UNIVERSITY OF CALIFORNIA 
 AT LOS ANGELES
 
 THE 
 
 MICRO-ORGANISM 
 
 OF 
 
 FAULTY RUM 
 
 BY 
 
 V. H. VELEY, M.A., F.R.S. 
 
 AND 
 
 LILIAN J. VELEY (NEE GOULD) 
 
 Melius autem est naturam secare, quam abstrahere.' BACON. 
 
 JSonfcon 
 HENRY FROWDE 
 
 OXFORD UNIVERSITY PRESS WAREHOUSE 
 AMEN CORNER, E.G. 
 
 1898 
 
 [ All rights reserved ]
 
 HORACE HART, PRINTER TO THE UNIVERSITY 
 
 M5WC, DIPT.
 
 TT 
 601 
 
 DEDICATED 
 
 BY PERMISSION 
 TO 
 
 EMIL. CHR. HANSEN, PH.D., 
 
 PROFESSOR AND DIRECTOR OF THE CARLSBERG LABORATORY, 
 
 COPENHAGEN, 
 AS A TOKEN OF GRATITUDE F$lf*', 
 
 HIS ADVICE, 
 HIS SCIENTIFIC INVESTIGATIONS, 
 
 AND 
 HIS PRACTICAL STUDIES IN FERMENTATION. 
 
 220639
 
 PREFACE 
 
 IN this monograph it is intended to give an account of 
 a micro -organism which, if the views herein brought forward 
 are correct, has caused damage to the extent of many thousands 
 of pounds, but yet has hitherto escaped observation, mainly 
 because it was supposed upon a priori grounds that no 
 organism whatever could live in liquids of such alcoholic 
 strength. No person previously looked for any organism, or 
 even suspected that masses observed under the microscope 
 could be anything but lifeless matter ; it was therefore left 
 for the authors to make the discovery of life in strong spirit. 
 
 As certain doubts were expressed upon the matter, on the 
 one hand by those with experience who had propounded rival 
 theories, and on the other by persons either without practical 
 qualifications to judge of bacteriological questions in con- 
 nexion with fermentation industries or personal inquiry into 
 our results, we consulted Professor Emil. Chr. Hansen, of the 
 Carlsberg Laboratory, Copenhagen, before venturing to lay 
 this monograph before the public. His examination of our 
 materials, cultures, and preparations confirmed our results, 
 though not our conclusions in their entirety as to the identity 
 or position of the organism or organisms, discovered by us, 
 in any scheme of classification. For Professor Hansen's kind 
 assistance and courtesy, as also for much time spent by him
 
 v i PREFACE 
 
 in helping us to elucidate the matter, we render him our most 
 
 cordial thanks. 
 
 Further our thanks are due to Dr. J. Ritchie, Lecturer of 
 Pathology in this University, for advice and assistance ; to 
 Mr. G. J. Burch for conducting a number of optical tests, some 
 of which he had previously invented, while others were 
 specially devised for this investigation ; to Mr. J. J. Manley, 
 for certain optical measurements ; to Dr. Gustav Mann, for 
 skilful micro-photographs ; and finally to certain firms and 
 individuals, for providing us with a variety of material with- 
 out which an investigation of this character would have been 
 impossible. 
 
 In conclusion we cannot but trust that we may have 
 opened out a new field of research in connexion with the 
 micro-organisms of spirit distilleries, which have not. as those 
 of breweries and wine industries, received much attention. 
 In consequence of the large capital and the many interests 
 involved, we have spared neither time, nor labour, nor expense 
 to elucidate the problem of the faultiness of rum, and to make 
 known its solution. 
 
 We have purposely entered somewhat fully into a descrip- 
 tion of all the experiments performed by us, whether resulting 
 in success or failure, in order that our work may be repeated, 
 and perhaps advanced in other laboratories, as we are well 
 aware that the life-history of the organism herein described 
 may continue for some time to be a matter of reasonable 
 controversy. 
 
 V. H. VELEY, 
 L. J. VELEY. 
 
 OXFORD, Nov. i, 1898.
 
 TABLE OF CONTENTS 
 
 PAGE 
 
 PREFACE .... v 
 
 CHAP. I. INTRODUCTORY i 
 
 II. GENERAL EXAMINATION OF SAMPLES .... 6 
 
 III. THE 'RESIN' THEORY 16 
 
 IV. THE FLUORESCENCE OF COLOURED RUM AND ITS 
 
 REFRACTIVE INDEX } 22 
 
 V. METHODS OF CULTURE AND EXPERIMENT ... 27 
 VI. LIFE-HISTORY OF THE MICRO-ORGANISM AS. DEDUCED 
 
 FROM CULTURES 34 
 
 VII. BIOLOGICAL AND CHEMICAL CHARACTERISTICS . 46 
 
 VIII. DISCUSSION OF CONCLUSIONS 53 
 
 IX. SUMMARY OF RESULTS 59 
 
 BIBLIOGRAPHY 61 
 
 EXPLANATION OF PLATES 63 
 
 PLATES.
 
 CHAPTER I 
 
 INTRODUCTORY 
 
 IN the past few years the importations of rum from the 
 colony of British Guiana, and to a less extent from certain of 
 the West Indian islands, have not unfrequently exhibited the 
 condition technically known as ' faultiness,' whereby a consider- 
 able reduction of value and consequently a pecuniary loss, 
 amounting to several thousands of pounds per annum, has 
 resulted, so that the manufacture of this spirit has hardly 
 been remunerative. Under these circumstances the Agricul- 
 tural Committee of the Royal and Commercial Society 
 requested Mr. J. B. Harrison, the Government Analyst of 
 British Guiana, to inquire into this matter, who issued 
 a report dated Georgetown, February 16, 1897, from which 
 the following extract is made by way of a preliminary 
 explanation : 
 
 ' Rums have been and are classed as " faulty " when on 
 admixture with water in such proportions as to reduce the 
 strength to 25% under proof, which in the case of our rums 
 means approximately their dilution with an equal bulk of 
 water, the diluted spirits, either at once or after standing 
 for some hours, become cloudy, either depositing on longer 
 standing a more or less copious precipitate or showing the 
 presence, in greater or less abundance, of floating flocculencies. 
 This so-called test appears to be made at times with cold 
 water, at times with boiling water, but in both cases the 
 rum is left to stand for several hours before a final decision 
 as to its quality is arrived at. 
 
 ' Very many samples of so-called " faulty " rum have been, 
 from time to time, sent back to this colony, and have here
 
 2 THE MICRO-ORGANISM OF FAULTY RUM 
 
 been retested with ordinary rain-water or with distilled 
 water. As a rule almost all of these samples have remained 
 perfectly clear, and the reason for their having been classed 
 as " faulty " has appeared to be a mystery. 
 
 4 On a few occasions rums have been found to become 
 cloudy upon dilution with pure water, and the causes of 
 this have been proved in some cases to be the presence of 
 a caramel in the colouring matter, easily soluble in alcohol 
 of a strength of 40% over proof, but insoluble or with 
 difficulty soluble in alcohol of 25% under proof, whilst in 
 other cases the cloudiness has been traced to empyreumatic 
 products, derived from gas liquor, present in the sulphate 
 of ammonia which had been added to the wash to accelerate 
 the fermentation. 
 
 ' Both these causes are easily overcome.' 
 
 Without at present entering fully into the various points 
 raised in the report, it will suffice to state that Mr. Harrison 
 ascribes two further causes for faultiness, namely, firstly, the 
 precipitation of substances contained in the rum by the lime 
 and magnesia salts present in the Liverpool well-water (from 
 which port the complaints have almost all been derived), 
 as also the precipitation of part of the calcium sulphate 
 and carbonate present in the well-water by the alcohol ; and 
 secondly, the extraction of resinous and other matters from 
 the staves of the casks which produce ' flocculencies ' and 
 precipitates when diluted with water. The second point 
 will be fully discussed in a subsequent chapter ; as regards 
 the first point, Mr. Harrison does not make clear the nature 
 of the substances contained in the rum which are precipitated 
 by the saline constituents of the Liverpool water. As the 
 conclusion of the whole matter Mr. Harrison writes as follows : 
 'At present I am unable to discern any additional line of 
 laboratory investigation likely to be of service in this matter, 
 and must now leave it in the hands of the planters,' and 
 ' If these precautions ' (namely, those proposed) ' do not 
 prove successful on the large scale, it will be necessary to 
 carry on this investigation on other lines.' 
 
 It would appear from information received that other 
 advice was subsequently taken, but as the report was of 
 a private nature, we do not feel ourselves at liberty to make
 
 INTRODUCTORY 3 
 
 any quotations therefrom. Within two months after the 
 publication of Mr. Harrison's report our attention was called 
 to the matter as a problem still unsolved. From the 
 information which we had received in the course of private 
 correspondence, we came to the conclusion that the difficulty 
 was possibly not of a chemical nature. 
 
 Notwithstanding the apparent improbability that any 
 micro-organism could survive in strong spirit, hitherto con- 
 sidered to be one of the best materials for preserving anatomical 
 specimens, the phenomena described to us as attending 
 the production of the turbidity led us to infer the possible 
 presence of some bacteria, and consequently our examination 
 of faulty samples sent to us was directed to this point. Our 
 investigations have shown that neither the cloudiness nor the 
 ' flocculent precipitates ' are due to any particles of a chemical 
 nature, whether suspended or deposited, but to the presence of 
 a micro-organism which we believe to be hitherto undescribed. 
 
 We were informed that the first samples of coloured rum 
 received by us direct from a bonded warehouse had been 
 assessed by the Excise as at 42% over proof, equivalent to 
 74-63% per weight of alcohol. Our determinations, using 
 standardized instruments, gave a value of 42-23^ over proof, 
 or 74'88/ o per weight of alcohol. A sample of white rum 
 was found to have a value of 38% over proof, equivalent to 
 72-09% per weight of alcohol. In all cases we observed a 
 small quantity of sediment at the bottom of the bottles. 
 Mr. Harrison in his report alludes to such sediments in the 
 following terms : 
 
 * The diluted " faulty " rums were next examined, and the 
 sediments separated. 
 
 ' Microscopically these sediments were found to consist 
 principally of amorphous matters mixed with occasional 
 depolarizing crystals, probably of sulphate and of carbonate 
 of calcium. 
 
 ' By analytical examination the major portion of the pre- 
 cipitate was found to be organic, whilst the inorganic 
 matters consisted of, after ignition, calcium sulphate and the 
 carbonates of calcium and magnesium. The bulks of these 
 sediments were far greater than if they had consisted only 
 B 2
 
 4 THE MICRO-ORGANISM OF FAULTY RUM 
 
 of the calcium and some of the magnesium salts present 
 in the water used for dilution.' 
 
 Mr. Harrison in another passage expresses himself as 
 follows : 
 
 'It was found that the amorphous organic matters pre- 
 cipitated by the addition of the well-water to the " faulty " 
 rums were largely soluble in solutions of caustic soda or 
 potash, or of the hydrate or carbonate of ammonia.' 
 
 Our microscopical examination of the sediments of faulty 
 rum led us to a result wholly unforeseen by Mr. Harrison ; 
 the deposit was certainly organic, but only ' amorphous ' in 
 the chemical sense of the word. For with a magnification 
 of 1,200 diameters this sediment was found to consist of 
 a micro-organism of coccus form occurring more generally 
 in pairs and groups, but also singly and in chains ; the cocci 
 were yellowish in colour, surrounded by a very thick, 
 highly refractive colourless envelope, and appeared to be non- 
 motile [PI. I. Fig. i, a, b]. 
 
 It is not altogether surprising under these circumstances 
 that Mr. Harrison could not obtain much residue 'after 
 ignition,' and it would appear to be a somewhat strong 
 measure to digest an organism in ' solutions of caustic soda 
 or potash, or of the hydrate or carbonate of ammonia.' 
 
 It was obvious that we had before us a bacteriological 
 problem of considerable interest and importance, and it 
 appeared most desirable to ascertain, firstly, if the organism 
 was living in the strong spirit ; secondly, if it was really 
 the cause of the faultiness ; and thirdly, its origin. As regards 
 the first point continuous microscopical examination showed 
 that the organism, in samples of recent importation, was 
 dividing actively, and cultures, hereafter to be described, were 
 made from it with success.. This fact alone of the existence 
 of life in alcohol of such concentration is, we believe, quite 
 new to science ; the only record in literature appears to 
 be the work of Bokorny [i], who showed that bacteria 
 survived in alcohol of not more than i/ o . 
 
 The second point will be fully discussed in subsequent
 
 INTRODUCTORY 5 
 
 chapters, and the evidence therein brought forward will, 
 it is hoped, be of a convincing nature. 
 
 As to the third point, we can only adduce such information 
 as we have been able to obtain from the planters, trade- 
 circles, and private sources without a personal visit to the 
 infected colonies. 
 
 Considerable difficulty was experienced in obtaining exact 
 data and various statistics from the planters, which would 
 have thrown much more light upon the subject. 
 
 It appears that the faultiness of rum was first noticed 
 about sixteen years ago, and it has been manifest inter- 
 mittently, but with increasing frequency, since that date. 
 The following abstract of returns obtained from a private 
 source will serve to illustrate these points: 
 
 Estate B. 
 Percentage of 
 Year. faulty rum. faulty rum. 
 
 1892 38-5 Nil. 
 
 1893 8-0 3-4 
 
 1894 Nil. 9-4 
 
 1895 Nil. 10-4 
 
 1896 50 90 (about). 
 
 The proprietors of one of the estates, who received from 
 us a report upon the subject, have advised us that their 
 crops since made have been practically sound, while those 
 of other manufacturers have been even more faulty than 
 before. Further comment appears superfluous. 
 
 Preliminary accounts of our investigation have been pub- 
 lished in Nature, vol. Ivi. p. 197 (1897); Sugar Cane, 1897, 
 p. 350, and 1898, p. 381 ; Natural Science, vol. xi. p. 87, 
 (1897); The Argosy, Demerara, July 3, 1897 ; and noticed in 
 Die Umschau, Jahrgang i, p. 542 (1897). Cultures and micro- 
 scopical preparations of the organism were exhibited at a 
 Conversazione held at the Oxford University Museum, May, 
 1898, by the Oxford University Junior Scientific Club.
 
 CHAPTER II 
 
 GENERAL EXAMINATION OF SAMPLES 
 
 I. Bacteriological Examination. 
 
 WITH a view to elucidating the problem we obtained through 
 various manufacturers and brokers a number of samples of 
 crops from British Guiana and various West Indian islands, 
 comprising importations of the years 1887 to 1897, of different 
 brands, consignments to different ports, from the same ship in 
 different years or different ships in the same year, diluted and 
 undiluted, coloured and uncoloured, all certified as sound or 
 faulty respectively by the brokers. Samples were also 
 obtained independently through shops, without giving any 
 information as to the purpose for which they were required. 
 The results of the examination are given below in the form of 
 short notes, which are collected together in a table. 
 
 Sample I, 1887. Coloured, undiluted, certified as sound, 
 contained debris of sugarcane but no micro-organism. 
 
 Sample II, 1891. Coloured, undiluted, certified as sound. 
 Good clean sample. 
 
 Sample III, 1893. Same as No. II. 
 
 Sample IV, 1894. Coloured, undiluted, certified as sound. 
 Same as No. I. 
 
 Sample V, 1897. Coloured, undiluted, certified as faulty. 
 Large colonies of micro-organism found, not only in the sedi- 
 ment, but throughout the liquid. 
 
 Sample VI, 1897. Coloured, undiluted, certified as faulty. 
 Drawn from bottom of puncheon. Numerous colonies of large 
 cocci in thriving condition in drops taken from the bottom,
 
 GENERAL EXAMINATION OF SAMPLES 7 
 
 identical cocci of smaller size in drops taken from the top. 
 More colonies in one field of the microscope than in any other 
 sample examined. 
 
 Sample VII, 1897. Coloured, undiluted, certified as faulty. 
 A drop taken from the bottom showed numbers of cocci, both 
 singly and in chains. 
 
 Sample VIII, 1897. Coloured, undiluted, certified as faulty. 
 Large cocci from bottom drops, small cocci from top drops. 
 
 Sample IX, 1897. Coloured, diluted ^ spirit,! water, certified 
 as faulty. Millions of cocci in all stages of development, also 
 debris of sugarcane with colonies clustered upon them. 
 
 Sample X, 1897. Coloured, diluted spirit, f water, certified 
 as faulty. Similar as IX, but a few motile bacilli found. 
 
 Sample XI, 1897. Coloured, diluted spirit, f water, cer- 
 tified as faulty. Large masses of cocci clustered upon debris 
 of sugarcane. 
 
 Sample XII, 1897. Coloured, undiluted, certified as sound. 
 Small colony found in one of many drops. 
 
 Sample XIII, 1897. Coloured, undiluted, certified as faulty. 
 Drawn from bottom of puncheon. Chiefly small cocci in drops 
 taken from the top and bottom. 
 
 Sample XIV, 1892. Coloured, undiluted, certified as faulty. 
 Plenty of large groups of cocci. 
 
 Sample XV, 1897. Coloured, undiluted, certified as sound. 
 Contained no trace of the organism. 
 
 Sample XVI, 1897. Coloured, undiluted, certified as faulty. 
 Drops taken from the bottom contained enormous quantities 
 of cocci collected in masses. 
 
 Sample XVII, 1897. Coloured, undiluted, certified as faulty. 
 Large cocci in bottom drops, small cocci in top drops. 
 
 Sample XVIII, 1897. Uncoloured, diluted A spirit, f water, 
 certified as faulty. Millions of small cocci aggregated together 
 with a tendency to rise to the top of the liquid in the form of 
 a scum. 
 
 Sample XIX, 1897. Uncoloured, undiluted, certified as 
 faulty. Same organism as that present in No. XVIII and the 
 coloured samples. 
 
 Sample XX, 1897. Uncoloured, undiluted as taken direct
 
 8 
 
 THE MICRO-ORGANISM OF FAULTY RUM 
 
 from still in an infected works. Free from any trace of 
 the organism. 
 
 Sample XXI, 1897. Coloured, undiluted, certified as faulty. 
 In this sample, which had been subjected to some process of 
 fining, large colonies were found at the bottom only. The 
 cocci were of enormous size, but dead, as was proved not only 
 by their abnormal appearance [PI. I. Fig. I, c], but by 
 failure to obtain growths from them when transferred to 
 favourable media. 
 
 It is clear from the following tabulation of the results that 
 the micro-organism was present in the faulty, but absent from 
 the sound samples, with the solitary exception of No. XII, in 
 which the quantity present was not sufficient to cause cloudi- 
 ness when examined by the broker's test of dilution. 
 
 organism. 
 Absent. 
 
 Present. 
 
 No. of 
 Sample. 
 
 Year of 
 Importation. 
 
 Dilution. 
 
 Condition. 
 
 I. 
 
 1887. 
 
 Undiluted. 
 
 Sound. 
 
 II. 
 
 1891. 
 
 ,, 
 
 
 
 III. 
 
 I8 93 . 
 
 M 
 
 f> 
 
 IV. 
 
 1894. 
 
 M 
 
 M 
 
 V. 
 
 1897. 
 
 
 
 Faulty. 
 
 VI. 
 
 B 
 
 w 
 
 
 VII. 
 
 " 
 
 " 
 
 
 VIII. 
 
 
 
 
 IX. 
 
 }> 
 
 Diluted. 
 
 '^ 
 
 X. 
 
 M 
 
 
 
 XI. 
 
 
 
 
 XII. 
 
 i 
 
 Undiluted. 
 
 Sound. 
 
 XIII. 
 
 
 
 
 
 Faulty. 
 
 XIV. 
 
 1892. 
 
 M 
 
 M 
 
 XV. 
 
 1897. 
 
 M 
 
 Sound. 
 
 XVI. 
 
 
 
 
 
 Faulty. 
 
 XVII. 
 
 M 
 
 M 
 
 
 XVIII. 
 
 n 
 
 Diluted. 
 
 
 XIX. 
 
 n 
 
 Undiluted. 
 
 
 XX. 
 
 n 
 
 
 Fresh fro 
 
 XXI. 
 
 
 
 n 
 
 Faulty. 
 
 Trace present. 
 Present. 
 
 Absent. 
 Present. 
 
 Present. 
 
 These collected observations afford, to our minds, strong 
 prima facie evidence that there is a relation between the 
 presence of the micro-organism and the ' faultiness.' As the 
 fact that the presence of the micro-organism always accom-
 
 GENERAL EXAMINATION OF SAMPLES g 
 
 panies the turbidity or faultiness is of itself no proof that the 
 former is the sole cause of the latter, the scientific method 
 of Concomitant Variations was applied, namely, experiments 
 were made with a view to converting samples of both 
 undiluted and diluted faulty rum into sound samples by 
 removing the organism, and conversely of converting sound 
 into faulty rum by its addition. 
 
 The first experiment consisted in passing a diluted faulty 
 sample through a sterilized Miincke's filter at a reduced 
 pressure of about 650 m.m. ; though the turbidity disappeared 
 for the most part after the first filtration the process was 
 repeated, the filter being meanwhile brushed and cleansed 
 inside and outside and again sterilized. After the second 
 filtration the liquid was not only quite bright and clear, but 
 completely free from the micro-organism ; optical tests were 
 applied as described in Chapter IV, The same experiment 
 was repeated with an undiluted faulty sample with the same 
 result, and further the twice filtered liquid when diluted in the 
 proportion of spirit and f water still remained clear, as 
 evidenced by the optical tests. 
 
 Conversely, a pint bottle of undiluted faulty rum was passed 
 twice through another and new sterilized Miincke's filter ; the 
 sediment left thereupon was transferred to an equal bulk of 
 sound rum contained in a similar bottle. As, however, there 
 was a considerable slimy deposit of the organism left on the 
 walls of the first bottle, the latter was cut in half, the deposit 
 detached by a sterilized scoop, the whole completely cleared 
 out and added to the sound rum mentioned above. The liquid 
 thus obtained had all the appearance of faulty rum, and sub- 
 sequent dilution in the usual proportions rendered it more 
 apparent, and by the optical tests the characteristic pheno- 
 mena were obtained. 
 
 The necessity for more than one filtration, and for repeated 
 cleansing and sterilizing of the apparatus, would be obvious 
 to experienced bacteriological investigators ; it does not appear 
 that Messrs. Harrison and Scard (communication to Sugar 
 Cane, vol. xxx. pp. 410 et seq.) performed the operation 
 more than once in any case. The following quotation from
 
 io THE MICRO-ORGANISM OF FAULTY RUM 
 
 Professor Hansen's well-known work [2] will show that he is 
 in accord with us upon this matter. ' In the course of these 
 experiments, however, I perceived that the Chamberland 
 filters would be of great service in preparing different 
 sterile liquids for laboratory experiments. If an absolutely 
 germ-free filtrate is required, the tubes must be sterilized 
 at short intervals ; they must not be kept in action for an 
 indefinite period, as was formerly imagined, for the bacteria 
 pass through the walls after a longer or shorter period V 
 
 As the faultiness is dependent upon the presence of the 
 organism in considerable numbers, as illustrated from Sample 
 XII, it would have been useless to have inoculated sound rum 
 with one cell, or even a few colonies, especially as it was found 
 that its power of reproduction by division was either lost or much 
 impaired after prolonged life in spirit. Hence the bulk for bulk 
 experiment above described appeared the most practicable. 
 
 Another method would have been to have cultivated the 
 organism on a large scale in sterilized solutions of caramel or 
 brown sugar, filtered it off, and transferred the colonies in 
 such bulk as that present in faulty rum to an equal bulk 
 of sound rum. 
 
 At an early part of the inquiry we inoculated samples of 
 sound rum with a few colonies of the micro-organism, but 
 since its power of multiplication by division soon becomes 
 latent in the spirit, the results, as was to be expected, proved 
 negative; if, however, the organism was transferred, even 
 after a length of time, to a suitable medium, growth took 
 place in the course of a few days, thus proving that the 
 organism was not dead, but merely in a resting stage. It 
 appears that Messrs. Harrison, Scard, and Daniels (loc. cit.) 
 performed similar experiments with the same negative result, 
 and arrived at the hasty conclusion that the organism was 
 dead, but there is no evidence in the paper above cited that 
 
 1 Cf. Migtila, Separatabdruck Jahresber. Ver. Nattir, Mannheim, 1894, 
 ' Wollen wir nun die Wirksamkeit eines Filters priifen, so bleibt uns dazu 
 nur das Mittel, die Zahl der in unfiltrierten und die der in filtrierten 
 Wasser vorkommenden Bakterien festzustellen und mit einander zu 
 vergleichen.'
 
 GENERAL EXAMINATION OF SAMPLES n 
 
 these writers, either in this case or in any other, attempted to 
 grow the organism in a suitable medium, such as a saccharine 
 solution ; it cannot be supposed that the organism is existing 
 in favourable conditions in rum of such alcoholic strength. 
 No person of experience in horticulture would sow seeds on 
 a cinder-path and expect to obtain a good flower-bed. The 
 production of disease in animals by inoculation with a single 
 cell of a pathogenic organism could not be cited as a parallel 
 case, as here the nidus or medium is suitable for growth and 
 reproduction. It will be obvious that the effects are to be 
 measured in the case of faulty rum by the relative proportion 
 of the micro-organism, and not by the absolute quantity of the 
 liquid, and therefore the addition of ' io/ o ,' or even more, of 
 faulty to sound rum would not necessarily render the latter 
 faulty, as of course in an experiment of this kind, described 
 by Mr. Harrison, he was not only increasing the quantity of 
 liquid by admixture, but actually decreasing the relative 
 proportion of the organism. 
 
 Experiments of the kind made by Messrs. Harrison and 
 Scard are not such as to satisfy the elementary requirements 
 of bacteriological investigation, since there is no mention of 
 any sterilization of vessels and materials used, and one set 
 of experiments was conducted with ' stoppered ' bottles ; 
 hence the most obvious precautions against error were 
 neglected. This is the more striking in view of the following- 
 passage in Messrs. Harrison and Scard's communication, 
 which we cannot but regard as naive in the extreme : 
 ' Any person acquainted with the interiors of distilleries in 
 the tropics would not be surprised by the presence of the 
 remains of different organisms in the sediments of the casks, 
 although he might be surprised if they were entirely absent. 
 We are not aware that any ' surprise ' has been expressed as 
 to the existence of micro-organisms in tropical distilleries ; it 
 would, however, be a matter of great surprise if, after the 
 investigations of Pasteur and Hansen on the micro-organisms 
 of fermentation, no steps were taken for their removal. The 
 increased difficulty of obtaining a germ-free product in the 
 tropics does not relieve the manufacturers from the necessity,
 
 12 THE MICRO-ORGANISM OF FAULTY RUM 
 
 if they do not wish to be left behind in the trade struggle for 
 existence. 
 
 As one of us is a director and part proprietor of a large 
 and successful brewery, conducted on scientific principles, and 
 the other is not without practical experience in the bacteriology 
 of fermentation, we feel ourselves to some extent entitled to 
 express our opinion on this point. 
 
 We think that Mr. Harrison is sanguine in expecting that 
 ' the removal of the microbe by death ' should cure the rum of 
 its faultiness, especially as he finds its ' apparent remains ' in 
 the sediment ; nor can we enter into Mr. Harrison's frame of 
 mind, when in one passage he definitely states (p. 412) that 
 he found the organism described by us, while in another 
 (p. 414) he refers to it as the ' alleged bacterium.' 
 
 Examination of Caramels used for colouring. 
 
 Two samples of caramel were obtained, one direct from and 
 recently manufactured in an infected works, the other made 
 in England by a well-known London firm. The former was 
 very strongly acid, and a small quantity partially dissolved 
 in sterilized distilled water and filtered through muslin showed 
 innumerable swarms of cocci, identical with those in faulty 
 rum, as well as an actively motile rod form. The latter was 
 neutral and contained only debris of vegetable fibre, and when 
 used for certain colouring experiments of redistilled rum, gave 
 perfectly bright and clear spirit. 
 
 One source of infection is therefore the caramel, which 
 would form a very suitable nidus for the micro-organism, 
 especially if it is kept, while cooling, in open vessels ; though 
 it is clear that this is not the only source of infection, since 
 the micro-organism is absent, as we should expect, from the 
 rum taken fresh from the still (Sample No. XX), but present 
 in much of the uncoloured rum, which must have been kept 
 in infected vats and puncheons. 
 
 Examination of Liverpool water used for dilution. 
 Three samples of Liverpool water as used by the brokers in 
 their test were sent to us ; no organism in the least resembling
 
 GENERAL EXAMINATION OF SAMPLES 13 
 
 that of faulty rum was found, the few bacteria present were 
 common water forms such as might occur in any sample of 
 tap-water. 
 
 II. Chemical Examination. 
 
 On fractional distillation of coloured undiluted spirit it was 
 found that all the volatile portion came over below 100-5 C-> 
 the first and greater portion distilled at 78-5-79, the boiling 
 point of ethyl alcohol, the second portion, from 80 to 98, was 
 milky in appearance and probably consisted of compound 
 ethers with water, and the third portion of 99-ioo-5 of water 
 only. This suffices to prove the absence of any substance of 
 the nature of a terpene which might be extracted from the 
 wood by the spirit, and precipitated therefrom on dilution. 
 
 In order to determine the materials other than alcohol, 
 traces of ethereal salts, and water, determinations were made 
 of the solid residue at 100, and the residue on ignition in the 
 case of two samples, .one coloured and the other white ; in the 
 former case 86-06 grams yielded on evaporation over a water- 
 bath -938 gram solid residue, which on ignition gave a residue 
 of -0635 gram ; in the latter case 86-7 grams gave -0145 gram 
 of residue dried at 100, and an inappreciable residue on 
 ignition. The food material in the first case consisted, there- 
 fore, of i'OJ/ o carbohydrates, and -O74/ inorganic salts, 
 reckoned as anhydrous, and in the latter case of -oi6y o 
 carbohydrates only. 
 
 It was thought, at the outset, that there might be some 
 relation between the faultiness and the proportion of free acid, 
 but analyses of various samples showed that sound spirit 
 might contain a greater quantity of free acid than ' faulty ' 
 spirit. It is, therefore, clear that the free acid produced in 
 the original fermentation and volatilizing over on distillation 
 might be in excess of that produced by the subsequent decom- 
 position of the sugar contained in the added colouring matter 
 by the micro-organism. 
 
 But for the better purpose of comparison the amount of 
 acid per unit weight was determined in the same faulty 
 spirit, (i) undiluted and (2) diluted, some months previous to
 
 i 4 THE MICRO-ORGANISM OF FAULTY RUM 
 
 examination in the proportion mentioned above. The 
 following determinations are selected to illustrate this point ; 
 the values are given, according to the usual convention, in 
 terms of acetic acid, and though it is most probable that this 
 is not the only acid present, yet the conclusion deducible is 
 not materially affected. 
 
 Proportion of acid 
 
 Sample, to 100 grams spirit. 
 
 ( Diluted . . . -1216 gram 
 
 A I Undiluted . . -0961 
 
 B 
 
 Diluted . . . -1176 
 
 Undiluted . . . -0849 
 
 Diluted . . . -1418 
 
 Undiluted . . . -0946 
 
 Coloured Samples. 
 
 In all cases the amount of free acid in the diluted was 
 greater than that in the undiluted samples, thus showing that 
 some decomposition of the caramel or alcohol had taken 
 place by the agency of the organism, when living in the 
 diluted spirit. 
 
 In this connexion there is an experiment described by 
 Messrs. Harrison and Scard (loc. cit. p. 414) in which it is stated 
 that the sediment from faulty rum was soluble in alcohol of 
 40 to 5o/ o over proof, and the solution after filtration through 
 a Pasteur-Chamberland filter became opalescent on dilution. 
 From the terms used it would appear that the sediment is the 
 same as that which we find in undiluted faulty rum of 
 4O/ o over proof ; if this is the case, the authors have succeeded 
 in finding a substance, bacterial or resinous, which is at once 
 precipitated out as a sediment, in spirit of 4Q/ o over proof, 
 but is also soluble in the same menstruum. If this experiment 
 had not been described in print, we should hardly regard it as 
 worthy of the serious attention of scientific persons. 
 
 III. Physical Examination. 
 
 These phenomena will be dealt with in detail in the two 
 succeeding chapters, wherein a full discussion of Mr. Harrison's 
 theory will be found; meanwhile we would desire to point 
 out that in the recent paper above quoted no tests seem to
 
 GENERAL EXAMINATION OF SAMPLES 15 
 
 have been applied for (i) opalescence and (ii) fluorescence ; 
 this would seem to be the more necessary as the authors 
 evidently confuse four totally distinct phenomena, namely, the 
 two just mentioned, (iii) turbidity, and (iv) alteration of 
 refractive index. 
 
 It may be convenient to remind those of our readers who 
 are interested in the methods of physical science, that the three 
 words turbidity, opalescence, and fluorescence are employed to 
 represent three separate phenomena, each with its special 
 significance and connotation. 
 
 We denote a liquid as turbid when it contains suspended 
 particles which disturb the passage of light through it, 
 rendering it thick without causing any alteration in the 
 character of the light they reflect. Turbidity indicates the 
 presence of particles visible at any rate under the microscope. 
 
 The term opalescence we have restricted entirely to that 
 phenomenon as originally described by Tyndall, especially 
 with reference to the blue waters of the Alpine lakes and blue 
 sky, which owe their colour to the presence of innumerable 
 particles so fine as to be invisible under the highest magnifica- 
 tion. Light reflected by such particles, when passed through 
 a Nicol's prism at right angles to the line of incidence, is 
 invariably polarized. 
 
 Fluorescence consists, as is well-known from the investiga- 
 tions of Sir G. Stokes [3], essentially in the emission by the 
 substance of a light of a different colour from that which 
 falls upon it. Many resins have the property, such for 
 example as a solution of catechu in alcohol, which gives 
 a green fluorescence, and guaiacum, which gives a blue 
 fluorescence. But when such solutions of resins are mixed 
 with water the fluorescence disappears, but opalescence appears. 
 It will be our purpose to demonstrate that the faultiness of 
 rum is properly described primarily as a turbidity, but also 
 as an increased fluorescence, due to the fact that the organism 
 extracts and assimilates that particular ingredient of the 
 caramel, which is fluorescent ; dilution of rum with ^vater in 
 no case causes an opalescence.
 
 CHAPTER III 
 
 THE 'RESIN' THEORY 
 
 IN the report of Mr. Harrison alluded to above the follow- 
 ing passage occurs : 
 
 'When the spirits were kept in contact with the split-up 
 staves [of casks used by manufacturers] for two or three 
 weeks, the alcohol took up from them considerable quantities 
 of resinous and other matters, so as to produce flocculencies 
 and precipitates when mixed with distilled water or with rain- 
 water. 
 
 ' Similar experiments were made with split-up staves, using 
 coloured rum which had successfully stood the test with the 
 Liverpool well-water, and it was found that after being in 
 contact with the pieces of staves for a few days, the coloured 
 rum acted in a manner not distinguishable from that in which 
 the faulty rum acted when mixed at 50 F. with the Liverpool 
 well-water. 
 
 'From this it appears that the deleterious substances pro- 
 ducing cloudiness when rum is mixed with Liverpool well-water 
 probably are derived from the staves of the cask by the solvent 
 action of the alcohol upon certain of the constituents of the 
 wood.' 
 
 Mr. Harrison does not bring forward any direct evidence 
 that the matters extracted from the staves of the casks were 
 of a resinous nature ; it does not even appear that he applied 
 any of the well-known physical or chemical tests for resins, 
 neither does he offer any proof that the ' flocculencies ' and 
 ' precipitates,' which he obtained after extraction of the wood 
 with the spirits and subsequent dilution, were identical in 
 character with the ' floating tiocculencies ' which he found in 
 faulty rum.
 
 THE 'RESIN' THEORY 17 
 
 Further, it appears from information obtained from the 
 manufacturers that the faultiness, though known for at least 
 fifteen years, has been of an intermittent character, whereas 
 the wood used for puncheons, vats, &c., has been constant ; 
 again, at certain times, some Demerara manufactories were 
 attacked and not others, yet there is good ground for believing 
 that all the firms were supplied with the same timber. 
 
 Notwithstanding the weakness of the evidence brought 
 forward to support the view that the faultiness is due to 
 resinous matter extracted from the wood, it seemed desirable 
 to ascertain if the presence of such matters could be detected 
 by microscopical, chemical, or physical tests, and as it will be 
 shown in the sequel that negative results were obtained, the 
 method of difference was applied, namely, that certain resins 
 were added both to distilled and coloured spirit, and the 
 effects produced compared with the faultiness of rum. 
 
 Microscopical Tests. 
 
 In the course of the investigation many hundred drops 
 were examined under the microscope ; these included all the 
 deposits of every character, both from diluted and undiluted 
 samples. But apart from the micro-organism, only de'bris of 
 definite vegetable structure, probably sugarcane, and in a few 
 cases crystals resembling calcium sulphate in appearance, 
 were found ; the magnification used varied from 600 to 1000 
 diameters. No amorphous substance, which might be of the 
 nature of a chemical precipitate, was ever detected. 
 
 The above evidence, though pointing to the improbability 
 of Mr. Harrison's views, yet cannot be accepted as definite, 
 as Picton and Linder [4] have observed that certain so- 
 called pseudo-solutions of mercury and other metallic sulphides 
 showed a clear field with a magnification of 600 diameters, and 
 even with 1000 diameters the resolution of such solutions 
 into its constituent particles was not always very apparent. 
 These writers further showed that though such fine particles 
 were not always retained by a dialyser of vegetable membrane, 
 yet they in no case passed through a porous cell ; this point 
 will be alluded to subsequently, 
 c
 
 i8 THE MICRO-ORGANISM OF FAULTY RUM 
 
 Chemical Tests. 
 
 About three-quarters of a pint of undiluted rum, classed as 
 faulty, was passed through a bacterial filter and subsequently 
 distilled to dry ness in a capacious flask with a long neck ; 
 the distillate was reserved for experiments to be described. 
 The brown residue obtained was first extracted with ether, 
 which removed a faint trace of a yellow colouring matter, 
 probably one of the constituents of the caramel ; the solu- 
 tion was evaporated and tested with stannic chloride for 
 a violet coloration (Renard's process) but with a negative 
 result. The residue was then extracted with absolute alcohol, 
 and the solution; after filtration, was tested to ascertain if 
 any turbidity was produced on addition of (i) ether and 
 (ii) ammonia, but also with negative results (Hirschsohn's 
 methods) ; alcoholic solution of lead acetate precipitated, of 
 course, the brown colouring matter of the caramel. 
 
 Lastly, a portion of the alcoholic extract and also the 
 remainder of the residue was digested with light petroleum 
 spirit, but no colouring matter was extracted nor any green 
 fluorescence observed in the solution (Durand-Claye method). 
 
 From the above it is evident that all the well-known 
 chemical tests for resins gave negative results ; further, the 
 small amount of residue, less than i/ o (vid. supra), obtained 
 on evaporation of a sample of undiluted faulty spirit would 
 equally point to the same conclusion that no appreciable 
 quantity of a resin is present. 
 
 Physical Tests. 
 
 As it has been shown that the faultiness is not due to the 
 presence of lifeless particles visible under the microscope, three 
 possible causes remain : (A) Turbidity, due to the presence of 
 the micro-organism ; (B) Opalescence, due to the presence of 
 particles so fine as to be separately invisible under the 
 microscope, but which polarize all light reflected by them at 
 right angles to the incident rays ; and (C) Fluorescence. If 
 a resin were present faulty rum would always show an
 
 THE ' RESIN 3 THEORY 19 
 
 opalescence on dilution, and in any case it might be more or 
 less fluorescent. Experiments to be described show that faulty 
 rum is not always opalescent, and at any rate the opalescence 
 is no measure of the faultiness, but all coloured rum, sound or 
 faulty, is more or less fluorescent, and the degree of fluor- 
 escence is a measure of the faultiness. The following methods 
 were applied for the detection of the three above-mentioned 
 phenomena. 
 
 (A) Turbidity and (C) Fluorescence. Light from an arc 
 lamp of about 2,000 candles, or direct sunlight, was con- 
 centrated on the surface of the liquid in an open glass 
 vessel; it was examined both for A and C by noting the 
 appearance at the surface of the liquid, -where the light 
 was more diffuse, and also at the focus. Simple turbidity 
 shows most light at the focus, but fluorescence, unless very 
 slight, is stronger at the surface than at the focus. The 
 two diagrams given below serve to illustrate this point. 
 
 Simple Turbidity 
 
 B. Opalescence. Parallel rays from an arc lamp or the 
 sun were thrown into the solution, and the reflected light 
 examined with a Nicol's prism held at right angles to the 
 line of incidence [5] ; if on rotating the prism a change 
 of luminosity could be detected, opalescence was considered 
 to be present. 
 
 C. Fluorescence. A converging beam from the arc-light 
 or sunlight was passed through a vessel containing a solution 
 
 C 2
 
 20 THE MICRO-ORGANISM OF FAULTY RUM 
 
 of ammonio-sulphate of copper of sufficient concentration 
 to completely stop all rays of longer wave length than G', as 
 determined by the spectroscope. The light reflected by the 
 liquid was also examined with the same instrument. 
 
 If only the violet end was visible, and if when sunlight was 
 used Fraunhofer lines appeared, then fluorescence was con- 
 sidered to be absent ; but if, on the other hand, green light 
 was reflected, showing no Fraunhofer lines, fluorescence was 
 considered as proved. 
 
 As an example of a fluorescent resin guaiacum was selected, 
 not of course as a substance likely to be present, but as 
 partly soluble in alcohol, forming a solution indistinguishable 
 in appearance from coloured rum. Two solutions were made, 
 one containing i/ o guaiacum in alcohol, and the other one 
 part per thousand of guaiacum in redistilled and coloured rum ; 
 both were filtered through paper, and then passed through 
 a bacterial filter in order to make the comparison with faulty 
 rum complete ; both were tested for A, B, and C before and 
 after dilution. The results are given below. 
 
 Solution 1. i/ o guaiacum. 
 Before dilution. 
 
 A. Turbidity none ; B. Opalescence none ; C. A blue fluorescence. 
 After dilution. 
 
 A. Turbidity none ; B. Beautiful opalescence ; C. No fluorescence. 
 
 Solution II. One part per thousand of guaiacum in re- 
 distilled, coloured rum. 
 Before dilution. 
 
 A. Turbidity none ; B. Opalescence none ; C. A blue and green 
 fluorescence. 
 After dilution. 
 
 A. Turbidity none ; B. Well-marked opalescence ; C. No fluor- 
 escence. 
 
 The general result of these experiments tends to show that 
 alcoholic solutions of fluorescent resins on dilution with 
 water lose their fluorescence entirely and become entirely 
 opalescent. 
 
 Similar experiments were conducted with various other 
 non-fluorescent resins in very dilute solutions, and in all
 
 THE 'RESIN 3 THEORY 21 
 
 cases such solutions, after passage through a bacterial filter 
 and subsequent dilution, became opalescent. It will be shown 
 in the sequel that faulty rum, when dealt with in a similar 
 manner, never becomes more opalescent. 
 
 To sum up, therefore, firstly, there is no evidence from 
 microscopical, chemical, and physical tests of the presence 
 in faulty rum of any resinous matter, and secondly, when 
 a trace of a fluorescent resin is added to redistilled coloured 
 rum, its presence can be detected both before and more 
 markedly after dilution. 
 
 It would appear somewhat difficult of explanation that 
 fragments of a resin could be treated with Hermann's fluid, 
 which contains platinum tetrachloride and osmic acid, could 
 then be washed with water and alcohol, stained with an 
 aniline dye, and subsequently treated with xy]ol, but yet 
 withal retain their original shape. 
 
 We have reason to believe that Mr. Harrison recently 
 received large masses of the organism from works which 
 (contrary to his belief) we, some thousands of miles away, 
 declared to be infected ; we are surprised that Mr. Harrison 
 does not allude to this occurrence in his subsequent com- 
 munication. 
 
 In conclusion, Mr. Harrison does not seem to have realized 
 that reiteration is not proof, and that it is incumbent upon 
 him to extract and isolate the resin from the timber used, 
 and to determine its chemical properties and constitution, as 
 also its physical properties, especially as regards solubility 
 in alcohol ; he must then show that the alleged resin in 
 faulty rum is identical either with the substance as extracted 
 from the wood, or those products of it which are dissolved 
 out by alcohol. Otherwise he is in the position of a person 
 who comes into a law court with a claim to the real estate 
 of a property, but cannot produce the title-deeds.
 
 CHAPTER IV 
 
 THE FLUORESCENCE OF COLOURED RUM AND ITS REFRACTIVE 
 INDEX 
 
 IN the introduction allusion was made to the turbidity 
 described as apparent in the coloured spirit when diluted, 
 for which the purchasers have claimed a rebate from the 
 importers. It was at once observed that such samples, when 
 viewed by transmitted light, appeared bright and clear, but 
 opaque to a greater or less degree by reflected light, and 
 also that this opacity could be diminished by the addition 
 of a further quantity of spirit. As the phenomena seemed 
 to be mysterious alike to the manufacturer and writers on 
 the subject, but were evidently due to some optical effect, 
 it was determined to ascertain if they could be traced to 
 either or both of the following causes : (i) Alteration of Re- 
 fractive Index. If the refractive index of the gelatinous 
 envelope surrounding the micro-organism were nearly equal 
 to that of the undiluted rum, then, on dilution with water, 
 the refractive index of the spirit would be altered (as its 
 density is increased) according to the well-known formula 
 
 = C, while that of the envelopes would remain un- 
 altered ; consequently the turbidity would become more 
 apparent. (2) Fluorescence, which is known to be charac- 
 teristic of many bacterial forms. 
 
 The result of the experiments tend to show that the phe- 
 nomena observed is the mixed effect of both causes, as the 
 presence of the micro-organism both alters the refractive 
 index of diluted spirit and increases the fluorescence of the 
 coloured spirit, whether dilute or undilute.
 
 THE FLUORESCENCE OF COLOURED RUM 23 
 
 I. The Alteration of Refractive Index. 
 
 The refractive indices of four samples of rum were deter- 
 mined, two of which were undiluted and two diluted ; of the 
 former, one had been classed as faulty and contained a large 
 quantity of the micro-organism, the other as sound ; of the 
 latter, one was the faulty sample mentioned above which 
 had been diluted in the proportion of one-third spirit to 
 two-thirds water, and the other, the same sample freed from 
 the micro-organism by filtration through a bacterial filter. 
 
 Values for m> at 19. 
 
 . f (i) Faulty rum . . . 1-36545 
 
 Undiluted samples] ,..' *; 
 
 I (11) Sound rum .... 1-36597 
 
 ,,.,,, , f (i) Faulty rum. . . . 1-34273 
 
 Diluted samples \ ,..; m , J , . .. ., . 
 
 ( (n) The same bacterially filtered 1-34132 
 
 Two other determinations were made of the undiluted 
 faulty rum with a view of studying the effect of temperature, 
 all other conditions remaining the same ; the values obtained 
 were at ai-6 p D = 1-36492, and at 23-6 p. D = 1-36491. It will 
 be evident from the inspection of the above figures that the 
 refractive index of both sound and faulty rum, when undiluted, 
 is the same, and therefore the presence of the micro-organism 
 produces no effect ; but if, as supposed above, the refractive 
 index of the envelopes differs from that of diluted rum, then 
 there should be a slight variation in the refractive indices of 
 the latter when containing and when free from the micro- 
 organism. 
 
 II. The Fluorescence. 
 
 The earlier experiments upon this subject were conducted 
 in accordance with the well-known methods of Sir G. Stokes, 
 and with one sample of diluted faulty rum ; the subsequent 
 experiments by the methods described in the previous chapter, 
 and with both undiluted, diluted, faulty, and sound samples, 
 as also with various samples of caramel and black sugar. 
 The results obtained are described below.
 
 24 THE MICRO-ORGANISM OF FAULTY RUM 
 
 First Series of Experiments. 
 
 Firstly, the solar spectrum was projected by means of 
 a heliostat and prism upon a screen in a dark room ; when 
 a small flat glass cell containing the spirit was passed along 
 the spectrum from the red to the violet, the phenomenon of 
 fluorescence appeared most marked in the latter. Secondly, 
 a converging beam of sunlight, when passed through a rec- 
 tangular glass vessel filled with spirit, was of a beautiful 
 green tint, but when passed through a second and similar 
 vessel, also containing the spirit, the green tint disappeared. 
 Thirdly, two large quartz prisms were substituted for the 
 glass prism used in the first experiment in order to obtain 
 a great dispersion ; when the cell was placed in the violet 
 part of the spectrum the green fluorescence was observed, 
 and was intensified in the ultra-violet when the green fluores- 
 cence became of a violet or blue tint. 
 
 By way of comparison the experiments described above 
 were repeated with a solution of aesculin, as one of the sub- 
 stances originally used in the classical investigations of 
 Sir G. Stokes. 
 
 Second Series of Experiments (by methods 
 described above, p. 19). 
 
 Sample I. Faulty Rum undiluted. 
 
 A. Turbidity, considerable. 
 
 B. Opalescence, roughly about 5% of the turbidity. 
 
 C. Fluorescence, very marked from orange to blue green. 
 
 Sample II. Rum, undiluted, which passed the brokers' test, 
 but which contained a small quantity of the micro-organism. 
 
 A. Turbidity, negligible. 
 
 B. Opalescence, very slight. 
 
 C. Fluorescence, not so marked as in No. I. 
 
 Sample III. Another sample of faulty rum, undiluted. 
 
 A. Turbidity, considerable. 
 
 B. Opalescence, none. 
 
 C. Fluorescence, very marked.
 
 THE FLUORESCENCE OF COLOURED RUM 25 
 
 Sample IV. Sound rum, undiluted. 
 
 A. Turbidity, none. 
 
 B. Opalescence, none. 
 
 C. Fluorescence, much less than in above samples, but evident. 
 
 Sample V. Faulty rum, diluted, not filtered. 
 
 A. Turbidity, well marked. 
 
 B. Opalescence, a trace. 
 
 C. Fluorescence, well marked. 
 
 Sample VI. No. V passed through a bacterial filter. 
 
 A. Turbidity, none. 
 
 B. Opalescence, none. 
 
 C. Fluorescence, slight. 
 
 Sample VII. Faulty rum passed through a bacterial filter. 
 
 A. Turbidity, none. 
 
 B. Not tested. 
 
 C. Fluorescence, evident. 
 
 Sample VIII. Sound rum, made faulty. 
 
 A. Turbidity, marked. 
 
 B. Opalescence, marked. 
 
 C. Fluorescence, very marked. 
 
 All the above experiments were conducted with samples 
 which had been coloured in the usual manner with caramel ; 
 the next two were made with uncoloured samples. 
 
 Sample IX. Rum fresh from still. 
 
 A, B, C. All gave negative results. 
 Sample X. Rum, not coloured, but containing the organism. 
 
 A. Turbidity, considerable. 
 
 B. Opalescence, slight. 
 
 C. Fluorescence, absent. 
 
 As these experiments showed that all coloured samples, 
 whether faulty or sound, undiluted or diluted, were more or 
 less fluorescent, though the phenomenon was much more 
 marked in the faulty than in the sound samples, further 
 experiments were conducted with various samples of caramel 
 and brown sugar, in order to determine if solutions of these 
 samples were also fluorescent.
 
 26 THE MICRO-ORGANISM OF FAULTY RUM 
 
 Sample XI. Solution of caramel, obtained from manu- 
 facturers. 
 
 A and B not tested. 
 
 C. Fluorescence, very marked. 
 
 Sample XII. Solution of canesugar. 
 A, B, and C gave negative results. 
 
 Sample XIII. Sample of caramel made from XII, and dis- 
 solved in water to give a solution of tint resembling undiluted 
 rum. 
 
 A. Turbidity, none. 
 
 B. Opalescence, not tested. 
 
 C. Fluorescence, resembling sound rum. 
 
 Sample XIV. Solution of brown sugar, as used for culture 
 experiments. 
 
 A. Turbidity, very slight. 
 
 B. Not tested. 
 
 C. Fluorescence, slight. 
 
 The above experiments show that solutions of caramel are 
 fluorescent per se, a fact which, it is believed, has not as yet 
 been observed ; but, again, the phenomenon was much more 
 marked in the solution of the caramel, which had been 
 obtained directly from one of the works, and was full of the 
 micro-organism. One further experiment was conducted with 
 a gelatine culture of the micro-organism in a Petri-dish, which 
 showed that the gelatine, originally non-fluorescent, had 
 become fluorescent even though sown from white rum. 
 
 The general conclusions to be drawn from the results 
 described above are as follows : 
 
 (1) Faulty rum is turbid both before and after dilution, 
 when tested with a sufficiently strong light. 
 
 (2) Faulty rum is never more opalescent after dilution. 
 
 (3) Sound rum is slightly, but faulty rum very fluorescent, so 
 that it appears that the micro-organism takes up the colouring 
 and fluorescent matters. 
 
 (4) Caramel, even when prepared from pure cane sugar, is 
 fluorescent, and here again the presence of the micro-organism 
 increases the phenomenon.
 
 CHAPTER V 
 
 METHODS OF CULTURE AND EXPEEIMENT 
 
 THROUGHOUT our experiments choice was made of various 
 saccharine media for cultural experiments in preference to 
 bouillon, agar, serum, &c., because it was desirable to imitate 
 as nearly as possible the conditions which might obtain in the 
 manufactories, and thus to avoid the possible production of 
 involution-forms by the use of highly artificial media. 
 
 Solutions were made as follows in sterilized distilled 
 water : 
 
 I. Five per cent, and ten per cent, solutions of very coarse 
 black sugar. 
 
 II. Five per cent, solution of cane sugar, with a trace of 
 microcosmic salt. 
 
 III. Five per cent, solution of milk sugar, with -^/ micro- 
 cosmic salt. 
 
 IV. Five per cent, solution of grape sugar, with -5/ 
 microcosmic salt. 
 
 V. Five per cent, solution of cane sugar, with i / o calcium 
 chloride. 
 
 VI. Five per cent, solution of cane sugar, with i/ o calcium 
 chloride and a trace of microcosmic salt. 
 
 VII. Five per cent, solution of milk sugar, with i/ o 
 calcium chloride and a trace of microcosmic salt. 
 
 VIII. Five per cent, solution of milk sugar only. 
 
 IX. Five per cent, solution of cane sugar with weighed 
 quantities of calcium carbonate. 
 
 X. One per cent, cane sugar, with T V% Qach of calcium 
 nitrate and gelatine.
 
 28 THE MICRO-ORGANISM OF FAULTY RUM 
 
 XL One per cent, milk sugar, with -&/ o each of calcium 
 nitrate and peptone. 
 
 XII. One per cent, starch disseminated in water. 
 
 XIII. Five per cent, cane sugar only. 
 
 XIV. Five per cent, milk sugar, with T V/ peptone. 
 
 XV. English caramel. 
 
 Cultures were also made in gelatine as follows : 
 
 I. Deep (not stab) cultures in test tubes (a) with, and 
 (6) without sugar. 
 
 II. On microscope slides ; (a) with, and (6) without cover- 
 
 Ill. Shake-cultures in Petri's capsules, without sugar. 
 Gypsum blocks were also soaked with certain of the solu- 
 tions mentioned above and sown. 
 
 Materials for Inoculation. 
 
 In the course of the investigations sowings were made 
 from : 
 
 I. Undiluted, coloured rum of six different brands as 
 received from the manfacturers and brokers. 
 
 II. Ditto, uncoloured rum. 
 
 III. Previous cultures, namely, after a growth had been 
 obtained in solutions, &c., when sown from I and II. 
 
 Methods of Sterilization and Inoculation. 
 
 Throughout our experiments all liquid media were sterilized 
 by long boiling and subsequent discontinuous heating for two 
 days in a steam sterilizer, but subsequently experiments were 
 repeated with these liquids bacterially filtered (under the con- 
 ditions laid down) before inoculation. 
 
 The gelatine used was obtained ready sterilized, through 
 the kindness of Dr. James Ritchie, from the Pathological 
 Laboratory at the University Museum, Oxford. Gypsum 
 blocks were sterilized by passing through the flame of a Bunsen
 
 METHODS OF CULTURE AND EXPERIMENT 29 
 
 burner. Flasks and all utensils used were first steeped in 
 a solution of mercury perchloride (i : 1000), and subsequently 
 washed, boiled, and discontinuously steam sterilized with 
 their cotton-wool stoppings. Our blank controls showed 
 these methods to be effectual, but, in the case of cultures 
 direct from rum, the small quantity of strong alcohol which 
 was of necessity introduced with the inoculation proved 
 antiseptic for other organisms. 
 
 As regards liquid media, ninety-six cultures were made in 
 Freudenreich-Hansen flasks [see PI. IV. Fig. xv] of 20 c.c. 
 capacity. Inoculations were made with pipettes of i c.c. 
 capacity, divided into hundredths, which were heated in a flame 
 immediately before use, and kept in a T ^VTT solution of mercury 
 perchloride when not required ; other inoculations were made 
 with a sterilized platinum loop. Various quantities from -i cc. 
 the minimum, to -6 c.c. the maximum, of the samples of rum 
 were introduced at a sowing, or a loopful of the cocci from 
 a previous culture. In all cases the flasks to be inoculated 
 were held in the hot air above the flame of a Bunsen burner, 
 a method which, failing a germ-free chamber, is the most 
 practicable and efficient; two operators worked simultaneously, 
 and the processes of opening the flasks, inoculating, and closing 
 them were performed as quickly as possible. 
 
 After having once obtained a growth, all cultures were 
 made in sets of threes, viz. (i) from rum direct, (2) from 
 previous cultures, (3) a blank sample as control. These were 
 incubated (a) in a Hearson's incubator at a constant tempera- 
 ture of 35 C., in the dark, (6) in the same exposed to light, and 
 (c) under glass in direct sunlight in a south window, at 
 a varying temperature. 
 
 In the case of gelatine cultures the test tubes were inoculated 
 according to Hansen's method, by sowing a measured quantity 
 of the cocci-containing rum on the solid surface ; the gelatine 
 liquefied at first, so that the cocci became suspended in it, but 
 soon solidified again.
 
 3 o THE MICRO-ORGANISM OF FAULTY RUM 
 
 Methods for Single-Cell Cultures. 
 
 With a view of studying the development of individual 
 cells the hanging drop method in a moist chamber was applied, 
 but after repeated trials was not found to be practicable. 
 
 The coccus, when placed in a hanging drop of the usual 
 dimensions, remained perfectly quiescent, and did not even 
 divide within reasonable limits of time. Other experiments 
 showed that this was probably owing to the fact that a very 
 considerable bulk of the nutrient medium was essential to 
 development ; the attempts, therefore, were repeated, using 
 large moist chambers completely filled with liquid, but in 
 these the cocci, as soon as they had become large and heavy 
 and had begun to divide, invariably dropped off the cover- 
 glass, and completed the cycle of development at the bottom of 
 the liquid out of reach of observations by high powers. 
 
 Many experiments were then made with chambers specially 
 constructed so that both the top and bottom consisted of cover- 
 glasses, in order that the chamber could be reversed when the 
 falling-off took place ; it was hoped that some at least of the 
 cells might attach themselves to either glass. These too 
 proved unsuccessful, as also subsequent experiments, in which 
 films of the cocci were dried on the cover-glass, but the nutrient 
 liquid always soaked them off again with the same result as 
 before. 
 
 The original method of the hanging drop, stiffened by the 
 use of a variety of substances, such as gelatine, soluble glass, 
 &c., was repeated, but also with unsatisfactory results. 
 
 The isolation of cells by the dilution method was not avail- 
 able where it was required to keep the individual cells under 
 direct observation, because the cell could not be transferred 
 from a large body of the liquid to the stage of the microscope 
 until its development had already proceeded so far that the 
 speck of growth became visible to the naked eye. It appeared, 
 therefore, that the simplest method was the isolation of cells 
 on a thin layer of a solid medium. 
 
 For this purpose microscope slides and large cover-glasses 
 were covered with the thinnest possible layers of warm liquid
 
 METHODS OF CULTURE AND EXPERIMENT 31 
 
 gelatine; when this had just solidified, a small quantity of the 
 rum containing the organism was poured on the surface from 
 a pipette, and immediately washed off again with sterilized 
 distilled water. 
 
 Some cocci remained sticking to the gelatine, and in this 
 way we obtained cells sufficiently isolated for microscopical 
 observation of the development. Sowings were also made 
 in Petri's capsules with the same view, but in this case 
 inoculation was made while the gelatine was still in a liquid 
 state, and isolation secured by shaking. As, however, our 
 investigations indicated that on solid media certain stages in 
 the normal cycle of development could be rendered latent or 
 suppressed entirely, we devised a method, which may be con- 
 sidered as a modification of Koch's, for isolating cells on solid 
 media, and subsequently transferring them to liquids for the 
 purpose of growth. The thinnest possible layer of liquid 
 gelatine was spread over a slide, the cocci-containing rum 
 was poured on and washed off in the manner described above. 
 The slide was then searched under the microscope for well- 
 isolated cells, and one selected which appeared in every way 
 normal, and which, so far as determinable by the highest 
 powers, had no impurity either adhering to or immediately ad- 
 joining it. The position of the selected cell was then marked 
 by drawing a square on the gelatine with the point of a fine 
 sterilized knife, while the slide was still in position. The 
 slide was now removed, and the size of the square reduced by 
 four more transverse cuts made just within the first. The 
 single cell was then searched for and found again under the 
 microscope, to ensure that no injury had been done in the 
 process of cutting round it, and the size of the surrounding 
 square was again reduced in the same manner. 
 
 This process of alternately cutting squares and finding the 
 enclosed cell under the microscope was repeated, the knife 
 used being sterilized in the flame of a Bunsen burner between 
 each cut. When the square was so small that high powers 
 revealed nothing within it but the required coccus, the gelatine 
 external to the square was removed very cautiously with a 
 hot knife, leaving the coccus isolated on a minute island of
 
 32 THE MICRO-ORGANISM OF FAULTY RUM 
 
 gelatine. A sterilized flask containing nutrient liquid was 
 then brought close to the slide, the portion of gelatine con- 
 taining the coccus was transferred to it on the point of the 
 freshly sterilized knife ; the flask was closed instantly with 
 sterilized cotton- wool, and placed in the incubator. 
 
 The flask used contained a solution of 5/ o cane sugar, with 
 a trace of black sugar, which had been boiled, steam sterilized, 
 and twice bacterially filtered, and had been incubated at 35 C. 
 for six weeks without showing a sign of any growth ; hence 
 it was incontestably sterile previous to inoculation. 
 
 Professor Marshall Ward [38] has laid down the dictum 
 that the only method of diagnosis between Schizomycetes and 
 Hyphomycetes is that of the hanging drop ; he does not seem 
 to have contemplated the possibility of a case in which this 
 method by the nature of things is not applicable. When, as 
 in the present instance, such a case does arise, then either the 
 organism must remain unclassified as not electing to conform 
 to the regulations of Professor Marshall Ward, or the proposed 
 method of diagnosis cannot be accepted as final. In company 
 with such observers as Zopf and Migula we prefer to adopt 
 the latter course. 
 
 Stains used, and Method of 'making Permanent 
 Preparations. 
 
 Fifty-four permanent microscopical preparations were made 
 not only of the organism as in rum, but as cultivated in 
 various stages. The stains used were as follows : 
 
 IX. Aniline green. 
 X. Gentian violet. 
 XL Orange G. 
 XII. Fuchsin. 
 
 XIII. Fuchsin S. 
 
 XIV. Biondi-Haidenhain's triple 
 
 stain. 
 
 XV. Ziehl-Neelsen's carbol-fuch- 
 sin. 
 
 I. Dahlia. 
 II. Methyl-green. 
 
 III. Safranin. 
 
 IV. Eosin. 
 
 V. Haidenhain's iron-haemat- 
 
 oxylin. 
 VI. Gram's stain (iodine with and 
 
 without gentian violet). 
 VII. Aqueous corallin. 
 VIII. Iodine green. 
 
 All these stains, and other reagents used, were obtained for
 
 METHODS OF CULTURE AND EXPERIMENT 33 
 
 the purpose of this investigation directly from the well-known 
 firm of Grubler & Co., Leipzig. 
 
 The method pursued in making preparations was as 
 follows: The usual rather rough-and-ready bacteriological 
 method of fixing by heat was entirely avoided, as liable to 
 cause distortion and artefact appearances. In the case of 
 preparations made from rum direct, a drop of the spirit 
 containing numbers of cocci was added to a drop of Hermann's 
 fluid on a slide, and allowed to evaporate in the cold ; it was 
 found necessary to smear previously with vaseline all parts of 
 the slide except that to be covered by the drop, as otherwise 
 the strong spirit in evaporating would rush all over the slide, 
 and the cocci would thus be carried away to the edges, 
 instead of remaining in situ. The cocci gradually sank to 
 the bottom of the drop, and were not only physiologically 
 ' fixed ' by the Hermann's fluid, but also caused to adhere 
 closely to the slide. When the drop had evaporated nearly 
 to dryness, and the crystals were beginning to form round 
 the edge, every trace of vaseline was removed, and the slide 
 washed thoroughly with distilled water to get rid of the 
 crystals, the sugar, and as much of the ddbris as possible. 
 The slide was then placed in stain in a stoppered tube, con- 
 taining enough of the reagent to cover three-fourths of the 
 slide, and large enough to hold two slides placed back to back ; 
 the slide was then thoroughly washed again with distilled 
 water. As the use of alcohol over 3O/ o strength was found 
 to decolourize instantly, dehydrating was effected by drying 
 very completely in the cold. The preparation was then 
 treated with xylol and mounted in a solution of Canada 
 balsam in xylol. 
 
 In the case of preparations from culture fluids, the same 
 process was used with the omission of the vaseline, and more 
 care taken to remove all traces of sugar by washing.
 
 CHAPTEE VI 
 
 LIFE-HISTORY OP THE MICRO-ORGANISM AS DEDUCED FROM 
 CULTURES. 
 
 (a) The Coed as living in Spirit. 
 
 THE cocci occurred most frequently in pairs ; even in the 
 large fused groups and in chains two individuals were 
 generally seen to be more closely connected. Single cocci 
 were, however, also abundant, each surrounded by a thick 
 gelatinous envelope ; they were round in shape and appeared 
 perfectly homogenous. They varied considerably in size, 
 ranging from I /u, to 5 p. in diameter ; the gelatinous envelope 
 averaged f /x extra in thickness. In coloured rum the cocci 
 were always yellowish-brown, but, from our observations on 
 the cultivated form, we considered the tint of the spirit-living 
 cocci to be due to their taking up caramel from the colouring 
 matter. This view was confirmed by the fact that in un- 
 coloured spirit the cocci were all colourless, while a sample of 
 the colouring matter used was found to contain the brown 
 form only. 
 
 The refractive envelope surrounding the cocci, whether 
 singly or in groups, only attained great thickness in spirit, 
 and reminded one strongly of the similar envelope of 
 Leuconostoc mesenteroides, a bacterium described by Van 
 Tieghem and Cienkowski [6] as occurring in the beet-sugar 
 factories of Java. 
 
 The fact that the only reagent which would stain the 
 envelope was the same in both cases, viz. corallin, rendered 
 the resemblance more striking, and at first led us to infer a 
 relationship, especially as the optimum temperature for the 
 culture of both was the rather unusually high one of 35 C.
 
 LIFE-HISTORY OF THE MICRO-ORGANISM 35 
 
 Another point noticeable about the cocci in spirit (and in 
 certain cultures) was that large groups were often seen to 
 be surrounded by a yellowish cloud of slime (PI. I. Fig. in). 
 This cloud was not resolvable into elements by a magnifica- 
 tion of 1,200 diameters, but from observations on cultures 
 in which the same thing occurred, we came to the con- 
 clusion that the slimy cloud always contained, though it 
 did not consist of, cocci of infinitely small but variable sizes, 
 differing only from the ordinary kind in size and in their 
 degree of affinity for stains. The most minute hardly stained 
 at all, those slightly larger were less and less resistent as they 
 approached the full size. 
 
 Whether these minute cocci are to be regarded as spores is 
 a point to be considered hereafter. 
 
 Reproduction by transverse division in equal halves in a 
 plane at right angles to the direction of growth was the sole 
 method observed in the cocci while living in spirit ; this 
 frequently occurred with such rapidity that chains were formed, 
 also large groups, the units composing which were so imperfectly 
 separated from one another as to present an appearance not 
 unlike drops of some oily substance partially run together 
 (PL I. Fig. i, a, b). Division took place by a simultaneous 
 constriction of the gelatinous envelope and the enclosed coccus. 
 It is probable that this method of reproduction was the only 
 one practicable during life in spirit, on account of the necessity 
 for retaining a protective envelope of the necessary thickness. 
 
 (b) The Cocci as cultivated in Liquid Media. 
 The cocci as cultivated lost their brown colour in the course 
 of a few days ; they were then colourless and refractive, and 
 measured from i ^ to 4\ p in diameter ; in form they varied 
 from perfectly round to oval ; some were slightly pyriform. 
 No structure definitely distinguishable as a nucleus could 
 be detected ; some of the cocci appeared homogeneous, but 
 more generally they contained granules which, from their 
 staining reactions, were considered to be chromatin. The 
 gelatinous envelope was present in those grown in liquid 
 media, but was thinner and difficult to see ; cocci cultivated 
 D 2
 
 36 THE MICRO-ORGANISM OF FAULTY RUM 
 
 in gelatine (deep cultures) were entirely without the envelope. 
 These last-mentioned cocci were always surrounded by a 
 yellowish cloud, which was very slimy in consistency, and 
 exactly like that described above as sometimes surrounding 
 the cocci in rum. 
 
 Our culture media and methods of inoculation have been 
 fully described above ; the following is an historical account 
 of the results obtained. For inoculation purposes we always 
 used the cocci (i) from undiluted ruin, (2) from cultures, and 
 from both obtained the same results, namely, the production 
 firstly of a rod-like motile form, and secondly of a filamentous 
 growth, exhibiting true dichotomous branching, bearing cocci 
 which were subsequently constricted off and were apparently 
 capable of reproducing the same cycle. The controversial 
 questions entailed by the record of such a life-history are of 
 great scientific interest, and this part of the subject will be 
 found fully discussed in succeeding chapters. At present it is 
 our intention to confine ourselves to an exact relation of the 
 facts observed by us. 
 
 The rate of growth was remarkably slow ; at least six weeks, 
 and under unfavourable conditions a longer time, was required 
 to complete the cycle of development from coccus to coccus. 
 The sequence of events observed was as follows : in flask 
 cultures in liquid media, inoculated with more than one cell, 
 the cocci increased in number by transverse division in equal 
 halves in a plane at right angles to the direction of growth, 
 until a thick sediment, amounting in some cases to heaped 
 colonies, was formed. After a few days the liquid, originally 
 quite clear, presented a turbid appearance, accompanied, in 
 the case of coloured sugar solutions, by strong fluorescence 
 resembling that of faulty rum ; by the sixth day the culture was 
 swarming with actively motile rods, as figured in Plate III. 
 Fig. xiv. The length of the rods varied from 5-9 /z, single seg- 
 ments averaging a|/x and their thickness nearly i p. they had 
 a peculiar and characteristic appearance due to differentiation 
 into highly and less refractive portions, and it is notable that 
 in this respect they corresponded in appearance and staining 
 reactions both with the cocci and the filaments.
 
 LIFE-HISTORY OF THE MICRO-ORGANISM 37 
 
 At first we considered these rods to be an impurity, and 
 threw away every culture which contained them ; we were, 
 however, struck by the fact (i) that they persistently occurred 
 in every bacterially filtered liquid medium used except milk 
 sugar, (2) that they were developed in liquids inoculated both 
 from previous cultures and from rum direct, (3) they appeared 
 in diluted uncoloured rum some weeks after dilution, though 
 not in the boiled water used for dilution nor in the undiluted 
 spirit, but (4) never in blank controls. In consequence of these 
 observations, instead of throwing away the cultures which 
 developed rods, we put them aside in the incubator, apart from 
 the others, and allowed the development to take its course. 
 
 Observation then showed that, in the course of a few days, 
 during which the rods increased both in length and thickness 
 and resembled single floating filaments, sometimes branched, 
 they became entangled in groups, which at first remained 
 suspended in the liquid, but ultimately, as they became larger 
 and heavier, sank to the bottom of the flask. When this took 
 place, the liquid became clear. 
 
 The filaments, matted together, grew radially, thus giving 
 rise to the ball-like growth seen in Plate IV. Fig. xv. In every 
 case such coccus- bearing filaments were ultimately developed, 
 and were identical with those grown in milk sugar, in which 
 medium no rods occurred. The inference, therefore, seemed 
 unavoidable, that we were dealing not with an impurity, but a 
 stage in the life-history of the micro-organism of faulty rum ; 
 but this inference was set aside until a successful method of 
 single-cell culture could be devised, or until we could prove by 
 direct microscopical observation of a single individual that the 
 rod-like form belonged to the normal cycle. At the earlier 
 stage of the investigations, owing to the special difficulties 
 described above, we had not been successful with single-cell 
 methods, and these difficulties were further increased by the fact 
 that we were dealing with a motile form; our ultimate success 
 will be described under the head of single-cell cultures. 
 
 In the case of milk-sugar cultures, in which no rods 
 appeared, the cocci increased in number by division until they 
 formed a thick sediment and eventually round heaped colonies. 
 
 2206G9
 
 38 THE MICRO-ORGANISM OF FAULTY RUM 
 
 Under favourable conditions, viz. in not too concentrated 
 solutions, these heaped colonies developed filaments, which 
 grew very slowly, ultimately attaining a length of nearly half 
 an inch. The earliest period at which filaments began to 
 develop was ten days after inoculation ; they were first 
 observed in cultures which had been kept under glass in 
 direct sunlight, and consequently at a varying temperature. 
 In the case of cultures kept in the dark at a constant tempera- 
 ture, the filamentous growth generally developed later and 
 more sparingly. In certain of the cultures kept under glass 
 and exposed to sunlight, but inoculated with a double dose of 
 the coccus-containing liquid, the filamentous stage was entirely 
 suppressed, though the stage immediately preceding it (marked 
 by the formation of heaped colonies) was reached. After seven 
 weeks' growth sufficient nutrient medium was added to bring 
 these cultures up to the same proportion as those which had 
 received the single dose at inoculation, and in all cases a 
 growth of filaments then took place immediately, and the 
 normal cycle was completed. The filaments almost always 
 remained submerged, and generally anchored to the parent 
 heap, but portions artificially detached continued to grow 
 radially while floating freely in the nutrient medium. In one 
 culture only (in a 5/ o solution of milk sugar) the cocci, instead 
 of becoming heaped at the bottom, formed a scum on the 
 surface, and in this case the filaments grew downwards into 
 the liquid, and the exposed surface of the colony assumed 
 a smooth waxy appearance. 
 
 The filaments, as seen when removed from the nutrient 
 medium into distilled water, were of the purest white when 
 fresh ; old cultures sometimes acquired a blackish-green tinge. 
 They exhibited true dichotomous branching (PI. II. Fig. v), 
 possessed a distinct double-contoured sheath, and were divided 
 by transverse septa into segments of varying length, which 
 contained or bore cocci. These septa were straight in young 
 branches, but in older filaments showed a tendency to become 
 concave, the concave surface being invariably directed towards 
 the terminal end of the filament. The contour of a full-grown 
 colony was almost globular, the filaments radiating towards
 
 LIFE-HISTORY OF THE MICRO-ORGANISM 39 
 
 the periphery from an apparently denser central mass, and 
 presenting somewhat the appearance of a ball of swansdown. 
 The filaments were slimy to the touch, and interlaced closely 
 and tenaciously, but never anastomosed, so that they could 
 hardly be said to form a true mycelium. Nor was there any 
 real morphological difference between those filaments which 
 bore cocci and some which did not, though the goblet-like 
 swelling of some filaments, when cocci were being constricted 
 off from their tips, gave rise to an appearance resembling 
 the conidiophores in certain moulds. The cocci did not lie 
 loose within the filaments, for pressure on the cover-glass 
 did not cause them to move along, nor could they have been 
 extruded at the tip, as in Crenothrix, since the septa would 
 stop the egress of all but the cocci in the terminal segment. 
 The substance of the filament was perfectly continuous with 
 the envelope of the cocci, as was plainly evident where the 
 latter were being constricted off. This process took place at 
 any point in the length of the filament as well as at the tip 
 (PI. II. Figs, vin, ix, xn), and varying numbers of cocci, from 
 single individuals to chains of six or more, were nipped off at 
 a time from one point. 
 
 The cocci thus thrown off were very large, had very thick 
 envelopes derived from the substance of the filament, and 
 generally contained from one to five minute colourless roundish 
 bodies, which were often extruded afterwards by the bursting 
 of the envelope surrounding the parent-coccus (PL II. Fig. ix, c). 
 These bodies were homogeneous and highly refractive in a 
 fresh preparation, but stained very deeply and then appeared 
 granular. The filaments were tapering in form towards the 
 tip, and extremely variable in thickness, viz. from i to 3 /u in 
 different branches of the same growth. The youngest colony 
 removable on the point of a needle is figured on Plate VII 
 Fig. xx. 
 
 Of liquid media other than milk sugar, the solution of coarse 
 black sugar, boiled, steam sterilized, filtered once through 
 paper, and twice through a Muncke's filter, was found to be 
 unquestionably the best, and our most successful cultures were 
 made in the 5/ solution.
 
 4 o THE MICRO-ORGANISM OF FAULTY RUM 
 
 The filaments were abundantly developed in this, markedly 
 the best in flasks incubated in the light at 35 C., next best 
 in those exposed to direct sunlight under glass at a varying 
 temperature, while in flasks incubated at 35 C. in the dark 
 there was very late development of filaments and much slower 
 growth. 
 
 In every culture without exception, and consequently in 
 every medium used, as well as from inoculations of the most 
 varied character possible, the sequence of events was as 
 described, and filaments were developed. 
 
 The Cocci as cultivated on Solid Media. 
 
 The following- results were obtained in deep cultures in 
 test tubes inoculated as described on p. 29. 
 
 Growth was marked by the appearance of a slight cloud, 
 which became more and more defined, and consisted of cocci 
 surrounded by a yellowish slime, so glutinous in consistency 
 that it was difficult to remove portions for examination with- 
 out extracting the whole mass. The gelatine without sugar 
 gave, contrary to our expectation, the best growth, but the 
 cocci were devoid of any envelope, yellowish in colour, and 
 very small, and no filaments were ever developed. 
 
 Transplanted with a very thin layer of gelatine on to 
 microscope slides, growth took place freely, and the colonies 
 formed presented very curious and interesting features. When 
 thus growing on the gelatine without a cover-glass, the young 
 colonies exhibited a raised corrugated surface, water-grey in 
 colour, with elevations of a most peculiar crater-like shape 
 (PI. III. Fig. xin), which we believe to be characteristic 
 of no other micro-organism hitherto described. The crater- 
 shaped central hollows afterwards tended to fill up, so that 
 older colonies assumed a more rounded contour, the corrugations 
 of the surface gave a convoluted or arborescent appearance 
 (ibid.). The whole formation was perfectly hard and glassy ; if 
 scratched with a sharp-pointed knife, both the sound and the 
 scratch were like those made by a skate on ice. The colonies 
 first became visible as minute rosette-like specks, with a 
 central 'crater-' like hollow ; always surrounding the periphery
 
 LIFE-HISTORY OF THE MICRO-ORGANISM 41 
 
 was a fringe of highly refractive processes with peculiar three- 
 knobbed growing-tips (PL III. Fig. xni, a, b) ; these knobs later 
 grew out as branches. As this fringe grew from underneath the 
 colony, and the latter was opaque, it was not possible to deter- 
 mine the earliest origin of these processes, but they were con- 
 sidered as corresponding to the filamentous stage in sugar. Nor 
 was it easy to determine with exactness the mode of formation 
 of the ' crater,' because the layer of gelatine, though thin, was 
 semi-opaque, and the cocci were embedded in it ; also because, 
 from the nature of the colony, a cover-glass (and consequently 
 an oil-immersion objective) could not be applied. It appeared, 
 however, from observation with the highest powers possible 
 under the circumstances (800 diameters), that the ' craters ' 
 were formed by the juxtaposition of cocci, cemented together 
 by a slimy secretion, which hardened into the glassy ice-like 
 substance above described. Probably the latter substance 
 and the slime previously observed were identical in nature, 
 and should be considered as different states of the same 
 secretion. Slide-cultures made under cover-glasses yielded 
 exactly the same results as test-tube cultures, but where some 
 of the cocci accidentally extended beyond the edge of an 
 unsealed cover-glass, crater colonies were again formed. With 
 the view of investigating this formation more closely, a great 
 number of cover-glasses were spread with the thinnest possible 
 layer of gelatine by pouring the fluid gelatine on them while 
 hot, draining off again, and then allowing the remaining 
 gelatine to set, and inoculating with cocci from various rums. 
 Some of these cover-glasses were then placed, gelatine side 
 downwards, over glass cells such as were used for moist 
 chambers, so that the development of the craters might be 
 observed from the under side, with the oil immersion. As the 
 gelatine was in contact with the air in the cell craters were 
 formed, and our observations showed that they arose by the 
 hardening of the slime secreted by, and immediately surround- 
 ing, each coccus. For some reason the coccus was not at 
 first covered completely by the slime, hence the formation 
 of the crater-like central hollows in young growths, which 
 corresponded to the position of the coccus. The slime, how-
 
 42 THE MICRO-ORGANISM OF FAULTY RUM 
 
 ever, hardened more quickly, and consequently more craters 
 were formed on cover-glasses, which were kept surface-upwards 
 under a large glass vessel, where they had access to more air. 
 
 Knobbed processes were not observed under these conditions, 
 perhaps owing to the very small amount of nutrient medium, 
 which was insufficient for their development. 
 
 Surface-cultures were tried on gypsum blocks moistened 
 with (i) 5% solution of black sugar, (2) 5% solution of cane 
 sugar with a trace of microcosmic salt, and (3) 5% solution of 
 cane sugar without salts. These were inoculated, as in other 
 experiments, both from rum direct and from previous cultures, 
 but in no case was any growth obtained. 
 
 CULTURE FOE THE OBSERVATION OF SINGLE CELLS. 
 I. On Solid Media. 
 
 For shake-cultures in Petri's capsules a measured quantity 
 of the infected rum was shaken up in a small quantity of 
 liquid gelatine, which was then allowed to solidify in a very 
 thin layer. Various brands of rum, both white and coloured, 
 were used for inoculation. From all we obtained a growth of 
 filaments identical with those grown in black sugar ; and were 
 for the first time able to trace under the microscope the 
 development of these from a single cell (PI. I. Fig. iv), and 
 thus to complete the life-history from coccus to coccus. 
 
 The shaking after inoculation was done very thoroughly, in 
 order to separate the cocci as much as possible. After the 
 gelatine had set, the Petri-dish was placed under the microscope 
 and searched for cells which were sufficiently isolated for each 
 to develop without interfering with others. When a cell 
 thus isolated was found, its position was marked by a circle 
 drawn round it with a needle on the surface of the gelatine, 
 and it was kept under direct observation under the microscope. 
 
 The mode of development was as follows : the coccus became 
 of a long oval in shape, then from one end (or sometimes both) 
 a tube-like filament was developed, in which septa were visible 
 as transverse lines (Plate I. Fig. iv, a, c) ; when the filament
 
 LIFE-HISTORY OF THE MICRO-ORGANISM 43 
 
 was about three segments long, it began to branch by dicho- 
 tomous division of the terminal segment (Plate I. Fig. iv, c), 
 the branches divided again, and more were developed by out- 
 growths from some one or other of the basal segments of the 
 main stem. The order of succession was thus quite irregular, 
 and there was nothing like acropetal growth. The develop- 
 ment was the same throughout as that in milk sugar. 
 The filaments, by interlacing closely, soon formed a dense 
 white circular mass on the surface of and embedded in the 
 gelatine (PI. VII. Fig. xxi). Old cultures tended to become 
 a blackish-green, and in every case the colonies produced a 
 very strong and disagreeable smell, resembling that of valeric 
 acid, a property not observed in any previous cultures. 
 
 II. In Liquid Media. 
 
 A. Transplanted to Flasks. 
 
 The method by which single cells were isolated on gelatine 
 and transferred to flasks containing various liquid media has 
 been described in Chapter V. The results obtained with these 
 cultures agreed in every respect with those obtained in flasks 
 containing similar media inoculated with more than one cell, 
 and thus afforded tolerably satisfactory evidence of the purity 
 of our earlier cultures. In every case the sequence of events 
 observed was the same as in the earlier liquid cultures, other 
 than milk sugar ; the liquid became turbid, and by the sixth 
 day contained numbers of the characteristic motile rods, which 
 became entangled in groups, sank to the bottom, and gave rise 
 to balls of filaments identical with those previously obseryed. 
 Preparations were made both of rods and filaments from 
 single-cell cultures, and stained with the same reagents. Other 
 flasks were inoculated from the produce of the single cells. 
 
 B. Sown in Drops. 
 
 In order to obtain, if possible, direct proof that (i) the 
 individual cocci developed into rods, and (2) the rods into 
 filaments, the following series of cultures were made and kept
 
 44 THE MICRO-ORGANISM OF FAULTY RUM 
 
 under direct observation with the microscope. Slides with a 
 large central cavity were used, and the hollow of each was filled 
 with the nutrient medium, and inoculated with as few as 
 possible of the cocci, obtained by the dilution method, and 
 either introduced directly into the drop or dried on the cover- 
 glass. The drop was covered with a cover-glass ruled in 
 squares, and the squares in some cases were further subdivided 
 by marking with paint, in order to facilitate orientation. The 
 cover-glass was carefully sealed with soluble glass on to the 
 slide to prevent currents of evaporation, and the slide then 
 examined under the microscope for isolated cocci. One was 
 selected and its position noted by the squares on the glass ; 
 the slide was then clipped firmly in position and kept under 
 observation for several days consecutively, the whole micro- 
 scope, while not in use, being covered with a glass shade to 
 prevent disturbance. The result of this experiment was that 
 the coccus remained in statu quo when the last observation 
 was taken at night, but by the next morning it was not 
 visible in the field of view. Repeated trials always led to the 
 same result. Yet the coccus is not motile. 
 
 As we desired to avoid all possible source of error, a slightly 
 different method was adopted. A plain cover-glass was used, 
 not ruled in squares, and the position of the coccus at the 
 beginning of the experiment was noted by means of a Leitz 
 camera lucida. The advantage of this particular camera for 
 the purpose was its consolidation with the eye-piece, and as 
 both together could be screwed into place, there was no possi- 
 bility of moving after adjustment ; further, its use obviated 
 the necessity of a sloping board for the drawing paper, which 
 could be pinned flat upon the table and thus could not slip 
 out of place. 
 
 When the hollowed slide had been clipped firmly in position 
 the isolated coccus was drawn on a card pinned to the table, 
 and a small circle drawn around it. If there were any move- 
 ment or alteration in shape of the object drawn, obviously its 
 contour then would no longer coincide with the outline 
 drawn, and thus the slightest displacement would be visible. 
 The whole apparatus was left in place for several days, and
 
 LIFE-HISTORY OF THE MICRO-ORGANISM 45 
 
 as a result it was found that when the last observation was 
 taken one night the coccus had become slightly elongated, and 
 by the next morning it had disappeared from within the 
 pencilled circle, and immediately outside the latter there 
 appeared a small rod in active movement, which was not 
 there at the outset of the observation. This experiment was 
 repeated eight times with single cocci, and once with five 
 cocci drawn in the field at once. On every occasion there 
 was the same sequence of events, namely, the cocci became 
 elongated, and after a certain number of hours no cocci were 
 visible, but a corresponding number of motile rods were found 
 close to the positions previously occupied by the cocci. The 
 inference, therefore, that a transformation of cocci into rods 
 took place, was very strong, but owing to the extremely 
 slow development of the organism, the uninterrupted watch 
 necessary for absolute proof of the transformation would have 
 required a number of skilled observers taking turns for several 
 hours apiece during several days. The camera method above 
 described was also adopted for the investigation of the further 
 question whether rods developed into filaments. For these 
 experiments the cavities of the slides used were larger, as it 
 was necessary to provide the rods with a large bulk of 
 nutrient medium. It was thus observed that the rods 
 increased in length and thickness, became entangled in 
 groups, and sank to the bottom, where, owing to the depth 
 of the hollow, the further development was beyond reach of 
 observation with high powers. As far, however, as the 
 sequence of events could be followed, it will be obvious that 
 the results accorded with the earlier observations.
 
 CHAPTER VII 
 
 BIOLOGICAL AND CHEMICAL CHARACTERISTICS 
 
 Conditions of Life. 
 /. With relation to Oxygen. 
 
 As this bacterium survives for a length of time at the 
 bottom of vats or puncheons of spirits, it is clear that it must 
 be potentially anaerobic, as the amount of oxygen dissolved 
 in the spirit is quite insignificant ; our experiments indicated 
 that it is preferentially aerobic, as it developed rapidly upon 
 the thin surface of gelatine on a microscope slide without 
 cover-glass or in Petri-dishes, but did not advance beyond the 
 coccus stage when immersed in deep layers of gelatine, or in 
 undiluted rum. 
 
 II. With relation to Light. 
 
 It has already been observed above that direct sunlight, 
 instead of being inimical, appears rather to favour the 
 development of this bacterium ; this is probably to be 
 expected of an organism of tropical habitat. Further, it 
 appeared that in the rod stage it was even heliotropic, 
 though at present we are unable to decide whether this 
 condition results from luminous or non-luminous rays. 
 Some observers (Migula) [7] have arrived at the general 
 conclusion that though sunlight prevents the development 
 of strictly pathogenic bacteria, yet the ordinary species of 
 saprophytes develop as readily in diffuse daylight as in 
 darkness.
 
 BIOLOGICAL AND CHEMICAL CHARACTERISTICS 47 
 
 ///. With relation to Alcohol. 
 
 In the previous chapters we have alluded to the fact at 
 once novel and remarkable of the survival of life in alcoholic 
 liquids containing upwards of 7o/ of spirit. 
 
 Strictly speaking, the organism survives not in the alcohol, 
 but in its gelatinous envelope, thus living, as it were, in 
 a state of siege in its own castle, through the walls of which 
 it can obtain its necessary supplies of food in the form of 
 sugar, while keeping out its enemy alcohol. 
 
 It has been known since the work of Dutrochet [8], in 
 the year 1827, confirmed by Graham [9], that of the com- 
 moner carbon compounds alcohol possesses the least, but 
 sugar the most, power of endosmosis or exosmosis through 
 animal membranes. In this connexion the well-known 
 observation of Sommering [10] and others may be men- 
 tioned, viz. that aqueous alcohol contained in a bladder 
 and hung up in a warm room loses water by osmosis and 
 consequent evaporation, nearly absolute alcohol remaining 
 behind ; this method was even formerly applied commercially, 
 and we believe it to be still in use in some hot climates. 
 We are not prepared to state that the bacterium, even though 
 protected, can survive indefinitely in spirit, but we are still 
 obtaining successful cultures from faulty rum, which has been 
 in our possession for eighteen months. 
 
 Two experiments were conducted to ascertain the propor- 
 tion of alcohol fatal to the bacterium ; saturated solutions 
 of cane sugar in absolute and 9O/ o alcohol respectively were 
 prepared, and 20 c.c. of each solution were inoculated with 
 25 c.c. of rum containing an abundance .of cocci. After incuba- 
 tion for a few days the cocci were found to be dead, as they 
 were shrivelled up, and no growth could be obtained when 
 transferred to favourable media. It would thus appear that 
 the limiting proportion of alcohol in which the organism will 
 continue to survive even for a short time is intermediate 
 between 75 and 9o/ o , and this limit is confirmed by the 
 experience of the manufacturers, who have found occasionally 
 that if the spirit is brought over at a rather higher strength
 
 48 THE MICRO-ORGANISM OF FAULTY RUM 
 
 the evil, arising from the reintroduction of the organism by 
 the colouring matter or otherwise, is to a greater or less 
 degree overcome. 
 
 Pathogenicity. 
 
 In order to determine if the bacterium possessed any 
 pathogenic properties, small portions of two separate cultures 
 in gelatine were rubbed up with sterilized distilled water ; 
 the extract was injected subcutaneously into a guinea-pig. 
 Up to the present time, viz. some months after the experi- 
 ment, no effect has resulted, as the animal has remained in 
 a normal state of health throughout the whole of the period ; 
 it is, therefore, improbable that any effect would be produced 
 upon the human organism. 
 
 We are indebted to the kindness of Dr. J. Ritchie for 
 performing the above experiment for us. 
 
 Habitat. 
 
 Unfortunately we have been unable to obtain from the 
 planters sufficiently definite information to enable us to trace 
 out the original habitat of the micro-organism. 
 
 We consider it probable that it is not truly indigenous, but 
 was accidentally imported ; it has certainly found a nidus in 
 the sugarcane extracts, and is reintroduced after distillation 
 with the caramel used for colouring or from infected vessels. 
 Possibly it may occur in the water supply, but in our opinion 
 the latter is not the most probable source. We have found 
 that the cocci may be desiccated and air-borne, as our great 
 difficulty, at the time when samples were being examined 
 and cultures made, was to keep out the organism from any 
 vessel in the laboratory, which was exposed to air. 
 
 The fact that some cocci, which had dropped off microscopical 
 preparations while these were being placed in the staining- 
 tubes, gave rise to vigorous growths in several aqueous 
 stains, showed that the organism possessed extraordinary 
 powers of resistance, even after having been dried and treated 
 with Hermann's fluid.
 
 BIOLOGICAL AND CHEMICAL CHARACTERISTICS 49 
 
 Chemical Changes. 
 Production of an Acid. 
 
 In the course of the preceding chapters it has been shown, 
 firstly, that the organism probably increases to a slight extent 
 the proportion of acid in diluted faulty rum ; and, secondly, 
 that a sample of caramel containing the organism was acid, 
 while another free from it was neutral. 
 
 Further, it was frequently observed in the course of the 
 investigation that the various culture media, originally of 
 neutral, became markedly of an acid reaction. In order to 
 test this point weighed quantities of calcium carbonate were 
 introduced into two (5/J solutions of sterilized cane sugar, 
 one of which was inoculated with filaments and the other 
 with cocci from previous cultures in raw sugar, both being 
 kept in the incubator at a constant temperature of 35 C. for 
 nineteen days. In both cases there was an abundance of 
 growth, and a proportion of the calcium carbonate was found 
 to be used up by neutralization, as shown by the following 
 
 results : 
 
 Experiment I. Experiment II. 
 
 Cocci. Filaments. 
 
 Weight of calcium carbonate introduced . -508 gram. -502 gram. 
 
 recovered . -465 '4559 
 
 Percentage neutralized. 8-47 9-19 
 
 The results obtained in the two experiments are fairly 
 concordant, and though in the process of inoculation a trace 
 of free acid was of necessity introduced, yet this may be con- 
 sidered as negligible. 
 
 A similar experiment was conducted on a larger scale, 
 namely, with 10 grams of calcium carbonate and half a litre 
 of a 5^ sugar solution, with a view of isolating the acid or 
 one of its salts ; this was inoculated with cocci taken direct 
 from the rum, and the whole mixture allowed to stand for five 
 weeks. In this case there was a certain development on the 
 side of the vessel nearest to, but comparatively little on 
 the side removed from, the light, and the solution had become 
 fluorescent. The mixture was then evaporated to dryness 
 over the water-bath, and the residue frequently extracted 
 E
 
 5 o THE MICRO-ORGANISM OF FAULTY RUM 
 
 with hot water to dissolve out any calcium salt of an organic 
 acid, and the solution subsequently evaporated, but it was 
 not found possible to obtain a sufficient quantity of salt for 
 identification. 
 
 The odour of the gelatine cultures in the Petri-dishes 
 mentioned above tended to show that the acid produced was 
 probably valeric, and not lactic, butyric, or acetic acids, which 
 are more commonly formed. 
 
 Reduction of Nitrates. 
 
 In order to examine if the micro-organism possesses the 
 property of reducing nitrates to nitrites or ammonia, the 
 culture liquids were made up according to those used by 
 P. F. Frankland in his investigations upon the subject 
 [iij. It was proved that the liquids, previous to inoculation, 
 were free from nitrites by the metaphenylene-diamine 
 reaction, and from ammonia by Nessler's solution. After 
 inoculation for forty days, and production of a vigorous 
 growth, the liquids were again examined ; no nitrites were 
 found, but plenty of ammonia in the first portions which 
 came over on distillation. As the quantity of ammonia was 
 out of proportion to the peptone used, it is probable that 
 its formation is due to the reduction of the nitrate by the 
 organism, and not wholly to the decomposition of the 
 peptone. 
 
 Hydrolysis of Starch. 
 
 Experiments made with this view led to negative results. 
 
 Production of Colouring Matter. 
 
 It has already been pointed out that the bacterium assimi- 
 lates the fluorescent colouring matter from caramel and black 
 sugar, but it appears that under certain circumstances it can 
 of itself form a colouring matter. Thus all the growths in 
 gelatine, even when sown with cocci from white rum, became 
 yellow or reddish yellow with a distinct green fluorescence, 
 these being the characteristics of Migula's [12] first group of 
 colouring bacteria. In order to afford further confirmation,
 
 BIOLOGICAL AND CHEMICAL CHARACTERISTICS 51 
 
 a gelatine culture, sown with white rum, was extracted 
 with water, when a yellow solution was obtained with 
 a slight green fluorescence ; the solution was divided into 
 two equal portions, to one of which a solution of potash was 
 added, which produced a deep moss-green colour, while the 
 other portion was diluted with an equal bulk of water by 
 way of comparison (Thumm's [13] reaction). Zopfs [14] 
 lypocyanine reaction with concentrated sulphuric acid gave 
 a negative result, thus showing that the colouring matter 
 was not a lipochrome, hence the organism did not belong to 
 Migula's second group. In another old culture containing 
 peptone a bright yellow colouring matter was found which 
 turned a deep red on addition of alkali; it is merely 
 desired to place this last observation on record without 
 however wishing to lay stress upon it. 
 
 Staining Reactions. 
 
 The cocci in coloured rum stained well with gentian violet, 
 and with Gram's method, but, owing to the deep yellow 
 colour already present, only such very powerful stains were 
 at all effectual on these ; the colourless cocci from uncoloured 
 rum stained more intensely with Gram's method. The 
 bacteria parted with stains, however, as readily as they took 
 them up ; treatment with clove oil or alcohol of over 30% 
 strength decolourized them instantly. The only exceptions to 
 this rule were preparations which had been purposely much 
 overstained with gentian violet, and those treated with 
 a strong mordant, as in Gram's method. The cultivated cocci 
 stained readily with nearly all the stains mentioned on p. 32, 
 except with eosin, fuchsin, and fuchsin S, which were 
 practically found to *be useless, however long applied. The 
 best results were obtained with a double stain of gentian 
 violet and orange G, and with Haidenhain's iron-haematoxylin ; 
 the gelatinous envelope of the cocci remained unstained, 
 except with aqueous corallin. It was not found possible to 
 obtain a contrast stain for the cocci, on account of the readi- 
 ness with which the corallin was extracted by other reagents. 
 Two points were noteworthy with regard to the behaviour of 
 
 E 2
 
 52 THE MICRO-ORGANISM OF FAULTY RUM 
 
 the bacteria with reagents, viz. (i) preparations of cocci and 
 filaments had the effect of precipitating many stains especially 
 eosin, iron-haematoxylin, and methyl-green so rapidly that 
 these could seldom be used a second time without filtering ; 
 and (2) the bacteria invariably stained a beautiful blue with 
 iodine green and methyl-green, both of which should give 
 a green stain ; the blue appeared on the first washing with 
 distilled water. The inner substance of the filaments stained 
 lightly with most of the reagents used, but the sheath was 
 quite unstainable except with Gram's strong iodine, and with 
 aqueous corallin ; the latter stained this structure only, 
 leaving the contained cocci untouched. Gram's strong iodine 
 stained the sheath of the filaments bright yellow, but sub- 
 sequent treatment with sulphuric acid produced no blue 
 colouration. 
 
 The cocci constricted off from the filaments, whether 
 terminally or laterally, were identical in size and appearance 
 with the original cocci, and stained in precisely the same 
 manner with the same reagents. The small bodies extruded 
 by these cocci stained intensely with almost all reagents 
 used (PI. II. Fig. ix, d) and then appeared granular. Cocci 
 which had extruded such bodies stained much paler than 
 those which had not (ibid., a, 6). 
 
 This affinity of the small granular bodies for stains indicated 
 that they were not of the nature of spores. 
 
 The rod or bacillar forms stained intensely violet with 
 Gram's method, and also with Ziehl-Neelsen's carbol-fuchsin, 
 applied hot. With the former the stain was uniform, and too 
 heavy to show any structural details, but with carbol-fuchsin 
 the rods showed very well their characteristic division into 
 highly refractive and less refractive portions, the latter staining 
 purplish brown, while the former remained unstained (see 
 PI. III. Fig. xiv). Iron-haematoxylin showed a similar 
 differentiation. Loffler's method for staining flagella was 
 also used for the rods, but though the modifications of this 
 method suggested by Winogradsky [15] and Moore [16] 
 were both tried, we did not succeed in showing the presence 
 of flagella.
 
 CHAPTER VIII 
 
 DISCUSSION OF CONCLUSIONS 
 
 IT will be seen from the preceding chapters that if the life- 
 history of the micro-organism is as described by us, it presents 
 points of resemblance to various groups, and that consequently 
 the determination of its true systematic position is very 
 difficult. The question depends wholly upon the purity of our 
 cultures, and we have described in the fullest detail every ex- 
 periment performed and every method used, in order that those 
 of our readers, who are bacteriologists with practical experience, 
 may be enabled not only to judge whether these methods 
 were reliable but also to repeat them, if so desired. 
 
 Meanwhile it is clear that, with regard to the results of our 
 investigation, three views could be held legitimately. 
 
 (1) The cocci are the conidia of a mycelium-forming 
 hyphomycete (mould) and the rods are bacterial impurities. 
 
 (2) The cocci are bacterial in nature and become rods as 
 described by us, but the filaments belong to a hyphomycete. 
 
 (3) The cocci, the rods, and the filaments are phases in 
 the life-history of a single organism belonging to the higher 
 bacteria, and probably allied to the Streptothrix group. 
 
 The arguments for and against each of these views will be 
 fully discussed seriatim, though, owing to the special diffi- 
 culties often alluded to above, positive proof has not here been 
 brought forward. 
 
 First View. 
 
 In favour of this it may be said that (i) the cocci as in rum 
 are variable in size, and generally large as compared with 
 the majority of bacterial cocci, (2) they are able to reproduce 
 the cycle without the interpolation of spores, (3) there is the
 
 54 THE MICRO-ORGANISM OF FAULTY RUM 
 
 direct microscopical evidence of their continuity in develop- 
 ment with the filaments, (4) there is also the possibility 
 (always present) of the introduction of the spores of a foreign 
 bacillar organism either at the moment of inoculation or in 
 the inoculating substance itself. 
 
 Against these points the following arguments may be 
 advanced : (i) the cocci, though large for bacteria, are certainly 
 somewhat small for conidia ; (2) the character of their division ; 
 (3) the ability to reproduce the cycle directly from conidia- 
 like bodies is equally characteristic of the higher bacteria of the 
 Streptothrix group, and the strong inference i'rom facts observed 
 that the cocci as in rum become transformed into rods ; (4) the 
 difficulty of imagining the rods to be impurities, considering 
 the circumstances under which they occur, the precautions 
 taken, as well as the absence of any other direct evidence 
 of the existence of a second organism, unless as invisible 
 spores, in the undiluted spirit. 
 
 Second View. 
 
 The arguments in favour of this may be summarized as 
 follows (i) The reproduction of the cocci by transverse 
 fission, the products being at the time of division of equal 
 size, this mode being characteristic of the Schizomycetes 
 (bacteria) ; (2) their staining reactions in which they resemble 
 bacteria ; (3) the observations above alluded to, showing that 
 the probability of the transformation of cocci into rods almost 
 amounted to a certainty. 
 
 This point has further been confirmed by Professor Emil. 
 Chr. Hansen, who very kindly made a few experiments with 
 some of our materials (namely, coloured and uncoloured un- 
 diluted faulty rum and some of our cultures) with which we 
 presented him. He wrote to us as follows ' Your observation 
 of the coccus becoming elongate and going into the form of 
 rods is probably quite correct,' and added that in the few 
 trials he was able to make with our materials, the ' rods and 
 the coccus developed only bacteria.' Professor Hansen did not, 
 however, agree with our conclusions as to the filaments, and
 
 DISCUSSION OF CONCLUSIONS 55 
 
 as his opinion is the only strong evidence we have in favour 
 of the second view, and against the genetic connexion of these 
 filaments with the rods and cocci, we think it best to quote 
 his experiments as they were made known to us in corre- 
 spondence. After having invited our attention to the fact 
 that the filamentous growth ' might represent a phase in the 
 development of a Penicillium species,' he wrote to us later, 
 * An experiment with filaments from the cultures you left 
 here (cultivation on wort gelatine 25 C.) showed that the 
 filaments really formed a Penicillium form.' In a second 
 letter he wrote that in his experiments 'the hyphomycetous 
 fungus (the mycelium form) only developed this form but not 
 bacteria.' 
 
 On the other hand, he was willing to admit that under the 
 circumstances the problem, whether one or two organisms 
 were present, was very difficult, would require a long time, 
 and might even prove insoluble, as far as attaining positive 
 evidence was concerned. 
 
 The Third View. 
 
 The idea of a genetic connexion between the bacteria and 
 moulds (Schizomycetes and Hyphomycetes) is, pf course, not 
 novel, but has been advanced at different times by several 
 investigators. It must be admitted that hitherto, when sub- 
 jected to strict experimental proof, sufficient evidence has not 
 been forthcoming. A work recently published by A, Klocker 
 andH. Schionning [17] shows that the'moderne Pleomorphie- 
 Wut,' to use their not inapt phrase, has even resulted in 
 assertions that certain moulds can produce both yeasts and 
 bacteria, though the originator of these views failed to satisfy 
 the legitimate inquiries into his materials and methods. 
 
 The question, however, of a possible bacterial link between 
 truly branching filamentous forms such as Streptothrix 
 (originally known as Actinomyces), now regarded by most 
 writers as true bacteria, and the Hyphomycetes is, we venture 
 to think, still an open one, and the study of new and aberrant 
 forms, such as the organism now before us, may furnish
 
 56 THE MICRO-ORGANISM OF FAULTY RUM 
 
 possibly some means for arriving at a decision. The 
 resemblances between our organism and the group associated 
 by Migula under the title of Chlamydobacteriaceae, in which 
 are included Streptothrix and others, are in many respects 
 most striking. The following passage in his work (loc. cit.) 
 may not inaptly be quoted : 
 
 ' Bei der am hbchsten entwickelten Gruppe der Bacterien, 
 den Chlamydobacteriaceen, scheint eine Endosporenbildung 
 vollkommen zu fehlen, dagegen kommt eine Form der 
 Fortpflanzung vor, die zwar bei den einzelnen Gattungen sehr 
 verschieden entwickelt ist, doch aber allgemein an die 
 Abschniirung von Gonidien bei den Pilzen erinnert und 
 offenbar physiologisch dieselbe Bedeutung hat. Als allge- 
 meines Charakteristicum der Bakteriengonidien kann man 
 ihre Ablosung von dem Mutterfaden zum Zweck der 
 Verrnehrung der Individuen ansehen ; sie stellen keine 
 Dauerformen dar, sondern wachsen auf demselben Nahrboden, 
 resp. in derselben Fllissigkeit, in der Regel schon nach kurzer 
 Zeit zu neuen Zellfaden aus. Sie unterscheiden sich von den 
 Endosporen sowohl dadurch, dass sie nicht im Innern von 
 vegetiven Zellen gebildet werden, als auch dadurch, dass sie 
 keinen mit Abstreifung oder uberhaupt Abhebung einer dis- 
 tinkten Sporenrnembran verbundenen Keimungsprozess durch- 
 machen und wie erwahnt nicht als Dauerzellen zu betrachten 
 sind.' 
 
 The production of slime, the occurrence of motile rods, and 
 of true branching are further points of agreement. On the 
 other hand, though there are great resemblances in our 
 organism to certain known Hyphomycetes, and especially to 
 that described years ago by Fresenius [20] under the name 
 of ' Torula rufescens,' which would now be called a species of 
 Penicillium, yet there are also marked points of difference, 
 especially in the branching of the conidia-chains in Torula. 
 As far as we could observe, neither budding nor conjugation, 
 nor formation of zygospores occurred in our organism ; further, 
 the mycelium is not divisible into sterile and fertile portions, 
 nor can it be said to possess distinct organs of fructification, 
 since the cocci are often produced laterally at any point as 
 well as terminally upon the filaments. We are informed, how- 
 ever, by Professor Hansen that he has observed the latter
 
 DISCUSSION OF CONCLUSIONS 57 
 
 peculiarity in the case of true Hyphomycetes, when growing 
 under abnormal conditions. 
 
 But the mode of branching is not in all respects similar to 
 that of any Hyphomycete, a septum seldom being found at the 
 actual point of junction of the branch with its stem, whether 
 the branch arises by the forking of a single terminal cell or 
 by an outgrowth from a proximal segment. Further, the 
 filaments never anastomose, however much they may inter- 
 lace ; their growth is also distinctly not acropetal, and does not 
 correspond to any one of the several types of acropetal growth 
 figured by Zopf [18 and 38], Marshall Ward [19] uses 
 acropetal and non- acropetal growth respectively as an abso- 
 lute distinction between Schizomycetes and Hyphomycetes ; 
 Professor Hansen, on the other hand, finds that certain Hypho- 
 mycetes do certainly, under abnormal conditions, present 
 non-acropetal growth. The bright yellow coloration of the 
 gelatine taking place in deep cultures in which the develop- 
 ment never proceeded beyond the coccus stage, and also 
 accompanying the formation of filaments as well as of rods, is 
 a bacterial characteristic, and appears to us a further indication 
 that the three forms are phases in the life of one organism. 
 
 The reactions with Gram's and Loffler's stains, as also with 
 corallin, eosin, and carbol-fuchsin, are especially bacterial, and 
 tend to show that the envelope of the coccus, the refractive 
 portions of the rods, and the sheath of the filaments are 
 identical in nature, while the staining reactions of the inner 
 substance of all these also correspond ; this would be the case 
 if the inner and outer substances were respectively derived, 
 as we believe, from the same sources. The youngest growth 
 of filaments of which stained preparations were made 
 corresponded in thickness with the largest rods. It appears 
 to us noteworthy that the rods never appeared in our blank 
 control-flasks ; hence it would seem that if they were 
 impurities, they entered our cultures in the inoculating sub- 
 stance, and not from the air, especially as during the investiga- 
 tion the study of all other micro-organisms of fermentation 
 was completely set aside. But even supposing the rods were 
 impurities, it is difficult to explain their invariable absence in
 
 5 8 THE MICRO-ORGANISM OF FAULTY RUM 
 
 milk sugar, whether with or without nitrogeneous substances, 
 as also in those of our gelatine cultures which received the same 
 inoculating medium supposed to contain spores. Finally, it 
 appears to us extremely difficult to avoid the conclusion that 
 the rods and filaments are genetically connected, when on the 
 one hand direct microscopical observation proved continuity 
 between the coccus from undiluted rum and the filament, and 
 on the other both our observations and those of Professor 
 Hansen tend to prove that exactly identical cocci, even from 
 the same sample of rum, produced rods in another medium. 
 
 If the steps from coccus to rod, coccus to filament, and 
 filament to-coccus are thus placed almost beyond doubt, then 
 the missing link is the rod to filament stage ; this is only 
 represented by our preparations, a number of which show the 
 rods to occasionally exhibit true branching (PI. VI. Fig. xix), 
 when the conclusion that they are single floating filaments is 
 almost forced upon the observer. It should also be mentioned 
 that the appearance of preparations of the youngest and smallest 
 filamentous colonies obtainable indicates that they are not, 
 at any rate, radial growths from a common point of origin. 
 
 In conclusion, we have not been able to find any descrip- 
 tion, in the literature of Schizomycetes or Hyphomycetes of 
 any organism exactly resembling that discovered by us in 
 faulty rum, hence we must consider it as new to science. If 
 our view is correct, our organism is excluded by one or other 
 of its described characteristics from any group in any classi- 
 fication except that of Migula's Chlamydobacteriaceae, and from 
 the genera at present included in this, namely, Streptothrix, 
 Cladothrix, Crenothrix, Phragmidiothrix, and Thiothrix. 
 
 As, therefore, the organism, or organisms, has, or have, 
 a real existence, it, or they, obviously cannot go forth into 
 the world without a name, hence we propose provisionally to 
 call it Coleothrix methystes \ though we repeat what we have 
 said in our preface, that the life-history of the organism may re- 
 main for some time to come a subject of legitimate controversy. 
 
 1 KoXeds, a sheath, /i0uorfo a drunkard. We are indebted to Mr. A. 
 Sidgwick, M.A., of Corpus Christ! College, for the ingenious suggestion 
 of this name.
 
 CHAPTER IX 
 
 SUMMARY OF RESULTS 
 
 IT has been shown in the foregoing chapters that the 
 ' faultiness ' of rum, first noticed about sixteen years ago, 
 which has caused a great pecuniary loss to the manufacturers, 
 is due neither to any resinous or other matters extracted from 
 the casks, nor to saline or other substances precipitated, but 
 to the presence of a micro-organism, hitherto undiscovered, 
 which we have named provisionally Coleotlirix methystes. 
 
 This organism, in the coccus stage of its existence, produces 
 in rum primarily a turbidity, but also an increased fluor- 
 escence, owing to the assimilation by the organism of the 
 fluorescent colouring matter of the rum. These phenomena 
 have been proved, by optical tests, to exist in undiluted 
 faulty rum, but become more apparent after dilution, in con- 
 sequence of alteration of the refractive index of the liquid. 
 Hence 'faultiness' has been rendered apparent by the 
 brokers' test of dilution in the proportion of | spirit to 
 f water. It has further been shown that faulty rum can be 
 made sound, and sound rum faulty, by the removal and the 
 addition of the organism respectively. The organism has 
 been successfully cultivated in various media, and consider- 
 able evidence has been brought forward in support of our 
 belief that it possesses three forms, viz. coccus, rod, and 
 filament. The methods of culture and experiment have been 
 fully described, as we hope, in such a manner that subsequent 
 observers may repeat our observations ; to this end the life- 
 history of the organism under various conditions has been 
 fully detailed, and illustrated by drawings and photographs.
 
 60 THE MICRO-ORGANISM OF FAULTY RUM 
 
 An account is also given of the biological and chemical 
 characteristics, as also of the staining reactions of the 
 organism. The three possible views which might result from 
 our investigations are discussed seriatim, and reasons assigned 
 for our own view that the cocci, rods, and filaments are 
 phases in the life-history of a single organism, belonging 
 to the higher bacteria, and probably allied to the Streptothrix 
 group. 
 
 In conclusion, we claim to have established the new facts 
 
 (1) that life can exist in alcohol of nearly 75/ strength, and 
 
 (2) that the organism discovered by us is the cause of ' faulti- 
 ness ' in rum. With regard to the minor question of whether 
 the forms cultivated belong to one or more species, positive 
 proof is not at present obtainable. 
 
 Again, we would record our thanks to colleagues and 
 fellow-workers for their advice and assistance in this investi- 
 gation, wherein nearly all the various branches of natural 
 knowledge are involved.
 
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 1882. 
 
 35. Hueppe, Die Methoden d. Bakterienforschung, 1891. 
 
 36. Brefeld, Botan. Untersuchungen uber Schimmelpilze, Heft 1-4. Leipzig, 
 
 1872-1881. 
 
 37. Winter, Die Pilze in Rabenhorsfs Kryptogamenflora, Lfg. I. 
 
 38. Marshall Ward, Annals of Botany, vol. xii. p. 316, 1898.
 
 EXPLANATION OF PLATES 
 
 Plate I. 
 
 FIG. 
 
 I. Cocci as in rum, (a) chains, (6) group, (c) dead cocci as in 
 rum after fining. Camera lucida, oc. I. obj. 7. Leitz ; tube 
 not drawn out. 
 
 II. Cultivated cocci from sugar-solutions, stained methyl-green, 
 x 1000. Cam. luc. 
 
 III. Cultivated cocci, fresh state (with surrounding slime), before 
 
 loss of yellow colour. Cam. luc. oc. I. Oil imm. ^, 
 finished oc. 5. Leitz ; tube not drawn out. 
 
 IV. Cocci developing filaments, from gelatine single-cell cultures. 
 
 Cam. luc. oc. I. obj. 7. Leitz ; tube not drawn out. 
 
 Plate II. 
 
 V. Outline drawing of filaments from sugar, fresh state, to show 
 true branching. Cam. luc. oc. i. Oil imm. -^ Leitz ; tube 
 at 1 6, finished oc. 5. 
 
 VI. Portion of similar filament, fresh state. Magnification as V. 
 VII. Single coccus, pair, and chains newly constricted off, fresh 
 
 state. Magnification as V. 
 VIII. Filament, fresh state, with cocci constricting off terminally 
 
 and laterally. Magnification as V. 
 
 IX. Penicillium-like filament, with cocci constricting off (a, c, d) ; 
 cocci extruding granules. Stained gentian violet and 
 orange G. x 1000. Cam. luc. 
 X, XI. Other filaments, fresh state. As V. 
 
 XII. Typical filament, with cocci constricting off. Stained safranin. 
 x looo. Cam. luc. 
 
 Plate III. 
 
 XIII. Culture on gelatine plate, showing crater- formation. Cam. 
 
 luc. oc. I. obj. 5. Leitz ; tube at 16, finished oc. 5. 
 
 XIV. Rods from single-cell culture in sugar. Stained Ziehl- 
 
 Neelsen's carbol-fuchsin. x 1000. Cam. luc.
 
 64 THE MICRO-ORGANISM OF FAULTY RUM 
 Plate IV. 
 
 FIG. 
 
 XV. Freudenreich-Hansen flask containing filamentous growth 
 fully developed. Nat. size. 
 
 Plate V. 
 
 XVI. Cultivated cocci, stained methyl-green, x 500. 
 XVII. Rods from single-cell culture, stained Gram, x 500. 
 
 Plate VI. 
 
 XVIII. Filaments with cocci, x 500. 
 XIX. Rods and developing cocci, x 500. 
 
 Plate VII. 
 
 XX. Very young filamentous growth from sugar, x 30. 
 XXI.. Old filamentous growth on gelatine. Nat. size
 
 1
 
 PI. I. 
 
 (OQ) 
 
 
 i 
 
 !\ ' 
 
 Werner & Winter, Fnuikfortm.
 
 17. 
 
 vm. 
 
 LJ. Veley del-
 
 PL. II.
 
 XIII. 
 
 m 
 
 "I ' '' ! '--'^^^^'^4\ : ^L- 
 
 
 LJ.Vtlty dtLetnin
 
 XIV. 
 
 i 
 
 PL III.
 
 PL. IV 
 
 FIG. XV
 
 . *r 
 
 I 
 
 &* % 
 
 A- ' 
 
 
 
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