter
cells need not be considered here, since the experiments made do not throw
any new light upon their mode of origin, beyond the fact that mitoses of the

553

1907.] Implantation of Actively Proliferating Epithelium.

somatic type are frequent among these cells, showing that they multiply

The giant cells, which are a striking feature of sections of the area

of implantation, do not resemble the giant cells of tubercular lesions, but are

iW.

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Apart from
Both the giant cells

being placed centrally.
and the single large mononucleated cells exhibit granular cytoplasm, which

1
the débris which they exhibit (figs. 5 to 8), the sections recall very vividly

the aspect of sarcoma tissue of the round celled variety.

like those of myeloid sarcoma, the nucle

554 Implantation of Actively Proliferating Epithelium.

often contains numerous pigment granules (fig. 8), and sometimes presents
a vacuolated character (fig. 7).

It may here be observed that, when epithelial proliferation is produced in
the rabbit’s ear by the injection of scharlach R (fig. 4), the nuclei of the
corlum are increased and mononuclear cells rich in cytoplasm become
observable. That this is not due to the action of scharlach R, is shown by
the fact that repeated attempts to obtain such cell infiltration by injection of
this dye, dissolved in olive oil, into the deeper layers of the subcutaneous
tissue and also into the testis of the rabbit failed. It follows, therefore, that,
as the result of the more active metabolism of the prickle cells under the
influence of scharlach R, a chemiotactic action is set up, leading to the
appearance of these cells.

The implanted normal corium in the second and third series of experiments
did not excite any marked chemiotactic response, no invasion of new cells into
it being noted. The cartilage survived in most implantations; when it
underwent necrosis, however, it did not become invaded by new cells, its
hyaline substance remaining as an inert body, and its nuclei becoming
shrivelled and staining indistinctly.

Summary.

1. During the first 7 to 14 days after implantation of actively proliferating
epithelium of the rabbit’s ear, mitosis proceeds apparently undiminished in
amount in such portions of the prickle layer as escape necrosis. |

2. In contrast to living prickle cells, dead prickle cells, when implanted
under the skin, exert a powerful chemiotactic action, determining at first the
appearance of polynuclear cells and later of large mononuclear and giant
cells; before the latter the dead prickle layer slowly disappears. Even
living prickle cells, when actively proliferating under the influence of
scharlach R prior to implantation, induce chemiotactically, through their
increased metabolism, the appearance of numerous cells, mostly mono-
nucleated, in the subjacent corium; this is, however, much slighter in
degree than that induced by dead proliferated epithelium. |

3. No metaplasia of actively proliferating epithelium occurs as a result of
implantation. |

4. Horny epidermic scales and hair shafts remain intact, behaving as inert
bodies.

555

The Rate of the Assumption of Chloroform by the Blood during
Anesthesia.

By G. A. Buckmaster, Assistant Professor of Physiology, University College,
University of London, and J. A. GARDNER, Lecturer on Physiological
Chemistry, University of London.

(Communicated by Dr. A. D. Waller, F.R.S. Received March 7,—Read
April 18, 1907.)

(From the Physiological Laboratory of the University of London.)

Although it is obvious that the essential condition of anesthesia is one in
which chloroform is associated with the cells of the body, and among these
the cells of the central nervous system, the gradual storing-up of the drug
must depend upon the supply available in the blood, the red corpuscles of
which are, as we have shown, the chief agents for the transport of chloroform
either from or to the pulmonary alveoli.

A definite threshold-value for the percentage of chloroform in arterial
blood must be reached in order that anesthesia shall occur, and the state be
maintained. The drug, as Tissot, Nicloux, and ourselves have found,
is eliminated at first rapidly, and subsequently more slowly, on the cessation
of the administration of chloroform. During anesthesia the drug does not
simply accumulate, since the processes of intake and output go on side by
side. Though chloroform is eliminated at first with great rapidity, the last
traces of the drug take a long time to slowly leak out of the tissues, but
apart from this fact the elimination of a high percentage of chloroform in the
blood takes place nearly as rapidly, and perhaps at times even as rapidly, as
the assumption of a high percentage.

The following scheme probably represents what occurs during anes-
thesia :—

( B. Portion which
| does not reach the
alveoli, but is expired.

A. Mixture of chloro-

D. Portion which is
form vapour and air,

associated by the
tissues.

!
constantly supplied in }

€XCess.

C, Portion which
reaches the alveoli and
lenters the blood.

E. Portion which is
eliminated at the pul-
| monary epithelium.

The relative amounts of B and C will, for a given percentage of chloro-
form, depend upon the rate and depth of the respiratory movements. This

556 Messrs. G. A. Buckmaster and J. A. Gardner. [Mar. 7,

is modified as the anesthetic state progresses to asphyxia, owing to the
increasing feebleness of the respiratory centres or to the gradual effect of the
drug on the muscles concerned in respiration, a factor which probably is of
small importance, since Sherrington has shown that striped muscles, unlike
the heart, are not readily poisoned by chloroform. In the case of cats,
during the very early stages of anzsthesia by chloroform, the rate of
respiration diminishes, and is followed by a marked increase. This variation
of respiratory movement would no doubt affect the relative proportions of B
and C. Of the chloroform passing inwards by the pulmonary epithelium
and which is carried by the blood, part is associated by the cells, D, and a
part is eliminated, KE. As anesthesia progresses D will be a decreasing factor,
while E will be an increasing factor, owing to the augmentation of chloroform
in the blood returning to the lungs. The rate of elimination, E, may be even
greater than the rate of assumption, C, though the total amount of C exceed
that present in the returning blood. Although the rate at which chloroform is
tending to pass out may be greater than the rate at which it tends to pass in,
the actual amounts going either way will depend, other things being equal, on
the mass of the chloroform on either side of the pulmonary epithelium. If,
therefore, the nerve centres remained unaffected, we should expect that with
a constant normal type of respiration a condition of equilibrium would
be reached between factors A and B and C and E, with any given percentage
of chloroform inhaled, which would effect a perfect anesthesia and not
rapidly kill.

During the progress of aneesthesia the nerve centres become paralysed, but
not necessarily in a regular order, as we have often noticed that various
reflexes vanish or reappear in different sequence in animals of the same
species. We should expect, therefore, that the curves indicating the
variations of the percentage of chloroform in blood with time would not
necessarily be smooth.

The determination of the amount of chloroform which is present in the
aneesthetised animal has been attempted in three different ways: (1) By
ascertaining the percentage of chloroform in the blood of animals anzesthetised
or killed by chloroform. (2) By determination of the amount of chloroform
in the blood and various organs of the body post mortem.* (3) More
frequently by reckoning the difference between the amount of chloroform in the
inspired and expired air. Waller,f Vernon Harcourt,t Collingwood,§ and

* Nicloux, ‘Comptes Rend.,’ 1906, vol. 60, p. 206.
+ Waller, ‘ Lancet,’ November 28, 1903.

{ Harcourt, ‘Brit. Med. Journ., June, 1906.

§ Collingwood, ‘ Physiol. Soc. Proc.,’ vol. 28, 1905.

1907.| Rate of Assumption of Chloroform by the Blood. By

Brodie and Widdows* have published the results of work undertaken on the
third line, and the last observers have ascertained that the assumption of
chloroform is much more rapid in the earlier than in the later stages of
anesthesia. We believe that by this method only a very limited amount of
information can be gained as to the physiological processes which are
concerned in anesthesia. With the desire of throwing further light on this
question, we have undertaken a number of experiments on the chloroform-
content of the blood during the stages of anesthesia. A very large number
of experiments have been made, of which we quote three of the most
detailed ones, which are typical of the whole.

Mode of Expervment.

The general mode of the experiment has been described in detail in
a former paper.t The method we have now followed is similar, except that
nitrous oxide was used instead of ether as the first anesthetic. The effect
of this gas was allowed to pass off and then chloroform was administered.
We found some difficulty in maintaining the chloroform-content of the
inspired air absolutely constant. The best mode of attaining this is by
means of goldbeater’s skin bags filled with a definite percentage of
chloroform vapour by means of the Dubois apparatus, and this was the
method adopted in some of the experiments. The apparatus gives fairly
good results within the limits for which it is constructed, i.e., up to
percentages of 2, but above this value it is difficult to obtain constantly
good results. It is also necessary that the goldbeater’s skin bags shall be
new and made of fresh skin, as it was found that with old bags, even if these
were perfect, the contents became gradually weaker in chloroform, and
direct experiments showed that this was due to the absorption of chloroform
vapour by the material. With fresh goldbeater’s skin this did not occur.}
All the tubes used in making connection with the bags or cannula in the
trachea should also be of wide bore, not less than ? inch, and rubber

* Brodie and Widdows, ‘ Brit. Med. Journ.,’ June, 1906.

t ‘ Roy. Soc. Proc.,’ B, vol. 78, p. 422. :

t Aspiration of a mixture of chloroform vapour and air from an old goldbeater’s skin
bag. Weight of chloroform determined densimetrically.

In one experiment the weight of chloroform progressively fell in three-quarters of an
hour from 0:045 to 0:032, and with a larger bag the progressive fall was from 0°023 to
0:01 in 20 minutes. 1:1 grammes of the skin of this bag, suspended in air containing
chloroform vapour, increased in weight 0°026 gramme, while a similar control in air
increased only 0°0015. An experiment with fresh skin showed practically no change in
weight. A new bag, 36 inches in circumference, the content of which was analysed at

regular intervals, showed no variation in an hour, though a slight decrease in the amount
of chloroform took place on prolonged standing.

558 Messrs. G. A. Buckmaster and J. A. Gardner. | Mar. 7,

junctions should be avoided as far as possible. In other experiments
a Woulff’s bottle of the following description was used instead of bags.
Capacity of the bottle 080 ¢c.; volume of chloroform 170 cc.; surface
area 43 sq.cm.; volume of air-space 410 ¢.c.; diameter of inlet-tube, which
was surrounded by a water jacket of the same temperature as the main
bath, 2°5 cm.; diameter of outlet-tube 1:5 cm. The bottle was placed in a
large bath so that it could be kept at any desired temperature. The
apparatus was so arranged that the distance of the lower end of the inlet-tube
from the surface of the chloroform could be varied at will. The outlet-tube
was connected with the Chauveau-valve apparatus by means of a densimeter
bulb 250 c.c. in capacity. By this means the percentage of chloroform in the
inspired air could be ascertained. This apparatus proved in many respects
satisfactory ; not the slightest drag on the respiratory movements was noticed,
a matter of great importance in any experiment of this kind, and particularly
so when the duration of an experiment is prolonged. We have found that
when the normal movements of respiration were mimicked by means of a
bellows apparatus, which aspirated a mixture of air and chloroform vapour
from the Woultt’s bottle, considerable variations, amounting to as much as
50 per cent. of the higher values, were noticed when the rate or amplitude or
both were varied. Actual experiments on cats showed that the variations
which occur during anesthesia were much less marked. Thus, during one
experiment lasting 20 minutes, the following percentages were obtained: 1°8,
271, 2, 1:6, 1°7. In another experiment of 24 minutes’ duration, the values at
intervals were as follows: 4:1, 3°6, 4:5, 3°1, 2°95, 3°8, 3°-7o. For the first
experiment the average was 1°8 and for the second 3°8.

In the experiment (No. I), asphyxial convulsions began within a minute
of the cessation of respiration, and with the continued inhalation of chloro-
form the convulsions recurred at intervals until 4.23. The heart ceased to
beat at 4.28.

The data obtained from this table are given in a graphic form in Curve I,
while in Column 7 of the table the type and frequency of the respirations, at
the time each sample was taken, are given.

In the experiment (No. II), chloroform was commenced at 2. 20 P.M., but
before the end of the first minute the animal ceased to breathe. The inhala-
tion was at once stopped, the animal recovered and the experiment restarted
at 2.45, by which time the chloroform would have been eliminated. This early
and sudden cessation of respiration we have repeatedly noticed with cats, and
it is not a mere holding of the breath, as in other experiments it was often
found necessary to use artificial respiration in order that the animal should
recover. After this has happened, under apparently precisely similar

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1907.] Rate of Assumption of Chloroform by the Blood. 561

conditions, with the same percentage of chloroform the subsequent anesthetisa-
tion will generally proceed in a perfectly normal manner for a prolonged period
of time. We consider this is a fact of much importance which will be
subsequently considered.

The results of this experiment are shown in Curve II. The type and
frequency of the respirations at the times the samples were taken are given
in Column 7 of the table.

Experiment III.—Cat. Weight 3°5 kilogrammes. Chloroform, between 3 and
4 per cent., administered by a Woulff’s bottle. This experiment is of a
special interest, as the early cessation of respiration mentioned in connection
with the last experiment took place, but the animal recovered naturally,
without it being necessary to discontinue the chloroform.

Differences
Weight of : in percentages
Time. Anesthetic. St of hoon eka: Sr ue of chlorine, | Remarks.
ood taken. reckoned as
CHCl.
12. 15 N,O on
12. 32 3 3 3454 0 -0456 0 °337 — Control
12. 40 Chloroform
3-4 per cent.
12. 41 A, 2-444 0 -0377 G 3814 0 :0498
12. 43 ‘ 2 °4221 0 :0388 0 3961 0 -066
2.44 5 — — — — Slight eye
2. 46 Bs 3 °3243 0 ‘0497 0 -3696 0 0366 ies.
2. 48 % — — — — Reflexes
2. 50 i 2 -4079 0 :0361 0 °3707 0 0378 gone
2. 55 “ 2 °8451 0°0441 0 *3832 0 -0519
1.0 33 2 -954 0 -0467 0 -3909 O :0604
1.10 af 2 °7349 0 0434 0 °3923 0 :0621
1. 20 x 2 °7826 0 0441 0 3918 0 -0615
1. 30 ; 2 °9142 0 0466 0 -3954 0 -0655
1, 353 5 2 °4282 0 0389 0 -3961 0 -0663 Respiration
ceasin
1. 38 a 1 °8758 0:03 0 -3954 0 -0656 Ist Sauk:
ial convul-
| sion

In Curve III the results of this experiment are illustrated graphically, and
we append a portion of the respiration tracing :—

562 Messrs. G. A. Buckmaster and J. A. Gardner. [Mar. 7,

Milligrammes of chloroform per 100 grammes of arterial blood.
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Respiratory Tracing of Experiment III.

564 Messrs. G. A. Buckmaster and J. A. Gardner. [Mar. 7,

Conclusions.

From the tables and curves that have been given, we consider that the views
expressed in the earlier part of the paper, which were based on a much larger
number of experiments undertaken with various other objects, and
with which this paper does not deal, are justified. The chloroform-content
of the blood rises in the initial stages of anesthesia with great rapidity to a
value which approaches a maximum. During this period the quantity of
chloroform in the blood appears to affect particularly the respiratory centres,
so that breathing becomes slower and often ceases during the first few
minutes of anesthesia, and it is necessary to resort to artificial respiration in
order to prevent the animal dying. With the percentages of chloroform that
have been employed, we have never noticed failure of the heart at this stage.
The cessation or slowing of respiration at this stage is, as might have been
expected, more liable to occur with high than with low percentages of
chloroform. Sudden failure of the heart’s action has never been noticed by
us in cats under the conditions of our experiments, where no percentages of
chloroform above 5 have been employed. We have found that a definite
danger-point occurs in the first few minutes of anesthesia, owing to paralysis
of the respiratory nervous mechanism.

If the animal naturally passes this stage, which is marked on the curves
between X and Y, as in Experiment III, or is restored either by stopping the
chloroform inhalation or by artificial respiration, then on continuing the
anesthetic the amount of chloroform in the blood quickly rises again towards
a maximum value. An equilibrium between the factors which determine
the amount of chloroform in the blood subsequently appears to be obtained,
the processes of intake and output at the surface of the lung going on side
by side. This stage extends from point Z onwards.

This state of equilibrium is reached, and may persist for a considerable
length of time. The exact length of this period differs with different
individuals, and even in the same individual in different experiments.
During this period the animal may die at any moment should any disturbing
influences come into play, and therefore the state is not one of safety.
During this period the animal can always be killed with chloroform, even
though the percentage of chloroform in the blood rises only very slowly.
We would emphasise the point that the difference between the amount
of chloroform in the blood throughout this anesthetic stage and death is
very minute. It will be noticed that the difference between the percentages
of chloroform in the blood at the point where the corneal reflexes vanish
and the maximum values attained are by no means large in actual value,

1907.| Rate of Assumption of Chloroform by the Blood. 565

but are relatively so, averaging, in the experiments quoted, about 50 per cent.
After the corneal reflexes have vanished, even though the percentage of
chjproform administered be kept absolutely constant, there is no criterion
other than analysis by which we can judge of the progress of anesthesia in
these animals. | .

In our experiments, death always occurred by failure of the respiration
succeeded by asphyxia. Under the conditions of our experiments under-
taken on a very large number of cats, we have never noticed death by heart
failure preceding cessation of respiration.

We take this opportunity of expressing our thanks to the Government
Grant Committee of the Royal Society for assistance in carrying out this
work ; and also our appreciation of the valuable assistance of Mr. G. W. Ellis,
who has helped in a large number of the laborious analyses.

to
M

VOL. LXXIX.—B.

566

Function of the Red Corpuscles in Chloroform Anesthesia.

By GrorcE A. BUCKMASTER, Assistant Professor of Physiology, Univegity
College, University of London, and J. A. GARDNER, Lecturer on
Physiological Chemistry, University of London.

(Communicated by Dr. A. D. Waller, F.R.S. Received March 12,—Read -
April 18, 1907.)

(From the Physiological Laboratory of the University of London.)

It has been conclusively shown by Pohl,* Nicloux,+ Moore and Roaf,t and
ourselves§ that when chloroform is associated with the blood, the bulk of
the drug is held by the red corpuscles. It would appear, therefore, highly
probable that in chloroform anzsthesia the transport from and to the surface
of the lungs is a function of the red corpuscles. In order to throw more
light on this point, we have investigated the effect of the variations in the
volume of blood in circulation on the duration of anesthesia and on the
percentage of chloroform in the blood.

Removal of blood, other conditions being constant, should not alter the
percentage of chloroform in the blood. Several causes might separately or
together prevent the conditions being constant, and therefore bring about
deviations from the above conclusion.

1. The gradual entrance into the blood of extra-vascular liquid. According
to Starling,|| “ the fluids contained in the tissue spaces possess the same
tonicity and the same composition in salts as the blood-plasma.” If, there-
fore, the extra-vascular fluids entered the blood-stream instantly to an amount.
equal to that of the blood withdrawn, the percentage of chlorine in the blood
would only be slightly raised, because the corpuscles contain a somewhat
lower percentage of chlorine than the plasma.{/ The restoration of the
volume after hemorrhage is known to be a gradual process, and during the
time and under the conditions of our experiments could not have taken place.
to any marked extent. Therefore the variations in the chlorine-content of
the blood would be practically negligible, and well within the errors of
experiment. This conclusion was repeatedly confirmed in the course of our
experiments by analyses made at the beginning, before and after bleeding.

2. An entrance of red corpuscles into the blood-stream, consequent on.

* © Archiv f. exp. Path. u. Pharmak.,’ vol. 28, 1890—1891.
+ ‘Comptes Rendus,’ Nos. 2, 3, 7, 1906.

t ‘Roy. Soc. Proc.,’ vol. 73, 1904.

§ ‘Roy. Soc. Proc.,’ B, vol. 78, 1906.

\| ‘Journal of Physiology,’ vol. 19, No. 4, 1896.
4 Buckmaster and Gardner, ‘ Roy. Soc. Proc.,’ B, vol. 78, 1906.

Function of Red Corpuscles in Chloroform Anesthesia. 567

hemorrhage, is known to be a very slow process, and one dependent upon the
activity of the bone-marrow. The evidence which exists on this point, there-
fore, negatives the idea that any appreciable effect on the chlorine-content of
the blood can be due to this cause. |

3. Abnormal depth and frequency of respiration, combined with a high
percentage of chloroform, might conceivably cause abnormality in any set of
parallel experiments made before and after bleeding by the chloroform
entering the plasma to an excessive amount.* The possibility of this factor
coming into play necessitated a large number of experiments. |

Finally, the peculiar nature of the anesthetic process, to which we have
drawn attention in another paper,f as an examination of the published
curves will at once make clear, renders it unlikely that any definite con-
clusions could be drawn from a small number of experiments as to the effect
produced by alteration in the volume of circulating blood. In the paper
referred to we showed that the percentage of chloroform in blood rapidly rises
to a point approaching a maximal value. When this point is reached, which
may occur within the first few minutes of an experiment, the animal often
ceases to breathe. This we have alluded to as the first danger-point of
anesthesia. When this point is passed naturally, the chlorine-content of the
blood falls, but again rapidly rises. In most experiments, when breathing
stopped at this point, we found it better to allow the animal to resume
breathing and restart the experiment. In either case the maximal value was
quickly reached again, when an equilibrium occurred between the intake and
output of the drug. This state of equilibrium, when the percentage of
chloroform only rose very slowly, was often maintained for a considerable
period of time. During this period the respiration may stop at any moment
from slight causes which cannot be controlled. This further rendered it
necessary to make a large number of experiments.

The operative procedures for the administration of chloroform and for
withdrawing blood were identical with those described in former papers.

Experiments in which the Asphyxial State was rapidly reached.

The percentage of chloroform used in these experiments was above
3 per cent. and under 5 per cent. Sometimes bags were used, sometimes
a Woulffs bottle such as we have employed in experiments previously
described, so that a slight drag on the respiratory movements was produced.
These experiments may be fairly taken as representative of those in which
death occurred at the first danger-point of aneesthesia.

* Buckmaster and Gardner, zbid.
t ‘Roy. Soc. Proc.,’ this volume.

568 Messrs. G. A. Buckmaster and J. A. Gardner. [Mar. 12,

The general plan of the experiment was to anesthetise the animal with
ether or nitrous oxide, allow the anesthetic to be disengaged from the body,
and then administer a known percentage of chloroform. The determination
of the chloroform-content of the blood was made at the asphyxial point.
The animal was then allowed to recover from the full effect of the
chloroform, either naturally or by artificial respiration, and sufficient time
was allowed for practically all the chloroform to be eliminated from the
blood. One hour to one and a-half hours was usually sufficient for this, and
it was generally necessary to give the animal a little ether or nitrous oxide in
order to preserve unconsciousness. During this period the animal was bled
from the carotid artery to a known amount. The quantity of biood
withdrawn varied with the weight of the animal. Sometimes the whole
amount was taken at once, but generally in successive small quantities. The
animal was re-aneesthetised and another determination of the amount of
chloroform in the blood made at the asphyxial point, but before re-anees-
thetisation a sample of blood was always taken as a control. .The details of
the experiments are recorded in Table I.

These are the only experiments in which the respiration-curves definitely
indicated that asphyxia occurred both before and after bleeding in the
initial stage of anesthesia. In five of the seven experiments, comparative
determinations were made before and after bleeding. Of these, in two cases
the percentages of chloroform found in the blood were practically identical.
In two cases the percentage was higher after than before bleeding, and in
one case lower. In the other two experiments in which values after bleeding
were obtained, the figures are fairly normal.

Before bleeding the average percentage of chloroform in 100 grammes of
blood was approximately 0°043 gramme and after bleeding 0045 gramme.
It would appear, therefore, that in the above experiments the percentage of
chloroform in the blood is not altered by bleeding.

Hzperiments wn which Asphyxia took place during the Second Stage of
Anesthesia.

In these experiments chloroform was inhaled from the Woulff’s bottle
described at length in a former paper “On the Rate of the Assumption of
Chloroform by the Blood during Anesthesia.” In those cases where
respiration stopped within the first few minutes, the animal was brought
round artificially or allowed to recover naturally and the anesthetic again
administered continuously. In other respects the procedure was the same
as in the first group of experiments. The details of the experiments are
recorded in Table II.

1907.| Function of Red Corpuscles in Chloroform Anesthesia. 569

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1907.] Function of Red Corpuscles in Chloroform Anesthesia. 571

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572. Messrs. G. A. Buckmaster and J. A. Gardner. [Mar. 12,

It will be seen, therefore, that the average percentage of chloroform in the
blood at the asphyxial point is 0-048 before and 0-051 after bleeding, or
eliminating the first two figures in Column 9, quoted in Experiment XTII, and
the first figure in same column in Experiment XIV, as these values were
determined in samples taken considerably before asphyxia, the corrected
averages are 005 before and 0:0497 after bleeding. In one experiment the
figures were sensibly the same before and after withdrawal of blood, and in four
experiments there was a slight increase after the withdrawal of blood. In
the two remaining experiments a decrease was observed after a hemorrhage.

In the following table (No. III), we quote two experiments in which the
respiration tracings showed that asphyxia took place before bleeding in the
second stage of anesthesia, and after bleeding at the initial stage.

Haperiments in which comparisons were made of the percentage of chloroform
in the blood at the asphyxial stage after hemorrhage and after the replacement
of the blood which had been removed by the blood of another animal of the same
spectes. All the observations were made in the second stage of ancesthesia.

The replacement of blood was effected in the following way :—A cat was
anesthetised with ether and a cannula placed in the carotid artery. The
cannula was connected by a short rubber tube fitted with a clip, with the
outflow end of a burette. This was warmed and smeared inside with olive
oil, and before making connection with the cannula, the cannula, rubber tube
and about an inch of the burette were filled with warm olive oil, so that the
blood could be received from the animal without coming in contact with the
air or glass, and without cooling to any serious extent. A small cannula had
been previously placed in the femoral vein of the animal which was the
subject of the anzesthetic experiment. This cannula was filled with a warm
solution of sodium sulphate of a strength isotonic with that of blood plasma.
The burette was now disconnected from the cannula in the carotid artery, and
connected with the cannula in the femoral vein, care being taken that no air
entered the circulation. The requisite amount of blood was then allowed
slowly to flow during the course of two or three minutes into the animal.
The whole transfer of blood was effected in less than four minutes. In none
of our experiments did we notice any disturbing effects produced by the
introduction of the blood. The animal was then re-anesthetised with
chloroform, after recovering from the first anzesthetic, precisely as in the first.
part of the experiment. Details of these experiments are recorded in
Table IV. |

In two out of the three complete experiments the percentage of chloroform
in the blood at the asphyxial point after bleeding and after replacement.

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1907.| Function of Red Corpuscles in Chloroform Anesthesia. 575

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EXPERIMENT XXIV.—Respiratory tracing taken after hemorrhage

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1907.| Function of Red Corpuscles in Chloroform Anesthesia. 577

of blood is practically the same. In the third experiment a small increase
is noticed after restoration of blood. In the case of the two incomplete
experiments the percentages of chloroform in the blood after replacement are
of the same order of magnitude as is found in normal animals at the stage
when respiration ceases.

Experiments in which comparisons were nade of the percentage of chloroform
in the blood at the asphysial state in the normal animal, after a hemorrhage, and
after the replacement of the blood which had been withdrawn by the blood of
another animal of the same species.

The mode of experiment was the same as that already described; the
results are given in table (No. V).

The results given in Table V confirm generally the results of the experl-
ments quoted in the previous tables. Closer results cannot be expected when
the prolonged and complex nature of the experiment is taken into considera-
tion. In connection with Experiment XXIV we have thought it advisable to
give the tracings of the respiratory movements which were obtained before
bleeding the animal, after withdrawal and after replacement of blood,
together with curves indicating the rate of the respirations during these three
conditions. These tracings and curves are broadly typical of a large number
of experiments. In the case of all the »experiments in which Woulff’s bottle
was used, the figures given for the percentage of chloroform administered
were obtained by a densimetric estimation at the end of the experiment, and,
therefore, only represent the strength of the chloroform-air mixture for the
particular time they were taken. They probably indicate, however, within
narrow limits, the average value during the experiment.

Conclusions.

The experiments we think show that the percentage of chloroform in the
blood does not. suffer any variation corresponding to differences in the
volume of circulating blood, and the results are therefore in accord with
what we should have expected would be the case if the red corpuscles were
the essential agents for the transport of chloroform.

As to the duration of time which elapses between the commencement
of the inhalation of chloroform and cessation of respiration, it will be noticed
that the length of time does. not vary according to the volume of blood in
circulation ; sometimes this was shorter, sometimes longer after than before
a hemorrhage. A somewhat greater equality in time is apparent in those
‘experiments where the respiration ceased at an early period of anesthesia.

578 Function of Red Corpuscles in Chloroform Anesthesia.

These results are in accordance with the views expressed in a former paper.*
We may mention here that a large number of experiments besides those
described were made with a view to ascertain this particular point,

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EXPERIMENT XXIV.

whether a loss of blood had a direct influence on the duration of anesthesia,
taking as the end-point the cessation of respiration. The results are in

* ‘Roy. Soc. Proc.,’ bid.

Rate of Elumination of Chloroform srom the Blood. 579

agreement with those we have described above, and therefore do not think it
necessary to publish them in detail.

We take this opportunity of expressing our thanks to the Government
Grant Committee of the Royal Society for assistance in carrying out this
work. We also desire to express our indebtedness to our assistant,
Mr. G. W. Ellis, for the care he has taken in carrying out many of the
analyses.

On the Rate of Emination of Chloroform from the Blood after

Anasthesia.

By GEORGE A. BUCKMASTER, Assistant Professor of Physiology, University
College, University of London, and J. A. GARDNER, Lecturer on Physio-
logical Chemistry, University of London.

(Communicated by Dr. A. D. Waller, F.R.S. Received July 19, 1907.)

(From the Physiological Laboratory of the University of London.)

In a paper* on the rate of assumption of chloroform by the blood, we
showed that the percentage of chloroform rises very rapidly to a maximal
value. This is the cause of an early danger-point in anesthesia. Subsequently
a rapid fall takes place, followed by a more or less rapid rise towards a
maximal value, which is maintained during the rest of anesthesia.

The following remarks will illustrate our conception of the anesthetic
process. The blood at first rapidly becomes charged with chloroform, which
is held almost entirely by the red corpuscles. The respiratory centre or
centres in the cat become affected quite early, and discharge impulses less.
frequently than the normal. As a result of this the intake of chloroform
is lowered, and consequently the percentage of chloroform in blood falls,.
either owing to the tissues rapidly storing up the drug at the expense of
the blood, or because the elimination of chloroform is as rapid or more
rapid than the assumption, or to both these causes. If the first danger-point
is safely passed, the respirations improve in frequency and become rapid..
It is known that many chemical substances, for instance, the group of
alcohols, ether or chloroform, which are lethal, primarily act as exciting
agents on living cells. Thus those bodies which in a given concentration are.
lethal for protoplasm, in lower amounts check its activities, but in still legs.
amount, so long as this is above the indifferent point, inversely will act as a
stimulus and augment the energy-discharges of protoplasm. In the case of.

* ©Roy. Soc, Proe.,’ this volume, p. 555.

580 . Messrs. G. A. Buckmaster and J. A. Gardner. [July 19,

chloroform it would seem that there is an initial stimulating effect which
may approach the lethal value, but that if this is not actually attained the
stimulating effect, which was masked, persists as a recognisable after-effect
when the first danger-point has been passed. Thereafter the chloroform-
content of the blood approximates to a maximal value, which is maintained
until asphyxia takes place (maximum value).

If the rate of elimination were more rapid than the rate of intake, or even
equal to this, it would not be easy to understand why this maximal value is
so rapidly reached or maintained, and for these reasons it appeared important
that we should study the rate at which chloroform was eliminated during
-ancesthesia. |

Method.

All the experiments were performed upon cats, which were anes-
thetised by ether, and the necessary operations performed after an
injection of hirudin into the femoral vein in quantity sufficient to delay the
coagulation of the blood during the time .of the experiment. After the
animal had so far recovered from ether that the reflexes had reappeared but it
was still under the influence of the drug, chloroform of about 2 to 3 per cent.
was administered from a Woulff’s bottle of the form described in our former
papers. The inhalation was stopped in some cases when the asphyxial point
was reached, and in other cases at an earlier point during the period we have
described as the second stage of anesthesia. Immediately the inhalation of
chloroform was stopped, samples of blood were abstracted from the central
end of one carotid artery or from the cephalic end of the external jugular
vein, or from the side tube of a short T-piece connecting the cephalic and
thoracic end of the vein, or by the introduction of a tube several inches
‘in length along the vein, so that a sample of blood could be taken from the
right auricle at definite intervals of time. In some experiments only arterial
blood was examined, in others only venous, and in other cases samples were
‘taken simultaneously from both the artery and vein. The methods of collecting
and analysing samples of blood were those fully described in our former
papers. In some experiments, however, it was found more convenient to
abstract the venous blood from the cannula by means of a syringe. During
the entire course of any experiment the respirations were recorded by a
tambour placed on the ensiform cartilage.

From all our experiments we have selected tive typical examples, details

of which are sani
Expervment I.

Cat. Weight. 3°7 kilogrammes. Respiration. racatiied by tambour on
‘abdomen. Samples from carotid artery.

581

iminat

1907.] Rate of El

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VOL. LXXIX.—B.

582 Messrs. G. A. Buckmaster and J. A. Gardner.

[July 19,

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Curve A.—Constructed from Data given in Experiment I.

Ordinates = milligrammes of chloroform in 100 grammes of blood ;
minutes after the cessation of chloroform inhalation ;

(eleven).

Experiment LI.

abscissee = times in

X = samples of arterial blood

Cat. Weight 3°5 kilogrammes. Respiration recorded by tambour on
abdomen. Samples taken by a syringe from a long cannula introduced into

the external jugular vein, so that blood was withdrawn from the circulation
close to the right auricle. It was ascertained post mortem that this was the
case. The first cubic centimetres of blood withdrawn were rejected so as to

avoid taking a sample of stagnant blood in the vein.

40
: EE EEE ee
se SOARS SRSRRRREEER SSS
8 nee ao BEcwe as
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=

C) ky PAW ey ZN TN (aie lie Moke <5) TOA I5 16 17 18 19 20 21 22 23 2425 26 27 28 29 40 3t 52 55 34.35 30 ST 35 39 40 Al

Minutes.

Curve B.—Constructed from Data given in Experiment II.

Ordinates = milligrammes of chloroform in 100 grammes of blood ;
minutes after the cessation of chloroform-inhalation ;

withdrawn close to right auricle.

Expervment LILI.

abscisse = time in

x = samples of venous blood

Cat. Weight 3 kilogrammes. Samples taken simultaneously from the

carotid artery and from the central end of the external jugular vein. The

venous blood was withdrawn by a syringe from a cannula pushed down the
vein so that the blood was aspirated from the superior vena cava close to the
right auricle. The exact position was ascertained post mortem. In order to
obtain the sample as close to the heart as possible, the blood stagnant in the
vein was rejected. The details of this experiment are given in Table III,
from which Curve C was constructed.

A number of other experiments were also performed, for example, some in

583

ion of Chloroform from the Blood.

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1907.) Rate of Elimination of Chloroform from the Blood. 585

which the right external jugular vein was severed and the ends connected by
means of a glass tube with a lateral branch from which the blood was
withdrawn without interfering with: the flow of blood, samples of blood
being taken at the same time from the carotid artery. In other experiments,
samples were taken simultaneously from the carotid artery and central end
of the external jugular vein. In the following Tables IV and V we give the
results of two types of these experiments. When the blood was taken
without much disturbance to the circulation (Table IV) and was really a
sample of particular venous blood coming partly from muscles and partly from
the brain, obvious variations in the chloroform-content of the blood occur
which may be due to variations in the rate of discharge from the tissues but.
of the exact significance of which we have no knowledge. Similar variations
in the content of venous blood are noticed in the data given in Table V.

; 50;

USS SEES
60

aad CEPR rrr H+
330 Sete

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Curve C.—Constructed from Data given in Experiment ITI.
Ordinates = milligrammes of chloroform in 100 grammes of blood ; abscissee = times in
minutes after the cessation of chloroform-inhalation; X = arterial blood ;.
KOE = venous blood.

Milligrammes
al
°

General Conclusions.

The rate of elimination appears to depend upon the physiological state of
the individual animal, but when all the experiments described in this paper
are considered generally and compared with those in our previous papers, the
rate of output is found to be at first comparatively rapid, and then
subsequently becomes slower. The initial rates of elimination are, however,
much less rapid than the initial rates of the intake of chloroform, and,
therefore, on the whole, elimination is a much slower process than
assumption, a view which is borne out by a comparison of the times at which
the various reflexes disappear and reappear. |

As the results of his experiments on dogs, Nicloux* shows that the
arterial blood loses about half its chloroform-content five minutes after
inhalation is stopped, and at the end of one hour about one-third to
one-quarter of what the blood originally held is eliminated. At the end of

* ‘Comptes Rendus,’ 1906, vol. 60, p. 14.
PAR EE 5

Messrs. G. A. Buckmaster and J. A. Gardner. [July 19,

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588 Messrs. G. A. Buckmaster and J. A. Gardner. [July 19,

seven hours the blood still may contain 1 to 2 milligrammes per cent. of
chloroform. He also observed that the palpebral reflex reappeared within
15 to 2 minutes after anesthetic periods which lasted 38 and 67 minutes.
In one experiment he mentions that the animal got up and walked off the
table 30 minutes after the chloroform was stopped, and the animal appeared
to be quite normal, though at this period there were 10 milligrammes of
chloroform per cent. in the blood. |

In the case of cats, the curves for arterial blood show a general similarity
in form to curves which we have constructed from the data given in Nicloux’s
paper. The initial falls in our curves are not so rapid as in the case of the
dog, and the chloroform-content of arterial blood was only reduced by 50 per
cent. in about 15 to 20 minutes. In our longest experiments, about three-
quarters of the chloroform was eliminated in about 30 minutes, when the
animal was breathing normally. When, however, an increased ventilation of
the lung is brought about by artificial respiration, a very much shorter space
of time is required, as is evident from the analyses quoted in former papers.

Reflexes in our experiments reappear later than was noticed by Nicloux
for dogs; but though the pinna reflex, corneal reflex, tendon reflex, and tail
reflex were noted, there is no definite order in which they reappear, though
as a rule the tail reflex is the last.

Not infrequently an animal goes to sleep during recovery from anesthesia,
but we have never noticed the rapid recovery of volition to which Nicloux
has drawn attention.

According to Tissot’s observations, it would appear that during recovery
from anesthesia the amount of chloroform in venous blood constantly exceeds
the amount in arterial, as may be seen from the following table constructed
from his paper. He also considers that a study of the chloroform-content of
arterial blood should be made during the induction of anesthesia and of
venous blood during the disappearance of anesthesia.

| Cessation of 45 minutes 2 hours
| chloroform administration. later. later.
|
Arterial blood ... 53-2 5-8 0:0
Venous blood | 48 ‘1 | aa 4-9

All these results we cannot confirm, though in Tables III, IV, and V,
which are those of the chloroform-content of samples of arterial and venous
blood taken simultaneously, we are in agreement with Tissot’s observations
that at the moment when chloroform is stopped, arterial blood contains an
excess of the drug when compared with venous blood.

1907.| Late of Elimination of Chloroform from the Blood. 589

For the sake of argument, we will assume that during recovery from
aneesthesia the blood circulates in the body, and that no exchange of chloro-
form takes place between the blood and tissues. Under such conditions it is
clear that, given a steady elimination at the surface of the lungs, the amount
of chloroform in arterial blood would be constantly less than in venous, and
this difference would probably be a decreasing one. During the initial rapid
elimination, this difference would conceivably be marked and easily detectable
by experiment; but in the later stages, where the elimination is slow, the
difference would possibly lie within the errors of experiment. But during
recovery from anesthesia there will be superimposed on this simple con-
ception of the circulation a discharge of chloroform from the tissues into the
venous blood. If this took place steadily, it would tend to increase the
difference between arterial and venous blood. The problem, however, appears
to be more complex. First of all, the blood comes from regions such as the
muscles, viscera, etc., which are probably not charged to any great extent
with chloroform, compared to what is believed to be the case for the central
nervous system. The nerve centres are differently affected, and doubtless
discharge at different rates, as the evidences of their activity or paralysis
appear and disappear irregularly. Further, it is not inconceivable that
during the elimination one region may be discharging chloroform while other
cell-districts are relatively inactive in this respect, or even actually con-
tinuing to absorb chloroform. The question will be further complicated in a
very marked degree by the nature of the respiration at any moment, for the
depth and frequency of respirations naturally govern the elimination at any
particular moment. On the whole, therefore, we should expect the
chloroform-content of venous blood to be higher than that of the arterial, but
this difference would be slight in the later periods of elimination. In actual
experiments one would expect the result would depend markedly on the
particular source whence the venous blood was taken. A comparison with
Curves A and B shows a general similarity in the rate of elimination both in
the case of arterial and venous blood. Both curves show irregularities in the
rate. In Curve C, the chloroform-content of arterial blood is contrasted with
the general venous blood of the body (sample taken close to the heart). At
the moment of cessation of respiration, the arterial blood is markedly higher
than the venous, but after regular respiration is established, the two curves are
practically identical, the difference being within the errors of experiment.

The 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 now express our thanks. |

. 1 i 5 : 4 ; é es a ¢

(har

INDEX to VOL. LXXIX. (B)

Address of the President, Lord Rayleigh, November 30, 1906, 83.

Adelea ovata, life-history of (Dobell), i55.

Air, solubility in fats, and relation to caisson disease (Vernon), 366.

Albinoes and pigmented animals, intravascular coagulation in (Mudge), 103.

Algal Flora, subaérial and fresh-water, of Ceylon (Fritsch), 197.

Amyedalase, a new enzyme in yeast (Caldwell and Courtauld), 350.

Antirrhinum majus, inheritance of flower colour in (Wheldale), 288.

Antisera, precipitin, inactivation and inhibition exhibited by (Welsh and Chapman), 465.

Armstrong (H. E. and E. F.) Studies on Enzyme Action. X.—-The Nature of Enzymes
360.

Auld (8S. J. M.) See Dunstan.

Bacillus dysenterie, acquisition of new fermenting powers by (Twort), 329.

Bacteria derived from sewage, presence of, in air of sewers (Horrocks), 255 ; —— of
typhoid-coli group, fermentation of glucosides by (Twort), 329.

Bainbridge (F. A.) and Beddard (A. P.) The Relation of the Kidneys to Metabolism:—
Preliminary Communication, 75.

Barometric pressure, influence of increased, on man (Hill and Greenwood), 21, 284.

Barratt (J. O. W.) On Opsonins in Relation to Red Blood Cells, 1 ; On Mitosis
in Proliferating Epithelium, 372 ; Implantation of Actively Proliferating
Epithelium, 546.

Bashford (K. F.), Murray (J. A.), and Cramer (W.) The Natural and Induced Resistance
of Mice to the Growth of Cancer, 164.

Beale (L. 8.) Obituary notice of, lvii.

Beddard (A. P.) See Bainbridge and Beddard.

Benson (M.) Miadesmia membranacea, Bertrand; a new Paleozoic Lycopod with a
Seed-like Structure, 473.

Bile secretion and absorption in obstruction of bile duct (Herring and Simpson), 517.

Blanford (W. T.) Obituary notice of, xxvii.

Blood, estimation of chloroform in (Buckmaster and Gardner), 309 ;
tion of (Shattock and Dudgeon), 427;

| (Buckmaster and Gardner), 555, 566, 579.

Blood-cells, red, and opsonins (Barratt), 1.

Blood-serum, increase in complement-content of (Henderson-Smith), 378.

Bolton (C.) On the Pathology of the Dropsy produced by Obstruction of the Superior
and Inferior Venze Cave and the Portal Vein.—Preliminary Communication, 267 ;

Further Studies of Gastrotoxic Serum (Progress Report), 533.

Bone, regeneration of, réle of various elements in (Macewen), 397.

Buchanan (F.) The Time taken in passing the Synapse in the Spinal Cord of the Frog,
503.

VOL. LXXIX.—B. re

fatty degenera-
in chloroform anesthesia, studies of

lxvi

Buckmaster (G. A.) and Gardner (J. A.) The Estimation of Chloroform in the Blood of
Anesthetised Animals, 309 ; The Rate of the Assumption of Chloroform by
the Blood during Anesthesia, 555 ; Function of the Red Corpuscles in Chloro-
form Anesthesia, 566 ; On the Rate of Elimination of Chloroform from the
Blood after Anesthesia, 579.

Buckton (G. B.) Obituary notice of, xlv.

Burdon-Sanderson (Sir J. 8.) Obituary notice of, 11.

Caisson disease, relation to solubility of air in fats (Vernon), 366.

Caldwell (R. J.) and Courtauld (S. L.) Studies on Enzyme Action. IX.—-The Enzymes
of Yeast : Amygdalase, 350.

Cancer, natural and induced resistance of mice to growth of (Bashford and others), 164.

Carmichael (E. 8.) and Marshall (F. H. A.) The Correlation of the Ovarian and Uterine
Functions, 387.

Cathcart (E. P.) and Leathes (J. B.) On the Relation between the Output of Uric Acid
and the Rate of Heat Production in the Body, 541.

Ceylon, algal flora of (Fritsch), 197.

Chapman (H.G.) See Welsh and Chapman.

Chitin, identification of, by physical constants (Sollas), 474.

Chloroform in blood, estimation of (Buckmaster and Gardner), 309 ; rate of
assumption by blood during anesthesia (Buckmaster and Gardner), 555 ;

aneesthesia, function of red corpuscles in (Buckmaster and Gardner), 566 ;

rate of elimination from blood (Buckmaster and Gardner), 579.

Clarke (C. B.) Obituary notice of, xlix.

Coagulation, intravascular, in albinoes, and relation to nucleo- proteids of testes (Mudge),
103.

Courtauld (8. L.) See Caldwell and Courtauld.

Cramer (W.) See Bashford (E. F.).

Croonian Lecture. See Hardy ; Farmer.

Cyanogenesis in plants, VI (Dunstan, Henry, Auld), 315.

Dean (G.) An Experimental Enquiry into the Nature of the Substance in Serum which
influences Phagocytosis (Second Communication), 399.

Dixon (H. H.) On the Transpiration Current in Plants, 41.

Dobell (C. C.) Observations on the Life-history of Adelea ovata, Aimé Schneider, with a
Note on a New Gregarine from the Gut of Lithobius forficatus, 155.

Dropsy produced by obstruction of venz cavee and portal vein, pathology of (Bolton), 267.

Dudgeon (L. 8.) See Shattock and Dudgeon.

Dunstan (W. R.), Henry (T. A.), and Auld (8S. J. M.) Cyanogenesis in Plants.
Part VI.—-On Phaseolunatin and the Associated Enzymes in Flax, Cassava, and the
“Lima Bean,” 315.

Enzyme action, studies on (Caldwell and Courtauld), 350 ; —— (Armstrong), 360.

Epithelium, implantation of proliferating (Barratt), 546; —— mitosis in proliferating
(Barratt), 372.

Ewart (A. J.) The Ascent of Water in Trees (Second Paper), 395.

Farmer (J. B.) Croonian Lecture.—On the Structural Constituents of the Nucleus, and
their Relation to the Organisation of the Individual, 446.

Fermentation of glucosides by bacteria of typhoid-coli group (Twort), 329.

Fritsch (F. E.) A General Consideration of the Subaérial and Fresh-water Algal Flora
of Ceylon. Part I.—Subaérial Algee and Algze of the Inland Fresh-waters, 197.

lxvil

Gardner (J. A.) See Buckmaster and Gardner.

Globulin, chemistry of (Sutherland), 130.

Globulins, Croonian Lecture on (Hardy), 413.

Gotch (F.) Capillary Electrometer Records of the Electrical Changes during the Natural
Beat of the Frog’s Heart (Preliminary Communication), 323.

Greenwood (M., jun.) See Hill and Greenwood.

Gregarine, note on a new, from Lithobius forficatus, 155.

Groom (P.) Longitudinal Symmetry in Phanerogamia, 305.

Hardy (W. B.) Croonian Lecture.—On Globulins, 413.

Heart, electrometer records of electrical changes during natural beat of (Gotch), 323.

Heat production in body, relation to uric acid output (Cathcart and Leathes), 541.

Henderson-Smith (J.) Increase in the Complement-content of Fresh Blood-serum, 378.

Henry (T. A.) See Dunstan.

Herring (P. T.) and Simpson (8.) The Pressure of Bile Secretion and the Mechanism of
Bile Absorption in Obstruction of the Bile Duct, 517.

Hill (L.) and Greenwood (M.) The Influence of Increased Barometric Pressure on Man,
21, 284,

Horrocks (W. H.) Experiments made to determine the Conditions under which
“* Specific” Bacteria derived from Sewage may be present in the Air of Ventilating
Pipes, Drains, Inspection Chambers, and Sewers, 255.

Howes (T. G. B.) Obituary notice of, xxxi.

Hutton (F. W.) Obituary notice of, xl.

Ince (W. H.) See Mann and Ince.

Inheritance of flower colour in Antirrhinum (Wheldale), 288 ; —— of invisible characters
in peas (Lock), 28.

Innervation, reciprocal, of antagonistic muscles (Sherrington), 337.

Kidneys, relation to metabolism (Bainbridge and Beddard), 75.

Leathes (J. B.) See Cathcart and Leathes.
Ledingham (J. C. G.) The Inhibitory Action upon Subsequent Phagocytosis, exerted
on Active Normal Serum by Inactive Normal Serum through which Bacilli have
been passed, 482.
Leucocytes, life-history of (Walker), 491, 495.
Lock (R. H.) On the Inheritance of certain Invisible Characters in Peas, 28.

MacBride (E. W.) Some Points in the Development of Ophiothrix fragilis, 440.

Macdonald (J. 8.) The Structure of Nerve-fibres, 12.

Macewen (Sir W.) The féle of the Various Elements in the Development and
Regeneration of Bone, 397.

Mann (E. A.) and Ince (W. H.) Contributions to our Knowledge of the Poison Plants
of Western Australia, 485.

Marshall (F. H. A.) See Carmichael and Marshall.

Martin (W. B. M.) See Muir and Martin.

Medlicott (H. B.) Obituary notice of, xix.

Miadesmia membranacea, a paleeozoic Lycopod (Benson), 473.

Minchin (E. A.) On the Occurrence of Encystation in Trypanosoma grayi, Novy, with
Remarks on the Method of Infection in Trypanosomes generally, 35.

Mitosis in proliferating epithelium (Barratt), 372.

lxvill

Mudge (G. P.) On Intravascular Coagulation in Albinoes and Pigmented Animals, and
on the Behaviour of the Nucleo-proteids of Testes in Solution in the Production of
Intravascular Coagulation, 103.

Muir (R.) and Martin (W. B. M.) On the Combining Properties of the Opsonin of an
Immune Serum, 187.

Murray (J. A.) See Bashford (E. F.).

Muscles, antagonistic, reciprocal innervation of (Sherrington), 337.

Nerve fibres, structure of (Macdonald), 12.
Nucleus, structural constituents of, relation to organisation of individual (Farmer), 446.

Obituary notices of Fellows deceased, i.

Ophiothrix fragilis, some points in development of (MacBride), 440.

Opsonin of immune serum, combining properties of (Muir and Martin), 187.
Opsonins in relation to red blood cells (Barratt), 1.

Ovarian and uterine functions, correlation of (Carmichael and Marshall), 387.
Oxygen, possibility of, being set free in body (Hill and Greenwood), 284.

Peas, inheritance of invisible characters in (Lock), 28.

Phagocytosis, nature of substance in serum which influences (Dean), 399 ; inhibitory
action upon, exerted on active normal serum by inactive normal serum through
which bacilli have been passed (Ledingham), 482.

Phaseolunatin and associated enzymes in flax, etc. (Dunstan, Henry, Auld), 315.

Plimmer (H. G.) Further Observations on the Effects produced on Rats by the:
Trypanosomata of Gambia Fever and of Sleeping Sickness, 95 ; and Thomson
(J. D.) A Preliminary Summary of the Results of the Experimental Treatment of
Trypanosomiasis in Rats, 505.

Poison plants of Western Australia (Mann and Ince), 485.

Precipitin antisera, inactivation and inhibition exhibited by (Welsh and Chapman), 465.

Rayleigh (Lord) Presidential Address, November 30, 1906, 83.
Reid (G.) Nitrification of Sewage, 58.
Richthofen (Ferdinand, Baron von) Obituary notice of, xxxv.

Sanderson (Sir J. Burdon) See Burdon-Sanderson.

Serum, gastrotoxic, further studies of (Bolton), 533 ; immune, combining properties:
of opsonin of (Muir and Martin), 187 ; —— nature of substance in, which influences.
phagocytosis (Dean), 399.

Sewage, nitrification of (Reid), 58.

Sewers, conditions in which “specific” bacteria may be present in the air of (Horrocks),
255.

Shattock (S. G.) and Dudgeon (L. 8.) Fatty Degeneration of the Blood, 427.

Sherrington (C. S.) On Reciprocal Innervation of Antagonistic Muscles.—Tenth
Note, 337.

Simpson (S.) See Herring and Simpson.

Sollas (I. B. J.) On the Identification of Chitin by its Physical Constants, 474.

Spinal cord of frog, time taken in passing synapse in (Buchanan), 503.

Sutherland (W.) The Chemistry of Globulin, 130.

Symmetry in phanerogamia, graphic method of recording (Groom), 305.

Testes, nucleo-proteids of, and production of intravascular coagulation (Mudge), 103.
Thomson (J. D.) See Plimmer and Thomson.
Transpiration current in plants (Dixon), 41.

lxix

- Trees, ascent of water in (Ewart), 395.

Trypanosoma grayz, encystation in (Minchin), 35.

Trypanosomata, effects produced on rats by (Plimmer), 95.

Trypanosomes, method of infection in (Minchin), 35.

Trypanosomiasis in rats, summary of results of experimental treatment of (Plimmer and
Thomson), 505.

Twort (F. W.) The Fermentation of Glucosides by Bacteria of the Typhoid-coli Group
and the Acquisition of New Fermenting Powers by Bacillus dysenterie and other
Micro-organisms. Preliminary Communication, 329.

Uric acid and heat production (Cathcart and Leathes), 541.
Uterine functions, correlation of ovarian and (Carmichael and Marshall), 387.

Vernon (H. M.) The Solubility of Air in Fats, and its Relation to Caisson Disease, 366.

Walker (C. E.) Observations on the Life-history of Leucocytes. Part I1.—On the
Origin of the Granules, 491 ; - Part ITI, 495.

Welsh (D. A.) and Chapman (H. G.) On certain Phenomena of Inactivation and of
Inhibition exhibited by Precipitin Antisera, 465.

“Western Australia, poison plants of (Mann and Ince), 485.

‘Wheldale (M.) The Inheritance of Flower Colour in Antirrhtnum majus, 288.

‘Yeast, new enzyme in (Caldwell and Courtauld), 350.

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Meeting on November 30, 1906 . Epa : j : ; 83

e £ urther Observations on the Effects produced on Rats by the Trypanosomata
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Series B. Vol. 79. No. B 534.
BIOLOGICAL SCIENCES.
CONTENTS.

Page
Fatty Degeneration of the Blood. By S. G. SHATTOCK and L. S. :
DUDGEON : : 3 . ; jo AZT

Some Points in the Development of Obes fragilis. ‘By E. W. MACBRIDE,
D.Sc., F.R.S., Strathcona Professor of Zoology in McGill University,
Montreal . : 440

CROONIAN LECTURE. Sst ihe Stenepie Gage. of ie Muchas

and ther Relation to the Organisation of the Individual. By

J. BRETLAND FARMER, D.Sc., F.R.S. ; 446
On Certain Phenomena of Inactivation and of Inhibition exhibited ©

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Pathology, and H. G. CHAPMAN, M.LD., BS., Damouetrator of

Physiology in the University of Sydney : : 465
Miadesmia membranacea, Bertrand; a New Paleozoic [eccead wae: a

Seed-like Structure. By M. BENSON, D.Sc., F.L.S. (Abstract) . 5 ARF
On the Identification of Chitin by its Physical Constants) By IGERNA

B. J. SOLLAS, Fellow of Newnham College, Cambridge ; ; 474

The Inhibitory Action upon Subsequent Phagocytosis, exerted on je

Normal Serum by Inactive Normal Serum through which Bacilli have

been passed. By J. C. G. LEDINGHAM, M.A., M.B. (Aberdeen),

Assistant Bacteriologist, Lister Institute . : . 482
Contributions to our Knowledge of the Poison Plants of Waders pale

By E. A. MANN, Government wae and W. H. INCE, Ph.D., F.LC.

- (Abstract) . : j ; ; : . 485
Observations on the Life- pees Be re ‘Pan II.—On the Origin of

the Granules. By C. E. WALKER, Assistant-Director of the Cancer

Research Laboratories, University of Liverpool. (Plate 5) . A . AQI
Observations on the Life-history of lLeucocytes.— Part III. By

C. E. WALKER, Assistant-Director, Cancer Research ce ie

University of Liverpool. (Plate 6) . . . 405
The Time taken in passing the Synapse in the Spinal Cord # Ay: Frog. By

FLORENCE BUCHANAN, D.Sc., Fellow of University College, London.

(Abstract) . : : . 503
A Preliminary Summary of the Beate se the ee cease of

Trypanosomiasis in Rats. By H. G.-PLIMMER, F.L.S., and J. D.

THOMSON, M.B., C.M. < A ; 4 : : : . 5 eS

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BIOLOGICAL SCIENCES.

CONTENTS.

“ ‘The ae of Bile Secretion and the Mechanism of Bile Absorption in
Obstruction of the Bile Duct. By PERCY T. HERRING, M.D., and
‘SUTHERLAND SIMPSON, M.D., Disc. é , ;

oe Further Studies of Gastrotoxic Senin (Prosrsss Report.) By CHARLES
fe, BOLTON, M. D., Research Scholar of the Grocers” Company. (Plate 7)

! On Be eda becca ihe Output of Uric Acid and the Rate of ‘Heat
Production in the Body. By E. P. CATHCART, M.D., and 1B.
_LEATHES, M.B. ee : : ago a : ; Rs : :

{
By J..O. WAKELIN

° -%

=a Gr Ae BUCKMASTER, Aceciant Professor of Physiology, Cenc ae
College, University of London, and J. A. GARDNER, Lecturer on
Physiological Chemistry, University of London

Be iction of the Red Corpuscles i in Chloroform el thea By GEORGE A.
__ _BUCKMASTER, Assistant Professor of Physiology, University College,
University of London, and J. A/ GARDNER, Lecturer on Physiological

i

- Chemistry, University of London . see ; : f ! ;

ry LO: the Rate of Elimination of Chloroform from the Blood after Aneesthesia.
_ By GEORGE A. BUCKMASTER, Assistant Professor of Physiology,
ae College, University of ae and. vA. GARDNER, Lecturer

e « . ry . e e .

(

e, Contents, etc.

No. B 535.

Page

817

533

555

(560

ere

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EXPERIMENTS ON THE LENGTH OF THE CATHODE DARK SPACE
WITH VARYING CURRENT DENSITIES AND PRESSURES IN DIFFERENT iene a

Francis Witit1am Aston, A.I.C., Research Scholar at the University of Birmingham .. 80 |
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Cambridge. (Abstract) > \ SGP te

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ON THE PRESENCE OF EUROPIUM IN STARS. By Josten Lise B. Se. a ae oe
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NOTE ON THE APPLICATION OF VAN DER WAALS’ EQUATION TO Sepa
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THE THERMOMAGNETIC ANALYSIS OF METEORIC AND ARTIFICIAL

ey IRON ALLOYS. eo Se eae ene M.A., A.R.CS., Be College of oe London . ae
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A RECORDING CALORIMETER FOR EXPLOSIONS. iy eee HorkiNson,
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ELECTRIC FURNACE REACTIONS UNDER. HIGH GASEOUS PRES-
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ADDRESS OF THE PRESIDENT, LORD RAYLEIGH, ON Dele ‘AT
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CALCIUM AS AN ABSORBENT OF GASES FOR THE PRODUCTION OF
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