vie “3 ‘ va We Bie § arya is hey ; 2 \ as od ve e ‘ ’ ‘ ‘ 4 We int ‘ ' ; » by Cte A ‘< ae : 1 A iy t ie A 3, be Yess pe Was ) ede ery ¥en ah f i ' a PR ea aT Th Pht ‘ wos my vi Vat Sy , th \ vie ts ‘ '. ‘ vat ‘ Nee - ‘ Beye ; 4 ; La ‘ ‘ - y a r ad hie ’ ’ Lis Ses es deen fl wh i iz, ion . oe Rea as : , aoe ‘ ” ‘ Wid. : Mat . i oF i F * was nar bs ot Af Boi es ; f ’ tye elder ; 2 , f ever aah ' 1 a tes 3 : ‘ ee x 3 ¥ meee ues Beri, e Bian ody Te ae te Babe aya # a eet AO Bae a ais ie va 4 oi Sie) of al ab peda at 5 ees te enagee ri THE JOURNAL OF THE LINNEAN SOCIETY. ZOOLOGY. VOL. XIV. LONDON: SOLD AT THE SOCIETY’S APARTMENTS, BURLINGTON HOUSE, AND BY LONGMANS, GREEN, READER, AND DYER, AND WILLIAMS AND NORGATE. 1879. | Dates of Publication of the several Numbers included in this Volume. No. 738, pp. 1-64, was published October 24, 1877. 7, (4, 5, G5=186; es May 23, 1878. » 7, 4, 187-811, Ve August 31, 1878. » 46, ,, dll—-4l7, is October 31, 1878. » 77, 4, 417-505, eee January 31, 1879. ,, 78, ,, 505-606, A April 23, 1879. 79, 607-688, ,, May 20, 1879. ,, 80, ,, 689-761, # September 2, 1879. bite \ PRINTED BY TAYLOR AND FRANCIS, RED LION COURT, FLEET STREET, ohidy , \ eeUN JAMO! \ \ F t \ Bs aR, LIST OF PAPERS. Page ALLMAN, Professor G. J., M.D., LL.D., F.R.S., &c. The Anniversary Address of the President for 1878.—Recent Progress in our Knowledge of the Structure and Development onthe Phylactolematous Polyzoal . 4.225. 00022 -+)20e es. 489 On the Relations of Rhabdopleura. (With awoodcut.) ...... 581 Armit, Capt. WittiAM H., F.L.S. Note on Australian Finches of the Genus Poéphila .......... 95 Notes on the Presence of Tachyglossus and Ornithorhynchus in Northern and North-eastern Queensland...............05. 411 Bastian, H. Cuaruron, M.D., F.RS., F.L.8., &c., Professor of Pathological Anatomy in University College, London, and Phy- sician to University College Hospital. On the Conditions favouring Fermentation and the Appearance of Bacilli, Micrococci, and Torulee in previously Boiled Fluids. (NMED 8) WOOCOHUS)) “Goes scoeppenpoo doce pooeSOGeoonoUN 1 Baty, Josrpu §., M.R.C.S., F.L.S. Descriptions of new Species and Genera of Eumolpide ...... 246 Descriptions of new Species of Phytophagous Coleoptera...... 336° Busk, Professor Grore®, F.R.S., F.L.S. On Recent Species of Heteropora. (Plate XV.) .........-4. 724 Burier, ArTuur G., F.L.S. &c., Assistant Keeper in the Zoolo- gical Department of the British Museum. On the Butterflies in the Collection of the British Museum hitherto referred to the Genus Euplea of Fabricius........ 290 _ Cosson, T. Spencer, M.D., F.R.S., F.L.S., Professor of Botany and Helminthology, Royal Veterinary College. The Life-History of Filaria Bancrofti, as explained by the Dis- coveries of Wucherer, Lewis, Bancroft, Manson, Sonsino, THREE, CINCH Apa cnac no ndoce one Momence) O00 00.0 os. 306 CoLLINGWooD, CuruBERt, M.A., M.B., F.L.S. New Species of Nudibranchs from the Eastern Seas. (Abstract.) 737 lv Page + Day, Francis, F.L.S. &e. Geographical Distribution of Indian Freshwater Fishes.— Partly, (Conclusion. 2% oc ite asm sa merarinmen Moonee 534 On the Occurrence of Morrhua macrocephala at the Mouth of — the Mhames: (Gelato! INV a) ee ck tedera detent cn leet r-tckeinie atten 689 Duncan, Professor P. Martin, M.B. (Lond.), F.R.S., &e. On some Ophiuroidea from the Korean Seas. (Communicated by W. Percy Siapen, F.L.S.) (Plates IX.—XI.) ........ 445, HANLEY, SyLvanvs, F.L.S. Description oftwomew. Sitells eres eie nee nee 580 HawxksuHaw, J. Charxe, M.A., F.G.S. “On the Action of Limpets (Patella) in sinking Pits in and abra- ding the Surface of the Chalk at Dover ................-. 406 JEFFREYS, J. Gwyn, LL.D., F.R.S., F.L.S. Notice of some Shells dredged by Capt.-St. John, R.N., in Korea Straith. occ es ace sits as oa lores ole a eee ake eee 418 Note as to the Position of the Genus Seguenzia among the Gas- EVOP OMB: cis hya bs + Pepetve wa tise crane lane toads fone cre er SETS a eRe ee rene 605 Luszock, Sir Joun, Bart., M.P., F.R.S., F.L.S., D.C.L., Vice-Chan- cellor of the University of London. Observations on the Habits of Ants, Bees, and Wasps.— Part Vieevnts: (With onwioodcubs))anninr ise : ae .... 265 parpaVile Amisha en ae ee Mi erste toc ee 0:50 ee 607 On the Anatomy ob Amts: (Aibstract.) cnet cei nae 138 Manson, Parricg, M.D. On the Development of Filaria sanguins hominis, and on the Mosquito considered as a Nurse. (Communicated by Dr. COBEOnD WHERAS SHUIinS» yaa bee eee PS us 304 M‘Intosu, W. C., M.D., LL.D., F.R.S., F.L.S. On the Annelids of the British North-Polar Expedition. (With Sketch Map.) js os wile Moke ahs iesus «9059 tee 126 On a Remarkably Branched Syliis, dredged by H.M.S. ‘Chal- Venger’ 6 oc vais ays ga u's wae eitie sles are alta er 720 M‘Lacutan, Ronert, F.R.S., F.LS., &e. Report on the Insecta Grclading Arachnida) collected aby Capt. Feilden and Mr. Hart between the Parallels of 78° and 83° North Latitude, during the recent Arctic Expedition. (With Sketeh: Map.) (ici kiss ce Vie Sete ‘nin eae M‘Lacuian, Roper, and Pastor H. D. J. WaLLENGREN. 3 An Analysis of the Species of Caddis-flies (Phryganea) de- seribed by Linnzeus in his ‘Fauna Suecica’ .............. 726 \ \ WY Page Miers, Epwarp J., F.LS., F.Z.S., Assistant in the Zoological Department of the British Museum. sevisionjol the Buppided. ’ (Plate Vo) ven nceuceessaseecc se 312 On the Classification of the Maioid Crustacea or Oxyrhyncha, with a Synopsis of the Families, Subfamilies, and Genera. Gibites 2SUG yo UID peensnos coo bbe cos oo ody dren ataateece 634. Mivart, Prof. St. GrorGx, F.R.S., Zoological Secretary, Linnean ‘Society. Notes touching Recent Researches on the Radiolaria. (With WGEVOOCCULSS) Paras sta cvasd slcusna’slars vise cupveisis guelslc syatsls/e)s'eislase;'< aus 136 Moss, Epwarp L., M.D., late Surgeon H.M.S. ‘ Alert.’ Preliminary Notice on the Surface-Fauna of the Arctic Seas, as observed in the recent Arctic Expedition .............. 122 Morig, Dr. J., F.L.S., Assistant-Secretary of the Linnean Society.. Remarks on the Skull of the Hchidna from Queensland. (With WOCKEM,))\ooganobednosounsnon HOM AC oR pools ose Oe Ro oes 418 Morir, Dr. J., F.L.S. &c.,and Professor H. ALLEYNE NICHOLSON, F,L.S. &e. On the Minute Structure of Stromatopora and its Allies. (Plates LW Co OOM a onde 656. tobe paOmC oC odo beDooeon 187 NicHoxson, Professor H. ALLEYNE, F.L.S. &c., and Dr. J. Muriz, F.LS. &e. On the Minute Structure of Stromatopora and its Allies. (Plates SDV ERS On WO OCCULS: Wires cle sues stecvesieteiancta coele a) cee Gc ti ene one ara « 187 Parker, Professor W. K., F.R.S., F.L.S., &c. On the Structure and Development of the Skull in the Urodelous Prsriago tale ee CATS ELACES)) ior cers! esete on wie va cla ansperter wea le # ecu e) 61 717 SAUNDERS, Howarp, F.L.S., F.Z.S. On the Geographical Distribution of the Gulls and Terns (STUD, Boi 6.02 ROO AOI OO ORR Os a aao 390 SHarpz, R. Bowpier, M.A., F.LS., F.Z.S., &c., Senior Assistant, Zoological Department, British Museum. Contributions to the Ornithology of New Guinea :— Part V. On recent Collections from the Neighbourhood of Port Morseby, S.H, New Guinea .............. 626 Part VI. On Collections made by the Rev. W. G. Lawes in Solin, New Grutieai saree aucrenae ase erc ps aay ei de cre ronsay 685 SLADEN, W. Percy, F.LS., F.G.S. On the Asteroidea and Echinoidea of the Korean Seas. (Plate AN PUTET Ber recromme rrite oe ety nee Me) ails Voreiiel uA RR es 424 val Page SMITH, FREDERICK, F.Z.8., Assistant Keeper, British Museum. Descriptions of new Species of Aculeate Hymenoptera collected by the Rey. Thomas Blackburn in the Sandwich Islands. (Communicated by A. G. Butter, F.L.8.) ............0. 674. WALLENGREN, Pastor H. D. J., and Ropert M‘Lacuuay, F.R.S. An Analysis of the Species of Caddis-flies (Phryganea) de- scribed by Linneeus in his ‘Fauna Suecita’ ..,........... 726 WATERHOUSE, CHar_zs O., Assistant in the Zoological Department of the British Museum. . Report on a small Collection of Insects obtained by Dr. J. C. Ploem in Java, with a Description of a new Species of Hoplia 134 WarTERHOUSE, FREDERICK H., Librarian to the Zoological Society of London. Descriptions of new Coleoptera of Geographical Interest, col- lected by i@harles Marwanls o-nyt a eye earl eee eee ee 530 Watson, Professor Morrison, M.D., and A. H. Youna, M.B., Owens College, Manchester. On the Anatomy of the Elk (Alces malchis). (Plates VI. & VEL, and: a woodcuta)in. ies sae cere pee eee eae 371 Warson, Rev. Roprrt Boog, B.A., F.R.S.E., F.L.S., F.G.S., &e. Mollusca of the ‘ Challenger’ Hepedcn _ I, Preliminary Report to Prof. Sir C. Wyville Theeneee F.R.S., Director of the Civilian Scientific Staff, on the Mollusca dredged during the Exploring Voyage ...... 506 I. The Sorzenoconcuta, comprising the Genera Dentalium, Suphodentalium, and Cadulus ...0......c00s. sere ene 508 Ill. Trocuipm, viz. the Genera Seguenzia, Basilissa, Gaza, ON: emo ois 5 hae vce tains nie sete ain neo eee ee eee 586 IV. Trocuip# (continued), viz. the Genera Basilissa and Tro- . chus, and the TURBINIDZ, viz. the Genus Turbo. (With B WOOMCUE) bers w atonal etn ceoeo lee ies eRe een 692 Wuire, F. Bucwanay, M.D., F.L.S. Descriptions of New Hemiptera Cl) ism. cecil tenet 482 Youne, A. H., M.B., and Professor Morrison Watson, M.D., Owens College, Manchester. On the Anatomy of the Elk (Alces malchis). (Plates VI. & VIL. and a woodcut.) ...... o's ie 0. SERGE gare ee ctarave een 371 vil EXPLANATION OF THE PLATES. PLATE ( STROMATOPORA.—Microscopical structure of species of, as well as of the genera Cenostroma, Caunopora, Clathrodictyon, Stylodictyon, { Stromatoceriwm, Pachystroma, doubtful Stromatoporoids, and fossil Hschara nobilis. To illustrate Prof. H. A. Nicholson and *\ Dr. J. Murie’s paper on Stromatapora and its allies. V. Hrerrpea.—Exterior and details of examples of genera and species of this peculiar group of the Anomurous Crustacea, as revised by Mr. Edw. J. Miers. VI Exx.—Tllustrations of larynx, digestive organs, and male genitalia : of Cervus malchis, as described by Prof. M. Watson and Dr. A. Wade H. Young. VIII. Korran Ecurroprrmata.— Exterior and minute structure of genera and new species illustrative of Asteroidea and Hchinoidea described by Mr. W. P. Sladen. IX. ; KorEan OpHivROIDEA.—Magnified structural peculiarities of disk X. | and arms of new species of Ophiurans, illustrating Prof. P. M. XI. Duncan’s paper thereon. OxyriynoHa.—Hnlarged views, structure of orbital and antennal XII. regions of the Maioid Crabs (Oxyrhyncha), illustrating Mr. Xill. Edw. J. Miers’s classification and synopsis of these forms of Brachyura. XIV. Morruva MACROCEPHALA.—Reduced figure of this rare Cod-fish captured at mouth of Thames, and described by Mr. F. Day. XV. HrtErororsa.—Exterior and microscopical sections of recent species of this genus of Polyzoa as described by Prof. G. Busk. ERRATA. Page 151, line 22 from top, for ‘‘ Acanthodermia” read Acanthodesmia. — 170, lines 22 and 31 from top, for “ Haliodiscus” read Heliodiscus. — 482, line 16 from top, for “pore-arms” read pore-areas. — 434, lines 8 and 9 from top, — 4835, line 18 from bottom, | for MUSINGS GBH Lani — 440, — 19 from bottom, delete “ HoninANTHUS TESTUDINARIUS, Gray.” — 448, — 14 from bottom for ‘“ Oputorricupz{ ” read OPHIOTRICHID®. — 482, — 9 from bottom, for “Limogonus” read Limnogonus. — 486, — 10 from top, “antennis gracillimis corporis squilongis ” should be antennis gracillimis ? corporis equilongis. — 688, — 9 from top, for ‘Camropnaca” read CARPOPHAGA. THE JOURNAL OF THE LINNEAN SOCIETY. NOTICE. In consequence of unavoidable delay in connexion with Plates and Index, the present Number, No. 73 (Vol. XIV. Part 1), is issued. before No. 72 (Vol. XIII. Part 8), which will be published shortly. It is requested that all communications forwarded to the Society to be read before the Scientific Meetings during the ensuing Session, be written out complete, with proper references, fit for publication ; otherwise there is possibility of their rejection. -It is to be regretted that difficulties and delay have already occurred through want of attention to the above matter. Theory of Diseases 1,02 ene eee tee er 83 I. Introductory Remarks. My object in the present communication is fourfold. First, I wish to make known in detail certain experimental conditions which I LINN. JOURN.—ZOOLOGY, VOL. XIV. i THE JOURNAL OF THE LINNEAN SOCIETY. Onthe Conditions favouring Fermentation and the Appearance of Bacilli, Micrococci, and Torule in previously Boiled Fluids. By H. Cuariron Bastian, M.D., F.B.S., F.L.8., Professor of Pathological Anatomy in University College, London, and Physician to University-College Hospital. [Read June 21, 1877:] Contents. uy Page I. Introductory Remarks... .......0.05....s.0s+s0esrsnorecesene 1 II. Heat as a Promoter of Fermentation .................. 6 III. Oxygen as a Promoter of Fermentation ............... 9 TV. Liquor Potassz as a Promoter of Fermentation ...... 12 V. Interpretation of Experiments with Urine and Liquor Potassze...... Peles casisatnaita ae aaa one otasiec ote 43 VI. Experiments with Superheated Fluids .................. 50 VII. Signs of Fermentation in the Boiled and Superheated Fluids employed in the foregoing experiments...... 53 VIII. General Interpretation ; present State of the Ques tion in regard to Archebiosis ..........sc...s0e-seee 6] IX. Bearing of the Experimental Evidence upon the Germ Theory Of Disease.......0....0..0ccosesereens-sernec secon 83 I. Introductory Remarks. My object in the present communication is fourfold. First, I wish to make known in detail certain experimental conditions which I LINN. JOURN.—ZOOLOGY, VOL. XIV. 1 2 DR. BASTIAN ON THE have found to be highly favourable to the development of fermen- tation in boiled fluids; secondly, to record fresh instances of the occurrence of fermentation in boiled acid fluids; thirdly, to bring forward certain crucial evidence bearing upon the disputed inter- pretation of the fertility of boiled neutral or faintly alkaline fluids ; and fourthly, to record fresh instances of the fermentation of guarded neutral fluids after they have been heated to 110° C. and upwards. The investigations recorded in the present communication have been made principally with boiled urine, which will, when securely guarded and kept at a temperature of 77°-86° F'. (25°-80° C.), remain free from all signs of turbidity from the appearance of organisms, as Pasteur, Lister, Roberts, Tyndall, and others have ascertained. ‘The testimony of previous investigators on this sub- ject is unanimous. M. Pasteur, speaking of sweetened yeast-water and of urine, says* :—‘ Nous avons reconnu que ces liquides, portés 4 la tem- pérature de l’ébullition 4 100° pendant deux ou trois minutes, puis exposés au contact de l’air qui a subi la température rouge n’éprouvent aucune altération.” The latter of these fluids may remain, he says, indefinitely, “sans éprouver d’autre altération qu’une oxydation lente de la matiére albumineuse,”’ and this even «4, la température de 25° 4 380°, température si favorable 4 la putréfaction de urine.” Prof. Lister} calls forcible attention to experiments with boiled urine in support of the germ theory, its continued barrenness, when protected after boiling, being the invariable result in his hands. In regard to any organisms and their germs which it might contain, he says :—“ It is necessary to maintain the elevated temperature (212° F.) for about five minutes to ensure complete destruction of their vitality.”’ Dr. William Robertst mentions healthy and diabetic urine as bemg amongst the easiest fluids to sterilize, “ three or four minutes’ boiling ” sufficing, as he says, to bring about this result and cause the liquid to remain permanently barren when kept at tempera- tures ranging between 60° and 90° F’. Prof. Tyndall§ also, in 1875, found five minutes’ boiling inyari- * Ann. de Chimie et de Phys. 1862, t. lxiv. pp. 58 et 52. + Introductory Lecture delivered in the University of Edinburgh, 1869, p. 19. { Phil. Trans. vol. clxiv. pt. 2, p. 461. § Phil. Trans. 1876, vol. clxvi, pt. 1, p. 42. CONDITIONS FAVOURING FERMENTATION. 3 ably sufficient to sterilize urine when it was subsequently exposed only to a “ moteless air.” Indeed, in regard to experiments made with “ wrine, mutton, beef, pork, hay, turnip, tea, coffee, hops, had- dock, sole, salmon, cod-fish, turbot, mullet, herring, eel, oyster, whiting, liver, kidney, hare, rabbit, fowl, pheasant, grouse,’ amount- ing in all to several hundreds, five minutes’ boiling was always found sufficient to produce complete sterilization. Tf, then, we omit from consideration those instances of “ smoul- dering fermentation’’* in which, whilst the bulk of the fluid remains clear, organisms are found mixed with sedimentary matter slowly increasing in amount at the bottom of the vessel, and confine our- selves solely to cases of well-marked fermentation characterized by the supervention of unmistakable general turbidity due to the multiplication of ferment-organisms, we shall have to deal with a comparatively simple problem. There will in such cases be no room for doubt as to whether or not the experimental fluids con- tain organisms; in the great majority of instances these will be so numerous that even a tyro with the microscope could find them. Neither will there be room for the supposition that the organisms which are found are “dead and have been there all the time.’ Dead organisms cannot by any stretch of fancy be supposed to multiply so as to make a previously clear fluid turbid. If, then, taking the fresh acid urine of a healthy person, and boiling it so as to kill any organisms and germs which it may contain, one is able, merely by subjecting this sterilized fluid to certain physical and chemical influences, to cause it to ferment in an unmistakable manner and swarm with living Bacteria, such a procedure and its sequence could scarcely be otherwise regarded than as a demonstrable proof of the truth of the physico-chemical theory, and as an equally cogent disproof of M. Pasteur’s exclu- sive “oerm theory”’ of fermentation. The same experiments would coincidently afford clear evidence as to the occurrence of so-called “spontaneous generation”’ f. * Proc. Roy. Soe. vol. xxi. (1873) pp. 333 & 337. + This term will, I hope, after a time be discarded, because under it two distinct processes have been included, which are liable to be improperly confounded with one another. One process, which I designate by the word archebiosis, includes the actual origination of living matter, its de novo forma- tion ; whilst the other, heterogenesis, signifies a particular transformation of some already existing living matter. 1* 4, DR. BASTIAN ON THE In the experiments of this kind now to be described I have had recourse to the aid of a stimulating physical influence (viz. heat), which has been much more sparingly resorted to by other inves- tigators, as well as by myself, on previous occasions ; I have also made use of certain chemical agents (viz. oxygen and liquor po- tasse) under conditions as novel as they are stringent. In several respects, indeed, the experiments about to be recorded differ much from those hitherto made for the purpose of throwing light upon the two correlated and much vexed questions, as to the conditions of origin of fermentations, and as to the present occurrence or non-occurrence of archebiosis. Whilst I have been careful to call to my aid all those conditions and influences which were admissible and might, within the nar- row boundaries of a strictly trustworthy experiment, be supposed to favour the process of fermentation, I have also neglected no precaution, however trivial, which has hitherto been insisted upon as needful for the completeness of the preliminary destructive process. I have stedfastly sought to destroy every trace of pre- existing living matter within the glass-bounded field of experi- ment, without unnecessarily deteriorating the mere organic matter. With this end in view, in a large proportion of the experiments the precaution has been taken, after boiling the fluids and hermeti- cally sealing the vessels, to immerse them in an inverted position — in acan of boiling water for 5”-15". By this means the portions of the retort- or flask- walls which, during the boiling over the flame, are only exposed to brief contact with the boiling fluid or to steam, come during the boiling in the can continuously into contact with the infusion itself heated to 212°F. An interval of three quarters of a minute must, in these cases, be allowed to elapse after the sealing of the tip of the retort or flask before it is inverted and plunged into the boiling water, in order that this over-heated tip may not crack by coming into contact with the fluid within, This accident will also be diminished in frequency by long practice, and by careful sealing in such a manner as to avoid any inbending of the glass. When a minute crack has occurred, it is always rendered obvious, during the period thas the vessel is cooling, by a line of small air-beads starting therefrom, Such a vessel must of course be rejected, or only kept for observation as an air-con- taminated specimen. A few words are desirable as to the best mode of subjecting the experimental fluids to any given generating temperature. ——————— CONDITIONS FAVOURING FERMENTATION. 5) In my earlier experiments, as well as in a few of those which were made in connexion with this research, I had recourse to the method of immersing the experimental vessels in a large beaker or pan containing water and a thermometer. The temperature of the water in such a vessel was raised to the required extent by a spirit-lamp or gas-flame. But without frequent watching and great care this method is almost sure to entail greater fluc- tuations of temperature than are at all desirable. I have there- fore now for some time had recourse to the ordinary incubator employed in physiological laboratories, supplemented by one of the ingenious and valuable gas-regulators* devised by Mr. F. J. Page, B.Sc. This combination of apparatus gives us a warm chamber which may be maintained almost indefinitely at any given temperature. The variations, extending over several weeks, may with care never exceed one degree Fahrenheit. In carrying out this research I have latterly found it convenient to employ two incubators, in one of which the experimental fluids could be ex- posed to a very high generating temperature, and in the other to moderately high temperatures. It is more than ever necessary to employ an efficient heat- regulator when the generating temperature to which the fluid is to be subjected is very high, because an accidental rise even of a few degrees might prove detrimental to the initiation of fer- mentative changes—more especially if the fluids remained exposed to this unduly elevated temperature for several hours. A caution is needed, however, as regards the mode of using the incubator in these experiments. A thermometer whose bulb is exposed to the air of the chamber does not afford a correct indication of the the temperature of an experimental fluid contained in a closed glass vessel which has been resting for several hours upon its floor. The temperature of the fluids would probably always be higher than that of the air, which the thermometer registers. A much more correct means of judging of the actual tempera- ture of any experimental fluids contained in the incubator is obtained by allowing the end of the thermometer, like the experi- mental vessels, to rest upon the floor of the incubator. It is of importance to regulate the temperature of the incubator in accordance with the reading of a thermometer thus disposed, since in the absence of such a precaution the experimen- * Described in Proceed. of Chem. Soc., Jan. 1876, vol. i. p. 24. 6 DR. BASTIAN ON THE tal fluids would mostly be exposed to temperatures higher by 7°_10° F. than had been intended—as I have ascertained by actual trials. Il. Heat as a Promoter of Fermentation. The great dependence of the processes of fermentation upon heat is one of the commonplaces of science. Jt is known, for instance, that nearly all such processes, if not all, cease at about 41° F. (5° C.), and, speaking generally, that they increase in energy with successive increments of heat till a temperature of about 86° F. (80° C.) is reached. It has hitherto been considered that temperatures between 77° and 95° F. (25° and 36° C.) were those most favourable for fermentations. The upper limits of favourable temperature, however, had not been carefully defined; and this was the case especially in regard to the occurrence of fermentation in previously boiled fluids. In previous experiments of this class no one had, so far as I am aware, designedly made use of a generating temperature above 100° F. (88° C.); the heat employed by some investigators has indeed been only too frequently below 77° F. (25° C.). Previous to the month of August 1875, I had myself never purposely employed a generating temperature above 100° F.; but early in that month I discovered that some boiled fluids which remained barren at a temperature of 77°-86° F. would rapidly become turbid and swarm with organisms if maintained at a temperature of 115°F. (46° C.). This important fact was ascertained whilst ex- periments were being made with hay-infusions and milk which had previously been subjected to destructive temperatures consi- derably higher than 212° F. Soon after I discovered that an incubating or generating tem- perature as high as 122° F. (50° C.) may be had recourse to with ad- vantage in dealing with some fluids. Organic infusions which would otherwise have remained barren and free from all signs of fermen- tation, have under its influence rapidly become corrupt and turbid. But although the high temperature proves to be so favourable for initiating chemical changes of a fermentative type in some, it must not be assumed that it would be equally provocative in respect to all organic fluids. The conditions most favourable for the initiation of such changes must be separately studied for each kind of fluid with which experiments are being made, since important specific differences may be encountered. I have already, however, ascer- CONDITIONS FAVOURING FERMENTATION. if tained that this high temperature of 122° F. (50° C.) is just as favourable for the fermentation of milk and of hay-, turnip-, and other vegetable infusions, as it is for urine. Shortly after my first announcement of this fact in June 1876*, it was made known by Professor Cohn} that Dr. Hidam*had also discovered that certain organisms would grow and multiply rapidly at this high temperature in infusions of hay, though it was one which proved fatal to Bacterium termo, Torule, and other allies. He moreover stated that the organisms found under these con- tions were invariably Bacillz. To this latter point I yhall have to return in a subsequent part of this communication. What I have now to say concerning the simple influence of 122° F. as an initiator and promoter of fermentation in boiled fluids may be comprised in a very few words. Where the initial acidity of urine, is such that it requires less, or not more than 6 minims (17 per cent.) of liquor potasset to the ounce (of 480 minims) to ensure its neutralization, I have found that such a fluid after it has been boiled 2” over the flame and 5''-10" in a can of boiling water, will almost invariably fer- ment in 15-48 hours if kept at a temperature of 122° F.$, though it will rarely or ever undergo this change at a temperature of 77--86> EF. Where the acidity corresponds to 7 minims of liquor potassze per ounce, a specimen of urine, boiled as above, sometimes ferments and sometimes does not. A urine whose acidity equals 8 minims of liquor potasse per ounce (nearly 1? per cent.) has only been known to ferment on two or three occasions out of numerous trials ; and where the acidity has been higher than this, the fluid has in- variably remained barren under the stimulus of a temperature of 122° F. acting alone—that is, without the additional aid of other promoters, such as oxygen or liquor potassze. When a urine whose initial acidity equals seven or eight minims of liquor potasse per ounce has fermented after boiling, this has been sometimes attributable to the fact that the specimen in ques- * Proceedings of Royal Society, No. 172, vol. xxv. p. 149. + Beitrage zur Biolog. der Pflanzen, Bd. ii. Hft. 2, 1876, p. 268. + A 5:84-per-cent. solution (see p. 16). § I have, however, found a diabetic urine of five minims of acidity (sp. gr. 1040) invariably remain pure after a short boiling, even when kept at a heat of 122° F. 8 DR. BASTIAN ON THE tion has deposited phosphates before it reached the boiling-point, and thus has had its acidity lowered*. The behaviour of a specimen of urine prepared in the manner above indicated has several times been tested, first under the in- fluence of a lower temperature and afterwards under that of the higher. Thus, to take an example from my note-book, two speci- cimens of a urine whose sp. gr. was 1025, and whose acidity was equivaleat to five minims per ounce of liquor potasse, were kept at a temperature of 80° F. for eight days without undergoing any change; but within twenty-four hours after they had been trans- ferred to a temperature of 122° I. they were in full fermentation. The powerfully stimulating influence of a temperature of 122° F. may also be easily seen in another way. In addition to causing certain fluids to ferment which would otherwise remain barren at ordinary temperatures (77°-86° F.), it shows its influence upon those fluids which will ferment at these lower temperatures, by bringing about such a change with much greater rapidity. No fluid serves better for showing these relative effects than urine which has been neutralized with liquor potasse before the process of boiling, because, though it will mostly ferment at the lower in- cubating temperatures, it does so with difficulty and only after many days. Thus I have found that a urine whose acidity re- quired ten to twelve minims of liquor potasse per ounce for neu- tralization, would (after such admixture and an ebullition of five minutes’ duration) not ferment under 12-15 days, if kept at a temparature of 70°-73° F., though such a change would show itself in 15-30 hours at a temperature of 122° F.+ In previous paragraphs, when speaking of the degrees of acidity of urine which would permit of its fermenting after ebullition at a temperature of 122° F., I have always referred to its initial aci- dity—its acidity, that is, previous to the process of ebullition, not * See p. 53. + Whilst such comparisons are so easily to be made by others, and will so plainly show the superior efficacy of a temperature of 122° F. in initiating fer- mentation, one can only marvel at the attempts of Prof. Tyndall and of Dr. Roberts to throw discredit upon my statements on this subject. It is to me surprising that Dr. Roberts (see Proceed. of Royal Soc, No. 176, vol. xxv. p. 456) could have resorted to so unscientific a method of testing the truth of such a simple statement. The method adopted by Prof. Tyndall was perhaps not at all more appropriate, though, as usual, he is very sparing in his narration of details (Phil. Trans. 1876, Part 1, p. 57), so that it is more difficult to be quite cer- ain what he did. CONDITIONS FAVOURING FERMENTATION. 9 after this event. Thisis a distinction of considerable importance. If we take a urine whose acidity equals 10-15 minims of liquor po- tasse per ounce, if we boil it and subsequently keep it for a long time in the incubator at 122° F.,it remains barren; and yet on opening the vessel and testing its acidity we may find that this has been reduced to five, four, or two minims per ounce; it may even be neutral*. The occurrence or not of fermentation in any given specimen of urine at 122° F. is, therefore, not a question of its less or greater acidity at some period subsequent to the process of boiling, but of its degree of acidity at the time of ebullition itself. Effects are produced by the heat plus the large amount of acid, which are not produced by the heat and a smaller amount of acid; and these effects may be merely germicidal, or they may be more purely chemical in their nature f. IIt. Oxygen as a Promoter of Fermentation. Harly in the present century Gay-Lussac assigned to oxygen an all-important role in the initiation of fermentative changes. He and his followers regarded the oxygen of the atmosphere as the “primum movens”’ in all fermentations—a doctrine which, though it is in the present day generally admitted to be too exclusive, was for a long time almost universally accepted. But even now no one questions the fact that oxygen acts in common with other agencies as a powerful inciter of fermentation and putrefaction. I freely admit this latter proposition, although I have brought forward some evidence tending to show that certain fermentative processes may be initiated just as freely (or rather more so) in closed vessels from which almost the whole of the air has been ex- pelled by boiling, as in others in which atmospheric air, and con- sequently oxygen, is present in much larger quantity f. * See further on, at page 47, footnote *. t The fact itself is shown by the different influence of potash upon an acid urine according as it is added before or after the process of boiling. A urine of twelve minims’ acidity to which six minims per ounce of liquor potassz has been added before boiling, will ferment freely under the influence of 122°F. ; but if this same urine had been boiled in its fully acid state, and the six minims per ounce of liquor potassze were added afterwards, no such result would follow ; or if it ever did ferment, it would be only rarely and after an interval of many days. This is an anticipation ofa subject to be considered further on, but which it is useful to note here. t ‘Beginnings of Life,’ Appendix C, expts. viii., ix., xiv., XV., XVIli., xx., xxVi., XXX., XXXI11., and xxxvi. 10 DR. BASTIAN ON THE The explanation of this fact is probably to be found in the sup- position that, in starting the fermentation of some fluids, diminu- tion of pressure may be of as much or even of more importance than contact with free oxygen. This appears to hold good for hay- and turnip-infusions. With some other fluids the influence of oxygen seems to be decidedly more potent as a co-initiator of fer- mentation than that diminution of pressure which is brought about by hermetically sealing the vessel before the fluid within has ceased to boil. Urine is an example of this latter class. Fig. 1. Retort with platinum electrodes. I have made only a few experiments bearing upon the effect of adding oxygen to boiled urine contained within retorts from which air has been expelled; but to these I now refer, partly because of the nature of the results obtained, though principally because T shall be able to call the attention of other investigators to a method which may hereafter prove capable of throwing much additional light upon the conditions favouring the fermentation of other boiled fluids, and perhaps upon the morphological varia- bility of ferment-organisms—since it enables us at will to modify the constitution of the fluid and the pressure to which it is sub- jected, whilst we also expose it to varying amounts of oxygen. In order to ascertain the effect of the addition of oxygen gas to boiled urine contained in a sealed vessel from which air has been expelled by boiling, I have made use of a retort (fig. 1), into the CONDITIONS FAVOURING FERMENTATION. 11 shoulder of which two platinum electrodes have been inserted. Such a retort may be charged with acid urine ; the urine may be boiled for the requisite time ; and its drawn-out neck may be her- metically sealed whilst the fluid is still boiling. After the fluid has cooled, or after an interval of some days, we can easily liberate a quantity of oxygen and hydrogen gas within the closed vessel by connecting its platinum electrodes with the poles of a suitable galvanic battery. We are thus able, by comparison, to ascertain whether the addition of these gases and the other alterations in- volved, exercise any appreciable effect in hastening the process of fermentation or in otherwise modifying its course or its products. From the few experiments which I have already made with the view of throwing light upon this point, it would seem that the addition of oxygen exercises the most marked influence when it is allowed to operate in conjunction with liquor potasse liberated from its tube almost immediately afterwards. I have as yet made only a few experiments in which the in- fluence of oxygen without liquor potasse was tested with acid urine. In these, closed retorts were employed which had been sealed after the fluids had been boiled for six minutes, oxygen was liberated by electrolysis, and the vessels were subsequently kept at 122° F. The results of the experiments were as follows :— In one of these trials with a urine whose spec. grav. was 1025, and whose acidity was capable of being neutralized by five minims of liquor potasse to the ounce, the fluid became turbid after the expiration of six hours, although, before the oxygen had been liberated, this retort had been exposed to a temperature of 80°- 90° F. (21-26°C.) for eight days without its contained fluid under- going any appreciable change. In three experiments with a specimen of urine whose specific gravity was 1026, and whose acidity was equivalent to eight minims of liquor potassz to the ounce, no general turbidity was noticed, though towards the end of the second day a very distinct amount of flocculent deposit was seen in each of the vessels*. On open- ing one of these retorts at once (after shaking its contents), Bacilli of different sizes, with progressive and rotatory movements, were found, some of which had grown into filaments, though they were not very numerous in each field. The fluid in the two other retorts underwent no very appreciable change during the next * No cloudiness of the fluid had been produced during the process of boiling. 12 DR, BASTIAN ON THE four days; and when these vessels were opened their fluids were also found to contain a sparing number of active Bacilli of dif- ferent lengths. In two other experiments, in which the urine was slightly more acid, requiring 9 minims of liquor potasse to the fluid-ounce for neutralization, a deposit formed more slowly and was smaller in amount. It was ascertained to consist principally of abortive crystalline matter; and the Bacilli, though present, were scarcer still—not more than one or two being seen in each field of a No. 9 “immersion ”’ object-glass of Nachet, with a No. 8 eyepiece. Finally, in two experiments with a urine.of extremely high acidity (represented by 20 minims of liquor potassz to the ounce) and a specific gravity of 1026, there was no appreciable naked- eye change after eight days, other than the presence of a very slight amount of sediment in each. On subsequently opening the retorts, no organisms were found in their respective fluids, and the scanty sediment was ascertained to consist of more or less abortive crystals together with amorphous mineral matter. A few other experiments in which the liberated oxygen was brought into play almost simultaneously with liquor potasse, will be subsequently referred to (p. 25). IV. Liquor Potasse as a Promoter of Fermentation. It has been well known for some time that the presence of alkalies, and especially potash, favours the occurrence of fermen- tation or putrefaction in suitable media. Gerhardt, for instance, in his ‘ Chimie Organique,’ said*, “‘ Many bodies which, alone or in the moist state, do not oxidize on exposure to air, undergo com- bustion as soon as they are subjected to the influence of an alkali. Thus pure alcohol can be kept exposed to the air indefinitely without becoming acid; but when itis mixed with a little potash, it quickly absorbs oxygen and becomes converted into vinegar and a brown resinous matter. Itis clear from this that potash ought to favour certain fermentations.” A little further ony, the same eminent chemist thus gives expression to a more familiar, though related, fact :—“ It is known that meats and vegetable substances pickled in vinegar are preserved from decomposition at least 1018 2) GSAAMIO THINS 5 595 be The majority of acids produce the same effect as vinegar.” The action of alkalies and of acids in favouring and retarding * Tome iy. 1856, p. 547. t Loc. cit. p. 556. CONDITIONS FAVOURING FERMENTATION. 13 fermentative changes has of late years become familiar to many workers, and is very generally recognized, more especially since attention was prominently called to one side of the subject by Pasteur in 1862*. He found that some neutral or slightly alka- line boiled fluids would ferment more easily than otherwise similar boiled acid fluids, though he made no observations as to the com- parative influence of acids and alkalies upon unboiled fluids. Taking into consideration a limited group of facts only, he en- deavoured to account for them in a manner which, if not adverse to, did not sufficiently appreciate, the wider point of view of Gerhardt. Yet this wider point of view and the relative influence of acids and alkalies may be demonstrated with the utmost ease, as I pointed out in 1870+. Thus, if two portions of an acid infusion are exposed side by side at a temperature of 77° F. (25° C.), fer- mentation may be made to occur earlier, and to make more rapid progress in either of them at will by the simple addition of a few drops of liquor potasse ; and, on the other hand, if a neutral infu- sion be taken and similarly divided into two portions placed side by side under the same conditions, fermentation may be retarded and rendered slower in either of them at will, by the simple addi- tion to it of a few drops of some strong acid. A neutral or faintly alkaline organic solution can in this way be demonstrated to possess a higher degree of fermentability than an otherwise similar acid organic solution. It seems there- fore obvious that the higher tendency to undergo change of these fluids might be less easily stifled than the lower tendency pos- sessed by acid infusions, and consequently that the changes ca- pable of taking place in boiled neutral and acid infusions respec- tively might be very different; the previous boiling, that is, might not prevent the higher fermentability of neutral infusions from still issuing in fermentation, though it might much more frequently extinguish the lower fermentability of acid infusions. Numerous experiments by different observers have now demon- strated the correctness of this inference. Boiled acid infusions, guarded from contamination, mostly remain pure and. barren if kept at or below 77° F. (25° C.), though some of the same infu- sions similarly treated, except that they have been rendered neu- tral by the addition of an alkali, will oftentimes become corrupt * Ann. de Chimie et de Physique, tome Ixiv. p. 58. t Nature, July 14, p. 227. 14 DR. BASTIAN ON THE and swarm with organisms even at this comparatively low tempera- ture. When subjected to a high temperature (122° F.), these previously boiled neutral infusions will still more frequently fer- ment, though this very strong stimulus will (as we have seen) also cause some otherwise barren acid infusions to ferment and swarm with organisms. In the summer of 1875 I first made experiments with urine to ascertain whether it followed the rule above alluded to—that is, whether, like other acid fluids, its fermentability would be in- creased by previously neutralizing it with liquor potasse. This preliminary inquiry was soon answered in the affirmative. Then came the more important question as to the cause or mode of production of such increased fermentability. For two reasons urine seemed to me to be a fluid specially favourable for use in attempting to throw light upon this problem:—/irst, because of the unanimity of experimenters as to the fact that, when boiled in its acid state and subsequently guarded, it invariably remained barren *; and secondly, because the marked acidity of urine would necessitate the use of liquor potasse in easily measurable quanti- ties, even when providing for the neutralization of such small por- tions of fluid as are commonly employed in these experimentst. Two alternative views are possible as to the cause of the fact in question:—(1) It may be due to the “ survival of germs ” of some of the ferment-organisms in the boiled neutral infu- sions, as Pasteur asserts; or (2) it may be due to the mere chemical influence of potash in initiating and favouring the molecular changes leading to fermentation, as originally sug- gested by Gerhardt. This important question would seem to be so capable of being settled by crucial experimentation as to make it not a little re- markable that no such attempt was ever made by M. Pasteur. Thus, the fluids may be boiled in their acid state so as to kill all their contained germs and organisms, and to these fluids boiled liquor potasse may be added in suitable quantity. The results of a number of such experiments should be sufficiently decisive to * This was my own experience also at the time. It wasnot till a later period that I began to obtain the results with high incubating temperatures, which have already been recorded (p. 7). + Mostly from 1 to 14 fluid-ounce has been made use of by me. { A few experiments of this nature were first made by Dr. William Roberts with hay-infusion (Phil. Trans. 1874, vol. clxiv. pt. 2, p. 474). CONDITIONS FAVOURING FERMENTATION. 15 enable us to fix upon the true mode in which liquor potassxe operates in determining fermentation. If the fluids to which boiled potash is added in suitable quantity still remained barren, then such experimental results would unquestionably favour the first interpretation, viz. that given by M. Pasteur and adopted by other germ-theorists. If, on the other hand, the addition of the boiled liquor potassze to the urine which has been boiled in its acid state suffices to convert this previously pure fluid into a turbid liquid teeming with ferment-organisms, then it would be conclusively shown that the increased fermentability of neutra- lized urine was ascribable to the second cause, viz. to the che- mical influence of the liquor potasse in initiating fermentative changes, whatever the precise nature of these early changes may be, whether (a) partly vital, or (6) at first purely physico-chemical. Some preliminary experiments were made with an apparatus closely similar to that employed by Dr. Roberts in the very few Fig. 2, Plugged Flask with liquor-potasse tube. 16 DR. BASTIAN ON THE experiments which he undertook with hay-infusion. Small narrow- necked flasks were taken capable of holding nearly 8 oz. of fluid ; and each of these was about half filled with a measured amount of fresh unfiltered urine, whose degree of acidity had been previously ascertained by carefully finding the exact number of minims of the liquor potasse of the ‘ British Pharmacopeia’ which were needed to neutralize 1 ounce of it*. Quantities of liquor potasse just sufficient to neutralize the amount of urine intended for each flask were then enclosed ina number of glass tubes, each of which had a small bulb at one extremity and a similar bulb near the other end, beyond which it was drawn out as a thin prolongation and bent at an obtuse angle. Hach of these tubes was charged by heating it in a flame before immersing its open capillary ex- tremity in the requisite quantity of liquor potassz, contained in a minute porcelain capsule. When the whole of the measured amount of alkali had thus been forced into the glass tube, this was in- verted, and its capillary extremity was sealed in the spirit-lamp flame. Its neck was then wrapped round with cotton-wool, and the tube itself was inserted into one of the flasks in such a manner that the cotton-wool might act as a plug thereto, whilst the capil- lary extremity of the tube just touched the bottom of the vessel. The flasks being thus charged and arranged, the urine in its altered acid state was boiled over a flame for five minutes. When the fluid had cooled, the tube was pressed down slightly so as to break off its capillary extremity ; and immediately afterwards a flame was applied to the external bulb of the tube, so as to expand its contained air. The measured amount of liquor potasse was thus expelled into the sterilized urine; and the flask was then placed in an incubator and maintained at a temperature of 104°-113° F. (40°-45° C.)+. Some tentative experiments were made in this manner with fresh urine whose specific gravity varied from 1020 to 1025, and whose acidity was such that 7-15 minims of liquor potasse per ounce were required for neutralization. In nearly every case it * In these first experiments the liquor potasse was delivered from a subcuta- neous injection-syringe, minim by minim when the point of saturation was nearly reached. It may be well to mention that the solution of potash above named has asp. gr. of 1058, and that it contains 27 grains of caustic potash to the fluid- ounce of water (5:84 per cent.). What I have used has always been purchased from Mr. W. Martindale, of 10 New Cavendish Street, London. No higher incubating-temperatures were used in these particular expe- riments. CONDITIONS FAVOURING FERMENTATION. 17 was found that the urine became lighter-coloured and turbid in two or three days. Other experiments showed that a slight excess of liquor potasse tended to retard or even prevent the occurrence of fermentation, though a quantity of liquor potasse notably below that needed for neutralization was found to be efficacious in inducing it, and that, too, almost as rapidly as if the neutrali- zation had been complete. Even when the liquor potasse was added in quantity only sufficient for half-neutralization, fermen- tation still took place in many instances, though in such cases the result was usually delayed for five or six days. In all these trials it was found that the fluid, when turbid, was not foetid ; its odour was for the most part scarcely at all altered, though at times it was rather more marked than usual. The organisms found in the fermenting urine were in all cases the same, viz. Bacilli, either short, medium size, or in the form of long threads—and not the ferment thought by Pasteur to be the invariable cause of the conversion of urea into ammonic car- bonate and water*. Sometimes only the short unjointed rods were found, though more frequently these were mixed with varying amounts of longer Vibrio-like bodies, and with threads such as I and others have generally spoken of as Leptothrix. The results of the foregoing preliminary experiments induced -me to seek other, stricter methods, free from two possible sources of fallacy which might be thought to have influenced the results. Thus, as the whole of the tube containing the liquor potasse was not immersed in the boiling fluid, it was possible that the heated vapour within the tube was not certainly sufficient to sterilize the small quantity of air also contained within it above the level of the liquor potasse. It would have been easy to meet this source of uncertainty by boiling the closed liquor-potasse tubes in a vessel of water for a time before inserting them into the expe- rimental flasks containing the urine. But the other possible source of fallacy would still have remained. It might be said by some that the cotton-wool plug, which hitherto had been deemed to be thoroughly efficacious as a protective barrier between the im- purities of the outside air and the boiled fluid, was itselfa nidus for germs, some of which, unkilled by the steam of the boiled fluid (by which, of course, the wool has been saturated), subsequently found their way into the fluid within the flask. This objection has been urged by Prof. Tyndall against some experiments made by Dr. William Roberts ; and if itis avalid objection (which I very * Ann. de Chim. et de Phys. t. lxiv. (1862) p. 50, LINN. JOURN.—ZQOLOGY, VOL. XIV. 2 18 DR. BASTIAN ON THE much doubt), there would be an end to the long-established reputa- tion of cotton-wool as a protective filter in such experiments. To get rid, however, of all doubt Fig. 3. of this kind, I determined to repeat the urine and liquor-potasse expe- bd riments with hermetically sealed a vessels from which air had been ex- | pelled by boiling, and to take the = further precaution of boiling the E liquor-potassee tubes before insert- | atl!) oy eg TTT 1 hi | = ing them into the experimental ves- sels. It was safe at once to resort to such a method, because I had previously ascertained that urine neutralized before boiling would fer- ment in such closed airless vessels almost, though not quite, as freely as in flasks plugged with cotton- wool. There was, therefore, nothing unduly restrictive in the proposed conditions. The new mode of procedure which I devised was conducted as fol- lows :— In the first place a stock of liquor- potasse tubes had to be prepared beforehand containing convenient amounts of liquor potasse. -Some were charged with 8, others with 10, and others with 12 or more minims. Those containing the same quantity were kept together in separate batches duly labelled and ready for pa use, as occasion required, according | to the degree of acidity of the urine with which experiment was to be made. In order to ensure perfect accuracy in the measurement of the liquor potasse, I have of late made use of a small burette-tube (fig. 3) Buretie:tubo (or mewn graduated to minims and fitted with liquor potasse. CONDITIONS FAVOURING FERMENTATION. 19 a stopcock, with which even half-minims can be delivered with ease™. Having prepared a number of small glass tubes closed at one end and drawn out at the other, I proceed to charge them with measured amounts of liquor potasse. As in the previous experi- ments, the liquor potasse is delivered into a little porcelain pot; and the open capillary extremity of the glass tube, previously well heated in the flame of a Bunsen’s burner, is immersed therein. When no more suitable rest is at hand the little por- celain vessel may be placed in the angle between two bottles, so that the upper end of the heated tube inclines against them, partly for support and partly that it may cool more quickly. In two or three minutes, when the whole of the liquor potassee is forced into the tube, this is to be inverted, and its shoulder, where Fig. 4. 9 | TT =A Liquor-potassx tubes with capsule and stand. it begins to narrow (fig. 4,*), is heated in a spirit-lamp flame, so that the tube may be drawn out still more in this situation. * This little instrument was made for me by Cetti and Co., of 11 Brooke Street, Holborn, London. oD 20 DR. BASTIAN ON THE Subsequently the end of tube is bent, in the manner shown in the figure (¢), and then hermetically sealed. Thus prepared, the tube should be just half full of liquor po- tasse. Its length will have been diminished as much as possible ; and its tip is so arranged that it may be easily broken off by a slight mechanical shock. These last steps in the preparation of the liquor-potasse tubes are best carried out with the aid of a very small spirit-lamp flame, as they require to be done slowly and with care. On the one hand, it is necessary that the bent part of the tube should be weak enough to break readily when jerked against the wall of the experimental vessel in which it is afterwards enclosed; and, on the other, it must not be so much weakened as to make it break too easily or be unable to bear the internal strain which it will have to undergo during its immer- sion in boiling water. This, in fact, is the final stage in the pre- paration of the liquor-potasse tubes. A number of them, after. they have been hermetically sealed, are to be placed in a suitable vessel containing warm water; and they are then to be raised to the temperature of 212° F. (100° C.) for the period fixed upon. As in these experiments I soon found that the longer or shorter duration of the period of boiling of the liquor-potasse tubes did not appreciably influence the results, they were, for the most part, boiled for 15'—-20” only—though in many cases it was much longer, and in two or three they were boiled for 2 hours. Thus prepared, the tubes were set aside in compartments labelled ac- cording to the number of minims of liquor potasse which they contained. A stock of such tubes being ready to hand, experiments may be made at anytime. A suitable specimen of fresh urine is to be taken, whose specific gravity is to be ascertained and whose degree of acidity is to be most carefully estimated. This latter process I have carried out by taking exactly 1 fluid-ounce of the urine and adding liquor potassz to it, minim by minim, from the burette- tube till the point of saturation is nearly reached. ‘Thereafter the alkali has been added in half-minims at a time, and tested between each addition with litmus and turmeric paper so as to make quite sure of the time of complete neutralization*. In order * It is not unimportant here to add that the test-papers which I have used have been those sold by Mr.W. Martindale, of 10 New Cavendish Street, London. They are similar to the papers used in the wards of University-College Hos- pital. By careful trial I have ascertained that } of a minim of liquor po- CONDITIONS FAVOURING FERMENTATION. al to facilitate this part of the process, I have made use of a lipped measure (fig. 5) having a rather narrow orifice, which can be easily covered by the thumb so as to allow its contents to be shaken for the thorough admixture of each quantity of liquor potassee with the urine to which it has been added. These experiments have hitherto been exclusively conducted with my own urine; and I have generally found that which was passed in the morning before breakfast very suitable for use. This fluid has remained clear after boiling, no phosphates being deposited during the process; its acidity has usually been neutralized by 10-14 minims of liquor potasse per ounce; and its specific gravity has varied from 1020 to 1025. When the acidity of the urine with which experiment is to be made has & thus been accurately determined, one = : can easily settle which set of the Lipped measure for admixture already prepared liquor-potassx tubes of potash. it will be most convenient to employ. I have generally made use of about 1-13 ounce of urine for each experiment, and, after tasse in an ounce of distilled water may be recognized by the previously red- dened litmus paper, whilst + of a minim in the same quantity of distilled water may be detected by the yellow turmeric paper. ‘The latter, though less delicate, gives the most certain indication, especially when a drop of the fluid to be tested is allowed to fall on dry turmeric paper. As the fluid is absorbed by this partly bibulous paper, a faint brown circle is seen for a moment or two when the fluid is very faintly alkaline, The importance of such details as this will be obvious when I say that, last July, urine which I tested in M. Pasteur’s labo- ratory and found to have an acidity equivalent to'74 minims of liquor potasse per ounce, was pronounced by M. Pasteur to he “ légérement alcalin,” according to the indications of a slightly reddened litmus paper recently used in his labo- ratory. This wasa most surprising difference; and I cannot say yet how far our very different indications hitherto as to degrees of acidity and alkalinity may account for some of the discrepancies between the results of M. Pasteur and myself in the performance of these urine and liquor-potassz experiments (see Note 1, p. 180, of the ‘Comptes Rendus’ for July 23, 1877, where M. Pasteur has also something to say on the same subject).—Sept. 1877. 22 DR. BASTIAN ON THE numerous trials, have found it best in this series not to provide liquor potasse sufficient to neutralize the quantity of fluid in the unboiled state, but to make use of liquor potasse in a closed tube to the extent of two thirds or three fourths of this amount—the former being, on the whole, the safest proportion*. An illustration will make the mode of procedure at this stage clearer. If the urine to be employed has an acidity of 12 minims of liquor potasse to the ounce, then 1 ounce of it should be placed in each experimental vessel (retort or flask of about 2 ounces Fig. 6. Retort used in experiment, as described in text. capacity); and with it a liquor-potasse tube containing 8 minims of this fluid should be also inserted, with its narrowed and bent extremity downwards. If, on the other hand, the urine had an acidity of 8 minims of liquor potassz to the ounce, and only tubes containing this amount of liquor potasse were at the time avail- able for use, we should then have to place in each experimental vessel 13 ounce of the urine and one of these 8-minim tubes. When properly charged, the neck of the retort or flask is to be * There is reason to believe that conditions other than the acidity of the fluid mney subsequently have to be taken into account, since, although 2 may seem quite favourable for one specimen, in another # of the amount of liquor potassee which would have been requisite for full neutralization before boiling appears to produce more speedy results. Such or analogous differences may haye to be ascertained also in regard to the urine of different individuals. CONDITIONS FAVOURING FERMENTATION. 23 heated and drawn out to a narrow extremity, after which the urine is gently boiled for about two minutes over a flame, great care being taken to avoid any waste of the fluid by spurting. During the continuance of ebullition the extremity of the vessel is hermeti- cally sealed. Some little practice is required to do this properly— that is, on the one hand to seal the vessel whilst there is a gentle outpouring of steam, and, on the other hand, to do it in such a way that there is no inbending of glass at the sealed extremity. Hyen a small amount of such inbending is very apt to lead to a minute crack at the next stage. After allowing an interval of ? of a minute for the sealed tip to cool a little, the vessel is inverted, and in this position is at once immersed in a can of boiling water prepared, and ready to hand, for this purpose. Here the experimental vessel is left for 8 minutes or more. Three purposes are served by this double process of heating. In the first place, it simplifies the experimental conditions to get rid of the air by boiling; secondly, the speedy closure of the vessel and the prolongation of the heating in a can of boiling water reduces the loss of fluid by boiling to a minimum; and thirdly, and principally, the inversion of the experimental vessel during the second period of heating brings those upper portions of the internal surface, as well as the outer surface, of the liquor- potasse tube (which, during the boiling over the flame, may only have come into contact with steam at 212° F.), into conti- nuous contact with the heated fluid itself *. After the urine in the boiled retort has become cool, the liquor potasse is allowed to mix therewith. This is easily brought about by shaking the retort or flask so as to jerk the bent capillary extre- mity of the liquor-potassz tube against its internal surface. The neck of the previously closed tube is thus broken off, and the liquor potasse itself, owing to the comparative vacuum within the experimental vessel, is forced out and mingles with the steri- lized acid urine. If six or ten vessels have been prepared in this way charged with the same stock of urine, some one or two of them may be selected for ‘“‘ control’’ experiments. In these the liquor-potassz tubes are not broken, whilst in all the others they are ; so that when subsequently placed in the incubator together at a temperature * The whole of the internal surface of the liquor-potasse tube is similarly exposed to the influence of its heated and caustic fluid during these different modes of heating. : 24. DR. BASTIAN ON THE ot 122° F., the two sets of retorts constitute crucial experiments capable of testing the influence of liquor potassz upon the steri- lized urine. What I have found to happen almost uniformly in about two hundred of such experiments is this :—If suitable fluids are dealt with—that is, specimens of fresh urine whose acidity before boiling does not require less than 8 minims of liquor potassz per ounce for neutralization, and which do not deposit phosphates on boil- _ing—the urine in the control experiments remains clear and appa- rently unaltered for an indefinite time; whilst where the potash. has been allowed to operate upon the sterilized fluid, it becomes turbid, lighter in colour, and swarms with organisms in from 18 to 36 hours, onan average. The period with different urines is some- times less and sometimes more, though no great prolongation occurs except through some alteration having been brought about in the proper ratio which should exist between the acidity of the boiled fluid and the amount of liquor potassze which is added thereto. Such delays in the occurrence of fermentation were common enough during my earlier trials with this method; but now that I have more carefully studied and ascertained some of their causes, Lam generally able to obviate them and ensure the super- vention of fermentation within two or three days*. This fermentation of urine to which liquor potasse is added after boiling, unquestionably takes place more readily in a flask plugged with cotton-wool than in a sealed retort from which air has been expelled by boiling. And that the slightly dimi- nished readiness of the fluid to ferment in the airless retort is attributable to the absence of atmospheric oxygen, seems to be confirmed by other experiments now to be recorded, in which an increased readiness to change is exhibited by urine and liquor potasse under the influence of nascent or less-diluted oxy- gen, liberated by electrolysis. I have also in two experiments with closed flasks containing urine and ordinary atmospheric airt liberated the liquor potassz after the flask and its contents had been boiled, with the effect of finding fermentation take place several hours earlier than it did * Tn a few cases this has occurred even when the urine has been heated in the can for 1-2 hours. Thus, I have seen it happen three times where the urine has been boiled for one hour, and once where it has been boiled two hours. With potash added beforehand almost to neutralization, I haye twice seen urine fer- ment even after three hours’ boiling. + The flasks haying been hermetically sealed whilst the urine was cold. CONDITIONS FAVOURING FERMENTATION. 25 in other companion retorts whose contents were similar, except for the fact that they contained no atmospheric air. The libera- tion of the liquor potasse from the tube with a capillary neck can only be brought about with great difficulty in a retort con- taining air; and this was the reason of my giving up the condi- tions more favourable to the fermentation of the boiled urine, in order to avail myself of the facile and automatic emptying of the liquor-potasse tube which takes place in the retort from which air has been expelled by boiling. In a trial of the combined effects of oxygen and alkali upon urine whose specific gravity was 1028, and whose acidity was neu- tralized by 18 minims of liquor potassz to the ounce, distinct tur- bidity of the fluid of the retort to which oxygen had been added made its appearance in thirteen hours; whilst in a companion vessel similarly treated, except for the absence of free oxygen and hydrogen, turbidity did not show itself till the expiration of the forty-first hour. On another occasion, when experimenting with urine whose specific gravity was 1023 and whose acidity was neutralized by 5 minims of liquor potasse to the ounce, I found that the fluid in two retorts into which liquor potassz had been liberated, and also a quantity of oxygen and hydrogen, became distinctly turbid in five and seven hours respectively ; whilst the fluids in companion vessels similarly treated, except for the absence of oxygen and hydrogen, did not show signs of turbidity before 223 hours. This result was very remarkable, since, under the combined influ- ence of liquor potassx, oxygen, and a temperature of 122° F., a specimen of boiled urine became turbid much more rapidly than a simple specimen of unboiled urine would have done, exposed to a temperature of 77°-86° F. It seems quite useless for me, in the present state of inquiry in regard to these questions, to dwell upon the fact of the number of times I have produced this or other results, or to describe them in more detail. What I seek to do now is, by careful description of my methods, to enable others, who will exercise proper care, to obtain similar results. With this object in view, I shall, in the first place, refer to certain causes of failure, and to certain causes of variation in the time of supervention of fertility in sterilized urine under the influence of liquor potassz, to which I have already drawn attention in a previous paper*, in order to show why * Now deposited in the Archives of the Royal Society. 26 DR. BASTIAN ON THE failure has as yet attended the efforts of others to reproduce these very delicate though crucial experiments. At an early stage of this investigation experiments were made by me to test the relative effects of different amounts of liquor potasse in such experiments as I have just described. I found that if only two or three drops of this fluid were added from a similar tube to a quantity of boiled urine when, in accord- ance with the directions previously given, it would have required 10, 12, or more minims, such a specimen of urine was never fertilized. This convinced me fertilization in the other cases was not due to the survival of germs either in the liquor potasse or in the small amount of air within the liquor-potasse tube, and consequently that the heat to which this had been submitted had been quite suflicient to sterilize its contents. One drop of this, as of any other fluid, if it really contained living germs, would always suffice to infect many ounces, a gallon or more of urine. The same conclusion, viz. that the contents of the liquor- potasse tube had been completely sterilized, was also forced upon me by the fact, which I frequently verified, that an excess of liquor potasse to the extent of 1 or 2 minims beyond the proper proportion would also invariably cause the urine to re- main sterile in these experiments with airless vessels—a result which would have been impossible if living germs had really been contained within the liquor-potasse tubes. A slight excess of liquor potassz was, in fact, soon found to be rather more inhibi- tory in dealing with airless flasks than it had been when experi- menting with flasks whose necks were plugged with cotton-wool— as in those of my tentative first series, in which the fluids were, of course, exposed to the influence of atmospheric oxygen. It was in consequence of this, and because on trial I have found that a slight deficiency of liquor potassee was comparatively harmless, that in the paper sent to the Royal Society last year, of which an abstract only has been published, I counselled the addition in the plugged-flask experiments of ~ of the quantity of liquor potassex which would have been needed for neutralization before boiling, and in those with closed airless vessels its addition to the extent of € of such quantity. In relation to the variable period of fertility, to which I also called attention in the paper above mentioned, as occurring in my earlier experiments, it may be well to quote the following re- marks :—‘ This variability might, I think, be in a great measure : CONDITIONS FAVOURING FERMENTATION. 27 obviated in the future, first, by a more strict attention than I at first bestowed upon obtaining or providing for the precise degree of neutralization desired—secondly, by attention to a fact of the full significance of which I way not at first aware, viz. that the acidity of the urine in certain cases becomes notably diminished by boiling. This diminution in acidity seems invariably to occur where acid phosphates are precipitated by boiling. The result is, that, wherever this occurs, the liquor potassz is liable to be added to the boiled urine in a harmful excess, since the quantity sup- posed to have been required, and enclosed within the liquor- potasse tube, was calculated upon the degree of acidity of the fluid before it had been lessened by boiling. In cases of pro- longed heating this diminution of acidity may have been very ap- preciable. In a specimen of phosphatic urine whose acidity was equal to 53 minims of liquor potasse to the fluid-ounce, I found the acidity only equivalent to 4 minims to the ounce after the urine had been boiled for only 2 minutes. In all cases, therefore, in which, after a preliminarytrial, phosphates are found to be deposited by boiling the urine, it would be proper to estimate the acidity with a specimen of the fluid which had already been boiled for the period determined upon as that to be employed in the expe- riments about to be made. This is a precaution which should in future never be omitted’”’*.... “It must not be forgotten also that in boiling the measured quantity of urine in the retort before its neck is sealed, any excessive spurting away of fluid may cause an alteration in the proper ratio between urine and liquor potasse.”’ Another cause of variation in the time of fermentation in spe- cimens of the same set is the different rate of boiling to which the fluids have been subjected during the first period—that is, whilst the fluid is being boiled over the flame. Rapid boiling in a retort or flask with a capillary extremity will very frequently cause the temperature to rise as much as 33° C. (nearly 7° F.) above the boiling-point, as I first ascertained in 18737. Acci- dental variations in the rate of boiling, causing the temperature to be raised to different points, may thus affect successive fluids of the same stock differently. This, indeed, is another reason why it is desirable to curtail the period of boiling over the flame and * T would rather say now that it is better not to use such a fluid at all in these particular experiments. t See ‘ Nature,’ vol. ii. (1870) p. 227. 28 DR. BASTIAN ON THE finish the heating process after the experimental vessel has been hermetically sealed. Since the first announcement of my new experiments, and before the publication in detail of my method, two English inyes- tigators have attempted to repeat them. Their method of pro- cedure, however, has been inaccurate and probably vitiated by the three causes of error above mentioned as having been pointed out in my original paper now in the possession of the Royal Society ; it was certainly, according to their own statements, vitiated by two of them, as I will now proceed to show. After referring to what appeared to him to be two sources of fallacy in my mode of conducting these experiments (to the men- tion and consideration of which I shall presently return), Dr. William Roberts * says he determined to repeat them, but taking care to avoid these alleged “defects.”” He proceeded as fol- lows :—“A flask with a longish neck was charged with an ounce of normal acid urme. The due quantity of liquor potassx requi- site to exactly neutralize this (as ascertained by previous trials) was enclosed in a sealed glass tube drawn to a vapillary portion at one end. The tube was then heated in oil up to 280° F., and maintained at that temperature for fifteen minutes. The tube was then introduced into the body of the flask. The neck of the flask was next drawn to a narrow orifice ; then the urine was boiled Sor five minutes, and the orifice sealed in ebullition.” Now, having regard to the two passages which I have italicized, it will be seen that Dr. Roberts’s supposed repetition of my experiment was no repetition at all; that is, he followed the very method most cal- culated to ensure failure, and which even what was said in my published “Abstract ”’ should have warned him against adopting f. As I shall presently explain, potash sufficient to render the fluids distinctly alkaline was added ; and this, together with the boiling for five minutes before the vessel was sealed, was doubtless the cause of Dr. Roberts’s failure. The same Number of the ‘ Proceedings of the Royal Society ’ contains a “ Note” by Professor Tyndall, in which he likewise announces the fact that he has failed to reproduce my results. His mode of repeating the experiment was also erroneous, at all events, in those of his trials in which he makes any mention of the * «Proceedings of the Royal Society,’ No. 176, vol. xxv. p. 455. t See ‘ Proceedings of the Royal Society,’ vol. xxv. p. 503. CONDITIONS FAVOURING FERMENTATION. 29 method employed. All that he says on this essential point is as follows :—“ In some of the experiments the procedure described by Dr. Roberts was accurately pursued, save in one particular. . His potash-tubes, however, were exposed to a temperature of 280° Fahr., while mine were subjected to a temperature of 220° only.” These experiments of Professor Tyndall, therefore, like those of Dr. Roberts, will only tend to confirm my statement that the addition of potash in excess leads to negative results. They have no other bearmg upon my experiments, and they conse- quently afford no evidence whatever as to the eflicacy of the two precautions, the necessity for which they were destined to illus- strate, and to whose discussion I shall presently return. One other experimenter has also questioned my results, though I cannot say that he has repeated my experiments. This is none other than the illustrious French chemist himself, M. Pasteur. In reply to a brief note of mine on the subject of these experi- ments, which was sent to the French Academy, and in which my exact method of procedure was not described, M. Pasteur accepted the fact as true, but denied the interpretation. He, however, instead of adding to the sterilized urine a quantity of liquor potasse almost sufficient to neutralize it, added, as he says, solid potash which had been heated to redness, or a solution of pot- ash heated to 230° F. (110° C.), and this in quantity sufficient to render the urine “ alkaline.” The result was (as I should have ex- pected), that the urine so treated remained barren. This barrenness I attribute to the fact that the potash had been added in excess ; M. Pasteur, on the other hand, attributed it to the higher tempera- ture to which the potash had been heated, and proclaimed his modi- fied experiment as a triumphant vindication of the truth of his previous theory. And yet it was not even this. His previous po- sition was, that in neutral or slightly alkaline organic liquids cer- tain germs were not killed at a temperature of 100° C. which were killed at 110°C. Here, however, was liquor potasse, a very strongly alkaline fluid, so caustic as to be capable of dissolving protoplasm even when cold; and M. Pasteur would have us believe that germs can, when immersed in it, resist a temperature of 100° C.—because he thought they did so in the very much weaker fluid. Much evidence would be needed to bring conviction to the minds of physiologists ‘on this point; and as yet none has been offered. At present, therefore, my experiments have not been repeated 30 DR. BASTIAN ON THE by either of these investigators. The trials they have made most closely resemble those experiments of mine which illus- trate the effects of adding liquor potasse-in excess; and the results they have obtained tend to confirm mine, and illustrate the restrictive influence of even a slight excess of this agent. I now turn to the subject of Dr. Roberts’s criticism of my method, because in pointing out the untenability of his positions J shall be able to throw some light upon the subject generally. Two objections have been raised by him to the mode in which I conducted my experiments, seemingly on the ground that in the five experiments of this order which he had previously made, two procedures were adopted which I did not imitate. Dr. Roberts thinks that I ought (1) to have superheated the liquor-potassz tubes ; and he thinks (2) that I ought to have allowed an interval of some days to elapse before breaking them and permitting the potash to mix with the boiled urine. Both these objections are indorsed by Professor Tyndall ; and, as we have seen, an objection - very similar to the first of them had previously been urged by M. Pasteur; they require, therefore, to be critically examined*. (1) Is tt necessary, or does any difference result from superheat- heating the potash-tubes?—To the first part of this question I had, previously to the date of my first communication on this subject, given an answer in the negative, and that for the follow- ing reasons :— (a) Quite early in this investigation I made comparative exper1- ments to test whether any or what influence over the result would be produced by prolonging the period for which the closed liquor- potasse tubes were heated; so that Dr. Roberts is in error in supposing that they had been raised only for “an inconsiderable period to the heat of boiling water.” In the majority of the ex- periments they were heated to this extent for over twenty minutes ; and in several of them they were boiled for one and two hours. It was only when I found that this prolongation of the boiling of the liquor-potassz tubes did not in the least affect the result that I contented myself with the shorter period of twenty minutes. * M. Pasteur urged the superheating of the potash-tubes on the ground, as above pointed out, that a temperature of 110° C. was needed to ensure the death of germs even in a strong solution of caustic potash. Dr. Roberts does not ven- ture so far as this. He considers the high temperature needful in order to ensure sterilization of the air within the liquor-potassz tubes. CONDITIONS FAVOURING FERMENTATION. ol (6) But I had, and I made known even in the abstract of my paper, a convincing proof that this comparatively short period of exposure to the temperatnre of boiling water was in fact sufficient to sterilize the contents of the liquor-potasse tubes—as well the liquor potasse as the small quantity of air which they con- tained*. This was to be found in the fact that one of these tubes charged with a very small amount of potash, or one charged with a slight excess, never, when broken, caused the urine to ferment, even though it had only been heated to 100° C. for twenty minutes. This seemed to me, and still seems, a convincing proof that no living germs existed within the tubes. But when such prominence was given to this part of the ques- tion by M. Pasteur—when, accepting the crucial nature of the experiment, he challenged me to produce the results which I had announced with sterile urine and a solution of potash, “ a la seule condition que cette solution sera portée préalablement 4 100 degrés pendant vingt minutes, ou 4 130 degrés pendant cing minutes,” I was very glad to meet his views and perform some experiments under these conditions prescribed by M. Pasteur f. The results even with liquor-potasse tubes heated to 110° C. for twenty howrs were in no way different from those previously procured with tubes heated only for a short time to 100° C.t After this I also repeated my experiments with liquor-potasse tubes which had been heated in oil to 140°-150° C. (284°-802° F.) for one hour §. The results were similar. In fourteen experiments with a urine of 1020 specific gravity, whose acidity was equivalent to 10 minims of liquor potassee per ounce, and which did not deposit phosphates when boiling or, except to the slightest extent, after the addition of the potash, fermentation took place in all. * See ‘Compt. Rend.’ Jan. 22, 1877. t The reality of my results M. Pasteur admits. Thus, in the ‘Comptes Rendus,’ July 17, 1876, tome lxxxiii. p. 178, he says, ‘Je m’empresse de déclarer que les expériences de M. le Dr. Bastian sont, en effet, trés-exactes ; elles donnent le plus souvent les résultats qu’il indique.” The only question between M. Pas- teur and myself is as to the interpretation of results now common to both. His interpretation (Comp. Rend. 29 Jan. 1877, tom. Ixxxiv, p. 206) is that germs are added with the liquor potassze. + See ‘Comptes Rendus,’ Feb. 12 (1877), tome lxxxiv. p. 306. § A most troublesome process and method, because of the subsequent difficulty in cleaning the potash-tubes. 32 DR. BASTIAN ON THE This first criticism is, therefore, quite invalid; it was directed towards a requirement which I had borne fully in mind from the commencement of these experiments, and had fully met. (2) Is the method of the “ control” experiment legitimate in such investigations ? or must we in each case allow an interval of some days to elapse before breaking the liquor-potasse tubes ?—Dr. Roberts says *:—‘‘It is not sufficient to rely in such a case on a control-flask or retort. Hach flask or retort should have its own individual sterility tested, because it is practically impossible to apply the heat exactly in the same degree in any two cases.” To this I demur, and maintain that where the results are checked and verified by a large number of trials, the method of the control experiment is a more accurate and scientific method than that to which he and, following him, Professor Tyndall had resort. This position is based upon the following considerations. First, it may be premised that the method of the “ control” ex- periment has hitherto been habitually practised by many eminent investigators, and that it has always been thoroughly relied upon in cumulating evidence which was believed to be in favour of the “germ theory.” Secondly, Dr. Roberts’s objection to its use in these experi- ments carries all the less weight with it because, as I have many times ascertained both before and since the publication of my first results, an exposure of two or three minutes’ duration to the temperature of 212° Fahr. always suffices to sterilize a urine of average acidity, whatever the incubating temperature to which it may subsequently be exposed. Moreover, previous to the an- nouncement of my results the undeviating verdict of other experimenters with urine—the verdict of Pasteur, of Sanderson, of Lister, of Tyndall, and even of Roberts himself—had been that after boiling it for two or three minutes, urine kept in a warm place, and free from extraneous contamination, always remained puret. Such an amount of heat had invariably been found suffi- cient to sterilize it. Why, therefore, when dealing with such a fluid, could it be necessary to wait in each case before the liquor- potasse tubes were broken P ~ In further support of the efficacy and trustworthy nature of the method adopted by me, I may state that I have several times repeated it in this manner :—Selecting any one at random from a * «Proceed. of Royal Society,’ No. 176, vol. xxv. p. 455, note. t See quotations to this effect ‘at p. 2. Qg9 CONDITIONS FAVOURING FERMENTATION. oo batch of 6-10 retorts or flasks whose contents had been boiled over the flame for 2” and which were sealed during ebullition, I have correctly predetermined that this particular fluid should re- main sterile by simply omitting to break its associated liquor- potasse tube, and that the others should ferment even after boiling them for an additional period four times as long—this result being induced simply by breaking their properly charged and superheated liquor-potasse tubes. It seems, therefore, super- fluous to urge that when such experiments are multiplied with essentially similar results, as they have been, they afford evidence of the most crucial and unmistakable character. Dr. Roberts, however, impeaches this well-tried experimental “method of difference ;”’ and Professor Tyndall countenances the impeachment. Let us see what kind of method they would put in its place. Dr. Roberts thinks it absolutely necessary to wait several days, or even a fortnight, in order to make perfectly sure that each in- dividual fluid is sterilized before adding the superheated potash thereto. Having shown that the grounds on which he rejects the evidence of the control experiment are peculiarly weak in -regard to a fluid so easily sterilized as urine, I will now endeavour to show that the alternative procedure which he recommends to replace this legitimate method is, on the contrary, open to the most serious objections. Dr. Roberts, like Professor Tyndall, is never unmindful of “the plain indications of the germ theory ;” and though he does not persistently ignore, still he is not always equally mindful of the plain indications of the opposite theory. From the point of view of the germ theory, it is true, there is not so much to be said against this delay in the liberation of the liquor potasse ; but from the point of view of the opposite theory, the serious question arises as to whether any or what chemical changes would take place in the experimental fluids during this period of probation. Other questions, as will be seen, have also to be considered in regard to their method. It is almost difficult to believe that Dr. Roberts could have been speaking seriously when he said* that from the point of view of the spontaneous-generation theory “there was no reason why the alkali should not have been equally effective in promoting germination, whether added before or after the short preliminary * Phil. Trans. vol. clxiy. (1874) pt. 2, p. 474 LINN. JOURN.—ZOOLOGY, VOL. XIY. a By) DR. BASTIAN ON THE boiling,’—when under the word “after” was included a period of fourteen days’ exposure “in a warm place.” The validity or degree of cogency of this statement may, perhaps, be best appreciated by translating it into a symbolic formula :— Let A represent ... The experimental fluid (urine). Kee a ... The total chemical changes induced by boiling it in its acid state. aces - ... The total chemical changes induced by boiling it after neutralization by potash. BS ay 5 ... The chemical changes which the boiled acid fluid may have undergone during the period of probation. Pes a3 ... The influence of potash upon this boiled acid fluid after the probation-period. The statement of Dr. Roberts is, therefore, tantamount to this :— Because A-+