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TECHNICAL MYCOLOGY

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TECHNICAL MYCOLOGY

THE UTILIZATION OF MICRO-
ORGANISMS IN THE ARTS
AND MANUFACTURES

A PRACTICAL HANDBOOK ON

FERMENTATION AND FERMENTATIVE PROCESSES FOR THE USE

OF BREWERS AND DISTILLERS, ANALYSTS, TECHNICAL

AND AGRICULTURAL CHEMISTS, PHARMACISTS,

AND ALL INTERESTED IN THE INDUSTRIES

DEPENDENT ON FERMENTATION

DR. FRANZ LAFAR

Professor of Fermentation-Physiology and Bacteriology in the
Imperial Technical High School, Vienna

With an Introduction by DR. EMIL CHR. HANSEN

Principal of the Carlsberg Laboratory, Copenhagen

Translated by CHARLES T. C. SALTER

IN TWO VOLUMES
VOL. L— SCHIZOMYCETIC FERMENTATION

WITH PLATE AND NINETY FIGURES
IN THE TEXT

LONDON
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EXETER STREET, STRAND
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\All rights reserved]

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LlStE

PREFACE

x

^ Da. LAFAR has paid me the compliment of forwarding me a copy of the

^ first volume of his " TECHNICAL MYCOLOGY," with a request that I should

write a preface to the work. A perusal of the book gives me the impres-

sion that its contents will in themselves be a sufficient recommendation,

•* and ensure the success of the work through its own inherent value ; con-

^ sequently, an Introduction by me is so far superfluous. Should, however,

3 a few words of mine be the means of helping to secure for the work of my

•:] young colleague a readier introduction, here and there, than it would

^ perhaps otherwise find, I shall be exceedingly pleased.

The First Volume treats of BACTERIA. In a series of chapters we are

shown the predominant roles — both useful and antagonistic — played by

to these organisms in Distilling and Brewing ; in the preparation of Wines

$2 and the Manufacture of Vinegar ; in the Dairy ; in Farming ; in the

"- preparation of Agricultural Fodder ; and in the manufacture of Tobacco

_ and of Sugar. Then follows an account of the relation of Bacteria to

— ^ sundry transformations occurring in Nature, particularly the important

facts recently established in connection with the combination of free

nitrogen by bacterial agency, with the iron and sulphur bacteria and the

bacteria of nitrification.

It might be feared that, in a work aiming at objects so decidedly
| practical, the theoretical side of the subject would possibly be overlooked.
ffi This is, however, not the case in the present instance, as a glance at the
Table of Contents will suffice to show.

That the Author possesses a grasp of the historical development of the
subject has already been evidenced in his previous treatises, and the same
feature often appears in the present volume.

In the majority of the Text-books and Manuals published in recent
years, great confusion exists with regard to the appending of authors'
names to the Illustrations. In one and the same book, for example, \ve
meet with instances where the name of the author of the original work
whence the copy has been taken is given — as it should be — and also

vi PREFACE

with other cases where the actual author is ignored, his Dame being
replaced by that of the compiler of some text-book from which the copy was
obtained — i.e., some one who himself has done nothing more than copy.
Such a mode of procedure is in a high degree calculated to produce a
misty conception of the actual circumstances in the mind of the reader,
the more so because, as stated, no importance is attached to the occurrence.
DR. LAFAR has, however, set vigorously to work to combat this bad habit
by taking all his reproductions direct from the original sources, so that they
are clear and accurate representations of these originals.

The subjects included in the present work have been dealt with in a
many-sided manner, the Botanical as well as the Technical and Chemical
aspects having been borne in mind, although preference has throughout
been accorded to the two latter. The style is flowing and clear, in many
places lively and picturesque, and I have read with interest even those
portions wherein I am not at one with the opinions of the author. The
attention devoted to the most recent developments of the subject gives a
special value to the book.

Within the last two decades the study of Microbiology has made gigantic
strides, both in the pathological and the technical branches of the subject ;
and just as investigations into the Physiology of the higher plants gave the
first impetus to the establishment of Agricultural Experimental Stations in
all countries, so, in like manner, have the Physiology of Fermentation and
Technical Bacteriology called into existence, within the last few years, a
number of Stations and Laboratories for the development of those branches
of industry wherein micro-organisms play an important part. Formerly,
Chemistry exercised an undisputed sway over the whole of this realm, but
now Biology has won for itself a co-ordinate position therein — a fact which
is now being recognised (although not yet to an adequate extent) in the
filling of professorships at the various Technical High Schools. An army
of eager workers has arisen, new technical journals have sprung into
existence, and a great number of treatises and books are published on the
subject every year. However cheering this may be in itself, the fact can-
not be gainsaid that a portion (unhappily much too large) of these publica-
tions ought properly never to have seen the light. It is true that an
intimate connection with practical conditions sets fresh tasks before the
investigator, and exerts on the whole a sufficiently stimulating influence ;
but, on the other hand, the same circumstance gives rise to the danger of
diverging into by-paths, and neglecting the strict scientific conditions of

PREFACE vii

investigation. Since these Stations and Laboratories are, as a rule, main-
tained by the circle of practical men for whom they work, the investigators
appointed thereto are often subjected to regrettable pressure. Even
though, otherwise, a certain amount of freedom is allowed them in these
institutions, they labour under the great difficulty of being obliged —whilst
engaged in the task of scientific investigation — to be ready at any moment
to give assistance — coupled with analyses and any wished-for disclosures —
to the parties interested. Still further difficulties arise when practical
men foolishly intermeddle in scientific investigations, and especially when
results that shall be immediately available for practical utilisation are
impatiently demanded — results which, however, are only attainable by
scientific investigation, and cannot be forced on at pleasure.

Under circumstances of this nature it requires great strength of
character not to give way to outside pressure, and many examples are
met with in the literature of the subject where this firmness has been
lacking.

The result of these vexed relations between Scientists and practical
men has been to call into existence a quasi-scientific literature by which
neither Science nor Practice has benefited — a result which every one who
has the healthy development of this subject at heart must greatly deplore
and endeavour to improve according to his ability. These conditions are,
however, in existence, and we must take them into account. Amongst the
chaff which occupies a large part of the aforesaid technical journals, there
is, nevertheless, some really good grain to be found, and he who undertakes
to write a work on Technical Mycology must not content himself with
gathering from purely scientific sources alone, but must, at the same time,
work through the technical journals as well. This (by no means easy) task
has been accomplished by DR. LAFAR with commendable discernment and
ability.

In the last few years, certainly, we have had various Text-books and
Manuals giving a summary of larger or smaller sectionst of Technical
Microbiology ; none of them, however, has treated the whole of this exten-
sive field from so comprehensive a point of view. To prepare a work like
the present requires not only many-sided discernment, but also enthusiasm
for the task, combined with courage and endurance — properties with which
the book shows the author to be endowed. The work will be welcomed,
not only by those for whom it is primarily intended — viz., Technical
Chemists, Chemists dealing with food-stuffs, fermentation, and agriculture,

viii PREFACE

Pharmacists, and Agriculturists — but many a professor also will derive
benefit from its pages for his lectures and researches. In this respect
the copious bibliographical references will also be of good service.
In the present volume we have unfortunately only the numbers of the
references, it being intended that the Bibliography shall be published as
an Appendix to the second volume. This increases the desirability of the
early appearance of the latter.

The Publishers have produced the work in a handsome and substantial
manner, and in this respect also the impression produced is of the best.

EMIL CHR. HANSEN.

CARLSBERQ LABORATORY,
COPENHAGEN,

September 1896.

TABLE OF CONTENTS.

INTRODUCTION.

I. — THE THEORY OF SPONTANEOUS GENERATION.

PAGE

§ i. Definition i

§ 2. Discovery of Fermentative Or-
ganisms 2

§ 3. Needharn's Demonstration in favour

of " Generatio JSquivoca " . .3

§ 4. Spallanzani's Experiments . . 4

§ 5. Franz Schultze's Experiment . . 4

Foundation of the Science of Anti-
septics by Schwann ... 5
§ 6. Labours of Schroder and Dusch . 5
§ 7. Pasteur's Examination of the

Theory 6

§ 8. Bechamp'a Microzyme Theory . 7
§ 9. Spontaneous Generation only Un-

proven, not Impossible . .8

II. — THEORIES OF FERMENTATION.

§ 10. The Alchemists— Stahl's Theory

of Fermentation . . . .10
§ n. Gay-Lussac's Opinion . . .10
§ 12. Cagniard-Latour's Vitalistic

Theory , . . . .11
§ 13. Th. Schwann's Researches . .12
§ 14. Fr. Kiitzing's General Theory . 13
§ 15. Liebig's Decomposition Theory . 14
§ 16. Pasteur's Theory . 16

§ 17. C. Nageli's Physico-Molecular

Theory 16

§ 1 8. The Enzymes and M. Traube's

Ferment Theory . . .17

§ 19. General Definition of Fermenta-
tion 18

§ 20. So-called Spontaneous Fermenta-
tion of Sweet Fruits . . .19

§ 21. Decompositions effected by Light

and Air 19

III. — THE ORGANISMS OF FERMENTATION.

§ 22. Their Position in the Botanical

System 21

§ 23. Classification of the Fungi. . . 22

§ 24. Schizophytaa 22

§ 25. Assimilation of Carbon Dioxide

without the Aid of Chlorophyll . 23
§ 26. Saprophytes and Parasites . . 23

DIVISION I.

SCHIZOMYCETIC FERMENTATION.

Section I.— General Morphology and Physiology of the Sehizomyeetes.
CHAPTER I. — FORM AND DIMENSIONS.

27. Forms of Growth .

28. Dimensions of Bacteria

25 i § 29. Mutability of Form
27 | § 3O. Involution Forms
ix

CONTENTS

CHAPTER II. — STRUCTURE AND CONSTITUTION OF THE BACTERIAL
CELL.

§ 31. Chemical Composition of the Cell

Wall 30

§ 32. Optical Properties of the Cell Wall 30
§ 33. Zoogloea Formation . . . 31
§ 34. Plasmolysis . . . .32

§ 35. Structure of the Cell Contents . 33
§ 36. Elementary Composition of the

Bacterial Cell .... 35
§ 37. Quantitative and Qualitative Se-
lective Power . . . .36

CHAPTER III. — POWER OF INDEPENDENT MOVEMENT IN BACTERIA.

§ 38. Molecular Movement and Loco-
motion ..... 37
§ 39. The Flagella or Cilia ... 37

40. Histology of the Cilia

41. Chemotaxis .

CHAPTER IV. — VEGETATIVE REPRODUCTION BY FISSION.

§ 42. Division in One Direction . . 42
§ 43. Division in Two Directions . . 43
§ 44. Division in Three Directions . 43

§ 45. Form of the Daughter-Cells . . 44
§ 46. Division of the Nucleus . . 44
§ 47. Rate of Reproduction . . -44

CHAPTER V. — THE PERMANENT (REPRODUCTIVE) FORMS OR SPORES.

§ 48. Formation of Endospores . . 46

§ 49. Alterations in the Form of the

Mother-Cell . . . .46

§ 50. Number of Spores . . . .48

§ 51. Form and Size of the Spores . 49

§ 52. Conditions Influencing Spore For-
mation * . . . -49

§ 53. Resist ing Power of the Endospores 49

§ 54. Behaviour of the Endospores to-
wards Dyes . . . -50

§ 55. Arthrospores .. .. . .51

CHAPTER VI. — THE GERMINATION OF THE ENDOSPORE.

56. First Type i 52

57. Second Type 52

58. Third Type S3

§ 59- Importance of this Process in the

Classification of Bacteria . . 55

Section II.— General Biology and Classification of Bacteria.

CHAPTER VII. — THE BACTERIA UNDER THE INFLUENCE OF PHYSICAL
AGENCIES.

60. Influence of Electricity . . 57

61. Influence of Temperature — Cold

and Heat-loving Bacteria . 58

§ 62. Influence of Light— Self-Purifica-
tion of Hirers .... 60

§ 63. Influence of Mechanical Shock,

Gravity, and Gaseous Pressure 63

CONTENTS

CHAPTER VIII. — BACTERIA IN THEIR RELATION TO ONE ANOTHER.

§ 64. Symbiosis, Metabiosis, Anta-
gonism

66

§ 65. Mixed Cultures .

PAGE
. 67

CHAPTER IX. — CLASSIFICATION OF THE BACTERIA.

§ 66. First Attempt by 0. F. Miiller . 69
§ 67. Cohn's Classification ... 69
§ 68. Billroth's Coccobacteria Septica. 70

§ 69. De Bary and Hueppe's Classifica-
tion 71

§ 70 Pathogenic, Chromogenic, and

Zymogenic Bacteria 73

Section III.— Principles of Sterilisation and Pure Cultivation.
CHAPTER X. — METHODS OF STERILISATION.

§ 71. Sterilising 75

§ 72. Freeing the Air from Germs . 75
§ 73. Filtration of Drinking Water . . 77
§ 74. The Bacterium Filter in the Ser-
vice of Enzymology ... 78

§ 75. Beer Filters 78

§ 76. Destroying Germs by Dry Heat . 79

§ 77. Destroying Germs by Moist Heat 80

§ 78. Intermittent Sterilisation . . 82
§ 79. Mineral Antiseptics : Corrosive
sublimate, sulphurous acid, car-

bon dioxide, chlorine and chlo-
ride of lime, ozone and hydro-
gen peroxide, milk of lime . 84

§ 80. Organic Antiseptics : Phenol,
cresol, creolin, lysol, sapocarbol,
solveol, solutol, saprol, salicylic
acid, antinonnin, ethyl alcohol,
ethyl ether, formaldehyde, iodo-
fonn, chloroform, benzoic acid 87

§ 8 1. The Combined Method of Sterili-
sation 91

CHAPTER XI. — METHODS OF PURE CULTURE.

§ 82. Nutrient Solutions ... 94
§ 83. The Dilution Method and Frac-
tional Cultivation ... 96
§ 84. Liquefiable Solid Media . . 99

§ 85. Koch's Plate Cultures— Streak
and Puncture Cultures — Pre-
serving Cultures — Auxano-
graphy

Section IV.— Chromogenie, Photogenic, and Thermogenie Bacteria.

CHAPTER XII. — CHROMOPAROUS BACTERIA PRODUCING RED AND YELLOW
COLOURING MATTERS.

§ 86. Coluured and Colouring Bacteria 105
§ 87. Micrococcus Prodigiosus . . 105
§ 88. Lipochromes .... 107

§ 89. Red Coloration in Milk, Cheese,

and Stock-fish- . . .108

§ 90. Bacteria producing Yellow

Colouring Matters . . . 109

xii

CONTENTS

CHAPTER XIII. — PURPLE BACTERIA AND THEIR BEHAVIOUR TOWARDS
LIGHT.

91. Their Morphology

92. Influence of the Individual

Colours of the Spectrum

PAGJt
III

§ 93. Assimilation and Oxygen Elimi-
nation 113

CHAPTER XIV. — CHROMOPAROUS BACTERIA PRODUCING BLUE, GREEN, AND
VIOLET COLOURING MATTERS.

§ 94. Blue Coloration of Milk . . 115

§ 95. Blue Coloration in Cheese . . 117

§ 96. The Fermentation of Indigo . 119
§ 97. Varieties of Bacillus Pyocyaneus

— Bacteria producing Violet
Pigments . . . .121
98. Green Bacteria — Green Colora-
tion of Cheese . . .122

CHAPTER XV. — PHOTOGENIC BACTERIA.

99. The Genus Photobacterium . 123 | § 101. The Luminous Bacteria as Tests
100. The Food Requirements of Phos- for Enzymes . . . .125

phorescent Bacteria . . 124 | § 102. The Phosphorescents . .125

CHAPTER XVI. — THERMOGENIC BACTERIA.

103. Spontaneous Combustion . .127

104. Spontaneous Heating of Hops . 128

§ 105. Tho Fermentation of Tobacco . 128
§ 1 06. The Preparation of Burnt Hay . 129

Section V.— The Heat-Resisting Bacteria : Their Place in Nature and their
Importance in the Fermentation and Food-Stuff Industries.

CHAPTER XVII. — BACILLUS SUBTILIS AND ITS CONGENERS.

§ 107. Roberts' Heat Method . .131
§ 108. Morphology of Bacillus Subtilis 132
§ 109. Influence of the Mode of Nutri-
tion on the Form of Growth . 133

134

§ no. The Potato Bacilli .

§ in. Bacillus Fitzianus — Production

of Alcohol by Fission Fungi 136
§ H2. Bacterial Content of the Soil . 137

CHAPTER XVIII. — ^BUTYRIC ACID FERMENTATION AND ALLIED
DECOMPOSITION PROCESSES.

§ 113. Anaerobiosis . . . .139

6 114. Methods of Cultivating Anae-
robic Bacteria . . . .140

9 115. Clostridium Butyricum (Praz-

mowski) and Bacillus Buty-

ricus (Hueppe) . . .143

§ 116. The Genus Granulobacter . . 145

§ 117. The Equation of Butyric Fer-
mentation . . • .147

§ 118. The Fermentation of Cellulose . 148

§ 119. The "Betting" of Flax and

Hemp 151

§ 120. The Rancidity of Fats . .152

CONTENTS

xiii

CHAPTER XIX. — THE PRESERVATION OP MILK.

§ 121. Dirt- and Germ-Content iu Milk 154
§ 122. The Part played by Milk as a

Carrier of Infectious Diseases. 155
§ 123. Boiling Milk . i . .156
§ 124. The Soxhlet Bottle . . .157
§ 125. Germ-Content of Milk treated

by the Soxhlet Method . .158

PAGE

§ 126. The Method of Neuhauss, Gron-

Avald, and Oehlmann . .159

§ 127. The Content of Pathogenic
Germs in Various Dairy Pro-
ducts— Preserving Milk for
Analysis 160

§ 128. Condensed Milk . .161

CHAPTER XX. — THE PRESERVATION OF MEAT, EGGS, VEGETABLES,
AND FRUIT.

129. Storage in Cold Chambers . 163

130. Dried Meat and Salted Meat . 164

131. Smoked Meats and Corned Beef 165

132. Preserving Eggs . . .166

§ 133. Desiccating and Preserving
Vegetables and Fruit — Con-
centrated Wine-Must . . 167

Section VI.— Lactic Fermentation and Allied Decompositions.
CHAPTER XXI. — GENERAL CHARACTERISTICS.

§ 134. Discovery of the Lactic Acid

Bacteria 170

§ 135. Bacterium lactis Lister, and

Bacillus acidi lactici Hueppe . 171

§ 136. The Equation of Lactic Fermen-
tation 172

CHAPTER XXII. — THE PRODUCTION OF OPTICALLY ACTIVE ORGANIC
COMPOUNDS BY FERMENTATION.

§ 137. Isouiers of Lactic Acid . . 174
§ 138. The Isomeric Tartaric Acids . 175
§ 139. The Division of the Racemic

Compounds . . . .177

§ 140. The Production of the Stereo-

isorneric Lactic Acids . .178

CHAPTER XXIII. — THE ARTIFICIAL SOURING OF CREAM.
§ 143. Defects in Butter

141. The Acid Generator .

142. The Aroma of Butter .

1 80
181

182

CHAPTER XXIV. — THE COAGULATION (CURDLING) OF MILK.

§ 144. Acid Curdling and Rennet

Curdling 184

§ 145. Characteristics and Activity of

Lab 185

§ 146. Lab-Producing Bacteria
§ 147. Casea?e

1 86

1 86

CONTENTS

CHAPTER XXV. — LACTIC ACID BACTERIA IN DISTILLING, BREWING,

AND YlNIFICATION.

;I48. The Spontaneons Acidification
of Distillery Yeast-Mash . 188
149. Artificial Souring by the Aid of
Pure Cultures of Lactic Acid
Bacteria, Bacillus acidificans
longissirnus . . . .189

§ 150. Effront's Hydrofluoric "Acid

Method 191

§ 151. The Lactic Acidification

(" Zickendwerden ") of Wine . 193

§ 152. The "Turning " of Beer . . 194

§ 153. White Beer, Lambic, Ginger-

Beer 195

CHAPTER XXVI. — THE LACTIC ACID BACTERIA IN THE PREPARATION
OP FODDER.

154. Brown Hay

155. Sweet Ensilage .

• X99 j I I56. Sour Fodder
. 200

CHAPTER XXVII. — THE PART PLAYED BY BACTERIA IN TANNING.

§ 157. The Fermentation of the Plump- I § 158. The Souring of Bark Liquor

ing Soak 204 |

205

Section VII.— The ormation of Mueus and Allied Phenomena of
Decomposition.

CHAPTER XXVIII. — THE IMPORTANCE OF BACTERIA IN THE
MANUFACTURE OF SUGAR.

§ 159. The Zoogloea of Leuconostoc

Mesenterioides
§ 1 60. Physiology of Leuconostoc

§ 161. Mucinous Fermentation and In-
version . . .212

CHAPTER XXIX. — ROPINESS IN MILK, WINE, BEER, AND OTHER LIQUIDS.

§ 162. Ropy or Viscous Milk . . 215

§ 163. Ropy Whey and Thick Milk . 216

§ 164. Ropinees in Wine . . . 217

§ 165. Ropiness in Infusions . . 218

§ 166. Ropiness in Wort and Beer . 219
§ 167. So-called Sarcina Turbidity in

Beer ..... 221

Section VIII.- Decompositions and Transformations of Organic
Nitrogenous Compounds.

CHAPTER XXX. — THE PHENOMENA OF PUTREFACTION.

§ 168. The Degradation of the Albu

minoids ....
§ 169. The Putrefactive Bacteria .
§ 170. Proteolytic Enzymes.
§ 171. Ptomaines and Leucomaines
§ 172. The Albuminous Poisons .
§ 173. Liberation of Nitrogen, and De-

223
226
230
232
234

nitrification — Nitric Fermen-
tation of Molasses . . . 235
§ 174. Loss of Colour( Umschlagen) in

Wine 239

Fermentation of Organic Acids

by Schizomycetes . . . 240
Mannitic Fermentation of Wine 241

CONTENTS

xv

CHAPTER XXXI. — THE FERMENTATION OF CHEESE, AND ALLIED
DECOMPOSITIONS.

§ 175. The Composition of Ripe Cheese 243
Conversion of Albumen into Fat 244

| 176. Duclaux' Studies on Cantal

Cheese 244

§ 177. Changes in the Bacterial Flora
of Ripening Cheese — Aromatic
Cheese 245

§ 178. Pure Culture Ferments . . 246

§ 179. Natto and Miso . . . . 247

§ 180. The Normal Pitting of Cheese . 248
§ 181. The Cause of Puffy (" Blown ")

Cheese 249

§ 182. Cheese-makers' Recipes . . 250
§ 183. Counteracting Puffiness in

Cheese 251

§ 184. Bitter Milk and Bitter Cheese . 251

CHAPTER XXXII. — THE FERMENTATION OF UREA, URIC ACID, AND
HIPPURIC ACID.

§ 185. Urea, the Final Product of

Animal Metabolism . . . 253

§ 186. Urea Unassimilable by Higher

Plants 254

§ 187. Discovery of the Urea-Ferment

by Pasteur . . . . 254

§ 1 88. Researches of P. Miquel . . 255

§ 189. Urase 257

§ 190. The Decomposition of Uric Acid

and Hippuric Acid . . . 257

CHAPTER XXXIII. — THE FIXATION OF FREE NITROGEN BY BACTERIA.

§ 191. Accumulators and Consumers of

Nitrogen 259

§ 192. Discovery of the Leguminous

Nodules . . . .261

§ 193. Formation and Functions of the

Nodules . . • . . .262

§ 194. The Nodule Bacteria . . .264
§ 195. The Bacteroids — Infection

Thread 266

§ 196. Clostridium Pasteurianum . . 269

Section IX.— Oxidising Fermentations.
CHAPTER XXXIV. — THE IRON BACTERIA.

§ 197. Morphology of the Genera

Crenothrix and Cladothrix . 272

§ 198. Physiology of the Iron
—Smell of the Soil .

Bacteria

275

CHAPTER XXXV. — THE SULPHUR BACTERIA.

§ 199. Morphology of the Genus Beg-
giatoa — Artificial Cultivation
! cvero of the Sulphur B£cteiia . . 278
§ 200. The Species of the Genus Thio-

thrix 280

§ 201. Morphology of the Non-Fila-
mentous Sulphur Bacteria —
Bacterio-purpurin . . . 281

§ 202. Physiology of the Su>phur Bac-
teria— The Limanea . . 283

CHAPTER XXXVI.— THE NITRIFYING BACTERIA.

§ 203. The Recognition of Nitrification

as a Physiological Process . 288

§ 204. Nifroso-Bacteria and Nitro-Bac-

teria 289

§ 205. Nitrosomonas and Nitroso-

coccus 290

§ 206. The Nitre-Bacteria . . .291
§ 207. Assimilation in the Dark . . 292
§ 208. Wall-Saltpetre and Plantation-
Saltpetre ... 293

CONTENTS

CHAPTER XXXVII. — ACETIC FERMENTATION.

PAGE

§ 209. Discovery of the Acetic Acid

Bacteria 295

§ 210. Morphology of the Acetic Acid

Bacteria 296

§ 211. The Morphological Influence of

Temperature . . . 298

PAGE

§ 212. The Equation of Acetic Fermen-
tation 302

§ 213. Pure Culture Ferments in the

Manufacture of Vinegar . . 305

CHAPTER XXXVIII.— THE OXYDASES.

§ 214. The Browning of Wines . . 308 I Vegetable Juices — Laccase,

§ 215. The Rapid Discoloration of Fresh Malase, Tyrosinase, Olease . 309

| § 216. The Bittering of Wine . .311

TECHNICAL MYCOLOGY:

THE UTILISATION OF MICRO-ORGANISMS
IN THE ARTS AND MANUFACTURES.

INTRODUCTION.

I.

THE THEORY OF SPONTANEOUS GENERATION.

§ 1.— Fermentation Physiology is the Science of the Character
and Activity of Fermentative Organisms.

Fermentative Organism is the name given to any minute being of
vegetable nature capable of exciting fermentation. Whether any given minute
organism is to be considered as a " fermentative organism " or not depends, there-
fore, on the answer to the question : " Does it possess the power of causing
fermentation ? "

In studying this subject, the first task that lies before us is to obtain a
definition of the term fermentation, or, in other words, to establish the common
factor of all the manifold processes classified under that general title.

This is, however, as will soon be apparent, no light task ; and the probability
of our attempts being crowned by a successful result will be greater if we limit
the scope of the question at the outset, and for the moment consider the term
" fermentation " as applying merely to those phenomena with which it is associated
in colloquial language, viz., the conversion of must into wine, wort into beer,
wine into vinegar, and fresh milk into sour, &c. To these may also be added the
process of putrefaction.

Adhering to this restriction of the term, let us follow in imagination the
path of investigation which has led to the knowledge that all the above-named
phenomena are occasioned solely by the activity of minute living organisms, and
constitute a manifestation of their vitality ; that fermentation and putrefaction
are, in short, not purely chemical molecular transformations, but physiological

Moreover, history shall be our instructor, and lead us on further, to the com-
prehension of those other processes, which, for the present, we assume as standing
without the pale of the term " fermentation," but which, nevertheless, should
actually be included therein. Such processes are, inter alia, the transformation
of ammonia into nitric acid, occurring in the soil of our fields ; the decomposition

2 THE THEORY OF SPONTANEOUS GENERATION

and dissolution of dead vegetable matter; the ripening of cheese ; the formation
of bog (iron) ore, &c. &c.

§ 2.— Discovery of Fermentative Organisms.

The organisms taking part in the processes of fermentation are so minute
that only a few can be detected, and that very imperfectly, by the unassisted eye.
The term microbe, introduced into the vocabulary of science by C. SEDILLOT (I.) l
in 1878, belongs to them of right. Their examination could not be carried on
anterior to the invention of appliances for observing minute bodies under high
powers of magnification, and therefore the inventors of the microscope deserve
to be held in grateful remembrance in the domain of fermentation. These were
Hans and Zacharias Janssen, father and son, spectacle-grinders, of Middelburg,
in Holland, who, about the year 1590, constructed a combination of lenses
which, although, of course, very imperfect when, compared with the instrument
of the present day, must be regarded as the first compound microscope made.

Nevertheless, however great this step undoubtedly was, both from a theoretical
and practical point of view, and however fruitful it proved in results, seeing that
it rendered possible later discoveries in the world of the " infinitely little," and
especially of the fermentative organisms ; still the fact remains that the first
fundamental observations were made, not with the compound, but with the
simple microscope, which then, as now, was little more than a magnifying glass
or bi-convex glass lens.

The honour of having discovered the presence of extremely small and hitherto
undetected organisms in putrescent and fermenting liquids belongs to another
native of Holland, by name ANTONY VAN LEEUWENHOEK. Born at Delft in 1632,
he acquired during his apprenticeship to a linen or cloth merchant in Amsterdam
some skill in grinding small glass lenses. Of this skill he made further use after
his final return to his native town, and succeeded in producing lenses capable of
magnifying from 40 to 100, and even to 150 times. With these he examined
various minute objects, and frequently, amongst others, all kinds of vegetable
infusions in a state of decomposition. He discovered therein sundry extremely
small creatures, many of them capable of motion, which he therefore regarded as
animals, and named from their habitat infusoria. He died in 1723. The modern
world has entitled him "the father of micrography," i.e. that science which treats
of the most minute forms of life.

This newly-discovered field of research was at first regarded by Leeuwen-
hoek's successors from an almost exclusively medical standpoint, as it is a
natural instinct in man to try and maintain health and to prevent disease. At
that particular period, too, a special impetus was given to the study of medicine
by the ravages of the plague, which only too frequently pursued its destructive
course throughout Europe.

On the other hand, the study of the phenomena of fermentation derived
little or no benefit from Leeuwenhoek's discovery. The first investigator whom
we meet with in this domain is the Viennese physician, Marcus Antonius
Plenciz, who in his work "Opera medico-physica," issued in 1762, applied the
results of Leeuwenhoek's discoveries, not only to the field of medicine, but also
to that of fermentation and putrefaction. In the latter connection he arrived
at the noteworthy conclusion that "a body undergoes putrefaction when the
germs of vermicular creatures begin to develop and multiply ; because these
animals excrete numerous precipitations consisting of volatile salts, by which
the liquids are rendered turbid and malodorous."

i The Roman numerals given in brackets after the names of investigators refer to the
Bibliographical References forming an appendix to the second volume.

DEMONSTRATION OF "GENERAT1O .EQUIVOCA" 3

However alluring a closer acquaintance with these minute creatures may
have been to the investigators who succeeded Plenciz, and however useful,
from a practical point of view, might be to observers the processes of de-
composition which they induced, these questions were nevertheless forced
temporarily into the background by another, namely, the origin of these
minute organisms.

How do the minute creatures so copiously developed in infusions originate ?

Some opined that these organisms were produced from certain unorganised
(and therefore inanimate) substances — chemical compounds— present in the
liquid in question, their formation being therefore considered as spontaneous
(generatio spontanea), or arising from elementary substances (primary genera-
tion). Or, whilst proceeding from elementary substances, as differing therefrom
(heterogeneous), or dissimilar thereto (equivocal) ; hence the name Heterogenesis
or generatio cequivoca : all of which terms, as well as that immediately to be
noted, have the same import.

The party opposed denied, on the other hand, the possibility of a transition
from a lifeless to a living condition (abiogenesis), and asserted that when " infu-
soria" are detected in an infusion, a liquid or matter undergoing decomposition,
their existence is due to living germs present therein.

Which view is correct ? On this point there arose, about the middle of the
eighteenth century, what formed one of the liveliest disputes agitating the
domain of natural science at that period, and which, after occupying the most
earnest attention of several successive generations of scientists, only terminated,
after numerous fluctuations, about the middle of the present century. From
among the numerous investigators who took part in this controversy, mention
can here be made of but few — Needham 011 the one side and Spallanzani on the
other being entitled to the first place.

§ 3.— Needham's Demonstration in Favour of
"Generatio £Jquivoca."

The most energetic champion of the theory of spontaneous generation was
the English divine, NEEDHAM (I.). This theory was in existence long before
his time, and had had renowned supportei-s — among them the chemist Van
Helmont, who proposed a method for producing artificial mice — but until then
had not progressed beyond the stage of indefinite assertion and unfounded
hypotheses. The cause of the extraordinary support and approval accorded
to the assumptions put forward by the English divine is, on the other hand,
attributable to the novel manner in which he arrived at his theory (pub-
lished in 1745), viz., not by untenable hypotheses, but by well-directed experi-
ments.

He set to work, for example, in the following manner : An aqueous meat
extract was boiled for a short time in a flask, which was then made air-tight and
left to stand for several days or weeks. When opened at the end of this time,
the contents proved to be plentifully infested with "infusoria," from which
Needham concluded that as the "eggs" originally present in the liquid were
killed by the boiling and the entry of fresh ones from the outside was precluded,
therefore the living infusoria discovered in the liquid on re-opening the flask
must have originated spontaneously, not from eggs (germs), but from the lifeless
constituents of the liquid.

The great impression produced on his contemporaries by these statements
can be appreciated by reference, for instance, to Buffon's work on the " System
of Generation."

4 THE THEORY OF SPONTANEOUS GENERATION

§ 4.— Spallanzani's Experiments.

Of the two hypotheses forming the basis of Needham's deduction, the
accuracy of the second, i.e. that relating to the exclusion of outside germs, was
examined first. Some twenty years after the appearance of the English theory,
the Abbe SPALLANZANI (I.) published a dissertation in which he combated the
doctrine of spontaneous generation. In this work the Italian divine detailed
the experiments which had led him to the conclusion that a development of the
animalculsB in question, in an infusion maintained at boiling-point for three-
quarters of an hour, was only possible provided air, which had not been pre-
viously exposed to the influence of fire, had been admitted. This position was
also maintained by Spallanzani in a second treatise (II.).

Nevertheless, the supporters of the spontaneous generation theory were still
far from regarding their cause as lost. They characterised these experiments as
inconclusive, since (so they said) " by the immoderate heat Spallanzani chose to
employ, the air in the vessel is so unfavourably changed, and rendered so
unsuitable for the maintenance of life, that it is no occasion for surprise that
all development was lacking." This objection was curtly rejected by Spallanzani,
but an experimental confutation was only ariived at much later. The next step
in this direction was accomplished in 1836 by —

§ 5.— Franz Schultze's Experiment.

In order to avoid under-estimating the value of the very short treatise (I.)
published by this investigator, regard must be had to the influence attained by

Chemistry in all branches of
natural science during the sixty
years that had elapsed since
Spallanzani's demonstration, an
influence which will be eluci-
dated, in so far as it refers to
the theory of Fermentation, in
subsequent sections. The idea
that ordinary air acts as an
inducer of fermentation or
putrefaction by reason of its
content of living germs was
first called into existence by

FIG. i.— Franz Schultze's Experiment. Schultze.

He described his experi-
ment as follows: "I filled a glass flask half full of distilled water (Fig. i),
with which I bad mixed various animal and vegetable substances, and closed
it with a sound cork, through which were passed two tight-fitting glass
tubes bent to elbow joints. 1 next placed it in a sandbath and applied heat
until the water boiled briskly, so that all parts were exposed to a temperature
of 100° C. Whilst the hot water vapour was still issuing from the two tubes,
I attached to the end of each an apparatus employed by chemists, in the
course of organic analyses, for the absorption of carbon dioxide. That on the
left-hand side was filled with concentrated sulphuric acid, the other with a
solution of potassium hydroxide." After cooling the apparatus, air was drawn
through twice every day during the ensuing two months, in such a manner that
it had to pass through the sulphuric acid before entering the flask. The results
confirmed the expectations of the investigator, the contents of the flask when

THE LABOURS OF SCHRODER AND DUSCH

5

opened being found free from living organisms, which, however, soon made their
appearance when the open flask was freely exposed to the air. This proved that
previous exposure to the influence of fire is not an essential condition for
depriving air of the power of inducing fermentation or putrefaction.

Three years later, TIIEODOR SCHWAJTN (If.) entered the field as an opponent
of the theory of spontaneous generation. Of his labours in this direction, a
slight modification of the Schultze experiment, consisting chiefly in the substi-
tution of a heated metal tube for the bulb tubes (see Fig. 2), occupies merely a
secondary position. More
important in the attack on
the theory of the spon-
taneity of the phenomena
of fermentation was the
establishment by him of
the fact that a resort to
heat is unnecessary in the
prevention of such decom-
position, but that the same
result can be attained by
the addition of some toxic
substance to the liquid :
" Fermentation is arrested

.~:,.'.:'!::: '^M:!.':' •____

by any influence proved
capable of killing the fungi,

FIG. 2. — Theodor Scbwanu's Kxpcrimeiit.

especially by heat, potas-
sium arseniate, &c." He
was, therefore, the founder of the science of antiseptics. Concerning his
fundamental researches in the narrower field of alcoholic fermentation, mention
will be made in a subsequent chapter.

The adherents of spontaneous generation applied to Schwann's method of
purifying the air the same objection (referred to above) which they had previously
lodged against Spallanzani. They did not even consider themselves confuted by
the results of Schultze's experiment, but asserted that here also the treatment of
the air, although by no means so violent, unfavourably modified its composition.
The refutation of this doubt was only accomplished after a lapse of seventeen
years, and that by

§ 6.— The Labours of Schroder and Dusch (I.).

Instigated by the researches of Loewel, who found that ordinary air could be
deprived of its property of inducing crystallisation in a supersaturated solution
of sodium sulphate by filtration through cotton-wool, the two investigators
named above modified, in 1855, the arrangement of Schultze's experiment, by
allowing the incoming air to pass through a glass tube packed with cotton-wool
before entering the flask. It was found that by means of this (decidedly not
" violent ") treatment the air also lost its power of causing decomposition and
the formation of minute organisms in extracts which would remain unchanged
when air was excluded.

The importance of this demonstration must not, however, be over-estimated,
for it only proves the presence in the air of a " something " capable of giving
rise to living creatures in inanimate nutrient media, and of exciting substantive
changes (fermentation and putrefaction) therein. Concerning the nature of this
active "something," the experimenters could give no satisfactory account; they
even left it an open question whether the something was gaseous or not. It

6 THE THEORY OF SPONTANEOUS GENERATION

may be considered that they were unduly diffident, since the action of the
cotton-wool filter proves that this something must necessarily be a solid body
and not a gas. But, on the other hand, both investigators could point to experi-
ments wherein the previously boiled test liquid afterwards underwent decom-
position, notwithstanding the fact that all the air which was allowed access to it
had been filtered through cotton-wool. Milk they had, in their first treatise,
recognised as such a liquid, and to this were added, in a second communication
by SCHRODER (I.), yolk of egg, meat, and meat broth, in all of which cases the
filtration of the air proved useless. This led Schroder to separate the pheno-
mena of decomposition — characterised as fermentation and putrefaction — into
two groups : the one, which he designated " voluntary decomposition," requiring
only oxygen for its inception, whilst the othei", e.g. the fermentation of wort,
required, in addition, the collaboration of that unknown constituent of the air,
which could be destroyed by fire or arrested by a cotton-wool filter. " Whether
this active substance should be regarded as germs floating in the air, or as some
hitherto unknown chemical substance modified by high temperature and sepa-
rated andi fixed by the influence of contact with the cotton fibres, must remain
undecided."

Glancing back for a moment at the work of Schultze, one would be only too
readily disposed to consider the results of Schroder and Dusch's experiment as a
retrogade step, since they not only did not afford us any further information
beyond that established by Schultze as to the nature of the germs in the air, but
also called in question the accuracy of Schultze's results. And, in fact, repeti-
tions of the Schultze experiment by many other workers, with various modifi-
cations, especially with regard to the kind of test liquid employed, confirmed the
results of Schroder and Dusch. In numerous instances decomposition ensued,
even in the boiled liquid, when purified air (filtered or heated to redness) alone
was admitted ; whilst in other cases, under precisely similar conditions, the
boiled sample remained unaltered for any length of time. Thus the state of the
question at the commencement of the sixth decade was just about as far
advanced as at the beginning of the century, and the adherents of the spon-
taneous generation theory were more certain of triumph than ever.

§ 7. The Examination of this Theory by Pasteur.

However, the day of refutation was close at hand, though the proof was
not obtained by the methods which had generally been favoured hitherto, but
which had led to no definite issue.

Experimenters had so concentrated their attention on keeping the air
admitted to the boiled liquid perfectly free from active germs, that it had not
occurred to any one to ask if the sterilisation of the liquid could not be equally
ensured by simply boiling it, either momentarily or continuously for a short
time.

Reasoning from the fact that all known forms of created life (animal as
well as vegetable) were incapable of resisting the temperature of boiling water,
even when exposed thereto for merely a short time, the conclusion was arrived
at that the same effect was produced on the small germs in question. It was
therefore considered, humanly speaking, certain that every liquid could be
rendered free from active germs by boiling for a short time. This was agreed
to both by those who accepted and by those who rejected the doctrine of spon-
taneous generation. Still such belief was based on a mere assumption, as CH.
BONNET (I.), a contemporary of Spallanzani's, implied when he inserted the
following query in his work opposing the theory of spontaneous generation :
" Is it, then, certain that there exist no animals or eggs capable of supporting a

BECHAMFS MICROZYME THEORY 7

temperature equal to that of hot ashes without losing their life or reproductive
power ? "

Pasteur called to mind this doubt of Bonnet's when he began to subject the
theory of spontaneous generation to experimental examination in response to
the offer made in 1860 by the Paris Academy of Science of a prize for "an
attempt, by means of suitable experiment, to throw new light on the question of
spontaneous generation." From the report of his researches, which appeared
early in the year 1862, in the form of a comprehensive treatise (I.), well
deserving perusal, only the most important result can be referred to here: viz.,
the demonstration of the possibility, by the assistance of sufficiently prolonged
heating at an adequately high temperature, of sterilising (i.e. freeing from living
germs) any substance whatsoever ; and of the fact that a sample so sterilised will
not subsequently undergo decomposition, but will remain unaltered so long as
care is taken to prevent the access of germs from the external air.

The objection raised by the heterogenists, viz., that decomposition is pre-
vented by the strong heating having rendered the sample unsuitable for the
production of germs, can be easily disposed of by inoculating the liquid with a
few germs ; these will be found to develop rapidly and luxuriantly. The sub-
stantiality of these germs was demonstrated by Pasteur in a very beautiful
experiment, for which he employed a culture vessel similar to that described by
H. Hoffmann (I,) in 1860, and now generally known by the name of Pasteur
flask ; a glass flask (fitted with a tubulus at the side for facilitating inoculation)
the neck of which is drawn out small and bent twice like a swan's neck. The
external air is obliged, in order to gain access to the contents of the bottle, to
pass through this neck, and as the direction of movement is changed at the first
bend, all the germs are deposited there.

Thus was laid the foundation on which the edifice of Fermentation Physiology
was gradually raised. The possession of perfectly sterile culture media, and the
power of protecting them from the intrusion of unauthorised germs, is a sine qud
non for a successful and reliable study of the organisms of fermentation.

§ 8. — Bechamp's Microzyme Theory.

Pasteur's investigation and elucidation of the causes of the tenacity of life
exhibited by many germs thenceforward occupied the earnest attention of
mycologists, and finally led to the acknowledgment that this power of resistance
is possessed by the reproductive organs known as spores. The morphology and
physiology of these organs forms the subject of §§ 48 to 55. At present, the only
point to be emphasised is that when these life-retentive organs are once killed,
no spontaneous development of germs can occur in the liquid harbouring them ;
hence such liquid will remain sterile until it is artificially re-inoculated.

It might be supposed that the adherents of the doctrine of spontaneous
generation would have responded to these demonstrations by abandoning their
previous attitude of opposition. This, however, they did not do; they merely
changed the field of combat without altering their opinion. As they could no
longer maintain that organised creatures could be spontaneously derived from
unorganised substances, they contended that the dead cells had the power of
liberating organised living matter capable of development into the various
species.

In a subsequent paragraph we shall learn that in the cell contents of most
fungi, e.g. yeast, small, highly refractive bodies, known as microsomata, may be
frequently observed. On applying pressure to the cover -glass placed on a pre-
paration containing cells that exhibit such enclosures, the membranes are ruptured
and the microsomes are liberated. If, now, the latter be transferred to another

8 THE THEORY OF SPONTANEOUS GENERATION

nutrient solution, there will most assuredly be a development of organisms, if we
omit those precautions which are considered essential by the bacteriologist, but
superfluous by those who believe in spontaneous generation. Such development
is, however, due, not to the microsomes, but to the germs introduced during the
transfer. Although this is so evident, it is strange that this view .should have
had its opponents, as, for instance, the botanists H. Karsten and A. Wigand
(I. and II.), and, with still greater pertinacity, A. Bechamp. The last-mentioned
designates these microsomes (" granulations moleculaires ") microzymes, and
attributes to them such tenacity of life that they are able to remain dormant,
not only for years, but even for entire geological periods, since, as Bechamp asserts,
he has found microzymes of cells which were buried in the strata formed during
the Cretaceous period still retaining their vitality and reproductive power. A
full account of this microzyme theory, which many amateur bacteriologists have
considered to be indisputable — communications respecting which have been
incessantly intruded upon the notice of the Academy of Science at Paris — is given
in a bulky volume which BECHAMP (I.) laid before his sceptical contemporaries
in 1883.

§ 9.— Spontaneous Generation only Unproven, not Impossible.

Omne vivum ex ovo (every living creature from an egg) ; omne vivum ex vivo
(every living creature from living creatures) — was the watchword elevated to a
dogma by the triumphant opponents of the theory of spontaneous generation.
Were they correct? or did they encroach beyond the limits of the facts they de-
monstrated ? Let us devote a few moments to a critical review of the question.

One thing is established beyond doubt, namely, that all the instances of sup-
posed spontaneous generation brought forward by the adherents of the theory
have been vitiated by numerous errors. It is, moreover, established that the
occurrence of spontaneous generation has not been proved, no unassailable experi-
ment being known in which living creatures were produced from inanimate
substances. Spontaneous generation is therefore unproven.

Whether it is also an impossibility is a point still to be decided. If the theory
of evolution, as presented by Lamarck and Darwin, be traced towards its origin
in the lowest organisms, we come to a standstill with the question : "And from
whence then comes the ultimate and lowest creature? — How did organic life
originate on our globe ? "

The reply furnished by the English physicist Thomson 1 — that our earth was
fertilised in its youth by meteors bringing the germs of organisms from other
heavenly bodies — affords no solution, but merely transfers the question to another
scene and to a more distant period of the past, and at once suggests the further
question : " How did life originate on these unknown, extra-mundane sources
of creative messengers ? " There are only two possible answers to these questions,
viz., spontaneous generation, or a miracle.

As a matter of reason, we are therefore obliged to assume that, at some
definite moment in the past, organised living beings were produced from un-
organised potentially organic substances ; and further, that such creative power
may still be operating, may perhaps be performing at present. The possibility
cannot be gainsaid.

That bacteria are the result of this primary creation of living beings is very
questionable, and even improbable, since their structure is much more compli-
cated than is consistent with their presumed origin directly from chemical
elements, unmodified by changes in passing through simpler intermediate
organisms.

i Lord Kelvin,

UNPROVEN, NOT IMPOSSIBLE 9

Many investigators, and amongst them C. NAGELI (I.), assumed that these
lowest forms really exist, although undiscovered at present, and in his important
and highly suggestive work on the Theory of Descent — which also contains a
fine chapter on "the limits of knowledge in natural science" — this author
touches upon the question under consideration. He calls these presumptive
connecting links Probien (pre-existing), on the ground of their being the pre-
decessors of all known forms of living beings. Such a Probion resulting from
spontaneous generation would be " merely a drop of homogeneous structureless
plasma, devoid of any definite form and composed of albuminates, associated
only with the compounds necessary for nutrition."

"We must assume" — says de Bary — "that organisms must at one time
have originated from organisable but unorganised substances, without pro-
genitors. . . . To prove such a primary creation of a living being is of the
highest interest, and exercises the same fascination on the investigator as the
expectation of the homunculus in the phial did on the alchemist. The expe-
rience of centuries has, however, shown that the homunculus when it actually
appeared was simply a small imp which had been secretly passed into the flask
by sleight of hand. . . . Therefore — admitting all imaginable possibilities —
the law, based on experience, of origin from ancestors, corresponds with the
enlightened state of our knowledge, and this is the starting-point that must be
taken in a work which has to deal with the exact sciences."

II.

THEORIES OF FERMENTATION.

§ 10.— Stahl's Theory of Fermentation.

WHOEVER was the first to leave the juice of sweet fruit to itself in storage for
a few days had the pleasure of observing a phenomenon hitherto unknown —
the incipient decomposition of the mass — which we nowadays term alcoholic
fermentation. This observation was made at so early a date that we have no
record of it beyond myth and tradition. The Greeks feted the deity Bacchus
as the inventor of wine, and the Egyptians ascribed to Osiris the first introduc-
tion of brewing.

Acquaintance with the nature of this phenomenon was, however, of an
extremely superficial character for a very long time. Even in the later Middle
Ages the word fermentatio (fermentation) was employed as synonymous with
digestio (digestion), the latter word being also currently used to denote any form
of chemical reaction ; and the word " ferment " was applied to any body capable
of producing such reaction.

At an early date it would necessarily be noticed that the " must " when in a
state of fermentation became covered with a froth, and that at the end of this
operation a copious deposit, viz., yeast, was left at the bottom of the vessels.
Fermentation was therefore looked upon as a process of purification, by which
the initially turbid and discoloured liquid was so improved and freed from dirt,
that the purified alcohol exhibited its true properties. For this reason the
deposit was described as the faeces vini or faeces cerevisice, i.e. the excrement of
the wine or beer. This view was held by, e.g. Basilius Valentinus, a German
monk and alchemist, who lived at Erfurt early in the fifteenth century.

It was also noticed that this sediment was a powerful ferment, i.e. it was
capable of rapidly exciting a brisk fermentation in still unfermented liquids,
such as wine-must or beer-wort. This idea was adopted in other branches of
chemistry, so that any reaction was considered as elucidated when the body
acting as " ferment " therein could be identified. Moreover, the " philosopher's
stone," the goal of the labours and aspirations of the alchemists, was nothing
but the much sought for, but never discovered, universal " ferment " for every
possible chemical process !

Among the disciples of the alchemic school, one other, viz., STAHL (I.),
deserves mention, because his views on the nature of fermentation were adopted
by Liebig a hundred and forty years later. Stahl extended the definition of
fermentation to all forms of decomposition, his theory being expressed verbatim
as follows : " Putrefaction (and also fermentation) is internal movement. A
body undergoing such internal movement may easily induce the same in any
other body, which, though still quiescent, is susceptible of such movement."

§ 11.— Gay-Lussac's Opinion.

Stahl's view remained in vogue until the commencement of the nine-
teenth century, when Gay-Lussac, in 1810, enunciated a new theory to a new

GAY-LUSSAC'S OPINION 1 1

age. The discovery by Lavoisier that combustion is a process of oxidation, a
combination of oxygen with the combustible substance, was an event the
influence whereof extended over the entire domain of chemistry. The assign-
ment to oxygen of a part in the process of fermentation was therefore opportune ;
but Gay-Lussac was especially prompted by another circumstance.

A Parisian confectioner and cook, named Appert, had made practical use of
the experiment devised by his contemporary Spallanzani for the refutation of
the heterogenists and, after some preliminary trials, perfected his process for
preserving meats, vegetables, spirituous liquors, &c. To this end he exposed
them, in hermetically closed vessels, to the temperature of boiling water for
some time — a process which had somewhat earlier (1782) been recommended by
the Swedish chemist SCHEELE (I.) for the conservation of vinegar. In this way
APPERT founded a new branch of industry — the manufacture of conserves —
which brought him both wealth and fame. He published a volume (I.) which
comprised the results of his experience. It was widely circulated and ran into
several editions, the first of which appeared in 1810, and the fourth in 1831.

It is therefore little matter for surprise that the attention of the Parisian
chemist was directed (whether from the culinary or the literary side) to the
productions of his enterprising fellow-citizen. GAY-LUSSAC (I.) now examined
conserves prepared according to Appert's process, and found them to be free
from gaseous oxygen. This incited him to make fermentation experiments
with wine-must, <kc., the results of which led him to assert that the presence
of oxygen is necessary to the inception of fermentation. A number of over-
zealous colleagues, in expounding their master's opinion, added new features to
it, and subsequently credited him with the assertion that oxygen is the actual
ferment — a statement as unfounded as it is inaccurate. Gay-Lussac only claimed
for the gas'a single function, the inception of fermentation ; once the process was
in operation the stimulus was no longer required. With regard to the nature
of this stimulating action he was, however, unable to report more definitely.

Among the observations which led Gay-Lussac to adopt this view, mention
may be made of one which appeared to him particularly conclusive, namely, the
sterilisation of wine-must by sulphuring. When wine-casks, before filling, are
thoroughly sulphured — i.e. the internal air contained in them is heavily charged
with sulphur dioxide by burning sulphur in the casks — the grape juice thereafter
introduced remains quiet and passive, without fermenting. This circumstance
is now unanimously ascribed to the vitality of the yeast cells in the must being
destroyed by the sulphurous acid. Gay-Lussac, on the other hand, viewing the
matter differently from his standpoint, held the opinion that as the sulphurous
acid had a strong affinity for the oxygen, the two combined, and as no oxygen
was available for starting the fermentation, the must necessarily remained inert.

The experiments made by Schwann in 1838, and described in § 13, refuted
the opinion of Gay-Lussac, by demonstrating that the role of exciting fermentation
is set up by certain microscopic living creatures which perform their functions
in the absence of oxygen. Subsequent research proved that the presence of this
gas is altogether superfluous, so far as the progress of alcoholic fermentation is
concerned, although it is not without influence thereon. PASTEUR (II.) in 1861
established it as a fact that this progress is more satisfactorily effected when
the fermenting liquid is subjected to brisk aeration.

§ 12.— Cagniard-Latour's Vitalistic Theory of Fermentation.

The French apothecary ASTIER (I. and II.) has generally been credited with
being the next individual, after Leeuwenhoek, who gave his attention to the
nature of yeast. An examination of his published works shows, however, that

12 THEORIES OF FERMENTATION

his investigations into fermentation were conducted without the aid of the
microscope, so that he did not bring to light any actual facts concerning the
nature of yeast, but — as was 'pointed out, though in vain, by QUEVENNE (I.) as
far back as 1838 — based his assumptions on hypotheses devoid of foundation.

In the same way another Frenchman, viz., Desmazieres, the reputed pioneer
of the founders of the vitalistic theory of fermentation, cannot permanently retain
this title. Like Astier, he is said to have recognised the part played by yeast in
fermentation, but, as a reference to his treatise, published in 1826 (in pages 42
to 67 of vol. x. of the Ann. des Sc. Nat.\ will show, this assertion is incorrect.
In these observations Desmazieres viewed the matter simply as a naturalist.
His investigations of the fungoid growths covering the surface of moist substrata
were conducted from this point of view, and it was in the course of this study
that he examined the mycelia that develop on beer, &c. These consist of masses
of elongated cells, to which he gave the name Mycoderma cerevisice. As he
fancied they exhibited powers of locomotion, he considered them as belonging to
the animal kingdom (animalcida monadina), but, true to his purely descriptive
inclinations, he disregarded their physiological properties, and especially their
influence on the substratum. Thus the reputation attributed to Desmazieres of
having, in 1826, microscopically studied the morphology of the yeast-like cells, to
which Persoon had definitely alluded four years earlier, is dissipated by facts.

On the other hand, a German worker, viz., EKXLEBEK (I.), had already, in
1818, correctly estimated the importance of yeast, in that he asserted it to be a
living organism, the vital functions of which are the cause of fermentation.
Unfortunately he did not follow up this idea, which was thrown out as a mere
occasional remark in his treatise on practical analytical experiments. Otherwise
he would, in 1818, have anticipated what was only accomplished twenty years
later, viz., the establishment of the fact that (alcoholic) fermentation is oausa-
tively connected with the life (vita) of certain organisms. This was determined,
almost simultaneously, by three investigators working quite independently of
each other : Cagniard-Latour in France, and Theodor Sehwann and Friedrich
KUtzing in Germany.

The paths by which these three arrived at their common goal differed. The
versatile French technicist is known by name to the majority of educated people
on account of the siren he invented, and which is largely used in the science of
acoustics. He also devoted some attention to brewing, and compiled a work on
the fermentation of beer. The preliminary studies undertaken in this connection
led him to more closely investigate the nature of the " yeast," of which — notwith-
standing the observations of his two compatriots already mentioned — practically
nothing was then known. This material he examined with the assistance of the
microscope, and laid the results of his researches before the Parisian Academy on
June 12, 1837, in a short paper (II.) containing the following chief points :

1. Beer-yeast, instead of being an inanimate chemical substance, as previously
supposed, actually consists of small globules which possess reproductive power,
and are therefore living organisms.

2. These bodies appear to belong to the vegetable kingdom, and to reproduce
themselves in two ways.

3. They seem to act upon sugar solution only whilst still living ; wherefore it
may, with great probability, be concluded that, by their vital activity, carbon
dioxide is liberated, and the sugar solution transformed into an alcoholic liquid.

§ 13.— The Researches of Theodor Schwann.

As the words printed in italics in the two preceding sentences show, and as
a closer examination of the original treatise will more clearly reveal, Cagniard

THE RESEARCHES OF THEODOR SCHWANN 13

did not indubitably establish the vegetable nature of yeast. The accomplish-
ment of this task, and the attribution of this organism to its proper position in
the system of Botany, formed the subject of a treatise published by SCHWANN (I.)
in the first half of 1837, i.e. contemporaneously with Cagniard's paper.

In following up the results of his researches on spontaneous generation,
Schwann studied beer-yeast, and found that the individual globules, of which the
mass was seen under the microscope to consist, frequently became united into
chain-like or laterally branching bands, and presented to the eye an appearance
greatly resembling that of many already well-known multicellular fungi. It
was not this discovery alone, however, but rather their mode of reproduction,
which induced Schwann to consider these bodies as of a vegetable nature. In
this process the globule pushes out from its interior a small nodule, which
Schwann was able to observe develop to its normal dimensions. " Observation
of its growth leaves no doubt as to its vegetable nature, since animals do not
reproduce themselves in this manner." The rate of reproduction of the globules
kept pace with the increasing briskness of the fermentation, so that Schwann
came to the opinion that it was highly probable that the development of the
fermentation was induced by that of the organism.

" Vinous fermentation must therefore be regarded as the decomposition
occasioned by the sugar fungus extracting, from the sugar and a nitrogenous
body, the materials necessary to its nutrition and growth, whereby such elements
of these bodies (probably among other substances) as are not taken up by the
plant unite, by preference, to form alcohol."

This discovery was communicated by Schwann to his friend and colleague,
Professor Meyen, who tested and confirmed it, " .stating with reference thereto,
that the only doubt arising was whether the organism in question was an alga or
a thread fungus, the latter seeming the more likely by reason of the absence of
green pigment." Thus yeast was recognised as a fungus, and, from its capacity
of fermenting sugar, was designated sugar fungus : whence the current generic
name, Saccharomyces Meyen.

According as such a sugar fungus was found active in beer- wort or wine-must,
it was called by the specific name of S. cerevisice or S. vini, which names remained
in general use in their original significance until REES (I.) in 1870 proposed a
system of differentiation which will be more fully noticed in a subsequent
paragraph.

As follows from the remarks already made, the name "yeast" applied merely
to one particular group of ferments, viz., those producing alcoholic fermentation.
For a considerable period after Cagniard's discovery, however, it was used indis-
criminately for all ferments. Thus, for example, Pasteur speaks of the " yeast "
of lactic fermentation, meaning thereby Bacteria ; and even in 1879 Nageli, the
investigator of the fission fungi, refers in his " Theory of Fermentation " to the
" yeast " of putrescent urine. This misuse of the term has been abandoned, and
the name " yeast " is now only employed when speaking of the budding fungi
that excite alcoholic fermentation.

§ 14.— Friedrieh Kutzing-'s General Theory of Fermentation.

The views promulgated, by this German worker in the field of Vegetable
Physiology and the Algae were in harmony with the spirit manifested in the
" Elements of Philosophic Botany."

Published almost simultaneously with the above-mentioned communications
of Cagniard-Latour and Th. Schwann — though actually compiled at a much
earlier date (before 1834) — Kutzing's treatise (I.) on this subject surpassed
those of his two colleagues in more than ore particular. The value of his

14 THEORIES OF FERMENTATION

actual determinations is not, in our opinion, lessened by the fact that he was an
advocate for spontaneous generation, since at that time (in 1837) there existed
no decisive and unassailable proofs to controvert this theory.

KUtzing did not restrict his researches solely to alcoholic fermentation, but
also instituted comparisons with a number of other similar phenomena, regard-
ing them all from the same point of view. Even though he must share with
others the credit of having discovered the organised structure of yeast, that of
determining the vegetable nature of the " mother of vinegar " and recognising
its mode of action belongs to him alone. With these discoveries are associated
a number of others of minor importance, such, for example, as the physiological
basis of the method (propounded by Scheele) of preparing gallic acid by allowing
a solution of pyrogallic acid (e.g. gall-nut extract) to become infested with
mould. The numerous phenomena he brings under our notice constitute so
many proofs of the theory that fermentation cannot be regarded as a purely
chemical process. " It is well known that chemistry explains vinous fermenta-
tion by the reaction of the so-called gluten on the amylum (starch) and sugar.
I must firmly maintain that the explanation does not give me a clear idea of
the process, and I am inclined to doubt whether others are more fortunate in
this respect. It is, however, certain that the entire process of alcoholic fermen-
tation is dependent on the formation of yeast, and the acid fermentation on the
formation of the vinegar plant. . . . Along with the increased growth of these
organisms the reproductive impulse also increases, and,* concurrently, their
reaction on the liquid present. ... In so far as fermentation is synonymous
with a reciprocal reaction of oi'ganic and inorganic bodies on the constituents of
a given liquid which may be regarded as forming the nutrient medium of the
organic product, so is it necessarily synonymous with every organic vital func-
tion : wherefore organic life = fermentation. On the other hand, such pro-
cesses as lead to the production of vinegar from alcohol by the use of platinum
black or other similar methods, cannot be compared with fermentation, being
purely chemical, whilst fermentation is an organo-chemical process, as is also the
life process of any organic body." "

One of the three members of the committee appointed by the Academie des
Sciences of Paris to report on the memoir presented by Cagniard — namely,
TUEPIN (I.) — took the opportunity thus afforded of experimentally dilating
upon his compatriot's work, and of amalgamating these new " discoveries "
with the revelations of Schwann and KUtzing. In this way a volume,
containing more pages than Latour's communication had columns, came into
existence, without, however, adding to our knowledge in the slightest degree.
Turpin seems, however, to have thoroughly known his public, since he is even
now regarded as one of the founders of the vitalistic theory of fermentation,
not only by compilers of text-books, but also by actual investigators, from whom
one might more reasonably expect a more thorough study of the original works
of their predecessors.

§ 15.— Liebig's Decomposition Theory.

Two years subsequent to the publication of the works of Cagniard, Kutzing,
and Schwann, Liebig placed before his colleagues a new theory, according to
which fermentation was a purely chemical reaction.

In order to avoid judging this chemist unreasonably, one must bear in mind
the age wherein this theory was promulgated. Synthetic organic chemistry
had just been founded. Eleven years previously (1828) Wbhler had succeeded
in artificially preparing urea, to the astonishment of his contemporaries, who
had hitherto considered as impossible the artificial production of organic

LIEBIG'S DECOMPOSITION THEORY 15

substances outside the animal or vegetable body of whose vital functions they
are the outcome. That organic substances could not be produced without the
concurrence of vital power was up till that time an established maxim. To
overthrow this dogma, and to prove that any desired organic substance can be
prepared without the assistance of vital action, was the endeavour of the
majority of the chemists of the age, Liebig being one of the foremost, most
industrious, and stubbornest workers in the cause. It is, therefore, small
matter for astonishment that — Cagniard-Latour, Kiitzing, and Schwann to
the contrary, notwithstanding — he could conceive a theory of fermentation
wherein the action of living organisms had no place and their vital force was
ignored.

The struggle against what he considered to be the objectionable theory of
these three physiologists was opened by Liebig in 1839 with an anonymous
treatise (I.), in which the new observations of the microscopists were covered
with highly amusing satire. In the next year he returned to the charge in
earnest in his work on " Organic Chemistry in Relation to Agriculture and
Physiology," on pp. 202-299 of which his new theory is enunciated. This is, as
already mentioned, akin to that put forward by Stahl more than a century
before.

Liebig considered all fermentation as molecular movement, which a body in
a state of chemical movement, i.e. decomposition, transfers to other substances
whose elements are not very firmly combined. Between fermentation (in its
limited sense) and putrefaction there is the following difference : In the latter —
putrefaction — the decomposition is transmitted by the decomposing material —
viz., the albuminoids — so that putrefaction, once begun, is continued by inherent
movement, even though the initial cause has been rendered inactive. With
fermentation it is otherwise. In this process the body (sugar) in a state of
incipient decomposition cannot transmit the movement to the still undecom-
posed substance. Consequently this function has to be performed by an extra-
neous causative agent, a ferment, which in this case is necessary not only for
commencing (as with putrefaction), but also for continuing the decomposition.

It must be admitted that, at first sight, this definition, as also the differentia-
tion between fermentation and putrefaction, is very attractive. Nevertheless
it will not bear the light of keen criticism. Take, in the first place, the character
on which the distinction between fermentation and putrefaction is based, viz.,
that the former will not go on without the presence of the ferment, whereas,
on the other hand, putrefaction, when once started, continues spontaneously,
the ferment being no longer needed. The reason why Liebig was induced to
make this distinction is easy to fathom. In the case of fermenting beer-
wort — which Liebig usually had in view when speaking of fermentation
— the ferment (beer-yeast) was discernible to the naked eye, and experience
taught that without this ferment the fermentation could not be satisfactorily
carried on. On the other hand, the presence of those minute organisms which,
as we now know, insinuate themselves into all substances liable to putrefaction,
and decompose the same without, as a rule, giving rise to such a multiplication
of the deposited ferment as can be remarked by the inexpert eye, is not so
immediately apparent as in alcoholic fermentation.

Thus, even in Liebig's opinion, yeast is essential to the continuance of
fermentation ; only, the ferment is degraded to a simple albuminoid substance.
To enter nowadays into a further onslaught against this theory would be
merely storming an undefended position. Moreover, as the subsequent editions
of the aforesaid work demonstrate, it was gradually modified by its author, so
that the form in which it was presented in his latest exposition (II.) in 1870
differs in many particulars from the original.

1 6 THEORIES OF FERMENTATION

The ocular demonstration, in individual instances, of the untenable nature
of the hypotheses supporting this theory, to those whom the representations
of Latour, Kutzing, and Schwann had not succeeded in convincing, was the
congenial task undertaken by Pasteur, and brought by him to a successful
issue with great experimental skill.

§ 16.— Pasteur's Theory of Fermentation (III.).

The victorious antagonist of the theory of spontaneous generation was not
content with controverting the views of Liebig ; he also sought to erect a better
theory in its stead. According to this doctrine, it is the lack of free oxygen that
leads to the fermentation being set up by the organism as a means of supplying
itself with the energy it requires by seizing upon the oxygen thereby obtain-
able. " Fermentation is life without air." A very slight experience in this
matter suffices for the recognition of the fact that this theory takes no account
of the several kinds of fermentation in which the presence of oxygen is a neces-
sary condition, viz., the so-called oxidation fermentations — the best example of
which is afforded by the acetic fermentation. In this respect the theory cannot
be further alluded to in the present chapter, which is devoted to general con-
siderations. It will be fully dealt with in a subsequent chapter. The only
remark to be made now is that this theory also has proved untenable.

The permanent value of the services rendered to Fermentation Physiology
by Pasteur are not diminished by the disproval of his theory of fermentation,
since they have their root in the successful endeavour, by means of careful and
extensive experimental demonstration, to bring into universal recognition the
theory originated — but only imperfectly formulated — by Cagniard, Kutzing, and
Schwann, of the causative connection between fermentation and the vital
activity of the microbe.

§ 17.— C. Nag-eli's Physico-Molecular Theory.

Although it was by this time indubitably established that without the vital
activity of micro-organisms no fermentation could occur, no clear account had
been given as to how the activity itself was exerted. Several explanations
were possible. According to one which was especially advocated by Kutzing and
Pasteur, a decomposition was effected within the cells of the organic ferments,
which obtained their nourishment from the fermenting material (e.g. sugar) and
discharged the fermentation products as waste matter.

According to another, the decomposing force simply emanated from the cells
and became the direct cause of the decomposition of the fermentable matter
around them.

The physico molecular theory, proposed by NAGELI (II.), expresses this view
in the following words : " Fermentation is, therefore, the transference of con-
ditions of movement in the molecules, atomic groups, and atoms of the various
compounds constituting the living plasma (which compounds remain chemically
unchanged) to the fermentative material, whereby the equilibrium of its molecules
is destroyed and their dissociation induced." The radius of the sphere of influence
of the individual yeast cells is estimated by Nageli as from 20 to 50 /*.

This definition differs from that formerly given by Liebig merely in a single,
though important, consideration, viz., it regards the living cell as the source of
action, whereas the other definition speaks of inanimate albuminoid substances.
.Nageli was, however, unable to prove the correctness of his theory, and the calcula-
tions deduced from other observations which he brought forward in support
thereof have in course of time proved inapplicable.

THE ENZYMES AND M. TRAUBE'S THEORY 17

§ 18.— The Enzymes and M. Traube's Ferment Theory.

There is still another possible explanation of the power of living cells to act
at a distance. This regards the cells, not as centres of radiating molecular
movements, but as forming centres of production of metabolic products which
penetrate through the cell membrane into the surrounding liquid. There they
become widely distributed by diffusion, and by their influence bring about the
decomposition of certain constituents of the solution, but do not undergo any
chemical change themselves. These active bodies are called enzymes, and their
behaviour is, as will be observed, different from that of ordinary chemical agents,
since the latter effect alterations in other groups of atoms by their chemical
affinity, whereby the old combination is broken up, and the separated portion
enters into a new atomic grouping with a part of the active agent. Accordingly,
a definite weight of the agent can only displace a definite quantity (known as the
"equivalent weight") of other compounds; whereas the enzymes behave differently,
their activity being practically illimitable. They do not combine with the pro-
ducts of the reaction, but continue to act on the residual undecomposed substance.

The first enzyme was discovered by PAYEN and PEESOZ (I.) in 1833, who
detected in malt extract a substance — which they termed diastase — capable of
converting starch into sugar. They were, however, unable to isolate it in a pure
condition. Three years later THEODOR SCHWANN (III.) discovered in gastric
juice pepsin, subsequently also named peptase, which in faintly acid solutions
resolved undiffusible albumen into assimilable dissociation products. Since that
time the same enzyme has also been detected in various vegetable organisms,
many varieties of bacteria in particular having the power of elaborating it.
There will be ample opportunity for reference to this point along with the other
known enzymes at a subsequent stage. At present we have only to consider them
as the basis of a theory of fermentation, the formulation of which dates back as
far as 1858, but has come to the front more of late years, and — so far as can be
judged from the data at present available — will acquire still greater importance.

As we have observed in § 10, the meaning attached, under the influence of
alchemical views, to the word ferment was, until the close of the eighteenth
century, very comprehensive, and it was only then that the restriction of the
term to bodies inciting fermentation began. Contemporaneous with the develop-
ment of positive knowledge with regard to these bodies was the discovery of the
enzymes, the behaviour of which resembled that of the former, in so far that
they exhibited a capacity of inducing decomposition. Moreover, the obscurity
in which these organisms were still enshrouded was equally mysterious in both
cases ; and, since the organic nature of the true instigators of fermentation was
either unknown or was not considered worth attention by the chemists of the day,
it happened that the name " ferment " was also applied to the newly discovered
enzymes. With an increasing insight into the true state of the matter grew the
conviction that two very different things had been grouped under orue name, and
this conviction found expression in the distinction thenceforward of the true
instigators of fermentation as organised or structural ferments, whilst the enzymes
were designated unorganised or structureless ferments. These terms are still
current in chemical text-books, whereas in Fermentation Physiology it is customary
to speak merely of fermentative organisms on the one hand and of enzymes on
the other.

M. TRAUBE(!. and II.) in 1858 made the origin and influence of these enzymes
the basis of a new conception (Ferment Theory) of fermentation, according to
which this process is not instigated by the organisms themselves, but by the
enzymes formed as products of their vitality and excreted by them.

1 8 THEORIES OF FERMENTATION

This theory was accepted by many persons, e.g. by Hoppe-Seyler, who con-
sidered it as being so self-evident to chemists as not to require any demonstra-
tive evidence. Nageli, on the other hand, advocated its rejection, mainly for the
reason that he was not convinced as to the existence of any fermentative
enzymes. Numerous workers have, however, since investigated this point, with
the result that Traube's opinion has again found general acceptance. The
investigations of Miquel with regard to urase, i.e. the enzyme excreted by the
bacteria of uric fermentation, merit special mention in this place, because they
brought our ideas into closer harmony with the ferment theory, at least so far as
regards the ammoniacal fermentation of urine. Moreover, they have greatly
strengthened the position of the ferment theorists by proving that this urase
cannot be placed amongst inorganic chemical substances, in the ordinary sense of
the term, but is really an intermediate stage between these and living pi^otoplasm.
Miquel goes so far as to say that his urase is actually protoplasm, chiefly differing
from that of the cell contents in that it dispenses with the protection of the cell
wall, and remains and works on the outside.

The reader desiring fuller information on the properties of the enzymes than
can be obtained from the present work is referred to the comprehensive treatise
published by E. BOURQUELOT (I.) in 1896.

§ 19.— General Definition of Fermentation.

We will now pass in review all the preceding explanations, and attempt to
extract from each of them whatever can possibly afford us assistance in finally
arriving at a satisfactory definition of the term fermentation. In the first place,
Liebig's explanation certainly does not call for further consideration in this con-
nection. In the results of the remaining researches we find one factor common
to all, and that is the certainty that, for the inception and continuance of the
process, which — in harmony with the limitation expressed in § i — we have
hitherto entitled " fermentation," the presence and active collaboration of low
types of living organisms is a prime essential. Concerning the nature of the
influence, whether direct or indirect, of these organisms, opinions are, however,
divided.

If, now, the instigators of fermentation be examined seriatim according to
the method outlined later on, it will be found that not only are they vegetable,
but also that all belong to the same class, and that, too, the lowest in the vegetable
kingdom, namely, the fungi. The power of inciting fermentation is, however,
restricted to comparatively few of the genera of this class. Nevertheless, these
latter are so intimately connected with the others, from a botanico- morphological
point of view, that it is impossible- to classify the fungi into two sub-groups,
characterised by the presence or absence of this faculty, without introducing
serious systematic anomalies.

The limitation that can be given to the definition of fermentation may be
thus expressed : " Fermentation is a decomposition effected by the vital activity
of fungi." Nevertheless, as is evident from what has already been intimated,
no greater precision can at present be imparted to such part of this definition as
refers to the nature or mode of action of the ferment. On the other hand, as
will soon be apparent, the word " decomposition " must give place to a term
that is both more accurate and more comprehensive.

The phenomena of fermentation forming the starting-point from which the
workers from Cagniard to Nageli began their researches, and which up to the
present have been the sole subject under our consideration, possess one charac-
teristic in common, i.e. they are always attended by the degradation of complex
organic compounds into simpler ones. By regarding this characteristic by itself,

SPONTANEOUS FERMENTATION OF SWEET FRUITS 19

fermentation might be defined as a decomposition of organic substances by the
agency of fungi.

The researches of the past fifteen years (1880-1895) have, however, neces-
sitated considerable modifiations of the words italicised. The study of the
bacteria of the nodules of leguminous plants has taught us that their main func-
tion consists in bringing about the combination of free atmospheric nitrogen :
hence their action is not a decomposing (analytical) one, but is synthetic or
constructive, so that in respect of these organisms our definition will have to
include the word " transformation." The adjective " organic " may still remain,
since the microbes in question require organic nutriment in addition to the free
nitrogen.

We shall, however, find ourselves constrained to reject this latter term when
we come to the study of nitrification, and make the acquaintance of another group
of microbes able to dispense with organic nutriment, and indeed thriving and
acting most effectively only when surrounded by inorganic substances exclusively.
Consequently we arrive at the following final and conclusive definition : Fermen-
tation is a decomposition or transformation of substances of various kinds
brought about by the vital activity of fungi.

§ 20.— The So-called Spontaneous Fermentation of Sweet Fruits.

The point of the preceding definition lies in the concluding words, which
restrict the term fermentation to such decompositions or transformations as
are produced by the vital activity of fungi. It is, however, not impossible for
similar decompositions to be effected in other ways, especially by the aid of other
vegetable cells differing from fungi. An example of such a reaction, resembling
fermentation but not induced by fungi, is afforded by the so-called spontaneous
fermentation of fruit.

• The first reliable data with regard to this phenomenon were collected by
LECHARTIEB and BELLAMY (I.) in 1869, all previous observations having to be
disregarded because they do not show that the activity of yeast-cells was pre-
cluded. Starting with the notes made in 1821 by their compatriot BERARD (I.),
these French investigators succeeded in establishing the fact that sweet fruits,
e.g. cherries, when kept in an uninjured condition and free from yeast in an
atmosphere of carbon dioxide, consume a portion of their sugar content, gaseous
carbon dioxide being evolved in the process and alcohol formed. The presence
of this latter substance in the cells of the fruit substance can be proved by
distillation, as much as one per cent, by weight having been detected in this
way.

PASTEUR (IV. and V.) also studied this phenomenon, which he employed as
one of the main buttresses of his previously formed theory asserting fermentation
to be a universal phenomenon, not dependent on certain organisms, but occur-
ring in every living vegetable cell debarred from a supply of oxygen. This
spontaneous fermentation appeared to form a striking proof of the correctness of
this theory, but the hopes thereby raised proved vain, since Pasteur was himself
the one to discover that alcohol (though in merely minute quantities) is also
formed in fruits when they are exposed to the air.

§ 21.— Decompositions Effected by Light and Air.

To the chemist — who is obliged to store in the dark and in properly stoppered
receptacles various inorganic reagents and normal solutions which he desires to
maintain in an unaltered condition — it will not be surprising to learn that sterile

20 THEORIES OF FERMENTATION

solutions of organic substances — such, for example, as are stored for use as
nutrient media for bacteria — also gradually undergo slight modifications when
air and light find admittance thereto. The proof that these changes are of a
purely chemical nature lies in the fact that they do not occur when the causes
indicated are absent.

In many instances the oxidation of the medium effected in this manner exerts
a favourable influence on the development of the organism subsequently inoculated
therein. Such, for example, is the case with beer-yeast. In wort through which
air has been blown ("roused ") for some time, the yeast sown therein develops
more rapidly, and deposits more quickly and satisfactorily (" breaks " better) at
the conclusion of fermentation, a circumstance highly desired by the brewer.
Sunlight possesses an even greater decomposing power than that of atmo-
spheric oxygen. On this point we are indebted to E. DUCLAUX (I.) and W.
SEEKAMP (I.) for exhaustive researches, the former of whom found that, in
presence of air, a sterile solution of tartaric acid is split up by sunlight into
formic acid, carbon dioxide, and water, according to the equation —

C4H606 + 30 = 2CH202 + 2C02 + H20.

In a second communication (II.) on this subject the same worker showed that
glucose and lactose in a sterile alkaline solution gradually decompose into alcohol
and carbon dioxide on exposure to sunlight, even when oxygen is excluded. The
same products are yielded by them when fermented with yeast. When baryta
(BaH202) or lime (CaH2O2) was substituted for the alkali, lactic acid was produced
instead of alcohol. Under the same treatment maltose yields dextro-lactic acid ;
levulose, levo-lactic acid ; and invert sugar the optically inactive acid. A similar
observation was made by WEHMEE (I.) with respect to a sterile solution of oxalic
acid.

These observations are of great interest to the bacteriologist, from a theo-
retical as well as a practical point of view, as they convey a special warning to
protect his stores of nutrient media from the influence of sunlight. G. Roux, as
the result of his adverse experiences, had already given the same warning as to
the prejudicial influence of the changes produced by sunlight on bacterial growth,
prior to the more exhaustive research by Duclaux.

These facts have a further interest, more nearly connected with our definition
of fermentation, since they demonstrate the occurrence of decomposition processes
by purely chemical means, apart from the intervention of micro-organisms. We
will therefore modify our general definition of fermentation, and, in place of
stating that fermentation is a process accomplishing transformations of matter
with the aid of micro-organisms only, will reverse the phrasing, and say that only
such changes as are effected exclusively by the vital action of ferments come within
the meaning of the term fermentation. The point of the definition, as already
mentioned at the commencement of the previous paragraph, lies in the words
italicised.

However equivalent the action of purely inorganic force on the one hand,
and of living organisms on the other may appear, it is so in regard to quality
only, the quantitative effects, the amount of substance decomposed in unit time,
being widely different. Regarded from this point of view, the minute ferments
appear as centres for the accumulation of high-tension energy, by the release of
which force the decomposition in view can be effected, not only in a shorter time,
but also in a more restricted space, than is possible by the action of purely
chemical forces.

III.

THE ORGANISMS OF FERMENTATION.

§ 22.— The Position of the Organisms in the Botanical System.

THE study of Mycology, or the science of Fungi, can be pursued from several
standpoints. The purely scientific position is assumed by the botanist, who
accords to each kind just as much importance as its morphological and physio-
logical considerations warrant. If, however, the standard of interest adopted
be that of the importance of the part played by the fungi in practical life — i.e.
Applied Mycology — then the number of species to be studied is reduced in a
very gratifying manner. The degree of attention bestowable on those remain-
ing is determined, not by their systematic position, but by the influence they
exert on their environment, the nutrient medium.

If the object subjected to the influence of the fungi is a living creature, i.e.
an animal or a plant, it is thereby brought into the condition universally known
as diseased. Fungi endowed with this power are designated pathogenic, and
their study is entitled Pathogenic Mycology, which is subdivided into two
branches, according to the natural classification of the infected organism. In
the case of human beings or animals, we have the study of Medico-Patho-
logical Mycology, and in the case of plants, Phyto-Pathological Mycology.

On the other hand, inanimate objects, such as milk, wort, vinegar, manures,
leather, indigo, &c., on which the influence of the fungi is manifested by
symptoms of decomposition, constitute the subject of Technical Mycology,
which differs from the pathological branch in another characteristic, namely, in
the nature of the influence suffered by the object. Pathological Mycology is
exclusively concerned with pathogenic, and therefore noxious, fungi, and its
object is to bring about their exclusion and annihilation. The aim of Tech-
nical Mycology is different, being to effect, by the aid of fungi, useful decom-
positions and transformations which, without the use of such living tools,
could only be accomplished incompletely, or in a more roundabout and costly
manner.

It is, therefore, with the influence of the fungi on their environment, i.e.
the manifestations of their vitality, that technical mycology has to do. Hence
it is principally the study of the vital functions of ferments, and may therefore
be also termed Fermentation Physiology. With botanico- physiological con-
siderations it is concerned only in so far as they either afford assistance in the
comprehension of the physiology of the organisms, or facilitate the differentiation
of the various species from one another; and to this extent a knowledge of the
morphology of the fungi is of essential assistance to the technical mycologist in
the attainment of his objects. Before going more minutely into this matter, it
will, however, be advisable to have a general view of the position occupied by the
fungi in the botanical system.

As every reader will be aware, the sub-kingdom Cryptogamia, which com-
prises all non-flowering plants, is divided into three chief sections, or seven
classes, viz. :

22 THE ORGANISMS OF FERMENTATION

I. Thcdlophyta.-?hMou* plants, with- f Class '" ^'--Fungi, devoid of chloro-
outl aves, stems, roots, or vascular J. 2 Algv.-Mg*, containing chloro-

bundles. ^ phyl]

II. Bryophyta— Mosses, with leaves and ) 3. Hepatirup.— Liverworts.

stems, devoid of true roots and
vascular bundles.

IIT. Ptcridophyta. — Vascular cryptogams,
with leaves, stems, true roots, and
vascular bundles.

4. Musci. — Feather mosses.

5. Lquisetince. — Horse-tails.

6. LycopodincE. — Lycopodiurn.

7. Filicince. — Ferns.

To the first of these three main divisions belong all those plants designated
Thallophytae on account of the absence of any specialisation of parts, such as
stem, leaves, &c., and from the comparatively simple form (Thallus) of the
individual. This section is subdivided into the two classes fungi and algae.

The body of all the remaining plants, from the mosses upwards, shows, on
the other hand, a differentiation of parts into stem and leaf, and is generally
designated Connus, all the higher plants being therefore generally grouped under
the title Cormophytes.

Of the seven cryptogamic classes, only one, the first and lowest (Fungi),
comes under consideration in Fermentation Physiology. In accordance with the
preceding scheme, these fungi are definable as : cryptogamic plants, devoid of
chlorophyll, roots, stems, leaves or vascular bundles ; or, expressed in a more
concise form : the fungi are thallous growths devoid of chlorophyll.

§ 23.— Classification of the Fungi.

The fungi are arranged, according to the individual mode of growth, into
two main groups, namely, Schizomycetes, or fission fungi, and Eumycetes, or higher
fungi. The latter consist in the main of thread-like cells, grow by acrogenesis,
form true branches, and reproduce by special organs called spores. Conversely,
the multplication of the (always unicellular) fission fungi is effected by sub-
division or fission, whence their name, so that we have the following scheme :

p . (Schizomycetes: (Fission fungi).— Fission.
u y1 \Eumycetes : (Higher fungi). — Acrogenous, Branching.

No doubt many readers will miss from this classification a third sub-group,
viz., the Myxomycetes, or mucus fungi. In order to at once disarm any objection
on this score, we will here mention that the organisms in question have been
shown by recent researches to belong, not to the vegetable, but to the animal
kingdom, of which they constitute the lowest type of development. For this
reason modern systematology has applied to them the name bestowed by De
Bary, viz., Mycetozoa, animal fungi, or fungoid animals.

§ 24.— Schizophytse.

As their systematic juxtaposition would lead one to conclude, the fungi and
algse exhibit many traits in common, and, in fact, the only important character
by which they can be differentiated is the absence of chlorophyll in the first-
named.

As will be readily understood, it is especially the lower and more simply
constructed species of algae that, apart from the characteristic difference just
mentioned, approximate closely to the fungi. This is particularly the case
with the fission algae or Diatomacece, which, with respect to their method of
reproduction by fission, bear no slight resemblance to the fission fungi.

SAPROPHYTES AND PARASITES 23

Owing to their greater size, and consequent discernibility, the algte formed
the subject of investigation at a much earlier date than the very much smaller
fission fungi, which necessitated the employment of more perfect methods of
examination, so that, by the time the latter began to be studied, the green algae
had already been systematised. The temptation to include the newly-discovered
bacteria among the analogous algse was therefore great, and thus it was that the
meritorious German algologist FR. KUTZING (I.) -was induced to ascribe the
acetic acid ferment discovered by him, and now known as Bacterium aceti, to the
algse, under the name of Ulvina aceti.

This inclination to regard fission fungi and algse as belonging to the same
class was also manifested by subsequent workers, and the more so since the
greater insight gained in the interim spoke more conclusively in its favour than
was possible in the initial, imperfect stage of knowledge. Hence FERDINAND
COHN (II.) in 1875 was obliged to discard his own (IV.) term for the bacteria,
viz., Schizosporce, as also that (Schizomycetes) proposed by N'ageli (V.) in 1857,
and to classify these organisms with the lowest of the algse (Nostoc, Chroococcus,
Merismopedia, Oscittaria, <fec.) into one group, which, from their common and
characteristic property of reproduction by fission, he called Schizophytce (fission
plants), and set up as an independent division.

§ 25.— Assimilation of Carbon Dioxide without the
Aid of Chlorophyll.

The classificatory basis for arranging the Thallophytes under the two groups
Algce and fungi, viz., the presence or absence of chlorophyll, is not of a
morphological, but of a physiological nature, as its connection is not with the
form, but with the vital activity of the cell. Now, it has been proved by much
research that chlorophyll plays an important part in the life of green plants.
The chlorophyll in the cells, aided by sunlight, splits up the carbon dioxide which
the plant has absorbed from the air. The oxygen of the CO2 is exhaled, whilst
the carbon is retained and utilised in the elaboration of the various organic
substances of which the body of the plant is composed. This operation is known
as the assimilation of carbon dioxide. Until recently, the opinion was generally
held that this process could not go on without the assistance of light and
chlorophyll, and as the fungi are, without exception, devoid of the last-named
substance, it has been laid down as a law that the fungi are incapable of
assimilating carbon dioxide and of constructing their cells of inorganic substances
like the algse do. The researches of Winogradsky have, however, shown that
there are fission fungi capable of splitting up carbon dioxide in the dark and
without chlorophyll, so that the above law has lost its universal applicability.
This point will be more completely treated in the chapter on the nitrifying
bacteria.

§ 26.— Saprophytes and Parasites.

Plants that are unable to obtain their necessary supplies of material by
drawing on the resources of inorganic nature exclusively are termed parasites.
As is evident from the statements in the preceding paragraph, all the fungi (with
some exceptions to be hereafter mentioned) are therefore to be characterised as
parasites. Incapable of elaborating the highly complex molecule of their cell
substance from the elements or the simplest atomic compounds (CO2, H2O, NH3,
&c.), they depend for their supply of nutrient material on ready-formed organic
substances, which they have then merely to rearrange according to their needs.

If this semi-prepared nutriment is obtained from a living creature (animal or

24 THE ORGANISMS OF FERMENTATION

plant), such fungi are designated parasitic ; but if, on the other hand, they
utilise inanimate (or defunct) organic material, they are known as saprophytes.

The parasites proper may be divided into two groups : strict or obligate
parasites, which are restricted to living animals or vegetable bodies, and facul-
tative parasites, which will also thrive on suitable inanimate substrata, and can
therefore be cultivated on artificial nutrient media.

This latter group forms the connecting link between Pathological and
Technical Mycology. The former science regards these, together with the
obligate parasites, as the causes of disease, whilst conversely Technical Mycology
is interested in the facultative parasites on account of the decompositions they
induce in artificial media, i.e. outside the animal or vegetable economy. This
interest is, however, purely scientific, since, for hygienic reasons, the use of
parasitic ferments for practical technical purposes is precluded.

The true interest of the technicist in the domain of Mycology is exclusively
centred in the non-parasitic ferments, and these alone form more especially the
subject of the present work, the first volume being devoted to the Schizomyceles,
and the second to the Eumycetes.

DIVISION I.
SCHIZOMYCETIC FERMENTATION.

SECTION I.

GENERAL MORPHOLOGY AND PHYSIOLOGY OF
THE SCHIZOMYCETES.

CHAPTER I.

FORM AND DIMENSIONS.

§ 27. — Forms of Growth.

THE cell forms of all the Schizomycetes may be typified by a small rod ; hence
these organisms are generally known by the name of bacteria, from the Greek
rendering of this descriptive term, bakterion, a rod.

When the dimensions of a bacterial cell are equal in all directions, it is
termed a coccus or micrococcus, monas or coccobacterium. On the other hand,

FIG. 3. — Spirochaeta and Spirilla. Mag. 950. (After P. Baumgarten.)

i. Spirochaete Obermeieri, the cause of relapsing fever (Febris recurrens). '
2 Spirochaeta from human dental mucus.

3. Denecke's Spirochaete from putrescent cheese.

4. Spirochaetal form of Koch's Cholera asiatica bacillus

5. Spirillum volntans, Cohn.

when tl.e cell is not iso-diametrical, but exhibits a difference between length and
breadth, the name bacillus, or elongated rod, is applied, provided the length is
at least double the breadth ; but when the former does not attain this rela-
tive size, we then speak of "bacteria. This latter term has therefore a twofold
application : a general one, in so far as all the fission fungi are briefly called

26 FORM AND DIMENSIONS

"bacteria: " and a special one, referring only to a particular form of growth, as
just mentioned.

A bacterial cell of the bacillus type which, during a certain physiological

yW

rixv

^ tffa $u

FIG. 4. — Illustrations of the manifold variety in size and form of different bacteria.

Kxcept A4 and A5, all the other illustrations are representations of equally magnified
bacteria from a single drop of putrescent blood. (After P. Baumgarten.) Mag. 950. Those in
A are all symmetrical cells, those in B are elongated.

A. — i. Cocci (micrococcns) of various sizes.

2. Diplococci of various sizes.

3. Streptococci of various sizes.

4. Micrococcus tetragonus (from a pure culture). Mag. 950.

5. Sarcina ventriculi. Mag. 700.

6. Staphylococci.

B. — i, 2, 4. Separate long rods of various lengths and breadths.
3. Short rods, partly of biscuit form.
5- Chains, composed of either short or long rods.
6. Long threads.

process (to be studied later on under the name of " spore formation "), swells out
like a spindle or club, is generally termed a clostriclium.

If the rod is arched like a bow or bent in the form of a comma, it is spoken
of as a vibrio, and if the bends be repeated several times, then a wavy kind of
growth denoted spirochaete ensues. When the bend departs from the level
plane and shapes itself in such a manner that it can be applied to the surface of
a cone or cylinder, the type of growth becomes spiral, and is designated spirillum.

DIMENSIONS OF BACTERIA 27

An illustration of these forms is given in Fig. 3. According to a remark of
Emmerich's, these arcuate forms of growth are more especially abundant in
muciferous substances, e.g. in the excrement of snails, which is rich in mucin,
as also among the algae (already in a state of decomposition, and therefore
abounding in vegetable mucus) frequently covering the sides of drains and
watercourses.

E. WEIBEL (I.) has published a series of observations on these forms of
growth, while fuller information concerning the spirilla frequently to be found
in the drainings from manure heaps can be obtained from KUTSCHER'S (I.)
treatise on this subject.

When the longitudinal development is excessive, then the name of thread
bacteria is given to the cells, which are further distinguishable into the forms
to be noticed later ; Cladothrix, Streptothrix, Crenothrix, and Leptothrlx being
described in chapter xxxiv., and Beggiatoa and Thiothrix in chapter xxxv. More
detailed notices of the Diplococcus, Streptococcus, Pediococcus, Staphylococcus, and
Sarcina forms of growth will be found in chapter iv.

§ 28.— Dimensions of Bacteria.

Notwithstanding our previous statement that the bacteria are the smallest
of all known living organisms, it can be established that the differences (Fig. 4)
in their size hitherto observed must be characterised as very considerable. In
the smallest kinds the dimensions are under i p., i.e. are less than o.ooi m.m.
For instance, the diameter of a lactic-acid-producing coccus examined by P.
Lindner was 0.6-1.0 p.. By way of contrast, mention may be made of Clos-
tridium butyricum, this bacillus measuring from 3 to 10 p in length, with a
breadth of i p.. The giants among the bacteria are to be found in the sub-group
of chromogenic bacteria, e.g. the genera Chromatium and Ophidomonas, which
have, therefore, formed the subject of exhaustive
investigation with regard to the internal construction
of the bacterial cell.

We may here briefly allude to a fission fungus
which, although unimportant from a practical, techni-
cal point of view, forms, thanks to its large size, an
especially favourable object for the exhibition of pro-
portional dimensions, spore formation, &c., viz., the
Bacillus megatherium, found by DE BARY (I.) on FlG. 5._BacilluS megatherium,
cabbage leaves. As the specific name would imply,

we have here an organism which excels in size all "' r^^ItS moment of
other fission fungi, as much as the prehistoric reproduction by fission. (After
Megatherium surpassed his contemporary congeners. De £arv) Ma£- 600-
This form is further illustrated in Fig. 5 ; the indi-
viduals h, r, k, I, will be described in chapter vi. (treating of spore gel-mination),
so that only m and b need be considered at present. These are rods 2.5 p.
wide and 10 /j, in length, each of which would easily hold about ninety of the
previously described cocci.

It will be useful to remember that the wave-length of light (corresponding to
the spectral line D) radiating from the sodium flame is about 0.6 p, i.e. about
equal to the diameter of the above-named lactic acid coccus. Bearing this in
mind, the remark already made, that the smallest of the bacteria are almost
invisible, becomes comprehensible.

28 FORM AND DIMENSIONS

§ 29.— Mutability of Form.

The question as to whether any given species of bacterium assumes only one
of the forms of growth already described, or has the power of appearing in
various shapes, is one of wide interest. A reliable answer could, however, only
be given when the necessary appliances for studying the separate species in
the form of pure cultures, i.e. cultures practised upon an isolated single cell
protected from subsequent contamination by other organisms, had been invented.
This possibility was first achieved early in the " eighties," since which time the
investigation of this question has proceeded with energy, and the results have
shown that the theory of uniformity of growth (monomorphism) is untenable for
bacteria, and must give place to the theory of multiformity (pleomorphism).

The remark made in a previous paragraph that the names bacillus, coccus,
&c., simply indicate certain forms of growth, will now first become perfectly
clear. When, in future pages, one or other of the various bacteria is qualified by
the generic name Bacillus (for instance, Bacillus urece), it is not meant that the
said species appears only in the form of rods. On the conti-ary, it may possibly
present itself in the form of cocci, threads, &c., and has only received the generic
name of Bacillus on account of its generally having this form, and especially
when the culture has attained the acme of its development.

This capacity — commonly known as mutability — of assuming a variety of
shapes is not possessed in an equal degree by all bacteria, and in a few of them
it even appears to be altogether lacking. Abundant pleomorphism chiefly
prevails in the arthrosporic bacteria, whilst, conversely, the kinds capable of
terming endogenous spores are mostly endowed with a smaller number of
mutation forms. This difference will be reverted to in a subsequent chapter.

The inciting cause of mutability is generally external, depending on the
conditions to which the culture is subjected. Since these can, to a certain
extent, be arbitrarily determined and controlled, a means is thus at hand of
exercising a formative interference in the existence of these organisms. Among
the morphological forces thus available, two are particularly powerful, viz., the
influence of temperature and the composition of the nutrient medium. The
former has been elucidated by the studies of E. Chr. Hansen on the acetic
bacteria, which will be exhaustively discussed in chapter xxxvii. On the other
hand, H. Buchner convincingly demonstrated the influence of the mode of
nutrition on the cell form of the hay bacillus (reported in chapter xvii.).

A large number of the species of bacteria noticed in the following paragraphs
are pleomorphic, and there will therefore be ample opportunity of becoming
acquainted with this phenomenon in all its details and varieties.

§ 30. —Involution Forms.

The remarks made in the preceding paragraphs with regard to the influence
of the composition of the nutrient medium require a not unimportant supple-
mentary explanation. When we arbitrarily bring about an alteration in the
form of a cell by modifying the conditions of the culture, a suitability of the
medium to the evolution of the species of bacterium in question is presupposed,
so that the vitality and reproductive power in the new form of cell thereby
produced is preserved.

This is, however, no longer the case in the modifications of form which result
when the medium is exhausted of nutrient materials, and consequently enriched
with injurious metabolic products. These degenerations of the cell have received
from Nageli the name of involution forms. They do not enter into the series of

INVOLUTION FORMS

29

types already drawn up (§ 27), and have no regular outline, but frequently
exhibit the most surprising shapes; generally they are strongly bulged and

FIG. 6. — Involution For

A, of Lactic acid bacteria, after Maddox.

B, of Clostridium Polymyxa, after Prazmowslci.

C, of Bacterium Zopfli, after Kurth.

D, of Bacillus subtilis, and

E, of Bacillus authracis, after H. Buchner.

F, of Vibrio rugula, after Warming.

distended, as can be seen in the examples given in Fig. 6. The faculty of form-
ing spores is no longer one of their attributes, and they must be regarded as
a diseased condition preceding dissolution. In the following pages frequent
occasions will arise of calling attention to similar degeneration forms.

CHAPTER II.

STRUCTURE AND CONSTITUTION OF THE BACTERIAL CELL.

§ 31.— Chemical Composition of the Cell Wall.

NAKED cells, i.e. those wherein the protoplasma is unprotected by an envelope
are unknown amongst schizomycetes, the body of the cell being in all fission
fungi shut off from the outer world by a membrane or cell wall. Little is as
yet known regarding its chemical composition, but the few investigations that
have been made in this direction demonstrate that we have to do with a structure
which not only differs in different species, but also undergoes alteration according
to the dietary of the cell.

The proximate assumption that the cell wall of the fission fungi consists of
cellulose cannot withstand searching criticism except in rare instances. At
present only one single species, viz., the Bacterium xylinum, examined by O.
LOEW and A. J. BROWN (I.), for which this assumption is justified, is known.
The cell walls of this acetic acid bacterium exhibit, after a suitable purification,
the following chemical composition, ascertained by ultimate analysis : —

Calculated
Fouud. for [C6Hi0O5]H.

Carbon 44.26 44.44

Hydrogen 6. 25 6. 1 7

Oxygen 49.49 49-39

The cell wall of this microbe is stained blue by aqueous or alcoholic solutions
of iodine, by iodine and sulphuric acid, and with iodo-chloride of zinc. In a
work from the pen of DREYFUSS (I.), dealing, however, chiefly with higher fungi,
and therefore preferably to be considered only in the second volume, a tew species
of fission fungi were included in the scope of investigation (e.g. a culture of hay
bacillus obtained according to Roberts' directions). These when tested for
cellulose by iodine yielded affirmative results. The amount was, however, only
very small. According to NENCKI and SCHAFFER (I.), the cell wall of certain
putrefactive bacteria contains nitrogen ; but they do not give the names of the
species.

§ 32.— Optical Properties of the Cell Wall.

Yet another means has been employed for obtaining an insight into the
chemical nature of the cell membrane of the fission fungi, namely, its optical
behaviour.

The cell walls of the higher plants are known to be anisotropic (doubly
refractive), a property also employed by F. v. Hohnel as a means of differen-
tiating textile fibres. Jf a couple of cotton fibres be placed on the stage of a.
polarising microscope, the Nicol prisms of which are crossed, the plane of oscilla-
tion of the polarised daylight issuing from the lower Nicol undergoes, in its
passage through the doubly refractive threads, a rotation, in consequence of
which the fibres appear bright and coloured against the dark background. Jf
the fibres are themselves coloured or dyed, they assume the corresponding
complementary colours,

30

ZOOGLCEA FORMATION 31

The attempt to utilise this means in the service of bacteriology was success-
fully made by AMAXN (I.). Since, under otherwise identical conditions, the
size of the angle through which the plane of polarisation is rotated — and conse-
quently the degree of illumination of the field of vision — is proportionate to the
thickness of the anisotropic object in question, it follows that, by reason of the
small dimensions of the cells under examination in this case, their double refrac-
tion cannot be observed when they are in a colourless condition. Now the eye
is better capable of appreciating differences of colour than degrees of brightness,
and therefore Amann, calculating on this peculiarity, stained his bacteria, assum-
ing that if the membranes were anisotropic and doubly refractive, an appearance
similar to that mentioned above in the case of cotton fibres would be produced.
The results confirmed his expectations, the stained bacteria (Bacillus tubercu-
losis and B. anthracis) exhibiting pleochroism, and being therefore doubly
refractive.

§ 33.— Zoog-lcea Formation.

Similar to the behaviour of the cell membrane of higher plants is the ten-
dency to swell up manifested by the membrane of many of the fission fungi, the
cell wall becoming distended to such an extent, through the absorption of water,
that its thickness often far exceeds the internal diameter of the cell. If to
such (previously killed) bacteria be added a solution of an aniline dye, the latter
will be absorbed by the cell contents, which, when examined under the micro-

TIG. 7. — Bacillus Pneumouiie
cruposse.

A and B, elongated rodsTC, D, E,|:short
rods; G-I, cocci. All the cells exhibit
highly distended membranes. Mag.
about 1500. (After W. Zopf.)

FIG. 8.— Bacterium Pasteurianum.

Zoogloea formation in an old film on the
surface of lager beer. Fixed and stained
by Lceffler's method. Mag. 1000.
(After Hansen.)

scope, will then be visible, enclosed in a colourless, or merely faintly coloured,
slightly refractive, and, consequently, paler cell wall.

Among the pathogenic bacteria the Pneumobacillus (Fig. 7), recognised as
the cause of croupous pneumonia, was the first in which this property was
observed — by P. FKIEDLANBER (I.) in 1883. He gave the name of capsule to
the mucinous integument, a term still remaining in use, especially among
medical bacteriologists. Consequently we understand by capsule bacillus one
wherein the cell wall is found to be in a distended condition under normal con-
ditions of vitality. The Bacillus diatrypeticus casei (Fig. 2 of Plate I.), described
in a subsequent section, may be mentioned as a second example.

As already stated, these mucinous envelopes are not affected by the ordinary
method of staining. ]f, however, they are previously treated with a suitable
mordant, the colour will be readily absorbed and fixed. Recipes for capsule

32 THE BACTERIAL CELL

staining are given by Friedlander, Ribbert, Lceffler, and others. The annexed
illustration (-Fig. 8) is drawn from a preparation stained in this way. It repre-
sents Bacterium Pasteur ianum, an acetic acid bacterium discovered by Hansen.
The chain seen at the left-hand side differs from the remaining portions of the
figure. The upper half presents no special peculiarities, and shows (like the
remaining chains) three darkened cells held together by the swollen membrane ;
whereas in the lower moiety the dark parts are wanting, the three cells formerly
present therein having been accidentally crushed in making the preparation, so
that only the mucinous envelope remains behind. In a more closely investigated
case (not, however, with this bacterium) the substance composing the capsule was
identified chemically as related to mucin, or probably identical therewith.

If the gelatinisation of the cell walls proceeds to a little greater extent, it
causes the individual cells to become joined or cemented into a coherent mass,
called zoogloea by COHN (V.), the size of the agglomerations being greater or
smaller according to the degree of development.

In a few species of bacteria, colouring matter or ferric oxide is stored in the
mucinous envelope, further particulars of which will be found in chapter xxxiv.

§ 34.— Plasmolysis.

The difficulty of detecting the cell envelope of bacteria can be removed by
immersing the organisms in a solution of salt, which exerts a hygroscopic action
on the cell contents, in consequence whereof they shrink and retreat from the
cell wall, whereupon the latter is readily recognisable, even in unstained
preparations.

This kind of influence, called Plasmolysis by H. de Vries, was first observed
in bacteria by De Bary, in connection with his Bacillus megatherium.

The experiment is simple in performance. An ordinary preparation of the
bacteria under examination is made, a few cotton fibres being immersed to pre-
vent the escape of the organisms. The salt solution is then allowed to flow in
at the edge of the cover -glass, its tranfusion being facilitated by absorbing the
water with blotting-paper held at the opposite side. Provided the solution is
not excessively concentrated, and that it contains no toxic substance, the resulting
plasmolysis in no wise destroys the vitality of the cells. It can be made to
disappear again by washing out the hygroscopic reagent.

The concentration of the cell contents caused by plasmolysis, which is also
generally accompanied with an increased refractive power, leads very often, as
BUCHXER (I.) pointed out, to agglomerations of material, which, unless more
closely examined, might illusively indicate the presence of endogenous spores.

In such bacteria as are endowed with the power of locomotion, the rate of
movement is retarded by increased concentration of the plasmolytic solution.
WLADIMIROFF (I. and II.) entitled the smallest percentage capable of arresting
locomotion the critical solution. The values of this for the various salts —
on any determined species of bacteria — show an unmistakable regularity, the
chloride of any given metal (K, Na, &c.) having the weakest, and the bromide
and sulphate the strongest retarding effect, whilst the nitrate occupies an
intermediate position. As far as the bases are concerned, potassium, sodium,
and ammonium rank in the order given. E. OVERTON (I.) has published several
communications with regard to a number of substances which ai^e unable to
effect plasmolysis owing to their passing through the plasma as rapidly as
water.

The weakest solution of sodium chloride (NaCl) capable of effecting plasmo-
lysis is, according to A. FISCHER (I.), 0.5-0.75 per cent, for Cladothrix dichotoma,
and for Crenothrix Kiihniana 0.75-1.0 per cent. These limits are, however, not

STRUCTURE OF THE CELL CONTENTS 33

unconditionally applicable, as the following example will show. A one per cent,
solution of common salt produced no plasmolysis in Clostridium buttjricum when
the cultures were only two days old, and the cells consequently young ; whereas
after a further four days they were, without exception, completely plasmolysed.
It will perhaps be useful to recall that a solution of 0.5 gram of NaCl in 100
grams of water is usually known as physiological salt solution, and is frequently
used under the supposition that it neither takes up nor gives up water from or
to the cells, — an assumption that, in view of the preceding figures, cannot be
universally justifiable.

If it is desired to render permanent ("fix") the plasmolytic condition of the
cells, they must be killed, a result easily obtained by the use of sublimate or
iodine solution. Ten per cent, lactic acid is also an excellent fixing medium,
instantaneous in action. After fixing, staining can be effected. More detailed
information on the practice of fixing will be found in a work by A. ZIMMER-

MANN (I.).

§ 35.— Structure of the Cell Contents.

This study, which presents no small difficulty on account of the minuteness
of the organisms to be examined, has been closely followed up during the past
several years only. All that was previously known was
that the cell contents of the bacteria consisted of a
homogeneous invacuolate plasma, in which small, highly
lustrous granules were frequently seen embedded.

BUTSCHLI (I.) in 1889 discovered in a few large
chromogenic bacteria (e.g. Chromatium Okenii and
0 phidomonas jenensis), as also in /Spirochcete serpens
and Beygiatoa, that their cell contents could, as a rule,
be distinguishable into two parts, viz., a central body
and a parietal layer, the latter being adjacent to and
surrounded by the cell wall.

The parietal layer may either surround the central
body on all sides, so that the latter nowhere touches
the cell wall, or may be restricted to one side only, in
which event it is generally, in the case of rod-shaped
bacteria, found at the two poles. This differentiation
of the cell contents can be rendered visible (Fig. 9)
by suitable stains, e.g. haematoxylin, which is most
readily taken up by the central body, thereby render-
ing it easily distinguishable from the more slightly
coloured parietal layer.

This treatment brings out a second and much
more important fact : the central body appears as a
complicated structure, reminding one of that seen in
honeycomb. A number of granules of red-violet
colour — called by Biitschli " red grains" — are stored
in a reticular framework which is coloured blue by the
staining dye. These granules do not occur in every
cell, and no cell has more than one. These enclosures
are detectable, even in the unstained preparation, on
account of their high refractive power. They — the bodies, not their enveloping
framework — were first observed by V. BABES (I.), then studied by P. ERNST
(I. and II.), and were regarded as the starting-point of spore formation, being
on that account designated sporogenic granules ; but this assumption has with
good reason, been contested by later workers. For a more accurate examination
i C

FIG. 9. — Cliromatium Okeuii.

A. Longitudinal section.

B. Cross section.

First killed, then freed from
bacterio-purpurin and sul-
phur granules by solvents,
and finally stained with hae-
rnatoxylin. The reticulated
structure of the (hatched)
central body (c), as also of
the parietal layer (b), and tlie
dark chromatin granules (s),
Biitschli's "red grains," can
then be detected. Magn.
2000-2500. (After Biitxchli.)

34 THE BACTERIAL CELL

we are indebted to SCHEWIAKOFF (I.), according to whom each of these small
enclosures contained in the honeycomb cells possesses a thin skin, the presence
of which can be convincingly demonstrated by pressing the preparation under
a cover-glass, whereupon the skin bursts, without, however, its contents being
dispersed. The latter can therefore only be of a solid and not of a fluid nature.
The chemical nature of the skin is unknown ; it does not give the cellulose
reaction. So far as the composition of the granular contents is concerned, the
last-named investigator has identified therein by micro-chemical means potas-
sium, calcium, and oxalic acid, in addition to the indeterminate organic matter.
In their behaviour, more closely investigated by WAHRLICH (I.), towards
colouring and solvent reagents, these enclosures in the central body of the
bacterium resemble those granular constituents of the cell nucleus of higher
plants known as chromatin granules, on account of their high power of absorbing
colouring matters. On the other hand, the reticular mass of the central body
resembles in point of structure and micro-chemical reaction the lignin of the
nuclear framework of the cells of higher plants.

These observations led BDTSCHLI (II.) to the opinion that the central body of
bacteria should be regarded as the (comparatively large) nucleus thereof Avhilst,
on the other hand, the above-named parietal layer corresponded to the cytoplasm
of the cells of higher plants — a conception which has not withstood the test of
criticism.

The structure of the parietal layer has not yet been determined with
certainty. Sundry observations, however, indicate that it exhibits a radial
honeycomb appearance. It is in this layer that the colouring matter of the
chromogenic bacteria is lodged, whereas the granules of sulphur in the sulphur
bacteria are located in the central body. The individual species of the genus
Granulobacter established by Beyerinck — to which belong the instigators of the
butyric acid and butyl alcohol, &c., fermentations — under certain conditions of
culture store up in their interior copious supplies of granulose, owing to which
circumstance they are stained a deep blue by iodine. It should be mentioned
that W. MIGULA (I.) could not detect any honeycomb structure of the cell plasma
during his researches into the structure of the Bacillus oxalaticus, discovered by
Zopf.

It may perhaps be useful, though not exactly necessary, to remind the reader
of the well-known botanical fact that the living plasma strives to prevent the
access of colouring matters. In this connection, also, the behaviour of the various
kinds of fission fungi differs. Some of them exhibit merely a slight resistance,
and absorb colouring matter without their vitality being impaired, as Birch-
Hirschfeld established with respect to phloxine red in the case of the typhus
bacillus. In the majority of instances, however, this resistance is so great that
the cells must be killed before they can be stained. On this account most of the
staining solutions employed for bacteriological purposes contain additions (e.g.
alcohol) destructive to the vitality of the cell, which being accomplished, the
plasma readily absorbs the colouring matter. According to the researches of
DREYFUSS (I.), it is not the true albuminoid matter, but the nuclein (also a
constituent of plasma) which fixes the colour.

Thanks to the care bestowed on the subject by medical bacteriologists specially
interested therein, the art of staining bacteria has reached a high degree of
perfection during the last few decades. Exhaustive directions thereon are to
be found in HUEPPE'S (I.) handbook and EISENBERG'S (I.) treatise, and BERNHEIM
(I.) has issued a very cheap book highly useful for laboratory work. In
Fermentation Physiology the examination of the bacteria being usually performed
on the living, unstained organisms, the aid of staining is seldom required ; the
principal occasions being when so-called cover-glass preparations have to be kept

COMPOSITION OF THE BACTERIAL CELL 35

for purposes of future microscopical comparison. The preparation of such is
described in all the books just mentioned.

§ 36.— Elementary Composition of the Bacterial Cell.

Determinations on this point by micro-chemical analysis were first attempted
by KAPPES (I), whose attention was principally devoted to Micrococcus prodigiosus,
cultures of which were made on solid nutrient media, and then, when they had
arrived at sufficient development and had expanded into mass cultures, examined
for the amount and composition of the dry matter therein. Cultures of this
fission fungus on agar-agar contained on an average : Water 85.5 per cent., and
dry matter 14.5 per cent., the latter yielding ethereal extract 0.7 per cent.,
nitrogen 1.7 per cent., and ash 2.0 per cent. Calculated on the dry substance,
the following percentage composition was arrived at for the microbe in question :

Ethereal extract (fat, &c.)
Albumen (N x 6.251
Ash ....
Undetermined substances

71.2

13-5
10.5

An examination made by NISHIMURA (I.) of a pure culture of a water bacillus
gave the following as the constitution of the dry matter in this microbe :

Per Cent.
• 63.5

12.2

Albumen
Carbohydrates
Alcoholic extract
Ethereal extract
Ash

Lecithin
Xanthin
Guanin .
Adeuin .

3-2

II. 2

0.68
0.17
o.i4
o.o8

The most noticeable figures here are those relating to the content of nitrogen,
which show that, in this respect, the bacteria are excelled by but few organisms,
while they have no compeers in the vegetable kingdom. To demonstrate this
fact more clearly the subjoined figures have been selected, showing the mean
nitrogen content in the dry substance of :

Nitrogen per cent.

Lean
beef.

14-3

Cow's
milk.

4-4

Soja
beans.
6.0

Kussian
wheat.

3-5

Micrococcus
Truffles, prodigiosus.
5-6 11.4

The earliest researches on the nitrogenous constituents present in bacteria
were carried out by NENCKI and SCHAFFER (I.), both of whom isolated from
putrefactive bacteria a nitrogenous substance to which they gave the name of
mycoprotein. The highly concordant results of a series of ultimate analyses of
this substance gave the following mean values :

C : 52.32 H : 7.55 N : 14.75 ° + S : 25.38

The percentage (14.75) °f nitrogen found is remarkable, and shows that
mycoprotein must have a very different constitution from that of ordinary
albumin or protein, which, as is well known, contains about 16 per cent, of
nitrogen. The amount of this compound in the dry substance of bacteria is at
least 40, and sometimes as much as 50 per cent. Differences are also apparent
as regards their behaviour towards reagents, nitric acid, for example, converting
albumin into a bright yellow compound, named, on the proposition of MULDER
(T.), xanthoproteic acid, whereas mycoprotein does not give this reaction.

36 THE BACTERIAL CELL

§ 37.— Quantitative and Qualitative Selective Power.

The proportions of the constituents present in the available nutriment
or nutrient medium supplied seldom correspond to [the requirements of the
organism which has to make good therefrom losses of'substance or energy. It
will then take from the supply the substances of which it has need, and leave the
residuum entirely untouched, only when the former are present in abundance.
This faculty, entitled quantitative selective capacity, is possessed by all living
organisms, and among them bacteria. Kappes, in his treatise already referred
to, gives very instructive examples of this as well. He compared the composition
of the bacterial crop with that of the soil (nutrient medium) in which it was
grown ; in this case peptonised meat extract, agar-agar. This contained, apart
from the 1.5 per cent, of agar-agar, which does not come under further con-
sideration here, altogether 2.5 per cent, of actual nutrient materials, and yielded
0.3 per cent, of dry bacterial substance; that is to say, only 12 per cent, of the
total nutriment was extracted. The relative proportions of the individual
constituents in the medium on the one hand and in the crop on the other proved
very different. Thus, for instance, the ratio of CaO : MgO was in the medium
0.70 : 0.44; in the crop, 0.56 : 1.05. Of nitrogenous substances (N x 6.25) the
former contained 42.5 per cent., calculated on the dry substance, and the latter
71.2 per cent., and so on.

The requirements of bacteria in respect of ash constituents were first
investigated by NAGELI (IV.), in 1879. SPRENGEL (I.) was admittedly the first
to demonstrate that higher plants (Phanerogamia) absolutely require for the
construction of their cells a number of mineral substances, viz., K,0, CaO, MgO,
FesO3, PZO5, SO3, all of which must be present, and in sufficient quantity, before
the phanerogamic plant can thrive. With the Cryptogamia the case is, however,
different. According to N'ageli, the fission fungi (tested by him in this connection)
are less exacting, since potassium can be replaced by rubidium or caesium without
detriment, so far as the fungi are concerned, though not by the alkaline earths.

Of the latter it is sufficient when one of the following, CaO. BaO, SrO, MgO,
is present ; iron can be dispensed with. It follows therefrom that not only
quantitative but also qualitative powers of selection are possessed by bacteria.
An authoritative confirmation of N'ageli's discovery is highly desirable, and
would prove a very thankworthy task if conjoined with observations on the
formative influence of the individual ash constituents. A typical example of
this kind of study, alike instructive, stimulating, and worthy of imitation, has
been made by Winogradsky on a film yeast, and will be referred to in the second
volume. A preliminary step in this direction was taken by A. K. FEDOROLF (I.)
in 1895, who, in continuing an investigation commenced by Gamaleja, found that
while lithium chloride caused abnormal cell forms in Bacillus megatherium, B.
typhi abdominalis, Vibrio cholerce asiaticce, and Bacterium coli commune, it had no
apparent influence on the cells of Bacillus subtilis, B. anthracis, and other microbes.

The quantitative selective power referred to above must not be understood
in the sense that the organisms inhabiting the nutrient medium always have
the same composition, irrespective of the relative proportions of its nutrient
constituents. E. CRAMER (1.) studied this matter exhaustively in relation to
several pathogenic bacteria and Micrococcus prodigiosus, and arrived at the con-
clusion that a typical composition of any species is out of the question. Accord-
ing to the kind of medium, the temperature and age of the cultui-e, and other
conditions, it may happen that one crop will contain twice as much of a particular
constituent as another crop. Thus, for example, the amount of dry matter varied
from 15.9 to 26.0 per cent., and of ash from 1.60 to 3.21 per cent.

CHAPTER III.

POWER OF INDEPENDENT MOVEMENT IN BACTERIA.

§ 38.— Molecular Movement and Locomotion.

LIKE his predecessors, Chr. Ehrenberg considered the bacteria as animalculse,
and that, chiefly, because in not a few of them he discerned active locomotive
powers. For the same reason Pasteur was inclined in 1861 to regard his
" vibrion butyrique" as an infusorial animalcule. In opposition to this was
established the fact that the faculty of voluntary movement is not peculiar to
the bacteria, but is also possessed by many motile spores belonging to the algse ;
that is to say, by organisms unanimously admitted to be of vegetable nature.

This motile power will now be more closely considered, both as regards its
nature and causes.

The movement known as the Brownian or molecular movement — which can
frequently be observed in small particles held in suspension in liquids, whereby
each particle describes a small orbit (sometimes rectilinear, sometimes circular or
oval) outside its horizontal axis of rotation — is not included in this consideration.
The cause of this Brownian movement has not yet been examined with sufficient
accuracy ; but it is a purely physical one and in no wise physiological, since it is
manifested not only by living cocci, but also, as already stated, by emulsions of
many inanimate substances both of inorganic and organic nature. Ali-Cohen,
in his treatise referred to below, describes a very useful means by which one can
decide in doubtful cases whether the movement is independent or merely mole-
cular. According to the researches of Exner, the latter diminishes in briskness
as the viscosity of the circumambient liquid increases. If then a little of the
doubtful sample be mixed with a lukewarm liquefied 5 per cent, solution of
gelatin, both locomotive and molecular movement will at the outset remain un-
affected, but in proportion as the surrounding medium cools and becomes more
viscous, the latter movement will diminish, and finally cease altogether, whilst
the voluntary and independent movement will continue.

By long-continued practice and observation the faculty will be gradually
acquired of distinguishing, without such aid, the so-called molecular from the
true voluntary bacterial movement, which we will now describe. This movement
is of two kinds, the first of which is occasioned by alternate contraction and
re-expansion of the plasma canal. This kind of movement occurs in the case of
such thread bacteria as attach themselves to a support by one of their" poles, and
then swing with a pendulous motion from this point of suspension, either
remaining in the same plane the while or describing a cone.

§ 39.— The Flag-ella or Cilia.

Much more frequent is the second or roving movement, noticeable in free
unattached bacteria, and produced by special locomotive organs termed flagella
or cilia. These were first noticed by EHRENBERG (I.), in 1836, in a spirillum which
he discovered in a brook near Jena, and named Ophidomonas jenensis. Closer
attention was first bestowed on these organs by COHN (I.).

37

38 POWER OF INDEPENDENT MOVEMENT IN BACTERIA

PLATE I.

DESCRIPTION OF THE DRAWINGS.

FIG. I. FIG. II.

Micrococcus Sornthalii. Bacillus diatrypeticus casei.

Without cilia. Magn. 650. Without cilia. Capsule bacillus.

After Adainetz. Magn. 1300.

After Fr. Baumann.

FIG. III. FIG. IV.

Bacillus cyanogenus. Large Ciliated Bacilli.

Cilia staining. Magn. noo. From a vegetable infusion. Accompanied

After Locffler. by other non-ciliated fission fungi. Magn.

IOOO.

After Loeffler.

FIG. V. FIG. VI.

Spirillum TJndula. Spirillum rubrum, Esmarcb.

From a vegetable infusion. With tufted With polar cilia. Magn. 1000.

polar cilia. Magn. 800. After Loeffler.
After Loeffler.

PLATE I.

FIG. IV.

'*
'v»

. <**

. **•*>

FIG. VI.

To face p.

HISTOLOGY OF THE CILIA 39

VAN TIEGHEM (I.) was of opinion that these locomotive organs were peculiar
to bacilli ; but similar locomotive powers were at a later date observed in the
cocci, such as the Micrococcus tetragenus mobilis ventriculi, discovered by MENDOZA
(I.) in 1887 ; the Micrococcus agilis, found by ALi-CoHEN (I.) in 1889 ; a coccus
(unspecified) studied by LOEFFLER (II.) in 1890 ; later, the Micrococcus agilis
citreus of MENGE (I.); and finally, the Sarcina mobilis of MAUREA (I.).

The position of these organs in the bacilli is either polar or lateral. The
polar flagella are either single — as, eg. in Chromatium — or in tufts, the latter-
consisting, in the case of Bacterium termo, of three or four, and in various spirilla
of eight to twelve, cilia. In Spirillum undula they are often plaited into the form
of a queue. The lateral cilia are, as was found by A. FISCHER (II.), evenly
distributed over the entire surface of the bacterial cell, their number being given
by Loeffler as twelve in the case of the typhus bacillus.

Starting with the assumption that the number of the cilia and their distri-
butive arrangement on the cell are constant for each kind, A. MESSEA (I.)
endeavoured to make this character the basis of a classification of the bacteria.
L. LUKSCH (I.) proposed the same method for readily differentiating Bacterium
coli commune from Bacilhis typhi abdominalis, which is very important in the
bacteriological examination of water. He found the former microbes to be pro-
vided with at most three cilia apiece, whereas the bacillus had from eight to
twelve. Subsequently, however, it was ascertained by FEBRIER (I.) that the
number, form, and length of the cilia depend on the conditions of the culture.
From the bacterium in question cultures can be obtained the individual cells of
which exhibit as many as ten cilia ; by this determination, therefore, the system
of Messea, as also the hopes of Lukscb, were deprived of support. Moreover,
Messea had been forestalled, as in 1864 DAVAINE (I.) proposed to separate the
fission fungi into two groups ; the one, forming his genus Bacteridium, comprised
all the species in which he could not detect independent movement under any
circumstances ; whilst the others, his genus Bacterium, included all the motile
species. In J. Schroeter's work (published in 1870) on pigment bacteria, of
which a notice is given in a subsequent section, this method of classification was
adopted, but later workers have abandoned it, and the term Bacteridium is now
perfectly obsolete.

When a bi-polar ciliated bacillus divides in two in the act of reproduction,
the new-formed poles are, naturally, without such locomotive organs at the
outset, but they quickly develop, and thenceforward each of the two cells is
ciliated at both poles. That these organs are extremely minute need not be
emphasised. Frequently they are undetectible by the ordinary means of
observation, even with objectives of the highest power and clearest definition,
since it is difficult to see the cilia, not only because of their extreme minuteness,
but also because their refractive power is almost the same as that of the liquid
in which they are immersed. In order to make them more readily recognisable,
use is made of the special methods cf staining devised by LOEFFLEK (I.). Some
directions relative to these will be found in UNNA'S (I.) historic-critical review
of the development of bacterium-staining, drawn up in 1888, and continued by
L. HEIM (I.) up to the year 1891. Plate I. shows four photographs of motile
bacteria taken by Loeffler from preparations stained in this way.

§ 40.-Histology of the Cilia.

This subject has hitherto received little attention. Van Tieghem considered
the cilia to be gelatinous elongations of the cell envelope, and their movement
as merely passive, the locomotive power being ascribable to contractions of the
plasma in the cell. He found that the cilia of Clostridium butyricum gave the

40 POWER OF INDEPENDENT MOVEMENT IN BACTERIA

cellulose reaction with ammoniacal copper oxide. Zopf, on the other hand,
explained these organs as contractile plasma-threads, which could be alternately
protruded from, and wihdrawn into, the central cell mass through aper-
tures in the cell integument, which apertures, however, have hitherto been
unobserved.

This assumption was combated by A. Fischer, who found that when motile
bacteria were subjected to plasmolysis, and the cell contents therefore caused to
contract, the cilia were not drawn in, as should be the case if they were
continuations of the plasma (pseudopodia). For arresting the movement of the
bacteria examined by him, the strength of the solution of salt had to be higher
than the minimum capable of producing plasmolysis. Fischer's observations
favour the view that the cilia are appendages of the cell, consisting of a mem-
brane enveloping the protoplasmic contents, which have an immediate connection
with the substance of the bacterial cell.

Adverse influences stop the movement, and the cilia become motionless and
torpid. According to the cause, this condition is said to be one of torpidity
through cold, heat, darkness, light, hunger, desiccation, or poison. Bearing
this in mind, it must, not be concluded that any species of bacteria which may
not exhibit movement under ordinary microscopic examination is therefore
necessarily non-motile ; but it should be further examined under various con-
ditions, and, in extreme cases, tested for the presence of cilia by staining, since
it may be in the torpid condition.

§ 41.— Chemotaxis,

The extended researches of ENGELMANN (II. and III.) teach us that certain
roving bacteria (i.e. those endowed with spontaneous movement), and, in par-
ticular, various putrefactive bac-
teria, have a great need for oxygen,
while other species do not require
it. If a drop of liquid containing
a mixture of these two kinds be
brought under the microscope, it
will quickly be seen that the one
species hastens to the edges of the
cover-glass, where oxygen pene-
trates by diffusion and is most
abundant, whilst the individuals of
the other species gradually retreat,
and collect at the centre, where the
(to them) unwelcome or obnoxious
gas does not penetrate. Repeating
Engelmann's experiment by insert-
ing a thread of green (i.e. oxygen-

FIG. 10. — Oxygen-loving bacteria infesting a thread of
alga lying in the micro-spectrum. The chlorophyll
granules contained in the alga cells are not shown, but

me aiga cens lire not shown, but PXPrpfinc\ al,rj, Tn tv,0 J,.™ ~ ri

the spectrum lines are given to denote the position of excret.1DgJ alga in tne diop, and
the spectrum. Magn. 200. (After JSnyeimann.) directing a small solar spectrum

thereon, then the oxygen-loving

bacteria will be seen collected around these alga threads, and surrounding those
spots in the micro-spectrum (Fig. 10) where the maximum evolution of oxygen is
taking place ; that is to say, between the spectrum lines B and 0 in the red, and
also at F. Oxygen, therefore, exerts an attractive and stimulating action on
many bacteria, and may thus be employed as an isolating and separating
agent therefor. Conversely, motile bacteria may also be employed as a delicate
reagent for oxygen. BEYERINCK (I.), to whom we are indebted for a very

CHEMOTAXIS 41

useful treatise on this question, examined more closely the methods of topo-
graphical arrangement adopted in liquid media by various motile bacteria under
the influence of oxygen, which arrangement he called respiration figures. Some
of these are shown in Fig. n.

From the researches of Stahl and De Bary, and especially those of W.
PFEFFER (I.), we learn that not only oxygen, but also various other substances,,

FIG. ii.— Respiratiou figures of motile bacteria. (After Beyerinck.) Natural size.

The three figures are horizontal projections of bacterial preparations, each in a large drop of water.
The three large circular cover-glasses only are shown, the slides not being reproduced. A small
platinum wire (not shown in the figures) is placed at the part represented by the top of the drawing,
between the cover-glass and slide, so as to form a wedge-shaped space, which is occupied by the drop of
water, the base of which lies in m ( = meniscus).

The three figures represent :

I. Respiration figure of the aerobic type. The roving individuals collect in the oxygenated
border zone («), whilst the quiescent ones (r) remain in the interior, leaving a vacant space {/) between
the two.

II. Respiration figure of the spirillum type. These organisms require and tolerate only
traces of oxygen. They therefore collect, not at the circumference of the drop, but at a little distance
therefrom (sp), where the tension of the penetrating gas is lower.

.III. Respiration figure of the anaerobic (air-shunning) type. These migrate to the centre
(an) of the drop, as being the place of lowest oxygen content.

are capable of attracting or repelling bacteria and other micro-organisms, a
faculty to which the name of positive or negative chemotaxis has been given.
Use is made of this property in order to capture the motile species in a
bacterial mixture by introducing therein a capillary tube filled with a solution
of a substance which exerts an attractive influence on one or other of the motile
species.

Among the inorganic compounds the salts of potassium have the greatest
power of attraction, and are therefore most frequently employed for this
purpose. Of the organic compounds, asparagin is particularly effective. The
sap or juice of raw potatoes contains both these lures, and is therefore highly
efficacious. More particular information on this method of isolation is given by
ALI-COHEN (II.).

The attractive power of such agents is not limited to motile bacteria alone,
but also extends to higher sessile fungi. If such an attracting agent be
brought sufficiently near to a culture of the latter description, the growth and
extension of the cell threads on that side of the culture will show marked
exuberance. This attempt on the part of sessile fungi to turn towards a point
of chemical attraction is entitled Chemotropism. Its cause is identical with
that of Chemotaxis ; the difference in the effect being due to the difference of
the object influenced. It may be mentioned that MIYOSHI (I.) has performed
exhaustive experiments of this kind.

CHAPTER IV.

VEGETATIVE REPRODUCTION BY FISSION.

§ 42.— Division in One Direction.

IN order to reproduce by fission in one direction, the cell becomes elongated,
and a partition (septum) is developed in the interior of the cell at right angles to
the length. This septum then divides into two lamellse, thus effecting the
separation of the daughter- cell from the mother-cell. If the organism is living
under conditions favourable to its vitality, each of these cells will soon undergo
a similar process of division. In many instances the new-formed cells of the
second, third, fourth, &c., generations do not become entirely
detached, but remain connected one with another ; and if — as
is most often the case — the division takes place continuously in
the same direction, chains of cells are formed. When the
/ members composing the chain consist of cocci, the fission
fungus is frequently designated a streptococcus, instead of
merely coccus. Hallier and Itzigsohn proposed to apply the
term Mycothrix to these rosaries of cocci. When the mem-
bers are united chiefly or exclusively in pairs, the organism
is termed diplococcus ; and such of the cocci as incline to
no • —Bacillus grouP themselves in grape-like agglomerations are frequently
tuuiescens. designated, in medico-bacteriological literature, by the name

a and 6, chains of short staphylococcus, first bestowed on them by OGSTON (I.) in

members. (After A. jggo

agn.noo. jj. ^e members of a chain are not iso-diametric cocci,
but rods, they are mostly termed thread (filamentous) cells, of which the hay
bacillus affords an excellent example.

In an iso-diametric cell, the separation of the new cells — and therefore the
preliminary expansion of the mother-cell — may occur in one of two directions :
either lengthwise or crosswise, the former case — wherein the position of the
dividing septum is transverse, i.e. perpendicular to the longitudinal direction —
being the most usual. On the other hand, only a few examples of the second or
longitudinal separation are as yet known. One of them is afforded by the
Bacillus tumescens, discovered by ZOPF (I.), which will nearly always be found
infesting slices of boiled carrot, when the latter are left to themselves for some
time in a not too damp condition. According to the conditions of vitality pre-
vailing, this microbe develops either chains of long ctlls formed by transverse
fission, or cell bands the members of which are short and joined broadside on,
the attached sides measuring 2.1 p each, whereas the length of each is only 1.3 /*.
This microbe, therefore, exhibits both styles of fission. On the other hand,
longitudinal fission alone is manifested in a fission fungus discovered by METSCH-
NIKOFF (I.), and named Pasteuria ramosa, which grows in the ventral cavity of
certain water-fleas (Daphnia pulex and D. magna), where it produces a fatal
disease. In these microbes the new septum is always longitudinal.

In the separation process now under consideration, wherein the division
of the mother-cell and the casting off of the daughter-cell take place in one

42

DIVISION IN TWO AND IN THREE DIRECTIONS 43

direction only, the number of mother- and daughter-cells is always equal ; the
total number (e) of cells obtained from the initial cell (a) at the end of the n*h
reproduction being :

e = 2 "a.

A much greater rate of increase is attained in a given time when the sub-
division is effected in several directions simultaneously. Examples are known of
both possible events, viz., separation in two, and separation in three directions at
right angles to one another.

§ 43.— Division in Two Directions.

In this case the cell contents subdivide into four parts by the formation of
two septa intersecting each other at right angles, and each splitting into two
lamellee, whereupon the mother-cell becomes four daughter-cells. The latter
again undergo subdivision, whereby 4x4=16 cells are formed. At the end
of n subdivisions, the total number of cells from a individuals will be —

In this case, where the separation goes on continuously in two directions
perpendicular to one another, and so always in the same plane, there results —
provided the gradually extending cells retain their connection — a mosaic-like
stratified plate which has been named Merismopedium (divided plate). A coccus
exhibiting this method of reproduction is known as a pediococcus. To this
group belongs the lactic-acid-producing Pediococcus acidi lactici discovered by
Paul Lindner.

§ 44.— Division in Three Directions.

Cocci, wherein the reproduction of the cell is effected by division in all the
three directions of space, are designated Sarcina. In this case the contents of
the mother-cell divide into eight equal parts by the for-
mation of three flat septa, perpendicular to each other, jSjp)
which subsequently split up into two layers, whereby ^*^
each of the eight daughter-cells is surrounded on all
sides with cell-membrane. *v^

When these, as they generally do, remain attached ^^
together, their appearance resembles a corded bale of
goods, or a cubical packet. See Fig. 13. ©

In the course of reproduction an initial number of ^
cells represented by a will increase by n processes of riG- J3- — Sarcina veuuicuii.

division to a total of — From the contents of a diseased

stomach, a. Individual cell;

e - 8"a = 23»«. b. the same divided in one

direction only ; c. the same

The Sarcina maxima, frequently met with in malt divided in three directions;
mashes, may be cited as an example of this form of d- packet colony of cells-
growth. A comprehensive classification and accurate characterisation of all
known species of the genus Sarcina has been given by TH. GRUBER (I.).

Subdivision in more than one direction, and therefore the formation of sheet-
like or packet-shaped aggregations of daughter-cells, has hitherto been observed
solely among iso-diametric and a few thread bacteria. The first group (of coccus
form) is constituted by Sarcina. The behaviour, in this connection, of the
second group (e.g. Crenothrix) will be subsequently discussed.

44 VEGETATIVE REPRODUCTION BY FISSION

§ 45.— Form of the Daughter-Cells.

Cocci, when observed immediately after their production by subdivision,
exhibit a more or less angular outline, since the surface of separation between
the mother- and daughter-cell is flat, appearing to the eye as a straight line.
This shape, however, very soon undergoes alteration, the cell-membrane being
caused to bulge outward by the pressure of the plasma, whereby the plane
surfaces are rounded and the cell assumes the customary appearance of the non-
facetted coccus. By the same cause the initially plane circular outline of the
smaller side of a cylindrical bacillus, newly formed by fission, becomes dome-
shaped.

§ 46.— Division of the Nucleus.

As is known, the division of the nucleus can be effected, in the higher plants
as well as in animals, in two ways. The simpler of these, generally known as
segmentation, or as direct or amitotic division of the nucleus, occurs, in the
higher plants, only in such cells as have ceased to subdivide, and whose
multiplication has therefore ceased. The nucleus elongates, becomes con-
stricted at an intermediate point, and finally falls apart in two halves. More
complicated, however, is the other process known as indirect or mitotic
division of the nucleus. In this case the final separation of the nucleus into
two portions is preceded by a far-reaching transformation of the substance
of the nucleus of the mother-cell, an operation entitled karyokinesis ; and it
is in this manner alone that the subdivision of the nucleus occurs in the
segmentation cells of higher plants and animals. Fundamental researches
into this process have been made by FLEMMING (I.), and in the narrower
domain of the Thallophyles the investigations of SCHMITZ (I.) merit attention.

In so far as the Schizomycetes are concerned, BUTSCIILI (I. and II.) made
various observations, from which Zacharias concluded that the division of
the central body of fission bacteria is amitotic or direct. WAHHLICH (I.),
FRENZEL (I.), and SJOBRING (I.) studied the matter more closely, and, according
to them, the chromatin granules of the central body (§ 35) are tirst dissipated,
and then the latter stretches and subdivides. Concurrently, the new septum
interposes itself between the two moieties of the cell and then splits up into two
lamelke, thus effecting the separation of the two cells.

As will be remarked, the Schizomi/cetes assume an exceptional position as
regards the behaviour of their nucleus during cellular subdivision, the operation
being in this case alone direct, whereas in all other plants karyokinesis occurs.

§ 47.— The Rate of Reproduction

is, naturally, influenced by external circumstances, especially by the method of
nutrition and the temperature. It also varies under the same external con-
ditions in the different species. The time required for the formation of one
bacterial cell from another (a new generation) is known as the period of gene-
ration. This period was determined by BREFELD (I.) and by PRAZMOWSKI (I.)
as twenty minutes at 35° ; thirty minutes at 30°; forty-five minutes at 25°;
ninety minutes at i8f-°, and four to five hours at 12.5° C., for the hay bacillus
(Bacillus subtilis). Similarly rapid is Koch's Vibrio cholerce asiaticce, the
period of generation for which under favourable conditions is only twenty
minutes, as ascertained by BUCIINER, LONGARD, and RIEDLIN (I.). If the number
of cells present in a bacterium culture at the commencement of an experiment

THE RATE OF REPRODUCTION 45

be represented by a, and at the end of a given time, t, has increased to b; then,
according to FE. BASENAU (I.), the period of generation is

t log 2
" log 6 -log a

Cohn, starting with the assumption that the period of generation is half an
hour, made the following calculation. If we take a single bacillus measuring
2 p in length and i p. in breadth, with a weight of 0.000000001571 mgrm., it
will increase, according to the aforesaid assumption, at such a rate that in two
days' time its progeny will amount to 281 billions, and will occupy a volume
equal to about half a litre (30.51 cub. ins.). Within a further three days the
quantity would increase to a mass sufficient to completely fill the beds of all the
oceans on the globe, and the number of the progeny would be expressible only
by 37 places of figures ! That such an inordinate development does not occur is
mainly owing to the repressing effect of external influences, and especially to the
enmity existing between the various species themselves. A no less powerful and
inevitable retardation is caused by the transformation products excreted as a
result of the vital activity of the reproducing cells, which finally arrest further
growth, even though a sufficiency of nutriment is still available.

CHAPTER V.

THE PERMANENT (REPRODUCTIVE) FORMS OR SPORES.

§ 48.— The Formation of the Endospores.

THE cell forms, hitherto considered, produced by fission, and generally designated
vegetative forms of growth, have only relatively low powers of resisting the
multifarious dangers to which bacteria are exposed in Nature. The fact that,
nevertheless, these tender organisms hold their ground is due to their faculty
for producing special forms, which, on account of their physiological function,
are known as permanent forms (spores). These may be of two kinds, viz., endo-
genous spores, and arthrospores. Any account relative to the continuation of
the species will therefore have first to deal with the formation of the endospores.
When a bacterial cell commences to develop such a new form, it condenses
its cell contents into a smaller space, and then surrounds them with a tough,
smooth, colourless membrane (probably composed of two
layers). The form thus produced is enclosed on every side
by the membrane of the mother-cell (Fig. 14), in which it is
developed, and is therefore called an endogenous spore or
endospore. The greater density of its contents is evidenced
by their greater refractive properties, which, were they
confined to the spores, would enable these to be detected
I m r with certainty by the optical method alone. This is, how-
FIG. 14.— Bacillus ever, not the case ; large, highly-lustrous drops of a fatty
megatherium. nature, and which cannot, without other means, be accu-

Spore formation. rately identified, occurring frequently in the cell plasma
r. chain of four cells of the fission fungi. In such cases germination tests — dealt

31^ ripl>' tou«h' with in the following chapter— must be resorted to for the

wauea enaospores. ,• •

(After De Bary.) purpose of differentiation .

Magn. 600. With regard to the transformations undergone by the

individual parts of the contents of the mother- cell prece-
dent to spore formation, uncertainty still prevails. According to the observa-
tions of P. Ernst (§ 35), the chromatin granules of the central substance
appear to play an important part in these changes, on which account this
worker entitled them " sporogenic granules."

Soon after spore formation has terminated, the membrane of the mother-cell
is dissipated, swelling up and dissolving in the surrounding liquid, and thus
leaving the spore free. This is the ordinary course, but in many instances
deviations occur, one of them being in the case of the Spirillum endoparagogicum,
discussed rather more fully below. In this case the membrane of the mother-
cell encloses the spore long after the latter is mature, and is still present when
the spore germinates. This is described in the next chapter.

§ 49.— Alterations in the Form of the Mother-Cell.

In many instances the mother-cell undergoes alterations of form during the
process of spore formation, and swelling occurs.

46

ALTERATIONS IN THE FORM OF THE MOTHER-CELL 47

When this happens at one of the polar terminations of a rod-shaped cell, the
latter then assumes the form of a nail or drumstick. Bacteria exhibiting this
peculiarity are styled nail-bacteria or helo -bacteria by Billroth, or Urocephalum
by Trecul ; and in medico-bacteriological literature they are also frequently
called pin-head bacteria. The earliest known example of this kind was the
Vibrio rugula (Fig. 15), frequently encountered in pools, and another is the

FIG. 15.— Vibro rugnla. FIG. 16. — Clostridium butyricum.

Seven rods, eacb with .1 ter- Spore formation.

minal spore. (After Praz- w 6 pure,y vegetative cells ; d. commencement of spore

formation ; c-e. progress ; f-h. completion ; «-/. contain
granulose stained blue by iodine ; h. devoid of this car-
bohydrate, unstained by iodine ; g. cell with two spores.
(After Prazmowsln.) Magn. 1020.

"drumstick bacillus," found in human faeces by BIENSTOCK (I.). The author
has often found morphologically similar fission fungi in the skin developing on
the surface of boiled infusions of hay ; and as a matter of general interest an
example from the pathogenic bacteria may be cited, viz., Bacillus tetani, by
which tetanus is produced.

Not inferior in number are the species wherein the sporogenic rod ordinarily
swells up in the middle and gives rise to a spindle-shaped outline resembling
that of a lemon or cop of yarn. TKECUL (I.),
who in 1865, in the course of his studies in
butyric fermentation, first became acquainted
with this bacterial form, gave it the name of
Clostridium, a term adopted as a generic name
by Prazmowski. The two species described by
this worker, viz., Clostridium butyricum (Fig.
1 6) and Cl. Polymyxa, were supplemented by
LIBORIUS (I.) with Clostridium fatidum, a fission
fungus, which is isolated from old cheese, and
produces a repellent odour in artificial nutrient
media. A fourth species is the Bacillus alvei,
discovered by CHESHIBE and UHEYXE (I.), which
causes the so-called " foul brood " in bees.
The Bacilhis inflatus (Fig. 17), discovered by
ALFKED KOCH (I.) in 1888, also belongs hereto.
Beyerinck investigated the conditions under
which the Clostridium form is assumed by a
number of species, having close affinities with Clostridium butyricum, which
have been grouped under the genus Granulobacter.

FIG. 17.— Bacillus inflatus.

Spore formation.

a, b, e. cells of Clostridium form, each
containing1 one elongated cylindrical
endospore ; c,d,f,ff. cells with two
spores of unequal size. (After A.
Koch.) Magn. 2100.

48 THE PERMANENT FORMS OR SPORES

§ 50.— The Number of Spores

produced in a single mother-cell exceeds unity iu but few species. The first
communication on this subject was made by Prazmowski, who found that in
exceptional cases Clostridium butyricum developed two spores in a cell. A re pre-
sentation of this is given in Fig. 16. ED. KERN (I.) observed in Caucasian
kephir granules a bacillus to which, on account of its faculty of producing two
spores, the name of Dispora caucasica has been given. This bacillus produces a
spore at each of its two poles, without any alteration of size or shape being
undergone by the latter. The contrary report, met with in many books, viz.,
that this microbe during spore formation swells up in such a manner that it
assumes the form of a dumb-bell, is a pure invention. The doubt raised by
MAC£ (I.) in 1889, and shared by many others, against the sporous nature of
this form, is also groundless, since a perusal of Kern's treatise shows that this
inquirer confirmed by observation the germination of the doubtful spores into

CL

FIG. 18. — Spirillum endopavagogicum. FIG. 19.— Bacillus tumescens.

C, vegetative cells ; A, two cells, one with two Chain ol seven cells, six of which have de-

and the other with three endospores. (After veloped one spore apiece, whilst the seventh

Sorokin.) and central cell has remained barren. Its

plasma is granular. (After A. Koch.) Magn.

1 100.

new rods. A third species in which this unusual fruitfulness has been observed
is the above-named Bacillus inflatus, in which, however — as is shown by Fig. 1 7
— the situation of the spores is not polar, but central. E. KRAMER (I.) reports
that the Bacillus saprogenes vini III., isolated by him from turned wine, swells
up at first at one of its poles and develops an endogenous spore therein, another
spore being then formed in the handle of the drum-stick form thus produced ;
so that two spores are developed in the same cell.

The formation of more than two spores in a single cell has hitherto been
noticed in but one species of fission fungus; the Spirillum endoparagogicum.
This was repeatedly observed by SOKOKIN (I.) in a small pool of rain-water
collected in the cavity of an old black poplar tree. A representation of this
microbe is given in Fig. 18. In A is seen a cell containing two, and another
with three endospores, and Sorokin found as many as six in a cell. The attempt
to obtain artificial cultures of this organism was as little successful as in the case
of so many other spirilla, there being (it may be mentioned en passant) up to the
present only a few known species wherein attempts of this kind have succeeded.
The first of these species is that which was isolated as a pure culture by ESMARCU
(I.) from putrescent fluid, and which formed rose-red colonies (Spirillum
rubrum) ; the second is the Spirillum desulfuricans, discovered and thoroughly
investigated by BEYERINCK (II,), which readily reduces sulphates to sulphides.
A third is the Spirillum luteum — developing a citron-yellow colouring matter —
obtained by H. JUMELLE (I.) from a bog; and the fourth is the Spirillum
itiarinum, described by H. L. RUSSELL (II.) as a frequent inhabitant of the mud

THE CONDITIONS INFLUENCING SPORE FORMATION 49

and water of the Bay of Naples. The spirilla, as MUHLHAUSER (I.) has shown,
are extremely susceptible to variations in the temperature and nutrient medium.

With these exceptions, only one spore is produced in the bacterial cell, in
which event the spore formation does not result in an increase in the number of
the cells. It is scarcely necessary to remark that it is not every cell that develops
a spore, it being a matter of frequent observation that individual cells in a
bacterial chain are sterile, leaving their neighbours on either hand to care for the
maintenance of the species. An attempt is made to represent this state of
things in Fig. 19, which is a drawing of Bacillus tumescens made from nature by
A. KOCH (I.).

§ 51.— Form and Size of the Spores.

These characteristics vary of course in different species, the spores of Bacillus
subtilis, for example, being ellipsoidal and measuring 1—2 p in length by about
0.6 p, in breadth, whilst the similarly shaped endospores of Clostridium butyricum
are 2-2.5 f- l°n£> an(^ 1>o V- broad. The general shape is oval, but there are
noteworthy exceptions to this rule. One of these is exhibited by the Bacillus
injlatus, which has already been frequently referred to. As can be seen from
Fig. 17, the spores of this microbe have the form of an elongated cylinder, and
are often curved in the shape of a bean. With a breadth of about 0.7 /* the
largest of them attain a length of 3.8 /*. in which respect this species is as yet
unrivalled. We may here mention that in A. Koch's work, as also in Eisenberg's
treatise, already alluded to, a number of spore measurements are given. The
duration of spore formation has been determined by Prazmowski for Clostridium
butyricum as 10 to 18 hours at 3o°-35° C.

§ 52.— The Conditions Influencing- Spore Formation

have been frequently investigated, but no generally satisfactory elucidation has
yet been obtained. H. BUCHNER (II.), on the basis of his studies, sought for
the explanation in the exhaustion of the supply of nutriment; but this is
contested by OSBORNE (I.). TURRO (I.), on the other hand, sees the cause in the
accumulation of noxious transformation pi-oducts, against which the vegetative
form seeks protection and the maintenance of the species by developing the
hardier reproductive spores.

A. Koch established the fact that Bacillus inflatus in hanging-drop cultures
forms spores when a i to 2 per cent, solution of meat-extract is employed as
nutrient medium, but that they are not formed if grape-sugar be added thereto.
Clostridium butyricum forms spores only in the absence of oxygen, whilst the
morphologically similar Cl. Polymyxa, on the other hand, produces them only in
presence of this gas. KOTLJAR (I.) found, in a microbe named Bacillus pseud'
anthracis, that spore formation was influenced favourably by violet light but
unfavourably by red light.

Past experience has shown that the formation of endogenous spores is
confined to the rod-shaped species (bacilli). This observation has been utilised
in the classification of bacteria, as will be seen in § 69. The reports to the
contrary found in the literature of the subject lack the force of proof, since they
ignore the fact that the sporous nature of the growths seen to originate in the
cocci has been demonstrated by germination tests.

§ 53.— Resisting- Power of the Endospores.

The forms in question are endowed with the character of reproductive cells,
since they are able to withstand those adverse conditions which would inevitably

50 THE PERMANENT FORMS OR SPORES

be fatal to the vegetative forms. As in the following sections — especially that
dealing with sterilisation — occasion will often arise for a closer investigation of
this faculty, so important for the maintenance of the individual species, an
exhaustive and tedious list of individual cases need not be given here ; it will
therefore suffice if we cite one example, viz., Bacillus subtilis. According to the
researches of BREFELD (I.), which were confirmed by M. GRUBRR (I.), a con-
tinuous exposure of twenty minutes to the action of boiling water suffices to
destroy the sporeless rods of this microbe ; whereas to kill the spores requires
three hours' boiling at 100° C., or a quarter of an hour's exposure at 105° C., or,
finally, the action of a temperature of 110° C. during five minutes. The
assertion made by Koch, that the continuous action of steam at 100° C. for
fifteen minutes will destroy the spores of any of the bacteria, was subsequently
negatived by his pupil E. VON ESMARCH (II.). According to SWAN ([.), spores
of Bacillus megatherium, dried on a cover-glass, retained their vitality and
germinating power for more than three years.

Use may be made of these powers of resistance for separating the sporiferous
from associated non-sporiferous bacteria. By skilful handling, e.g. by the aid of
sufficiently high temperatures, the weaker species in a mixture of bacteria can
be killed off, leaving only the spore-producing individuals. A process based on
this mode of procedure, and known as the boiling method, was introduced into
bacteriology by ROBERTS (I.) for obtaining pure cultures of the hay bacillus,
and the same method was employed by Prazmowski for preparing cultures of
Clostridium butyricum.

The seat of this high power of resistance has already formed the object of
numerous researches. One school looks for it in a peculiar modification of the
spore plasma — for instance, in the presumably low water-content thereof, as
suggested by LEWITH (I.). Others, again, attribute to the spore membrane an
exceptionally low heat-conducting power, and a very slight degree of permeability
by noxious substances. This latter opinion seems the more probable one,
considered from a physical point of view, and is further supported by

§ 54.— The Behaviour of the Endospores towards Dyes.

As already observed in a previous chapter, the dead plasma of the bacterial
cell absorbs colouring matters greedily and copiously. The staining of the
endospores is, however, more difficult, and consequently they have to be exposed
to the dye a much longer time before they will absorb any of it. However, the
colour thus taken up is retained by them more firmly than by the vegetative
forms.

This property has been utilised in microscopy to obtain a differential staining
of the spore-bearing bacterial cells, for which purpose the latter are treated
with a suitable (e.g. red) colour solution until the spores are thoroughly im-
pregnated therewith, the preparation being then steeped in a decolorising liquid
(generally slightly acidified alcohol), wherein it is left until the vegetative cells
are deprived of the colour. These latter are thereafter stained anew by a short
immersion in a second colour (e.g blue) solution, a two-colour preparation
(double-staining) being thus obtained, the spores in this case being red and the
rods blue. More detailed directions for double-staining will be found in Hueppe's
handbook, Eisenberg's treatise, and in Bernheim's " Taschenbuch " (Pocket-book).
Fig. 20 gives a black and white reproduction of a cover-glass preparation of
spore -bearing Bacillus subtilis stained only once, so that the spores are unchanged,
and appear colourless (white).

The aforesaid behaviour of bacterial endospores towards colouring matters is
characteristic of all. From this fact it is not infrequently, though erroneously,
assumed by medical bacteriologists that any formation, in the interior of the

ARTHROSPORES 5 1

cell, that behaves similarly towards dyes is to be considered as an endospore ;
whereas it is not yet proved that spores alone
exhibit this power. A general report concerning the
spore formation in any bacillus must therefore be received
with due reserve when it rests merely on the result of
staining experiments. The sole decisive proof of the
sporous nature of such bodies is afforded by their ger-
minating power alone, a subject discussed in the next
chapter. When this property has been observed, the
staining flask is no longer needed, its use in such case
being confined to the preparation of a coloured slide, FIG. 20.— Bacillus subtiiis.
which, in itself, is now valueless as a criterion. Cover -glass preparation

The property of offering considerable resistance to fr°m. an fight-days-oid

, , . f Oj , , , . , gvlatm culture grown

decolorising agents, possessed by the endospores, is also at room temperature,
shared by the vegetative forms of a few species of bac- stained with Vesuvine.

teria, among which are the tubercle bacilli and the leprosy T,he 8P°rc8; n°l ha™s'
...'. m° ii_i- i <• -i- i • absorbed the dye, show
bacilli. This unusual behaviour greatly facilitates their up as WMte specks
detection by microscopical examination alone, and is of against the (dark-
particular utility in this respect in the examination of JjJJ^ ^"^mglrSn)
milk and of the sputa of consumptive patients. Hagn. 950.

The differential staining of tubercle bacilli, also ex-
perimentally applied to non-pathogenic bacteria by many bacteriologists, will be
found dealt with in each of the above-named books.

§ 55.— Arthrospores.

As already remarked, the capacity for forming endogenous spores is not
universal among the fission fungi. The question then arises as to the means
whereby those species not endowed with this faculty protect themselves against
adverse external influences.

In many cases the resistance of such cells, and consequently the maintenance
of the species, is secured by the development of a protective wall of cells. This
is most frequently met with in zooglo3a-masses of bacteria.

In other cases actual spore formation occurs. This, of course, takes place
not within the bacterial cell, since that would imply endospore formation, but by
a thickening of the membrane of the individual cell in question, which thereby
plays the part of a reproductive cell. This procedure is known as arthrospore
formation, since the spore detaches itself from the chain of moribund cells, encysts,
and becomes dormant until conditions are once more favourable for its germina-
tion, when the cell increases in length and subdivides in the same manner as the
vegetative form.

The thickening of the cell membrane of the incipient arthrospore proceeds,
in many instances, to such an extent as to form spiny excrescences on the exterior
surface. This was observed by HANSGIRG (I.) in two species of bacteria, viz.,
M ycacanthococcus cellaris and Mycotetraedron cellare, found by him on the walls
of a cellar at the Castle of Pleissen, at Leipzig. The arthrospores of the latter
species, which are tetrahedral in form, exhibit at each of the four angles a spiny
thickening of the membrane 2 p, in length.

The name arthrospore will be understood when it is remembered that this kind
of spore is met with particularly in the thread bacteria, from which it bcomes, as it
were, actually dismembered. Examples of this are exhibited by the Grenothrix
observed by Cohn, Cladothrix by Zopf, Leptothrix by Miller, and Streptothrix Fuer-
steri by GASPERIST (I.). The discovery of these organs in species of cocci, &c. —
such as the urea bacterium found by JAKSCH (I.), in B. vernicosumby ZOPF (II.),
in Bacterium Zopfii by KUHTH (1.). and so on — was only a secondary matter.

CHAPTER VI.

THE GERMINATION OF THE ENDOSPORE.

§ 56.— First Type.

WHEN the spore is exposed to favourable conditions as regards nutrition, it
abandons its dormant state and begins to germinate by commencing to absorb
liquid from the surrounding medium. It then becomes distended, its high
refractive power gradually diminishing in the same proportion. Further
development can then proceed in three ways.

The first type of spore germination was accurately observed by H. BUCHXER
(III.) in Bacillus anthracis, the producer of anthrax, and afterwards discovered

'•«( »oiO<*c<f*<3rt

***

FIG. 21. FIG. 22. — Clostridiuin butyricuui.

Bacillus anthracis. Spore formation.

Germination of spores. „ ripe 8porei

s. the ripe spore before b. ditto expanding in nutrient solution.

germination begins; i, c. final dimensions attained, and separation of

2, 3. three successive exosporium from endosporium visible,
stages of germination ; rf, e. young rod escaping from pol-ir extremity

3. the fully developed of spore capsule.

rod. (After De Bary.) (After Pra~mowski.) Magu. 1020.

Magn. about 600-700.

in other kinds ; as, for instance, by PRAZMOWSKI (II.) in a fission fungus named
by him " mistbakterie " (dung bacterium), and in a second species isolated from
fermenting urine. The progi-ess of germination in this type is very simple
(Fig. 21). The spore, gradually acquiring the normal dimensions and functions
of the vegetative form, soon divides and reproduces by fission. According to
several observations made by Brefeld, the external layer of the spore-membrane
(" exosporium ") separates during this germination process and swells up. This
harmonises well with the remark made in a previous paragraph, that the spore-
capsule probably consists of two layers, distinguished as exosporium and endo-
sporium.

§ 57.— Second Type of Spore Germination.

Microscopically the initial stage of the process is identical with that described
in the foregoing paragraph ; the refraction of the spore diminishes and an in-
crease in size occurs. Then, however, the contents of the spore are elaborated
into a new rod, which is surrounded by a thin membrane, and which, by its
further growth, bursts the spore capsule.

This rupture is effected at the point of least resistance, the position of which —
and consequently the mode of escape of the germ— varies in different species.

In Clostridium butyricum, Cl. Polymyxa, and a few others, the spore capsule
opens at one of the poles, so that the direction taken by the young rod in its
escape is in a line with its length, as shown in Fig. 22, d. The expulsion is

52

THIRD TYPE OF SPORE GERMINATION 53

effected by the spore membrane, the tension of which is gradually increased to
such an extent, through the expansion of the spore contents, that it finally
squeezes out the mature spore. The capsule then shrinks at once to its original
size, and gradually disappears from view by swelling up and dissolving in the
surrounding liquid. If the spore has originated from any species of motile
bacteria, the liberated rod begins to rove directly one of its extremities is free.

It not unfrequently happens that the empty spore capsule is not completely
detached from the germ, but rests as a well-defined cap on the rearward
pole for some time, until finally it disappears. In a few species the persistence
of this membrane is very considerable, whilst the force of contraction is small
and insufficient to expel the matured rod. An example of this is afforded by
the Bacillus sessilis, discovered by L. KLEIN (I. and II.), which — as its distinctive
name implies — remains embedded in the spore membrane. This captivity,

A

200

30

SO

lOVhrM.

B,

?00

&

7/K

1&

,'&

12 U.

s

61'.

n:

3V-1E**

> V

AWe. — 10 to 12 Uhr M. (U.) = 10 A.M. to 12 noon ; i to 7 U. = i to 7 P.M.

FIG. 23. — Germination of spores of Bacillus sessilis.

Progressive observation of the germination of five cndospores (1-5) under the
microscope at 3O°-35° C. Hanging-drop culture in meat-extract solution. The time
of the observation (ironi 10 A.M. to 7 P.M.) is given under the illustrations of the
separate stages of development (A-K) of the germination. Spore 3 had not
germinated even at 7 P.M. (After L. Klein.) Magn. about 1000.

nevertheless, in nowise retards its nutrition and reproduction, since by fission it
forms new rods, which it protrudes through the polar openings at both
extremities in the muff-shaped capsule, the said rods quickly forming spores in
turn. An illustration is given in Fig. 23.

§ 58.— Third Type.

In Bacillus subtilis (Fig. 24), Bacillus megatherium (Fig. 25), and ft few other
specie.", the spore membrane does not burst at the poles, but along a line coincid-
ing with the equator of the spore. This line, however, extends only part of the
way, not right round the spore, so that the two halves of the membrane still
remain attached together at one point. The rod then makes its exit by bending
somewhat at the centre, and, by turning one of its extremities, pushes it out of
the capsule, one half of which often remains on the other pole for some time like
a cap, the other half hanging down empty. Sometimes the germ cannot liberate
even one end from the capsule, both poles remaining wedged between the two
halves of the membrane (these acting like a pair of tongs), and the central
portion alone projecting. This position, however, does not prevent reproduction,

54 THE GERMINATION OF THE ENDOSPORE

but gives rise to horse-shoe chains .(Fig. 26), which only separate into their
individual members when the spore membrane has become swollen and flaccid.

FIG. 24.— Bacillus subtilis.
Spore germination.

a. ripe spore ; l>. placed in a nutrient solution,
the refraction disappears ; e. enlargement
begins ; rf. the equatorial fissure is formed
and the young germ begins to escape ; e. in
the npper row the central portion of the
germ is just protruding, in the lower row
one pole is already freed ; /. the young rod
is at liberty ; g. it grows to its normal size ;
/i. reproduces by subdivision. Extra long
cells are seen at g and h in the lower row.
(After J'razmoirski.) Magu. IO-JQ.

FIG. 25. — Bacillus megatherium.
Spore germination.

//]. two dried ripe spores enclosed
by the walls of the mother-cell.

7;2. the same spores after forty-
five minutes' immersion in a
nutrient solutiou.

/•, /. the spore contents have in-
vested themselves with a new
membrane and are escaping
from the old capsule.

m. two full-grown rods.

(4fter 1)e Bar;/.) Magu. 600.

FIG. 26.— Bacillus subtilis.
Impeded germination.

1. Cells with ripe spores causing
the mother-cell walls to bulge.

2. Commencement of spore ger-
mination, capsule fissured equa-
torially.

3. Ordinary unimpeded escape of
the germ.

4. Exit somewhat impeded, one
pole being eventually liberated.

5. Both poles of each germ re-
main fixed; germ divides into
two cells.

(After lit liar a.) Magn. 600.

FIG. 27. — Spirillum endoparagogicum.
Spore germination.

A, purely vegetative cells in brisk motion.

B, three spirilla with four to six spores, those
in the central cell being ripe.

D, mother-cell with germinating spores, from
which proceed

E, branched forms, subsequently dismembered
into single cells.

C, moribund spirilla, one with three spores.

(After Sorokin.) Magn. 1375.

The time occupied by Bacillus subtilis in germinating is, according to
Pra^mowski, generally 3-4^ hours at 3o°-35° C., but frequently much longer.

IMPORTANCE OF PROCESS IN CLASSIFICATION 55

A unique procedure is manifested in the germination of the spores of Spiril-
lum endoparagogicum, the membrane of the mother-cell remaining unimpaired
(as already mentioned) after the spores are formed, so that the germs proceeding
from the spores have to penetrate the membrane of the mother-cell in order to
attain their freedom. Not infrequently they remain attached by the one end,
thus giving rise to a branched form, as shown in Fig. 27.

§ 59.— Importance of this Process in the Classification of Bacteria.

Since, according to observations made thereon, the course of spore germina-
tion differs in the various species, it may be utilised, in the characterisation of
species, as an invariable and therefore reliable indication. One example, serving
for Bacteriology in general, may here be cited. H. BUCHNER (II.) ascertained
that by a suitably modified method of culture it was possible to deprive the
anthrax bacillus of its virulence and render it harmless. Sundry other experi-
ments (subsequently found to be defective and deceptive) with the hay bacillus
(B. subtilis) induced him to assert that these two
species were identical, the hay bacillus being an
anthrax bacillus that had lost its virulence, and vice
versd. Now it has already been shown that the
germination of the endospores of B. anthracis
follows a different course to that occurring in B.
subtilis; consequently, if the former were by
Buchner's treatment not only rendered harmless,
but also actually converted into B. subtilis, then the
course of germination must also have become corre-
spondingly changed. Starting with this assump-
tion, PRAZMOWSKI (III.) subjected a weakened
anthrax bacillus (presumably transformed into B.
subtilis) to examination with regard to the nature
of the formation and germination of its spores.
He found that both operations pursued exactly
the same course as in the virulent (unweakened)
anthrax bacillus, thereby disproving the assump-
tion of the identity of the two species.

The first observation of the production of de-
veloping reproductive cells within the bacterial cell
was made by PERTY (I.) in 1852, who designated
the rods he found to possess this property Sporo-
nema gracile (Fig. 28). His communication,
however, met with no recognition and fell into
oblivion. About fifteen years later PASTEUR ("VI.),
unaware of Perty's observation, re-discovered the

same fact in the course of his researches on the causes of lethargy in silkworms
(gattine). The fission fungi which he found in large number in the alimentary
canal of the diseased (lethargic) worms, and which he experimentally ascertained
to be the cause of this generally fatal epidemic, frequently exhibited internal
lustrous enclosures, the formation and function of which he explained to be
reproduction by endogenous germs ("reproduction par noyaux interieurs"),
without, however, observing them more closely in order to ascertain the accuracy
of this hypothesis.

Only one proof was needed to set this beyond doubt, namely, the
demonstration that these forms have the faculty of germinating and of develop-
ing into new individuals. FERD. COHEN (VII.) first succeeded in doing this, in

FIG. 28.
Spore formation according to Perty.

b-g. Sporonema gracile ; b. with one
terminal spore ; c. with two spores ;
in d the spore has escaped from the
mother-cell ; e-g shows the gradual
development of the spore until
ripe. In a is shown the (formerly)
so-called Metallacter, a chain of
short threads which when viewed
under a low power is apt to be
mistaken for a long rod, hence its
name.

56 THE GERMINATION OF THE ENDOSPORE

1876, with a bacillus isolated by him from an infusion of hay. This fact defi-
nitely proved that the bacteria belong to the vegetable kingdom, since spore
formation exclusively characterises the sub-kingdom Thallophyta, and is unknown
in animals. A more accurate account of this important process was given two
years later by OSKAR BREFELD (I.) for Bacillus subtilis ; two years subsequently
by|A. PRAZMOWSKI (I.) for Clostridium butyricum; in 1882 by HANS BUCHNER
(II.) for B. anthracis ; then by DE BARY (I.) for his B. megatherium, and by
others.

SECTION II.
GENERAL BIOLOGY AND CLASSIFICATION OF BACTERIA.

CHAPTER VII.

THE BACTERIA UNDER THE INFLUENCE OF
PHYSICAL AGENCIES.

§ 60.— Influence of Electricity.

THE effects of this agency were first recorded by SCHIEL (I.) in 1875, the earliest
exhaustive researches being carried out by COHN and MENDELSOHN (I.) in 1879,
succeeded in following years by the labours of APOSTOLI and LAQUERRI^RE (I.),
PEOCHOWNIK and SPATH (I.), and DUCLAUX (III.)- The same method of experi-
menting was followed by all these observers, and consisted in passing an electric
current through the culture. Conn found that, to produce an appreciable
weakening effect by this means, a battery of at least two cells was required, the
current from which, when passed for 12-24 hours through a nutrient solution
inoculated with bacteria, was unable to kill the germs, but nevertheless ren-
dered the medium unsuitable for further culture. This result, as explained by
Cohn, was due to the action of the current in forming decomposition products
inimical to fungi. Bearing this in mind, such a method of experiment is there-
fore unsuitable for affording a clear insight into the influence of the current
itself. These labours are, nevertheless, worthy of mention, since, having been
further pursued with a practical aim, they have led to the elaboration of a
process for the purification of sewage water (as developed and tested by WEBSTER
(I.) in particular). The water to be purified is led through a trough into which
dip large iron plates, acting as electrodes for a powerful current generated by a
dynamo machine and passed through the liquid. FERMI (I.) tested the process
from a bacteriological point of view, and ascertained that — under the conditions
of the experiment — a current of 0.5 to i o ampere reduced the number of germs
to between ^th and y^th of the initial quantity.

To as-certain the effect of the electric current, unaffected by secondary
chemical influences, BURCI and FRASCANI (I.) proceeded by drying the bacteria
(i.e. a small portion of inoculated nutrient solution) on a pad of glass wool at
37° 0., and then dipping the pad into mercury through which a constant galvanic
current was being passed. In this case the bacteria were killed ; but the method
of experimenting is not free from objection, since the dried constituents of the
medium were present along with the bacteria, and might retain moisture and
form decomposition products noxious to the latter.

These injurious secondary influences can only be perfectly excluded when
the electric current is prevented from coming into contact with the nutrient
medium, a condition first attained in the experimental method selected by
SPILKER (I.) and GOTTSTEIN (I.). The glass flask containing the bacterial culture
was enveloped by a coil of the line wire, and an induction current then passed.

57

58 THE BACTERIA UNDER PHYSICAL AGENCIES

Micrococcus jm-odigiosus — washed by sedimentation in pure water or in water
qualified by nutrient gelatin — was killed when the liquid (2500.0. in volume)
was exposed for twenty-four hours to the influence of a current of 2.5 amperes
and 1.25 volts. Other species of bacteria offered greater resistance: as, for
example, those occurring in milk, which are gifted with the power of forming
endospores capable of retaining their vitality under very adverse conditions.
For this reason the above-named observers never succeeded in thoroughly freeing
milk from living germs by electrical treatment, although the number of the
germs could be reduced thereby.

D'ARSONVAL and CHARRIN (I.) studied the influence of the electric current
on the blue pigment of the Bacillus pyocyaneus found in the pus discharged by
wounds. They placed a culture of this organism in the cavity of a solenoid
traversed by a current of 10,000 volts; an exposure of twenty minutes sufficed
to destroy the chromogenic power of the bacilli almost completely. A similar
decrease of virulence was observed by S. KRVGER (I.) in the case of a few
pathogenic bacteria ; and, finally, reference may be made to a research of this
nature performed by H. FRIEDENTHAL (I.).

At present, owing to the high cost entailed, the utilisation of the anti-
bacterial powers of electricity in the food-stuff industries is out of the question.
Use has, however, been made of these powers in the fermentation industries,
although the primary object of the process is not the destruction of germs, but
the chemical changes effected by the electric current. Alcoholic beverages (wine,
cognac) are artificially matured, and a slight esterification, and consequent
mellowing of flavour, produced by allowing the liquors to flow slowly through
an electrical field. A more detailed consideration of this process is, however,
beyond the scope of the present work. A review of the methods proposed for
this purpose and the experiments made therewith is given by A. SCHROHE (I.).

D'Arsonval and Dubois have made a few observations on the influence which
magnetism (so closely allied to electricity) has on bacteria, but unfortunately
these have not been followed up any further.

§ 61. — Influence of Temperature.

The ordinary conceptions with regard to the favourable or prejudicial influence
of certain temperatures on organic life cannot be applied, without modification,
to bacteria, and this is particularly the case with respect to the effects of cold.

J. FORSTER (I.) was the first to fiud (in 1887) that there are species of bacteria
which at a temperature of o° C. are not only alive, but actually reproductive.
This report is based on a luminous bacterium isolated by him from the surface
of a phosphorescent salt-water fish. B. FISCHER (I ) next discovered fourteen
other species, some in sea-wator, others in the soil, all of which were very
reproductive at o° 0. Returning to the subject, J. FORSTER (II.) then examined
more narrowly the natural habitat of similiar bacteria, and found that

Commercial milk contained up to 1000 per i c.c.
Dr.iin water contained up to 2000 per I c.c.
Garden soil contained up to 140,000 per I gram.
Street mud an innumerable quantity per i gram.

MIQUEL (I)., by keeping a sample of sea-water at o° C., found that an initial
number of 150 germs per i c.c. increased to 520 in twenty-four hours and to
1750 in four days. These facts indicate that glacier water, haiJ, and snow may
also contain bacteria. Quantitative researches on this point have been carried
out by L. SCHMELCK (I.), O. BUJWID (I.), W. FOUTIN (I.), TH. JANOWSKI (I.),
and especially P. MIQUEL (I.).

INFLUENCE OF TEMPERATURE 59

The resistance of bacteria to low temperatures extends considerably below
zero Centigrade, FRISCH (I.) having shown that some species will bear cooling
down to - 110° 0. for a short time without injury. R. PICTET and E. YUNG (I.)
found that bacteria (species unknown) could be kept at - 70° C. for 108 hours
and at - 130° C. for twenty hours without succumbing ; certain (unnamed)
species even withstanding the effects of a short exposure to -213° C. in
solidified oxygen. These facts are not merely of general biological interest, but
also, at the same time, important as regards the question of the suitable treat-
ment of stored food-stuffs. This will be discussed in a subsequent paragraph.

Antithetical to these cold-loving bacteria is the Bacillus thermophilus, dis-.
covered by MIQUEL (II.), which thrives and reproduces with great activity at
70° C., a temperature which instantly kills animal cells, coagulates egg albumin
and blood serum, and produces painful burns on the skin. When kept at 50° C.
this aerobic bacillus occurs as short rods, about i fi in thickness, which become
longer as the temperature rises, threads beginning to form at 60° C., and
constituting at 70° C. the sole occupants of the field. The lowest limit of
temperature at which development of this organism can be observed is about
42° C. ; above 72° C. the vegetative forms die off. This non-ciliated fission
fungus is but seldom met with in atmospheric dust, but is very frequent in
sewage, and therefore also in sewage-contaminated waters. It is likewise
present in the alimentary canal of human beings and mammals. This locality
seems to possess a highly suitable temperature for the growth of this saprophyte,
although accurate knowledge on the subject is still lacking. When the
temperature rises above 50° the medium undergoes putrefaction as a result of
the activity of the bacillus.

Between Bacillus thermophilus and the aforesaid cold-loving species there
are numerous species forming intermediate links in the chain. In the case of
Forster's microbe, already mentioned, the highest limit of supportable tempera-
ture is 35° C., and it cannot retain its vitality when exposed, even for a few
hours, to a temperature of 35° to 37° C. GLOBIG (I.) isolated from garden soil
twenty-eight species of bacteria, each of which still developed luxuriantly at
60° C., whilst the minority were able to grow at even higher temperatures. In
connection with their occurrence in nature the question of the limits of tempera-
ture— range of climate — within which they can grow is of interest. In this
respect great differences were observed, one of them growing as well at 15° as
at 68° C., whilst most of the others required a temperature of over 50° C., and
one exhibited signs of development only when the temperature exceeded 60° C.
It must therefore be concluded that, under natural conditions, the reproduction
of these organisms proceeds only in the height of summer, when the soil is
sufficiently heated by prolonged sunshine. These warmth-loving bacteria are
not found in the ground exclusively. LYDIA RABINOWITSCH (I.) isolated from
the excrement of various animals, as well as from manures, milk, &c., eight
widely distributed species, for which the highest limit of temperature at which
growth was possible was found to be 75° and the minimum about 39*" 0. These
organisms are therefore able to reproduce freely in the alimentary canal of
warm-blooded animals and human beings. Warmth -loving fission fungi are also
not infrequently encountered in sea-water. One example of this is afforded by
a phosphorescent bacterium found in the West Indies and described in chapter
xv. under the name of Bacterium phosphorescens. This inhabitant of the tropics
thrives best at 2o°-3o° C., and ceases growing at i5°C. Living bacteria have
also been found in boiling springs, e.g. that discovered by Cortes and Garrigon
in the basin of a mineral spring, the temperature of which was 64° C.
J. KARLINSKY (I.) in 1895 discovered in the hot sulphur springs at llidze, near
Sarajevo, in Bosnia, two species of Schizomycetes which he named Bacterium

60 THE BACTERIA UNDER PHYSICAL AGENCIES

Ludwigi and Bacillus Ilidzensis capsiilatus, the former developing only when the
temperature rose above 50° C., and the second producing endospores able to
withstand four hours' exposm-e in water at 100° C. without succumbing.

DIEUDONNK (I.) drew attention to the fact that, owing to the possession by
bacteria of a certain power of adaptation to climatic conditions, no hard and fast
lines can be drawn respecting the limits of temperature within which growth is
possible ; but that by carefully controlling the stages of transition it is possible
to somewhat extend these limits.

In ciliated bacteria spontaneous motion ceases when the temperature of the
environment approaches the lower or higher limit, and they fall into a state
of torpidity through cold or heat, from which they recover as soon as the
temperature once more becomes favourable.

Reference to the morphological influence of temperature has already been made
above (as also in § 29), and will be exhaustively described and illustrated, with
a particularly fine example, in a subsequent paragraph. The transforming and
modifying power of warmth also extends to other properties of bacteria ; for
example, to the virulence of pathogenic bacteria, i.e. their capacity for en-
gendering disease. In the present work, however, not more than a single one
(on account of its general interest) can be referred to, viz., Pasteur's process of
preventive inoculation for anthrax. If Bacillus anthracis be cultivated in
meat-broth for twenty-four days at 42°-43° C., a virus (premier vacciri) is obtained
the virulence of which is so attenuated that sheep (the animal most subject to
anthrax) inoculated therewith experience only a mild form of the complaint.
If then inoculated with a second culture prepared by exposure to the attenuating
influence of a temperature of 42°-^° C. for only twelve days (second vaccin),
the animals no longer sicken, even if inoculated by unattenuated B. anthracis,
and are therefore immune against inoculative anthrax.

§ 62.— Influence of Light.

The old empirical hygiean maxim concerning the disease-banishing power of
the sun's rays — which is well expressed by the Italian proverb, " Where the sun
does not enter the doctor does " — finds a full explanation in the bacteriological
discovery that the overwhelming majority of the fission fungi thrive much better
in darkness than in the light, and are, in fact, under certain circumstances,
killed by direct sunshine. This question of the influence of light on bacteria has
already formed the subject of innumerable researches, most of which, however,
are of purely medical and hygienic interest, on which account their consideration
here must be restricted to a mere recapitulation of the main points involved. A
summary review of the literature of the subject up to 1889 will be found in a
work by J. RAUM (I.), which in this particular is to some extent supplemented
by the more recent publications of TH. JANOWSKI (II.) and TH. GEISLER (I.).

Most of our knowledge of the question was obtained from the earliest
investigations therein, published in 1877 and 1878 by DOWNES and BLUNT (I. and
II.), who found that the growth of the bacteria is restricted by the influence of
diffuse white daylight and is completely stopped by sunshine. The blue and
violet rays proved the most injurious, the red and orange rays being weaker in
their action. The authors explained the injurious effect of light as an indirect
one, in that it strengthens the decomposing power of oxygen, the result being
the decomposition and destruction of the bacterial plasma. JAMIESON (I.), in
1882, gave another explanation of the phenomenon by attributing the injury
observed to the increase of temperature affected in the cells by the sun's rays.
The fallacy of this hypothesis — which had been rejected by DOWNES (I.) — was
demonstrated in 1885 by DUCLAUX (IV.), who was also the first to employ pure

INFLUENCE OF LIGHT 61

cultures — viz., of Tyrothrix scaber — in the study of this question. He proved,
at the same time, that the duration of exposure to sunlight necessary to kill the
microbe is dependent on the composition of the nutrient medium employed for
the culture, cells cultivated in bouillon proving less capable of resistance than
those of the same species grown in milk.

Several other explanations have been given regarding the particular and
more intimate reactions that occur in a bacterial culture exposed to the rays of
the sun. Some observers adhered to the opinions expressed by Downes, and
attempted to show that, by exposui-e to sunshine, decomposition products are
formed in the medium and act fatally on the bacterial cell. Support for this
view is found in the observation made by G. Roux (I.), that the destruction of
the germ goes on much more rapidly when there is a concurrent admittance of
air ; and an indication pointing in the same direction is afforded by the fact,
determined by RICHARDSON (I.), that hydrogen peroxide — a substance highly
poisonous to bacteria — is formed when sterilised urine is exposed to sunlight.
On the contrary, other observers — WARD (I.) in particular — have shown that
the presence of such oxidising agents is not essential, but rather that sunshine
alone suffices to destroy the vitality of even the strongest bacterial spores.
Probably in nature both agencies co-operate in producing the same results.

The last-named investigator also examined more closely the degree of influence
exerted by the individual colours of the spectrum, and found that, in the case of
red to green, this action is almost nil, increasing thence to its maximum at the
violet end of the blue, and then falling away again in the violet and ultra-violet
rays. According to the researches of SANTOUINI and GEISLER (I.), a similar,
though less powerful, injurious action is exerted by the electric light; and
F. MINCK (I.) has performed several experiments on the effect of the Rb'ntgen
rays on bacteria.

The anti-bacterial influence of sunlight is of the highest importance, especially
in regard to the self -purification of rivers. As is well known, the amount of
organic matter and the number of bacteria in river-water diminish in proportion
as the water increases its distance from the point of contamination. This
property, on account of its hygienic and technical importance, has already
formed the subject of investigation. HANS BUCHNER (IV. and Y.) in 1892
pointed out that all previous explanations of this occurrence had omitted one
factor, viz., the influence of light. He showed that a natural water to which
about 100,000 cells of Bacterium coli commune — an organism constantly and
abundantly present in faeces — had been added per i c.c., contained, after one hour's
sunlight, no living germs. To bring this action into specially prominent notice,
he poured peptonised meat-juice-agar-agar, inoculated with a copious supply of
typhus bacilli, into Petri basins, on the under side of which were affixed the
letters TYPHUS cut out of black paper. The basins were then exposed to the
sun's rays for one to one and a half hours, or to diffused daylight for five hours,
and afterwards left in a dark room for twenty-four hours. On the paper letters
being then removed, their form was found to be marked out bjr the thickly
clustered whitish colonies composed of the bacteria that had been protected from
the fatal effects of sunlight by the paper cover, and had consequently remained
alive, whilst the residual uncovered portion of the medium was destitute of any
such colonies. Fig. 29 is a reproduction of the photograph taken by Buchner
from one of the plates. The same result was obtained by illuminating the care •
fully-closed culture basin under water. Experiments made in the clear waters
of Lake Starnberg showed that the anti-bacterial influence of the sun's rays
extends to a depth of some two metres (about eighty inches) below the surface
of the water. Therefore, to the already known factors in the self -purification of
rivers — viz. sedimentation, oxidising influence of air, consumption of filth by

62 THE BACTERIA UNDER PHYSICAL AGENCIES

algae, &c. — all of which are more concerned with alterations of chemical com-
position— must be added the influence of sunlight in diminishing the number of
bacteria. A critical review of the most important labours and researches per-
formed in respect of the self -purification of rivers is given by E. DUCLAUX (V.).
All the pathogenic Schizoinycetes seem to succumb under the influence of sun-
light. This has been shown by Arloing and Ward in respect of Bacillus anthracis;
Gaillard for B. typhi abdominalis ; Pansini for Vibrio cholerce asiaticce and a

FIG. 29. — Thickly-sown plate culture of typhus bacilli on agar-ayar. Covered with
paper letters and exposed to the sun's rays for ij hours, then kept twenty-four
hours in the dark, whereupon development of thickly congregated whitish
colonies was found ouly at the parts covered by the letters. (After H. Buclmer.)
Nat. size.

fungus giving rise to white pus in wounds (Staphylococcus pyoyenes albus) ;
Chmiliewski for the organism which induces the formation of yellow pus (St.
pyojenes aureus), and the bacillus of erysipelas (Streptococcus erysipelatis) ; Hob.
Koch for Bacillus tuberculosis ; Charrin for the organism producing swine-
erysipelas ; and others. Most of the non-pathogenic fission fungi also succumb
to the influence of light. GALEOTTI (I.) arranged a number of chromogenic
species in the following descending series, the first member of which resists the
action of diffused daylight the longest : Bacillus ruber, Micrococcus prodiyiosus,
Sarcina rosea, Bacillus violaceus, B.pyocyaneus, B. lactiserythrogenes. According
to the researches of GKOTENFELT (I.), the last-named fission fungus does not
produce red colouring matter at all when strongly illuminated. K. DUBOIS (I.)
ascertained that the luminous bacterium, Photobacterium sarcophilum, found on

INFLUENCE OF MECHANICAL SHOCK 63

spontaneously phosphorescent flesh, temporarily loses iis light-producing power
on prolonged exposure in a light room.

Great differences in susceptibility to sunshine are also exhibited in the
Schizomycetes. At the extreme end of the series stand the purple bacteria,
examined more closely by Engelmann, which always seek out the more highly
illuminated positions. One of the species was named by ENGELMANN (I ) Bacterium
photometricum, on account of its variable susceptibility to the colours of the
spectrum and degrees of brightness. These organisms, which will be fully noticed
in a subsequent chapter, also display the phenomenon known as movement of
alarm. If a microscopic preparation containing one of them in large numbers
be illuminated in such a manner that the light rays can fall only on one sharply
defined portion, then all the roving bacteria collect within this space and bustle
about briskly therein. If now one of them in its onward career passes beyond the
circle of illumination into the dark portion, it stops instantly, and then returns
by the same road into the illuminated field. This is the phenomenon of the
movement of alarm. Consequently each sharply defined illuminated portion of
the field acts as a trap for the bacteria, from which they cannot escape until the
illumination has been altered. If a definite form be given to this trap, such, for
instance, as the shape of a W, and the closely congregated cells be fixed and
stained in this position, then a so-called bacterial photogram — i.e. a coloured
picture of the trap, composed of the organisms themselves — is obtained.

§ 63.— Influence of Mechanical Shock.

The first to inquire whether the vitality of lower organisms can be influenced
by agitation was A. HORVATH (I.) in 1878. He made his observations with
bacteria because he assumed that, on account of then- small size, the possibility of
mechanical injury (rupture) due to agitation would, in the case of these organisms,
be reduced to a minimum. On gently agitating bacterial cultures (in Cohn's
nutrient solution) he was unable to detect the manifestation of any retarding
influence on the growth of the organism. The results were, however, different
when the sample was made to undergo, by means of a shaking machine, about a
hundred movements — in a direct line and of an amplitude of about 10 inches
(25 cm.)— per minute. This treatment for a period of twenty-four consecutive
hours diminished the reproduction of the bacteria in question ; and when continued
for forty-eight hours the agitation proved fatal. On the basis of his researches
Horvath formulated the opinion that " for the development of the living organism,
or the physiological reproduction of the elements constituting the organism, a
certain degree of repose is necessary," meaning thereby that rest mainly favours,
whereas movement injures, reproduction. This generalisation was opposed by
NAGELI (II.) and E. Ch. Hansen, the former of whom drew attention more
particularly to the reproduction of algae living beneath large waterfalls and
exposed to much more violent agitation than was effected by Horvath's shaking
apparatus.

In 1879 E. CH. HANSEN (I.) instituted experiments in order to test Horvath's
assertions. Working with beer yeast (i.e. not bacteria), he ascertained that this
organism developed better when the liquid (beer wort) was set in motion by stirrers.
The probability of this favourable influence of movement being due to aeration is,
according to Hansen, inadmissible, this latter effect having been but slight.

A year later the question was taken up by J. REINKE (I.). An objection
raised by NAGELI (II.) led him to try the effects of movements more nearly
approximating in amplitude to molecular movements than were those produced
in Horvath's experiments. To this end he made use of sound waves, the end of
a metal rod, caused to emit sound by friction, being immersed in a glass filled

64 THE BACTERIA UNDER PHYSICAL AGENCIES

with Cohn's nutrient solution containing bacteria, and thereby transmitting the
wave-motion to the liquid. The experiments showed that a considerable restric-
tion, but not cessation, of growth occurred. From this Reinke concluded that " if
it be assumed that the molecules of living protoplasm are endowed with specific
vibratory movements, the idea appears feasible that when those specific molecular
vibrations are crossed by other molecular motions of external origin, the vital
functions of the protoplasm will be weakened."

The labours subsequently made public by L. Tumas, 0. Roser, H. Buchner,
H. Cramer, H. Miquel, H. Leone, A. Gartner, B. Schmidt, and others, did not
produce anything having a material bearing on this question. A treatise by
H. RUSSELL (I.), who workediwith Monilia Candida, Saccharomyces mycoderma, and
Oidium albicans, and found that the form and dimensions of the cells are but
little altered by agitation, and that the percentage of germs in agitated samples
is almost double that in samples left at rest for the purpose of comparison, is,
however, worthy of mention.

The results appear to contradict one another. It should, however, be re-
membered that the experimenters who obtained favourable results with agitation
subjected their cultures to comparatively gentle movements, whereas the motion
set up by Horvath was violent and prolonged. The conditions of his experiment
were first repeated by S. MELTZER (I.) in 1891, who worked chiefly with Bacillus
megatherium. He made numerous experiments, but we will only draw attention
to those that gave results in advance of those previously obtained. A New York
mineral water works placed at Meltzer's disposal their agitator, with which appa-
ratus he was enabled to subject the test samples to 180 reversed movements —
of an amplitude of 15^ inches (40 cm.) — per minute. The flasks employed were
only one-third full. Meltzer found that the number of germs (ascertained by the
plate method) in the agitated example in no instance amounted to as much as
one-tenth of those in the unshaken check samples ; and was, in fact, almost in-
variably smaller than at the commencement of the experiment. The restriction
of reproduction thus indicated increased with the duration of the treatment, so
that by this means the liquid could be completely freed from germs. The effect
was even more powerful when sterilised glass beads were added before commenc-
ing the operation, the complete annihilation of the germs being accomplished
under these conditions by ten hours' agitation. In addition to B. megatherium,
Meltzer also included a micrococcus (presumably M. radiatus, Fliigge) and a
short motile bacillus (albus ? ) in the scope of his investigations. A difference in
the degree of resistance to this kind of inhibition is inherent in these organisms,
since it was found possible to successively eliminate each form from a mixture of
the three species. B. megatherium, as the most susceptible, disappeared first, and
was followed, in order, by Micrococcus radiatus and Bacillus albus. The cells were,
as a result of the shaking, split up, not into visible debris, but into an indistin-
guishable fine powder, a circumstance showing that the destruction of the vitality
of the cell was not the result of a coarse mechanical disruption, but was due to a
much more refined process; as was, in fact, shown by the further researches
made by the same observer. He left several flasks containing cultures of B. mega-
therium or B. subtilis in solutions of common salt, to stand for several days in the
engine-house of a large New York brewery, wherein, in consequence of the unin-
terrupted working of the engine, an incessant vibration was produced throughout
the room. After four days all the germs in the several flasks were dead, whilst
energetic reproduction had proceeded in the check samples placed in a quiet spot.
Consequently, not only violent shocks, but also minute vibrations, exhibit the
power of retarding the growth of bacteria, and even killing the organism.

Motion may, however, also exert a favourable influence, and especially when it
is comparatively weak, reproduction being thereby accelerated, as has been moie

INFLUENCE OF MECHANICAL SHOCK 65

particularly demonstrated in the case of B. ruber. Meltzer therefore arrived
at the following conclusions : Slight concussion favours tho vitality of micro-
organisms and has a stimulative effect, the rate of reproduction being highest
when the optimum of vibration is obtained ; but from this point onwards the
restrictive effects of concussion become manifest. The constants of optimum and
maximum effect have different values for different organisms. That degree of
concussion which is injurious for one species may be favourable to a second, and
without any appreciable effect on a third. This explains the contradictory
reports made by the pioneers in this field, each of whom experimented on different
organisms.

The influence of gravity on the direction of growth, which comes into play
in the higher plants, and the effects of which are known in Vegetable Physiology
as geotropism, has also been observed in the Schizomycetes. BOYCE and EVANS
(I.) found that vertically disposed puncture-cultures of Bacterium Zopfii in
nutrient gelatin arranged themselves in the form of a feather, and in such a
manner that the individual rays grew in a slanting upward direction. When
the tubes containing the cultures were placed radially on a rapidly-revolving
horizontal glass disc, the vegetation then developing assumed an appearance
corresponding to that already described, the individual rays, which extended
from the axis of the puncture, formed acute angles therewith, the apertures of
which were reflected towards the centre of the disc. This species therefore
exhibits negative geotropism. BEYERINCK (III-) — erroneously, as the author
conceives — has denied this fact.

The lower fungi generally, and bacteria in particular, remain, within wide
limits, unaffected by high gaseous pressure. Thus, SCHAFFER and FREUDENREICH
(I.) and others have inoculated samples of milk with different bacteria (those of
anthrax and typhus among them), and then exposed them for seven days to
carbon dioxide at a pressure of fifty atmospheres, without being able to cause
any appreciable injury to the organisms. Similar behaviour was also observed
with oxygen under a pressure of twenty- one atmospheres, prolonged for a week.
There is, therefore, no reason for hoping that liquids which are injuriously
affected by heat can be sterilised in the cold by the aid of gas (C02, O, air)
under high pressure. For exhaustive experiments on the influence of high
gaseous pressure on living creatures generally, and the pathogenic Schizomycetes
in particular, we are indebted to Paul Bert.

CHAPTER VIII.

BACTERIA IN THEIR RELATION TO ONE ANOTHER.

§ 64.— Symbiosis, Metabiosis, Antagonism.

IT is only in exceptional cases that a sample of a natural liquid contains but
a single species of micro-organism when in a state of fermentation. Nearly
always we have to deal with a mixture of several species, the separation of which
one from another, and the reproduction of the isolated individuals, is termed
pure cultivation. A liquid or solid nutrient medium inhabited by a single
species is called a pure culture, the methods of preparing which will be considered
in the next section.

When two or more species are simultaneously engaged in the consumption
of a given nutrient medium, their association is termed Symbiosis. A couple
of examples will serve to make this clear, one of them being the Kephir
granules, which will be described in a later chapter. These granules chiefly
contain two classes of organisms, lactic acid bacteria and yeasts ; and when intro-
duced into milk the fission fungi generate acidity, whilst the yeasts decompose a
portion of the milk-sugar and produce alcohol and carbon dioxide. In this way
an acid, foaming liquor known as "kephir"is obtained. A second, cognate
example is afforded by the ginger-beer yeast, investigated by WARD (II.), and
used in England for making ginger-beer. This is another instance of symbiosis,
viz., the association of Saccharomyces pyriformis with a fission fungus, Bacterium
vermiforme, the latter of which— as is described in chapter xxv. — induces lactic
fermentation in (spiced) cane-sugar solutions.

The mutual relation of two or more species contained in the same culture
may, however, be such that the one species, by the exercise of its vital functions,
renders the nutrient medium suitable for the growth of the second species. This
preparatory function of the one 'species may consist either in the absorption and
elimination of certain constituents of the medium which retard the development
of the other species, or in the excretion of certain products otherwise lacking in
the medium, and either indispensable or highly favourable to the other organism.
This kind of dependence was styled by GARRK (I.) Metabiosis, an excellent
example of which is afforded by the decomposition set up in natural wine-must.
If this be allowed to stand in an open vessel as soon as it comes from the press,
a decomposition characterised as alcoholic fermentation rapidly sets in. The
skin of the grape is the habitat of an abundant flora of fungi, which are intro-
duced into the must in the operation of pressing. Of these (exceptional instances
apart), the organism exciting alcoholic fermentation is the first to develop,
because the constitution of the must favours it the most, the result being that
the sugar therein contained is split up, and carbon dioxide and alcohol are pro-
duced. When this decomposition is effected, the character of the liquid has
become changed, and now a new species, exciting acetic fermentation, comes into
play. This organism was already present in the must, but could not make head-
way against the predominant yeast, because, in the first place, the alcohol,
without which it feeds but indifferently, was lacking. Secondly, even had this
substance been present, it could not have been utilised, because of the atmosphere

66

MIXED CULTURES 67

of carbon dioxide, immediately above the liquid, preventing the free access to the
latter of the copious supply of oxygen without which the oxidation of the alcohol
cannot proceed. Now, however, that both substances are present, the liquid
commences to undergo a second alteration, and turns sour, the acetic acid bac-
teria being now on the surface ; and this condition endures so long as there is
any alcohol left. When this is exhausted, a third group of organisms comes
to the front, thread fungi establish themselves in the strongly acid liquid and
consume the acetic acid, carbon dioxide and water being found. This accom-
plished, the once again altered nutrient medium is attacked by putrefactive
bacteria, which have been carried into the vessel along with the dust in the
atmosphere, but can only develop now that the alcohol and acid, which are
poisonous to them, are wanting. The liquid is seized upon by these Schizomycetes,
and, with their activity, the series of metabiotic phenomena which the wine-must
presents to our notice closes.

The mutual influence of two or more species may be of such a nature that it
is impossible for them to live together, the presence of the one species retarding
the development of the other. This set of conditions is termed antagonism, a
number of examples of which will be given in subsequent sections.

§ 65.-Mixed Cultures.

When a nutrient medium is inoculated with two or more species of symbiotic
organisms, we obtain a mixed culture. Such a culture may, under certain cir-
cumstances, yield fermentation products that cannot be obtained from any of
the component species cultivated singly, but owe their origin partly to the
coalescence of the normal products of the individual species, and partly to the
reciprocal stimulative action exerted by the associated organisms. A few highly
instructive examples of this are given below.

The first of these — which was discovered by NENCKI (I.) — is afforded by the
bacillus of symptomatic anthrax (Rauschbrand) and Micrococcvs acidi paralactici.
Fuller information concerning the individual behaviour of these two Schizomycetes
will be found in subsequent paragraphs, which we will here anticipate in respect
of the fact now coming under consideration, viz., that the first-named bacillus
yields, in nutrient solutions containing cane-sugar, the following fermentation
products : hydrogen, carbon dioxide, normal butyric acid, and inactive lactic
acid. On the other hand, Micrococcus acidi paralactici forms, almost exclusively,
optically active paralactic acid, and that, too, in a quantity almost identical with
the theoretical yield from the sugar eliminated. If, now, both these organisms
be cultivated together in the nutrient solution aforesaid, fermentation proceeds
much more rapidly, and the final products consist not only of the already men-
tioned substances (yielded by the organisms singly), but also of a large amount
of normal butyl-alcohol. This substance, therefore, owes its production in this
case to the co-operation of two species of bacteria, neither of which singly is
capable of such power.

Interesting as this fact, that new fermentation products can be formed by
the association of organisms, may be, the following one, which was first estab-
lished by BUKRI and STUTZER (I.), is so in a still greater degree. In this case
two organisms are concerned, neither of which is capable singly of liberating
nitrogen from nitrates ; but, when acting conjointly, they decompose the sam<
nutrient medium with violent disengagements of gas. The one organism is the,
Bacterium coli commune, already mentioned, and very abundant in human fseces
and that of domestic animals, whilst the second microbe was named by the
above-named naturalists Bacillus denitrificans I. A bouillon containing three
grams of sodium nitrate per litre, inoculated with both these organisms and

68 BACTERIA IN THEIR RELATION TO ONE ANOTHER

then maintained at 32° C., began to disengage gas in a short time, the nitric
acid in the nitrate being reduced to nitrogen so completely that, even at the end
of forty-eijiht hours, the extremely delicate test with di-phenylamine-sulphuric
acid gave only negative results.

Apart from the purely scientific interest excited by these facts, new vistas
are also opened up in a practical sense. Up to the present, investigators have
contented themselves with the examination of the transformation products
resulting from the pure cultivation of single species of bacteria. In future
researches, however, the question whether or not a species can be spurred on to
more extended activity by the collaboration of a second, so as to give rise to the
development of powers which without such stimulant, would remain unobserved
and unutilised, cannot be neglected.

This claim is not restricted to the domain of schizomycetic fermentation,
but applies also to the ferments of the JEumycetes class. The mode of action
exhibited by mixed cultures of different species of yeasts is of great importance
in brewery and distillery practice. In this connection we are already in posses-
sion of several studies by E. Ch. Hansen and others, the results of which will be
considered in a subsequent section.

CHAPTER IX.

CLASSIFICATION OF THE BACTERIA.

§ 66.-First Attempt by 0. F. Miiller.

IT has already been mentioned in the Introduction (§ 2) that Leeuwenhoek
observed bacteria as far back as the end of the seventeenth century. For a long
time, however, nothing more was done than merely to admire the appearance
presented by these organisms under the microscope ; and since many of them
were observed to exhibit brisk movements, they were considered as animals and
denominated animalcula.

The first to study these organisms from a scientific standpoint, and to
arrange and systematise the multitude of forms, some of which were already
known, while others were discovered and described by himself, was the Danish
investigator Otto Friedrich Miiller of Copenhagen. In his important work
"Animalcula infusoria fluviatilia et marina," published in 1786, all the small
animals unsuitable for inclusion in Linnseus's sixth class, Vermes, were classed by
him under the name of Infusoria (infusion animalculae), and he divided these
into two main groups : those provided with external organs and those devoid
of same. He also originated the generic names Vibrio, Nonas, and Proteus,
still in use.

The next worker to whom we are indebted for important conclusions respect-
ing the character and species of bacteria is Christian G. Ehrenberg. In his
work " Die Infusionstierchen als vollkommene Organismen " (The Infusoria as
Perfect Organisms), published in 1838, the generic names Bacterium, Spirochcete,
and /Spirillum first occur. He also classed all these organisms with the animal
kingdom, by reason of their (frequently very active) spontaneous motion.

It was left to the Breslau botanist FERDINAND COHN (V.) to ascertain, in
1853, that the organisms we now know as bacteria are of a vegetable nature.
This he established by proving the lack of animal organisation, and also from
the fact that these creatures increase by subdivision after the manner of the
algas, from which they differ, as he says, merely in one characteristic : the
absence of chlorophyll. Four years later NAGELI (Y.) bestowed on these
organisms the name of Schizomycetes, which they still retain.

§ 67.— Cohn's Classification.

The first point was to bring the confusion of forms into order. What
characteristic should be taken as a guide thereto? Were there several at
disposal on which one could rely? These questions COHN (I.) may well have
asked himself when, in 1872, he felt himself impelled to attempt a classification
of the bacteria, and finally thought his object attained by the following
system :

I. Sphcerobacteria, globule bacteria
Genus i : Micrococcus.

II. Microbacteria, short rod bacteria.
Genus 2 : Bacterium.
69

;o CLASSIFICATION OF THE BACTERIA

III. Desmobacteria, thread (long rod) bacteria.

Genus 3 : Bacillus.
Genus 4 : Vibrio.

IV. Spirobacteria, spiral bacteria.

Genus 5 : Spirillum.
Genus 6 : Spiroch&te.

The sarcina organisms have no place in this system, because Cohn did not
consider them as belonging to the fission fungi.

As may be seen, the basis of classification employed was the form of the cells,
i.e. their form of growth. However, since methods of pure culture were then
undiscovered, the diagnosis of the individual species was as yet impracticable,
and the question whether the form of the cells in each species is definite and
unchangeable was, in particular, still unsolved. The answer to this question is,
nevertheless, of vital importance to the Cohn system, and, if negative, causes it
to break down (as was subsequently the case). The weakness of the system was
recognised by Cohn himself, and he particularly stated that his classification was
only a provisional one. A number of over-zealous disciples, however, overlooked
this reservation, and, by degrees, expounded the system as meaning that each
separate species has a single well-defined and invariable cell form ; the one
species appearing only as short rods, the second only as cocci, and so on. This
constitutes the theory of constant form, also known as Monomorphism.

§ 68.— Billroth's Coceobacteria Septica.

The exaggeration resulting from the misapprehension of Cohn's attempt at
classification soon brought about a coi responding reaction. In proportion as
assiduous microscopic research revealed the certainty that bacteria do undergo
changes of form, so the hasty assumption of monomorphism of species had to be
given up. In 1852, PERTY (I.) had already observed a short-rod bacterium,
which, on account of its faculty of changing into the thread form, he named
Metallacter. Twenty-one years later LANKESTER (I.) studied a species of red-
coloured bacterium, named by him Bacterium rubescens, and observed that, under
varied conditions of cultivation, its cells underwent different modifications of
form — an observation which led him to deny that specific constancy of form
existed. He would thereby have anticipated subsequent decisions had the basis
on which he relied proved free from objection. This was, however, unfortunate!)1
not the case, and, indeed, such a condition was at that time unattainable owing
to the lack of irreproachable and reliable methods of cultivation, without which,
and the resulting pure cultures, the problem in question cannot be solved. A
culture intended for modification experiments may, when examined under the
microscope, present a perfectly uniform appearance, and nevertheless contain a
few unnoticed individuals of another species, which by their rapid increase when
transferred to a medium favourable for their development may lead to the erroneous
supposition that a second and modified form of growth has been produced. By
another re-inoculation a third species may be brought into prominence, and so
forth.

A very instructive example of the possibility of similar self -deception is
afforded by LISTER'S (I.) striking experiment. He allowed ordinary milk to
become sour spontaneously, and then introduced a drop of the liquid into boiled
milk, beet-extract, and into urine; from thence into Pasteur's nutrient solution ;
thence into urine again ; and finally back again into milk. Finding, then, that
from identical sowings differently shaped cells made their appearance in the
various media, he concluded that be had to do with so many changes of form of

DE BARY AND HUEPPE'S CLASSIFICATION 71

one and the same organism, which, on account of its origin, he named Bacterium
lactis,

It must not be understood that similar errors were confined to the island of
Britain ; on the contrary, they attained their culmination on the Continent in
the assumptions of HALLIER (I.) concerning the metamorphosis of one fungus
into another. It need, therefore, be small matter for surprise that the Austrian
surgeon Tn. BILLROTH (I.), in a comprehensive work published in 1874, not only
attributed all infectious diseases to the agency of a single species of bacterium,
susceptible of multiform modifications, but also considered all known bacteria
generally as vegetation forms of this one species, viz., Coccobacteria septica. This
observer was supported by the botanist NAGELI (VI.), in so far that the latter-
declared that no necessity existed for the division of the bacteria even into only
two specifically different forms. This opinion he still maintained in 1882, not-
withstanding the appearance in the interim of a work by Cohn containing a
number of fresh data calculated to complete and support the theory of difference
of species in bacteria. This treatise has already been mentioned in § 24, because
its author upheld the relationship of the fission fungi to fission alga3 and advocated
their collection into one group, Schizophytes. As at present, however, we are not
concerned with the relationship of the Schizomycetes to other organisms, but with
the separation of the former into genera, we must confine ourselves to remarking
that the new classification in the said treatise rested too exclusively on morpho-
logical characters to be of practical value,

§ 69.— De Bary and Hueppe's Classification.

Gradually an accumulation of facts arose which afforded a basis whereon a
new system of grouping the fission fungi was attempted. Differentiation based
on cell form only was still considered justifiable up to 1878, but could no longer
be maintained in the face

of incontrovertible observa- egr^Tr^.... -,^v^\-.^-^.-^^;'-'.^^:t ;•:' V^'^TVV*^^^^*^
tions made, in the course of /

the following years, with
absolutely pure cultures of
various species of bacteria,
and all leading to the same
conclusion, that mutability,
i.e. modification of form,
unquestionably does occur

in the fission fungi. This ©©QOo©©©©®(ii©Q©(a9®(a©®©©O3©©s©@®©©©0
knowledge is the result of 9-

various researches, amongst
which may be mentioned :
in 1879, that of E. CH. •>'

HANSEN (II.) on Bacterium FlG. 30._Bacte. ium merismopedioiW

aceti and B. Pasteurianum ; Found in the mud of the river Panke (Berlln)>

in I 882 those of W. T_ A tliread form breaking up into : -2. long rods ; 3. short rods ;
ZOPF (III.) On Bacterium 4. cocci; 5. a chain formed of rods of different length?.

merismopedioides (Fig. 30), <-After z°Pf^ MaS'n- 7°°-

and by H. BUCHNER (VI.)

on Bacillus subtilis ; in 1883 that of KURTH (I.) on Bacterium Zopfii (Fig. 31),

afterwards also examined by H. SCHEDTLER (I.) ; in 1885 that of G. HAUSER (I.)

on a few species of putrefactive bacteria of the genus Proteus ; and others. The

adherents of Koch at first unconditionally opposed the theory of the pleomorphism

of bacteria ; but, not being able to sustain this view in the face of the facts

72 CLASSIFICATION OF THE BACTERIA

brought to light, they then asserted pleomorphism to be peculiar to the non-
pathogenic bacteria. Even this restricted assumption has, however, given \vay,
since undoubted pleomorphism was proved in 1882 by ARCHANGELSKI (I.) and
ROLOFF (I.) for Bacillus anthracis, and by Friedlander for Pneumobacilhis (§ 33) ;
in 1883 by TH. EHLERS (I.) for the Rauschbrand bacillus (of symptomatic
anthrax); in 1889 by E. METSCHNIKOFF (II.) for his newly discovered pathogenic
Spirobacillus Cienkowskii (of Daphnia magna) ; and in 1892 by F. FISCHEL (I.)
for Bacillus tuberculosis. It may be remarked en passant that Metschnikoff
prefaced the report of his discoveries with a short review (well worthy of perusal)
of the development of the pleomorphism theory.

At the end of the "seventies" Cohn had established beyond doubt the ability
of certain fission fungi to produce endospores, and thereby obtained reliable

B

FIG. 31. — Bacterium Zopfii, Kurth.

Gradual chaiigcs iu the same thread observed under the microscope.

A, thread without apparent articulation ; B, breaking up into rods which liually form

cocci in C ; a-e are corresponding cells. (After Kurth.) Magn. 740.

means of differentiation. Very soon after, De Bary showed that several of
the species which do not form endospores protect themselves from injurious
influences in another way, viz., by the formation of arthrospores. Hence a
classification was devised in 1883 by VAN TIEGHEM (II.), which was further
developed by DE BARY (I.) and HUEPPE (II.), in 1886, and in which two main
groups were recognised, viz., the endospore and arthrospore forming bacteria.
The second group also comprises all the species in which the formation of repro-
duce! ve cells has not yet been observed. Fuller details of this system can be
seen in Hueppe's treatise, but the system need not be further developed here, as
it has not yet been generally accepted in scientific circles.

For fuller information regarding Van Tieghem's system, as well as for parti-
culars relative to the systems proposed by P. Miquel and by Woodhead in 1891,
which may be properly designated as " diagnostic tables," reference may be made
to WARD'S (III.) readily accessible and comprehensive treatise. The new system
published by W. MIGULA (II.) in 1896 may also be simply referred to.

In this connection there remains only one remark to be made, arid this
concerns the term Bacillus. This word has been hitherto employed by us to
designate only a well-defined form-phase of cell, viz., the cylindrical bacterial cell,
the length of which is at least double the breadth. Hueppe's system, however,
applies the generic name Bacillus only to such rods as have been proved capable

PATHOGENIC, CHROMOGENIC, ZYMOGENIC BACTERIA 73

of developing endospores. This definition has not yet been accepted by the
majority of bacteriologists ; hence it happens that newly discovered rod-shaped
species of fission fungi are still occasionally assigned to the genus Bacillus,
although the describer may have no knowledge whatever as to their capability
of forming endospores. The author has not considered it within his province to
change this nomenclature, and therefore this fact must be borne in mind in
perusing the present work. It should also be remembered that in the following
paragraphs the generic name Bacillus — Hueppe's definition notwithstanding —
means nothing more than that the species of bacterium so entitled exhibits,
preferentially and under normal conditions, the bacillus form of growth.

A comprehensive collection of the relative dimensions and forms charac-
teristic of growth in various nutrient media, &c., of about three hundred species
of fission fungi was prepared by EISENBEUG (I.), and may be advantageously
employed as an aid to determining whether any species under examination is
identical with any known species. A descriptive table of eighty-seven of the
bacteria of most frequent occurrence in drinking and utilisable water is given
by ADAMETZ (I.). Reference may also be made here to the very valuable book
of TIEMANN and GAHTNER (I.) in connection with the bacteriological analysis
of water. FRANKLAND and WARD (I.) give a comprehensive account of the
literature published up to the year 1882 on the bacteria occurring in natural
and mineral waters, and a comparative investigation into the distribution of a
number (twenty-eight) of well-known bacterial species in various well-waters has
been made by W. MIGULA (III.).

§ 70.— Pathogenic, Chromogenic, and Zymogenic Bacteria.

The attempts hitherto made to obtain a method of classification of bacteria
have always been restricted to the morphology of the organisms themselves. It
will now be well to remember that the attention of Applied Mycology is pre-
ferentially directed to the influence exerted by the fungi on their nutrient
media. The interest aroused by these organisms has always, from the outset,
had its practical side. Bearing this in mind, it will be readily conceivable that,
long before the establishment of Cohn's first classification, there had appeared in
the literature of the subject a division of bacterial species into three main
groups : pathogenic, chromogenic, and zymogenic bacteria.

It is quite unnecessaty to remark that this grouping is just as faulty as the
division of the Schizomycetes into cocci, bacilli, and thread bacteria. Neverthe-
less it was exceedingly convenient, as it was based on some well-marked primary
characteristics. If the fission fungus in question excited any form of disease in
men or animals, it was referred to the pathogenic group ; if it possessed the
faculty of producing colours, it was relegated to the category of chromogenic
bacteria ; and if it exhibited a capacity for effecting those chemical changes which
were comprised in the term "fermentation" (§ i), it was considered as zymo-
genic. A strict adherence to this method of partition is impracticable, because
there are some bacteria which, on account of their range of activity, would
have to be placed in two, or even all three of these classes. A large number of
examples could be adduced in support of this assertion; it will be sufficient
to cite merely a single one, viz., Staphylococcus pyogenes aureus, the cause of
osteomyelitis (bone caries), and therefore pathogenic. However, since it also, as
its name implies, produces a golden-yellow colouring matter, it is also chromo-
genic ; and, finally, from its power of setting up lactic fermentation in suitable
nutrient media, it is therefore also zymogenic.

From this example it will be evident that the domains of Pathological and
Technical Mycology cannot be rigidly kept separate. On the contrary, their

74 CLASSIFICATION OP THE BACTERIA

further coalescence will undoubtedly result — and that soon, we hope — in pro-
portion as fermentation physiologists acquire a greater insight into the chemical
changes effected by bacteria, and pathologists determine the precise action the
bacteria exert on the organs of animals and plants. A fine, but unfortunately
still very isolated, example of the successful combination of these two fields of
research is afforded by the labours of L. NENCKI (I.) on the bacterium which is
the cause both of " blown " cheeses and of inflammation of the udder in the cow.

The distinction between chromogenic and zymogenic bacteria can also be
further maintained, not because there is any essential reason for it, but because
there are certain species of Schizomycetes which are interesting to the technicist
solely because they produce colouring matters.

So far as the zymogenic bacteria, in the narrower sense of the term, are
concerned, i.e. those either cultivated, or dreaded, on account of the chemical
changes they produce, there is the same need for a well-established consistent
classification as in the two groups just noticed. The changes effected by
them are expressed in terms having reference to the predominant fermenta-
tion products ; hence it is we speak of the bacteria of lactic fermentation,
acetic fermentation, and so on. This purely practical method of classification
will be adopted in the description about to be given. Before passing thereto it
will, however, be necessary to consider the methods practised in the examination
of these organisms, this knowledge being essential for the study of the organisms
themselves. This will form the subject of the two following chapters.

SECTION III.
PRINCIPLES OF STERILISATION AND PURE CULTIVATION.

CHAPTER X.

METHODS OF STERILISATION.

§ 71.— Sterilising.

To sterilise an object, e.g. a nutrient solution, piece of apparatus, &c., means to
treat it in such a manner that it no longer contains any living germs, and is
therefore sterile.

The reader must not expect to find in the present work a detailed description
of even the most important of the methods of working adopted for this purpose.
Those who have an opportunity of studying the methods of Technical Mycology
in a laboratory devoted to Fermentation Physiology will learn all they need
much more speedily and intelligibly from oral instruction than from a printed
book. On the other hand, those who have access to the latter only will attain
their object by the exertion of a little diligence in consulting the books referred
to later on, and especially

Hueppe, Ferdinand : Die Methoden der Bakterien-Forschung, 5th edition, 1891,
Wiesbaden (C. W. Kreidel).

Lindner, Paul : Mikroskopische Betriebskontrolle in den Garungsgewerben,
1895, Berlin (P. Parey).

In the first-named compendium the reader will find a better description than
the present author could give of all the methods used in general Microbiology.
The second, very useful, work treats, with great experience, a narrower field,
wherein it will afford reliable guidance and help to the student on all matters
relating to fermentation technology. In the newest edition (1895) of the work
on water-analysis by TIEMANN-GARTNER (I.), already referred to (and which
should be in every efficient chemical laboratory), the reader will also find
descriptions of the most important manipulations and methods employed in
sterilisation, pure cultivation, re-inoculation, &c. In selecting apparatus for
installing a new laboratory for Fermentation Physiology work, the beginner
should seek the advice of an expert, and should compare the illustrated catalogues
of such firms as make the supply of these appliances a speciality, e.g. U. Desaga,
of Heidelberg ; Erhardt and Metzger, of Darmstadt, &c.

It is not our purpose now to give a detailed initiation into the work of a
fermentation physiologist's laboratory, but rather to describe, in bold outline,
only so much as is necessary to facilitate the object of the present work, viz., the
study of the character and modes of action of the organisms of fermentation.

§ 72.— Freeing the Air from Germs.

There are two chief methods by which liquid substances and gases can be
sterilised, viz., either by killing the germs present therein, or by removing them

75

76 METHODS OF STERILISATION

by passing the liquid or gas through a suitable filter. The sterilisation of air on
a large scale is effected exclusively by the latter method, the prototype of which
was constituted by the tubes, plugged with cotton-wool, first employed by
Schroder and Dusch. The air is therefore passed through a cotton-wool filter,
as it is termed, such a one being used, for example, to purify the air admitted
to the sterilised wort in an apparatus for the pure cultivation of yeast. It will
not be out of place to lay stress on the fact that such a filter will only work
efficiently provided it be thoroughly dry ; otherwise the Eumycetes spores en-
tangled therein will germinate and develop into long-thread cells, which will
penetrate right through the filter and quickly form new spores, so that the air
at the end of the filter nearest the wort is not only not freed from germs, but is
probably richer therein than before. Attention to the air filters must, conse-
quently, not be neglected. E. CH. HANSEN (III.) has reported on experiments
made by Poulsen concerning the time during which such filters continue, under
normal conditions of practical working, to pass the air in a germ-free state.

The cotton-wool plugs with which, since the time of Schroder and Dusch, it
is customary to close test-tubes, bottles, and flasks in which cultures of organisms
or stores of nutrient media are kept, are simply small cotton-wool filters. They
are especially brought into action when currents of air pass into the vessels as a
consequence of the partial vacua formed within them by a lowering of tempera-
ture, the germs in which are retained by the plugs. The efficiency of the filter
depends on its being kept dry. Its reliability is not, however, permanent, since,
though the fission fungi are always retained, this is not the case with the spores
of mould fungi, which are so abundantly met with in the air. These latter are
very troublesome, as they often produce much mischief even when the mycologist
has taken the greatest care. If the room in which the cultures are kept be free
from moisture, then the cultures dry up very rapidly, which, in order to pre-
serve their vitality, necessitates their being frequently re-inoculated into fresh
media — a tedious and unpleasant task. On the other hand, if the surrounding
air be too moist, then it not infrequently happens that the spores of the mould
fungi on the surface of the cotton-wool stopper germinate, and the resulting
cell threads penetrate to the other end of the plug, and there form spores, which,
falling into the culture, contaminate and spoil it.

Various remedies have been proposed to overcome this evil, one of them
being a previously sterilised indiarubber cap, which is drawn over the mouth of
the vessel (test-tube, &c.) after the outer end of the stopper has been burnt away.
This latter operation must always be performed when one begins a re- inoculation,
since the germs resting on the surface of the cotton plug are thereby annihilated,
and consequently prevented from falling into the culture when opened. Instead
of the rubber cap, one can be made out of a double layer of filter-paper tied on
with a string ; many cultures specially requiring air are covered with a cap of
this kind only, the cotton-wool plug being dispensed with.

It is not essential that the working layer of the filter should consist of
cotton- wool, various other stuffs being employed for special purposes. Thus, for
example, Pasteur, in carrying out his researches (referred to in § 7) on the
organised bodies present in the atmosphere, passed the air through gun-cotton.
This was then immersed in a mixture of ether and alcohol, which dissolved out
the nitro-cellulose and left the entrapped organisms behind, so that they could
be more closely examined as to their size, form, and structure. This was the
first microbiological analysis of air. Of the numerous methods since proposed
for the estimation of the number of germs in the air, that given by FHANKLAND
and PETRI (I.), which is a successful modification of the Pasteur prototype, is
the most suitable for the purposes of the technical mycologist. These observers
deprive a measured quantity of air of its germs by passage through a filter

THE FILTRATION OF DRINKING WATER 77

charged with sterilised glass powder or sterilised fine sand, the contents of the
filter being then intimately mixed with a gelatinised nutrient medium, and the
whole poured into flat glass basins. The separate germs then develop into multi-
cellular families (colonies). When counted their number — referred to unit
volume— gives the germ content of the air. The difficulty in the way of study-
ing the cultures, caused by the presence of the powdered glass and sand, can be
overcome by substituting a soluble filtering medium, such as coarsely powdered
crystals of sodium sulphate of about 0.5 mm. in diameter. This is specially
recommended by MIQUEL (III.), to whom (be it remarked en passant) we owe
the most comprehensive experiments on the percentage of germs in the air.
Regular reports of his researches appear in the Year- Book (published annually
since 1879) of the observatory established, under his direction, for studies of this
kind, in the southernmost district of Paris. Readers are hereby referred to this
"Annuaire de 1'Observatoire de Montsouris." The percentage of germs in the
atmosphere of breweries was more particularly investigated by E. CH. HANSEN
(II.) ; and PETRI (I.) has summarised all the methods of examination proposed
up to 1887.

The method, originally performed by Th. Schwann, of purifying air by
exposure to a red heat is at present used by fermentation physiologists in one
instance only, viz., when working with the so-called Pasteur flasks. When
liquid is poured out of the lateral tube — whether for the purpose of taking a
sample or for inoculating a similar flask with the contents — the air coming in in
its place is purified by holding the aperture or the first bend of the swan-neck
tube in the flame, i.e. heating it to redness.

§ 73.— The Filtration of Drinking- Water.

The methods of sterilising liquids are various, but are not all equally suitable
for any given case. For example, the employment of poisonous substances is
precluded when the liquid to be sterilised is intended for human consumption,
and the use of heat — which next suggests itself — is frequently inapplicable on
account of the expense entailed. Such, for instance, is the case with the drinking
water of towns deriving their supply from a river. Under these circumstances
a so-called sand-filter is employed, the true filtering layer of which is not the
strata of sand and gravel, but the mud which is gradually deposited thereon.
A fuller consideration of this subject, which belongs to the domain of Practical
Hygiene, may be passed over the more readily since it has been treated in
Tiemann-G'artner's work already alluded to. This may be referred to, as also a
very practical investigation performed by A. REINSCH (I.), bacteriological adviser
to the Altona Waterworks.

The filtrate obtained from such filters intended for use on a large scale is,
when the service is carefully regulated under bacteriological control, found to be
very low in germs, though not perfectly free therefrom. If it be desired to
attain such perfection — which is necessary in times of epidemic — other filters, of
greater powers of retention and correspondingly diminished delivery, must be
resorted to, and employed solely for the water intended for human consumption.
The prototype of these is the apparatus invented by TIEUEL (I.) in 7871, and
subsequently (1884) improved, especially by Chamberland. In the form devised
by the last-named, the effective constituent of the bacterium filter consists
of a candle-shaped hollow cylinder of hard-burnt, porous, unglazed porcelain
(" biscuit "), with an effluent aperture at one end, which, before use, is sterilised
by dry heat. This candle (bougie) is enclosed in a somewhat wider metallic
cylinder, the liquid to be freed from germs (the suspected potable water) being
forced into the intervening space, and, finding its way through the porous wall

78 METHODS OF STERILISATION

of the caudle, collects in the interior of the latter and escapes through the
aforesaid aperture at the bottom. Fine kieselguhr (diatomaceous earth) is
employed by NORDTMEYEK (I.) and Barkefeld for making the candle, and GARROS
(I.) uses asbestos of fine fibre. The reliable working of this filter is, however,
not illimitable, and that for two reasons: first of all, the pores become gradually
obstructed by the fine, slimy deposits separated from the liquid, which necessi-
tates the cleansing of the filter from time to time ; secondly, the bacteria grow
by degrees through the pores of the filter, a circumstance first observed by
Bourquelot and Galippe. In some cases the pores of the filtering candle are too
large; consequently, a germ-free filtrate is unobtainable. For testing the
efficiency of the filter the photo-bacteria can. according to BEYERINCK (IV.), be
employed with advantage. A comparison of the capacity and efficiency of the
Chamberland and Berkefeld systems was drawn up by (inter alia) DACHNJEWSKI
(I.), and the columns of the Centralblatl fiir Bakteriologie contain numerous
articles respecting the advantages and defects of the aforesaid apparatus.
Mention should be made of the filter constructed by Breyer, which, according
to an investigation made by WICHMANN (I.), acts satisfactorily. PLAGGE (I.)
instituted exhaustive experiments in respect of the efficiency of all the known
water-filters designed for use on the small scale.

§ 74. — The Bacterium Filter in the Service of Enzymology.

In many instances the filter affords the sole reliable means of sterilising a
given liquid ; as, for example, when a species of bacterium is to be tested with
regard to its capacity of producing enzymes. For this purpose it is necessary to
free the culture, containing any such chemically active substance, from germs,
since otherwise it would be impossible to determine whether the chemical
reaction obtained by means of the sample is effected by the enzyme itself, or
primarily by the vital energy of the bacterium. The sterilisation admittedly
necessary in such case cannot be effected by heat, since this agency would at the
same time destroy the readily decomposable enzyme. There remains, therefore,
but one way open to us, viz., removing the germs by filtration ; and of the above-
named apparatus (filters), therefore, there is likewise only one that is reliable
and suitable for use for the purpose in view, namely, that of Chamberland.
This is, however, unfortunately expensive, and consequently not accessible in
every laboratory. For this reason the pattern described by A, KOCH (II.),
which is both efficient and cheap, forms a welcome substitute.

Whichever of the two appliances be employed, it must never be forgotten that,
in its passage through the filter, the bacterial culture under examination is not
only deprived of germs, but may also, under certain circumstances, part with
some of its chemical constituents, so that the equation, Filtrate = bacterial
culture - bacteria, does not always hold good. The filtering cylinder, especially
when used for the first time, retains varying amounts of the individual consti-
tuents of the liquid passing through it, a fact that was first recorded by FLtrGGE
and SIROTININ (I.) in 1888, and more closely examined by ARLOING (I.) in 1892.
We will, in this place, merely refer to the oxidising influence of the air, observed
more particularly by Miquel in the separation of urase from cultures of uric-
bacteria. It is therefore advisable to perform such filtering operations in an
atmosphere of pure hydrogen.

§ 75.— The Beer Filters

used in the brewery must also be briefly considered here. The object of these
appliances is to render the beer bright, i.e. perfectly clear and transparent, when

DESTROYING GERMS BY DRY HEAT 79

drawn from the storage cask and sent out to the purchaser. Under normal
conditions this clarification is effected in sufficient degree in the storage cask, and
recourse should therefore be had to the filter only in such cases where, by reason
of defective treatment or other unfavourable circumstances, a turbid lager beer is to
be made similar to a beer of standard quality. Such was the practice in Bavaria
until a few years ago ; but since the great breweries in that country began to
cater for the export trade, they have had to conform to the tastes of their foreign
customers, who judge the quality of beer by the eye, and would, without having
tasted it, set it down as inferior if it were not perfectly bright. Therefore, in
order to render it acceptable to this large and continually increasing clientele, the
beer has to be passed through the filter. The South German connoisseurs in beer,
who judge their beverage by the flavour, raised objections, and with reason,
since nitration causes— apart from the exception aforesaid— an uncalled-for
depreciation of quality. This applies primarily to the chemical composition, the
filter removing from the beer sundry mucoid substances, extremely minute in
quantity and of as yet undetermined composition, but which, nevertheless,
contribute to the fineness of the flavour, so that an experienced palate can
distinguish with certainty between a filtered and unfiltered beer. This defect,
regretted though it be by connoisseurs, is, however, the lesser evil when compared
with the dangers, from a biological point of view, that are obviated by filtration.

Two main types of beer filters are in general use. The one constructed by
Enzinger consists chiefly of a number of chambers, the walls of which are com-
posed of perforated plates lined with thick filter-paper, specially prepared for
the purpose, and through which the beer is forced by compressed air acting on
the storage cask. The second type of filter, recommended for brewery work by
Stockheim, contains as its acting ingredient purified (and therefore tasteless)
cellulose of a felty nature. No objection can be raised against the use of such
appliances in exceptional cases, since by this means a clear filtrate is obtainable
when all other methods of clarification have failed to remedy turbidity. This
decision must, however, be amended when it is a question of beer already in
good condition, this latter often suffering, under such treatment, a considerable
alteration (in certain circumstances) with regard to its flora, apart from the
depreciation of flavour already alluded to. The filter removes the yeast
cells, but allows the (much smaller) bacteria to slip through, so that the latter
appear in almost their original numbers in the filtrate, where, moreover, they
have free play, owing to their previous competitors, the yeast cells, having been
got rid of. This unfavourable modification in the relative condition of the two
classes of organisms becomes especially objectionable when a filtering material
that has already been in use before is employed, without- having been sufficiently
purified in the interim. In this manner the filtrate can be actually enriched
with bacteria, as the author ascertained by experiments with the Enzinger filter
in 1894.

Respecting the wine filter in continually extending use in cellar management,
a full report can be perused in the handbook issued by BABO and MAOH (I.).

§ 76.— Destroying- Germs by Dry Heat.

Strictly speaking, the term " germ-free " should be applied only to such objects
as have actually been devoid of germs from the beginning or have been brought
into this condition by filtration. In the language of bacteriological practice,
however, it is also applied to objects wherein all the germs have been destroyed
and are only present in a defunct condition. Hence it would be more correct to
say that the object in question is " free from living germs," but this distinction,
being practically unimportant, is not generally drawn.

8o METHODS OF STERILISATION

For the destruction of germs a number of methods are available, and may be
classified into two principal groups : the one physical and the other chemical.
The former may be subdivided into germ-killing by warmth, electricity, light,
mechanical concussion, or, finally, by gas under high pressure. We will confine
ourselves to the first of these five methods, the employment of the remaining
four being, for the purposes of the mycologist in general and of the fermentation
physiologist in particular, either too costly or too cumbersome. In so far, how-
ever, as their influence is of general biological interest, we have already reviewed
them in the preceding section.

On the other hand, sterilisation by heat is the method always resorted to,
unless found undesirable on other grounds. Before giving it more detailed con-
sideration, we must first ascertain which group of organisms exhibits the greatest
tenacity of life and is able to longest withstand influences adverse thereto. This
group alone has to be borne in mind in testing the etficacy and general applica-
bility of a method of sterilisation ; since if the same is capable of destroying the
organisms exhibiting the greatest power of resistance, it will certainly, and much
more quickly, deprive all the remaining weaker ones of life. On the other hand,
when the contrary is not proved, it must always be assumed that the object to
be sterilised is infested with organisms of the highest resisting power.

These hardy organisms we are already acquainted with, namely, the bacterial
endospores, which in this respect have no equal, and can therefore be made to
serve as test objects for determining the reliability of any germ-destroying pro-
cess coming under examination. It has been already stated, in § 53, that great
differences exist in the resisting powers of the spores of the various species of
bacteria ; but of course we have only to take the strongest into consideration.
According to the investigations hitherto made, these are : among the non-patho-
genic varieties, those species commonly known as the hay — and potato — bacilli ;
and among the pathogenic bacteria, the anthrax bacilli. Bearing this in mind,
ROBERT KOCH (I.), the eminent medical bacteriologist, employed as reagent for
testing the efficacy of various disinfectants spores of anthrax bacilli, which, for
greater convenience in application, he allowed to dry on silk threads.

The articles of metal or glass to be sterilised are placed in a case, formed on
the plan of the drying-ovens used in chemical laboratories, wherein they are
heated to 150° C. for an hour. During this time no diminution of temperature
is permissible, because if such a fall occurs, the labour will have been bestowed in
vain. KOCH and WOLFFHETGEL (I.) have shown that there are bacterial spores
that are killed only after an exposure to air at 140° 0. for three hours. How-
ever, by an exposure to 150° for one hour we may be sure that all the germs
present have been killed ; and air-filters fitted with cotton-wool (freed from fat)
are also sterilised by the same treatment, the cotton-wool assuming thereby a
yellowish to brownish coloration. Both the apertures of such a filter must have
been previously closed with plugs of cotton-wool, which must not be removed
until the filter is about to be used. It is necessary that glass articles should be
dry before they are introduced into the hot-air sterilising apparatus, since other-
wise they will crack.

Small metal instruments, such as forceps and inoculating needles, as well as
the glass stoppers used for closing Pasteur flasks, can be conveniently purified
in the flame of a Bunsen burner or spirit-lamp.

§ 77.— Destroying- Germs by Moist Heat.

The opinion expressed in a former chapter, that the seat of the high powers
of resistance enjoyed by bacterial spores is to be sought in their membrane, is
supported by their behaviour towards the influence of warmth, in so far — as has

DESTROYING GERMS BY MOIST HEAT 81

been ascertained by numerous experiments — that, under otherwise identical
conditions, moist heat, e.g. in the form of steam, exerts a more violent action
and kills them much sooner than dry heat at the same temperature. This
behaviour is explained by the unusually low heat-conducting power of the
unaltered spore membrane. By the influence of moisture, however, the struc-
ture of this protective envelope is loosened and its permeability to heat rays
increased.

Although the use of moist heat may thus appear preferable to the method
described in the preceding paragraph, it is nevertheless inapplicable in many
special instances. For example, air-filters must be sterilised by dry heat
alone, but when liquids have to be freed from living germs by the aid of
heat, then moist heat must be decided upon. This can now be employed
in one of two ways : either by boiling the liquid over a naked flame, or by
exposing it to the influence of water vapour heated to a sufficiently high
temperature.

That every liquid can be sterilised by simple boiling at 100° C. was shown by
HUEPPE (III.) in 1882 ; the time of exposure necessary in order to secure the desired
result with certainty being, however, very long. In this connection we may recall
the experience of Brefeld, mentioned in § 53, according to which the killing of
the spores of the species of hay bacillus examined by him necessitated their
exposure for full three hours in boiling water. However, the nutrient solutions
destined for the cultivation of organisms, and requiring to be sterilised anterior
to use, must not be treated in this manner, since they would be concentrated
too much by such prolonged boiling. Such solutions are generally sterilised by
exposure to low-pressure steam, for which purpose the so-called " steam steriliser,"
proposed by Gatfky, R. Koch, and Loftier, and resembling in arrangement an
ordinary potato-steamer, is employed. It consists principally of a high cylin-
drical tin pot, covered over with asbestos board or felt, and fitted with two
bottoms, the upper one, which is perforated, serving as the support for the
vessels to be sterilised by exposure to the steam evolved by the boiling water
below. This process is known as sterilising by direct steam ; it obviates the
inconvenience arising from the evaporation of the nutrient media, and also
prevents local overheating. The samples are surrounded on all sides by steam,
which drives away the protecting envelope of air and raises the temperature
uniformly throughout to that of the boiling water. This is, of course, dependent
on the prevailing atmospheric pressure, and generally ranges between 96° and
1 00° 0. A reduction of the time of exposure is not to be thought of, since here,
as before, we have to do with a temperature of only about 100° ; this must be
particularly emphasised, since the Koch school at one time fell into error on this
point, by promulgating the maxim that " the spores of bacilli cannot withstand
the temperature of boiling water for more than a few minutes." We have
already recalled a fact controverting this, and will now cite a second example,
given by GLOBIG (II.), viz., that the endospores of a species of bacterium, dis-
covered by this observer on potatoes, originating therefore in cultivated soil, and
named the " potato bacillus," resisted the influence of a current of steam at
100° C. for as much as six hours. This is the most powerfully resistant of all
organisms hitherto observed.

Numerous modifications have been made in Koch's steriliser, in accordance
with the special purposes for which it is intended. Thus, for example, the
water-chamber has been separated from the steam-chamber, and the steam
introduced into the latter from above. This pattern is specially preferred in
the case of the large apparatus employed for disinfecting invalids' linen,
hospital bedding, and the like. Readers desirous of obtaining full information
on this point are referred to a treatise by DUNCKER (I.), who subjected a

82 METHODS OF STERILISATION

number of steam-disinfectors to a careful examination. The simple and
inexpensive form described above is sufficient for the purposes of the fermenta-
tion physiologist.

In the fermentation industries the method of destroying germs by steam is
highly prized on account of its convenience and efficacy. In breweries, for
instance, all the piping is steamed out, as also the wort cooler, and so on. The
Enzinger filter, however, cannot be treated in this way, owing to the softening
action of moist heat on the filter paper.

The duration of exposure requisite for the destruction of germs by moist heat
can be considerably shortened by employing supersaturated high-pressure steam.
If, for example, the steam be used at a temperature of 1 20° C. (corresponding
to an extra-pressure of one atmosphere), an exposure of twenty minutes suffices
for sterilising liquids up to 50 c.c. in volume with certainty. For larger
quantities a corresponding additional exposure at 120° C. (five to ten minutes)
is given. The use of supersaturated high-pressure steam is attended with much
smaller outlay, but requires a strongly built autoclave. Laboratories already
possessing such an apparatus — which is required, for example, in the determina-
tion of starch in cereals, &c. — can also employ it to advantage for sterilising.
In many instances, too, a method of this kind is advisable, not only on account
of the saving in fuel, but also by reason of the fact that the chemical com-
position and nutritive quality of the liquid to be sterilised are less impaired
by fifteen minutes' exposure to 120° than by three hours' exposure in the Koch
apparatus.

On the other hand, there are liquids which are so readily decomposed that
neither of the above methods of treatment can be thought of. An example of
these is afforded by the medium so frequently employed in mycological labora-
tories under the name of nutrient gelatin ; a solution of bouillon, wort, &c.,
containing 8-10 per cent, of gelatin. This mixture, which sets at the ordinary
temperature of a room to the consistency of soft glue, and liquefies at about
25° C., would lose its pi-operty of setting if exposed to such degrees of heat, and
would thereby become useless. In such cases another method of killing the
germs must be employed — namely, that first proposed by Tyndall, and known
as —

§ 78.— Intermittent Sterilisation.

The powerful methods hitherto described have been considered necessary, for
the sole reason that the sample to be sterilised had to be regarded as presumably
containing highly resistant bacterial spores. In the absence of such forms, the
object in view is attainable by much milder means, and the liquids, &c., to be
sterilised can be converted into this more favourable condition by causing the
spores (possibly present therein) to germinate. It then becomes a much easier
task to deal with the resulting vegetative forms, since these latter perish at
temperatures below 100° C., and therefore so much the more certainly in a
current of steam. For this reason then the sample to be sterilised — which, as
before, is supposed to contain the most highly resistant types of bacterial spores,
in addition to the comparatively feeble vegetative forms — is exposed at first to a
temperature of 100° C. in the Koch steriliser for a short time. The duration
of ' his first treatment depends on the volume of liquid in the individual samples.
For flasks containing a charge of 10-15 c c- each, fifteen minutes will suffice ;
larger quantities warm through more slowly, and must be left in the steamer for
a correspondingly longer time. In every case the liquid should remain at a
temperature of 100° 0. for about fifteen minutes. By this treatment only the
vegetative forms and weaker spores are killed, and the next step is to ensure
that the still living spores germinate, which is generally effected by simply

INTERMITTENT STERILISATION 83

leaving the samples to stand at room temperature. At the end of twenty-four
hours the first treatment in the steamer is repeated, whereby the vegetative
forms that have in the meantime developed from the spores are killed. It
being, however, possible that, owing to the known irregularity of germination,
some of the spores have not developed, the samples are again left at rest for a
day, and thereafter steamed a third time to kill the residual cells proceeding
from these tardy spores. The medium, liquid, &c., will in this manner be
entirely freed from living germs without having been subjected to injury from
over-prolonged or excessive heating. This method is known as the intermittent
process of sterilisation, and is the only one in use for the preparation of nutrient
meat -juice gelatin. The success of this method of killing germs depends on the
whole of the spores being caused to germinate. Now we know, from statements
already made, that there are certain species of bacteria which will only develop
under high temperatures, and for whose germination the temperature of the air
of a laboratory is therefore insufficiently high. On this account it will be
evident that, under certain circumstances, the samples will have to be left to
stand at high temperatures. Since, however, these temperatures will, on the
other hand, retard the development of the spores of such species as thrive only
at low temperatures, it is therefore impossible to neglect either consideration.
The samples must, consequently, be kept for a certain time at room temperature,
and for another interval at higher temperatures. In no case, however, must
this be relied on without further examination, but it must be laid down as a
fundamental rule of conduct, that any nutrient medium apparently rendered
sterile by fractional sterilisation, may only be considered as actually sterile, and
used as such, when it is found that after a short storage, following the above
treatment, no spontaneous development has taken place. This regulation,
urgently necessitated by reason of the insecurity of the sterilising process in
question, must not be neglected. Nevertheless, if the work is cleanly done, it
will seldom be found necessary to reject samples on account of insufficient
sterilisation, since the highly resistant spores, now in question, are generally
absent in the majority of the substances employed in the preparation of artificial
nutrient solutions, and only creep in when the manipulations are performed
without due care. Cultivated soil is rich in such organisms, so that if such soil
is, by any means, introduced into these media, an unsuccessful result may readily
ensue, as was, for instance, observed by L. HEIM (II.). The occurrence of such
spores in meat-extract is no rarity, and the remarks just made should therefore
be recalled when such material is employed.

It may happen that a nutrient medium, which cannot be exposed to a
temperature of 100° C. without decomposing, will have to be sterilised. An
instance of this is afforded by the solution employed in the study of uric
fermentation, which, in addition to the nutrient substances, contains also an
admixture of urea. This body, as is well known, is gradually converted at
1 00° 0. (in aqueous solutions) into ammonium carbonate. In preparing a
medium containing this amide the directions of LEUBE (I.) should be fol-
lowed, the solution of the other nutrient substances (e.g. a bouillon) being
first treated by itself in the steamer, and the urea sterilised separately by
heating it in the dry state at 106° C. for half an hour. By this treatment
it is maintained unaltered, and may, when cooled, be added to the cold sterile
bouillon.

Under particularly favourable circumstances, exposure to a temperature much
below 1 00° C. can bring about either the complete mortification of the germs
present in a solution, or else render them so debilitated that their further
development is prevented, so that the liquid will remain for a long time (months
and years) without alteration. This result is attainable when the influence of

84 METHODS OF STERILISATION

warmth is seconded by suitable antiseptics, substances which we must first
consider before noticing the combined process of sterilisation to which we are
gradually leading up.

§ 79.— Mineral Antiseptics.

The substances exerting a toxic action on micro-organisms are still often
divided into two groups : those serving to annihilate the pathogenic bacteria being
termed disinfectants; whilst the substances capable of retarding fermentation
and putrefaction are denominated antiseptics. There are, however, no good
grounds for this distinction, since, as we know, there are bacteria capable of
originating both disease and fermentation.

Very exhaustive researches on the efficacy of the vai-ious antiseptics are
available. Those of R. KOCH (I.) were undertaken in the interest of medical
hygiene. As in the case of other agencies inimical to bacteria, so is it in the case
of toxic substances : the destruction of the life of vegetative forms of growth is
relatively the easiest to effect ; stronger means being necessary to prevent the
germination of the endospores, and the most powerful influences of all to kill
these latter.

The strongest antiseptic is corrosive sublimate, or mercuric chloride, HgCl, ;
but, unfortunately, this substance cannot, for hygienic reasons, be employed in
the fermentation industiy. In the laboratory, however, the fermentation
physiologist always keeps a stock of this reagent for disinfecting (inter alia) the
bell glasses used for storing fresh plate cultures. A sufficient quantity is also
put in vessels containing cultures that are no longer needed, but which should
not be placed in the hands of the cleaner until they have been killed. Again, in
the laboratory of the chemist in large works a solution of sublimate should always
be kept, along with materials for bandages, as being the first remedy to apply
when the workmen are injured or wounded. In washing wounds with this
solution, one should always be mindful of the fact that the first treatment has a
preponderating influence in the restoration of health. The strength of solution
employed, both in the laboratory and for this Samaritan service, is one gram
of HgCl, per litre of distilled water. Calcareous well-water must not ^be used,
and the author would recommend any chemist who cannot afford to purchase
distilled water to prepare his stock of sublimate solution in rainy weather, using
pure rain water for that purpose. Like most of the salts of mercury, sublimate
forms insoluble compounds with albuminoids (e.g. in the blood), and has then no
longer any effect on bacteria. This reaction is prevented by adding 5 grams of
sodium chloride per litre of solution, since this salt forms with the mercuric
chloride a double salt soluble in water. According to the researches of R. Koch,
the spores of Bacillus anthracis perish in an hour when immersed in this solution.
For the prevention of their germination the presence of i part of sublimate in
300,000 parts of water suffices.

The earliest disinfectant employed was sulphurous acid, the use of which for
sulphuring wine casks has been handed down from remote ages. In this process,
so-called sulphur threads are ignited and placed in the cask, being prevented from
falling by the bung. These sulphur threads are strips of linen about the breadth
of the finger, steeped in melted sulphur. The germicide properties of gaseous
sulphurous acid (sulphur dioxide) were examined by G. WoLFFHtiGEL (I.) ; and
G. LINOSSIER (I.) endeavoured to express in figures the relation between the
percentage content of a solution of this dioxide and the length of exposure
necessary to kill various germs. His experiments were not conducted with
bacteria, but with Eumycetes ; they are, nevertheless, given in the following
Table :

MINERAL ANTISEPTICS

Species.

Fatal dose of SO2 in c.c. per litre for an
exposure lasting —

15 min.

6 hours.

24 hours.

5 days.

Beer yeast . . ...
Wine yeast . . . ...
Mycoderma rini ....
Aspergillus nirjer . . . .-

200
100
2CO

50

100
20
IOO
20

20

20
IOO
10

10

40

With regard to the deadening of wine-must by sulphurous acid, referred to
in § n, mention may be made of the discovery of this observer that 25 c.c. of
SO., per litre sufficed not only to hinder the inception of fermentation in wine-
must, but also to bring it to a standstill when already in progress. The presence
of a small quantity — by itself inert — of another mineral acid was found to
increase the power of the sulphurous acid in a remarkable degree. The subse-
quent fate of this latter in sulphured wine varies : a small portion combines with
the aldehydes, a little (often merely a trace) of which is always present, to form
aldehyde-sulphurous acid, a compound of agreeable odour, but the bulk is converted
into sulphuric acid, and is then found as potassium sulphate. Several experi-
ments in this connection have been conducted by E. CHUARD and M. JACCARD (I.).
Apart from the cases already mentioned, this antiseptic is not used in a gaseous
form in fermentation industries, since it attacks the metal fittings, irritates the
workmen's lungs, ifcc. It is, however, employed in combination with lime, as
calcium bisulphite, Ca0.2SO2.H.)0 = Ca(HSO3)2, with which the fermenting
tuns, tfcc., in the brewery are purified. On the basis of his experiments on this
point with beer yeasts and film yeasts, H. WILL (I.) recommends an aqueous
solution of this salt containing 10 grams of SO2 per litre. As the commercial
salt contains 70-75 grams of SO., per litre, one part by weight of this liquid must
therefore be diluted with six parts of water.

The suitability of Pictet's solution (liquide Pictet) — a mixture of (JO2 and
SO, (i : i) — for disinfecting purposes has been i-eported upon by DE RECHTER
and LEGROS (I.).

As a rule, the gsrmicidal power of carbonic acid (carbon dioxide) is over-
estimated by non-professional people. The researches of CARL FRAENKEL (I.),
confirmed by C. STEINMETZ (I.), have shown that this acid has no power at all on
certain bacteria, these latter thriving even in an atmosphere of the pure gas. Other
species are less able to stand it, and the remaining kinds, though retarded in their
development, are killed by it only with great difficulty. The most important
literature on the subject has been arranged by P. FRANKLAND and WARD (I.).
The above-mentioned fact suffices of itself to destroy the hope that carbonated
mineral waters are necessarily devoid of germs (as was assumed by Leone some
years back), the researches of P. SIEDLER (I.) having shewn that this is not the
case. The influence of this acid on the vital activity of yeast and the progress of
alcoholic fermentation will be dealt with in the second volume.

Chlorine, also, is not employed in the gaseous state, but as chloride of lime
(calcium hypochlorite). This substance was recommended by H. WILL (II.) for
the disinfection of the sacks — made wholly or in part of wool — used for filtering
off the "cooler sludge" in the brewery. As these bags are rendered unusable
by hot-water washing, their purification has to be effected by a cold process.
That cold washing does not produce the desired effect was proved by Will, who
found the sacks to be strongly infected with bacteria and wild yeasts, especially

86 METHODS OF STERILISATION

around the stitches, a circumstance sufficient to account for the bad repute in
which wort- and beer-droppings are held. Disinfection experiments have,
however, shown that these germs can be killed by exposure to the action —
assisted by careful brushing — of a chloride of lime solution containing i per cent,
of active chlorine. As good commercial chloride of lime yields 30-35 per cent,
of chlorine, the solution may be prepared for use by mixing 3-3 \ kilos. (6.6-7.7
Ibs.) of the chloride with i hectolitre (22 gallons) of water — i.e. about 5 oz. per
gallon — stirring the mixture up frequently, and, after settling, pouring off' the
clear liquid from the (useless) sediment. According to R. Koch, 0.2 percent,
chlorine water will kill the spores of B. anthracis within an hour. Exhaustive
experiments — conducted chiefly from a medico-hygienic point of view— on the
anti-bacterial properties of chlorine and bromine haye been carried out by
BERNHARD FISCHER and B. PROSKAUER (I.).

Among the inorganic acids, hydrofluoric acid and its alkali salts have proved
to be particularly poisonous to bacteria. In the last few years this substance
has, by the labours of Effront, been utilised in distilleries ; on this head more
detailed reports will be given in a later section.

Boric acid, either per se or in the form of borax, is occasionally — in despite
of prohibitory regulations — used for preserving food-stuff's (e.g. milk). A per-
missible and useful application of this substance may be made in the preparation
of starch paste by employing an aqueous solution of borax as a substitute for
water. Paste prepared in this way can be recommended, for instance, for
affixing the labels on wine bottles kept in store, the occurrence of the uncleanly
formation of mould, otherwise intervening, being thereby prevented.

The effect of ozone and hydrogen peroxide on bacteria is due to a common
cause, viz., the decomposing power of the oxygen liberated. Accoiding to the
determinations made by H. SONNTAG (I.), ozone has only a weak germicidal
power, but other experimenters, e.g. OBERDORFFER (I.) and WYSSOKOWIT.SCH (I.),
obtained somewhat more favourable results. According to the researches of
OHLMULLER (I.), this gas acts more powerfully when it is passed, along with
oxygen, through the culture. When the volume of the liquid amounted to
500 c.c. an ozone-content of 90 m.grms. of O3 per 100 c.c. of the gas was requisite
in order to kill the germs of the spores of anthrax bacillus present. According
to the researches of CHRISTMAS (L), the germicide power of ozone sinks to ntZ
when its amount falls below 0.05 per cent, by volume ; so that no effect can be
anticipated from the much lower proportion (i-io m.grms. per 100 litres) of
ozone present in the atmosphere. With regard to the purification of river water
— intended for drinking purposes — by the aid of ozone, prepared artificially on
a large scale, an exhaustive report has been drawn up by E. VAN ERMENGEM (I.).

Owing to the great expense entailed, the utilisation of the anti-bacterial
power of hydrogen peroxide in the service of the fermentation industry is as
yet impracticable. The invention of a less expensive method of production
would, however, ensure it an extensive sphere of operation, since this bacterium
poison offers the advantage that during its action it is resolved into water and
oxygen. When the latter has killed the organism, nothing is left of the anti-
septic but harmless water. Great advantage might be derived from this property
in connection with the manufacture of conserves ; but hitherto its value does
not seem to have been sufficiently appreciated. A few experiments have, how-
ever, been made with it in connection with the freeing of drinking water from
germs. In partial improvement on the results reported by Van .Tromp, it has
been proved by ALTEHOEFER (I.) and P. SCHILOW (I.) that an addition of i part
per mil of H2OZ to drinking water will, within twenty-four hours, be fatal to
the common (innocuous) water bacteria, the microbes usually present in conduit
waters, and the organisms which produce cholera and typhus. No alteration in

ORGANIC ANTISEPTICS 87

flavour results from this application ; and an injurious influence on health is
the less likely, since the peroxide is quickly decomposed. A reduction of the
dose below i per mil would naturally interfere with the efficiency of the reaction,
a circumstance which explains the unfavourable results obtained by other
experimenters, reported by A. SCHROHE (T.). A proposal, worthy of being
followed up, has been made by A. GOTTSTEIN (II.). A sample of water contain-
ing 1000 bacteria per i c.c. was found to evolve bubbles of gas at its upper edges
fifteen minutes after the addition of H2O2, the gas being oxygen liberated from
the peroxide by the activity of the microbes. Since the extent of this evolution
of gas fluctuates in accordance with the number of living bacteria present, this
behaviour might perhaps be utilised in arranging a simple method for controlling
the efficiency of water niters at frequent intervals. No appliances beyond a
stock of hydrogen peroxide and sterilised test-glasses would be required. Of
course, this crude method neither could nor should be used to replace the
examination of the efficiency of the filter by bacteriological tests, but is intended
for the sole purpose of enabling the engineer in charge to convince himself, every
quarter of an hour (or at other selected intervals), that the filtrate has fewer
bacteria than the unfiltered water. According to the critical researches of
HUGO LASER (I.), the Gottstein method is not sufficiently reliable.

Milk of lime is, when fresh, a fairly good disinfectant, but loses its disinfect-
ing property as soon as the calcium hydroxide becomes converted into carbonate,
the latter being innocuous towards many organisms, and even favourable to
others (especially the acid-forming microbes). In the absence of other disinfec-
tants this liquid may be successfully used. According to the researches of E.
PFUHL (I.), it is sufficient to add two volumes thereof, and leave them to react
for an hour, to ensure the death of the typhus bacilli and cholera bacteria in
liquid faecal matter. L. STEUBER (I.) has made several experiments as to the
influence of milk of lime on yeast-cells, and on its suitability for disinfecting
brickwork in the brewery.

§ 80.— Organic Antiseptics.

The antiseptic most appreciated — next to sublimate — in surgery, viz.,
carbolic acid (Phenol, C6H.OH), which is used as a 4 per cent, solution for
washing wounds, is never employed for industrial purposes. Nevertheless, it
merits brief mention here because the discoverer of its antiseptic action, viz.,
J. LEMAIRE (I. and II.), established the interesting fact that this constituent of
coal-tar, whilst capable of restricting the development of organised ferments,
leaves the efficiency of the enzymes unimpaired, a differential behaviour which
afforded support to Pasteur in his campaign against the Liebig theory of fer-
mentation. The toxic action of phenol on the individual species of the bacteria
varies, a circumstance which is utilised in the bacteriological analysis of water.
In order to determine if the water under examination for impurities contains
Bacterium coli commune, a small quantity is, in accordance with Pere's sugges-
tion, placed in bouillon containing one part of carbolic acid per mil. This will
retard the development of most of the water bacteria, but not that of B. coli
commune, which will therefore increase in the culture, and can then be more
readily detected by supplementary means (plate cultures). Crude carbolic acid
is soluble with difficulty in pure water, but readily so in sulphuric acid, com-
bining therewith to form sulpho-acids, an aqueous sohition of which, under
the name of aseptol, is employed in surgery. According to the researches of
R. Koch, the strength of aqueous carbolic acid solution requisite to prevent
the germination of the spores of B. anthracis is I part in 850. In a 5 per cent,
solution the death of these spores is caused only after more than forty days.

88 METHODS OF STERILISATION

The three succeeding higher homologues of phenol, viz., the cresols,
06H4.OH.CH3, are also used in surgery. The so-called kreolin or creolin is a
mixture of soap with a tar-oil, containing a small quantity of phenols (cresol, &c.)
and a large amount of hydrocarbons. As the last are insoluble in water, a
milky emulsion is produced by pouring creolin into that liquid. Lysoland sapo-
carbol are mixtures of soap and tar-oils containing more phenols and a smaller
proportion of hydrocarbons than the substance last described ; both these
mixtures will dissolve in water without producing turbidity. The solubility of
the cresols in water is slight : about i part per 100 aq., but can be increased
considerably (as ascertained by Hueppe) by the presence of other substances.
Thus, when sodium cresotate is used, solveol is obtained. An alkaline aqueous
solution of sodium-cresol will absorb a very large quantity of cresol, thereby
forming solutol. By adding to a 50-60 per cent, crude carbolic acid about 20
per cent, of its weight of mineral oil, a mixture known as saprol is obtained,
which is lighter than water and floats when applied to fsecal matter. The
suitability of this preparation for the continuous disinfection and deodorisation
of the contents of cesspools and closets was tested by SCHEURLEN (I. and II.).
Mention of the foregoing seven antiseptics is only made here for the purpose of
stating their composition as a matter of interest to the technical chemist. They
are, however, unimportant so far as fermentation industries are concerned. A
derivative of orthocresol, viz., salicylic acid, C6H4.OH.COOH,is still occasionally
used, e.g. for the preservation of jams, to arrest the formation of mould on wine, &c.
The time when H. Kolbe (who held the first patent for the manufacture of this sub-
stance on a large scale) strongly recommended its employment has long gone by.

On the other hand, another derivative of cresol, viz., potassium orthodinitro-
cresol, C6H2.(N02)2.CH3.OK, finds extensive employment, its explosibility being
entirely done away with by the use of a small addition of gljcerin, soap, &c.
The red pasty mass thus obtained is put on the market, as a patented prepara-
tion, by the Bayer Farbenfabrik under the name of Antinonnin, this name
being given to it on account of its having been first used on a large scale in
practice in 1892, for the destruction of the " nonnen " (Monacha) larvae infest-
ing the forests of Bavaria and Wiirttemberg. This paste dissolves in water in
proportions up to 5 per cent., forming a clear solution, dark yellow in colour and
of a soapy smell, possessing no corrosive action and attacking neither metals
nor fabrics, but penetrating deeply into wood and other porous substances, and
remaining fixed thereiil without volatilising or imparting any odour to the
material. Reports on the applicability of this antiseptic are unanimously in its
favour. TH. STETTNER (I.), for example, has drawn up an exhaustive account
of its usefulness in preserving wood employed for building purposes, and it
forms a reliable means for the annihilation of the dreaded dry rot in timber
(respecting which, it may be casually remarked, a comprehensive monograph has
been written by R. HARTIG (I.) ). To prevent the spreading of this fungus, all
the woodwork (and especially that forming the floor joists) is treated, by
dipping or brushing the ends to be imbedded in brickwork, with a ^ per cent,
(i : 200) solution of antinonnin. Dipping is also recommended for preserving
railway sleepers and wood blocks for paving. The latter are at present steeped
in creosotic tar, and render the streets malodorous in hot weather by the
vapours they evolve. Antinonnin will equally counteract putrescence without
inconveniencing the olfactory organs. Telegraph posts, fencing, hop-poles, and
vine-props are treated by setting the butt ends in a 0.5 to i per cent, aqueous
solution of antinonnin for a day, whereby they will acquire great powers of
resistance against rotting. The packing for spaces between ceilings, for which
purpose building waste is generally employed, and which is so often the
breeding-ground of pathogenic germs (particularly tetanus bacillus), should be

ORGANIC ANTISEPTICS 89

impregnated with this disinfectant. Antinonnin is also a very suitable material
to employ when it is .a question of keeping the brickwork of a building dry and
arresting corrosion, the cause of which latter phenomenon is probably bacterial.
The evil may be remedied by brushing the walls with a i per cent, solution of
antinonnin. If it be desired to prevent the inception of such corrosion — as will
be specially the case when a wall is to be decorated with fresco paintings — then
the mortar applied directly to the wall should be mixed with about 5 per cent,
of antinonnin. The walls of hospital wards, &c., maybe cheaply and reliably
disinfected by brushing them over with a saturated (5 per cent.) solution of this
agent. Full information concerning its successful employment in the brewery
has been given by AUBRY (I.), who recommends its use for purifying all utensils
not brought into direct contact with the beer. The walls of the fermenting and
storage cellars, which are frequently damp and form the habitat of mucinous
and malodorous fungi prejudicial to the beer, may be dried and freed from
mould by brushing them over with antinonnin solution.

Ethyl alcohol, in an undiluted condition, behaves as a fairly powerful poison
towards bacteria, and, according to R. Koch, will hinder the germination of the
spores of Bacillus anthracis, even when diluted with twelve times its own
volume of water. The use of this compound — of 90-96 per cent, strength — is
strongly recommended to the fermentation physiologist, since it possesses the
advantage over sublimate of rapidly attacking the spores of those mould-fungi
that coat themselves with an excretion of fatty matter, owing to which they are
able to resist the influence of aqueous antiseptics for a long time. It is advisable,
before performing inoculations in Pasteur flasks, to wash the flasks all over with
alcohol, more particularly the part of the lateral tube covered by the caoutchouc
tubing, and the mouth closed by the glass stopper. The surface of the table on
which the inoculation is effected should also be cleaned with alcohol of about
50 per cent, strength.

The disinfection of the hands is, as shown in particular by FURBRINGER (I.),
a very tedious labour when it has to be absolutely efficient. This, however, is
necessary only in the case of surgeons, and the fermentation physiologist may
rest contented with simply washing them with soap and water, and finally with
alcohol, before undertaking a delicate inoculation. The latter precaution should
in no wise be omitted before handling the ends of the caoutchouc tubing of
Pasteur flasks. The susceptibility of the different species of bacteria to alcohol
is various, a few of them being able to resist it very well when dilute ; and some
even utilise it as a source of energy, e.g. the acetic acid bacteria, which still
thrive freely in presence of 10 per cent, by volume of this alcohol.

Ethyl ether is also a very powerful antiseptic, and is recommended by
R. WOLLNY (I.) for use in sterilising by the cold process. For this purpose
the ether is added in the proportion of 10 per cent, to the liquid, and then, after
the germs have been killed, removed by the air-pump. The advantage of this
method over that of heat is that it has no effect on the albuminoids coagulable
by the temperature of boiling water.

Formaldehyde, also known as formol (formalin), will in the near future enjoy
extended employment as a powerful disinfectant. Many objects, such as clothing
dyed with delicate colours, furs, &c., must not be disinfected with liquid anti-
septics or by steam, gaseous germicides alone being suitable. Among these there
is but little range of choice ; chlorine and sulphur dioxide not only destroy the
germs, but also the materials to which the latter adhere ; and the only other
resource at our disposal is in formaldehyde. The antiseptic properties of this
substance were indicated by 0. Low (I.) and by BUCHNER and SEGALL (I.), and
have since been thoroughly investigated by TRILLAT (I.). Meat-broth containing
one-twelfth part of formaldehyde per mil was found to be perfectly free from

90 METHODS OF STERILISATION

germs at the end of several weeks. AROXSON (I.) found that typhus bacilli,
Staphylococcus pyogenes aureus, and B. anthracis could not develop in bouillon
containing one-twentieth part per mil of this aldehyde. According to the
researches of J. STAHL (I.) and of E. VAN ERMENGEM and SUGG (I.), the spores of
B. anthracis and those (very tenacious of life) from garden soil were killed by an
exposure of one hour to the influence of a i per mil solution of formaldehyde,
and a solution containing i part in 750 proved fatal to the germs in a quarter
of an hour. This disinfectant is therefore on a par with the strongest mineral
(bacterium) poison, corrosive sublimate, as regards efficiency, and surpasses it in
point of general applicability. Moreover, unlike the mercury salt, formaldehyde
is but slightly dangerous to man and the higher animals. The air may be im-
pregnated with sufficient of the vapour for the purpose of disinfection without
causing any greater inconvenience than coughing, which, however, soon dis-
appears, since one quickly gets acclimatised to this reagent. Formaldehyde is
generally met with in commerce as a 40 per cent, solution known as formalin.
TRILLAT (II.) gives a few methods for testing its strength and disinfecting
value. — A few pads of cotton-wool or kieselguhr, &c., are moistened with the
liquid formalin and transferred to a box or other receptacle, wherein the articles
to be disinfected (clothing) are suspended ; or the same are laid between linen
cloths moistened with the liquid. By this means K. B. LEHMANN (I.) thoroughly
disinfected a complete suit of men's clothing, even when infested with anthrax
bacilli, by the aid of 30 grams (a fraction over i oz.) of formalin in twenty-four
hours. For the preparation of formaldehyde on a small scale R. CAMBIER and
A. BROCHET (I.) recommend a burner, and B. TOLLENS (I.) a lamp, both fed
with methyl alcohol. In the latter apparatus a dome or cap of platinum gauze
(2 cm. high and i cm. wide) is placed over the slightly projecting lighted wick,
and as soon as the gauze is red hot the flame is extinguished, whereupon the
formation of formaldehyde goes on uninterruptedly. It should not be forgotten
that — as pointed out by A. BROCKET (I.) — this incomplete combustion of methyl
alcohol also produces some 3 to 5 per cent, of carbon monoxide. An apparatus
constructed by Krell, and resembling the Barthel soldering-lamp, has been
described by A. DIEUDONNE (II.), by means of which a constant current of form-
aldehyde vapour can be produced from methyl alcohol and blown into crannies
and corners that require disinfecting. The different degree of susceptibility
exhibited by the various bacteria towards this poison has been utilised by E.
SCHILD (I. and II.) for the differentiation of typhus bacilli from the very
similar Bacterium coli commune, which, in the bacteriological analysis of water,
is both very important and difficult. The latter species develops freely in a
bouillon containing i part of formaldehyde in 7000, whereas the former will not
do so. Therefore, if a species of fission fungus isolated from the sample of
water, and suspected to be typhus bacillus, produces turbidity in such a medium,
this behaviour shows that it is not the bacillus which causes typhus. The
applicability of this method — which gives a negative characterisation — has been
confirmed by RUD. ABEL (I.). The researches above noticed deal only with the
action of formaldehyde on bacteria, but for the fermentation industry it is also
important to know how the higher fungi, and especially the alcohol yeasts,
behave towards this disinfectant. In this connection it has been established by
W. WINDISCH (I.) that yeast cells show much less susceptibility ; consequently
this aldehyde is not a suitable means for killing them. Fortunately, however,
they are readily affected by the influence of hot water vapour, chloride of lime,
&c., so that there is no lack of available remedies.

The antiseptic power of iodoform, CI3H, was studied by BEHRING (I.), with
the result that this compound was found not to injure (kill) bacteria, except in
the rare cases when iodine was liberated. In all other instances (which thus

THE COMBINED METHOD OF STERILISATION 91

constitute the rule), its favourable action in the healing of wounds is based
exclusively on the counteraction of the poison produced by the pus-forming
bacteria, without, however, the appearance of the latter being prevented. The
use of chloroform for disinfection is only, as a rule, resorted to when it is desired
to sterilise milk for use as a culture medium, in which case it is necessary to
dispense with strong heat. This will be discussed in a subsequent chapter.

The organic acids have a fatal effect, even in small quantities, especially on
putrefactive bacteria. Frequent and regular use is made of this property in
technical processes of sterilisation, as also in distillery work ("souring the
mash "), as will be frequently noticed in the course of the present work. On
the other hand, fairly high degrees of concentration are required for killing such
bacteria as are themselves active producers of acid. Benzole acid, though pro-
hibited by law, is occasionally employed for increasing the keeping properties of
milk. This acid — even in very small quantities — has a very restrictive influence
on alcoholic fermentation, and it is to this influence that the difficulty of
exciting fermentation in the juice of the whortleberry (Vaccinium Vitis Idcea)
is to be ascribed, considerable quantities of this acid being present therein, MAOH
and POKTELE (I.) having found 0.64 to 0.86 grm. per litre.

§ 81.— The Combined Method of Sterilisation.

The influence exerted on micro-organisms by the substances already con-
sidered is subject to the same fundamental law as has been established for
physical force, viz., that the effect produced varies with the intensity of the
causative influence. A solution containing so large a proportion of antiseptic
that it is capable of killing a given microbe, will, when sufficiently diluted, have
a merely restrictive influence on development, without, however, proving fatal.
Proceeding farther in the same direction, a condition of dilution will be
attained which will exert a favourable effect, stimulating the vital activity of
the organism ; and finally, if the degree of dilution be extended beyond this
point, no effect will be observable. This fact was expressed by HUGO SCHULZ (I.)
in the following phrase: "Each impulse exerts on each cell an action whose
effect on the activity of the cell is in inverse proportion to the intensity of the
impulse." A series of researches, which confirm this law, have been made on
microbe poisons, but it will be sufficient to simply mention two examples, viz.,
that of CH. RICHET (I.), treating of the bacteria of lactic fermentation, and that
of BIERNACKI (I.), which deals with alcoholic fermentation.

This law forms the basis of the theory of toxic action originated by O. Low(IL)
in a book the perusal of which is commended to the reader, and more especially
for the complete critical digest it contains of the literature, relating to the action
of poisons, published anterior to 1893. According to Low, the ultimate cause
of toxicity is to be sought in the lability of the albuminoid matter of the cell
protoplasm. The activity of the latter consists in a continuous chemical change
of the atomic groups composing the molecule, the briskness of which alteration
is increased by slight stimuli. Larger quantities of the irritant (poison) exert
such a strong preponderating influence on the change, that the lability of the
plasmic albuminoid is arrested, and the life of the cell is consequently destroyed.
Probably, then, toxic action may be the means of throwing light upon the obscure
problem of the chemical dynamics of the cell ; just as, in many other branches
of natural philosophy, the study of disturbing influences has afforded the deepest
insight into the normal course of phenomena.

A knowledge of the nature of toxic action — the progress of which depends
more or less upon chemists obtaining a clear idea as to the constituents of the
albuminoids — is of the greatest importance, both to the study of organic life in

92 METHODS OF STERILISATION

general, and to that of the pathogenic and fermentative microbes in particular.
It is also important, as we shall soon see, for the technique of sterilisation.

The destruction of germs by heat in certain nutrient solutions and food-stuffs
is often a very difficult task, because it necessitates temperatures that damage the
sample both as regards nutritive value and palatability. Success may, however, be
attained by combining the influence of heat with that of poison, although the iso-
lated action of either is incapable of killing the germs. This is the leading idea on
which is based the process of mixed or combined sterilisation, wherein the death
of the micro-organism is caused by the simultaneous application of two factors ;
one of which (the poison) is without influence on the chemical composition of the
sample, whilst the other (heat) is too low to set up any injurious decomposition.

At first sight it may seem that the presence of poison restricts the application
of the process to such cases as the sterilisation, pure and simple, of a liquid, and
precludes its use when such liquid is intended for the cultivation of micro-
organisms or for human consumption. On more mature deliberation, however,
a contrary conviction will be formed.

Many of the substances named in the preceding paragraph are in themselves
innocuous to the health of man, provided the quantity present is not too large ;
this is particularly the case with alcohol and the organic acids, and it is precisely
these acids that are generally employed for the preservation of numerous food-
stuffs. A fuller account of this subject will be given in a future chapter, so
we will simply refer to it here and pass on to the consideration of the second
question : Is the combined method also suitable for sterilising nutrient media
intended for mycological work ?

Let us recall the observation that has been frequently made in previous
paragraphs with reference to the behaviour of micro-organisms under the
influence of physical and chemical forces. Just as a certain degree of heat is
fatal to one species, simply retards the development of a second, is favourable to
a third, and insufficient to allow the cells of a fourth species to grow at all — so
given amounts of poison may be fatal to one species of organism, inert towards
a second, and even stimulating to a third. In other words, the constants of
influence of a given poison vary with different organisms.

We are indebted to TH. SCHWANN (II.) for the first observations on the varia-
tions in behaviour thus exhibited, but to PASTEUR (I.) for the first practical appli-
cation thereof. Attention has already been directed to the susceptibility of the
putrefactive bacteria to the influence of acids, a property of which Pasteur availed
himself to protect his cultures of 1'erments (in the restricted sense of the term)
against injury on the part of such interlopers. For example, in order to study
acetic fermentation, he first acidified the artificial medium with acetic acid. By
means of a skilful combination of various anti-bacterial forces, properly adapted to
each particular case, a given nutrient medium can be freed from germs without
diminishing its suitability for the culture in view. One factor of this combined
method of sterilisation is usually heat. Many examples of this will be given in
the course of subsequent paragraphs, so that we will now simply refer to that
afforded by the boiling of beer-wort.

At the moment when the still unhopped wort runs from the mash-tun into
the copper, it contains innumerable bacteria, chiefly derived from the malt. Not
only do these survive the mashing process uninjured, but their increase is such
that 0.07-0.12 per cent, of lactic acid is produced. The acidity of the wort is
somewhat further increased by the addition of the hops placed in the copper
before boiling is commenced. But, as a consequence of the conjoint influence
of the boiling temperature (ioo.5°-io3° C.), the lactic acid and the hops,
the germs in the wort are — as found by G. H. MORRIS (I.) — at the end of
fifteen minutes' boiling, partly killed and partly so far weakened that they are

THE COMBINED METHOD OF STERILISATION 93

incapable of further development; the wort is therefore practically sterile.
Sometimes — but, as E. CH. HANSEN (HI.) has shown, not always — the complete
destruction of all the germs (absolute sterility) is attained in this way. How-
ever, the residual living germs in the wort do not develop therein, though they
will do so if transferred to a more favourable medium — e.g. meat-broth. In this
case we have to do with relative sterility. The rapidity of the effect is chiefly
attributable to the influence of the hops, which, in turn, owe their germicidal
powers to the possession of certain resinous bodies, generally known under the
collective name of hop-resins. The chemical properties and biological effects of
these bodies have been investigated by M. HAYDUCK (I.), who found three different
resins in hops, all of which are soluble in alcohol, ether, and chloroform. One of
these, viz., the brittle, tasteless y-resin, insoluble in petroleum spirit, does not
interest us in the present instance, the germicidal properties of the hop not
being due to its influence, but to that of the two (extremely bitter) soft resins
the a-resin and /3-resin. These two act powerfully on the lactic acid- and
butyric acid bacteria, but are innocuous towards acetic acid bacteria, sarcina,
and higher fungi (especially yeast). The latter organisms are, however, subject
to the influence of the boiling temperature, so that the wort is delivered in a
sterile condition to the cooler, where it is infected anew. The attempts of all
discerning brewing technicists to abolish the cooler and to effect the rapid cool-
ing of the wort (as well as its aeration by the injection of germ-free air) in
closed vessels fitted with refrigerating appliances, are thus easily accounted for.
This method of procedure, which, from the fermentation physiologist's point of
view, is the only correct one, is, however, beset with a difficulty as regards the
separation of the sedimentary matter. Therefore the hot wort from the copper is
generally allowed to stand until the sediment has subsided, the still hot goods being
then carefully drawn off and conveyed to suitable cooling and aerating apparatus.
For a description of the latter, reference must be made to Handbooks on Brew-
ing, three of which are recommended : that of THAUSING (I.) studies the wants
of the practical brewer; whilst MORITZ and MORRIS'S (I.) work is intended for
the brewing chemist familiar with chemistry and microbiology, to whom it
presents a large amount of lucid information. These two books being supple-
mentary one to the other, the student will do well to leave neither unread.
Finally, the third work, C. J. LINTNER'S (I.) "Handbuch der landwirtschaft-
lichen Gewerbe," is adapted for imparting instruction in High Schools.

The sterilisation of wort in Pasteur flasks — the medium most frequently
employed in the fermentation physiologist's laboratory — will be briefly described
as an addendum to the preceding remarks. In order to produce a clear liquid, poor
in precipitated albuminoids, &c., the Pasteur flask is half filled with wort (not from
the hop copper, but from the cooler), which will now contain numerous germs,
several hundreds to thousands per c.c. The flask is then placed on a heated sand-
bath and the steam evolved is allowed to escape for ten minutes — counting from the
moment boiling begins — through the short caoutchouc tube on the lateral tube
of the flask, whereupon the former is closed by a glass stopper previously purified
in the flame. Then, for a further ten minutes, the steam is allowed to escape
through the swan-neck, and the flask is left to cool, being for that purpose placed
on a hollowed cork or a ring of millboard one inch in height. When the liquid has
again sunk to the temperature of the room, the moisture condensed in the swan-
neck is driven off by means of the gas-flame, and the neck is closed by a small plug
of asbestos, which subsequently serves as a germ filter. Any organisms capable of
passing through this are deposited in the first bend of the tube, which is then
freed therefrom, by heating it to redness in the flame, before proceeding to inocu-
lation. Concerning the sterilisation of the large copper apparatus for pure yeast
culture, detailed instructions have been given by E. CH. HANSEN (III.).

CHAPTER XL

METHODS OF PURE CULTURE.

§ 82.— Nutrient Solutions.

IN § 15 of the Introduction it was stated that Liebig's theory regarded the
disintegration of the albuminoids as the true active agency in fermentation.
PASTEUR (VII.), the active opponent of this theory, interested himself in the
preparation of artificial media which, though free from albuminoids, began to
ferment when inoculated with a minute quantity of fermentative organisms
(e.g. a trace of yeast). The oldest of these, generally known as Pasteur's fluid,
consists of —

Grams.

Water 100.0

Ammonium tartrate i.o

Cane-sugar 10.0

Yeast-ash (corresponding to one grin, yeast) . . . 0.075

and was intended preferably for the cultivation of the higher fungi (yeast in
particular). Its suitability for bacterial cultures was examined by Colin, who
found that for this purpose the sugar could be dispensed with. On the basis of
researches into the requirements of yeast as regards mineral matters, ADOLF
MAYER (I.) proposed to employ, in place of the yeast-ash, which is soluble only
with difficulty, an artificially prepared solution of the salts of which this ash is
known from experience to consist. Utilising this report, COHN (I.) prepared a
nutrient solution which he named " normal bacterial liquid," and which was
composed of —

Grams.
Water joo.o

Potassium acid phosphate (KH2P04)
Tribasic calcium phosphate (Ca3(P04)2)
Crystallised magnesium sulphate .

Ammonium tartrate

0-5

0.05

0.5

i.o

NAGELI (IV.), relying on the results of his researches (indicated in chapter ii.)
on bodies suitable for the nutrition of the lower fungi, prepared three " normal
liquids for fission fungi," one of them having the subjoined constitution :

Grams.

Water 100.0

Di-potassium phosphate (K2HP04) . . . . . o. i

Crystallised magnesium sulphate 0.02

Calcium chloride o.ot

Ammonium tartrate i.oo

The nutrient solutions hitherto described play a great part in earlier myco-
logical literature, on which account their constitution is now given, though at
present they are but seldom used.

On the other hand, a second nutrient solution given by Pasteur, viz., yeast-
water, is still frequently used. To prepare this solution, about 100 grms. of
thick brewer's barm (or 75 grms. of pressed yeast) are placed in a tin can with

94

NUTRIENT SOLUTIONS 95

one litre of water over the fire, and boiled for a quarter of an hour, and are then
passed through a folded filter. If the liquid passing through is turbid it is
returned to the filter, and in this way a clear, pale yellow filtrate is obtained,
which is made up to one litre by the addition of distilled water, and is then
sterilised (either in bulk or in portions) by exposure to 100° C. in a steamer on
three consecutive days, or by a single operation of twenty minutes at 120° C.
under pressure. By a preliminary addition of 5 to 10 per cent, of sugar a very
useful nutrient medium for yeast is obtained. When acidified with acetic acid
and qualified with alcohol, yeast- water rendered good service in Pasteur's studies
in acetic fermentation.

For the cultivation of beer-yeasts the most suitable medium is hopped beer-
wort, sterilised in the Pasteur flask as already described. The hop-resin in this
liquid exerts a toxic action on many organisms, and among them the lactic acid
bacteria, which play an important part in distillery work ; so that hopped wort
must not be employed to cultivate these organisms in the laboratory, unhopped
wort being advisable for this and sundry similar purposes. Unhopped wort is
an advantageous medium for numerous fermentative organisms, and therefore
requires special care in sterilising.

Wine-must serves for the artificial multiplication of wine-yeasts and fruit-
yeasts, and a concentrated form of it is kept in stock in the laboratory. On this
point fuller particulars will be found in chapter xx.

Saprogenic and most pathogenic bacteria thrive particularly well in meat-
juice. This is used in the form of so-called bouillon, and, following the lines
indicated by the researches of PETRI and MAASSEN (I.), is prepared as follows :
Half a kilogram (i.i Ib.) of finely minced beef, free from fat, is placed in a tin
pan or earthen crock along with one litre (if pints) of welUwater, and, after
standing for an hour at the ordinary temperature, is heated to about 60° C.
during three hours, with frequent stirrings. At the conclusion of this period
of extraction the mixture is boiled for half an hour and then filtered through a
folded filter. The pale yellow effluent liquid is made up to one litre when cold,
and exhibits an amphoteric reaction. Its primary salts of orthophosphoric acid
(e.g. KH2P04) redden blue litmus paper, whilst on the other hand the secondary
phosphates (e.g. K^HP04) also present behave in the contrary manner. In
presence of phenolphthalein, however, only the tertiary phosphate (K3PO4) acts
as a base, and consequently meat extract behaves as an acid towards both blue
litmus and phenolphthalein. As a general rule, 10 c.c. of this broth require
an addition of 1.8 c.c. of deci-normal alkali to prevent the colour change from
taking place with blue litmus, and an addition of 3 c.c. to enable it just to
redden phenolphthalein. This acid reaction of meat-broth being a hindrance to
the development of many bacteria, it is on that account rendered very slightly
alkaline, the resulting liquid containing a smaller or larger percentage of alkali
according to the indicator used, and which should be selected in accordance with
the requirements exacted of the medium in each case. After neutralisation,
i per cent, of dry peptone and a ^ per cent, of common salt are added to the
liquid, which is then boiled again for a quarter of an hour (but not longer),
and filtered hot ; the resulting liquid, generally known as nutrient bouillon, is
filled into small bottles (e.g. 5-10 c.c.) and sterilised by either a thrice-repeated
treatment in the steamer or once under pressure.

When, under particular circumstances, suitable meat cannot be obtained,
meat extract is used instead. Hueppe's formula for making meat-extract
bouillon is : 30 grms. dry peptone, 5 grms. grape-sugar, and 5 grms. meat
extract, dissolved in i litre of water, and boiled, filtered, and neutralised as
previously described. The sterilisation of the (once more boiled and filtered)
bouillon must be performed with scrupulous care, the meat extract being rich

96 METHODS OF PURE CULTURE

in bacterial spores which are very tenacious of life. If this or the previously
described bouillon refuses to filter clear, the white of an egg, previously beaten
to a froth, is added, and the whole warmed up, boiled, and filtered, whereupon
the liquid will run through bright.

The power of thriving in a solution of salts devoid of albuminoid matters
was first observed by DUJARDIN (I.) in 1841, in the case of a fission fungus
allied to Bacterium termo, and was afterwards decisively proved, as regards the
zymogenic fungi, by Pasteur. In 1893 USCHINSKY (I.) demonstrated that the
majority of pathogenic bacteria (of typhus, cholera, diphtheria, tetanus, swine-
erysipelas, &c.) could also be cultivated in a liquid containing ammonium lactate
and sodium asparaginate as its sole supplies of nitrogenous nutriment. Cultures
in such media are specially suitable for the study of the poisonous substances
(toxins) excreted by these originators of disease, the separation of the former
being easy on account of the absence of albuminoids. The fact that these
toxins (which are probably allied to the albumoses and peptones) can also be
elaborated in non-albuminous media proves that they are not derivatives of
albumen, but are the result of synthetical processes occasioned by the vital
activity of the organisms.

This matter has been investigated by FERMI and SCHWEINITZ (I.), PROSKAUER
and BECK (I.), C. FRAENKEL (II.), and others. Since bacteria rapidly increase
in such a solution, they are therefore also endowed with the faculty of effecting
the synthesis of albumen. Comparative researches instituted by E. CRAMER (II.)
with cholera vibrio showed, however, that the percentage content of albumen
(calculated to dry substance) in the cells cultivated in Uschinsky's solution is
lower than in the case of cultures grown in media containing albumen.

In the sixth and seventh decades of the present (nineteenth) century the
preparation of a medium suitable as a universal nutrient medium for all possible
bacteria formed the object of the repeated exertions of many bacteriologists.
Such an attempt is now regarded as hopeless on account of the knowledge which
has been gained of the very opposite conditions governing the vitality of the
several species.

§ 83.— The Dilution Method and Fractional Cultivation.

It has already been remarked in chapter viii. that it is quite the exception
for a natural bacterial growth to consist of merely a single species, but that, as
a rule, we have to deal with a mixture of several. To separate these from one
another, and to further multiply each species by itself, so as to obtain therefrom
a pure culture, forms the aim of the methods of pure cultivation.

We start with the assumption that we have to deal with a number of
different bacteria inhabiting a liquid, inasmuch as there is a second condition
possible, i.e. when the organisms are distributed within a solid body (such as
cheese, butter, soil, &c.). In the latter case a finely divided suffusion of the
sample must be made with sterilised water and treated in the same manner as
liquid bacterial samples.

Very often the mycologist is set the task of determining the germ content,
i.e. ascertaining how many individual cells are contained per unit of space in a
sample. This contingency is often met with in fermentation experiments with
yeasts, in order that, from the result of the counting, the extent of the cell
multiplication occurring during the fermentation maybe ascertained. For such
purposes a so-called counting chamber, such as supplied, e.g., by Carl Zeiss of
Jena, is used. The arrangement of this appliance is shown in Fig. 32, in plan
at A and in vertical section at B. On a thick glass slide there is mounted a
cover -glass (a) with a circular hollow, within which is cemented a second glass

THE DILUTION METHOD 9;

disc (c) o.i mm. thinner. On the upper side of this latter are etched two
systems (crossing each other at right angles) of twenty-one parallel lines at
regular intervals of 0.05 mm., and therefore enclosing compartments each of
which has an area of 0.0025 S<1- mm-

If now a sufficiently large droplet of the sample to be counted be laid on the
centre of c and covered with a cover-glass (6) about 0.5 mm. thick, then various
portions of the liquid, which has a uniform thickness of o.i mm., can be
examined under the microscope for the number of germs present therein.

A number (ten to fifty) of the square divisions are counted and the mean
of the resulting figures is taken. This being denoted as M, the germ content

of the liquid will then be o 00025 = 4°°° ^ Ver cubic millimetre.

In order to arrest the movement of motile forms or prevent the multiplica-
tion of rapid-growing cells (e.g. yeast), a portion of the sample, well shaken up,
is previously mixed with an equal volume of 10 per cent, sulphuric acid, which

r \ i J

IIIK'

S

A

FIG. 32.— Counting Chamber. Nat. size. Description in text.

will kill the organisms. This dilution must be taken into account in calculating
the germ content from the n umbel's found in the counting ; so that M must be
multipled by 8000 instead of 4000.

Assuming the germ content to have been ascertained in the way described
above in the bacterial mixture, the different species in which are being isolated ;
then, the number of cells present per unit of space being known, a portion of
the sample — but not that diluted with acid — must be thinned down with
sterilised water to such an extent that only one cell is present in two to five
drops. One drop of this diluted liquid is then placed in each of a series of flasks
containing a sterile nutrient medium, which flasks are subsequently "kept at a
suitable temperature, whereupon some of them will, after a while, exhibit
signs of development ; these will constitute the wished-for pure cultures. They
are, however, not unconditionally reliable, since it not infrequently afterwards
becomes evident that, in despite of calculation, some of the flasks contained more
than one germ. By this method, generally known as the dilution method,
Lister in 1878 prepared a pure culture of Bacterium lactis, which was (chrono-
logically) the first bacterial pure culture, and FRITZ (VII.) also employed the
same method in his studies on fermentation by fission fungi. The first six species
of the Saccharomycetes studied by E. Ch. Hansen, and which stand out so
prominently in the literature of fermentation physiology, were also isolated by

98 METHODS OF PURE CULTURE

the aid of an improved form of the dilution method, further mention of which
will be made in the second volume.

The so-called fractional method of culture, as it was afterwards styled by
Klebs, was employed in particular in Pasteur's experiments on fermentation.
It consists in taking from a sample of fermenting liquid that has attained its
maximum of development a small aliquot portion and transferring this to a new,
sterile medium. By recalling the remarks made in the paragraphs of Section
II. dealing with symbiosis, it will be understood that, at the period of highest
fermentation in a natural liquid — and therefore one rich in different species —
that species which is the cause of the fermentation in question will preponderate.
Therefore, if merely a single droplet thereof be placed in a medium analogous in
composition to the original habitat, this species will be favourably situated from
the outset, and will increase at a relatively quicker rate than its associates. By
repeating this transference (" re-inoculation ") several times over, cultures will
finally be obtained wherein impurities, i.e. extraneous species, can only be
detected by more searching methods of separation, such as are described in the
next paragraph.

These subjugated species will, however, come to the front again if the
(apparently pure) bacterial culture be inoculated in a different medium forming
a favourable environment for their development. Mention of this has already
been made in a previous section, when referring to the older evolutionary
labours of Lister, Lankester, Hallier, Billroth, and others. We are now in
possession of another more convenient method for the purposes of pure cultiva-
tion, which will be described in the succeeding paragraph, and consequently a
criticism of the dilution method can be omitted.

At present, only a couple of words will be devoted, as supplementary to the
remarks already made, to the examination of brewery water for the presence of
dangerous organisms. For the brewer those water bacteria alone are important
that develop in wort and beer and are capable of producing injurious changes
therein. Consequently, sterilised samples of both these liquids are employed
in the biological analysis of brewing water. The method employed was first
proposed by E. CH. HANSEN (IV.), according to whom fifty small Freudenreich
flasks are used, twenty-five of them being charged with 15 c.c. of sterilised wort
apiece and the remainder with a similar quantity of sterilised beer. In each of
the first fifteen flasks in both series is placed one drop (0.04-0.05 c.c.), and in
each of the remaining flasks 0.25 c.c., of the water to be tested. These fifty
flasks are then kept at a temperature of 24°-2^° C. for fourteen days, and are
examined to ascertain how many become turbid or throw up a skin, i.e. exhibit
signs of the development of organisms. The ratio of the number of flasks with
turbid contents to the total number is referred to i c.c. of water, and a standard
for determining the destructive capacity of the sample in question is thus
obtained. Assuming that, for instance, three out of the fifteen wort-flasks
(inoculated with o 04 c.c.) exhibit turbidity, then three growths have proceeded
from 15 x 0.04 c.c., or five from i c.c. of the water. Or, on the other hand,
suppose that of the beer-flasks only one has become turbid, and that this is one
inoculated with 0.25 c.c. In this case, then, there is but one growth per
10 x 0.25 = 2 5 c.c.

H. WICHMANN (II.) attempted to add, as a co-factor influencing the con-
clusion arrived at, the length of time required for the turbidity to develop ; of
this fuller particulars will be found in the reference just given. Hansen showed
that a largo number of species of water-bacteria are incapable of developing
in the two solutions last named, and this is particularly the case with beer, the
flasks charged therewith seldom becoming turbid after inoculation with water.
J. CH. HOLM (I.) has, for several consecutive years, regularly examined the well-

LIQUEFIABLE SOLID MEDIA 99

water and main-water of the breweries at Alt-Carlsberg, near Copenhagen, by
this method, and found that the spores of mould-fungi are comparatively the
most frequent, cells of bacteria capable of thriving in wort and beer being less
general, and yeast cells very rare. If the water be used merely for malting and
mashing purposes, its germs are unimportant, being, as we have already seen,
unable to withstand boiling in the hop-copper. There is, however, one un-
avoidable opportunity afforded for the contact of the beer with the water in its
unchanged condition, and that is in the washing out of the storage casks and of
the trade casks in which the beer is sent out to customers. In Germany these
casks, being lined with pitch, will not stand cleaning with hot water or steam,
and are cleaned with cold water, a small quantity of which is always left behind
in the casks ; so that, if this water be rich in organisms injurious to beer, serious
inconveniences may arise. Dr. Will has reported to the author an instance
coming under his knowledge where the beer from a brewery was constantly so
turbid that no customers would take it. After prolonged investigation the cause
was eventually discovered in the well-water, used for swilling out the casks,
which was found to be rich in the organisms producing turbidity in beer Subse-
quent examination showed that the well was connected with the drains by means
of fissures in the soil.

§ 84.— Liquettable Solid Media.

If a species is represented in a bacterial mixture by a few individuals only,
its isolation by the dilution method requires an inconveniently large number of
culture vessels. In order to overcome this difficulty (with others that need not
now be touched upon), Robert Koch, utilising a method practised by Schroeter,
devised a new method of separation, generally termed plate-culture. The essen-
tial part of the method consists in the addition of a gelatinising substance to
the nutrient solution, whereby the latter acquires the property of becoming
liquid at a moderate warmth but is solid at room-temperature. The medium
thus liquefied is inoculated with a little of the bacterial mixture to be separated,
and, after being well shaken up, is poured, whilst still fluid, on to sterilised glass
plates, on which it sets as a thin film. In this film (under favourable conditions)
each one of the cells inoculated therein is held fast and isolated from the others,
and can subsequently multiply, undisturbed, into an aggregation of similar cells
known as a colony.

Gelatin is the substance most frequently used for this purpose, and nutrient
media containing it are called by the generic name of nutrient gelatin, a distinc-
tion being drawn between wort gelatin, meat-juice gelatin, must gelatin, &c.,
according to the kind of nutrient solution used. The amount of gelatin added
is about 9 or 10 per cent., and this produces a medium that is liquid above
30° C. and solid below 24° C., so that inoculation can be conveniently performed
at 35° C., a temperature exerting no injurious influence on organisms. Bouillon
gelatin, often called peptonised bouillon gelatin, is prepared by making up the
meat extract — prepared as already described — to its former volume, i.e. i litre,
with distilled water, after boiling, filtering, and mixing it in a glass flask with
i per cent, of peptone, 0.5 per cent, of Nad, and 10 per cent, of gelatin. The
flask is carefully warmed in the water-bath or steamer until the gelatin liquefies,
and the liquid is then neutralised in the manner prescribed for bouillon. It is
next boiled in the steamer for half an hour and filtered hot through a moist
folded filter, to remove the precipitated albuminoid matters and those thrown
down in neutralising. Samples that clarify badly are improved by egg-albumen,
since the filtrate has to be perfectly clear and transparent. The liquid is filled
whilst warm and fluid into vessels for use (e.g. test-tubes holding 5-8 c.c.), and

ioo METHODS OF PURE CULTURE

sterilised by intermittent heat, being left for twenty to thirty minutes in the
steamer on three consecutive days, as explained in the preceding chapter. In
bacteriological treatises frequent mention is made of " nutrient gelatin " pure
and simple without any qualifying term ; in such cases the peptonised bouillon
gelatin referred to above is always meant. The tubes spoken of in the colloquial
language of the bacteriological laboratory as " gelatin tubes " are ordinary test-
tubes containing 5 to 10 c.c. of nutrient gelatin. The preparation of wort
gelatin is very simple, the unhopped or hopped wort (according to the purpose
it is intended for) being mixed with 10 per cent, of gelatin, melted, boiled for
half an hour in the steamer, filtered hot, filled into vessels, and sterilised by the
intermittent process. Must gelatin requires a little care in preparation, the
high acidity (= 0.7 to i.o per cent, of tartaric acid) of the must having to be
previously almost exactly neutralised by caustic potash, since otherwise the
setting power of the gelatin is impaired. After the acid has been neutralised,
10 per cent of gelatin is added, liquefied, cooled down to between 30° and 40° C.,
and mixed with the white of an egg beaten up to a froth ; then boiled for half
an hour in the steamer, filtered off, filled into the recipients and sterilised as pre-
scribed. During storage, numerous crystalline concretions (up to the size of millet
seed) of potassium tartrate separate out in the solid medium, and by presenting
the appearance of colonies, give rise to the supposition that the medium has been
imperfectly sterilised. Attention is therefore now called to this phenomenon.

As already stated, nutrient gelatin liquefies above 30° C., and therefore also
at the usual temperature prevalent in the incubator, viz., 38°— 39° C., on which
account it is unsuitable for use in the separation of such organisms as require
higher temperatures for their development. In such cases a medium tempered
not with gelatin, but with agar-agar, is used. This substance, obtained from
Eastern Asia, and fully described in a treatise by N. K. SCHULTZ (I.), is a dried
vegetable jelly prepared from various marine algse and put on the market in the
form of thin strips or as a powder. Its manipulation being more conveniently
effected in the latter condition, the use of agar-agar powder is recommended as
preferable. In French literature this gelatin is generally known as " gelose."
For use, not more than 2 parts of agar-agar per ioo of nutrient solution should
be taken. It dissolves very slowly and with great difficulty. For the prepara-
tion of peptonised bouillon agar-agar Hans Buchner recommends the following
process : The meat-broth (bouillon), prepared in the usual manner, is qualified
with peptone, Nad, and I or 2 per cent, of agar-agar, and boiled underpressure
at about 105° 0.; neutralised after cooling down to 100° C. ; then boiled up
again, filtered hot, and filled into vessels for use. It is sterilised by exposure to
120° C., under pressure, for a quarter of an hour. The agar-agar media do not
readily adhere to the glass walls of the vessels, a circumstance which in many
operations may be very troublesome, but may be obviated if the adherent
properties be increased by adding to the agar-agar employed (i to 2 per cent.)
the same amount of gelatin or gum. For the study of the lactic acid bacteria of
the distillery, which thrive best at about 48° to 50° 0., a i per cent, unhopped
wort agar-agar medium containing 2 per cent, of gelatin is used. Moreover,
these agar-agar media do not lose their power of solidification if stored for a long
time at 100° to 120° C. They are liquefied only at temperatures exceeding
40° C., and since this last-named temperature is for many organisms the highest
supportable maximum, the agar-agar is used in the following way when designed
for the separation of a bacterial mixture : The recipient tubes ai-e immersed in
boiling water to induce liquefaction of the contents, which are then cooled down
to 40° C. (at which temperature they are still just fluid), inoculated quickly,
shaken up and mixed thoroughly, and poured out on to the aforesaid glass plates,
which rest on a support warmed to 40° C.

KOCH'S PLATE CULTURES 101

For pure cultures at temperatures above 50° C. agar-agar cannot be used,
since it then begins to soften. For such (rare) cases, Miquel, when experiment-
ing with BaciUus th&rmophilust replaced agar-agar in the nutrient solution by
2.5-3.0 per cent, of Caragheen moss (Irish moss), from Chondrus crispus. — For
special purposes suitable indicators are also added to the nutrient media. For
example, if it is desired to separate merely the acid-forming species from a
bacterial mixture, then a little litmus is added to the medium before sterilising ;
the colonies of acid-forming bacteria in the subsequent plate culture will then
become surrounded by a red halo standing out conspicuously against the blue
background. For the same purpose BEYERINCK (V.) recommended an addition of
fine levigated chalk, which forms an opaque chalk nutrient medium, becoming,
however, clear at the parts of the plate culture occupied by acid-forming bacteria,
in consequence of their solvent action on the calcium carbonate.

Certain organisms, such, for example, as the nitrifying bacteria, do not thrive
in the solidified nutrient media hitherto described. Therefore, in order to pre-
pare cultures of the same by the aid of the plate method, recourse is had to the
medium prepared from precipitated silica, proposed by W. KtiHNE (I.). Silica
precipitated from water-glass (alkali silicate) and carefully purified will, when used
as a 3.4 per cent, aqueous solution, set within an hour to a firm mass if mixed
with 0.25 per cent, of NaCl. The salt is added to a sterilised solution which also
contains the other requisite nutrient substances. In this solution is distributed
a small portion of the bacterial sample to be separated, the cells of which will,
when the medium has set, be fixed and develop into colonies. Further particulars
concerning the preparation of this silica medium will be found in the above-
mentioned treatise, as also in one by Winogradsky which will be referred to
later.

The numerous nutrient media employed in practical mycology are more
fully described in the handbooks of Hueppe, Eisenberg, Tiemann-Gartner, and
Bernheim, but only one need be briefly noticed, viz., the potatoes employed for
the so-called potato cultures. The potatoes — the better sorts used (in Germany)
for salad-making — after being carefully cleaned externally, are steeped for an
hour in a i per mil solution of sublimate, then swilled with water and sterilised
in a wire basket by two hours' exposure in a current of steam. When this is
effected, and they are so far re-cooled as to be fit for handling (with disinfected
fingers), they are cut into halves by a sterilised knife and placed under a sterilised
bell-glass. When cold, inoculating streaks are drawn on the cut surfaces, and
subsequently develop into potato cultures.

§ 85.— Koch's Plate Cultures

are, as previously indicated, prepared by pouring out the liquefied and inoculated
medium (e.g. 5-8 c.c. in a test-tube) on to colourless glass plate s, rectangular in form,
about one-twelfth of an inch thick, 5 to 6 inches long, and 3 \ to 4 inches broad,
previously ^terilised in batches in a copper or iron box, from which they are taken
as required. The plates are laid on a plate-pouring apparatus arranged horizontally
— as described and shown in the above-named handbooks— and the distribution
of the stratum of gelatin or agar-agar is assisted by the aid of the rim of the test-
tube. To sterilise the latter, it should be held for a short time in the Bunsen
flame and allowed to re-cool sufficiently before proceeding to pour. When the
gelatin layer is set the plate is transferred to a sterile damp chamber, which is
placed in the thermostat and maintained at the constant temperature required.

These plates are rather inconvenient to handle, since, in following up the
development of the growing colonies the plate must be frequently taken out of the
chamber. During each observation the mould spores in the air are liable to fall

102 METHODS OF PURE CULTURE

upon the medium, where they rapidly develop into such masses of branched
threads that the bacterial colonies are smothered, thus rendering all the care
bestowed upon the preparation of no avail. In order to prevent this, the flat
plates are replaced by double shallow glass dishes, in which the cultures can be
examined under low powers without being exposed to the air. These dishes
were first introduced into bacteriology by Salomonsen, but are generally known in
Germany as Petri dishes, this latter worker having been the first to test them.
Their use for this purpose can be recommended. — Instead of pouring out the in-
oculated gelatin, the closed tube can be held almost horizontally under the stream
from the water-tap and slowly turned round on its axis, whereby the contents
are distributed uniformly over the walls, and will set as a thin stratum wherein
the germs then develop into colonies. These cultures are generally called Esmarch
tubes or roll cultures, and were first proposed by W. Hesse.

Pkte cultivation affords useful assistance, not only for the separation of a
bacterial mixture into its several species, but also for the determination of the
number of cells present therein, a few gelatin tubes being charged with various
quantities of the sample and poured on to plates. This method is of particular
importance in the quantitative bacteriological analysis of water, for which
reference should be made to Tiemann-Gartner's handbook. The counting of the
colonies grown on the glates is effected by the aid of special counting apparatus,
that of Wolffhiigel being used for the Koch plates. For counting the colonies on
gelatin plates in Petri dishes the author, in 1893, constructed a cheap counting-
plate, obtainable from F. Mollenkopf, of Stuttgart (10 Thor Strasse). The
number of germs thus found is always smaller than the living cells actually
present in the inoculating mixture, since only such as have developed into
colonies are enumerated, whereas a number of germs in the original have failed
to develop under the conditions prevailing, owing to the medium being unsuit-
able for some, and the temperature of the incubator, though favourable to the
majority, being too hot or too cold for a minority. The medium relatively most
suitable for the purpose of ascertaining the number of germs is, in most cases,
gelatinised meat-juice, and this is therefore the one most frequently used. Con-
siderable influence on the number of developing germs is exerted by the degree
of alkalinity of the medium, a fact first conclusively demonstrated by A. REINSCH
(II.) and confirmed by MAX DAHMEN (I.). Jf it is a question not of ascertaining,
as nearly as possible, the total germ content of a sample, but only how many of
the cells are capable of development in a given medium, then the latter is arranged
in a solidified condition as a plate culture. For example, wort gelatine is generally
used — unless the contrary be expressly stated — when determining the number of
germs in brewery water. It is important to know for certain whether the
colonies in a plate culture are each developed from a single cell, since it is only
in such cases that a pure culture can be obtained on re-inoculation. This aim is
attempted by thin sowing and thoroughly shaking the liquefied medium, in
order to separate the cells from each other. Nevertheless, there is always some
uncertainty, which we must endeavour to remove by discarding the first series
of plates and by preparing a second series wherein any impurities may become
manifest ; then, if the colonies are found to stand this test, the reinoculations
therefrom may be considered as pure cultures. This can be ensured from the
outset if the growth of the colonies, i.e. from the single cells, be followed by the
aid of the microscope from the beginning. This test is, however, feasible only
with largo cells (Eumycetes spores, yeast cells, <fec.), and will be enlarged upon
in a subsequent chapter in connection with the pure culture of yeast. On the
other hand, it is, as a rule, impracticable for bacteria, since their examination
necessitates the use of such a high (short focus) objective that the latter has to be
brought so near the plate as to impinge on the gelatin stratum.

KOCH'S PLATE CULTURES 103

Pure cultivation with solidified media is almost exclusively applicable to fungi
alone, its application to other groups having been successful in the case of a few
only of the lowest unicellular algae. In this manner BEYERINCK (VI.) prepared
pure cultures of the last-named organisms (frequently found in the microscopic
examination of river- water), viz., Chlorella vulgar is, Scenedesmus acutus, Chloro-
sphcera limicola, Chlorococcum (Cystococcus) humicola, Stichococcus major, and a
second species of Chlorella. WILHELM KRUGER (I.) prepared by the plate method
pure cultures of two algse, which he named Chlvrella protothecoides and Chlorothe-
cium saccharophilum, from the sap of the lesser maple. Quite recently BEYERINCK
(VII.), A. CELLI (I.), FR. SCHARDINGER (I.) and others, have also successfully
attempted the cultivation of Amceba in the same manner.

The solid nutrient media in question are, except those containing chalk,
transparent, so that the plates prepared therefrom can be laid on the stage of
the microscope and their colonies examined under a low power by transmitted
light. The differences thereby observable form valuable indications for the
identification of the individual species. A number of them, Bacillus subtilis for
example, excrete a peptonising enzyme, in consequence of which the gelatin is
liquefied as far as the solvent enzyme proceeding from the colonies is able to
diffuse, and thus a liquefactive colony is obtained. The converse was supplied
by such organisms as produce no enzyme capable of dissolving gelatin, and which
therefore do not liquefy the medium, but grow as solid colonies. The develop-
ment of these latter may proceed in various ways ; the colonies of Bacterium
aceti, for instance, gradually assuming a stellar form, whilst those of the lactic
acid bacteria have a circular outline. Bacillus ramosus — a fission fungus
frequently occurring in soil and in natural waters, and generally known as
wurzel (root) bacillus — which M. WARD (IV.) subjected to exhaustive
morphological and physiological examination, grows on agar-agar to colonies
built up of entangled, branched, and plaited threads resembling the roots of a
tree. — A comprehensive description of these characteristics, as presented by the
separate species of bacteria, will be found in Eisenberg's work.

If a platinum wire, previously heated to redness and then dipped in a
bacterial culture, be thrust into a solid medium contained in a test-tube, the
cells so implanted in the passage formed by the wire will develop to a so-called
puncture culture, the appearance of which also affords valuable indications for
the recognition of individual species. Organisms requiring air grow only on
the surface, whereas those shunning the air will develop only in the deepest
part of the channel, and those dissolving the gelatin will forma liquefied funnel.
This latter indication is one developed in a highly characteristic manner by the
cholera bacillus, and is, therefore, made use of in the bacteriological analysis of
water. If a test-tube containing about 8 or 10 c.c. of liquefied nutrient gelatin
or agar-agar be held at a sharp angle, the contents will set in the form of a
wedge, and if the plane surface be stroked over with a" small quantity of a
culture, then a so-called streak culture will be developed. This also in many
cases assumes a characteristic form that should be taken into consideration in
the identification of a species. The potato cultures already referred to are
nothing more than streak cultures on the cut surfaces of steamed potatoes. We
are indebted to C. FRAENKEL and R. PFEIFFER (I.) for an excellent atlas of
photographs of the colonies, streak cultures, cover-glass preparations, &c., of a
number of (mostly pathogenic) bacteria. An atlas of coloured plates of these
objects has been issued by K. B. LEHMANN and R. NEUMANN (I.).

It may be valuable, for purposes of instruction, to preserve in the cultures
the appearance they present at the time of their most vigorous growth.
According to G. HAUSER (II.), the preservation of cultures on gelatin or agar-
agar can be most conveniently ensured by means of formaldehyde. Cultures in

104 METHODS OF PURE CULTURE

test-tubes can be treated by moistening the cotton plug with formalin and then
covering it with a rubber cap to prevent desiccation. Plates and cultures in
Petri dishes can be kept for some time by covering them with filter-paper
moistened with the same antiseptic, which kills the cultures without destroying
their form. It also penetrates the medium, hardens the gelatin, and makes it
unsuitable for the further development of organisms, so that the preparations
thus treated are exceedingly durable. Fuller information on the preservation
of pure cultures of fermentative organisms, and practical hints concerning the
arrangement of mycological museums, which are very useful both for teaching
and for reference purposes, will be found in the treatises prepared by J. SOYKA
(I.), F. KRAL (I.), H. PLAUT (I.), E. CZAPLEWSKI (I.), and E. KRUCKMANN (I.).

The necessity will not infrequently arise for reliable (living) pure cultures
of authoritatively named species of bacteria, &c., either for use as a starting-
point for s£udy or for comparing, and, as far as possible, identifying some
newly-discovered species. Krai's Bacteriological Laboratory (n Kleiner Ring,
Prague I.) can be recommended as a source from whence to obtain them. This
institution supplies living pure cultures, streak-cultures on oblique solidified
agar-agar, in test-tubes at the moderate price of one to two marks ( = shillings)
per tube.

Koch's plates can also be used with advantage when it is a question of
ascertaining which nutrient media are suitable for a given microbe. For this
purpose BEYERINCK (VIII.) has devised a method which he calls Auxanography.
A 10 per cent, gelatin or 2 per cent, agar-agar in distilled water is prepared,
both of which substances in the pure state form very bad media, whether for
bacteria or higher fungi. Plate cultures of the micro-organism whose nutritive
requirements form the object of the investigation are then prepared. These, if
left to themselves, will not exhibit any appreciable degree of development, so
the surface of the plates is stippled with a few drops of aqueous solutions of the
substances whose nutrient properties are to be tested. These drops are absorbed
by the gelatin or agar-agar, and form circular fields of diffusion around the
spots in question. The thickly sown cells of the species under examination will
then develop into strong colonies on those spots only where the requisite nutrient
materials are encountered, so that the organisms inscribe, as it were, with their
bodies, the answer to the question propounded as to the suitability of the
nutrient substances at hand. Such a plate of colonies grown in this manner is
called by Beyerinck an Auxanogram. This method may also be employed for
testing the toxic action of various substances on given organisms. BEYERINCK
(IX.) also employed this process as a basis for the qualitative and quantitative
method of microbiological analysis proposed by him in the reference just given.

SECTION IV.
C.IROMOGEN1C, PHOTOGENIC, AND THERMOGENIC BACTERIA.

CHAPTER XII.

CHROMOPAROUS BACTERIA, PRODUCING RED AND YELLOW
COLOURING MATTERS.

§ 86.— Coloured and Colouring- Bacteria.

IN classifying the chromogenic (colour-producing) bacteria, the situation, as well
as the nature, of the colour has an importance that cannot be disregarded. An
examination for this first-named characteristic in individual species quickly leads
to the differentiation of the chromogenic bacteria into coloured bacteria on the
one hand and colouring bacteria on the other • the cells in the latter being
themselves quite "colourless, but excreting a coloured transformation product;
these species have been designated chromoparous by Beyerinck.

In the coloured bacteria, on the contrary, the colouring matter remains
within the cells. This group may be divided into two sub-groups, the one
comprising those coloured bacteria the colouring matter of which performs an
important physiological function, as in the case of the purple bacteria treated
of in the following chapter ; such bacteria are termed chromophorous. On the
other hand, the second sub-group includes those coloured bacteria in which the
colouring matter has no such function, and must be regarded as a purely passive
metabolic product, which is, nevertheless, not excreted (as in the chromoparous
species), but remains within the cell without manifesting any apparent activity.
These bacteria are termed parachromophorous.

The chemical properties of the bacterial colouring matters and their
importance for distinguishing one species from another were discussed by PAUL
SCHNEIDER (I.) after exhaustive experiments with thirty different species. His
results in this connection may be thus summarised : (i) The bacterial colouring
matters can to some extent be differentiated by their behaviour towards solvents.
(2) A given species, grown under identical conditions, always produces the
same colouring matter. (3) Two species, differing as regards form and con-
ditions of growth, may in certain cases produce the same colouring matter.
(4) Most of the species apparently producing the same colouring matter, and
also analogous in other respects, can be differentiated by the reactions of their
colouring matter.

§ 87. — Micrococcus Prodiglosus.

This being the oldest known chromogenic bacterium, will be dealt with first.

Many a victim of the proceedings taken against witchcraft during bygone
centuries must have been consigned to the stake on the charge of having fabri-
cated the blood-red spots that were occasionally found developed on the Host,

105

106 CHROMOPAROUS BACTERIA

and which filled the credulous mind of the masses with horror ; and even in 1819
the entire province of Padua was set in a commotion by the frequent appearance
of such spots and drops on various articles of food. This red, slimy coating
was examined by SETTE (I.), who recognised it as endowed with vitality and
named it Zoogalactina imetrofa. A small quantity applied to still unaltered
food-stuffs, &c., sufficed to produce red spots on these latter. This phenomenon
was first more closely investigated in 1848, when it was of frequent occurrence
in Berlin. Ch. Ehrenberg studied the spots and drops, and found them to consist
of minute oval cells 0.5 to i.o /* in length ; and bearing in mind their form and
observed powers of locomotion, he classified this wonderful organism as a new
species of his genus Monas, and called it Monas prodigiosa, a designation subse-
quently changed by Cohn to Micrococcus prodiyiosus. As this microbe (mostly
appearing as approximately spherical cells) will, under certain conditions of environ-
ment, assume an elongated form, it is also frequently named both Bacterium
prodigiosum and Bacillus prodigiosus, as was done by Fliigge in his handbook.
These names, therefore, indicate one and the same species of fission fungus, and
are also synonymous with the older names Palmella prodigiosa and Sacteridium
prodigiosum.

This fission fungus excretes a peptonising enzyme, and consequently liquefies
the gelatin medium. A temperature of 25° C. is the most favourable one for
its growth, and it thrives most luxuriantly on boiled potatoes, the formation of
trimethylamine becoming at the same time apparent. Starch paste, boiled rice,
boiled egg-albumen, boiled carrots, boiled meat, milk, and many other food-stuffs,
form suitable media for this microbe, which, however, will not develop on raw
potatoes, raw meat, or uncooked steeped rice. It is therefore evidently a true
saprophyte, occurring only in defunct or destroyed and converted nutrient media.
When cultivated in thinly fluid solutions it exhibits— as was established by
SCHOTTELIUS (I.) — brisk powers of locomotion.

The red colouring matter, which is produced in presence of air only, is,
according to the researches of this observer, at first diffusely distributed through
the young cells. It is then excreted, and collects into various-sized granules
which lie between the cells and so impart a red coloration to the culture. The
tone of the colour changes with the age of the culture, beginning as a pale rose
and passing through a bright scarlet stage into dark brown-red. According to
the researches of J. SCHROET.ER (I.) and SCHEURLEN (III.), the colouring matter
is insoluble in water, but readily soluble in alcohol, xylene, chloroform, carbon
bisulphide, and, to a slighter extent, also in ether and in fats (e.g. olive oil).
The alcoholic solution exhibits in the spectroscope three absorption bands : one
beyond D, the second just before E, and the third before F. The elementary
formula was determined by A. B. GRIFFITHS (I.) as CMH.6NO5, though the
analytical results obtained by Scheurlen are not in conformity therewith. The
opinion expressed by O. ERDMANN (I.) that the colouring matter generated by
M. prodigiosus is identical with fuchsine has been contradicted by OTTO
HELM (I.) and BORDONI-UFFREDUZZI (I.).

This fission fungus forms a very suitable object for the study of mutability.
E. WASSERZUO (I.) traced the changes of form which this species underwent
in consequence of alterations of the conditions of nutrition. By repeated
cultivation on faintly acid media — 0.3 to 0.4 grm. of tartaric acid per litre —
cultures are obtained the cells of which are no longer globular or oval, but
exhibit the form of actively motile long rods and threads ; the modifications
being the more pronounced as the number of inoculations is increased. As soon,
however, as an inoculation is made from such acid liquids into an alkaline
medium, the typical short cells reappear. This reversion also occurs when the
cells remain for some time in the original acid medium, after the reaction has

LIPOCHROMES 107

become alkaline from the tranbformation products (trimethylamine, &c.) excreted
by the microbe.

In addition to the form of the cells, the development of colouring matter is,
as Schottelius has found, also dependent on the nutritive conditions; since, if a
prodigiosus culture, grown at io°-25° C. and already red in colour, be inoculated
on sterile potatoes (steamed and cut in halves), and the temperature kept at
38°-39° C., the inoculating streaks develop into colourless streak cultures.
From these again a red culture can be once more obtained by suitably modifying
the conditions of the culture, i.e. reverting to a lower temperature.

§ 88.— Lipochromes.

With the organism mentioned in the last paragraph are classified a number
of other species also producing red colouring matter. One of these, Bacillus
erythrosporus, first discovered by ED. EIDAM (I.) in putrefying egg-albumen, is of
particular interest. This is a slender motile bacillus, which does not liquefy
gelatin. As is indicated by its second name, the seat of the dirty red colouring
matter is not in the vegetative form of growth, but in the endospores. The
so-called (motile) "Kiel bacillus," found by Breuning in the Bay of Kiel, occurs
generally as long rods (0.8 p. broad, by 2.5-5 /* l°ng)> which liquefy gelatin.
There is great similarity in the red colouring matter produced by this bacillus
and Micrococcus pi-odigiosus, but the former microbe is distinguished by its
greater susceptibility to direct sunlight, which, according to the researches of
E. LAURENT (I.) permanently destroys its chromogenic power. A similar
effect is produced by the presence of carbohydrates in the medium, the Kiel
bacillus, in such event, elaborating no colouring matter. The following chromo-
parous red species will only be briefly alluded to : Bacillus ruber, discovered by
Frank and described by COHN (II.) ; Bacillus indicus, discovered in the contents
of the stomach of an East Indian ape ; the Bacillus gramdatus of Babes ;
Bacillus corallinus, isolated by C. SLATER (I.) from atmospheric dust, and the
Bacillus rubellus, discovered by OKADA (I.), which forms endospores and thereby
assumes the clostridium form.

Greater interest attaches to several red and yellow species studied by ZOPF
(IV.), and especially as regards their colouring matters, which were named by
him lippchromes or fat-colouring matters. These are excreted from the cells
and collect between them to form dendritic crystalline aggregations, which are
luminous in the darkened field of the polariscope. The lipochromes known at
present are red and yellow, the former being styled liporhodine and the latter
lipoxanthine. The reagent for these is concentrated sulphuric acid, whereby
they are converted into deep blue acicular crystals of lipocyanine, which remain
isolated when derived from lipoxanthine, but arrange themselves in characteristic
groups when produced from liporhodine. Illustrations of these will be found in
OVERBECK'S (I.) work on this subject. These colouring matters can be extracted
from the cultures by means of ethyl alcohol, in which they are just as.soluble as
in methyl alcohol, chloroform, carbon bisulphide, and benzene. On evaporating
the solvent, a fatty mass, furnishing the acrolein reaction, remains behind. This
being saponified and salted out with a hot solution of sodium chloride, the liquid
underlying the soapy layer will contain the colouring matter, which can then be
extracted by shaking up with petroleum spirit and examined spectroscopically.
Of these species the following were more closely examined by ZOPF (V\) : Micro-
coccus rhodochrous, isolated from the contents of a goose's stomach, is about 0.9 p.
in diameter, and will grow on nutrient gelatin, potato discs, <fcc., to form deep
red masses. The absorption spectrum of the liporhodine extracted therefrom
shows an absorption band in F. The Micrococcus erythromyxa, obtained from

io8 CHROMOPAROUS BACTERIA

the town- water of Halle, has a diameter of 1.0-1.2 /*, and its colonies on the
nutrient media referred to resemble in appearance those of the first-named
species. In addition to liporhodine, however, it produces a yellow colouring
matter, soluble in water, but of unknown nature.

Antithetical to these two red- producing species are : Bacterium egregium,
obtained from atmospheric dust ; then Bacterium chrysogloia ; and finally the
Staphylococcus pyogenes aureus, already several times referred to in the previous
chapter. The.se three develop into yellow cultures producing lipoxanthine, the
absorption spectrum of which consists of two bands, one near F and the other
between F and G.

§ 89.-Red Coloration in Milk

may be due to various causes, one of them being an admixture of blood derived
from a broken blood-vessel in the udder. In this case the coloration of the
liquid is not uniform, but is due to scattered patches of red, fiocculent blood
coagulum. If, on the other hand, the milk is found to be uniformly coloured
red or reddish throughout the entire mass as soon as it is drawn from the udder,
then other causes are in operation. If the colour undergoes no change on stand-
ing, it is attributable to the fodder having contained a large quantity of madder
(Rubia tinctoruiii) or of Galium verum. Should, however, the red-drawn milk
precipitate a red sediment on standing, the colour of the liquid concurrently
decreasing, then the coloration is due to a transudation of haematin. This case
is analogous to that of " red water," and is a consequence of the consumption of
highly stimulating fodder.

When, however, a normal milk gradually becomes red after standing, then
micro-organisms are at work ( Micrococcus prodigiosus being frequently the agent),
and the colouring matter excreted by the organism is absorbed by the fat
globules of the milk. This microbe has already been more minutely characterised
in the preceding paragraph.

A second milk-reddening organism is the Bacillus lactis erythrogenes, dis-
covered by Hueppe and more closely examined by GROTENFELT (I.). The non-
motile rods of this organism are 0.3-0.5 p. in diameter, with, usually, a length of
1-1.5 M) but attaining to 4.3 fj. in bouillon. This fission fungus (which liquefies
gelatin) grows on solid media to yellow colonies, which excrete around their
periphery a red colouring matter. When inoculated into sterile milk it pro-
duces a gradual coagulation attended with a sickly-sweet odour, without affecting
the reaction of the liquid to any appreciable extent. The serum gradually clari-
fying from the deposited casein absorbs the resulting deep red colouring matter,
which develops most copiously in the dark and ceases to form if the culture be
exposed to light or the medium has an acid reaction. Its absorption spectrum
exhibits two strong bands between the lines D and E and a third in the blue.
A species of fission fungus allied to B. lactis erythrogenes has been isolated from
red milk by A. BAGINSKV (I.).

Of the sarcina group two species are known to be endowed with the faculty
of reddening milk. One of these was discovered by K. MENGE (II.), viz.,
Sarcina rosea Menge ; and the second was isolated from red milk by L. ADAMETZ
(II.), and was identified with the Sarcina rosea Schroeter, very frequently met
with in the air. Apart from the red coloration, Menge's sarcina produces
no noteworthy changes in milk, but Schroeter's sarcina, on the other hand, first
precipitates the casein which is subsequently gradually re- dissolved. Conse-
quently a milk-culture (growing dark brown in colour) kept at 25° C. for four
to five weeks no longer exhibits any deposit beyond a sediment consisting of
sarcina packets.

Red coloration in cheese may arise from various causes, those of a purely

BACTERIA PRODUCING YELLOW COLOURING MATTER 109

chemical nature being merely referred to here for the purpose of differentiation.
According to the researches of H. BURSTERT and F. J. HERZ (I.), a number of
rhodanate (thiocyanate) compounds develop in ripening curd, and when the ripe
cheese is cut the iron compounds therein oxidise and combine with the thio-
cyanates, the resulting ferric thiocyanate being in sufficient quantity to impart
a red or reddish tinge to the cut surface. A sample of cheese thus reddened is
decolorised by immersion in a solution of oxalic acid, so that this reagent should
be employed in doubtful cases. If, on the other hand, micro-organisms are at
work, the result is different. This disagreeable phenomenon is more rarely
encountered in hard than in soft cheeses. Red spots gradually form on the
surface and spread by degrees, but do not penetrate more than a few millimetres
into the substance of the cheese. The spots may be composed of red Eumycetes
or of pigment bacteria. ADAMETZ (III.) isolated two species of micrococcus from
red cheese, both of which develop a red colouring matter in milk and cheese ; but
more frequently Eumycetes, i.e. higher fungi, are the cause of this phenomenon in
cheese. These will be discussed in a subsequent section.

The cause of the reddening of dried codfish (stock-fish) was investigated by
LE DANTEC (I.). About one-third of this article put on the market is said to be
affected by this evil, and is thereby rendered unsaleable, not only by reason of
its salmon colour, but also because of a popular belief that reddened stock-fish is
poisonous. The annual loss thus entailed is estimated at ten millions of francs
(^£400,000). Le Dantec isolated three organisms from such spoiled fish ; the first
being a sporogenic red-producing bacillus, morphologically similar to tetanus
bacillus, and liquefying gelatin ; secondly, a coccus of 3-5 p. diameter, developing
on gelatin to solid red colonies, but not producing a red colouring matter in
the fish unless accompanied by a second species of coccus, which, by itself, is
incapable of developing colouring matter. In order to render the reddened fish
fit for sale again, it is brushed in cold water and re-dried. In America borax is
added to the salt used in curing the fish, in order to prevent the development of
the evil; and an addition of from 10 to 15 per cent, of sodium bisulphite or
potassium nitrate (saltpetre) is also said to be efficacious.

§ 90.— Bacteria Producing1 Yellow Colouring1 Matters.

These were first studied by C. J. FUCHS (I.) in 1841, the starting-point of
his researches being the so-called yellow milk, i.e. milk that on standing develops
a pale- to orange- yellow coloration.

The cause of this appearance was traced to a microbe named Vibrio
synxanthus by Ch. Ehrenberg, and afterwards known also as Vibrio xanthogenus
and Bacterium synxanthum. The same phenomenon was also investigated by
J. SCHROETER (I.) in 1870 ; according to whom it occurs in boiled milk only,
inoculations from a slightly yellowed milk into normal unboiled milk being
unsuccessful. The activity of lactic acid bacteria was presumably the cause of
this prevention. On inoculation with this microbe, boiled milk coagulated after
twenty-four hours, and the yellow coloration made its appearance after the lapse
of a second period of equal duration. Thereafter the precipitated coagulum
gradually disappeared and became re-dissolved, so that in six days the milk had
become converted into a citron-yellow, watery, strongly alkaline liquid containing
merely a few particles of casein in suspension. The pigment is insoluble in
alcohol or ether, but soluble in water ; it is unchanged by alkalies, but acids
combine with it to form a colourless compound. The absorption spectrum is
devoid of characteristic bands, and merely exhibits a darkening of the rays on
either side of the yellow. Schroeter proposed the name of Bacterium xanthinum
for the microbe investigated by him, which designation was converted in later

no CHROMOPAROUS BACTERIA

text-books to Bacillus synxanthus. It is desirable that the study of the
organisms of yellow milk should be taken up anew, since Schroeter's investigations
were not performed upon pure cultures in the present acceptance of the term.

With the species hitherto described (Bacterium egregium, B. chrysogloia,
Staphylococcas pyogenes aureus, B. synxanthuni) there can be associated a
number of others, equally characterised by the faculty of producing yellow
colouring matters. Of these, mention will here be made merely of the Micro-
coccus ochroleucus, discovered by 0. PROVE (I.) in human urine in a state of
incipient decomposition. When kept in the dark this microbe develops into
colourless cultures, but if exposed to diffuse daylight or the sun's rays it elabo-
rates a sulphur-yellow colouring matter. This may, therefore, be regarded as
a probable means of protection.

Many species of bacteria producing orange-yellow or orange-red colouring
matters are met with in air, water, and soil. Since they are of no particular
importance, it will be sufficient to merely mention a couple of them, viz., the
Micrococcus aurantiacus ( = Bacteridium aurantiacum), described by Cohn and
Schroeter, and the Bacillus aurantiacus, discovered by Frankland.

The group of globular Schizomycetes to which the generic name of Sarcina
has been applied is rich in species producing red or yellow colouring matters.
A few examples of the former having been already
given in previous paragraphs, it will now be sufficient
to consider merely those developing into yellow colonies.
The first observation of any sarcina species whatsoever
was made in connection with a species of this group.
JOHN GOODSIR (I.) in 1842 discovered, in the vomit
of a patient suffering from a diseased stomach,
colonies of microbes resembling bales of merchandise
in form, to which he applied the name Sarcina ventri-
culi (Fig. 33). His opinion that the microbe was a
vegetable organism led to a controversy only termi-
nated in 1847 by VIECHOW (I.), who agreed with his
English colleague. Sarcina ventriculi produces, how-
ever, but a faint yellow colouring matter. The cells
of the Sarcina Jlava, described by De Bary, which
measure 1-2 p in diameter, produce a yellow colouring matter, and this species
is distinguished from the Sarcina lutea, discovered by Schroeter, by its power of
liquefying gelatin. Paul Lindner prepared pure cultures of Sarcina aurantiaca
from Berlin " Weissbier " (white beer), the colouring matter of which organism
is, according to the researches of H. VON SCHROTTER (I.), allied to lipoxanthine.
No sarcina producing blue, violet, or green colouring matters are as yet known.

FIG. 33. — Sarcina ventriculi.

From the contents of a dis-
eased stomach, a-d. various
stages of development.
(After Zopf.)

CHAPTER XIII.

PURPLE BACTERIA AND THEIR BEHAVIOUR TOWARDS LIGHT.

§ 91.— Their Morphology.

WHEX in September 1826 the " Versammlung Deutscher Naturforscher und
Aerzte" (Association of German Naturalists and Physicians), founded by Lorenz
Oken in 1822, was held in Jena, Ch. Ehrenberg was among those present. In
the course of a stroll to the neighbouring village of Ziegenhain he observed in a
pool in the brook below the church a number of large red patches, about a
hand's-breadth across. These he found to be composed of enormous swarms of
a unicellular cylindrical organism provided with a single cilium and measuring
10-15 /* l°n£ ky 5 M broad, which he named Monas Okenii. Later on, PERTY (I.)
arranged this species, along with other similar ones, into a new genus, Chroma-

FIG. 34. FIG. 35.

Chromatium Okenii. Monas Wariningii.

Magn. 600. (After Cohn.) Magn. 600. (After Cohn.)

tium, and the Monas in question then received the name Chromatium Okenii,
which it still retains. This organism is shown in Fig. 34.

The main reason for considering this organism here, separately from the
other red bacteria previously noticed, is on account of its physiological action
rather than its morphological character, which action places this bacterium (and
many other similar species) in quite a distinct category from all other pigment
bacteria, and, in fact, all other bacteria whatsoever. Even in 1875 COHN (II.)
expressed a doubt as to whether the ciliated Chromatium Okenii and allied forms
could be classed as bacteria at all, since it was at that time assumed that the
latter organisms were not endowed with special organs of locomotion. However,
as explained in chapter iii., the improvements made in the methods of optical
and micro-chemical examination have led to this opinion being modified.

The chief characteristic of the Chromatia and their congeners is their
behaviour towards light ; but before considering this more closely we will throw
a glance over the multitude of organisms now in question, with some of which
we have already had an opportunity of becoming acquainted. Thus we know
from § 68 that Lankester studied the ciliated red monads and classified them all
as special forms of growth of a single species (" Bacterium rubescens "). This un-
proved pleomorphism we declined to recognise at the time, but must now revert

i i 2 PURPLE BACTERIA

to it in order to append the remark that, though we are not indebted to the last-
named worker for any revelations regarding the morphology of these organisms,
we have to thank him for an exhaustive study of their colouring matter, which
he termed bacterio-purpurin. A form similar to Chromatium Okenii was dis-
covered by E. WARMING (I.) on the coast of Seeland and afterwards examined
by Cohn, and by him entitled Monas Warmingii (Fig. 35), so that Perty's pro-

FIG. 36.

Spirillum volutans.
Mag;n. 600. (After F. Cohn.)

FIG. 37.

Ophidomonas sanguinea.
[Magn. 600. (After F. Cohn.)

FIG. 38.

Rhabdomonas rosea.
Magu. 600. (After F. Cohn.)

position with respect to the generic name Chromatium seems not to have
been accepted by the Breslau bacteriologist. Whilst the species hitherto
mentioned differ among themselves in point of size, but not appreciably in
form, and all more or less correspond with the plump, short rods shown in
the drawing, a second group of similar species exhibits the spirillum form of
growth. One example of this is afforded by Spirillum rubrum (Fig. VI.
Plate I.), a second being the Spirillum volutans, shown in Fig. 36, and a third
the Ophidomonas (Fig. 37), described by Ehrenberg. A third sub-group finally
comprises organisms of elongated spindle form, and therefore resembling a
whetstone in outline ; e.g. Rhabdomonas rosea (Fig. 38) — 4-5 /i wide and
20-30 p, long — first observed and described by Cohn. These organisms are not
infrequently to be found in ponds and lakes ; sometimes being so abundant
as to colour the water red. A series of observations respecting such occurrences
was made by CHARLES MORREN (I.).

§ 92.— Influence of the Individual Colours of the Spectrum.

The necessity for a separate consideration of these red species, grouped
together by ENGELMANN (V.) as purple bacteria, is soon apparent when an
attempt is made to study their physiology. The bacteria described in the
previous chapter, of which Micrococcus prodigiosus may serve as an example,
also produce red colouring matter ; but, in their case, the latter is a mere inert
waste product, appearing under certain conditions, or absent under others,
without the growth of the cell being thereby seriously affected. Contrary to
this, the purple bacteria do not excrete any colouring matter into the environ-
ment, but the pigment occurs exclusively within the cells (Fig. 39) in the part
of the contents which immediately adjoins the cell-wall, and which is described
in chapter ii. as the parietal layer. The bacterio-purpurin is not always distri-
buted throughout the whole of this layer, but is frequently restricted to isolated
spots therein, and, in exceptional instances, is altogether absent. It is not
present in any definite shape, such as granules, bands, or plates, like the chloro-
phyll of the higher plants, but occurs diffused in the plasma.

FIG. 39. — Chromatium Okenii.
Optical longitudinal section.

figure) immediately adjoins the
outer layer of protoplasm, which
carries the colouring matter —
shown as fine dots. Large sul-
phur granules are present in the
interior of the cell. Magn. about
1600. (After F. Forster.)

ASSIMILATION AND OXYGEN ELIMINATION 113

It has already been stated in chapter iii. that the purple bacteria exhibit
great avidity for light, and therefore always strive towards the sunlight.
Closer observation shows this behaviour to be intimately connected with the
presence of bacterio-purpurin. It was a happy
thought on the part of Engelmann to investigate
the nature and extent of the influence exerted on
the vitality of the purple bacteria by the several
colours of the spectrum.

If a preparation rich in such organisms be
placed in a drop of water, and a micro-spectrum of
a few millimetres in length be projected thereon,
a rapid movement towards certain parts of the
spectrum will be observed under the microscope,
the organisms collecting and resting there in micro-
scopically visible bands. By quickly killing the
cells they will remain in position and constitute a
permanent preparation, or, as Engelmann appro-
priately named it, bacteriospectrogram. This, when

submitted to examination, is found to correspond TLe cell-wall (the thicknessof which
with the absorption spectrum of bacterio-purpurin, is somewhat exaggerated in the
showing a sharply denned band in the ultra-red
(wave length X = 0.9 to 0.8 /*) ; a second, less power-
ful, small band in the orange (X = 0.6 1 to 0.58/1);
and, finally, a pale washed-out band in the green
(X = o.55 to 0.52 /i). Engelmann then determined
by an accurate quantitative photometric examina-
tion, with the aid of the bolometric method, that a remarkable ratio prevails
between the intensity of the physiological action and the extent of the absorp-
tion, i.e. the attractive force of a given colour of tha spectrum is greater in
proportion as the latter is retained by the colouring matter. From this is
deducible the further conclusion, that the purple bacteria have great need, not
merely of light in general, but of certain components thereof in particular,
and especially those corresponding to the lines A0, D, E, of the spectrum.

§ 93.— Assimilation and Oxygen Elimination.

This behaviour of the purple bacteria, unique in the bacterial kingdom,
reminds one so much of the connection between light and the activity of chloro-
phyll in the higher plants, that not only does the question naturally arise as
to the nature of the reactions occurring under the influence of the selected
rays, but also the idea that here likewise there is assimilation accompanied by
the elimination of oxygen. This is, in fact, the case, the purple bacteria
excreting oxygen in the presence of light.

ENGELMANN (VI.) proved this in a variety of ways. One of his experiments,
which demonstrates it in a very elegant manner, is based upon that already
given in § 41. Quiescent forms, resembling a zooglrea, of one or other of these
species are employed, a portion about 2 sq. mm. in size being placed in a drop
of water. To this are added a number of aerobic organisms (e.g. Spirillum
tenue, Sp. undula, various infusoria, &c.), capable of reacting on even small
quantities of oxygen, and the cover-glass is surrounded with vaseline to prevent
the admission of air. The oxygen dissolved in the water is very quickly
exhausted, and the organisms come to a standstill. If, now, the preparation be
illuminated, it will be seen directly that the wanderers scattered about rise and

I H

H4 PURPLE BACTERIA

hasten to the red skin where the oxygen they need is produced. On the other
hand, when replaced in the dark, they disperse again in all directions. That
oxygen is the attraction is demonstrated by the circumstance that the pheno-
menon just described does not occur when the experiment is performed on a
preparation the cover-glass of which is not made air-tight, and to which air can
consequently penetrate by diffusion.

This peculiarity gives the purple bacteria a unique position amongst the
Schizomycetes as a connecting link between them and the green plants. From
its capacity of converting the actual energy of light into potential chemical
energy, and of changing vibrations of light into force, bacterio-purpurin deserves
the title of a true chromophyll, since it plays in the purple bacteria the same
rdle as chlorophyll does in green plants. These two substances are antithetical,
accomplishing similar tasks by different methods of working. On more closely
examining the individual spectral colours for their power of eliminating oxygen,
this latter faculty is found to be proportional to the absorptive capacity of
bacterio-purpuriu for the colour in question. The maximum effect is produced
in the case of the aforesaid ultra-red rays (X = 0.8-0.9 p), whilst, on the other
hand, the rays between the lines B and C are inert. With chlorophyll the
converse is the case, this being quite inactive in the ultra-red rays, and exerting
its greatest effect in the red rays (between B and C).

The aforesaid ultra-red rays (\ = 0.8 to 0.9 /z) are well known under the
name of " invisible heat rays," being inappreciable to the eye as light. The
discovery that they are the rays that not only enable the purple bacteria to
exhibit activity, but also spur the latter on to their highest degree of efficiency,
allows the wider conclusion to be drawn that the elimination of oxygen through
the activity of the vegetable cell is not dependent on the co-operation of visible
light rays, but may also proceed in the dark.

So far for the facts ; but looking beyond them, it may be asked if the faculty
of absorption in the dark is inseparably connected with the presence of bacterio-
purpurin, or if there are also colourless bacteria similarly endowed. The answer
to this query will be found in chapter xxxvi., which treats of nitro-bacteria.
This is a matter of such great interest as to deserve special consideration ; at
present, therefore, we will merely review the facts hitherto discovered in the
case of the purple bacteria, their general importance being so great that we
shall certainly not regret having bestowed attention on these organisms, not-
withstanding that they are devoid of technical application. The scientific
harvest they are capable of yielding is, however, still far from being exhausted.
It will be remembered that it is in the outer layer of the inner substance of the
cell of these red organisms that this very influential bacterio-purpurin has its
abode, and that the central substance is surrounded by this layer. The study
of this from a morphological standpoint by Biitschli led to the conclusions
respecting the structure of bacterial plasma already recorded in an earlier sec-
tion. The investigation of the physiology of this central substance has yielded
a second series of weighty results, which will be given in chapter xxxv.

CHAPTER XIV.

CHROMOPAROUS BACTERIA, PRODUCING BLUE, GREEN, AND
VIOLET COLOURING MATTERS.

§ 94.— Blue Coloration of Milk

is a phenomenon known from time immemorial. The opinions as to its cause
are widely divergent, but the earliest of them are at present only of historical
interest. They may be found in MARTINY'S (I.) useful handbook, which (it may
be parenthetically observed) affords a rich supply of information respecting the
literature published on milk, butter, and cheese up to the year 1871.

The first to arrive at the opinion that the blue coloration of milk might
proceed from some external infection penetrating into the liquid, was STEINHOF (I.)
who, however, made no attempt to prove by experiment the correctness of his
hypothesis. This was only effected three years later, viz., in 1841, by C. J.
FUCHS (I.), who inferred from the results obtained during numerous inoculation
experiments and much microsopic research that the blue coloration of milk is
induced by the development of a pigment microbe, which he first named Vibrio
cyanogenus and then Bacterium syncyaneum.

Unfortunately, however, for the study of this question, Liebig just at this
time promulgated his theory of fermentation, and fetterad philosophers in his
dogmatic shackles. This explains why HAUBNER (I.) lost sight of the object for
which Fuchs had striven, and, by endeavouring in 1852 to adapt the result of
his numerous and careful experiments on this point to preconceived opinion, came
to the conclusion that the blue coloration of milk is not caused by vibrions, but
by a lifeless chemical ferment contained in the decomposing casein. If, then, this
work is mentioned now, it is not for the purpose of controverting its untenable
conclusions, but because it contains an instructive description of the development
of the milk disease in question. It runs verbally as follows :

"Under ordinary domestic conditions blue milk occurs only in the warm season,
and persists from early summer to autumn. In small households where the milk
is not kept in a separate chamber, but in warmer apartments (living rooms), the
evil may be prolonged through the winter. A case of this kind is recorded by
Steinhof as lasting for twelve years, without interruption, in the house of a farmer.
The blue coloration appears from twenty-four to seventy-two hours after the
milk is drawn from the cow, the process being accelerated by warm, clare weather,
and retarded by cold. At a temperature of i5°-2o° R. (i8|° -25° C.), it may
ensue in twenty hours — the shortest limit of time observed by H. — whilst, at
only a few degrees above zero, it may linger on to the seventh day. The colora-
tion always begins to form at the surface, never in the depths of the liquid, and
generally appears at first as isolated patches or dots, immovable, and increasing
in circumference and depth ; so that there are various stages, ranging from
single superficial patches to almost complete impregnation of the mass with blue
colour. "

F. NEELSEN (I.), in 1880, took up the work commenced by Fuchs. Convenient
and reliable methods of pure cultivation were, however, lacking at that time, and,
in fact, the cultures prepared by Neelsen, when subsequently examined analytically

u6 CHROMOPAROUS BACTERIA

at the laboratory of the Berlin State Board of Health, were found to consist of a
mixture of four species of bacteria, only one of which proved capable of develop-
ing blue coloration in milk. This one was named by HUEPPE (IV.) Bacittus
lactis cyanogenus, and consists of actively motile, spore-producing rods, o. 3-
o. 5 /x broad, 1-4 /z long, such as are shown photographically in Fig. III. of Plate I.
They are non-liquefactive towards gelatin, and are extremely sensitive to higher
degrees of acidity in the medium, a circumstance explaining the facts (noted by
Haubner) that sour milk does not turn blue, and that blue patches produced in
sweet milk cease to spread when the latter turns sour.

This fission fungus is highly aerobic, and consequently requires oxygen as an
essential factor for its development. In order that this need may be supplied,
the organism always grows on the surface of the liquid, so that the colour is
produced in that situation solely, and only becomes disseminated through the
bulk of the milk by diffusion.

This microbe grows not only on milk (and equally well on human milk as on
that of the cow, ewe, goat, mare, ass, and dog), but also on many other media.
On several of these (e.g. almond milk, boiled rice, boiled potatoes, vegetable
casein, Cohn's nutrient solution qualified with ammonium lactate) it also develops
colouring matter which, on the other hand, is not formed in cultures on animal
albumen (egg-albumen, blood serum), gum, and a few other media. In artificial
inoculations the period of incubation, i.e., the time elapsing between the in-
oculation and the visible appearance of the blue coloration, is found to be about
twenty hours, but, as Haubner ascertained, depends on the prevailing tempera-
ture. Milk with a tendency to this disease transmits the property to the butter
prepared from it.

The colouring matter is not stored up within the cell, but is merely produced
there and excreted into the surrounding medium ; Bacillus lactis cyanogenus
is therefore chromoparous. The constitution of the colouring matter has not
yet been determined with accuracy, but 0. ERDMANN (I.) was led by his com-
parative experiments to consideritas one of the aniline group, viz., triphenylrosani-
line. Neelsen's endeavours to prepare it in a pure condition were frustrated by
reason of its instability. Dilute solutions of acetic acid, hydrochloric acid,"phos-
phoric acid, sulphuric acid or nitric acid produce no noticeable alteration. Ammonia
gives rise to a violet tint, whilst under the influence of the hydroxide or the car-
bonate of potassium or sodium, conversion into a beautiful rose -red occurs, the
original colour being restored by acidification. Frequently the low degree of
acidity of the milk, under which condition the bacillus is still able to develop, is in-
sufficient to enable the colouring matter to assume a deep blue tint. The colour
of the surface of the liquid is merely greyish-blue, and only becomes a pure,
full blue when the lactic acid bacteria come into action and raise the acidity to
a sufficient degree. The colour shade in individual instances may exhibit any
intermediate tint between a delicate light blue and the deepest indigo. Accord-
ing to Neelsen, the absorption spectrum of this colouring matter, which consists
of the strong lines E and F and of a broad band in the yellow, is almost
identical with that of triphenylrosaniline. From the researches of C. GESSAED
(I.) and K. THUMM (I.), it appears that Bacillus cyanogenus also produces, in
addition to the blue colouring matter, a yellow fluorescent substance. L. HEIM
(III.) and P. BEHR (I.) have given an account of a variety of this bacillus that
had lost its faculty of producing colouring matter in nutrient gelatin,
nutrient agar-agar, and skim milk. W. ZANGEMEISTER (I.) found on milk that
turned blue spontaneously a fission fungus (B. cyaneo-fluorescens) allied to
Bacillus cyanogenus, further particulars of the properties of which will be found
in the treatise referred to.

Bacillus cyanogenus is not injurious to health. The poisonous properties

BLUE COLORATION IN CHEESE 117

attributed to blue milk by early observers — Steinhof and Mosler — have been
controverted by Haubner ; therefore, if illness has actually ensued on the con-
sumption of such milk, the fission fungus in question was not to blame. Harm-
lessness apart, blue milk is not a merchantable article, and its sale should be
prohibited, since its appearance is, to put it mildly, unappetising.

Since the causes of this blue coloration in milk have become known, its
occurrence has, as a rule, been very limited ; and when it is observed, many ways
of combating it are employed, chief among them being scrupulous cleanliness in
all appliances and utensils with which the milk comes into contact. The dairy
or milk-room is then thoroughly sulphured several times, and, finally, a little
salt or Glauber salt (sodium sulphate) is added to the cows' dietary. This last-
named remedy must, however, be employed with discretion, since, under certain
circumstances, it may prejudicially affect the health of the animals and the
composition of the milk, as was ascertained by E. HESS, F. SCHAFFER, and
M. LANG (I.). The milkers' hands and the cows' udders should be carefully
washed before milking. The sudden appearance of the evil, and its frequent
disappearance after a change of fodder, permit the conclusion that the bacillus
occurs not infrequently on ceitain vegetables, from which it finds its way into
the dung of the animal and thence into the milk.

As has already been stated, the blue coloration of milk by bacterial agency is
effected gradually when the liquid is left to stand, and first makes its appearance
on the surface. If, on the other hand, the milk is of a bluish or blue colour
when freshly drawn, this results from the cow having partaken freely of the
flowering rush (Eutomus umbellatus), which contains a blue colouring matter
(indigotin ?) that is taken up by the gastric juices, conveyed in an unaltered
condition into the arterial circulation, and thus finds its way into the milk.

§ 95.— Blue Coloration in Cheese.

This is an evil to which the Dutch dairy industry is particularly liable.
Since it very often makes its appearance only after the ripening stage is over —
that is to say, at a time when the article has already passed into the hands of the
salesman — it is the cau^e not only of monetary loss to the cheese-maker, but
also of unwelcome complaints on the part of the purchaser. The phenomenon
manifests itself in various forms. Either the whole bulk of the cheese is of a
bluish cast, or exhibits blue patches internally, or, finally, is interspersed with
blue spots (Dutch, "stipjes") from i to 2 mm. (^ to -^ inch) in diameter, the
latter form (blue grain : Dutch, " blauwstippigkeit ") being the most common.

The causes of blue cheese are twofold ; one of them being chemical, and due
to iron sulphide, as was demonstrated by M. SCHMOEGER (I.) and TH. KLARVER-
WEIDEN (I.). Normal cheese contains but a very small quantity of iron ; Cheddar,
for instance, having 0.009 Per cen^. ; Gouda, o.on per cent., and so on. If,
however, a larger quantity of iron obtains admittance to the milk or freshly
precipitated curd, it will then gradually, during the ripening process, enter into
combination with the sulphuretted hydrogen separated from the albumen by
bacterial action, aud will form iron sulphide, which, as is well known, exhibits a
blue shade when in a dilute condition. When the iron has been admitted in
the soluble form, then cheeses coloured a fairly uniform blue or bluish shade
throughout will result. If, on the other hand, the metal be present in coarser
particles, e.g. in the form of rust, then a patchy-blue or blue-grained cheese will
be obtained. This explains the more frequent occurrence of the phenomenon
since the introduction of the centrifugal machine into the dairy industry, the
numerous rivets of the iron cylinder of this machine being, as demonstrated by
Schmoeger, so many sources of contamination of the milk by rust. Careful

u8 CHROMOPAROUS BACTERIA

tinning of the interior of the cylinder is therefore advisable, and should be
renewed in good time. One observation made by Klarverweiden still remains to
be mentioned, viz., that the frequency of the occurrence of blue cheeses in Holland
is coincident in point of time (August and September) with the highest per-
centages of the iron bacterium, Crenothnx Kilhniana, in natural waters.

Blue cheese may, however, also result from the activity of micro-organisms.
H. DE VRIES (I.), in 1887, asserted that a microscopical examination showed
that the blue dots in question ought to be considered as colonies of a blue pig-
ment bacterium ; but he made no attempt to prepare cultures thereof. The
subject of his investigations was Edam cheese, a kind very frequently affected
by blue grain. BEYERINCK (X.) identified, as the cause of this disease, a fission
fungus which he named Bacillus cyaneo-fuscus. However, before proceeding to
the consideration of the properties and functions of this organism, we must
devote a little attention to the microscopy of cheese.

The structure of sound cheese, as exemplified by the appearance of thin
sections immersed in water under the microscope, is made up of the following
elements. The ground mass or matrix consists of amorphous casein enclosing
small drops of fat and bubbles of gas, which are, however, for the most part, not
spherical, but irregular in outline, owing to the pressure of the enveloping curd.
Along with these two enclosures — readily detectable by their optical and micro-
chemical behaviour — a closer examination will reveal crystalline spheroids of a
substance allied to or identical with tyrosine ; also yeast cells, and, finally, a
large number of bacteria. The crystalline spheroids are oval or kidney-shaped,
and about as large as big yeast cells (some 10 p long by 8 /* broad). Each of
these crystalline aggregations exhibits (like starch granules) a central nuclear
spot, around which the crystal needles are arranged radially, and constitute in
their- totality a crystalline spheroid. A thin section cut from a blue patch or
speck of a blue cheese reveals, in addition to these normal constituents, sundry
granules of colouring matter, mostly bluish-black, but frequently brownish in
colour, and forming the actual pigmentary substance. The crystalline spheroids
are also highly coloured. Bacteria will be found more plentifully assembled in the
centre of the blue patch or dot than in any other position. The (usually globular)
colour granules, the diameter of which varies from i to 5 /u, are an excretory pro-
duct of the £acillits c^aneo-fuscus, which can be isolated from the blue patches.

The dimensions of this motile microbe vary according to the conditions of
nutrition. On nutrient gelatin the cells grow to a length of 1.0-1.5 /* D7
0.2-0.3 fj, in breadth ; whereas in a solution of 0.5 per cent, of peptone in ditch-
water they only develop to 0.3-0.6 /z by 0.15 /*. If we follow up the alterations
gradually occurring in a culture of this fission fungus in the latter medium at
10° C., we find that after four or five days the liquid, which has hitherto been
pale yellow, assumes a more and more decided greenish cast. A sample taken
from the bacterial skin covering the surface discloses two constituents : a number
of colourless rods connected to form bands, and, secondly, lying between them,
the blue pigment granules, almost globular in form and 1.5-3.5 ^ in diameter.
The colour of the liquid then successively changes by degrees (beginning at the
surface) into brownish-grey, brown-black, and finally into persistent yellow, the
conversion being effected by the oxidising influence of the air, which slowly
decomposes the blue colouring matter and forms brown intermediate products,
all finally oxidised to a colourless compound. The micro-chemical analysis of
these pigment granules shows them to consist of a framework of albuminous
matter on which the pigment crystals rest, but the constitution of the colouring
matter itself has not been established, although Beyerinck, on experimental
grounds, consideis it to be allied to indigo. It resists the reducing action of the
bacteria in cheese, especially those productive of lactic acid.

THE FERMENTATION OF INDIGO 119

Bacillus cyaneo-fuscus is very susceptible both to desiccation — on which
account it is not found in atmospheric dust — and high temperatures, as also to
acids. This explains why the microbe cannot be cultivated from the blue granules
in old ripe cheese, since it is no longer alive therein, but has succumbed under
the influence of the lactic acid (of which Edam cheese, for example, contains
between 1.3 and 1.8 per cent.) produced from lactose by the other bacteria in
the cheese during the ripening process. Considered from this standpoint, the
good result obtained by so-called ropy whey (a culture of long thread and lactic
acid bacteria, more particularly described in chapter xxix.) as a preventive of
blue grain becomes obvious. Since, as already stated, oxidising agents destroy
the colour, it has been proposed by De Vries to decolorise blue cheese by ex-
posing it for some time to the action of oxygen in a closed vessel.

This Bacillus cyaneo-fuscus (not infrequently met with in natural waters and
soils) is the sole species that as yet has been positively identified as capable of
producing blue grain in cheese. It is uncertain whether this faculty is also
inherent in other organisms, though Hollman, who, in his " Handboek voor den
Kaasmaker " (•'' Cheesemaker's Handbook "), deals thoroughly with this disease,
ascribes the responsibility for its appearance to Bacillus cyanogenus — an assump-
tion which, however, according to the inoculation experiments conducted in this
connection by Adametz and Beyerinck, is untenable.

For the sake of completeness it should be recorded that, according to Beyer-
inck's report, Bacillus cyaneo-fuscus has been found to make itself unpleasantly
apparent in a glue factory by giving rise to black glue, which, by reason not
only of its undesirable colour (due to the pigment developed by the microbe),
but also of its diminished setting power, was thus seriously depreciated in value
and merchantable quality. The source of infection was discovered in a dirty
pipe previously used for the conveyance of ditch water, and afterwards for
delivering the finished glue into the setting pans. When this pipe was cleansed
the evil disappeared entirely.

§ 96.— The Fermentation of Indigo.

As is well known, the indigo so highly prized for dyeing, and constituting such
an important article of the world's commerce, is obtained from certain species of
the Leguminous genus Indigofera. The province of Bengal alone produces from
Indigojera tinctoria over twelve millions of pounds of indigo per annum, the
value being about ^2, 000,000 sterling. Some five hundred thousand workers
make a livelihood in that province by the cultivation and treatment of this plant.
The colouring matter does not exist ready formed in the plant, but is developed
therefrom by the fermentation of a glucoside constituent known as indican. The
plants are cut down shortly before flowering-time, and are left immersed in a
five to eight-fold quantity of water, to undergo a continuous fermentation for
eight to fifteen hours at an atmospheric temperature of 25°-35° 0., the liquid
gradually becoming yellow in colour, with an alkaline reaction, and throwing up a
blue-violet scum. The most important of the fractional processes constituting this
fermentation is the splitting up of the indican into sugar (indiglucin) and indigo
white, which remains in solution in the alkaline liquor. The liquor is then drawn off
into another vessel and beaten with rods, whereby numerous and fresh points of
attack are presented to the air and the indigo white (C16H12N2O2) is oxidised by
atmospheric oxygen into indigo blue or indigotin (C16H10N202), which is precipitated
as an insoluble body and deposited at the bottom of the vessel. The sediment is
then boiled in a pan, strained through cloths, dried, and brought into commerce
in the form of irregular fragments, cubes, balls, &c.

Several noteworthy observations on. the organisms taking part in this

120 CHROMOPAROUS BACTERIA

fermentation have been recorded by ALVAREZ (I.). The fermentation may be
performed on a small scale bv comminuting fresh leaves of the indigo plant and
leaving them to stand in water, whereupon fermentation, attended with evolu-
tion of heat, will ensue in twelve to twenty-four hours, the surface of the liquid
becoming covered with a thin blue skin, which, when broken, subsides to the
bottom and is succeeded by a new one. Microscopical examination shows that
this skin consists of bacteria surrounded by fine blue acicular crystals. A
sterilised extract of the leaves, when inoculated with a little of this skin,
exhibits normal indigo fermentation, whereas without inoculation it remains
unaltered even when air is freely admitted. Indigo fermentation is therefore
due to the activity of a fission fungus, the Bacillus indigogeniis, which is asso-
ciated in the said skin with other species of bacteria that need not be taken into
account here. This bacillus is of variable dimensions, but generally 3 p long and
1.5 p. broad, and is always surrounded by a gelatinous envelope. Its microscopic
appearance is almost the same as that of Friedlander's pneumonia microbe (see
Fig. 7), and being motile, it is thereby able to collect at the surface of the liquid,
where the desired supply of oxygen is found. It also produces a disengagement
of gas, to which the formation of a froth or head on the liquid is attributable.
When introduced into the blood of the guir.ea-pig, Bacillus indigogenus proved
pathogenic, and therefore belongs to the group of organisms endowed with both
zymogenic and pathogenic properties. A second observation made by Alvarez
is also worthy of note, viz., that the microbe of pneumonia can set up indigo
fermentation, whereas on the other hand many other Dathogenic bacteria proved
incapable of so doing.

Apparently unacquainted with these results obtained by Alvarez, C. J. VAN
LOOKEREN (I. and II.) expressed the opinion that the decomposition of indican
is not produced by micro-organisms, but by an enzyme present in the living
protoplasm of the leaf -cells. The reasons whereon this hypothesis is based do
not, however, carry conviction, since attempts made to isolate the alleged enzyme
proved unsuccessful. Apart from this, however, the treatise can be read not
without profit, and ife also affords several welcome supplementary additions to
the bibliographical references collected by G. v. GEORGIEWICZ (1.) in his mono-
graph on indigo.

Alvarez's researches ought to be regarded as a thankworthy preliminary work
incentive to further study of the subject, but they do not afford a closer insight
into the progression of the fermentation process in question. It is in this case
not merely a matter of determining the nature of the ferment or ferments, but
rather of the solution of a whole group of problems of both scientific interest
and technical importance. Commercial indigo, as is well known, contains, in
addition to indigotin, a number of other organic constituents, such, for instance,
as the indigo red or indirubin, isomeric with the blue and soluble in alcohol ;
also indigo brown, obtained by treating the indigo with alkalies ; and finally,
indigo gluten, soluble in dilute acids. The proportion of these subsidiary
constituents influencing the shade of the colour is variable in different samples
of indigo. An investigation of the conditions affecting their production is a
necessary preliminary to the establishment of the most suitable method of
fermentation, capable of yielding, on the one hand, the maximum quantity of
indigo (in Lookeren's experiments 0.2 per cent, of the weight of the plant),
and on the other, producing at will any desired variety of the pigment. A very
promising field of profitable enterprise is thereby opened up to mycologists
residing in the districts where the indigo plant is cultivated. Moreover, it is
incumbent on fermentation physiologists in the centres of consumption to study
the fermentation of the indigo dyeing-baths — the woad-vat, the so-called potash
bath, and, finally, the urine bath — the preparation and emplo}ment of which are

VARIETIES OF BACILLUS PYOCYANEUS 121

carried on according to old-fashioned rules and without any knowledge of the
internal reactions occurring therein. One thing is certain : these reductions are
not purely chemical operations, but are true fermentations, as already emphasised
by A. FJTZ (IV.) in 1878. Frequently they proceed in an undesired direction ;
two such maladies of the indigo bath being : destruction of the colour, and
blackening. However, as regards these, mycological investigations are still
lacking.

§ 97.— Varieties of Bacillus Pyocyaneus.

The number of bacterial species capable of producing blue colouring matters
is by no means exhausted by those mentioned in the three preceding paragraphs.
A fourth, which has been repeatedly referred to in earlier chapters, is the Bacillus
pyocyaneus, described by C. GESSAED (II.). This organism, conveyed in atmo-
spheric dust, comes in contact with the pus exuded from wounds, and developing
therein, produces, as its name implies a coloration ranging from blue to verdigris
green. This microbe, which really belongs to pathological and not to technical
mycology, is very mutable — a property which must also be briefly dealt with here.
In addition to the blue pigment designated pyocyanine — which may be separated
from the cultures byshaking them up with chloroform, and which was first prepared
in the pure crystalline state by FORDOS (I). — the parasite in question, when
cultivated in bouillon, produces a green fluorescent colouring matter. By skil-
fully modifying the conditions of nutrition GESSARD (III. and IV.) obtained three
varieties, morphologically indistinguishable, one of them producing only pyocya-
nine, the other only the fluorescent pigment, and the third no colouring matter
at all. Whilst these 'three varieties can be re-transformed into the typical
species, such change cannot be effected with a fourth variety, cultivated by
CHARRIN and PHISALIX (I.), which had permanently lost its chromogenic faculty.
Unaware of this variability, P. ERNST (III.) proposed to distinguish between two
species — Bacillus pyocyaneus a. and ft, a course which Gessard considers incorrect.
In contradiction to the results obtained by the latter worker, and confirmed by
HANS BUCHNER and ROHRER (I.), K. THUMM (I.) asserted that B. pyocyaneus
produces only a single pigment, the blue. Undoubtedly this investigator, as a
result of the system of cultivation preferred by him, unwittingly deprived the
bacillus of its property of elaborating the green fluorescent colouring matter.

During the past two decades there has been isolated from water, air, and soil a
number of blue-producing species of Schizomycetes, which, however, are of no
special importance, and can therefore merely be mentioned here, though fully
described in Eisenberg's work. They are : Bacillus janthinus, obtained by Zopf
from the river Panke, Berlin ; Bacillus berolinensis indicus, by H. CLAESSEN (I.)
from the Spree ; Bacillus lividus, by Plagge from Berlin town-water ; Bacillus
cceruleus, by ALLEN J. SMITH (I.) from the water of the Schuylkill river. It is
probable that these species are chromoparous, as was established with certainty
in the case of a fission fungus obtained by Voges from natural water in Holsteiii
(and also named Bacillus cceruleus), which excretes the blue colouring-matter
into the surrounding medium, where it collects into small granules.

At the present time a fairly large number of species producing violet
pigments are known ; they are found in the same places as the above-named, and
are equally of little practical importance. A few may be cited as examples, the
oldest species known being the Bacteridium violaceum, observed by Schroeter,
and named Micro-coccus violaceus by Cohn. This organism develops on solid
media (gelatin, potatoes, &c.) to solid-growing violet-blue colonies. In contrast
to this is Bacillus violaceus, which liquefies gelatin and produces a deep violet colour.

Bacillus membranaceus amethystinus was discovered by Jolles in Spalato well-
water, and produces a dark violet pigment exhibiting a metallic lustre.

122 CHROM OP ARGUS BACTERIA

§ 98.— Green Bacteria

were observed by Schroeter, and COHN (I.) named one such species (producing a
sap-green colour) Micrococcus chlorinus. VAN TIEGHEM (III.) introduced into the
literature of the subject two new green non-motile species under the names of
Bacterium viride and Bacillus virens. The Bacterium chlorinum, discovered by
ENGELMANN (VII.), is more highly interesting. It is endowed with powers of
locomotion, and when present in microscopic preparations where there is a lack
of oxygen it strives to reach such spots as are illuminated by white, yellow,
or red light. The three last-named species are not chromoparous, but chromo-
phorous bacteria. W. SYMMERS (I.) described a Bacillus viridans. Reference may
be made in this place to a treatise on the green bacteria by P. A. DAXGEARD (I.).
Green fluorescent transformation products are excreted by very many species
of bacteria, a number of which are described in Eisenberg's work. At present
mention of three will suffice, viz., the gelatin-liquefying Bacillus fluorescens ligue-
faciens, and the Bacillus fluorescens non-liquefaciens, which grows on the same
medium into solid colonies. Both organisms occur in natural waters. The
author in 1891 discovered a fission fungus of very frequent occurrence in Munich
butter, and named it Bacillus butyri fluorescens.

C. GESSARD (V.) recorded a beautiful observation in connection with the
formation of fluorescent pigment by Bacillus pyocyaneus. This substance is — in
the case of nutrient solutions prepared artificially from mineral salts — produced
only when phosphates are present in quantity equivalent to at least 0.25 grm. of
potassium phosphate per litre. When this supply is reduced, the organism
develops, but produces no fluorescence. This behaviour is also exhibited by
other fluorescent bacteria, so that these can therefore be regarded as very delicate
tests for potassium phosphate.

K. THUMM (I.) comparatively examined a number of bacterial species
yielding fluorescent cultures, viz., Bacillus fluorescens tennis, B. fl. putidus, B.fl.
albus, B. erythrosporus, B. viridans, B. pyocyaneus, Bacterium syncyaneum
(Bacillus syncyaneus). These species produce, in alkaline gelatin, a fluorescence
initially sky-blue, but afterwards moss green, caused by an excreted yellow
pigment, which is formed only when the medium contains magnesium sulphate
and potassium phosphate.

The green coloration of cheese is, as first determined by CARLO BESANA (I.),
due to the presence of copper, this evil being especially manifested by Lodisan
cheese, the Parmesan cheese produced in Lombardy — formagyio di grana
lombardo. The initially yellow cut surface of this cheese becomes green by
exposure to the air. For the successful preparation of this cheese a certain
fairly high degree of acidity in the milk — equal, according to J. RAY! (I.), to
0.22 per cent of lactic acid — is essential, and therefore it is the custom in
Lombardy to leave the milk to acidify -in untinned copper vessels, whereby it
takes up a considerable quantity of copper. In fact, the progress of the souring
is determined by the gradual disappearance of the metallic lustre from the
previously polished surface of the vessels. G. MARIANI (I.), who examined
twenty-five samples of these cheeses for their content of copper, found the
minimum to be 54 mgrms. of Cu per kilogrm. of cheese, and the maximum 215
mgrms., the average per kilo, of Lodisan cheese being 100-110 mgrms. of
copper. That this metal alone is actually responsible for the green coloration of
cheese is evidenced not only by comparative laboratory experiments with tinned
and untinned milk vessels, but also by the fact that the Parmesan cheeses made
south of the river Po (especially in Reggio), and brought to Parma for sale,
undergo no alteration in their yellow colour when cut. In that region, however,
the milk is left to acidify in wooden tubs.

CHAPTER XV.

PHOTOGENIC BACTERIA.

§ 99.— The Genus Photobaeterium.

THE first occasion of witnessing the phenomenon known as marine phosphorescence
will never be forgotten by the beholder. The boat cleaving the gleaming waves
inscribes its track in glittering lines, and every movement of the water causes
pronounced phosphorescence. A rain of sparkling drops of light trickles from
the poii-ed oar, each apparently becoming the centre of fresh evolutions of light.
This phenomenon, the sight of which is calculated to captivate the senses of the
coast-dwelier, and lead him to forget all trouble for a time, has a counterpart
which filled the mind of earlier races of mankind with terror and lent great
support to credulity, viz., the phosphorescence of meat, fungi, arid decaying wood
in forests. It is only in our own day that an insight into the cause of this
wonder has been gained, the microscope, in this case also, being the instrument
used to open the door to knowledge. To devote a few words to this phenomenon
would be in any event justifiable, on account of its general scientific interest;
and moreover, the matter cannot be avoided iu the present work, since some of
the facts established in this connection also deserve a brief consideration, both
from a chemical and a technical standpoint.

The first attempt made to investigate this long-known phenomenon in a
scientific manner was that of G. FABRIZIO (I.) in 1592. The treatises subse-
quently published thereon (up to 1887) are to be found included in a historical
and critical review of the subject written by F. LUDWIG (I.). This has been
supplemented (up to 1891) by a work issued by O. KATZ (I.) which may be
referred to in this place.

We are indebted to Pfliiger for the first microscopical examination of this
phenomenon. He examined, in 1875, the white mucus covering the surface of
fish exhibiting a silvery phosphorescence, and found it composed of globules,
frequently united to form chains. When these forms were mixed with water
and the mixture passed through a doubled layer of dense filter-paper, the
latter became phosphorescent, whilst the filtrate ceased to exhibit this property,
thus proving the phosphorescence to be due to the minute organisms themselves.
These, which were recognised as bacteria, were in 1878 named Micrococcus
phosphoreus by Cohn. Wht reas Pfliiger's studies were con ei ned with the
carcases of phosphorescent salt-water fish, the first microscopical examination of
the phc sphorescent flesh of cattle slaughtered for food was made by Niiesch, who
a'so founrl a fission fungus, which he named Bacteriiim lucens, to be the cause.
Ludwig, in 1882, showed that a transference of the mucus ot ph( sphorescent
sea-fish on to sound animal flesh rendered the latter phosphorescent in turn.
Unawaie at the time that the organism Lad already received two i ames,
he bestowed on it the title of Micrococcus Pflugeri. This was the first phos-
phorescent bacteria obtained (in 1885) as an undoubtedly pure culture. Three
years later B. FISCHER (II.) proved the existence of other species, three of
which he himself described, one of thim (from the West Indian seas) being
named Bacillus y hosphorescer/s, and the other two (found on Geiman shores)

123

124 PHOTOGENIC BACTERIA

he called respectively Bacterium phosphorescens and "native phosphorescent
bacillus."

In 1890 BEYERINCK (XI.) proposed the generic name of Photobacterium for
the entire group of phosphorescent bacteria, and more closely investigated the
six undermentioned species :

1. Photobacterium Pflugeri.

2. Ph. phosphorescens.

3. Ph. balticum = Fischer's "native phosphorescent bacillus."

4. Ph. Fischeri = Bact. phosphorescens, F.

5. Ph. indicum = Bacillus phosphorescens, F.

6. Ph. luminosum.

With these six (motile) species Katz in his above-mentioned treatise
associated a second half-dozen species collected on the coast of Australia, and
EIJKMANN (I.) added a thirteenth (Photobacterium jacanense), repeatedly found
by him on phosphorescent sea-fish in the market at Batavia

§ 100. — The Food Requirements of Phosphorescent Bacteria

formed the subject of comprehensive investigations by Beyerinck, a few of whose
multifarious results obtained therefrom may be given here.

A remarkable difference exists between the first four and the last two of the
six species named in the foregoing list. The former require at least two organic
nutrient materia's, the one (supplying nitrogen) being a substance resembling
peptone, and the second a compound supplying the carbon : peptones alone, or
amides and the like, by themselves producing neither growth nor phosphorescence.
Ph. indicum and Ph. luminosum behave differently, peptone (or any other
albuminoid substance) being of itself sufficient as an organic food-stuff therefor.

A slight addition of sugar to the medium increases the luminosity, but a
higher percentage arrests both growth and phosphorescence, a circumstance due
not to any injurious effect of the carbohydrate itself, but to the acids produced
therefrom by the vital activity of the organism, the luminous bacteria thriving
solely on neutral or faintly alkaline media. Concerning the extent of the
injurious content of sugar, and also the varying influence exerted by the different
eaccharides, Beyerinck arrived at noteworthy conclusions in respect of his six
species. Thus, for example, maltose is taken up by Ph. phosphorescens, but is
discarded by Ph. Pfluyeri ; Ph. Fischeri is very susceptible to cane sugar, a
content of 0.5 per cent, sufficing to retard growth and suppress phosphorescence,
whereas, on the other hand, Ph. balticum will stand 5 o per cent, without injury.
A similar relation in respect of glucose obtains between Ph. luminosum and
Ph. indicum, the luminosity of the former ceasing when the medium contains
i per cent, of this sugar, whilst the latter produces light even in presence of
4 per cent.

The six species of photobacteria examined by Beyerinck are halophil, i.e.,
absolutely require sodium chloride, of which the medium must contain at least
3.5 per cent. Consequently it follows that none of these species is able to thrive
on the flesh of land animals slaughtered for human food. The luminosity
appearing on this latter substance is caused by other species, among them being
the above-named Micrococcus Pfliigeri discovered by Ludwig. The presence of
oxygen is essential to the production of phosphorescence, but is not requisite for
mere growth. For preparing pure cultures a nutrient gelatin made from fish
bouillon is employed, and, for cultivation on a large scale, boiled salt-water fish
forms an advantageous medium.

THE PHOSPHORESCENTS 125

§ 101.— The Luminous Bacteria as Tests for Enzymes.

The different behaviour of Ph. phosphorescens and Ph. PJlugeri towards
maltose can be utilised when it is desired to ascertain whether this sugar is
produced in any diastatic process. For this purpose plate cultures of the two
organisms are prepared,_a mixture of sea-water with 8 per cent, of gelatin, i per
cent, of peptone, and J per cent, of boiled potato-starch being used. On these
cultures are placed small drops of a solution of the substance whose saccharifying
properties are to be tested. Then, if this elaborates glucose or levulose from the
starch, the (thickly sown) plates will shortly become luminous at the points
affected, whereas if maltose alone is formed, the culture of Ph. Pfliigeri will
remain dark. In point of delicacy this reaction has but one compeer, viz., the
Bunsen flame reaction, whilst in respect of the duration of the phenomenon it is
unequalled. The method may also be used with advantage in the solution of the
question whether any given microbe has the power of elaborating an enzyme,
and if so, of what nature. Thus, for instance, if Saccharomyces Kefyr, a higher
fungus occurring in Kephir granules, is to be tested on this point, a plate culture
thickly sown with Ph. phosphorescens is prepared in a medium composed of
sea-water, gelatin, and peptone. On several of these non-luminous plates are
laid a couple of drops of an aqueous solution of lactose, on others cane-sugar, and
on a third series raffinose, none of which sugars are taken up by the photo-
bacterium, and consequently the plat?s remain dark. The drops are quickly
absorbed by the gelatin and formed patches named by Beyerinck diffusion-fields,
in the vicinity of Avhich inoculating streaks of Saccharomyces Kefyr are then
drawn, and, on developing, finally enter the field of diffusion. After a short
time the colonies of Ph. phosphorescens gradually begin to become luminous at
the points where the diffusion-fields are in contact with the Saccharomyceles
cultures, this luminosity occurring in all three series, and thus proving that
Saccharomyces Kefi/r produces an enzyme (known as lactase) which penetrates
into the diffusion-fields of the lactose, saccharose, and raifinose and inverts these
di- and tri-saccharides to assimilable hexoses, which then cause the photo-
bacterium to become luminous.

§ 102.— The Phosphorescents.

The question whether the light proceeds from within the organisms, or
whether they are in themselves dark, but excrete luminoas metabolic products
into the surrounding medium, has been much disputed. The latter opinion
was upheld, notably by BR. RADZISCEWSKI (1.), according to whose exhaustive
researches the aldehydes and aldehyde-ammonia derivatives are, in general,
endowed with the faculty of becoming luminous in alkaline solution, and are
thereby gradually oxidised by atmospheric oxygen. The quantities coming into
play per unit of time are comparatively small ; a solution of 1.8 grins, of lophin
in 25 c.c. of caustic potash remaining luminous for over three weeks. If, then,
the fact be remembered that the photobacteria are luminous only in alkaline
nutrient media, and in presence of compounds which are partly already aldehydes
(especially the sugars) and partly exhibit a similar constitution (e.g. glycerin and
asparagin) ; and if it be also remembered that the luminosity only occurs in
presence of oxygen, and that in the cultures acids, i.e. oxidation products, are
formed from the said luminous materials, then it will be readily understood
why Radziscewski sought the source of this beautiful phenomenon, not in the
bacterial cell, but in aldehydic metabolic products, phosphorescents, which are
oxidised, with evolution of light, outside the organism. The same opinion is

126 PHOTOGENIC BACTERIA

held in the main by Quatrefages, Owsjannikow, Ludwig, and Dubois, the
observer last mentioned designating the assumed phosphorescents luciferin.
The most important effect of the photobacteria, viz., marine phosphorescence,
stands, however in the way of the general applicability of this interpretation,
the external conditions under which this phenomenon is produced being still
unknown. It is consequently uncertain whether the food stuffs (aldehydes and
ammonia bases) necessary for the production of the phosphorescents are at such
times exceptionally present in sufficient quantity in the waves.

The spectrum of the bluish-green light emitted by Micrococcus PjJugeri is,
according to Ludwig, a continuous one, and expends from the line b (green) into
the violet. Equally continuous and more extensive — occupying the entire breadth
from D to G — are the spectra of two European species discovered by B. Fischer,
and a luminous bacterium described by J. FORSTER (I.). Here the blue and violet
rays predominate, and, consequently, these organisms can be photographed by
their own light, a result first successfully attained by Forster in conjunction
with Van Haren Noman. One year later B. Fischer also demonstrated that the
light from streak cultures of these microbes is strong enough to illuminate and
photograph other adjacent objects, such, for example, as a watch.

Marine phosphorescence can be caused not only by photobacteria, but also
by a variety of low forms of animal life. When these latter come into play, then
the illumination of the water only becomes well developed provided the latter is
in motion and the necessary supply of oxygen is thereby copiously supplied to
the photogenic animals. If, on the other hand, photobacteria are in question,
the entire surface of the water glistens uniformly and continuously with a soft
lustre. Ludwig was the first to successfully produce marine phosphorescence
artificially and on a small scale, and the experiment was then repeated on
a larger scale by B. Fischer in the Berlin Aquarium. The demonstration
well repays the slight trouble involved. Cultures of photobacteria are prepared
on salt-water fish, on which they grow and form a mucinous coating, which, on
being stripped oft' in salt water and dispersed through it, immediately produces
marine phosphorescence capable of prolonged duration.

A beautiful observation made by A. GIARD (I. and II.) must be recorded
here, viz., his cultivation — from the luminous Talitrus — of a bacterium which is
both pathogenic a,nd luminous, inhabiting the abdominal cavity of the aforesaid
aquatic animal, fully permeating all its organs, and, finally, causing its death.
During the prevalence of this malady the victim shines with a green light,
visible nearly a dozen yards off, and persisting for a few hours after the death
of the animal. This fact, established as it has been by successful inoculation
experiments, induces the supposition that the luminosity of other small marine
animals (infusoria, polyps, and medusse) may also be due to photobacterial
infection.

CHAPTER XVI.

THERMOGENIC BACTERIA.

§ 103. — Spontaneous Combustion.

THE state of our knowledge on the thermic side of the process of fermentation
does not extend beyond a few crude, isolated determinations, so that nearly
everything in this department has still to be accomplished; even the primary
question — whether there exist; fermentative organisms with purely exothermic
and others with purely endothermic cell-activity — being as yet unsolved. One
thing only has been established for certain, viz., that many microbes under
certain conditions generate heat and give it off to the environment. Hence the
chemical changes then occurring are exothermic processes.

An example of this is affor led by the organisms effecting the so-called
spontaneous heating of hay and cotton. For sundry researches hereon we are
indebted to F. COHN (VIII.), from which it appears that fission fungi, allied to
the hay bacillus already several times referred to, are here concerned. That the
heating is actually the result of microbial activity was proved by Haepke, who
ascertained that sterilised cotton-waste, under otherwise identical conditions,
only became heated when moistened with washing-water from fresh unsterilised
waste. The heating only occurs in presence of oxygen, and comes tQ a standstill
when this substance is lacking, the action being the result of brisk oxidising
activity (respiration) on the part of the bacteria in question. The fluffy greasy
waste material formed during the cleaning and spinning of raw cotton, consisting
of cotton fibres, seed capsules, &c., spontaneously rises in temperature up to
67° 0., according to the observations of Haepke, and becomes gradually con-
verted into a humous mass with evolution of the vapours of trimethylamine.
This observer attributes the fermentation occurring in this case to various
species of micrococcus.

The heating of vegetable matter to high temperatures should not, however,
always be ascribed to the action of fissiun fungi. For example, the temperature in
badly managed heaps of germinating (malting) barley may rise to 60° C. and over,
the cause of which, according to the researches of COHN (IX. and X.), is a mould,
viz., the Asp'rgillus fumigatus, nearly allied to the common mould fungus.
That the diastase in the germinating barley is thereby greatly injured is cert dn.

In many instances the spontaneous heating of the aforesaid vegetable
matters may develop into spontaneous combustion, whereby barns and spinning
works have often been set on fire. Nothing definite is known of the precise
conditions concerned in this phenomenon. In the first place, a knowledge of the
igniting temperature of the various substances under consideration is necessary,
and as this temperature is probably higher than the maximum heat supportable
by living organisms, the actual ignition in such cases cannot be directly attributed
to their vital activity. Hence, the probable explanation of spontaneous ignition
is that certain micro-organisms, by their oxidising action, convert the vegetable
fibres into a humous porous mass, which is then (like finely divided iron, &c.)
capable of occluding oxygen, whereby ignition is induced. More detailed researches
into this phenomenon are still required.

127

128 THERMOGENIC BACTERIA

§ 104.— The Spontaneous Heating- of Hops

is well known to every brewer from wide personal experience. For a more
intimate knowledge of the subject we are indebted to J. BEHEEXS (I.), who has
not only discovered a cause for the phenomenon, but has also traced a con-
nection between the latter and the well-known presence of trimethylamine in
hops, first shown by GRIESSMAYER (I.). Behrens found, in hops that had
become warm, a fission fungus, in a condition of almost pure culture, which he
named Bacillus lupuliperda, and explained to be nearly allied to the Bacillus
fluorescens putidus described by FLUGGE (I.). The cells of this newly-discovered
motile microbe are about 0.7 p in breath, and vary in length, according to the
conditions of cultivation, from 0.7 to 2.5 /*. It liquefies gelatin. Peptone
alone is insufficient for its support, a second substance from which it can derive
carbon being required ; consequently it belongs to the group of peptone-carbon
bacteria established by Beyerinck. It thrives freely in hop extract and quickly
renders sugar-free media alkaline by excreting copious quantities of ammonia
bases, especially trimethylamine. In presence of sugar the reaction of the
medium at first becomes acid, butyric acid being formed, although in Behrens'
experiments only to the extent of o.i per cent, at most. This microbe, which
gives rise to spontaneous heating in hops, and causes them to give off an odour
of trimethylamine, was found by Behrens in all the samples of hops examined
by him. It appears to be chiefly domiciled in the soil, arid passes thence to the
hop cones, which, being fairly hygroscopic, attract moisture when bagged, and
thus enable the bacillus to develop, the hops becoming ;' warm " and commencing
to decompose, whereby they are reduced in value. Timely prevention may be
ensured by removing the bagging in which the hops are packed, and thus
admitting cool and dry air into the interior of the contents, in consequence
of which the activity of the microbe is lessened. Closer studies both on the
spontaneous heating of hops, and especially into the transformation products of
Bacillus lupuliperda and their relation to the production of rancidity in hops,
constitute a productive field for future research.

The above remarks on the spontaneous heating of stored vegetable substances
may now be supplemented by a few observations concerning

§ 105.— The Fermentation of Tobacco,

which cannot well be included in subsequent chapters. The tobacco leaves,
when gathered, are allowed to become somewhat withered, and are then arranged
in moderate-sized heaps, where they undergo a so-called " sweating." The rise
of temperature occurring during this process is, as determined by Miiller-
Thurgau, a consequence of the activity of the leaf-cells, which transpire their
store of carbohydrates and convert their albuminoid matters into amides, the
heat thereby liberated effecting the gradual drying of the leaves. The water
vapour evolved condenses into the matting employed to cover the heaps, which
are then said to " sweat." The alteration of the nitrogenous bodies in the leaves
can also be effected by " shed drying." On this point more will be said in a later
chapter dealing with Botrytis cinerea in the second volume.

As soon as the tobacco leaves have finished sweating and become "shed
ripe," they are made to undergo fermentation, for which purpose they are tied
in bundles and arranged in great heaps, containing as much as fifty tons of
tobacco. Hereupon active decomposition quickly ensues and the temperature
rises. NESSLER (I ) found this to be as much as 54° C. even on the second day,
but, as a rule, the heaps are not allowed to become warmer than 50° C., further

THE PREPARATION OF BURNT HAY 129

increase being prevented by turning the heap, so that the outer layers of the
first heap become the central portion of the new one. This fermentation is due
to the action of bacteria, and was studied thoroughly by E. SUCHSLAND (I. and
II.), who, however, furnished but scanty reports thereon. A patent was granted
to him in connection with the use of pure cultures of bacteria for the purpose of
favourably influencing the fermentation of tobacco. He prepared pure cultures
of bacteria from fine West Indian tobacco and transferred them to inferior
German tobacco in course of fermentation. By this means the flavour of the
latter was so greatly improved that it was no longer recognisable as such even
by connoisseurs and experienced smokers of native tobacco.

The chemical changes hereby produced have been investigated by J. BEHKENS
(II.), according to whom the loss of matter amounts to 4 to 5 per cent., and
consists principally of soluble carbohydrates and fixed organic acids, the former
disappearing almost entirely. Carbonic acid is evolved as the result of these
changes, while FESCA and IMAI (I.) ascertained that nitrates are no longer
present in the fermented mass. The amount of nicotine is also reduced,
only 70 per cent, of that originally present being afterwards found (in one
experiment) by Behrens.

The flora of the fermenting tobacco heap does not consist solely of bacteria.
For example, JOH. BEHRENS (III.) frequently met with the Aspergillus fumigatus
already mentioned, and Davalos and Behrens also very often detected Monilia
Candida, remarks on which will be given in a subsequent chapter.

A few researches have been made into the fermentation of snuff, the most
important being those carried out by TH. SCHLOSING (I. and II.) relative to the
chemical changes involved. With respect to the part played by micro-organisms
he expressed himself as follows: "The fermentation begins at the ordinary
temperature under the predominant influence of micro-organisms, but above a
certain (still to be determined) limit, which is over 40° 0. and below 70° C., and
is probably about 50° C., the changes become purely chemical reactions in which
the living organisms have no longer any share." When giving utterance to this
opinion Schlosing was unacquainted with the newer researches in connection
with the heat-loving organisms which thrive at 70° C. It is therefore desirable
that his experiments should be repeated and extended, with this fact borne in
mind.

The tobacco subjected to this fermentation is usually moistened with a liquid
containing sugar, syrup, honey, and the like, in addition to various aromatics,
and not infrequently alcohol as well. In many cases this "sauce" also has
added to it wine yeasts, particularly for the grades known as St. Omer, St.
Vincent, and Paris tobaccos. These additions (found by experience to be partly
essential and partly useful) probably furnish the material for, on the one hand,
a weak alcoholic fermentation, and, on the other, for the formation of esters.
More detailed knowledge is, however, lacking with regard to this primary
fermentation, and to the subsequent after-fermentation of the tobacco, either
packed tightly into casks or rolled up in " pigtail " form and wrapped in linen
cloths. The snuff yielded by the latter method is finer than that obtained by
the quicker cask-fermentation process. More definite researches on this subject
are highly desirable.

§ 106.— The Preparation of Burnt Hay

must also be briefly described in this place. There are two chief methods,
differing from one another, for the preservation of green fodder, viz., either by
acidifying it, allowing it to ferment, and so producing " sour " fodder or ensilage
—for which see also chapter xxvi. ; or it is dried, and then forms hay. The
i I

130 THERMOGENIC BACTERIA

removal of the water, of which the plants now under consideration contain some
85 per cent., can be effected in two different ways. Either the necessary heat
is applied from outside, i.e. they are exposed to the sun's rays, thus producing
air-dried hay, or else the same result is obtained by spontaneous heating, and
therefore by the action of the thermogenic micro-organisms dwelling on the
plants. Here again there are two possible methods of procedure, by following
one of which burnt hay is obtained. On this subject we are indebted to
BOHMER (I.) for a critical research. According to him, the mown grass is piled
up into heaps about 10 to 13 feet high and 13 to 16 feet in diameter, the mass
being trodden down as tightly as possible in order to prevent the admission
of air — which might favour the development of mould — into the interior. In
these heaps spontaneous heating goes on and becomes apparent, often within
twelve hours, but generally in twenty-four to thirty hours. The operation is
watched, and as soon as the temperature inside the heap reaches 70° C. — which
is mostly the case in forty-eight to sixty hours — the heaps are opened and the
contents spread out thinly, a single turning being then sufficient to complete the
curing of the hay. By this fermentation, about which nothing definite is known
from the physiological point of view, not only is the desired degree of dryness
attained, but the hay also becomes friable and acquires an aromatic odour.

As mentioned above, the heaps must be opened when the temperature has
risen to 70° C., since, if this be neglected, the spontaneous heating will quickly
become spontaneous ignition. If, as is possible, it is rainy when the hay is
ready for spreading out, such preliminary labour is futile. To this circum-
stance is due the fact that burnt hay is seldom prepared, as also that, in
districts where the weather is too uncertain to allow of ordinary haymaking
being conveniently practised, another method of drying has been developed, in
which the necessary heat is likewise generated by a process of fermentation, from
which a product known as " brown hay " is obtained. This will be dealt with in
the Section on Lactic Fermentation.

SECTION V.
THE HEAT-RESISTING BACTERIA.

THEIR PLACE IN NATURE AND THEIR IMPORTANCE IN THE
FERMENTATION AND FOOD-STUFF INDUSTRIES.

CHAPTER XVII.

BACILLUS SUBTTLIS AND ITS CONGENERS.

§ 107.— Roberts' Heat Method.

THE unfavourable conditions to which the bacteria inhabiting the soil are
(herein exposed result in the accumulation of such species as are capable of
developing reproductive forms endowed with great vitality. These are carried
from the surface soil on to plants, and in this way hay becomes infested with

FIG. 40.— Bacillus subtilis.

G, a fragment of the skin formed on hay infusion, magn. 200 times. Consists of tightly packed
filamentary groups of cells. B and C show the individual parts of these threads at an early stage.
D, a thread, each separate part of which contains an oval endospore. E, the mother-cell mem-
brane swells up and the spores are liberated. F, progress of spore-germination. B-F, magn. 600.
(After Brefeld and Zopf.)

the spores of highly resistant Schizomycetes, which can withstand the tempera-
ture of boiling water for several hours. Early observers, being unacquainted
with this property, noticed with astonishment that a development of bacteria
occurred spontaneously in vegetable infusions (especially infusions of hay) that
had previously been exposed to boiling heat for an hour. The cells were almost

i 32 BACILLUS SUBTILIS AND ITS CONGENERS

exclusively in the form of short rods, and were named Vibrio subtilis by Ehrenberg
and Bacillus subtilis by Cohn. To obtain them with certainty the following
process, known as the heat method, which was devised by EGBERTS (I.), and
already referred to in § 53, is employed: Dry hay is chopped up fine,
suffused with a little water, and then left to stand four hours, at about 36° C.,
the usually somewhat acid liquid being afterwards poured oft' (ivithout filtering),
exactly neutralised, and diluted until it shows a density of 1.004. This liquid is
then boiled gently for an hour over the sand-bath in a flask plugged with cotton-
wool. Since the number of heat-resisting spores present on the hay is fre-
quently but small, sufficient hay and water are taken at the outset to yield at
least half a litre after an hour's boiling, which quantity will be sure to contain
some living germs. The flask is removed from the sand-bath, left to cool down
to the temperature of the hand, and then placed in the incubator, the tempera-
ture of which is regulated to about 36° 0. during the ensuing twenty-four hours.
At the end of this time there will have appeared on the surface of the infusion
a thin skin, which subsequently thickens and develops to a typical zooglcea. A
little of this, examined under the microscope, will present the appearance shown
at G in Fig. 40, viz., a number of closely adjacent rows of short rods.

§ 108.— Morphology of Bacillus subtilis.

That, by the aid of Roberts' method, pure cultures (in the present acceptance
of the term) cannot be obtained, need hardly be insisted upon, all that is produced
being a culture of heat-resisting bacteria ; hence the "hay bacillus" prepared in
this way by different observers will vary. Really pure cultures may, however,
be obtained therefrom by modern methods of pure cultivation. The organism
examined and styled Bacillus subtilis by Brefeld is not identical with that of the
same name described by Prazmovvski, though allied thereto, and indeed so closely
that the physiologically important phenomena of spore- germination are alike in
both kinds. This circumstance has already been fully noticed in § 58, and the
reader is therefore referred thereto. A few supplemental morphological facts
will now be added, the opportunity being also favourable for remarking that a
reliable culture of B. subtilis can be prepared by mixing crushed malt and rye in
a flask with about four times their volume of water, inserting a plug of cotton-
wool, boiling up the mixture, and then leaving it to
stand at 35°-4o° C. A thick, wrinkled skin will
rapidly develop on the surface of the liquid, and the
contents of the flask acquire a characteristic sickly-
sweet odour. At a somewhat earlier stage, before the
surface is entirely covered with skin, the liquid (which
on this account becomes turbid) swarms with numerous
actively motile rods, Formerly, with the defective
instruments at tfommand, only a single cilium could
be discerned on each of the terminal poles (see A, Fig.
FIG. 41.— Bacillus subtilis. 40), but subsequent researches established the fact
Cilia staining. that Bacillus subtilis, like many other species, is richly

Magn. about 1500. (After endowed with cilia, as may be seen from Fig. 41,
A. Fischer.) which is reproduced from a photograph. The de-

velopment of the motile rods into the multicellular

chains constituting the skin must be regarded as a transition to the quiescent
stage. The formation of this wrinkled cover first becomes noticeable when the
nutrient medium is in an advanced state of decomposition. In most of the cells
composing the chain there will then be found a firm, brilliant endospore
(D, Fig. 40) producing an uncommonly beautiful and remarkable appearance.

INFLUENCE OF NUTRITION ON GROWTH 133

The walls of the mother-cell swell up, the chain is dissolved, and the endospores
thereby liberated. Some facts indicative of their power to resist adverse
influences have been given in a previous section (§ 53). The spore membrane is
capable of swelling up, so that when the spores are placed in water each of them
soon appears to be surrounded by a dull halo — the swollen external layer of the
membrane.

§ 109.— Influence of the Mode of Nutrition on the Form of Growth.

This factor was exhaustively described, for the bacillus in question, by HANS
BUCUNEU (IV.). A few of the forms of growth observed are shown in Fig. 42.
By employing a faintly
alkaline 5 per cent, solu-
tion of meat extract,
rods (as at ia) are ob-
tained, 0.5/4 broad and
6-10 p. long. If a neutral

solution of 5 per cent, of
sugar and o.i per cent.
of meat extract be taken,
then the forms shown in
2« appear, viz., short rods
0.8 /* broad and only 4-6
p. long. Finally, in an
infusion prepared from
hay in which woody
stems predominate, the
cells (yi) have a length
of 12 ji and a breadth
of i.o p. Under all the
above conditions repro-
duction goes on with
vigour, the fission being
very rapid. The new
partition walls formed
during the operation are
at first so thin and so
faintly refractive as to
escape the eye in the case
of unstained prepara-
tions. If, however, a solu-
tion of iodine be added,
then the apparently
uniform long cells are
seen to be divided into
short cells in the manner
diagrammatically
sketched at b and c in the
figure. All these shapes
belong to the cycle of

FIG. 42.— Bacillus subtilis under various conditions of cultiva-
tion. For explanation see text. Magn. about 4000. (After
H. Buchner.)

normal forms of growth, of strong vitality, and capable of reproduction. When,
however, the composition of the nutrient solution is, from the first, unfavourable
(e.g. a solution of o. i per cent, of asparagin or albumen, and 10 per cent, of sugar),
or becomes so subsequently in consequence of the excretion of injurious waste

134 BACILLUS SUBTILIS AND ITS CONGENERS

products on the part of the cells, then there result involution forms, that have lost
their reproductive faculty and must be regarded as diseased and moribund modi-
fications. A couple of these are shown in Fig. 6. Similar forms are also produced in
nutrient media containing a larger percentage of acids than usual, the hay bacillus
being very susceptible to these reagents. The locomotive powers, as well as the
form of the cells, are influenced by the method of nutrition. Thus, for example,
the cells grown in a i per cent, solution of asparagin at 25° C. are devoid of cilia.

Bacillus subtilis liquefies nutrient gelatin. Streak cultures on agar-agar
develop into a wrinkled white pellicle. This microbe must be classified among
the extremely aerobic organisms, i.e. those essentially requiring the presence
of oxygen for their development. Care must therefore be taken that air has
admittance to the cultures. In reference to this matter, a valuable observation
was made by LIBOBIUS (I.). If air be excluded, the reproduction of the cells
ceases, but the formation of a peptonising enzyme is not interrupted so long as
the medium contains sugar. As already remarked, this microbe is extremely
sensitive to acids, even the small quantity present in normal beer-wort and beer
— and which, expressed as lactic acid, amounts to only 0.09-0.12 percent, in the
former case, and up to 0.2 per cent, in the latter — sufficing to suppress the
development of the bacillus in question, so that the brewing industry is exposed
to no danger from this quarter.

The decompositions effected by this microbe were first studied by G. VANDE-
VELDE (I.) in 1884, who was, however, unable to make use of pure cultures. On
the other hand, such cultures were used in the researches carried out by ADRIAN
J. BROWN (II.), in 1895, which were specially directed to the decompositions
sustained by the various sugars under the influence of this fission fungus. It
oxidises dextrose to an (unspecified) acid, which is thereafter entirely consumed,
and a levo-rotatory volatile dissociation product, of unknown nature, but
exerting an exceptionally high reducing power on copper solutions (such as
those prepared by Fehling and others). The decomposition of the total sugar
supplied is effected completely when the acid, by repeated neutralisations, is kept
down below 0.04 per cent. Saccharose undergoes a preliminary inversion, and is
then oxidised.

§ 110.— The Potato Bacilli.

Before the great tenacity of life possessed by many of the bacterial spores
inhabiting the soil was recognised, it frequently happened that potatoes, which
had been presumably thoroughly sterilised, became (when employed for streak
cultures), infested with a spontaneously developed, wrinkled zoogloaa of rod-
shaped Schizomycetes, which, starting from the potato skin, rapidly extended
over the cut surface. These species, observed by different workers, are, with
reference to their habitat, named the "potato bacillus." This is naturally a
very comprehensive appellation, which has to be more narrowly defined in each
separate case. These uninvited guests have their origin in the soil, sufficiently
large quantities of which remain in the depressions (known as " eyes ") in the
potato ; and the germs adherent thereto will withstand any heating that is not
pushed too far. Of the species belonging to this group a considerable number is
already known, and a few of them will be referred to later on, e.g. in the chapter
treating of "blown" cheeses. A few others must, however, be briefly dealt
with in this place, namely, the three species of most general occurrence. These
possess in common the property of growing on solid media e.g. potato cuttings,
to form a pellicle, the surface of which becomes more and more wrinkled and
convoluted, and recalls the appearance presented by the mesentery.

The most common of all is the Bacillus mesentericus vulyatus, discovered by

THE POTATO BACILLI 135

Fliigge, a plump, actively motile rod, shown in Fig. 43. The colonies on
potatoes are dirty white, can be drawn out into threads, and develop equally
well in the incubator and at room temperature. This bacillus excretes several
enzymes, one being peptonising and producing liquefaction of the medium ;
another diastatic ; and a third resembling rennet, whereby the casein of milk is
at first precipitated, but is subsequently redissolved by the peptonising enzyme.
It forms endospores, the final stage of germination of
which is shown at 6 in Fig. 43.

Bacillus mesentericus fuscus was first described by
FLUGGE (I.). The cultures on agar-agar and on
potatoes are initially yellow, but as their age in-
creases deepen progressively into brown. It also pro-
duces a peptonising enzyme, which liquefies nutrient FlG _p0tato bacillus,
gelatin. The production of spores is in this species a. two motile rods ; ft. a newly

less Copious than in the foregoing and following germinated rod, just having
kinds, the spore capsule, and un-

The vegetative cells of the Bacillus mesentericus ^d^rawn f Jom a photo-'
ruber, discovered by GLOBIG (II.), are short rods, rather graph by Xetihauss.)
more slender in form than those of B. m. vulgatus.

Like the other two species, the vegetative cells are motile and produce a
peptonising enzyme. The colour of the streak cultures on potatoes is at first
reddish-yellow, but subsequently becomes rose-red. The tenacity of life ex-
hibited by the endospores of this species was minutely examined by Globig. A
i per cent, solution of sublimate kills them after ij hours' exposure; but they
resist the action of 5 per cent, carbolic acid for more than a fortnight. For
their destruction by a current of steam at 100° C. an exposure of 5^ to 6 hours
is necessary, and they will bear without injury an immersion of three-quarters
of an hour in high-pressure steam at 109°-! 13° C. On the other hand, they
perish in twenty-five minutes in steam at ii3°-ii6° C., in ten minutes at
1 22°- 123° C., in two minutes at 127° C., and immediately in steam at i3°°.C:

The great difficulty of sterilising articles contaminated with traces of soil is
due to the great powers of resistance possessed by the spores they contain of the
species just described, as also of a large number of their congeners, which are
the hardiest of all organisms. The preservation of numerous food-stuffs, milk in
particular, is thereby rendered more expensive, as will be sufficiently demon-
strated in the two next following chapters. At present, attention will be
drawn to a phenomenon which could not well be referred to- there, viz., a
disease in bread (due to the potato bacilli), which becomes manifest by the
crumb of the loaf gradually changing into a sticky mass, capable of being drawn
out into long threads, and having a repulsive sour sweet smell. EMIL LAURENT
(II.) was the first to examine this disease closely, and he attributed it to a fission
fungus, which presumably also plays a part in the normal fermentation of dough
(dealt with in the second volume), and has therefore received the name of
Bacillus panificans. This is undoubtedly a species of the group of potato
bacilli. A second case of this disease was investigated by KEATSCHMER and NIEMI-
LOWIGZ (I.) by the aid of the plate culture method, whereby B. m. vulgatus was
recognised as the exciting agent. According to the researches of Aime Girard,
reported by BALLAND and MASSON (I.), the temperature prevailing in the
interior of loaves of bread during the baking process ranges between 100° and
102° C. The duration of exposure to this temperature being insufficient to kill
the spores of potato bacilli, those originally present in the flour will still be
found alive in the finished loaf. The sites occupied by these organisms,
which germinate and reproduce immediately, will then become the headquarters
of active masses of microbes, as already described. Batches of bread containing

136 BACILLUS SUBTILIS AND ITS CONGENERS

much bran are especially liable to this disease, since it is on this part of the grain,
and not on the enclosed flour, that the bacteria reside ; and, as a matter of fact,
it is commissariat (military) bread and Graham bread that are most frequently
affected, as was pointed out both by Loeffler and UFFELMANN (I.). The latter
observer found the Bacillus mesentericus vulgatus to be associated in this disease
with a second allied species, viz., the Bacillus liodermos, discovered in cows' milk
by LOEFFLER (III.), which owes its other name of "gum bacillus" to the thick
gummy appearance of its zoogloea-like cultures on cut potatoes. That the bread
disease in question does not make its appearance so frequently as might be
expected from the (often very large) number of potato bacilli found in the flour
is due to the strongly acid reaction of the dough, which facilitates the extinction
of the germs.

§ 111.— Bacillus Fitzianus.

If a cold-prepared infusion of hay be left to stand at room temperature, there
quickly forms on the surface of the liquid a skin composed of various organisms,
including the bacillus named above, the chemical activity of which was first
examined by A. FITZ (III.). If a little of this skin be transferred to a
sterilised solution of 2 per cent, of meat extract and 5 per cent, of glycerin that
has received an addition of some 10 percent, of calcium carbonate, then the said
microbe develops and acts on the glycerin (C3Hg03) in such a manner that ethyl
alcohol and volatile acids are the chief products formed. Fitz obtained, in two
experiments, a yield of alcohol amounting to 25.7 and 25.8 per cent, respectively.

The fermentation is very brisk,

o O O *

and attains its maximum with-
in twenty-four hours.

At the time Fitz made his
experiments, no method of
preparing pure cultures had
as yet been devised. It is
therefore of interest to record
that his reports were tested
by H. BUCHNER (VI.) by the
aid of the dilution method.
The results were confirmatory,
not only of the fermentative
activity, but also of the pleo-
morphism of this glycerin-
ethyl bacterium, which was
later named by Zopf Bacillus
Fitzianus. As shown in Fig.
44, this fission fungus occurs
both as cocci and as short and
long rods, and is able to pro-
duce endospores. Though of
no practical importance, it
is mentioned here chiefly in
order to show that the pro-
duction of ethyl alcohol during
fermentation can be effected,
not only by the higher fungi (yeast in particular), but also by Schizomycetes.
This fact is overlooked by many chemists when they speak of " alcoholic fermen-
tation fungi," meaning thereby yeast alone.

Allied to the last-named bacillus as regards fermentative activity is the

OO CO

FIG. 44.— Bacillus Fitziunus.

a, b, f, g. Cocci gradually changing into short rods, then (c, e)
into long rods ; d. The same with an cndospore. Magn.
4030. (After H. Buchner.)

BACTERIAL CONTENT OF THE SOIL 137

Bacillus ethaceticus, cultivated from sheep-dung by P. FRANKLAND, J. Fox (I.),
and MACGREGOR (I.). This is a motile rod about 0.8-1.0 /u. broad and 1.5-5.1 /*
long, which, however, seems to lack the faculty of producing spores. It fer-
ments glycerin, mannite, and arabinose in such a manner that the chief
products are, in addition to small quantities of formic acid and succinic acid,
ethyl alcohol and acetic acid, the ratio found being, in the first instance
2. 1 1 : i ; in the case of mannite, 1.63 : i ; and in the last, i : 1.96. In a
subsequent communication FRANKLAND and FREW (I.) demonstrated that glyceric
acid, CH,.OH — CH.OH — COOH, is decomposed in the same way, the molecular
ratio of the ethyl alcohol to the acetic acid being about i to 4.

Whereas the last-named bacillus leaves dulcite unattacked, this hexavalent
isomer of mannite is fermented by Bacillus ethacetosuccinicus. This microbe was
discovered by P. FRANKLAND and W. FREW (II.) in a solution of ammonio-ferric
citrate, which, originally intended for photographic purposes, was found to
have spontaneously fermented with vigour. These observers give the following
equation as a deduction from their experiments :

3C6H1406 = 4C2H60 + 2CH202 + C2H402 + C4H604 + 2C02 + 2H2.
Dulcite, Ethyl Formic Acetic Succiuic Carbon Hydro-

Mannite. alcohol. acid. acid. acid. dioxide. gen.

Many other bacteria also produce ethyl alcohol, but only one more will be
noticed, and that a pathogenic organism, viz., Friedliinder's Bacillus pneumonice.
According to the researches of F. BRIEGER (I.), and of P. FRANKLAND, STANLEY,
and FREW (I.), this bacillus, when grown in nutrient solutions containing sugar
(saccharose, glucose, mannite), produces ethyl alcohol and acetic acid, together
with other fermentation products in smaller amount. These four examples may
suffice to support the assertion made above, that ethyl alcohol is producible by
bacterial activity. No practical application of this is made in the arts, the
higher fungi known as "yeasts" being exclusively used; consequently this
property of many Schizomycetes will not be referred to again. We will now
supplement the account given of the hay- and potato-bacilli by a few remarks on
the

§ 112.— Bacterial Content of the Soil.

To determine this quantitatively the procedure followed is to finely divide a
weighed quantity of soil in sterilised water and then prepare cultures from the
washings in the usual manner. P. MIQUEL (IV.), to whom we are indebted for
the first determinations made in this connection, fixed the unit of weight to be
taken as one gram, and it is to this unit that all the subjoined data refer.

It would, from the first, be expected that layers of the same soil at different
depths would exhibit differences, both quantitatively and qualitatively, with
regard to their bacterial inhabitants. The property of soils (especially clays) of
combining with the fertilising materials supplied in manures, prevents these
substances from penetrating in any quantity to great depths, and from this
circumstance alone one would expect the number of bacteria in the subsoil, at
considerable depths below the surface, to be but small. The filtering action of
the upper layers of the soil also conduces to the same end, these layers fixing not
only the fertilising substances, but also (and that in a purely mechanical way)
the bacteria applied to the soil in manures. Thus it happens that subsoil water
is perfectly free from, or at least very poor in, bacteria ; a fact established
by PASTEUR and JOUBERT (I.), and of which C. FRAENKEL (III.) has given
instructive examples. (A remarkable exception to this rule was reported by
S. ROHN and H. WICHMANN (I.)-) Finally, the influence of aeration should not
be forgotten. Thip, in the lower and comparatively undisturbed layers, is almost

138 BACILLUS SUBTILIS AND ITS CONGENERS

nil, and the qualitative nature of the inhabitants of the soil is greatly influenced
by this factor, the upper layers being relatively the richest in aerobic bacteria.

The greatest percentage of organisms should not be expected in the uppermost
layer, since this is exposed to a too rapid alternation of excessive moisture and dry-
ness, heat and cold, as well as to the anti-bacterial influence of the sun's rays.
For these reasons the largest content of germs is always found at a depth of

10 to 20 inches below the surface ; a fact first shown by ROBERT KOCH (I.). As
the depth increases beyond this, the finds become smaller and smaller, and
approximate to nil at about 60 to 80 inches.

It is manifest that the germ content of a soil is also dependent on the presence
of nutrient substances, and that a soil rich in humus will be much more thickly
infested than a poor sandy soil. Thus BEUMER (I.) found in dune sand only
some 1000 germs per gram, which is very few ; and an almost identical result was
attained by A. MAGGIORA (I.) in the examination of a sample of sandy soil from
a hill near Turin. On the other hand, he found in tilled agricultural soil some

1 1 millions of germs per gram, and in the same weight of a sample of soil taken
from a street in Turin no less than 78 millions of bacteria.

That the degree to which a soil is warmed, as also its condition as regards
moisture and meteorological factors, all influence its bacterial population needs no
further argument. It follows naturally that the percentage of germs is higher
in summer than in winter, and that it falls in dry, but increases in wet weather.

Any reader desirous of more closely studying the bacterial content of the soil,
especially from a hygienic point of view, will find a good introduction thereto in
Fodor's work, " Hygiene des Bodens '' (Hygiene of the Soil), forming the 4th part
of the useful " Handbuch der Hygiene " issued by Weyl (Jena, 1894 et seq.).

In addition to bacteria, the soil harbours a large number of higher fungi,
comprising not only numerous innocuous mould fungi, but also the spores of
phytopathogenic Eumycetes. These are of no immediate importance to agricultural
chemistry, and therefore do not need any further consideration here. E. Ch.
Hansen demonstrated that the wine yeasts winter in the soil, but on this point
reference must be made to the chapter devoted to Saccharomyces apiculatus in
the second volume.

CHAPTER XVIII.

BUTYRIC ACID FERMENTATION AND ALLIED
DECOMPOSITION PROCESSES.

§ 113.— Anaerobiosis.

THE first mention of the fact that butyric acid, discovered by Chevreul in 1814,
can also be produced by fermentation, was made by R. MARCHANB (I.) in 1840,
in connection with his researches on the composition of the milk of the South
American cow-tree (Galactodendron americanum). In the following year
Noellner described, under the name of pseudo-acetic acid, a substance which he
had found to result from the spontaneous decomposition (fermentation) of calcium
tartrate, and which was then recognised by Berzelius as a mixture of butyric
and acetic acids. Two years later TH. PELOUZE and A. GELIS (I.) observed that
the lactic fermentations instituted by them did not progress satisfactorily, butyric
acid and considerable quantities of hydrogen being produced. In following up
this observation they formulated the recipe, still current in many text-books on
chemistry, that to start butyric acid fermentation a solution containing about
10 per cent, of sugar should be mixed with chalk and a little cheese, and left
to stand at 2 5 "-30° C.

These observers did not endeavour to follow the progress of this fermentation
more minutely, as their attentio i was entirely devoted to the new fermentation
product ; the only remark they make is that the butyric fermentation is not set
up at once in their method, b it is preceded by a lactic fermentation " without
its being possible to influence the progress thereof." It was naturally far
from the thoughts of Liebig's former fellow-worker at Giessen to attribute the
decomposition to the activity of living organisms.

The discovery of the true state of the case was made in 1861 by PASTEUR
(VIII.), who showed that two successive processes are here involved: first, the
conversion of sugar into lactic acid or calcium lactate, and afterwards the trans-
formation of the lactate into butyrate. He demonstrated that each of these changes
is due to a special ferment, of which the second is the only one we have to deal with
now. The microbe (2 p. broad and 2-15 \t long) causing butyric fermentation,
and which we now recognise as a bacillus, was regarded by Pasteur, not as a
plant, but as an animal, one of the infusoria, because it was observed to possess
powers of locomotion. Nevertheless he laid but little stress on this distinction,
the point being one of minor importance in comparison with the property he
recognised in this " vibr ion butyrique" viz., the faculty of existing without air.
This observation formed one of the main supports on which this gifted philosopher
founded his theory of fermentation, mentioned in § 16, and with regard to which
a few additional remarks will now be made.

At present we have first to consider the said peculiarity by itself, irrespective
of the resulting decomposition effected in the nutrient medium. Creatures
requiring oxygen for the continuance of their existence are termed aerobiontes,
whilst the term anaerobiontes, or, shortly, anaerobes, is applied to creatures capable
of development in the absence of this gas. A distinction is drawn between two
sub-groups, viz., strictly anaerobic organisms, i.e. such as can live only in an

139

140 BUTYRIC ACID FERMENTATION

environment devoid of oxygen, and for which this gas is therefore a poison ; and
facultatively anaerobic organisms, i.e. those to which oxygen is neither

injurious nor essential, and which can con-
j sequently thrive either in presence or absence
of air. Thus, for example, the acetic acid
bacteria are strictly aerobic, certain lactic acid bacteria
facultatively anaerobic, and the majority of the butyric acid
bacteria strictly anaerobic.

At the present time numerous species of anaerobic fission
fungi are known. PASTEUR (IX.) himself, in 1863, associated
with the " vibrion butyrique " a second anaerobic species, viz.,
the microbe which sets up fermentation in calcium tartrate.
This process of decomposition, already observed by Noellner,
but which requires more careful investigation, often occurs
spontaneously in tartaric acid works, destroying the tartaric
acid and occasioning great loss. The different species of
butyric acid bacteria will be thoroughly discussed later on. At
present, by reason of their general interest, the known patho-
genic anaerobic species, three in number, will be considered.
The first of these, as regards priority of discovery, is the
" vibrion septique" found by PASTEUR, JOUBERT, and CHAMBER-
LAND (I.), and subsequently examined more closely by R. Koch
and Gaffky, and now generally known to bacteriologists as
Bacillus ozdematis maligni. According to an opinion expressed
by Pasteur, this bacillus is identical with the one effecting the
fermentation of calcium tartrate. The decompositions set up
by the bacillus of malignant oedema in nutrient media con-
taining carbohydrates were studied by R. KERRY and S.
FRAENKEL (I.). Grape-sugar yielded ethyl alcohol, ethylidene
lactic acid, and butyric acid. From calcium lactate were pro-
duced butyric acid, a little formic acid, and propyl alcohol.
Milk-sugar and also cane-sugar were gradually fermented to
ethyl alcohol, formic acid, butyric acid, and ethylidene lactic
acid. Starch was also attacked, and yielded the three last-
named acids. With Bacillus cedematis maligni have been
associated two other pathogenic anaerobic species of fission
fungi, namely, the bacillus of symptomatic anthrax, by Feser
and Bellinger in 1876-1878, and that which causes tetanus
— Bacillus tetani — by Nicolaier in 1885. The latter is of
somewhat frequent occurrence in arable soil, and was also
discovered by S. A. SEVERIN (I.) in horse-dung. The bacillus
of symptomatic anthrax, according to the researches of M.
NENCKI (I.), when cultivated in media containing grape-
sugar, produces chiefly normal butyric acid, along with acetic
acid and optically inactive lactic acid, accompanied by the
evolution of C02 and H,. The remarkable symbiosis of this
bacillus with Micrococcus acidi paralactici has already been
briefly mentioned in § 65.

FIG. 45. — Gruber's
tube for anaerobic
cultures. Ready for
use. Somewhat re-
duced in size. (After

Oruber.)

§ 114.— Methods of Cultivating* Anaerobic Bacteria.

Pasteur covered the nutrient liquid with a layer of oil in order to prevent
access of air. This method of covering up the medium with a protective stratum
has been variously modified in order to render it applicable to solid media as

METHODS OF CULTIVATING ANAEROBIC BACTERIA 141

well. Thus R. Koch, in 1884, proposed to cover the gelatin plates with a film
of mica, which, however, according to the ex-
perience of P. LIBORIUS (I.), is not always
sufficient in the case of strictly anaerobic
bacteria. On the other hand, a second
method (also emanating from the German
State Board of Health) has proved highly
suitable, viz., that of cultures in deep layers,
as prescribed by W. Hesse in 1885. For
these a puncture culture is made in test-
tubes containing nutrient gelatin or agar-
agar, the medium being covered, after
successful inoculation, with a sterile stratum
of the same liquefied substance.

Another method, which has also been
variously modified, is that first employed by
Pasteur in connection with his studies of
the " vibrion septique," which consists in ex-
hausting the air (oxygen) from the vessel
containing the nutrient medium inoculated
with an anaerobic organism. The modifica-
tion made by MAX GRUBER (II.) is con-
venient and reliable, and is in general use,
especially in the laboratories of Fermenta-
tion Physiologists. Strong test-tubes (Fig.
45), about 7 inches long, with a much con-
stricted portion in the upper third of their
length, are used. These are filled with
about 10 c.c. of nutrient medium, then
closed by a cotton plug, and after inoculation
are immersed in water at about 3o°-35° C.,
and then connected with an air-pump. The
air is all driven out by the water vapour given
off under the diminished pressure, where-
upon the narrow part of the tube is closed
by fusion and the upper portion removed.
By the use of nutrient gelatin this method
also facilitates the cultivation of colonies, so
that the individual anaerobic species in a
bacterial mixture can be isolated. For this
purpose the still warm liquid contents of
the tubes are converted into Esmarch roll-
cultures, as shown in Fig. 46.

In place of removing the air from the
culture vessel by mechanical means — pump-
ing or driving it out by vapour — recourse
may be had to oxygen-absorbing chemicals.
For this purpose a solution of pyrogallic
acid [v-C6H3(OH)3] in caustic potash, a
mixture that takes up oxygen with avidity,
and which, as is well known, has long been
in use in gas analysis, is employed. It was
introduced into physiological work by Nencki in 1880, as a test for the presence
of anaerobic organisms, but it was not until the publication of H. BUCHNER'S (VII.)

. ',' 4

<

/^^

»*^''

^^^^^^

.* ' '<* '

^^^^^1

• ' 'V

' r' ' **

<* V. ;

^

i * • ' *

» » *,*,

•Sfc

FIG. 46.

Gruber's anaerobic

tube exhausted ol air.

- ^

Upper portion removed

^- H

by fusing. Contents

- ^

arranged as an Es-

-

march culture where-
in the germs have

2k

developed to colonies.

^

Somewhat reduced in

~" =

size. (After Gruber.)

•

FIG. 47.

Buchner's anaerobic

tube.

The pyrogallol solution

fss^S

is at p, the wire sup-

port resting therein

and carrying tlie
test-tube with the

I1

inoc ila ed nutrient

medium (n). Some-

what reduced. (After

^a^sssjp

Bucliner.) ^r"M^

142

BUTYRIC ACID FERMENTATION

m

method in 1888 that it caine into general use in Bacteriology. The test-tube
containing the inoculated nutrient medium — as a roll culture if desired — is
placed in a large test-tube (Fig. 47) about i^ inches wide and 10 inches long, at

the bottom of which has just
previously been inserted i gram
of dry commercial pyrogallic acid
and 10 c.c. of deci - normal
caustic potash. The smaller
tube rests on a small wire sup-
port, in order to prevent it from
dipping into the liquid. The

" Buchner pyrogallol tube " is closed by a well-fitting
previously-moistened rubber stopper, and may then
be placed in the incubator. When kept at 37° C. the
absorption of oxygen is complete in twenty-four hours,
or in two days at 20° C. To treat plate cultures by
this method, the culture is placed over a basin contain-
ing a sufficient quantity of the said solution and resting
on a flat plate of ground glass, the whole being covered
with a well-sitting bell-glass, the edge of which has
been rubbed over with vaseline. Another method for
the culture of anaerobic organisms consists in expell-
ing the air from the culture vessel by another gas,
e.g. carbon dioxide, hydrogen, coal-gas, or nitrogen.
Carbon dioxide is frequently recommended by the
French school, and particularly by Pasteur, but its
employment is not without objections, since it is not
an inert gas, but is absorbed by the medium, which
it then renders acid, and hence has the power of
restricting growth. Moreover, according to the re-
searches of P. FKANKLAND (I.), it acts as a fatal poison
on many bacteria. Although experience shows that
hydrogen gas is not inert, still it may be accepted
as the best to use for anaerobic cultures. Ordinary
illuminating gas was recommended for this purpose
by R. WURTY and A. FOUREUR (I.), but, according to
the researches of TH. KLADAKIS (I.), it must be re-
jected, since he found it acting as a poison on many
bacteria. Nitrogen may be regarded as perfectly
innocuous, and would long ago have been employed
for anaerobic cultures were it not that the method of
preparation is too cumbrous and costly, at least for
the physiologist. Many methods have been proposed
for the expulsion of oxygen by one of the above
gases, but only two will now be briefly mentioned
here. That of C. FRAENKEL (IV.) is concerned with
the treatment of test-tube cultures, ordinary wide
test-tubes with two-holed stoppers being employed.
One of the glass tubes inserted therein reaches
almost to the bottom of the test-tube, whilst the second is cut off close below
the stopper. When filled with nutrient gelatin, agar-agar (or bouillon,
wort, &c.), the tubes are sterilised in the steamer in the usual way, and
inoculated, a current of hydrogen being introduced through the longer tube and
passed through. When all the air is expelled the small tubes are hermetically

v^*^
FIG. 48.

Fraenkel's anai-robic tube.
Contents arranged as an Esmarch
culture round the walls, and
developed colonies appearing
as black spots. Somewhat
reduced. (After Fraenkel.)

CLOSTRIDIUM BUTYRICUM AND BACILLUS BUTYRICUS 143

sealed by fusion (Fig. 48) and the stopper smeared with warm paraffin. Esmarch
roll cultures can then be prepared. If plate cultures (e.g. in Petri dishes) are
to: be exposed to an inert gas, they are (according to P. LIBORIUS (I.) ) placed
under a bell (of copper, <fcc.) which can be tightly fixed by screw clamps against a
caoutchouc plate. The gas (when hydrogen is used) enters through a tube fixed
in the crown of the bell, and leaves by way of another tube situated below.

It is impossible to refrain from mentioning that there is another method
capable of taking rank with those described, and fulfilling all the conditions
usually prevailing during anaerobic growth in Nature, viz., the simultaneous
presence of strongly aerobic organisms. It is certain that anaerobic organisms
can often be detected in liquids to which air has unrestricted access, and such
associations of aerobic and anaerobic organisms are not difficult to bring about
by artificial means, this having been successfully attempted by R. PENZO (I.),
BEYERINCK (II.), and others. The application of this method is, however,
somewhat limited, since, of course, only mixed cultures can be produced by
its aid.

It was remarked by Pasteur that the growth of anaerobic organisms could
be promoted by an addition of sugar to the nutrient medium. Now, an
alkaline solution of grape-sugar is well known to have a strongly reducing
action; hence these two facts induced KITASATO and WEYL (I.) to ascertain
whether other reducing bodies were equally efficient ; and they strongly recom-
mended the addition of 0.3-0.5 per cent, of sodium formate, or of o.i per cent,
of sodium indigo-sulphate. A solid nutrient medium, qualified and stained blue
by the last-named substance, is decolorised as far as the growth of the reducing
organism extends. The use of indigo-sulphuric acid as a test for reducing
action was first practised in 1858 by M. TRAUBE (I.) in his researches on
ferments, and A. SPINA (I.) was the first to employ the sodium salt as a reagent
for the same purpose.

§ 115.— Clostridium Butyrieum (Prazmowski) and Bacillus
Butyrieus (Hueppe).

Pasteur's discovery that organic life is possible without free oxygen, and that
certain organisms can obtain the energy they need by so breaking down organic
compounds as to liberate heat, is one of the highest importance for physiology
generally. The amount of heat so evolved is naturally much less than it would
be if the compounds in question were directly converted into carbon dioxide.
Pasteur, however, went somewhat too far in founding on this newly-discovered
fact a theory of fermentation which culminated in the assertion that : " Fermen-
tation is a universal phenomenon, and consists of life without air, life without
free oxygen," because, if this definition be accepted, we should be able to speak
of but few phenomena as fermentation, and, in particular, it would be necessary
to discontinue the application of the term to those decomposition processes
that from time immemorial have been, and are even now, principally borne in
mind in speaking of "fermentation," viz., the alcoholic fermentation excited by
yeast, an operation which proceeds both with and without free oxygen.

To return to the " vibrion butyriqite." In the years 1877 to 1880, A.
PRAZMOWSKI (I.) published a careful morphological investigation of a butyric
acid bacterium, presumably identical with the vibrion butyriqiie — though this
cannot be stated with certainty. Elevating the designation Clostridium (first
used by Trecul, and then only to indicate a form of growth) to a generic term,
Prazmowski named two new species of bacteria Clostridium butyricum and
Clostridium Polymyxa. The latter completely coincides with the former both in
morphology and life history, but differs from the strictly anaerobic Cl. butyricum

144 BUTYRIC ACID FERMENTATION

both by its inability to exist in the absence of oxygen, and also by its incapacity
to incite fermentation (in the restricted sense of the term). Fig. 49 reproduces
the vegetative forms of growth depicted by Prazmowski as those ot his butyric
acid bacterium. They are mostly plump rods, some i /* broad. The generation
period was determined by Prazmowski as about thirty to thirty-five minutes at
35° C., and forty-five to fifty minutes at 30° 0. Under certain conditions, and
especially whilst young, the rods store up in their plasma a substance which
resembles starch (amylum) in being stained blue by iodine. This phenomenon
had already been noticed by Trecul, who gave it expression in the generic name
of Amylobacter, which he applied to these Schizomycetes. During the formation
of spores the rods swell up, as related and shown in § 49. The power of with-
standing heat possessed by the endospores of Cl. butyricum was examined by
Prazmowski. They are able to remain in boiling water for five minutes without
injury ; but if the treatment be prolonged to twice that duration, then only the

FIG. 49. — Clostridiuin butyricum.
Vegetative forms of growth ; short rods of
different lengths, partly straight (c, d),
partly curved (a, b). Magn. 1020. {After
Prazmowski.)

FIG. 50.

Clostridiuin butyricitin.
With endospore.

Stained cilia. Magn. 2000.
(After A. Fischer.)

hardiest spores of all are left alive, and even these succumb if the boiling be
extended to fifteen minutes. The progress of the germination of the endospores
has already been described in § 57 (q.v.\ The brisk locomotion of the rods is
produced by a large number of cilia, shown in Fig. 50. Prazmowski did not
have pure cultures, in the present acceptance of the term, at disposal for his
researches, but was obliged to confine himself to an approximately pure culture
prepared by means of Roberts' boiling method (§ 107). The same remark
applies to a valuable treatise by A. FITZ (VII.), wherein the fermentative
activity of a fission fungus named Bacillus butylicus is reported upon.

After E. Ch. Hansen, in 1878-79, had established, in connection with acetic
fermentation, the new and important fact that this decomposition process is
effected by at least two different species of bacteria, F. HUEPPE (IV.) in 1884
found the same to be the case with butyric fermentation, and discovered a
Bacillus butyricus which exerted its decomposing activity in presence of air.
This fact was confirmed by MAX GRUBEB (II.), working with a reliable method
of pure culture in 1887, and it was at the same time demonstrated that the
Clostridiuin butyricum of Prazmowski consists of a number of closely allied, but
nevertheless distinct, species. Nearly related to this is a ferment isolated by
P. LIBORIUS (I.) from old cheese, and introduced into literature under the name
of Ciostridium ftetidum. This organism liberates very foul-smelling gases, in

THE GENUS GRANULOBACTER 145

addition to producing butyric acid, and forms one of the many connecting links
between the butyric acid bacteria (in the restricted sense of the term) and the
so-called potato bacilli. No sharply de6ned limit can be drawn between these
two groups. In the same category must also be included one of the two species
of bacteria which were isolated in 1894 by W. KEDROWSKI (I.) from a butyric
fermentation produced by a method approximating to that of Pelouze. In still
closer relation to the potato bacilli are a few anaerobic species isolated by
C. FLUGGE (II.) from boiled milk, as also the Bacillus liodermos, frequently
observed by LOEFFLER (III.) in imperfectly sterilised milk.

§ 116.— The Genus Granulobaeter.

As the reader will be aware, organic chemistry distinguishes between two
kinds of butyric acid, only one of which, viz., the propyl carboxylic acid, having
the subjoined formula —

CH3 -CH2- CH2— COOH,

is, in the present state of science, known to result from butyric acid fermenta-
tion, on which account it is also called fermentation butyric acid. On the other
hand, the isomeric acid, which, in accordance with its constitution —

CH, H CH3

.COOH

CH.(

CH3 COOH CH3

is also styled dimethylacetic acid or isopropyl formic acid, has not hitherto been
obtained by the aid of fermentation. However, not only the first-named acid,
but also the corresponding alcohol, viz., normal butyl alcohol,
CH3— CH2— CH2— CH2OH,

can be produced by the activity of fission fungi ; so that we may also speak of a
group of the bacteria of butylic fermentation. In this connection we are
indebted to M. W. BEYERINCK (XII.) for some thoroughgoing researches, which
have not only brought new facts to light, but also led to a more definite charac-
terisation and limitation of a number of species of butyric acid bacteria. This
observer has given to the bacteria of butylic fermentation the common generic
name of Granulobaeter, since they all possess the faculty of storing up granulose
in the interior of their cells, owing to which they are stained blue by iodine.
The characteristics of this genus are given by Beyerinck as follows : " Strictly
or temporarily anaerobic fermentative bacteria, which in a condition of complete
anaerobiosis become partly or entirely filled with granulose, and then assume
the clostridium form. In presence of traces of oxygen, short motile rods are
quickly produced, which are stained yellow by iodine. Endospores make their
appearance in the c'ostridia. They are able to remain uninjured for a few
seconds or minutes at a temperature of 95°-ioo° 0. Among the products of the
fermentations set up by individual species of this genus are : carbon dioxide
always and hydrogen generally, but methane is never found."

Granulobaeter butylicum is the species producing butyl alcohol. It is pre-
sumably identical with Gruber's Bacillus amylobacler /., and is frequently met
with in the flour of cereals. It is anaerobic, and produces from maltose normal
butyl alcohol, hydrogen, and caibon dioxide, but no butyric acid ; diastase is
formed concurrently, but not glucase. A spontaneous butyl-alcoholic fermenta-
tion can be set up by gradually adding to 100° c.c. of boiling water as much
coarsely ground, unsifted, fresh barley-meal (from Hordeum di&tichum nudum)

146 BUTYRIC ACID FERMENTATION

as will produce a thick gruel, and then cooling down to about 35° C. so quickly
that the final portion of barley-meal will have been exposed to 100° C. for a few
seconds only. The mixture is kept at a temperature of 35°-37° C. At the
expiration of twelve hours bubbles of gas will be perceptible, and the presence of
butyl alcohol will be manifest, by its odour, after a further twenty-four hours.
If the aforesaid temperature be strictly maintained, almost pure Gramdobacter
butylicum will develop in the liquid, and a pure culture can be obtained there-
from, unhopped malt-wort gelatin forming a suitable medium, and one of the
methods described in § 114 being employed. In this medium the fission fungus
in question will develop into milk-white, visco-rnucinous non-liquefactive colonies.
The fermentations induced therewith (e.g. in unhopped malt-wort of not more
than 10° Sacch.), and which must be carried out in the absence of air, progress
in two stages : so long as any free oxygen remains dissolved in the liquid, deve-
lopment will proceed but slowly, only carbon dioxide and hydrogen (no butyl
alcohol) being produced. When the liquid is finally purified, then not only can
the appearance of the alcohol be observed, but also an unusually vigorous increase
of the cells, which will be found to be so full of granulose that a drop of the
liquid will become stained quite blue-black by iodine. The endospores, which
soon make their appearance, attain, with a breadth of i /*, a length which may
be as much as 2 /x. This species is very sensitive to butyric acid.

A second species is Granulobacter saccharobutyricum, the true butyric acid
bacterium, generally so called, and presumably identical with the Bacillus buty-
licus examined by Fitz. It is more widely distributed and of more frequent
occurrence than the last-named species, with which it is associated on cereal
grains and in the green malt, groats, and flour prepared therefrom. It is this
species, also, which occurs, and gives rise to damage, in badly prepared distillery
yeast-mash. Glucose and (but with greater difficulty) maltose are decomposed
by this species, butyl alcohol, carbon dioxide, and hydrogen in variable propor-
tions being produced, in addition to butyric acid. From a morphological point
of view, it is closely allied to the first-named species, but the spores are some-
what smaller ; also, like the other, it does not liquefy gelatin. Probably identical
with, or at least very closely related to G. saccharobutyricum is an anaerobic
ferment (Bacillus butyrlcus), isolated by S. Botkin (I.) from Berlin and Breslau
milk, and also frequently noticed by FLUGGE (II.) in market milk.

Granulobacter lactobutyricum is probably identical with the organism causing
butyric acid fermentation in calcium lactate, described by Pasteur. When culti-
vated in the absence of air, it grows in the form of plump, short clostridia, which
stain violet-blue (not pure blue) with iodine and convert calcium lactate into
butyrate, hydrogen and carbon dioxide being liberated. The endospores are
smaller and shorter than those of the first-named species. When kept in
presence of air, this organism converts calcium lactate into large spheroidal
crystals of the carbonate, and in this case takes the form of slender short rods,
resembling those of Bacillus subtilis and staining yellow with iodine. From the
fact, recorded by Beyerinck, that this species in a state of pure culture dies out
after several re-inoculations, whether air be admitted or excluded, it may be
presumed that the organism needs for its prosperous development the symbiotic
association of another, still undetermined, species of fission fungus.

Granulobacler Polymyxa is frequently found on cereal grains, and is pre-
sumably identical with Prazmowski's Clostridium Polymyxa. This species develops
most satisfactorily in an unrestricted supply of air, and then assumes the form
of motile rods. When the aeration is deficient, spore- bearing clostridia appear,
and a weak fermentative action is noticeable, a small quantity (traces) of butyl
alcohol, together with carbon dioxide, being formed, but neither hydrogen nor
butyric acid. — The Leptothrix buccalis, very frequently found in dental mucus,

THE EQUATION OF BUTYRIC FERMENTATION 147

and whose thread chains are generally stained blue by iodine, is also classified by
Beyerinck along with the Granulobacteria.

§ 117.— The Equation of Butyric Fermentation

is set out in a very simple form in most text-books on chemistry, that for the
decomposition of the hexoses (glucose, &c.) being given as follows —

C6H]206 = 2H2 + 2C02 + C4H802,

or with lactic acid (or its lime salt) as the raw material —
2C3H603 = 2H2 + 2COa + C4H802.

In the preceding paragraphs we have, however, made the acquaintance of a
very large number of bacterial species with divergent methods of action, so that we
must at once admit that a general equation for butyric acid fermentation is not to
be thought of. All that can be hoped for is the discovery of more accurately
defined equations for each of the various species and their characterisation, as
e.g., the equation for the fermentation set up by Granulobacler saccharobutyricum,
and so on. Nevertheless, even this limitation is not sufficiently strict, as will be
evident from what follows.

L. PERDRIX (I.) examined the fermentative capacity of an anaerobic spore-
bearing butyric acid bacterium (closely allied to Botkin's Bacillus butyricus),
which he isolated from the water in the Paris mains and named Bacille
amylozyme by reason of its property of bringing starch into solution (sacchari-
fication). When grown in a meat-broth containing glucose and calcium car-
bonate, with exclusion of air, this fission fungus produces acetic acid and butyric
acid, in addition to hydrogen and carbon dioxide. The mutual ratio of these
four fermentation products changes with the increasing age of the culture. In
the first three days it can be approximately expressed by the equation

S6C6H1206 + 42H20 = is6H2 + II4C02 + 39C2H402 + 36G'4H802,
but later on by the equation

46C6H1206 + i8H20 = ii2H2 + 94C02 + I5C2H402 + ^SCJJ^Oy

Finally, the transformation becomes simplified, acetic acid being no longer
produced, and the sugar then splitting up very nearly as follows :

C6H12O6 = aH2 + 2CO2 + C4H802.

Similar ratios were established for the fermentation of saccharose and lactose,
which is not preceded by inversion. Starch is, as already mentioned, saccharified
by the Bacille amylozyme, and is then fermented, amyl alcohol and ethyl alcohol
being formed.

Along with these Schizomycetes must be ranked the Bacillus suaveolens,
described by SCLAVO and Gosio (I.), which converts starch into dextrin and
glucose, and ferments these with excretion of alcohol, aldehyde, formic acid,
acetic acid and butyric acid, which then partly unite to form sweet-smelling
esters. Butyric acid bacteria that produce aromatic substances as well are
important for the ripening of cheese, being essential for the development of the
characteristic odours of the various kinds of cheese. However, in this matter
our knowledge is still only in a rudimentary state. E. VON FREUDENREICH (I.)
separated from milk a Clostridium fcetidum lactis, which develops, in this
medium, an odour resembling that of Limburg cheese, and the same observation
was made by H. WEIGMANN (I.). The Bacillus saccharobutyricus, isolated from

148 BUTYRIC ACID FERMENTATION

so-called " Quargelkase " (small country cheese, a sour soft variety) by V. VON
KLECKI (I.), and examined by him for its fermentative power, also belongs
hereto.

The influence of the age of the organisms used as " seed " and the reaction
of the nutrient medium on the progress of butylic fermentation was shown by
L. GRIMBEET (I.) in the case of the anaerobic Bacillus orthobutylicus. This was
isolated as a pure culture from a fermenting aqueous liquid containing calcium
tartrate and leguminous seeds, the said salt being, however, as little affected by
the bacillus as is calcium lactate. On the other hand, saccharose, lactose,
maltose, invert sugar, glucose, and the like, form favourite nutrient substances,
normal butyric acid, acetic acid, normal butyl alcohol, and a little iso-butyl
alcohol, together with hydrogen and carbon dioxide, being the products of
fermentation. The ratio of these products is found to depend on the reaction
of the medium, the yield of butyl alcohol increasing and that of butyric acid
diminishing with the increased acidity thereof, whilst the amount of acetic acid
remains unaffected. In harmony with this determination is the further fact
that, as the age — and concurrently the acid content — of the fermenting liquid
increases, the amount of butyl alcohol produced per unit of time becomes larger.
So far as the age of the " seed " (i.e. the germs and organisms transferred in the
process of inoculation) is concerned, it is found that, as regards the produc-
tion of butyl alcohol, the fermentative power of young cultures is greater than
those of more mature age. — Sundry experiments in the technical preparation
of butyric acid by fermentation were made by L. LEDEREK (I.), but these leave
much to be desired from a bacteriological point of view.

The faculty of producing starch-dissolving enzymes is widespread among the
bacteria, and is in no wise restricted to the above-named species. Our know-
ledge of these amylases or diastases (in the general sense) is, however, still in
its infancy. In this connection we may refer to a treatise by BEYERINCK (XIII.)
on glucase, the enzyme of maltose. After J. WORTMANN (I.) had already, in 1882,
made a few investigations thereon, but only in bacterial mixtures, CL. FERMI (II.)
approached the matter more closely in 1890. According to his observations
(made exclusively with pure cultures), diastatic enzymes are excreted by the
following species : Bacillus subtilis, B. megatherium, B. anthracis, B. tetragenus,
B. ramosus, B. Fitzianus, Vibrio cholerce asiaticce, and others ; this faculty being,
on the other hand, lacking in Bacillus pyocyaneus, Nicrococcus jrrodiyiosus, &c.
According to the researches of A. YILLIERS (I.), there occurs among the fission
products of the action on starch paste of a fission fungus belonging (presumably)
to the group of Granulobacteria, a small quantity (0.3 per cent, of a new carbo-
hydrate, known as cellulosin, which has the formula CI2H,0010 + 3H,0. — The
starch-dissolving action of bacteria also probably comes into play in the prepara-
tion of the alcoholic beverage known in Central America as cliicha. V. MAR-
CANO (I.) states that this liquor is prepared by steeping maize for four to six
hours in water, then boiling for a short time, and afterwards leaving the mix-
ture to settle, whereupon a brisk fermentation quickly ensues. More accurate
information respecting the fission fungus concerned is still lacking.

§ 118.— The Fermentation of Cellulose.

To dissolve and get rid of what has ceased to live is (according to an
appropriate remark made by Pasteur) the task of the fungi in general, and of
the fission fungi in particular. Without their activity the circulation of the
elements of which the organic world is constructed would quickly come to a
standstill, and the surface of the earth become in a few years thickly covered
with the dead bodies of animals and plants. Respecting the constituents of the

THE FERMENTATION OF CELLULOSE 149

latter a few words will now be devoted to the fate of the Cellulose, of which the
greater part of the cell walls of plants is composed. Here the question arises as
to how the carbon in this substance is set free again, and the gradual, and finally
complete, accumulation of this element in a useless form prevented. In this
case once more assistance is afforded by bacteria, which split up the cellulose and
remove it out of the way.

E. MITSCHEELICH (I.) was the first, in 1850, to comment on the natural
decomposition of cellulose, by expressing his opinion that it was attributable to
the fermentative activity of vibrios. The probability of his view was increased
by Popoff's (I.) discovery in 1875, that this decomposition process can be
moderated or completely arrested by the addition of substances poisonous to
bacteria. A closer investigation of the organisms in question was undertaken
two years later by VAN TIEGHEM (IV.), who gave them the name of Bacillus
amylobacter, and (V.), from microscopical examination only, declared them
identical with Pasteur's vibrion butyrique.

It would be useless at the present time to argue on this assumption, since
both observers worked with what were probably complex mixtures of several
species, certainly not with pure cultures. On the other hand, Van Tieghem's
further demonstration that petrified cells of (morphologically) similar fission
fungi are also to be found in the fossil coniferse of the Carboniferous period is
worthy of mention.

A. H. 0. VAN SENUS (I.), in 1890, endeavoured to obtain a pure culture
of the organism giving rise to cellulose fermentation. According to him, a
symbiosis of two species is here in question, the one of them — which he named
Bacillus amylobacter — occurring in the form of rods 0.8-1 /z broad and 2—10 p in
length, which, under special conditions, are stained blue by iodine. When air is
admitted they form endospores, which then germinate only when air is excluded.
The second of these symbiotic species is of much smaller dimensions, and is
by itself, like the B. amylobacter, incapable of fermenting cellulose. For this
purpose the conjoint effect of both species is necessary, an enzyme being then
excreted which dissolves the cellulose. Van Senus isolated this enzyme, and
demonstrated its solvent power on cellulose by applying the alkaline solution
(containing chloroform in order to suppress bacterial growth) to the cell walls of
slices of beans.

V. OMELIANSKY (I.), in 1895, obtained very different results. He inoculated
a mineral nutrient solution (containing potassium phosphate, magnesium sulphate,
ammonium sulphate, and chalk), in which strips of Swedish filter-paper were
held in suspension, with a small quantity of mud from the Neva, and then kept
the whole at 3o°-35° C., air being excluded. Fermentation rapidly set in, the
strips of paper gradually becoming thinner, and finally disappearing altogether.
By repeated transferences of small portions of the fermenting liquid to fresh
sterile media the ferment was purified, and finally brought into a state of pure
culture by anaerobic cultivation on discs of boiled potato. Omeliansky describes
the organism as an unusually slender bacillus, measuring only 0.2 to 0.3 /* in
breadth for a length of 6-7 n, and forming terminal globular endospores i /i in
diameter, whereby the pole at which the spore occurs is swollen up.

As in many other directions, here also, in the case of the fermentation of
cellulose, the extension of our knowledge is dependent on the elucidation, still
to be made by chemists, of the composition of the substances subjected to
investigation for the products they yield on fermentation. At the present time
the essential requirement that the ferment shall be used in a state of pure
culture is almost fulfilled, but so far as the purity of the cellulose to be decom-
posed is concerned matters are by no means on a satisfactory footing. ERNST
SCHULZE (I.), in 1895, in a treatise setting forth the present state of our know-

150 BUTYRIC ACID FERMENTATION

ledge on the subject (and one well worthy of perusal) shows how very divergent
are the substances which now bear the name of " cellulose." He separated a
number of these, and collected them into the group of hemicelluloses, distinguished
by their solubility in hot dilute mineral acids, whereby they are converted into
glucose, whilst the remaining celluloses do not undergo this change. Considering
how divergent these substances are, it is not surprising that different investigators
do not always obtain concordant results as regards the products of cellulose
fermentation. This decomposition occurs on a large scale in the mud of marshes
where there is no lack of decaying plants, and where, moreover, the other
conditions are favourable. It has long been known to chemists that, in such
water, a somewhat copious discharge of gas bubbles rises out of the ground,
which discharge consists for the most part of methane (CH4), i.e. the gas to
which the name of marsh-gas has been given, from the places where it is found
in Nature. Carbon dioxide is also liberated at the same time. The proportional
quantity of the two products was reported in several analyses communicated by
POPOFF (I.) in 1875, and more accurate researches on the same point were
published by HOPPE-SEYLER (I.) in 1886. The latter kept, with exclusion of
air, either mud from marshes and rivers, or else clean paper inoculated with a
little mud and then distributed in water, and demonstrated that in the mixture
of gases evolved therefrom methane predominated greatly at the outset, but
thereafter gradually diminished to the proportion i : i, so that Hoppe-Seyler,
being unable to discover any other decomposition products, expressed the
opinion that the cellulose is hydrolised by the action of the bacteria — which from
microscopic examination he asserted to be the same as Van Tieghem's Bacillus
amylobacter — and is then split up into equal volumes of methane and carbon
dioxide, according to the equations —

C6H10Oe + H20 ^ C6H1206.
C6H1206 = 3C02 + sCH4.

He found the ratio different when sulphates (gypsum, &c.) or ferric salts
were present in the water or mud. In such case the nascent methane, by its
reducing action on these salts, converts them into carbonates, sulphuretted
hydrogen being liberated, according to the equation —

CaSO + CH4 = CaC03 + H2S + H20.

The sulphuretted hydrogen is, then, under natural conditions, acted upon by
the sulphur bacteria which are always present in such waters. This will be
dealt with in chapter xxxv.

The importance of cellulose fermentation in the physiology of nutrition
(especially of cattle) must also be briefly adverted to. The opinion long held by
Emil Wolff, that the vegetable fibres consumed with the food pass out of the
alimentary canal unaltered, was contradicted, in the case of ruminants, as far
back as 1854, by Haubner, who showed that even in the case of sawdust and
paper pulp mixed with the fodder, only a portion (less than half) was expelled
again in the excrement, a fact confirmed by the exhaustive researches of Henne-
berg and Stohmann. The difference between the amounts of cellulose (crude
fibre) taken in and rejected was highest in the case of ruminants (up to 75 per
cent.), being only 50 per cent, at most in horses, and still less in the human
subject and in swine. In carnivora (dogs), on the other hand, no such difference
could be detected. The quantities of cellulose thus disappearing in digestion
were, it was thought, digested, and were regarded as approximately equivalent
in nutritive value to the soluble carbohydrates. A number of animal physio-
logists maintained that the cellulose was dissolved by an intestinal enzyme,
which, however, was sought for in vain. The earliest reliable determinations

THE SO-CALLED "RETTING" OF FLAX AND HEMP 151

on this point were made by H. TAPPEINER (I.) in 1884, who showed the fate
of all cellulose taken into the body and not evacuated in the faeces to be, not
digestion and absorption into the arterial circulation, but fermentation into
marsh-gas. The operation, however, does not proceed exactly according to the
above equation, but yields, in addition to methane, volatile fatty acids (chiefly
acetic acid, then butyric acid, &c.). Bearing this in mind, this decomposition
process cannot be regarded as totally without value for the animal body, although,
naturally, the coefficient of digestibility, hitherto usually assumed, necessarily
suffered considerable depreciation. Moreover, this process is indirectly favour-
able, in so far that, by the solution of the cell-walls, the cell contents of the
vegetable nutriment are laid bare, and thus rendered more readily accessible
to the digestible fluids. Although by this discovery the chief source of the
intestinal gases so copiously evacuated by herbivorous animals is made manifest,
still it should not be assumed that the methane therein is exclusively derived
from the fermentation of cellulose, since Huge and Planer proved that, even in
cases of a purely flesh diet, methane is to be found in the intestinal gases (of
man and the dog).

Cellulose fermentation also plays a part in the preparation of brown hay,
sweet ensilage, and sour fodder, considered in chapter xxvii., where it forms
one of the causes of the great loss of matter inherent in these processes, in
connection wherewith reports have been made by Weiske, 0. Kellner,
M. Maercker, and others. This process also goes on — as shown in 1884 by
P. DEHERAIX (I.) and U. GAYON (I.) — in manures. If, favoured by special
circumstances, it proceeds more rapidly than the decomposition of all the other
(and especially the nitrogenous) constituents, then an irregularly fermented
product — deficient in the fibres necessary to impart porosity to the mass — and
known as fatty manure, is the result.

The evolution of marsh-gas and hydrogen by the agency of bacteria also
occurs, not infrequently, in other situations ; for instance, in the diffusers in
sugar-works, and very often so strongly that the amount of gas suffices to
produce powerful explosions when the diffusers are incautiously approached
with a naked light. The teaching of experience, that frozen beet is particularly
liable to such a form of decomposition, is probably explicable by the circum-
stance that such beet cannot be entirely freed from adherent particles of soil,
and the ferments present therein. It is also conceivable that, by the agency of
frost, the pectins in the beet are transformed into a more readily decomposable
condition. On this point a few observations have been made by Millot and
Maquenne, and, more recently, by P. DEHERAIN (II.) ; more accurate investiga-
tions thereon are, however, still lacking.

§ 119.— The So-called " Retting" " of Flax and Hemp.

The present forms the most fitting occasion for making a few explanatory
remarks on this matter. As is well known, it is the bast fibre of these plants
that is referred to when hemp or flax is spoken of in the textile industry. In
order to lay these fibres bare and obtain them in a pure state, it is necessary to
dissolve the intermediate intercellular substance (the so-called central lamella)
— which consists, not of Pectose, as stated by J. Kolb, but (according to the
researches of Mangin) of calcium pectate. This can be effected by chemical
means, by a (patented) process which Baur successfully introduced into Silesia
on a large scale in 1882, and which consists mainly in treating the plants with
very dilute sulphuric acid, and then neutralising the adherent acid by a weak
alkali bath. The solution of the cementing calcium pectate can, however, be
brought about by a fermentation, known as "retting," that has been practised

152 BUTYRIC ACID FERMENTATION

from remote ages without any special knowledge of the more delicate processes
involved. According as the moisture necessary for this fermentation (or for the
development of the fission fungus effecting the same) is imparted to the rippled
stalks by means of dew, sprinkling them with water, or by immersion, the
process is known as dew -retting, water-retting, or, finally, mixed retting.

With reference to the bacterial species taking an active part in this process,
VAN TIEGHEM (VI.) in 1879 expressed the opinion that they should be assigned
to his Bacillus amylobacter, which he, as already mentioned, also regarded as the
cause of cellulose fermentation. The inaccuracy of this view is evident, since,
if the retting (Fr. rouissage, Ger. rosten) of the flax were mainly a process of
cellulose fermentation, there would not be much of the fibre (which chiefly con-
sists of cellulose) left. It was V. Fribes who — working under the directions
of S. WINOGRADSKY (I.) — in 1895 clearly proved the true state of the case and
made known the active agent of this pectin fermentation. This organism is a
fairly large-celled species, occurring in the form of rods, which, when young,
have a length of 10-15/1 with a breadth of 0.8 p, but subsequently become
broader )i /*), and swell up to a thickness of 2 p. at the one end (tadpole shape),
where a long endospore (1.2 x 1.8 p.) is developed. This (anaerobic) bacillus will
not grow on gelatin, but when supplied with nitrogenous food in the form of
peptone will ferment glucose, saccharose, lactose, and starch, leaving these, how-
ever, untouched when the peptone is replaced by ammonia salts. On the other
hand, even in this latter case, any pectin bodies that may be present, i.e. pectin
and pectic acid, are fermented, and that, too, even more readily than the carbo-
hydrates already mentioned. The organism, however, has no action on cellulose
and gum-arabic. When clean portions of plants, previously washed, first with
acidified, and then with faintly alkaline, water (free from bacteria), are exposed
to the action of pure cultures of this bacillus, they quickly lose the greater
portion of their pectin content, the loss of weight they suffer being almost
exclusively due to this cause. As reported by E. PFUHL (II.), a patent has
been obtained in the United States by Allison and Pennington for the method
tested by them, whereby the retting of flax can be effected in a few days in any
class of water by the addition of salts promoting the growth of the desired
ferment. Perhaps an inoculation by water from a locality — such as the river
Lys, a tributary of the Scheldt — where flax-retting is extensively carried on is
also to be made.

§ 120.— The Rancidity of Fats, particularly Butter,

will be briefly dealt with here parenthetically, as there is no other appropriate place
for it. The characteristic indication of this well-known phenomenon is an increase
in the percentage of free acids (chiefly the volatile acids, butyric, caproic, &c.).
This may be attributed to four different causes : (i) The activity of lactic acid
bacteria converting the residual lactose in the butter into lactic acid, which latter
is then transformed into butyric acid by Granulobacter lactobutyricum ; (2) the
decomposition of albuminoids (casein) by bacteria capable of forming butyric
acid therefrom ; (3 and 4) the dissociation of the fats into glycerin and free
fatty acids, either by bacterial agency on the one hand, or by the action of light
and air on the other. The preponderance of one or other of these causes
depends on the attendant circumstances.

E. DUCLAUX VI.) rendered valuable service in demonstrating the influence
of air on the fatty matter of butter and cheese, and by showing that this
influence is twofold, viz., it first, by saponification, breaks the matter up into
glycarin and fatty acids, and then converts the latter (especially oleic acid) into
oxy compounds. In the dark, and in presence of a copious supply of air, saponi-

THE RANCIDITY OF FATS 153

fication predominates, the butter then smelling strongly of butyric acid ; bub if,
on the contrary, the oxidising action gets the upper hand, then a tallowy flavour
is the result.

A recognition of the influence of light and air does not necessarily imply that
bacteria play no active part in rancidity. This latter view was maintained by
H. SCHULZ (II.) in 1878, but was afterwards denied by E. RITSERT (I.), ARATA (I.),
and others. With reference to the careful work performed by Ritsert, it should
be remarked that the main point is in the treatment of the question as to the
occurrence of rancidity in the absence of micro-organisms. The affirmative
answer thereto does not, however, involve the conclusion that their activity is,
under natural conditions, unimportant for the development of rancidity. The
action of the lactic acid bacteria can be illustrated by a practical example
afforded by the so-called Paris butter. This is a particularly stable product, and
is prepared from sweet cream, which, before churning, is heated to nearly 100° C.
and then rapidly cooled again, whereby the fission fungi just mentioned are
killed. The bacteria which split up the glycerides in butter include one which
R. KRUEGER (I.) isolated from butter that had become cheesy, and which he
named Bacillus Jtuorescens non liquefaciens.

Investigations on the breaking up of fats by bacteria, which process is of
medic il interest in connection with the putrefaction of dead animals, were con-
ducted by G. vos SOMMARUGA (I.), chiefly with pathogenic species, only a few of
which were found to possess this power. Among these may be mentioned
Bacillus typhi abdominal-is, B. pyocyaneus, Micrococcus tetragenus, Vibrio cholerce
asiaticce, Deneckes spirillum, and others. On the other hand, Bacillus mega-
therium and B. subtilis do not possess it.

CHAPTER XIX.

THE PRESERVATION OF MILK.

§ 121.— Dirt- and Germ-Content in Milk.

THE sterilisation of milk is excessively difficult, because this liquid is particularly
liable to infection by very hardy germs. Even when yielded by a healthy cow,
the milk, on issuing from the udder, is already infested with bacteria. When
milking is ended, a small quantity of milk is left behind in the lacteal ducts,
and in this there settle a number of bacteria which make their way from outside,
are favoured in their development by the high temperature, and become in-
corporated with the subsequent flow of milk. In addition to these are the
innumerable bacteria originating in the dung and adhering to the udder. Both
groups consist mainly of species very tenacious of life, derived from the soil and
entering the alimentary canal along with the fodder. They pass through the
intestines unhurt, are conveyed with the dung on to the udder and the hands of
the milker, and then into the milk, where they flourish exceedingly. The dirt,
adhering to the cows, and itself infested with bacteria, is partly disseminated as
dust through the air of the cowhouse, so that this also is impregnated with
bacteria, and yields up no small quantity to the milk. For investigations on
this point we are indebted to G. J. LEUFVEN (I.), who held sterilised flat glass
dishes open, for a second, above the edge of a milking-pail into which milk was
being drawn from the cow, and then introduced into the dishes some liquefied
nutrient gelatin, wherein the germs present in the basins developed into colonies,
and could therefore be counted. In this manner it was proved that, in the space
of one second, from 47 to 1210 germs, according to circumstances, were deposited
in an area of i square decimetre (100 sq. cm., or about 16 square inches).

The amount of the germ-content is thus primarily determined by the degree
of contamination prevailing in the cowshed, a criterion of which is afforded by
the amount of dung constituents present in the milk. This estimation was first
attempted by RENK (I.), who found, for instance, in the market milk of Halle-
on-Saale, some 75 mg. per litre; in Berlin milk, 10 mg. ; and in Munich milk,
9 mg. of such milk-dirt, the highest quantity amounting, at Halle, to 0.362 grm.
per litre. If the milk be treated in the centrifugal machine, in order to remove
the cream, the dirt is separated, and collects, along with casein, bacteria, &c., as
a coating, sometimes granular, at others mucinous, on the walls of the inner
drum, and is generally known as milk-sludge. It consists of about 26 per cent,
of albuminoids, 67 per cent, of water, &c., and is relatively much richer in
bacteria than the milk. O. WYSS (I.) was the first to investigate this mud, and
the quantity separated was found, in a case examined by NIEDERSTADT (1.), to
amount to 43 grms. per hectolitre (slightly more than 30 grains per gallon).

The connection between the content of dirt and of germs in milk was shown
in particular by UHL (I.), a few of whose figures are now given :

Dirt. Number of Germs.
Sample No. Mg. per Litre. Per c.c.

1 36.8 12,897,600

3 ' . .20.7 7,079,820

6 • -5-2 3.338,775

MILK AS A CARRIER OF DISEASE 15$

Sundry researches on the germ-content of cow-dung, and on its dependence
on the dietary, were carried out by E. WUTHRICH and E. VON FREUDENREICH (I.).
They found up to as many as 375 millions of bacteria per i gram of fresh faecal
matter, the majority consisting of Bacterium coli commune, along with some
3 millions of hay bacilli and others. An equal weight of the hay used for fodder
contained about 7! millions of germs, of which about one-fourth were hay bacilli.
Sour brewers' grains (forty -eight hours old) yielded 375 million colonies.

The germ-content of freshly drawn milk increases very rapidly during trans-
port to the centres of consumption, as also during storage, e.g. in milk-shops. A
number of estimations have been made in this connection, a few of which, by
FREUDENREICH (II.), are subjoined. In these, the influence of the length of
storage in conjunction with the prevailing temperature is taken into account.

Number of Germs per c.c.

On arrival in the laboratory (two and a half hours after milking), 9300.

Stored at

Duriii"-

15°

25°

35° C.

3 hours . . .

10,000

18,000

30,000

6 „ . . . .

25,000

172.000

12,000,000

9 ,.

46,500

1,000,000

35,280 ooo

*4 >.

5,700,000

577,500,000

50,000,000

A milk with an initial germ-content of 9300 will be reckoned poor in bacteria
when it is known that J. v. GEUNS (I.) found 2.5 million bacteria per c.c. in fresh
samples of Amsterdam milk. The reproduction can be moderated, but not
arrested, by cold, because,, as we have seen in § 61, milk contains also sundry
species of bacteria capable of development at o° C. Therefore if it be desired
to prevent the decompositions set up by these organisms, it will be absolutely
necessary to kill the germs.

§ 122.— The Part Played by Milk as a Carrier of Infectious Diseases.

The aforesaid requirement is really imperative in view of the fact that diseased
cows — which the owners often fail (or refuse) to recognise as such — yield milk
containing pathogenic bacteria. And this applies particularly to tuberculosis,
from which complaint (on a moderate computation) one cow out of every five in
Germany suffers. As reported by J. CH. BAY (I.), on the authority of a compila-
tion made by the Danish pathologist B. Bang (to whom is due the honour of
clearing up this question), out of 132,294 head of cattle examined in the Copen-
hagen slaughter-house between 1891 and 1893, no less than 23,305 (some 17.7
per cent.) were recognised as tuberculous by macroscopic examination alone.
According to the researches of Dr. Martin, reported by R. CNOPF (I.), one out of
every thirteen samples of milk exposed for sale in Paris contains tubercle bacilli,
and from the results obtained by Dr. Schroeder in Washington, at least one in
every nineteen samples of milk sold in that city contains a sufficient number of
tubercle bacilli to produce infection. For the microscopic investigation of the
(specifically heavy) tubercle bacilli in milk, the sample, previously prepared by
skimming and clarifying, is separated, by centrifugal force, in strong test-tubes,
for which purpose special processes have been designed by THORNER ([.) and

156 THE PRESERVATION OF MILK

ILKEWITSCH (I.). K. OBERMULLER (I.) examined Berlin market milk in this way,
and recognised it as infected with tubercle bacilli in a high degree. In this
connection it should be remarked that, according to determinations made in 1896
by A. BULLING (I.), goats are also liable to this disease, and therefore cannot be
considered as immune.

The extent of the danger attendant on the consumption of unboiled milk is
not sufficiently illustrated by the foregoing particulars, which are only concerned
with the possibility of infection by such bacteria as are pathogenic for men and
animals, i.e. tuberculosis, anthrax, and so on. Milk is, however, also a frequent
carrier of typhus bacilli, which fission fungi (almost exclusively pathogenic for the
human subject alone) find their way into the milk, either directly from diseased
milkers or milk-dealers, or from the milk vessels being swilled out with water
containing these microbes. Farmyard wells are frequently very close to dung-
hills, cesspools, and closets, and if typhus breaks out on such farms, then the
well-water very soon becomes impregnated with typhus bacilli by means of fsecal
matter. Proofs of this exist by the dozen. The first reliable observation on this
subject was made by Ballard in 1870, when an epidemic of typhus broke out in
Islington, 67 houses and 167 patients being infected. A careful investigation of
all the cases led to its being traced to a farm-house from whence the milk supplied
to the infected families was derived, and there the closet cesspool was found to com-
municate (through rat-holes) with the well, the water from which was also used
for cleansing the milk-pails. To this first instance two others of recent date may
be added. One of them was investigated by PAUL SCHMIDT (I.), and arouses
interest because it treats of the inmates of a prison, where communication with
the outer world is much easier to trace. In two prisons in Strassburg (Alsace),
where typhus had not recurred since the Franco-German War, it broke out again
in 1890, and that, too, among a section of the inmates who had partaken of milk
derived from a neighbouring village where the disease was rife. The epidemic
died out when the supply of milk from this source was prohibited. A second
equally convincing instance was observed by REICH (I.) in 1892. ROWLAND (I.)
found living typhus bacilli in an Indian milk-comestible (known as " Dahi "). —
The so-called explosive occurrence of this rapidly extending pestilence in a healthy
neighbourhood is thus explained by the fact of its germs gaining access to the
system with the food. Finally, milk is also a carrier of certain diseases that are
recognised as infectious, but whose exciting agent has not yet been discovered,
e.g. scarlet fever — a case of which is recorded by W. H. POWER (I.) — and foot-
and-mouth disease.

§ 123.— Boiling1 Milk.

The particulars given sufficiently evidence the necessity for killing the germs
present in milk. Experience teaches that a short boiling suffices to destroy the
pathogenic organisms, the tubercle bacilli being — according to the researches of
J. FORSTER and C. DE MAN (I.), and of BONHOFF (I.) — killed by the action of a
temperature of

55° C. in 4 hours.
60° C. „ i hour.
65° C. „ 15 minutes.
70° C. „ 10 „

80° C. in 5 minutes.
90° C. „ 2 „
95° C. „ I minute.

The cholera bacteria and typhus bacilli are, as proved by GEUNS (II.), capable
of still less resistance, and are therefore killed much more quickly than the
tubercle bacilli by a treatment expressed by the above figures. Only a single
pathogenic species can withstand the short boiling to which milk is ordinarily
subjected in domestic management, and this is the anthrax bacillus (spores).

THE SOXHLET BOTTLE 157

The danger incurred on this account is, however, slight, since this microbe only
forms spores in presence of oxygen, and therefore not within (the arterial circu-
latory system of) the animal body. Even in the worst case, therefore, only the
vegetative forms (easily destroyed by boiling) of this microbe can find their way
into the milk from the body of the cow ; and, on the other hand, the intro-
duction of these germs from external sources is hardly to be feared. According
to the researches of O. CAHO (I.), the virulence of spore-free forms of growth of
Bacillus anthracis in milk disappears within twenty-four hours at a temperature
of 15° C., a circumstance attributable to the injurious influence of the lactic
acid gradually formed in that liquid. The spores, however, completely retain
their vitality under these conditions.

Among the bacteria present in unboiled milk, the species inducing lactic
fermentation are never lacking, and it is to these that the souring of milk is
due. The fact that they are destroyed by boiling explains why boiled milk will
keep, without alteration, a much longer time than is the case with unboiled milk.
In addition to the species already mentioned there is present a third group of
tSchizomycetes forming spores very tenacious of life, which withstand boiling,
and germinate when the milk is kept at a moderately warm temperature. The
resulting rods having, by the process of boiling, been freed from the presence of
sundry inconvenient associates of other species, then develop and increase
rapidly, setting up a brisk fermentation whereby a large volume of gas is
liberated. The importance and extent of this fact first becomes clear in the case
of suckling infants.

§ 124.— The Soxhlet Bottle.

With the continual extension of enervation the number of mothers unable
or unwilling to suckle their infants increases from year to year. How far the
natural nourishment thus withheld is superior, in point of chemical composition,
in the various periods of lactation to any artificial medley cannot be expounded
here. From the bacteriological standpoint it may be regarded as almost perfect.
If the mother be healthy in body, then the milk absorbed by the child at the
breast is, -according to the researches of T. RINGEL (I.) and others, almost entirely
free from bacteria of any kind. The expression "almost free" is used advisedly,
since the milk generally contains a small number, originating in the air and
making their way into the lacteal ducts of the mammary glands, where they
increase. The necessary hygienic treatment of this organ by the young mother
will greatly contribute to the child receiving the best nourishment both bac-
teriologically and otherwise. To return, however, to those other matrons who
bring up their infants on the bottle, filled with boiled and sufficiently cooled
milk. The stomach of the young child being small, whereas the amount of
material required for the growing body is large, the infant requires frequent
supplies of small quantities of nourishment. Generally, for the sake of con-
venience, a sufficient quantity of milk for the whole day is boiled at once, portions
of this being taken from time to time as required. Through ignorance on the
part of the mother, or by the carelessness of the nurse, it often happens that
this food is supplied to the infant in a partially decomposed condition. A parti-
cular fault, frequently committed, is that the bottle, which has been lying for two
or three hours in the warm nursery, is refilled from the vessel containing the bulk,
without the residual milk from the preceding meal having been removed. That
such carelessness (frequent, though as constantly denied) must conduce to diges-
tive disorders requires no further demonstration, the high rate of infant mortality
from intestinal catarrh being sufficient evidence.

For this reason it has been attempted to render milk stable by boiling it in
small bottles, holding just sufficient for a meal, and closing the same with a

i$8 THE PRESERVATION OF MILK

stopper (impervious to bacteria), which is removed only just before use. This is the
fundamental idea of the so-called Soxhlet method of sterilising milk, in which
several bottles are inserted in a movable frame and immersed in a tin pan con-
taining water, which is thereupon kept on the boil for forty minutes. SOXHLET (I.)
employs latterly, as automatic stopper, an indiarubber disc resting on the ground
mouth of the bottle, and prevented, by means of a loose-fitting tube, from
becoming displaced laterally. The gases and steam given off from the milk in
boiling escape into the air by forcing up the disc, and when the operation is
finished and the apparatus is removed from the fire, this clack-valve is kept
tight by the pressure of the outside air, the partial vacuum within the bottles
being generally equivalent to 100 mm. of mercury. This stopper not only pre-
vents access of air, but also debars dealers or purchasers from opening the bottle
with fraudulent intent, since it cannot be closed again. This ensures the pur-
chasing public receiving the milk in the same unadulterated condition in which
it left the dairy. Similar to Soxhlet's method are those of Egli and Escherich, a
short description of which (as also of those of Soltmann, Bertling, Gerber, and
Stadtler) will be found in a comparative treatise by EMMA STRUB (I.).

§ 125.— Germ-Content of Milk Treated by the Soxhlet Method.

The foregoing method would meet all requirements were the destruction of
the pathogenic and the lactic acid bacteria alone in question. However, as has
previously been mentioned, milk also contains very hardy bacterial spores, able
to withstand such a course of boiling as that specified . The number of such
spores varies, and is greater in proportion as .the degree of uncleanliness in the
attendance on the cows and in the operation of milking increases. These milk
bacteria (which resist the ordinary means of sterilisation) were investigated
with regard to their properties and action by C. FLTJGGE (II.)- A few of them
are very widely diffused, but they do not develop below 18° C. Milk sterilised by
mere boiling may therefore be rich in such bacteria and yet keep unaltered for a
long time at room temperature, though, if introduced into the alimentary canal
of the young infant, the hardy spores develop into rapidly-multiplying bacilli
which decompose the constituents of the milk. In such case, not only are
copious amounts of gas, giving rise to considerable flatulency, formed, but also
poisonous decomposition products of albuminoid matter, which when fed to
puppies produce diarrhoea attended with fatal results.

The danger incurred from this cause is much greater for the nursing infant
than for the adult, not only because the latter organism is stronger, but also for
the further reason that the dietary of adults is a mixed one, in consequence
whereof numerous other bacteria, inimical to those in question, are introduced
into the alimentary canal. On the other hand, the result of using such imper-
fectly and partially sterilised milk is that the digestive organs of the infant,
nourished on milk alone, are converted into a veritable breeding-ground for
these poisonous microbes.

These bacteria are closely allied to the Bacillus mesentericus vidgatus. Fliigge
himself described a number of such species; and S. STERLING (I.) added five
new ones to the series, naming them — with reference to their chemical activity —
as Bacillus lactis peptonans a, |3, y, 8, e. Attention was drawn at an earlier date,
by LOEFFLER (III.) and Emma Strub, to the frequent occurrence of B. mesen-
tiricus vu'gatus in milk.

Attempts have not been lacking on the part of dairy technicistsand bacterio-
logists to arrive at a method for annihilating these pests as well. A critical
examination of these methods cannot, however, be made here, but any reader
desiring fuller details is referred to a comprehensive exposition of the question

THE METHOD OF NEUHAUSS, ETC. 159

compiled by H. WEIGMANN (II.)- At present, merely a single example will be
given, namely :

§ 126.— The Method of Neuhauss, Gronwald, and Oehlmann,

which was tested by PETRI and MAASSEN (II.)- We have already, in a previous
section, demonstrated that it is not easy to render milk sterile in the strict
meaning of the word. The high and long-continued heating necessary thereto
is sufficient to alter the chemical constitution of the milk in such a manner that
it becomes almost unsuitable for nutrition. The lactose, as P. CAZENEUVE and
HADDON (I.) have shown, decomposes into dark brown fission products (contain-
ing formic acid), with an empyreumatic flavour; the fat loses its emulsified
condition and separates out as cream, which cannot be made to diffuse again
even by shaking ; and the albuminoids are converted into a form very difficult
of digestion.

Neuhauss, Gronwald, and Oehlmann, calling to mind the information afforded
by the fractional method of sterilisation, sought to induce the hardy spores to
germinate, in order that the end in view might then be attained by moderate
means. With this object the milk is placed in bottles with loose-fitting stoppers,
which are then put into a specially constructed case, where they are surrounded
by steam and allowed to remain for half an hour at a temperature of 8o°-95° ^
This so-called preliminary sterilisation is once repeated, with the result that the
pathogenic and the lactic acid bacteria are destroyed, and the milk is then
left to cool gradually, whereby it passes through the degrees of temperature
favourable to the germination of the surviving hardy spores. On the following
day the samples are subjected (in the same bath) to the so-called chief sterilisation
at 102° C., and when this is finished the stoppers of the bottles are immediately
and simultaneously tightened up by means of an arrangement manipulated from
the outside. The instructions given to adhere to a temperature of 102° C. prove
that the germination of the spores is not numerically complete, since, it' this
were the case, a maximum of 100° C. would suffice for the chief sterilisation, all
the vegetative forms quickly perishing at this temperature. If, h .wever, spores
be still present at the commencement of the chief sterilising process, the
probability is by no means small that they will also be able to withstand the
short exposure to 102° C., and it may be anticipated that even this method will
not always accomplish its object. As a matter of fact, reports are not wanting —
e.g. that of M. BLEISCH (I.) — to the effect that milk samples assumtd to have
been sterilised by the method in question have been subsequently discovered to
be in a state of decomposition ; whilst Fliigge was unable to confirm the
favourable reports given by Petri and Maassen, and PICTET and WEYL (I.).

In short, there is at the present time no practicable and certain method for
freeing milk (on a large scale) from germs without at the same time seriously
prejudicing its flavour and nutritive value. Since, then, the annihilation of the
hardy germ.s in this case is so difficult, attention is now directed to their
exclusion from the milk ; the greatest care is therefore taken — by washing the
udder, hands, and milk vessels— to secure extreme cleanline s in the preparation
of "nursery milk" intended for infant consumption. The so-called sterilisation
then becomes a much easier task, the milk, drawn with such precaution from the
cow, being very poor in the above-mentioned gas-forming bacteria. As a ready
means of detecting the presence of these organisms will often be useful to the
scientific adviser of a Dairy Association, the fermentation flasks described by
F. SCHAFFEE (I.),Tn. SMITH (1. and II.), and others, are therefore recommended
for the regular examination of the milk supplied by the individual farmers
as regards its content of the pests under consideration,

160 THE PRESERVATION OP MILK

§ 127.— The Content of Pathogenic Germs in Various
Dairy Products.

A few words must be devoted to the description of the treatment of skim-
milk in the factories of Dairy Associations. As is well known, a large portion
of the fresh milk sent to the dairy is not sold as such, but is employed for
butter-making. The skim-milk formed in large quantities during this process is,
in many instances, partly sold per se, and partly worked up into semi-fat and
skim-cheese, milk-bread, and the like. In other cases, the contracts made with
the membets of the association stipulate that each shall have returned to him,
for feeding purposes, a quantity of skim-milk proportional to his deliveries of
new. Now, since in these large dairies the cream is removed from the milk by
means of the centrifugal machine, it follows that the milk from all sources
becomes intimately mixed up together, and consequently if any one parcel of the
milk is contaminated, the whole of the skim-milk will become infected thereby.
In this manner an epidemic hitherto confined to a single farm may, by means of
the returned skim-milk, be rapidly disseminated to all the other cowkeepers.
This has actually been frequently proved in respect of the foot-and-mouth
disease. In this connection there is an increase in the number of supporters of
legislative action in favour of a compulsory heating of the skim-milk returned by
dairies to the farmers. As reported by P. VIETH (I.), a Ministerial ordinance
has been in force in Prussia since 1894, prescribing that the skim-milk from
cows suffering from infectious diseases shall be either kept at a temperature
of 90° C. for at least a quarter of an hour, or be heated up to 100° C. before
being allowed to leave the dairy.

It is imperatively necessary that the cream destined for butter-making
should be freed from pathogenic germs. According to the concordant results of
the researches of L. HEIM (IV.), G. GASPERINI (II.), and O. ROTH (I.), the active
organisms of cholera, typhus, and tuberculosis present in the butter long retain
their vitality and power. Now, a large proportion of the butter made is con-
sumed in a raw state in the form of bread and butter and the like, and if it has
been derived from milk or cream infected with pathogenic bacteria, its consump-
tion is attended with great danger. Consequently a reliable preliminary
treatment of the cream to ensure the removal of these germs is in the highest
degree desirable. That this can be practically accomplished will be shown in
chapter xxiii. which treats of the artificial souring of cream.

The same requisition should also be imposed in the case of milk den'gned for
cheese-making, but at present this can hardly be effected, because the treatment
required for killing the pathogenic germs lessens the suitability of the milk for
the purpose in view, and also modifies the flora of the milk to such an extent as
to unfavourably influence the ripening process of the cheese prepared therefrom.
In fact, until we are in a position to introduce and carry on this process to its
completion in a reliable manner by artificially added ferments, the above-named
requirement must necessarily remain unfulfilled. Fortunately the acid produced
by the ripening process forms an effective antidote, which checks the develop-
ment of the pathogenic organisms. H. WEIGMANN and G. ZIRN (I.) proved
that Bacillus (vibrio) cholerce asiaticce perished within twenty four hours when
artificially inoculated on cheese.

As will be gathered from the preceding observations, the sterilising of milk
samples destined for the cultivation of organh-ms in the laboratory is a very
troublesome operation, since this necessitates an absolute freedom from germs.
In order to obtain this result, the samples are exposed for ten to fifteen minutes
to steam under pressure, at a temperature of 120° C. The decompositions

CONDENSED MILK 161

hereby induced have no injurious effect in some cases; nevertheless when
delicate organisms are to be cultivated that would not thrive in milk thus
altered, a method of mixed sterilisation must be practised. A little ether or
chloroform is added to the sample, allowed to react for a short time, after being
thoroughly shaken up, and, at the end of two or three days, disinfection is
effected by placing the sample for twenty to thirty minutes in the steamer (at
100° C.). The statement, often met with in books, that milk may be sterilised
by exposure to the action of a current of steam at 100° C. for twenty to thirty
minutes on three successive days, is (according to the author's experience)
deceptive. If such " sterilised " milk be placed in the incubator, an effluvia!
decomposition, with copious development of potato bacilli and the like, will be
noticeable in nine cases out of ten.

A number of bacterium poisons — the suitability of which has been made the
subject of comparative investigation by J. NEUMANN (I.) and M. KUHN (I.) —
have been proposed for preserving milk that is to be sent to a laboratory for
the purpose of having its fat-content ascertained. The potassium permanganate,
formerly recommended, behaved badly under the ordeal, whereas, on the other
hand, potassium bichromate (for the use of which for the purpose in question
Alen has taken out a patent) proved reliable in cases where it was a matter of
preserving milk thab was still sweet. The sample is treated with an admixture
of 0.5 grm. of pulverised K2Cr207 (or with 5 c.c. of a 10 per cent, solution of
this salt) per litre, the dilution produced in the latter case exercising no appre-
ciable influence on the accuracy of the fat determination. If the milk at the
moment the sample is drawn is already somewhat sour, then an addition of
ammonia — 3 c.c. of a 27 per cent, solution of ammonia per litre of milk — will be
preferable.

Not infrequently milk intended for sale is qualified with substances acting
as poisons towards bacteria, with the idea of increasing its keeping properties.
Boracic acid, both in the free state and in the form of borax, is in great favour
for this purpose, and recourse is occasionally had to salicylic acid. F. M. HORN
(I.) records an instance where benzoic acid was used. All additions of this
kind are contrary to law, and therefore punishable.^

§ 128.— Condensed Milk.

It is not infrequently desirable to convert large quantities of milk into a
permanently stable condition for use as food, e.g. for provisioning ships. In
other cases, owing to local circumstances, the milk production of a district may
be far in excess of the local requirements, and consequently the necessity arises
for converting the product into a stable and readily portable condition for
export. Many of the Swiss Cantons, for example, are in this position. Such
milk must be capable of remaining entirely unaltered for any desired period
(several years), even when exposed to tropical temperatures — a condition un-
attainable by any of the means already described. In former years the opinion
was current that this could be effected by the method proposed by Appert, and
in the middle of the seventies Nageli also attempted to attain this object by a
similar (secret) method of treatment. However, the " preserved milk " produced
by him (and not a few of his successors) failed to fulfil expectations, and equally
unfavourable results attended the numerous attempts made to render milk
stable by so-called " Pasteurisation," i.e. by the prolonged action of a temperature
of about 60° to 65° C., in connection with which subject N. L. RUSSELL (HI.)
carried out a series of investigations (Fig. 51). All these methods, as well as
numerous others of allied nature — such, for example, as that devised by Soxhlet,
and which Loeflund attempted to technically utilise — have, however, been

162

THE PRESERVATION OF MILK

recognised as unreliable. At present there is only one single way of arriving at
the object in view, and that is by thickening the milk and adding sugar.

This so-called " condensed milk " — as manufactured in particular by the
" Anglo-Swiss Condensed Milk Company " in their chief factory at Cham, on

Lake Zug, and in a number of branch
establishments outside Switzerland — is
prepared in the following manner : The
fresh new milk is purified by centrifugal
force, and is then heated on a water-
bath until nearly boiling, and mixed (in
a wooden vat fitted with a steam coil)
with 1 2 per cent, of cane-sugar. When
this is dissolved the liquid is passed
through a fine sieve and transferred to
a vacuum pan, where the thickening
process is effected at a temperature of
5o°-6o° C. As soon as the requisite de-
gree of consistency is attained the milk
is run off, rapidly cooled, and packed in
clean tins, which are soldered air-tight.
Commercial condensed milk contains
about 25 per cent, of water and 50 per
cent, of sugar, the remainder consisting
of albumen (12 per cent.), fat (n per
cent.), and ash (2 per cent.).

Some of the germs present in the new
milk, especially the lactic acid bacteria,
are already killed by the aforesaid heat-
ing before and during the thickening process. A few, however, survive this,
and are found to be still alive in the finished product, but not in a condition
to do any damage, since the high concentration plasrnolyses the germs, retarding
their development and so preventing decomposition. By reason of its high con-
tent of sugar, however, this condensed milk is unsuitable for the nourishment of
infants.

Illustratior

FIG. 51.
of the effect of Pasteurising milk.

The black square represents the germ-content of
raw milk per unit of space, that of the same
sample after Pasteurisation being shown by
the small white square. (Aj'ter liussell.)

CHAPTER XX.

THE PRESERVATION OF MEAT, EGGS, VEGETABLES, AND FRUIT.

§ 129.— Storage in Cold Chambers.

IT has been established by the researches of MEISSNEE and ROSENBACH (I.),
G. HAUSEU (III.), F. ZAHN (I.), J. VON FODOR (I.), and others, that the blood
and flesh of healthy animals are entirely free from fungi. On the other hand,
the contents of the digestive organs are exceedingly rich in Schizomycetes, higher
fungi not being absent, though their number is quite subordinate to that of the
former organisms. As was shown by D. POPOFF (1.), the digestive canal of the
healthy new-born animal is, at the moment of birth, free from bacteria. These,
however, subsequently obtain access, principally in the food, and the contents
of the bowels become extremely rich in microbes. According to the researches
of NENCKI and FREY (I.), such species as decompose the carbohydrates predomi-
nate in the small intestine in man, whereas in the large intestine the microbes
productive of albuminoid putrefaction exert their sway* s .

If, now, the carcase of a slaughtered animal be left without being disem-
bowelled, these saprophytes will make their way through the capillary vessels
of the intestinal villi into the arteries, the alkaline contents of which (rich in
albumen), are unusually favourable for the development of these acid-shy putre-
factive bacteria, so that the entire carcase quickly begins to undergo decompo-
sition. This can be prevented by the excision of the entire length of the
alimentary canal from oesophagus to rectum inclusive, and if this long-known
and practised precaution be adopted, then the remaining flesh, &c., will be per-
fectly free from fungi. If putrefaction subsequently arises, it is due to the
bacteria from external sources (air, supports, butchers' hands, &c.) obtaining
access to and settling in the flesh. Their gradual penetration by way of the
blood-vessels into the interior of the flesh was studied by S. TROMBETTA (I.) and
by Gartner. The latter found them only in the external layers in the case of
meat three days old, but, at the end of another seven days, they had penetrated
to a depth of 2 cm. below the surface. Since the sources of this bacterial
infection cannot be entirely shut off, though they may be considerably reduced
by cleanly procedure, attempts are made to prevent the increase of these parasites
in the flesh.

The oldest known remedy is cojd, but in order to realise expectations, the
temperature must be kept several degrees below zero (0.), and this is the method
pursued in the large (export) abattoirs in America (Chicago in particular) and
Australia. Immediately a beast is killed and disembowelled, the carcase is
placed in a refrigerating chamber and then transported in cooled railway trucks,
and cold chambers on shipboard, to its destination in a frozen state. Thus, for
instance, there appear daily in the London market hundreds of carcases of
Australian sheep still frozen hard. In a similar manner Central Europe has
been for several years supplied with haddocks prepared for shipment in the north
,'of Norway (Vardb) by being frozen at - 40° R. ( - 50° C ) directly they are caught
and cleaned, and being then shipped in this condition in specially built steamers.
,The freezing of meat does not kill the germs present, but merely hinders their

163

1 64 THE PRESERVATION OF MEAT, ETC.

reproduction, and, as a matter of fact, A. KOCH (III.) found very many bacteria
in fish that had been treated in this way.

If the meat be not stored at low temperatures, but merely put in the ice-chest
or laid on ice, whereby it attains, in the most favourable instances, a tempera-
ture of o° C., then, as follows from the already reported labours of Forster and
others, an increase of the initial number of germs ensues. To the activity of
such cold-supporting organisms is attributable the peculiar, disagreeable taste
and smell acquired by edibles after remaining in the ice-chest for a few days.
Actual putrefaction is not, however, produced by these bacteria.

We must not lose the present opportunity of issuing a warning against
bringing food-stuffs in immediate contact with natural ice, since this substance
contains not only numerous putrefactive bacteria, but also, under certain circum-
stances, pathogenic germs (especially typhus bacilli) as well. In this connection
we may refer to the researches into the bacterium content of ice that have been
made by C. FEAENKEL (V.), BORDONI-UFFKEDUZZI (II.), F. PEUDDEN (I.), and
A. HEYROTH (I.). In the cooling chambers of large abattoirs — of the arrange-
ment of which there is an excellent description in a work by OSTHOFF (I.) — the
meat is not exposed to this source of infection.

The well-known fact that frozen meat, when thawed, undergoes decompo-
sition more rapidly than fresh meat is easily explained. The cellular structure
is loosened by freezing, and access to the interior is thereby facilitated for any
organisms present on the surface.

§ 130.— Dried Meat and Salted Meat.

The development and activity of the organisms exciting decomposition can
also be prevented by depriving them of the water necessary for metabolism.
The drying of meat has been practised, particularly in hot countries, from the
earliest times. The resulting conserve is known in South America under the
names of pemmican, charque, and tassajo. The process is as simple as it is
reliable, and has a great future in prospect, especially for the provisioning of
armies in the field. In the method of treatment hitherto practised, the meat
during drying suffers a great depreciation in flavour, but in recent years Hof-
mann and Meinert have devised and patented a process for the artificial drying
of meat without removing or destroying its flavouring matters. By this process
a Bremen firm manufactures a meat meal met with in commerce under the name
of Came pura.

Under the same name inferior meat meals only fit for cattle food are shipped
to Europe from Argentina ; these are prepared from waste materials and require
some care in handling. The drying of flesh does not, of course, result in the
killing of all the bacteria present therein, and if the flesh of cattle suffering from
epidemic diseases has been employed, then, under the defective conditions of
live-stock inspection in South America, disease germs will be disseminated
by means of such infected food. A case of this kind has been reported by
R. BURRI (I.).

The most important example of dried flesh is afforded by the dried cod-fish
(stock-fish), which forms the chief article of export from the Scandinavian
peninsula. It contains, in the dry state, nearly 80 per cent, of albumen, and
constitutes a favourite and cheap article of food among the poorer classes in
Central Europe.

The salting and pickling of meat is generally credited with great efficacy,
but a closer examination reveals that it is really only the hygroscopicity of the
salt that comes into play and that the sole power the latter possesses is that of
setting up plasmolysis in the germs present in, or subsequently conveyed to, the

SMOKED MEATS AND CORNED BEEF 165

flesh, and so preventing their reproduction. Consequently, the germs, especially
those of a pathogenic nature, cannot be completely killed by these processes.
C. J. DE FREYTAG (I.) has proved that the influence of concentrated solutions
of common salt is resisted by tubercle bacilli for three months ; by typhus
bacilli for six months ; and by the bacilli of swine erysipelas for two months, the
organisms remaining alive and virulent during these periods. F. PEUCH (I.)
examined the effect of salting on the flesh of animals succumbing to anthrax,
and found that a ham from such an animal, after lying in salt water for fourteen
days, still contained virulent anthrax bacilli, as was proved by direct experiment
on animals with the expressed juices of the meat. PETRI (II.) showed that
virulent " rothlauf " (erysipelas) bacilli were still present in the pickled flesh
from swine affected with swine erysipelas, after six months' immersion in brine.

If the inspection of meat is carried out with even only a moderate amount of
care, it will not be easy for animals suffering from anthrax to be slaughtered for
food ; so that there is not much danger to be dreaded from that source. The
case is, however, different as far as animals affected with tuberculosis or swine
erysipelas are concerned.

In a former paragraph the prevalence of tubercular affections among cattle
was mentioned, and this should be sufficient to deter the reader from indulging in
uncooked beef, whether in the form of "beefsteak a la tartare" or uncooked
pickled beef. It is well known that the flesh of swine that have been com-
pulsorily slaughtered on account of swine erysipelas is offered for sale ; hence it
naturally follows that many kinds of (" wurst ") sausages that are made from raw
flesh, and eaten in an uncooked state, will contain pathogenic germs.

§ 131.— Smoked Meats and Corned Beef.

Smoking forms a more reliable means of preserving meat from putrefaction, the
real active agents in the process being the vapours of phenol, creosote, and allied
compounds present in the smoke. Beechwood being found to yield the smoke
containing the largest quantities of these substances, is therefore held in
particular esteem for this purpose. The volatile distillation products of heated
wood chips are condensed on the pieces of flesh, and arrest the development of the
bacteria. Since, however — as A. SERAFINI and G. UNGARO (I.) have shown —
these antiseptics do not penetrate far into the flesh, and are therefore unable to
exert much action in the interior of the pieces, smoking can only be effectual
when it is a question of preserving fresh meat (from healthy animals) which is
only superficially infested with germs. The manner in which the process is
carried out in practice very often leaves much to be desired ; and thus it is — as
shown by the exhaustive researches of H. BEU (I.) and A. SERAFINI (I.) — that
the germ-content of commercial smoked meat varies considerably. The salting
which precedes smoking, though of such little efficacy in itself, is nevertheless
useful, and forms an essential part of the process, by withdrawing water from the
meat, and thus facilitating the penetration of the smoke. The certain 'destruction
of pathogenic germs is not effected by smoking, Petri having found that the flesh
of swine affected with swine erysipelas contained erysipelas bacilli in a state of
undiminished vigour, after immersion for a month in brine, followed by careful
smoking for fourteen days. A similar unfavourable result was obtained by
J. FORSTER (III.) in the case of smoked meat from tuberculous animals.

The best method at present available for the preservation of meat consists in
steaming the same in vessels which remain hermetically closed up to the time
the meat is eaten. Such a food is known as preserved meat (in the restricted
sense), or as tinned meat, the quality in most demand being corned beef (chiefly
obtained from Chicago). This should consist of the flesh of oxen, but, neverthe-

1 66 THE PRESERVATION OF MEAT, ETC.

less, occasionally originates at the horse-slaughterer's. As already explained in a
former chapter, we are indebted to the French confectioner APPERT (I.) for the
fundamental practical experiments made in connection with preservation by
steaming. In accordance with his process, the meat is boiled in any convenient
vessel, and pressed into tins. These are then closed, with the exception of a
small aperture; and placed in a bath of boiling water, the aperture being closed
by means of a little liquid solder, after steam has been given off for a short time.
Appert's successors subjected his process to numerous modifications, Fastier pro-
posing to heat the tins up to 1 10° C. in a bath of salt, and others recommending
additions of boracic acid, &c. Up to the present, no known antiseptic possesses
the dual property of, on the one hand, preserving flesh without depriving it
of valuable nutritive constituents or flavouring matters, and, on the other, of
having no injurious effect on health when the meat is eaten regularly.

Inventors have been, and are still, particularly active in this field, and the
different processes for preserving meat that have been proposed are very numerous.
To report exhaustively on these would, however, far exceed the limits of the
present work ; any reader wishing to be more accurately informed on this subject
is referred to a comprehensive treatise compiled by PLAGGE and TRAPP (I.).
Instructions intended for practical use in the preservation of meat, fruits,
vegetables, &c., have been given by L. E. ANCLES (I.) and J. DE BREVANS (I.).

§ 132.— Preserving- Eggs.

The contents of the freshly laid eggs of birds, especially of poultry, are not in
all cases perfectly free from fungi. In refutation of a widespread assumption to
the contrary, it was shown by U. GAYON (II.) in 1875, and confirmed by 0. E. R.
ZIMMERMANN (I.) in 1878, that the eggs, even of healthy birds, are exposed, even
during the time of their formation, to infection by bacteria. These organisms,
starting from the common anal duct of the bird, make their way into the ovary,
where they become mixed with the albumen of the embryo egg, and reproduce
themselves therein when the nutrient medium permits. The new-laid egg is
therefore already inhabited by bacteria, a circumstance that must be borne in
mind when it is desired to utilise raw eggs for the cultivation of bacteria,
according to the proposal made by F. HUEPPE (V.).

The obnoxious decomposition not infrequently set up in eggs is generally
attributable to the development of these early intruders. Their pure cultivation
was first attempted by J. SCHRANK (I.), and then on a larger scale by C. ZORKEN-
DORFER (I.), according to whom the so-called spontaneous stinking putrefaction
of eggs goes on in two ways.

Thej?r«5 type is characterised by the albumen (white) changing colour through
whitish-grey to grey-green, and by the yolk becoming gradually converted into
a greasy, blackish-grey mass. At a subsequent stage the yolk becomes mixed
up with the albumen, so that the entire contents of the egg form a pulpy ichor,
smelling strongly of sulphuretted hydrogen, which gas is not infrequently pro-
duced in such quantity that the shell of the egg bursts with a report. Of the
organisms taking part herein, Zorkendb'rfer isolated ten species, and distinguished
them as Bacillus oogenes hydrowljureus a, ft, y, 8, t, £, 17, 0, i, K, the first six of
which liquefy gelatin.

In the second type of (bacterial) egg-putrefaction this gas is not detected.
Here the yolk and the albumen quickly coalesce to form an initially thin, but
subsequently pulpy, mass of a pale ochreous-yellow colour, and with an odour
like that of human faeces. Zbrkendorfer described five species of organisms
causing this decomposition, and bestowed on them the name of Bacillus oogenes
fluoreucens a, ft, y, 8, f. The first of these liquefies gelatin, and they all elabo-

PRESERVING VEGETABLES AND FRUIT 167

rate a pale green pigment which imparts a beautiful blue fluorescence to the
medium.

All these bacteria are exclusively aerobic, i.e. oxygen is essentially necessary
to their development. This needful gas obtains access from outside by passing
through the eggshell, which is well known to be permeable thereto, since other-
wise the development of the embryo chicken could not proceed. This necessity
for air on the part of the egg-putrefying Schizomycetes supplies the explanation
of the practice currently employed for preserving eggs, viz., by simply immersing
them in milk of lime, which not only excludes air, but also — by its disinfectant
properties — acts on the organisms present on the eggshell and ready to penetrate
into the interior, killing some and restricting the development of others.

The aforesaid bacteria perish within the space of two days when exposed to
temperatures above 40° C., but at lower temperatures and in damp air they
develop rapidly. Bearing these facts in mind, eggs could be preserved by keeping
them for one or two days at 50° C. and then storing them in a dry place, were
it not that the quality is thereby depreciated. If, then, steeping in milk of lime
is not determined upon, the eggs can be preserved by coating them with lacquer
or varnish after a careful cleaning.

That bacteria penetrate through the unbroken eggshell has — contrary to an
opposite opinion expressed by Gayon — been placed beyond doubt by the ex-
haustive researches of Zorkendorfer. WILM (I.) showed that pathogenic bacteria
are also capable of so doing, cholera bacilli being found, in his experiments, to
penetrate to the interior of the egg within fifteen to sixteen hours. PIORKOWSKI
(I.) arrived at a similar conclusion with regard to typhus bacilli. On this account
none but clean chaff, free from pathogenic organisms, should be used for packing
eggs-

Bacteria are not to blame in all cases where eggs become spoiled during
storage, but sometimes higher fungi (Eumycetes) come into play, penetrating
the shell and growing freely in the interior. Further particulars on this point
will be found in the second volume, in the chapter dealing with Iformodendron
cladosporioides.

§ 133.— Desiccating and Preserving- Vegetables and Fruit.

In contradiction of the erroneous assumptions of M. GALIPPE (I.) and
H. BERNHEIM (II.), it has been shown by E. LAURENT (III.), A. FERNBACH (I.),
and H. BUCHNER (VIII.) that — apart from the exceptions to be considered in
chapter xxxiii. — the cells and cellular tissues of the higher plants are, whilst in
a healthy condition, free from fungi. In the preservation of vegetable food-
stuffs it is, therefore, merely a question of the destruction, or restriction of
development, of the germs of extraneous origin inhabiting the surface. The
oldest process for attaining this object is that of drying, and this is practised
more particularly on certain fruits. In warmer regions the rays of the sun
suffice, e.g. in the case of raisins and figs, but in colder climes recourse must be
had to artificial warmth, and, consequently, so-called kilns or drying ovens are
employed, wherein hot air at a temperature of 60^-65° C. is allowed to stream
over the fruit. American desiccated apples and Bosnian and Servian prunes
are prepared in this way. A description of the individual systems of kilns for
this purpose cannot be entered upon here. The dried fruit still contains a con-
siderable proportion of moisture (some 30 per cent.) and at least the same
amount of sugar : about 30 per cent, in the case of pears, some 40 per cent, in
apples and damsons, 50 per cent, in figs, and 60 per cent, in raisins. From i to
3 per cent, of free acid is also present. Only some of the organisms present on
the fruit are killed by drying, but the development and decomposing action of

1 68 THE PRESERVATION OF MEAT, ETC.

the rest are checked by the plasmolytic influence of the high sugar-content.
The putrefactive bacteria also suffer through the action of the free acid present.
Sundry vegetables, especially those employed for Julienne soup, are preserved
by drying, for which purpose they are cut into small pieces, and exposed in
special ovens to a hot air temperature of 5o°-6o° C. Not infrequently they are
then, in accordance with a proposal made by Masson, subjected to hydraulic
pressure, compressed vegetables being thereby produced.

Drying is a comparatively inexpensive operation, but cannot be resorted to
in every case, since many kinds of fruit and vegetables have their fine flavour
too much impaired thereby. Such articles are treated by the Appert process.
Green peas, cauliflower, asparagus, beans, and suchlike are manipulated as
follows : After being carefully cleaned, they are placed in glass jars or in tins,
which are then filled with water and set in a salt bath, the temperature of which
is maintained at below 100° C. for one or two hours, and thereafter raised to
boiling point (108° C.), at which it must be allowed to remain for some time, in
order to definitely destroy all the hardy spores of the hay and potato bacilli.
The temperature is then allowed to sink to 60° C., whereupon the small blow-
hole in the otherwise closed tin is sealed up by means of a drop of solder, glass
jars being closed air-tight by other suitable means. Preserves carefully pre-
pared in this manner are sterile in the strictest sense of the term ; but if perfect
sterility is, by reason of any oversight, not attained, the still-living germs sub-
sequently increase at a great rate. Their development is mostly attended with
the evolution of gas, in consequence of which the straight walls of the tin are
bulged outwards, and frequently even burst.

A drawback accompanying Appert's process is that the colour of the
vegetables so treated is generally destroyed. If the preservation of the colour
be desired, as is the case, e.g., with red beet, gherkins, and the like, then other
means must be resorted to, and the antiseptic properties of the acids be utilised.
The samples in question are boiled in vinegar, the liquor being then poured off
and replaced by fresh unimpaired vinegar. In this way mixed pickles, for
instance, are prepared. In many cases the boiling is omitted, pickled gherkins,
for example, being preserved by simple immersion in cold vinegar.

That no protection against the development of bacteria is afforded by
steeping in brine needs no further argument. As a matter of fact, an easily
observable decomposition occurs in the so-called salted gherkins prepared in this
way, the phenomenon proving to be lactic fermentation ; and it is to the acid
thereby produced, and not to the small proportion of common salt present, that
the retardation of decomposition is due.

The boiling of fruits and fruit-juices is an operation too well known to need
detailed description here. The added sugar employed herein restricts decom-
position by strongly plasmolysing and preventing the development of such germs
as are not destroyed by the boiling. In many cases, this action is assisted
by the addition of a certain quantity of whortleberries. A few particulars
respecting the high percentage of the strongly antiseptic benzoic acid present in
the latter have already been given in an earlier chapter (§ 80). The glass jars
destined to contain the finished jam, marmalade, &c., are sulphured previous to
use. — The preservation of fruit is greatly facilitated by a careful preliminary
cleaning, a precaution that should, moreover, not be omitted when the fruit is to
be eaten raw, since the usually sticky surface tenaciously retains the dust and
accompanying germs that are blown on to it. Thus, M. T. SCHNIRER (I.)
discovered virulent tubercle bacilli on the surface of grapes sold in the Vienna
market.

A word must be added with regard to the gelatinisation of fruit juice. As
is well known, the cells of mxny fruits are rich in pectin, which, when the cells

PRESERVING VEGETABLES AND FRUIT 169

are crushed, passes into the juice and causes it to coagulate. This phenomenon
was explained by Fremy, in 1840, to be due to the action of an enzyme, viz.,
pectase, whereby the pectin is converted into pectic acid. This opinion was
modified by the researches of G. BERTRAND and A. MALL^VRE (I.), in so far that
they showed that the enzyme can accomplish the transformation referred to only
when in presence of soluble salts of the alkaline earths (e.g. lime), with which
the pectic acid enters into combination, and forms insoluble pectates. The
presence of this gelatinising compound is indispensable for the preparation of
pure fruit jellies ; and the latter must not be too strongly boiled, or their setting
properties will be diminished or completely destroyed. In the preparation of fruit
juices, such as raspberry juice, it is necessary, on the other hand, to get rid of
these pectin substances, because they detract from the utility of the juice, which
should remain liquid. This end is attained by leaving the fresh juice to itself
for a time, fermentation soon ensuing, by which the pectin or pectate is
decomposed. The juice is then strained and boiled down after the addition of
sugar. — A few references to the literature of the subject will be useful to the
food-stuff chemist, who is not infrequently asked for advice concerning the best
means of turning fruit to account. Full particulars on the treatment of fruit in
general, as also of drying and preserving it, will be found in the handbooks of
FR. LUCAS (I.), KARL BACH (I.), and H. TIMM (I.). A brief introduction to the
preparation and treatment of fruit wines has been arranged by M. BARTH (I.),
and a pamphlet written by W. TENSI (I.) deals chiefly with currant wine (as the
finest of all fruit wines), as well as with gooseberry wine, &c.

The preservation of wine-must is practised on a large scale, particularly in
Sicily. To render the juice highly suitable for transport, it is (after a preliminary
filtration) concentrated in vacua at 40° C. to about one-fourth of its original
volume. In this manner a thick, syrupy mass is obtained, the composition of
which can be deduced from the following analytical figures furnished by TH.
OMEIS :

Water 35. 1 per cent.

Dextrose + levulose . ,,, . w . 62.2 „
Acid . . . . '.".'/ . 1.2

Ash . .0.7 „

Albumen, gum, &c. . . . . • ••. ' . 0.8 „

This concentrated wine-must, which is shipped in sealed tins, is not in any
case sterile, though the still living germs (particularly yeasts) present therein
are, by reason of the high concentration of the liquid, incapable of development.
If, however, the mass be diluted with sterilised water, then fermentation ensues
within a short time. According to the experience of J. WORTMANN (II.), and
also of the author himself, the employment of this must can be recommended in
laboratories dealing with Fermentation Physiology. The dilution of i part of
must with 4 parts of water yields a nutrient medium exceedingly favourable for
the cultivation of higher fungi (wine yeasts), the low percentage of nitrogen
being improved by an addition of i per cent, of ammonium tartrate.

The preservation of beer and wine by heating (Pasteurisation) will be dealt
with in a subsequent section of the second volume.

SECTION VI.
LACTIC FERMENTATION AND ALLIED DECOMPOSITIONS.

CHAPTER XXI.
GENERAL CHARACTERISTICS.

§ 134.— Discovery of the Lactic Acid Bacteria.

IN chemical text-books acetic acid is generally characterised as being the first acid
known to man. This assumption cannot, however, be considered as probable,
since, in order to obtain acetic acid, the previous preparation of alcoholic liquids is
necessary, and the human race in its earliest stage of civilisation, viz., nomadic life,
would hardy have attained that skill — the production of wine and vinegar, even
in the most primitive fashion, presupposing a settled mode of existence. On the
other hand, the flock-owning nomadic races must, at a very early period, have
noticed that the milk supplied by their animals very quickly underwent altera-
tion, and turned sour when left to itself. Lactic acid must therefore be regarded
as the earliest acid known to man, though not in a pure condition, since that
condition necessitates the employment of methods for removing all the other
constituents of the milk. This result was first accomplished by the Germano-
Swede Scheele in 1780. The earliest complete chemical investigation of the
souring of milk was instituted in 1833 by PELOUZE and GAY-LUSSAC (I.),
but from that date fully twenty-five years elapsed before the knowledge that
this process is a manifestation of vital activity on the part of sundry micro-
organisms assumed definite shape. It is true that already in 1701 Andry had
noticed that sour milk contained such organised microcosms. Nevertheless, this
observation remained at first as unproductive, as regards the comprehension of
the question, as did also the labours of sundry other subsequent workers. Among
these mention may be made of Blondeau, who in 1847 made a microscopic
examination of milk, and distinguished therein two types of micro-organisms :
the one (which he named Torula) was a yeast-like plant ; the other, a mould
fungus, which he assigned to Penicillium and held to be the cause of lactic
fermentation.

We may recall to mind that Pasteur, in his treatise (against spontaneous
generation) in 1862, pointed to the non-success experienced by the opponents
of this theory, especially Schroder and Dusch, when they employed milk for
their refuting experiments. Knowing, as we do, that, before Pasteur, no one
had succeeded in rendering milk absolutely free from germs, it is therefore easy
to understand that up till then nothing definite could be urged against the hypo-
thesis, put forward by chemists, of the purely chemical nature of the process of
lactic fermentation. Thus, for example, ROWLANDSON (I.), under the influence
of the Liebig and Gay-Lussac theories of fermentation, denned the preliminary
conversion of lactose into lactic acid in the souring of milk as an oxidation pro-
cess, and expressed the nai've opinion that a cow that had been running about
(and therefore breathing rapidly) before milking would yield a milk rich in

170

BACTERIUM LACTIS LISTER 171

oxygen, and consequently liable to turn sour with unusual rapidity. The
opinion of FREMY and BOUTRON-CHALARD (I.) (formed in 1841, under the
influence of Liebig's theory), that casein was the cause ("ferment") of lactic
fermentation, was revived, though with little success, by A. P. FOKKER (I.)
in 1889.

Pasteur (X.) was the first to describe (1857) an organism characteristic of
lactic fermentation, and to prove the same capable of producing acidification in
a sweet, sterile milk. This organism, which Pasteur named the "ferment, or
yeast, of lactic fermentation," was a bacterium. A pure culture of this, in the
present meaning of the term, was at that time unattainable, no suitable method
having then been devised. Pasteur demonstrated the difference existing between
this "ferment" and that of alcoholic fermentation, and proved that in nutrient
media containing sugar, the former organism always sets up lactic fermentation,
whilst the other invariably gives rise to alcoholic fermentation.

This discovery formed an important and welcome support to the theory of
specific ferments promulgated by Fr. Kutzing in 1837, and implying that
chemically different fermentations are carried out by physiologically different
species of organisms.

§ 135.— Bacterium lactis Lister, and Bacillus acidi lactici Hueppe.

The important work issued in 1873 by the English surgeon and founder of
the antiseptic treatment of wounds has already been noticed (§ 68). In that
paragraph the methods of working employed by him at that time were referred
to as defective and misleading. Jt was also stated that the name, Bacterium
lactis, employed by him was erroneous, the bacterial culture to which it was
applied not being a uniform species, but an indefinite (and very probably highly
diversified) mixture of different species.

LISTER (II.) himself very soon recognised the weakness of his arguments, and
sought for a remedy. This he found in the so-called dilution method, by the aid
of which, in 1877, he produced from sour milk a pure culture of a fission fungus
to which he applied the name of Bacterium lactis as before — this time correctly.
The twofold origin of this name should therefore always be remembered. Lister
was also the first to make the observation, subsequently confirmed by Cohn, that
lactic acid bacteria, though of frequent occurrence in the rooms of dairies, are
comparatively seldom found in the open air.

The introduction of gelatinised nutrient media into bacteriology also furthered
the study of lactic fermentation. By means of this new method of pure culti-
vation HUEPPE (IV.) in 1884 isolated from sour milk a microbe known as
Bacillus acidi lactici, which, in so far as can be gathered from the description
given, was identical with Lister's bacterium. Hueppe also made the more important
discovery that several different species of bacteria are capable of setting up lactic
fermentation. However, before noticing these other organisms, we will examine
more closely the Bacillus acidi lactici, which occurs in the form of non-motile
rods 1.0-1.7 P- l°ng and 0-3-0.4 p. broad, mostly in pairs and but rarely united
to form a four-cell chain ; it is aerobic and forms endospores. This ferment
acidifies milk between the temperatures of 10° and 40° C., the reaction being
accompanied by the precipitation of casein, and an evolution of gas. On gelatin
plates the organism forms white colonies which do not liquefy the nutrient
medium.

In addition to the five species of lactic acid bacteria discovered by Hueppe —
and to which Microcoscus procligiosus belongs — many others possessed of the same
property have been made known to us, by Maddox in England (1885), Beyer in
North America (1886), and FOKKER (II.) in Holland (1890). R. KRUEGER (I.)

172 GENERAL CHARACTERISTICS

isolated his Micrococcus acidi lactis (which liquefies gelatin) from cheesy butter.
G. MARPMANN (I.) discovered five species belonging to this group in Go'ttingen
milk, and named them Bacterium lactis acidi, Bacillus lactis acidi, Bacterium
limbatum lactis acidi, Micrococcus lactis acidi, and Sphcerococcus lactis acidi.
G. GROTENFELT (II.) isolated a lactic-acid-forming, anaerobic Streptococcus acidi
laclici from Finnish milk. In his communication, issued from Hueppe's labora-
tory, there also occurs the remark that Bacillus acidi laclici H. can be perma-
nently deprived of its acidifying power by cultivating it for some time in media
free from sugar. This attenuation of the cultures is also often noticed in patho-
genic bacteria, many of which lose their virulence — i.e. poisonous nature, and
consequent capacity of producing disease — when kept for some time under
unaccustomed conditions of nutrition, viz., outside the animal body. Bearing
this in mind, Grotenfelt speaks of a variable virulence of Bacillus acidi lactici,
meaning thereby the possibility of reducing its fermentative power.

The fermentative properties of the Bacterium lactis aerogenes, found by
ESCHERICH (I.) in the contents of the intestines of sucklings, and also in uncooked
cow's milk, were investigated by A. BAGINSKY (II.), who found that in artificial
media containing lactose it produces both acetic acid and lactic acid. The gas
liberated during the reaction consisted of C02, 22 percent.; H, 30 percent.;
CH4, 9 per cent. ; N, 39 per cent. R. WURTZ and R. LEUDET (I.) considered
this microbe to be identical with Pasteur's lactic acid bacillus, but their opinion
does not seem to be well founded. According to J. DENTS and J. MARTIN (I.),
B. lactis aerogenes is only a variety of the Pneumobacillus (§33) discovered by
Friedlander.

Respecting the Pediococcus acidi lactici discovered by P. LINDNER (I.) a few
particulars will be given in chapter xxv. ; and details concerning the part played
by the lactic acid bacteria in certain industrial fermentation processes, such as
distilling, dairying and cheese-making, tanning, &c., will be found in chapters
xxiii. to xxvii.

§ 136.— The Equation of Lactic Fermentation

is (when lactose or grape-sugar is presupposed as the raw material) generally
expressed in chemical text-books as follows :

CaHaOu + H20 = 4C3H603
Lactose. Lactic Acid.

C6H1206 = 2C3H603.

Actually the process is not so simple as here represented, a certain quantity
of the sugar employed being consumed by the organisms to enable them to dis-
charge their vital functions and bring about the fermentation in question. Con-
sequently the actual yield of lactic acid obtained is less than the theoretical
quantity calculated from the foregoing equations. Another proof of the complex
nature of the operation is afforded by the large quantity of gas liberated during
the fermentation, but which is not indicated in the reaction expressed by the
equations aforesaid.

According to the researches of R. WARRINGTON (I.), the amount of acid pro-
duced varies greatly in different species, and is so small with some that (as noted
by Conn) it is insufficient to curdle the milk. This difference is explicable by
the varying susceptibility of the individual species to the adverse influence of
the resulting acid. On this account alone, fermentation may come to a stand-
still notwithstanding the presence of sufficient unconsumed nutrient material .
The difficulty is easily met by opportunely neutralising the acid by an addition o f

THE EQUATION OF LACTIC FERMENTATION 173

the carbonate of calcium, magnesium, or zinc. In the latter case, the highly
characteristic lustrous acicular crystals of zinc lactate ([C3H503]2Zn + 3 aq.) are
obtained.

A few quantitative experiments made by ADOLF MAYER (II.) show that 100
parts of fermented lactose produce —

83.9 parts of lactic acid
3.7 „ „ acetic acid
12.4 „ „ unknown substances.

These results, however, were not obtained with pure cultures of lactic acid
bacteria, and therefore are not fully conclusive.

Pure cultures of lactic ferments were first employed by E. KAYSER (I.) in
1894, in an investigation of fifteen different species of lactic acid bacteria isolated
from French milk, Belgian beer, Danish cream, wine-must, rye infusion, sauer-
kraut, &c. Confirming the results of Mayer and Baginsky, he showed that
volatile acids, also, are produced in the course of lactic fermentation, their
amount depending on the composition of the nutrient medium as well as on the
species of ferment. Thus, for example, a greater quantity of volatile acids was
produced from a milk qualified with peptone than from a peptonised maltose
solution. Cultures grown at the bottom of the nutrient liquid (" lactic bottom
fermentation ") yielded less than surface cultures. This fact had been already
recorded in 1889 by OPPENHEIMER (I.), who found the ratio of acetic acid
to lactic acid produced from milk fermented by Bacterium lactis acrogenes to be
as 85 : 15 ; and in the case of Bacterium coli commune as 70 : 30. This ratio is,
however, not invariable, but is chiefly determined by the amount of oxygen
present. Hence, in the absence of air, only small quantities of the volatile acids
are found, lactic acid being almost the only acid present.

Attempts were made by G. KABRHEL (I. and II.) and H. TIMPE (I.) to
investigate the part played by casein and the phosphates in the lactic fermenta-
tion of milk. The optimum fermentation temperature is between 30° and 35° C.,
and the operation proceeds much more actively when air is excluded. Kayser
was unable to detect any lactic enzyme excreted by the bacteria and capable of
converting lactose into lactic acid.

In view of these results, it is hardly necessary to say that the establishment
of a satisfactory equation to represent the reactions occurring in lactic fermenta-
tion is highly improbable.

CHAPTER XXlt

THE PRODUCTION OF OPTICALLY ACTIVE ORGANIC COMPOUNDS
BY FERMENTATION.

§ 137.— Isomers of Lactic Acid.

THE details given in the preceding chapter with regard to the physiological
• activity of the lactic ferments require supplementing in one important particular.
Mention has been made of lactic acid, and always without qualification or
reference to the fact that there are several isomeric lactic acids. This question
can now be considered from a higher standpoint, and the occasion utilised for
dealing with the production of optically active substances in general, through the
activity of micro-organisms.

Stereo-chemistry teaches that all optically active organic substances contain
in the molecule at least one asymmetric carbon atom, i.e. one whose four atom-
fixing powers or affinities are connected with four different elements, or groups of
atoms (radicals). On the other hand, experience shows that there are; compounds
(such as racemic acid and mesotartaric acid) which contain one or more asym-
metric carbon atoms, but are nevertheless optically inactive. The structural
formula of mesotartaric acid is COOH— CHOH— CHOH— COOH. This same
formula, however, is also adopted for two other acids, named from their optical
properties respectively dextro-tartaric acid and levo-tartaric acid. To explain this
fact it is necessary to refer to the hypothesis promulgated in 1874 by VAN 'T
HOFF (L), that the four equivalents of a carbon atom are arranged at the angles
of a tetrahedron, in the centre of which the carbon atom itself is situated, whereas
the atoms or radicals combined therewith occupy the angles. For example, let
Cabcd be a compound containing one asymmetric carbon atom C, with which are
combined the four (different) atoms or radicals a, b, c, d. By employing the
tetrahedron scheme, the arrangement of these latter can be effected in two
different ways :

These two formulae are not identical, but stand in the mutual relation of an
image and its reflection. In order to get from b over c to d, it is necessary
(looking from a) in the one instance to move in the same direction as the hands
of a clock ; but in the other case the movement is reversed.

The compound Cabcd is thus obtained in two modifications which have the

i74

HO/

THE ISOMERIC TARTARIC ACIDS i;5

same structural formula, and behave similarly from a chemical poipt of view, but
differ in a physical sense (crystalline habit, solubility, behaviour towards polarised
light). Such compounds, whose different behaviour can only be explained by the
assumption of a difference of grouping in the molecule, are designated stereo-
isomers. Of the compound Caked there may exist one modification capable of
deviating polarised light towards the right hand, and a second producing a left-
handed rotation ; and one part by weight of the dextro-rotatory modification
gives rise to just as great a deviation towards the right hand as one part by
weight of the levo- modification does towards the left hand.

If now a compound containing such an asymmetric carbon atom be artificially
prepared from substances devoid of asymmetric atoms, the probability is that
just as many levo- rotatory as dextro-rotatory molecules will be produced ; and
assuming that each molecule of the one kind coalesces with a molecule of the
other class to form a double molecule, then an optically inactive compound will
result. There may therefore be presumably obtained from one combination,
Cabcd, containing a single asymmetric carbon atom, three different modifications,
two of which (one dextro-, the other levo-rotatory) are optically active and the
third inactive. An example of such a compound is afforded by ethylidene lactic
acid —

CH3 H CH3

C = CH.OH

I
COOH COOH

of which one optically inactive form is known, and in chemical text-books
is generally named fermentation lactic acid. The dextro-rotatory modification
has also long been known to chemists under the name of sarco-lactic or para-
lactic acid, whilst the third modification, levo-lactic acid, was only discovered
and produced a few years ago by fermentation physiologists. This will be again
referred to later on.

§ 138.— The Isomeric Tartaric Acids.

Of the compounds containing two asymmetric carbon atoms in the molecule
we will refer to only that group in which these two atoms are connected by a
single bond, as is represented in the subjoined typical formula —

I— C — C— a

/ ^

c 7

As regards the positional arrangement of the two groups of substitutes
(a, b, c, or a, ft, y) in the molecule, four possibilities are conceivable : (i) both
grouped in the dextro- position; (2) both in the levo- position; (3) the one
dextro- and the other levo- ; (4) or, finally, vice-versd.

Assuming the group — C — to possess a (numerically) greater optical

rotatory power (D or L) than — C— ft, which may be expressed in letters

1

thus —

D > d L > I,

176 OPTICALLY ACTIVE FERMENTATION PRODUCTS

Then the rotatory power of the entire molegule will have the following values —
I) + d, L + I, D - d, or L -I,

according to which of the four possible compounds is present.

The optical effect of the one semi-molecule will therefore be strengthened
by that of the other semi-molecule in the first two instances, or weakened
thereby in the other two cases. Consequently there result four stereoisomeric
active modifications : the first strongly dextro-rotatory ; the second strongly
levo-rotatory ; the third faintly dextro-rotatory ; and the fourth faintly levo-
rotatory. Furthermore, one molecule apiece of the two strongly rotatory
(D + d and L + Z), as also one apiece of the two faintly rotatory (D - d and L - I)
modifications, can coalesce to form inactive double molecules —

The total number of all conceivable stereoisomeric modifications of the com-

°\ /"

pound b— C — C — 13 is therefore six.

c y

Particular interest attaches to the special instance wherein the two semi-
molecules are equal, and which therefore comprises the compounds expressed by

the formula b— C — C— b. Tartaric acid,

c c

COOH

COOH COOH CHOH
! — C— H

\)

H— C

\

CHOH

COOH

is the type of this group.

In this case we have D = d and L = Z. The four above-named general
expressions for the rotatory power of the individual active forms are, in this
special case, resolved into

2D zL 0 0

The modifications zD and 2L correspond to the dextro- and levo-tartaric acids ;
and, in place of the two faintly rotatory modifications, we have a single optically
inactive form, which, in the tartaric acid group, is named meso-tartaric acid.

This last-named acid is inactive as a result of intramolecular compensation,
and is therefore distinct from the modification produced by the coalescence of
the two optically active forms | ^ }i which double molecule is also optically
inactive, and in the tartaric acid group is named racemic acid.

The existence of two fundamentally different groups of optically inactive
compounds containing asymmetric carbon atoms is thus theoretically possible,
viz. :

i. Monomolecular, indivisible, optically inactive in consequence of
intramolecular compensation of their optically active atom groups. Type :
D-cJ = oorL-Z = o. Example: meso-tartaric acid.

THE DIVISION OF THE RACEMIC COMPOUNDS 177

2. Bimolecular, optically inactive, appearing only as double molecules,
one of which belongs to the dextro- and the other to thelevo- modification
of the compound. Type : 2D - zL = 0. The inactive double molecule
can be split up into two optically active components, 2D and aL.
Example : racemic acid (Fr. acide racemlque, Ger. Traubensdure).

The first of these two modifications, viz., the monomolecular, indivisible kind,
is termed the anti- combination ; the second, or bimolecular modification, being
known as the para- form, or (from the best-known example) racemic modification.

On the other hand, it is found, both theoretically and in practice, that bodies
containing but a single asymmetric carbon atom can appear in only one inactive
form, namely, the divisible, bimolecular para- form.

§ 139.— The Division of the Racemic Compounds.

The correctness of the assumption that the racemic modifications of
stereoisomeric bodies are actually double compounds, consisting of an equal
number of dextro-rotatory and levo-rotatory molecules, can be demonstrated
in two ways. The first is by synthesis. If, for example, equal quantities
of equally strong (nearly saturated) solutions of dextro- and levo-tartaric
acid be mixed together, combination of the two, attended with the evolution
of heat, occurs, and the mixture solidifies in consequence of the crystallisa-
tion of a racemic acid which can be proved to be identical with natural racemic
acid. Again, if equal quantities of dextro-lactate and levo-lactate of zinc be
dissolved in water and mixed, the optically inactive zinc salt of " fermentation
lactic acid" — possessing all the properties of this substance — will crystallise
out. This synthetic construction of the inactive double molecule from the two
optically active components is, however, of merely theoretical interest, but is
without any practical value, no new and hitherto unknown compounds being
thereby obtained.

As a rule, it is the inactive para- form with which we have to deal, and
this has to be split up into its constituent molecules of the dextro- and
levo- modifications. This division can be effected in several ways. In some
cases the inactive substance decomposes spontaneously on crystallising out
from solution, and the components — which differ in their crystalline habit —
can be separated by hand (selecting the crystals according to their structure).
The earliest example of this was given by PASTEUR (XI.). If a solution of
sodium-ammonium racemate (C4H4(NH4)Na06 + 4 aq.) be allowed to evaporate
slowly, hemihedral crystals are obtained. Pasteur found that the hemihedral
surfaces did not occupy the same position in all the crystals, but that the
one class of crystals formed, as it were, the reflected image of the other, one
being dextro-hemihedral but levo-rotatory, and the other levo-hemihedral
but dextro-rotatory. On separating the two kinds, and preparing the acid
from each, he obtained dextro-tartaric acid in the one case and levo-tartaric
acid in the other, but in no instance was the inactive racemic acid produced.

The most usual method of division consists in allowing the inactive sub-
stance to unite with an optically active one. If the substance to be split up
is an acid, then an active alkaloid (e.g. morphine, quinine, strychnine, &c.) is
used ; if a base, it is combined with dextro-tartaric acid. Experience teaches
that the resulting compounds have very different degrees of solubility, on
which account the two optically active modifications can be separated without
much difficulty. Thus, it was shown by T. PURDIE and J. W. WALKER (I.) in
1892, that by combining the optically inactive "fermentation lactic acid " with

1 78 OPTICALLY ACTIVE FERMENTATION PRODUCTS

strychnine, it can be decomposed into its two optical components, viz., dextro-
rotatory sarcolactic acid and levo-rotatory lactic acid.

However, the most important method of separation is that in which the
activity of micro-organisms is called into play. This method, also, was proposed
by Pasteur in 1860. He sowed certain lower fungi — the precise species cannot
now be ascertained — in a solution of optically inactive ammonium racemate, and
found that the levo-rotatory properties of the liquid gradually increased ; and
that after a certain lapse of time ammonium levo-tartrate alone was detect-
able. It must therefore be concluded that certain ferments are endowed
with selective powers. In the present instance, the organism has separated
the racemic acid into its two optically active components, one of which (the
D-tartaric acid) it consumes, whilst the other (the L-acid) is liberated. Since
Pasteur's time this separating power has been utilised in various ways, two
examples of which are now given. In the first place, J. LEWKOWITSCH (I.) in
1882 dissociated the optically inactive mandelic acid, C6H5 — CH.OH — COOH,
into its two active components in this manner. On the other hand, P.
FRANKLAND and W. FREW (III.) allowed their Bacillus ethaceticus to react on the
calcium salt of the optically inactive glyceric acid, CH,OH — CH.OH — COOH,
whereby its dextro-rotatory component was obtained, the levo- component being
consumed.

§ 140.— The Production of the Stereoisomerie Lactic Acids

by fermentation merits closer attention. In the first place, it should be men-
tioned that only the three isomers of ethylidene lactic acid, CH3 — CH.OH — COOH,
are in question, since ethylene lactic acid, CH2.OH — CH2 — COOH, has hitherto
been obtained by purely chemical means alone. The sub-title, fermentation
lactic acid, so long borne solely by the inactive form of ethylidene lactic acid,
is now recognised as also appertaining to both its stereoisomers, so that the term
is now synonymous with ethylidene lactic acid generally. Of the two active
forms, the so-called paralactic acid was the first to be prepared by the fer-
mentation method, the discovery being due to M. VON NEXCKI and N. SIEBER (I.)
in 1889. These observers found that certain tumours in an animal affected
with symptomatic anthrax contained (in addition to the characteristic bacillus
of this disease) an anaerobic fission fungus, which produces large quantities of
paralactic acid (i.e. dextro-lactic acid) in saccharine media, and is on that account
named Micrococcus acidi paralactici. On the other hand, the aforesaid patho-
genic bacillus, under the same conditions, produces inactive lactic acid.

Levo-lactic acid was first prepared in the year 1890 by FR. SCHARDINGER
(II.), by means of the fermentative activity of a short-rod species found in a
Hungarian well-water, and to which the name of Bacillus acidi Icevolactici has
been given. In size this organism greatly resembles Hueppe's Bacillus acidi
lactici. It ferments dextrose, saccharose, lactose, and glycerin, the resulting
products being levo-lactic acid, a little ethyl alcohol, a quantity of carbon
dioxide, and an unspecified combustible gas. The zinc salt of inactive lactic acid
is obtained by crystallisation from a warmed solution of the zinc salts of this
levo-lactic acid and of paralactic acid.

So far as is known at present, the Schizomycetes species forming levo-lactic
acid are of less frequent occurrence in nature than those producing either
inactive or dextro-lactic acid. K. GIJNTHER and H. THIERFELBER (I.) examined
a large number of samples of sour milk, in none of which could they find levo-
lactic acid. Neither did they succeed in isolating from the milk any fission
fungus capable of forming this acid, but always obtained either inactive or
dextro-lactic acid, or a mixture of both. This, however, does not imply that

PRODUCTION OF STEREOISOMERIC LACTIC ACIDS 179

Schizomycetes producing levo- lactic acid are rare. On the contrary, a considerable
number of species (mostly pathogenic) endowed with this property are already
known. Investigations on this point were first made by J. KUPRIANOW (I.) and
repeated by B. Gosio (I.), and it was shown that the amount of this acid
produced per unit of quantity of the fermented sugar varies according to the
species employed.

Vibrio cholerce asiaticce (in addition to other vibrios) was found to produce
levo-lactic acid, whilst Spirillum tyrogenum (Deneke) produced dextro-lactic
acid. On the other hand, G. LEICHMANN (I.), in 1896, showed that when
ordinary milk is kept at 44°-5o° C. — instead of the lower temperatures
employed by Giinther and Thierfelder — levo-lactic acid is invariably produced.
The (long-rod) fission fungus concerned in this reaction was named by LEIGH-
MANN (II.) Bacillus lactis acidi,a, name (as we have seen in § 135) already in use
for another species of Schizomycetes as long ago as 1 886.

The kind of lactic acid produced under given circumstances by a certain
bacterium affords, in many instances, a valuable means for differentiating allied
species. For example, in the case of Bacillus typhi abdominalis and Bacterium
coli commune, the latter — as shown by BLACHSTEIN (E.) — produces dextro-lactic
acid from glucose, whilst the former, under identical conditions, gives rise to
levo-lactic acid.

Nevertheless, the faculty of a given species of bacterium for producing a
definite kind of lactic acid must not be regarded as unconditional. On the
contrary, a good deal depends on the conditions of nutrition. The necessity for
the maintenance of identical conditions during experiments of this kind has
already been emphasised, and it should be also mentioned that the results are
influenced not only by the kind of carbohydrate (sugar) subjected to fermentation,
but also by the constitution of the nitrogenous nutrient material. For this
discovery we are indebted to A. PERE (I.), who showed that when ammonia salts
alone (unaccompanied by peptone) are at the disposal of the microbe, both
B, typhi abdominalis and Bacterium coli commune produce levo-rotatory lactic
acid from glucose.

The separation of optically active compounds by means of Eumyceles will be
frequently referred to in the second volume, so we will only mention a treatise
by M. VON NENCKI (II.), which describes a valuable method for manipulating
fermenting liquids and determining their content of optically active lactic acid.
The characteristics of the salts of this acid have been described by T. PURDIE and
J. WALLACE WALKER (II.). According to F. HOPPE-SEYLER and FR, ARAKI
(I.), the lithium salts are the most suitable compounds to employ in experiments
for determining the rotatory power of the various lactic acids.

CHAPTER XXIII.

THE ARTIFICIAL SOURING OF CREAM.

§ 141.— The Acid Generator.

THE preferences exhibited by the various families of the human race for different
kinds of butter are very marked. China and Japan, for instance — to which
countries Denmark ships large quantities of this food-stuff — prefer sweet-cream
butter, i.e. that prepared from fresh, sweet cream ; whereas, in Scandinavian
countries, Denmark itself, North Germany, and England, a preference for sour-
cream butter prevails.

In order to obtain the latter product, the cream is allowed to turn sour
and undergo a fermentation, principally of a lactic character. Until within the
last few years the general practice was simply to leave the cream to become sour
spontaneously ; hence, in view of the fluctuation to which the bacterial flora of
milk and cream is exposed, it is not surprising that such a method of procedure
frequently resulted in the production of defective butter.

A reliable means of combating this adverse tendency is, however, now avail-
able, namely, the process — introduced into the dairy industry in 1890 by H.
WEIGMANN (III.-V.) — of artificially souring cream by the aid of pure cultures
of selected races of lactic acid bacteria. This process is divided into two
manipulations: the preparation of the acid generator and the preliminary
treatment of the cream to be soured.

The acid generator (or starter) brings the cream quickly into a state of
fermentation. According to Weigmann's recipe, it is prepared as follows:
Separated or skimmed milk — in the proportion of 2-3 per cent, of the cream to
be acidified — is warmed up to about 60° C. and then immediately re-cooled as
quickly and as much as possible. This treatment kills some of the bacteria in
the milk and weakens others to such an extent that they cannot offer more than
a feeble opposition to the development of the lactic acid bacteria, which are then
added to the treated skim-milk. For this purpose a pure culture of lactic fer-
ment, obtained from a Dairy Experimental Station, is employed. The vessel is
kept for twenty-four hours at a medium temperature (15° C.), by which time
its contents will be converted into acid generator ready for use.

The cream, also, requires a preliminary treatment to prepare the way for the
action of the acid generator. Sterilisation, or at least Pasteurisation, would
afford the best results, but as these are generally difficult to effect in a reliable
manner, a method of weakening the " wild " bacteria present, by cooling the
cream down to a low temperature and then quickly warming it up again to
i6°-2o° C., has to be resorted to. The acid generator is then added and well
incorporated by stirring, and the cream vat is left, at about 15°-2O° C., until
the following day, by which time the cream will be ripe for churning.

If it be desired to cultivate the acid generator further, a small portion is
taken from the bulk before use and employed in the same way as a pure
culture.

In spite of the adverse opinion of many practical men, the possibility of
producing good butter from Pasteurised, or even sterilised cream, has been

180

THE AROMA OF BUTTER 181

demonstrated by the researches reported by P. SCHUPPAN (I.); and BENNO
MABTINY (II.) has drawn attention to the hygienic advantages attending such a
method of working. Finally, it was proved by POPP and BECKEK (I.) in 1893
that butter prepared from sterilised cream keeps better than that from Pas-
teurised cream, and far better than that from cream which has not been heated
at all.

§ 142.— The Aroma of Butter.

The results of Weigmann's researches up to the present (according to a
private communication) indicate that probably only a single species — though
appearing as numerous varieties — of bacterium sets up the lactic fermentation
now in question. This organism is a coccus (described by W. STORCH (T.) ),
measuring about i p. in diameter, and uniting to form chains. The varieties
(also called races] of this coccus, which, from the point of view of the systematic
botanist, do not differ sufficiently to be classified as separate species, generally
exhibit one or other of the following good qualities : they either give rise to a
powerful aroma, which imparts a very fine flavour to the butter, or else the
product, without exhibiting any marked excellence of flavour, is endowed with
good keeping qualities. Consequently such races or varieties should be used in
the manufacture of butters for export. Whether there are races in which both
these advantages are combined cannot yet be definitely asserted. The aroma
produced by the bacteria cannot have originated in the volatile acids of the
butter, since it is also developed in cultures free from fat, and containing no
nitrogenous nutriment other than peptone. Comparative experiments on the
flavour of butters prepared by the aid of different varieties of lactic acid bacteria
were made by ADAMETZ and WILCKENS (I.) in 1892. — Owing to a noteworthy
discovery effected by H. W. CONN (I.) in 1895, the question has latterly taken a
new turn. He succeeded in isolating from a sample of South American milk
a fission fungus named Bacillus No. 41, which is not one of the acid bacteria,
but produces in milk and cream a fine aroma, identical with the highly-prized
flavour known in North America as " grass flavour " or " June flavour," because
it is produced only in the month of June, at a time when the cows are foddered
on tender grass rich with blossom and perfume. Cream inoculated with this
bacillus yielded butter endowed with this fine grassy aroma. By means of this
process butter ran now be produced with a uniform degree of excellence and
marketable value. The mode of working with this bacillus is simple. A culture
of the organism in milk is procured, the usual volume being about ^ litre (nearly
half a pint), and is poured into about 6 litres (1.32 gall.) of Pasteurised and re-
cooled cream. After a lapse of a couple of days the whole is transferred to the
bulk of the (fresh) cream, which is left for twenty-four hours and will then be
ripe for churning. About 6 litres of this ripe cream are reserved for inoculating
the next batch in the same manner as before. It will be noticed that the bulk
of the cream is not heated, and consequently the lactic acid bacteria therein will
be still living and capable of souring the cream, whilst the Bacilh's No. 41 acts
concurrently and develops the aroma. The latter organism, however, retains
the upper hand, having been initially present in excess. This flavour-developer
has now been tested and proved in more than a hundred North American dairies,
so that its employment can be recommended. Naturally, fresh cultures must be
introduced into the dairy at intervals (two to three months), since otherwise the
bacterium gradually loses its powers.

The discoverer of the organism attributes to it the additional faculty of
remedying defective butters, but on this point the data at hand are insufficiently
conclusive. It should be mentioned that the microbe appears in the form of
non-motile short rods, 0.7 /x broad and i.i (* long, generally united in pairs, but

1 82 THE ARTIFICIAL SOURING OF CREAM

never as chains. The optimum temperature is 23° C. Its acid-producing powers
are so slight that the milk is never coagulated. The aroma developed in the milk
is initially delicate, but becomes progressively stronger, and finally (after a lapse
of several weeks) resembles that of fine cheese. As a result of further researches
published in 1896, CONN (II.) was led to conclude that acidification and the
production of aroma are independent phenomena. He considers that aroma is
developed by the activity of peptonising bacteria which separate volatile bodies (of
agreeable or offensive smell and taste) from the albuminoid constituents of the
cream.

§ 143.— Defects in Butter.

The advantages offered by this artificial method of souring cream are only
appreciated at their true value when its application cures certain defects in butter,
to which we will now refer, and which were formerly attributed exclusively to
bad fodder. In this case also bacteriologists have been able to confute erroneous
opinions and render valuable assistance to practice.

There is probably not a single dairy in North Germany or Denmark whose
butter has not at some time or other been " oily," i.e. exhibited a flavour
recalling that of mineral oil. This malady appears with particular frequency in
dairies deficient in appliances for keeping the souring cream and finished butter
sufficiently cool. Weigmann showed that both the acid generator (prepared by
spontaneous acidification) and the butter-milk of such dairies are very impure,
in a bacteriological sense, and he was invariably successful in remedying the
complaint by the introduction of artificial souring.

A second and not less injurious defect is the so-called turnip flavour. Butter
suffering from this complaint has a repulsive sweet taste, recalling that of
turnips. To throw the responsibility on the latter is an obvious, but not always
justifiable, procedure, since cases are known where neither the cows nor any of
the dairy appliances had come into contact with turnips, notwithstanding which
the flavour still made its appearance in the butter. C. 0. JENSEN (I.) discovered,
in 1891, in the milk of several Jutland dairies where this complaint had long
been rife, a microbe which he named Bacillus fcetidus lactis, and which was
recognised as the cause of the malady. This motile bacillus has a breadth of
0.4-0.6 ft, its length varying usually between o.y and 1.5 /*, and often attaining
5.5 ft. No spore- formation has been detected, and the organism does not liquefy
nutrient gelatin. As its second name implies, this bacillus gives rise to stinking
decomposition in milk, but is not the only species producing the same complaint,
Jensen himself having grouped along with it a number of others possessing the
same power, among them being several species of micrococcus (not more
definitely named), a Merismopedium, &c. The employment of pure cultures of
lactic acid bacteria gave satisfactory results, producing a pure and fine-flavoured
butter in place of the previously almost unsaleable article.

A third evil which is probably (though not yet indubitably) attributable to
bacteria, is the so-called fishy or train-oil flavour in butter. Other defects, such
as greasy, tallowy, cheesy butter, have their origin in the inferior chemical com-
position of the cream ; whilst for the third group of complaints, e.g. stable smell
and smoky smell, the uncleanliness of the milker is responsible.

How firmly the injurious bacteria settle themselves in the rooms of the
infested dairies is evident from the observations made by Ronneberg, according
to whom the beneficial results accruing from the employment of pure cultures
of acid bacteria in infested dairies are only temporary, and disappear if fresh
supplies of the invigorating pure culture are not introduced in good time.

The advantages of this new method are so numerous that its employment
should not be delayed until the maladies in question appear ; in fact, the method

DEFECTS IN BUTTER 183

is designed as a protective rather than a remedial measure- The expense of
applying the method is small, the supply of pure culture needs, under normal
conditions, to be renewed only about once a fortnight, and the outlay at the same
time ensures protection from unwelcome surprises. Even when the continuous
employment of the method is not decided upon, it should at least be practised
at such times as a change from dry to green fodder, or vice versd, is made, this
change often becoming very unpleasantly manifest, not only in the cream-pan,
but also in the cheese-room, as will be explained in chapter xxxi.

The method is most extensively used in Denmark. From a report by FRTIS,
LUNDE, and STORCH (I.), it appears that whilst in 1891 only 4 per cent, of the
samples of butter exhibited at the butter shows (held annually in various parts
of the kingdom) had been prepared by the aid of pure cultures of acid generator,
the number had increased in 1894 to 84 per cent. This fact affords the best
recommendation of the method.

CHAPTER XXIV.

THE COAGULATION (CURDLING) OF MILK.

§ 144.— Acid Curdling- and Rennet Curdling".

THE amount of nitrogenous constituents in cow's milk fluctuates between 2.5
per cent, and 4.2 per cent, by weight, and is on the average 3.5 per cent. The
chemical composition of these nitrogenous matters has not yet been satisfactorily
determined, and can only be touched upon here so far as is requisite and useful
for bacteriological purposes. More precise information, accompanied by copious
bibliographical references, will be found in "W. FLEISCHMANN'S (I.) work on
dairying.

In refutation of the opinion expressed by DUCLAUX (VII.), that only a single
albuminoid body is present in normal cow's milk, the Swedish chemist Olaf
Hammarsten showed, in 1875, that at least three such compounds can be
distinguished therein, viz., casein, lactalbumen, and globulin. The first forms
about 80 per cent, of the total quantity, and the remainder is principally
lactalbumen (free from phosphorus), globulin being present in but very small
quantities ; both of these latter are soluble in water. The casein (containing
phosphorus) is acid in character, and consequently is not present in a free state
in the milk, but occurs as a salt of lime containing 1.55 parts of CaO per 100
parts of casein. This compound of lime and casein is not dissolved in the milk,
but is held in suspension as a swollen, colloid, finely divided mass. When the
milk is acidified the casein is liberated, and — being insoluble and incapable of
swelling — is precipitated in fine flakes ; in other words, the milk curdles. The
acid may be either artificially added or generated by fermentation in the milk
itself ; in either case the ensuing precipitate is known as acid curd (Ger. Quark).

Milk can also be curdled by another means, namely, by lab or rennet, an
enzyme secreted by special glands in the stomach of many animals. This rennet
is very plentiful in the stomach of the calf, from which it is prepared by drying
in the air and leaving to stand for a few months, then comminuting the mass
and extracting with a weak (5 per cent.) solution of common salt.

On adding a small portion of such a solution of rennet to sweet, unboiled,
lukewarm milk, the latter gradually curdles, the coagulum thus formed being,
however, not casein itself, but a derivative of that substance. Hammarsten
found that the casein is in this case split up into two portions differing greatly
in amount, viz., lacto-protein, small in quantity, soluble, and remaining in the
whey, and the insoluble paracasein. The latter, therefore, forms the chief
constituent of the coagulum separated (" set ") in cheese-making by the aid of
rennet, and known as rennet curd (Ger. Bruch), or crude cheese.

Casein, or paracasein, though the sole nitrogenous constituent of the coagu-
lum produced in any of these methods, is, however, by no means its sole
component, a number of other substances being precipitated and carried down
at the same time. If whole milk — i.e. unskimmed milk — is set for cheese,
almost the whole of the fat will be found in the curd, which will then subse-
quently produce rich cheese — skim-cheese being the result in the converse case.
Along with the fat, the calcium phosphate contained (in suspension) in the milk

184

CHARACTERISTICS AND ACTIVITY OF LAB 185

will also be thrown down only in the case of rennet curd, not in the curd
produced by acidification.

Not only are fat and (in this instance) calcium phosphate carried down by
the coagulum, but also a large part of the organisms present in the milk will be
found in the fresh curd, so that the latter is relatively as rich in organisms as
the milk from which it was precipitated. Here, again, a considerable difference,
from a biological point of view, exists between the two classes of curd, and
exercises a decisive influence on their subsequent career. The flora of the
rennet curd from sweet (i.e. almost neutral) milk is much more diversified than
that of acid curd. The latter, having been thrown down from a sour milk in
a state of vigorous lactic fermentation, consequently contains only a limited
number of species, and these endowed with a particular fermentative power.

Acid curd differs, therefore, from rennet, both in the method of production
and also in composition. Being devoid of flavour, both kinds are, however, un-
suitable for food ; their conversion into a form in which they both stimulate the
appetite and are also themselves more readily digestible, is the task of the
cheese-maker's art, fuller particulars of which, from the bacteriological point of
view, will be found in chapter xxxi.

§ 145.— Characteristics and Activity of Lab.

If the enzyme in question were exclusively a metabolic product of the animal
body, the foregoing details would suffice. However, since it is excreted by many
fungi as well, a few additional particulars will not be out of place in a work
dealing with technical mycology.

When and in what manner the attention of mankind was first drawn to this
enzyme cannot be determined, since even the oldest authorities, e.g. the Bible,
speak of its employment as an ancient practice. Its method of action, however,
was unknown even down to the commencement of the nineteenth century.
The prevalent opinion, based on the curdling of sour milk, was that the
precipitation effected by rennet was indirect, an acid being first formed, which
then caused the precipitation of the curd. The elucidation of the true state of
the case was reserved for BEEZELIUS (I.) in 1840, and his discovery was soon
afterwards supplemented by the labours of FR. SELMI (I.), 0. G. LEHMANN (I.),
HEINTZ (I.), and VOELCKER (I.), who showed that the action of rennet is quite
independent of the formation of acid.

The identity of this enzyme with the pepsin discovered by SCHWANN (§ 18)
was disproved by 0. HAMMAESTEN (I.) in 1872. This observer was the first to
successfully separate these two gastric secretions, an operation which DESCHAMPS
(I.) had failed to effect thirty-two years earlier. Hammarsten, however, did not
make use of the name chymosin, proposed by his predecessor, but adopted the
ancient appellation, lab. We are also indebted to the Swedish chemist for deep
researches into the activity of this substance. It acts on casein alone, not on
lactose or lactalbumen.

A divergence of opinion still prevails as to the nature and course of this
reaction, and we will therefore merely refer to the investigations made on these
points by the under-named workers: A. DANILEWSKY and P. RADENHAUSEN (I.),

W. EUGLING (I.), F. SCHAFFER (II.), E. DlJCLAUX (VIII.), M. ARTHUS and

C. PAGts (I.), A. S. LEA and W. S. DICKINSON (I.), S. RINGER (I.), P. WALTHER
(I.), and A. FICK (I.). SCHREINER (I.), in 1877, showed that milk when boiled
is no longer coagulable by rennet, but the reason for this behaviour was not
ascertained until eleven years later, when FR. SOLDNER (I.) found that the lab
reaction can only proceed in the presence of soluble salts of lime, which
latter are precipitated by boiling. For the same reason coagulation does not

1 86 THE COAGULATION OF MILK

occur in milk that has been rendered alkaline ; and the most favourable
condition is one of very slight acidity. The constitution of this enzyme is
still unknown. Its decomposing power is unusually high, one part by weight
being (according to Sbldner) sufficient to throw down at least one hundred
million parts of casein. As A. MAYER (III.) has shown, the optimum tem-
perature for the reaction is 37° C., the coagulation taking three times as
long at 25° 0. Above 45° C. the enzyme is paralysed, and is destroyed at
70° C. Coagulation does not ensue immediately upon the addition of the lab,
but only after a lapse of from several minutes to some hours, according to
the temperature.

The occurrence of this enzyme in nature is by no means rare. It was
found by Roberts in the stomach of birds, and in that of fishes by Benger,
and is also present in the cell-sap of various plants, e.g. of butterwort (Pin-
gulcula vulgaris and P. alpina), withania (Punceria coayulans), fig-tree (Ficus
carica), artichoke (Cynara scolimus), and others. It is also excreted by several
species of Schizomycetes, particulars of which will be given in the following para-
graph. For industrial purposes, however, there is only a single source (the
richest) worthy of consideration, viz., the stomach of the calf. The method of
production indicated above is now practised on a manufacturing scale, especially
in Copenhagen, whence most of the German and Dutch cheese factories derive
their supplies. The products are : Kennet solution, containing boracic acid
to improve the keeping quality ; rennet powder ; and, finally, rennet tabloids.
The efficacy and germ-content of these preparations were investigated by FRITZ
BAUMANN (1.).

§ 146.— Lab-Producing1 Bacteria.

Milk may also curdle without previous acidification or addition of rennet.
HAUBNER (I.) in 1852 was the first to record this fact, and the first explanatory
research was made by DUCLAUX (IX.) in 1882. From his studies in this matter
the latter concluded that this precipitation of casein (occurring with an alkaline
reaction) is due to the activity of certain bacteria which excrete an enzyme
lesembling lab; and CONN (III.), in 1892, succeeded in isolating this enzyme
from cultures of such Schizomycetes. At first sight the identity of this lab with
the active ingredient in the rennet solution from the stomach of the calf appears
probable, but the discovery that the bacteria in question (which include many of
the species belonging to the potato bacillus group, § in) are able to coagulate
boiled sterilised milk, goes against this view, rennet being incapable of producing
this reaction.

The presence or absence of this power affords, in many instances, a valuable
means of differentiation between two species of bacteria. This is particularly
the case with the organism producing abdominal typhus in man, viz., Bacillus
typhi abdominalis, discovered by Eberth in 1880, and already referred to in
preceding chapters. This microbe is not endowed with the faculty in question,
whereas, as JAK. URY (I.) has shown, a number of the putrefactive bacteria
collectively termed Bacterium coli commune, and greatly resembling the typhus
bacillus in form, &c., rapidly produce coagulation in milk. According to the
researches of C. GORINI (I. and II.), Micrococcus pi-odigiosus also produces this
enzyme in large quantities.

§ 147.— Casease.

Not infrequently the precipitation of casein effected by such bacteria
disappears again after a short time. Duclaux ascertained that this new
alteration is due to a second (albumen-dissolving) enzyme, to which he gave the

CASEASE 187

name of casease. The same observer also discovered, in the case of several
species of Tyrothrix isolated from Cantal cheese, and of which a description will
be found in chapter xxxi., certain bacteria gifted with the faculty of excreting
both enzymes, the casein precipitant and the casein solvent. R. WARINGTON
([I.) as well examined a number of such species, and observed that they all
liquefy nutrient gelatin.

The ratio between these two enzymes differs in the various species. A few
produce the casein solvent alone, and when sown in milk do not precipitate the
casein, but decompose it direct into soluble fission products, among which leucin
and tyrosin have already been identified. In proportion as the casein disappears
the milk becomes clearer, and is finally quite transparent.

The production and activity of both these enzymes are variously dependent
on external influences, the one resembling lab being able to act only within
narrow limits of temperature, whilst the other — the proteolytic enzyme — I. as a
wider sphere of activity. The same applies to the methods of nutrition of the
bacteria in question. A species examined on this point by Conn initially pro-
duced both enzymes, but subsequently — after prolonged cultivation on gelatin —
yielded the proteolytic one only ; and by increased interference the production
of the latter also can be restricted. WOOD (I.) attained this object by adding,
during several successive generations, a little carbolic acid to the nutrient
bouillon employed.

The constitution of casease has not yet been accurately determined, neither
has any one succeeded in ascertaining whether, and in what respect, this enzyme
differs from pepsin and trypsin — which it greatly resembles in action — nor
whether the casein-dissolving enzyme produced by different species of bacteria
is the same in all cases. WEIGMANN (VI.) states that he has isolated casease from
bacterial cultures, and that this substance favours and accelerates ripening when
added to fresh cheese.

The spontaneous coagulation of milk without the co-operation of micro-
organisms, the possibility of which was maintained by early workers, denied by
Lister, and finally established as a fact by Meissner, was more closely examined
in 1887 by A. LEVY (I.), who found that a very faint coagulation can be
detected in all milk that has been left to stand for some time. The sediment
deposited by such milk contains, however, only a small quantity of coagulated
casein, the bulk consisting of small fragments of decomposed colostrum. As the
cells of this latter substance die off a slight degree of acidification ensues, which
causes the precipitation of a certain quantity of casein.

The rapid curdling of milk so frequently observed during thunder-storms has
not yet been satisfactorily explained. The opinion expressed by J. LIEBIG (I.)
in 1890 will not bear investigation, and the assumption put forward, on
experimental grounds, by G. TOLOMEI (I.), that it is caused by the action of
ozone produced by electrical discharges, rests on insufficient foundation. The
same objection also applies to the views held by H. GEKSTMANN (I.) on this
point.

CHAPTER XXV.

LACTIC ACID BACTERIA IN DISTILLING, BREWING,
AND VINDICATION.

§ 148.— The Spontaneous Acidification of Distillery Yeast-Mash.

THE preparation of the pitching yeast for distillery work is not such an easy
matter as in the sister industry of brewing. In the regular course of the latter
no special labour is required for the production of the necessary quantity of
yeast, since in this case the yeast settles down, as soon as the fermentation is at
an end, to the bottom of the tun, and can then — after the immature beer is racked
off — be used at once for " pitching " (i.e. inducing fermentation in) a fresh
quantity of wort. The case is different in distillery work, where the liquid to
be fermented, instead of being thin and self-clarifying like wort, is a thick mash,
in which the yeast cannot settle down. For this reason the distiller is obliged
to prepare his pitching yeast in another way. He grows it artificially in special
vats, and, on this account, terms it "artificial yeast." For this purpose a sweet
mash is prepared in a small tun, the quantity amounting to about 10 per cent,
of the principal mash to be fermented. A more detailed description of the pre-
paration of this yeast-mash belongs to the domain of Chemical Technology, and
we will here content ourselves with briefly mentioning that crushed green malt
is mixed with water and gradually warmed to 6-j°-To° C., then mixed with a
variable) amount of sweet "goods" from the principal mash tun, and the mix-
ture left to saccharify for two hours at 70° C. Before this medium is pitched
with the yeast to be reproduced, it must, however, be subjected to another
preliminary treatment known as " souring."

The green malt is infested with a copious flora of various kinds of bacteria,
chief among which are the species of the hardy organisms causing butyric fer-
mentation. These spores are not killed by the aforesaid mashing temperature —
which, moreover, for reasons connected with the preservation of the diastase,
must not be exceeded — and therefore they afterwards germinate and increase,
and produce butyric acid. Now this acid, being a powerful yeast poison, would
injure the development of the pitching yeast ; but since the injurious bacteria
are themselves very sensitive to high degrees of acidity, their development may
be hindered by quickly making the fresh mash decidedly acid. To attain this
end the lactic acid bacteria are called in aid.

The question now arises, How can the latter be cultivated without allowing
the butyric ferments to gain the upper hand ? This can be secured by main-
taining the optimum temperature, which for the lactic acid ferments now under
consideration is between 47° and 52° C., whereas the butyric ferments thrive
best at about 40° C. The sweet yeast-mash is therefore kept at about 50° C. ;
consequently the lactic acid bacteria develop with vigour, and the increase in
their activity can be determined by titration.

When the operation progresses satisfactorily, the acid-content rises with
increasing rapidity and attains 2.2-2.5 degrees of acidity; i.e. 20 c.c. of the
filtered sour mash require 2.2 to 2.5 c.c. of normal alkali for complete neutralisa-
tion, a quantity corresponding to i.o-i.i per cent, of lactic acid. When this

1 88

ARTIFICIAL SOURING WITH LACTIC ACID BACTERIA 189

point is reached, the mash is heated to 70° C. in order to kill the lactic acid
bacteria, and is immediately re-cooled to i7°-2O° C. and pitched with yeast.
For the first yeast-mash of a new season a sufficient quantity (i kilo, per hecto-
litre of mash, i.e. at the rate of i Ib. per 10 gallons) of a pure culture of a
selected race of distillery yeast is employed. Such yeast can be obtained from
the Berlin Experimental Distillery Station (Versuchsstation fur Brennerei). At
the expiration of some fourteen to sixteen hours the development of the yeast
has so far progressed that the contents of the vat can be applied to their destined
purpose, a portion (about one-tenth) being, however, reserved, under the name
of mother-yeast, for pitching the soured yeast-mash on the following day. The
remaining nine-tenths of the prepared yeast are then transferred to the principal
mash, whereby the latter not only receives the requisite amount of active yeast,
but is also rendered acid, and is thereby better enabled to resist bacterial infec-
tion. This explains the whole dictum of the distiller, " The more acid in the
yeast, the less in the fermenting tun," because the greater the acidity of the
mature yeast-mash, the lower the possibility of injurious (acid-producing) germs
developing in the principal mash during fermentation. The reason for this is
that lactic acid reduces the vital activity of the microbes (butyric acid and acetic
acid bacteria) now under consideration. The increase of acidity in the mash is
employed as a measure of the progress of the fermentation. When the yeast is
first added, the sweet mash exhibits an acidity of o.5°-o.7°, corresponding to
0.2-0.3 Per cent, of lactic acid, and this increases during fermentation by 0.2°
when the management is first-class, 0.3° when good, and by as much as 0.4° and
more when the process is not properly carried out.

§ 149.— Artificial Souring- by the Aid of Pure Cultures of
Lactic Acid Bacteria.

The credit of recognising the utility of souring the mash is due to practical
distillers themselves, their experience on this point having been gained by repeated
experiments. It is only in recent years, however, that a closer insight into the
characteristics and actual value of this preliminary treatment of the yeast-mash
has been obtained. Until lately the generally accepted opinion was that
expressed by SCHULTE IM HOFE (I.), viz., that lactic acid is necessary, or at any
rate favourable, to the conversion of the (insoluble and undiffusible) albumi-
noids of the wort into peptones assimilable by yeast. Delbriick's researches on
this point failed, however, to reveal the presence of any appreciable quantity of
peptones in the soured yeast-mash, and it is now certain that the favourable
result is solely due to the relative toxic action of lactic acid. This acid acts
much more quickly and powerfully on the development of the bacteria than on
yeast, the latter being able to stand a fairly large amount of the acid without
appreciable injury.

The reader may well inquire from what source these lactic acid bacteria
which cause the souring of the yeast-mash are derived. Until recently the
answer was far from satisfactory, since it indicated that the matter was left to
chance. The initial temperature of 70° C. in the yeast-mash kills the lactic
acid bacteria already present therein, but not the spores of the butyric fer-
ment ; the subsequent development of the latter is, however, prevented by the
restrictive temperature of 50° C. maintained during the souring process. The
active lactic acid bacteria must, therefore, make their way into the mash from
outside sources, e.g. the air, the vessels, and utensils, &c., so that the inoculation
of the mash is left entirely to chance. Consequently it is not surprising to learn
that the operation frequently miscarries, failures being, under such circum-
stances, inevitable.

190 LACTIC ACID BACTERIA IN DISTILLING, ETC.

We are indebted to Morawsky for the first improvement on this point.
Instead of waiting for the yeast-mash to become infected spontaneously by lactic
acid bacteria, as in the ordinary course, he proposed to set aside about one-tenth
of the soured mash before applying heat, and to add this mother-acid to the next
day's mash as soon as the latter has been saccharified and cooled down to 50° C.
This modification, although constituting a valuable improvement on the older
method when once operations are in full swing, nevertheless does not positively
guarantee good souring ; and its deficiencies are most apparent at a time when
help is most essential, viz., at the commencement of a new season. During the
first few days after work is resumed, it often becomes apparent from the odour
permeating the yeast-room that the souring is not progressing satisfactorily, but
that the mash is rich in butyric acid. This is due to the fact that the lactic acid
bacteria in the distillery have more or less completely perished during the
summer while the works were shut down.

To completely overcome the difficulty, nothing must be left to chance, and
the souring must be properly regulated by inoculating the sterilised and cooled
yeast-mash with a sufficiency of a pure culture of lactic acid bacteria. Such a
method was first introduced by the author at the Hohenheim Distillery, where it
was tried with great success. The further treatment of this artificially inoculated
mash differs in no wise from the procedure already described, i.e. when the sour-
ing is completed the mother-acid is removed, the bulk of the mash is heated up
to 70° C., then cooled, and pitched with the prepared mother-yeast. Next day
a portion is taken to serve as mother-yeast for a succeeding mash, and the
remainder is added to the principal mash. If, through any mischance (unskil-
fulness or carelessness on the part of the distiller), the souring of a given mash
proves defective, then no mother-acid is reserved from it, but a pure culture is
used for pitching the yeast-mash on the following day.

Although the species of bacterium now in question shares with the milk-souring
bacteria the property of decomposing sugar and forming lactic acid, it nevertheless
differs from them in more than one respect. For example, the various species of
the lactic acid bacteria in milk, so far as they have been examined, are incapable
of developing in mashes and worts under the conditions prescribed above. Mor-
phological differences are also apparent at the first glance, the cells of the
distillery-bacillus being long, almost invariably more than 2.5 j*, and very
frequently attaining ten times this length, whilst the breadth remains uniformly
about i p. This microbe was isolated by the author in 1896 from a satisfactory
yeast-mash prepared by the old souring method in the Lietzen Distillery (in the
Mark Brandenburg), and received the name of Bacillus acidificans longissimits.

On account of its powerful fermentative activity, this bacillus can also be
utilised to advantage in the preparation of lactic acid for technical purposes. The
dyeing and cloth -printing industries in particular require continually increasing
quantities of this acid, the preparation of which by purely chemical means is at
present a rather costly process, and can be more cheaply effected by means of
lactic acid bacteria. For this purpose a sterilised unhopped beer wort, rich in
maltose and qualified with a sufficient addition of calcium carbonate, is inoculated
with a pure culture of the bacillus and maintained at 50° C. When the fermen-
tation is ended the liquid is concentrated, and the lactic acid separated by
decomposing the calcium lactate formed. G. JACQUEMIN (I.) proposed a similar
method, but gave no precise information concerning the nature of the ferment,
and it is therefore uncertain whether the organism is allied to, or identical with,
the above-mentioned bacillus. The method described by E. DELACROIX (I.)
utilises, by a similar course of treatment, the sweet whey formed as a waste
product in dairies.

EFFRONTS HYDROFLUORIC ACID METHOD 191
§ 150.— Effront's Hydrofluoric Acid Method.

It is found impracticable to protect the fermentation of distillery mash from
injurious by- fermentations by sterilising the mash before adding the pitching
yeast, since such treatment would also kill the diastase, the continuance of whose
saccharifying action during the fermentation cannot be dispensed with. More-
over, such sterilising would not be of much value, since it is practically impossible
to protect such large quantities of fermentable material from subsequent infec-
tion by extraneous germs during the fermentation. The only course, therefore,
is to devise some means of restricting the development of the invading organisms.
The souring of the yeast-mash is, as already explained, a method of this kind.
This method, however, was not based upon a recognition of the true nature of
the evil to be overcome, but is rather the result of multifarious experiments,
which finally demonstrated that a strongly acidified yeast-mash affords a
guarantee for the satisfactory progress of fermentation in the mash proper, and
protection from injurious bye-fermentations. Consequently, as soon as the
anti-bacterial action of lactic acid was recognised as the actual agency at work in
this process, investigation into the suitability of other bacterium poisons for the
purpose in question followed as a matter of course. Thus in 1886, U. GAYON
and G. DUPETIT (I.) ascertained that an addition of o.i gram of basic nitrate of
bismuth per litre of mash was able to keep the fermentation free from contamina-
tion. Many other investigators have occupied themselves with the same subject,
from which it is evident that the task is by no means an easy one. As a matter
of fact, the antiseptic sought must, to be suitable, unite in itself several properties.
For one thing, if must be able to restrict the development of the Schizomycetes
without injuring the yeast present at the same time. Furthermore, it must
not impart any evil odour or flavour to the alcohol produced, and must, therefore,
be non-volatile and remain behind in the distillation residue (grains) without
being — in its actual condition of dilution — dangerous to the animals subse-
quently fed thereon. Finally, the employment of the bacterium poison should
not entail any great expense. It is, however, difficult to find a substance capable
of fulfilling the whole of these conditions. The metallic poisons, such as the
aforesaid bismuth salt, must be at once dismissed from consideration. The acid
sulphite of lime (calcium bi-sulphite), which has been frequently recommended,
is rendered unsatisfactory owing to the partial reduction of its sulphurous acid,
by the fermentative organisms, to sulphuretted hydrogen, which spoils the odour
of the alcohol. An addition of artificially prepared lactic acid to the mash is
too expensive, and its substitution by mineral acids is, with a single exception,
impracticable, owing to their injurious action on the yeast. Up to the present
only a single reagent has proved useful, viz., hydrofluoric acid, which was intro-
duced into distillery practice by J. EFFKONT (II.).

This so-called hydrofluoric process — i.e. the use of this acid, either in a free
state or in the form of salts, especially as ammonium fluoride — has already passed
through two stages of development and given rise to a number of investigations
and treatises, which will be found epitomised in an essay by H. CHATELINEAU
and A. LEBRA.SSEUR (I.). EFFRONT (I.) commenced his publications on the
subject in 1890. His initial proposition was to add between 4 and 8 grams of
HF per hectolitre (22 galls.) to the mash (treated in the usual manner) before
pitching with the yeast, this quantity being sufficient to prevent the development
of injurious bacteria.

Hydrofluoric acid surpasses all other mineral acids in its anti-bacterial powers,
since, according to Effront, 25 mgrms. of this acid per 100 c c. of wort will
prevent the appearance of lactic or butyric fermentation, whereas 200 mgrms.

192 LACTIC ACID BACTERIA IN DISTILLING, ETC.

of hydrochloric acid or 300 mgrms. of sulphuric acid are necessary to produce
the same results. The butyric acid bacteria, being more susceptible to the
influence of acids, can be repressed by as little as 10 mgrms. of HF per 100 c.c.

This original hydrofluoric acid process entailed no alteration in the customary
method of preparing the yeast, and in particular the souring of the mash
remained unchanged. Effront, however, endeavoured to render this preliminary
treatment superfluous by modifying his method into the so-called new hydro-
fluoric process by adding a sufficient quantity of hydrofluoric acid or fluorides to
the sweet mash instead of leaving it to sour spontaneously.

Here naturally follows the question of the action of hydrofluoric acid on the
vital activity of yeast. It has been proved that the susceptibility of the various
races of yeast to the influence of this acid differs, a circumstance which explains
the irregular (sometimes good, sometimes bad) results yielded by the old process.
In distilleries using very susceptible yeast the prescribed addition of HF to the
mash might not only be without any good result, but probably even give rise to
unfavourable symptoms, such as sluggish or imperfect fermentation.

The discovery that cell-reproduction on the one hand and fermentative
activity on the other are affected in different degree is an important one.
According te EFFRONT (III.), the former is completely arrested by the addition
of 300 mgrms. of NH4F per 100 c.c., whereas the fermentative energy is merely
reduced, not stopped, by this quantity. The same authority has also showed
(IV.) that yeast can be gradually accustomed to large additions of fluorine. In
this manner a given yeast can be brought to withstand an addition of 300
mgrms. of HF per 100 c.c. without losing its reproductive power. Doses below
this limit — up to about 200 mgrms. per 100 c.c. — retard reproduction, but
stimulate the decomposing energy of the organism, and therefore lead to a larger
production of alcohol. The yeast becomes so much accustomed to this stimulant
that it is subsequently rendered incapable of unfolding its energies except when
pitched in a mash also containing fluorine, the yield of alcohol being otherwise
far below the normal standard.

In practice the new hydrofluoric process is, in the main, carried out as
follows : For the preparation of the yeast-mash 4 parts iper cent, (by volume)
are taken from the principal mash (previously saccharified and cooled down to
30° C.), and a sufficient amount of hydrofluoric acid (or fluoride) added, this
being followed by i volume of mother-yeast to each 4 volumes of mash taken.
Of course, at the commencement of the season a sufficiency of pure culture
yeast must be used instead. The amount of added acid is regulated by the kind
of yeast in use, i.e. by its susceptibility; but 10 grams (say £ oz.) of HF per
hectolitre (22 galls.) of yeast-mash will generally suffice. After the addition of
the mother-yeast the mash, which was pitched at 26° C., quickly warms up to
about 31° C., at which temperature it is maintained. In this procedure the
older process described in the preceding paragraph is somewhat modified : the
heating of the saccharified yeast-mash up to 70° C., which was there found
advantageous, being, in the present instance, abandoned (since the injurious
organisms are suppressed by the HF), as is also the separate addition of malt to
the mash. In addition to the properties already mentioned, hydrofluoric acid
is also credited with exercising a favourable influence on the diastatic action, in
that in presence of this acid a much smaller amount of the said enzyme suffices
tojhydrolise a given amount of starch in a given time. The ratio of maltose and
dextrin is also modified in favour of the former, as much as 96 parts of maltose
being sometimes obtained per 100 parts of starch, whereas in presence of HC1 (or
HjSOJ the highest percentage of maltose amounts to 75 (or 76) per cent., and in
the absence of mineral acids to 74 per cent. When the yeast-mash is matured
(in about twenty hours time), one-fifth is set aside to serve as mother-yeast for

THE LACTIC ACIDIFICATION OF WINE 193

the succeeding mash. The residual four-fifths are incorporated with the fresh,
saccharified principal mash, previously mixed with enough hydrofluoric acid (or
ammonium fluoride) to make the (percentage) content thereof equal to one-half
that of the yeast-mash. This proportion has been found to be suificient ; and
since high patent royalties have to be paid — to the " Societe Generate de Maltose
a Bruxelles," of which M. Effront is director — for the use of hydrofluoric acid for
this purpose, the distiller will naturally avoid employing more than is absolutely
necessary.

Although hydrofluoric acid undoubtedly affords a reliable means for com-
bating bacteria, and can be used with advantage to keep yeast free from these
objectionable organisms, the case is different when the purification of a yeast
from contaminating wild yeasts is in question. EFFJRONT (V.) prescribed a
method which he thought could effect this latter purpose, but the same was
shown by A. JORGENSEN and J. CH. HOLM to be unreliable. Some further
particulars on this point will be given in a suitable place in the second volume.
At present we will merely state that the hydrofluoric acid process in nowise
supersedes the employment of pure culture yeast ; on the contrary, the value of
such yeast has here been revealed in a new light.

§ 151.— The Lactic Acidification ("Ziekendwerden ") of Wine.

The souring of wine and beer is by no means a uniform phenomenon, but
may, on the contrary, appear in many forms. The most frequent is the vinegar
taint, i.e. a partial conversion of the alcohol into acetic acid by acetic acid
bacteria. Fuller particulars of this evil will be given in chapter xxxvii. The
subject of the present paragraph is the lactic; acid taint, i.e. the production of
acidity by lactic acid bacteria, and which is generally known in Germany as
" Ziekendwerden." This is a not infrequent malady, and usually makes its
appearance in company with other injurious changes. Even Pasteur classed it
along with the so-called turning or breaking of wine, and the expression " Vin
tourn6 " is still applied in France to both phenomena, other terms being " Vin
monte " and " Vin qui a la pousse." The course and characteristics of the
malady are as follows : It mostly attacks young vintages, occasionally appearing
even in the first year. The wine turns turbid, and the odour and flavour
gradually become irritating, like rancid butter. The turbidity increases by
degrees to such an extent that the wine has the appearance of diluted milk, this
white break passing over finally, in many instances, into the stage of black
break, the wine then being in the condition of a brown to inky black liquid.
Concurrently with this change of colour occurs a gradually increasing precipi-
tation of dark slimy masses — a phenomenon characteristic of this malady. The
presence of lactic acid in " Vins tournes " was detected by A. BALLARD (I.)
in 1861. J. BERSCH (I.) examined four samples of broken ("zickender ") wine
for their acid-content, and obtained the subjoined results :

—

Xo. I.

No. II.

NO. nr:

No. IV.

Free acid, cal. as tartaric acid
Carbonic acid . . . .
Acetic acid

Per Mil.
5-71
0.09
0.67
2 V7

Per Mil.

3-35
0.05

1. 00

086

Per Mil.
1-59
0.05

i-37

Per Mil.
8.2S
0.19
0.88
o 08

The production of the two last-named acids was ascribed by PASTEUR (XII.)
in 1866 to the action of fission fungi. Experience shows that vintages poor in

194 LACTIC ACID BACTERIA IN DISTILLING, ETC.

acid, e.y. 1893 wines, are particularly liable to the malady. MliLLEu-TnuiiGAU
(I.) found such wines were always infected with a bacillus 1.2-2.0 /i long and
0.3 /i broad, capable of forming lactic acid, not merely from sugar alone, but
also from tannic acid and another (unidentified) constituent of wine. When
inoculated in must, this bacillus sets up lactic fermentation.

Musts that from any accidental cause have been deprived of the whole or a
great part of their acid-content are therefore very susceptible to this kind of
infection. Thus, MACH and PORTELE (II.) report on a considerable occurrence
of lactic acidity in South Tyrol, where, in the autumn of 1882 and 1883, the
vineyards in the lowlands of Etsch were flooded, and the grapes became in-
crusted with the carbonates of lime and magnesia. Consequently a considerable
portion of the acid in the must became neutralised in the process of crushing,
the immediate result being complaints of the appearance of lactic acidity. On
the other hand, those grapes that had been freed from the incrustation of
carbonates, by treatment with dilute sulphuric acid before crushing, escaped the
malady.

Here, as also in most other maladies of wine, the true cause of the evil is to
be sought in the defective constitution of the liquid. If the presence of disease-
producing germs were the sole essential factor, there would be hardly ever any
good wine at all, because all grapes — and therefore all fresh must — are infested
with a variety of species of fission fungi, both harmless and injurious. The
researches of MULLER-THURGAU (II.-IV.), MACH and PORTELE, and MARTINAND
and RIETSCH (I.), conclusively proved this in many instances ; and yet, not-
withstanding the (often considerable) infection thus produced, the must under
normal conditions resists its foes so effectually that the matured and bottled
sound wine is free from bacteria. This fact, demonstrated by SCHAFFER and
FREUDENREICH (II.), is so decisive that it was regarded by both these workers
as an indication of purity, since the made wines examined by them invariably
exhibited a larger or smaller content of bacteria. In future investigations on
the subject of the diseases of wine, more attention will have to be paid than has
hitherto been the case to the natural susceptibility of the wine to infection.
No known remedy exists for the lactic taint in wine, but Pasteurisation may be
recommended as a preventive measure.

§ 152.— The " Turning: " of Beer.

Although the term " turning," as applied to wine, is not yet clearly defined,
still, in the case of beer, only a single malady is understood by this definition,
viz., the undesirable appearance of lactic fermentation. PASTEUR (III.) made
several observations on " biere tournee," and traced the cause to certain fission
fungi, which he described as long rods i p. broad and of variable length, fre-
quently joined together in chains. For a closer investigation of these we are
indebted to H. VAN LAER (I.), who in 1892 obtained pure cultures of this
ferment, and named it Saccharobacillus pastorianus.

The commencement of this malady in beer is evidenced by a gradual decrease
in the brightness of the (previously clear) liquid, which finally becomes quite
turbid, and gradually assumes an unpleasant smell and taste. If the sample be
shaken, delicate waves of a fine thread-like character appear in the liquid,
resembling in appearance the fine films produced at the plane of contact between
two liquids of unequal densities. This appearance is so remarkable that it
suffices of itself to characterise the malady. After a time there separates out a
deposit, which Pasteur reproduced in Plate II. of his above-mentioned work,
and which consists — apart from the yeast-cells, which may be disregarded — of a
nitrogenous precipitate thrown down by the lactic acid, and of single cells and

WHITE BEER, LAMBIC, GINGER-BEER 195

chains of Saccharobacillus pastorianus. The latter are the cause of the aforesaid
optical phenomenon exhibited when the liquid is shaken. Meat-broth gelatin
is unsuitable for the pure cultivation of this fission fungus, and it develops but
imperfectly in wort gelatin, so that slightly alcoholised malt extract agar-agar,
in which the organism thrives, has to be employed. The re-inoculations made
by Yan Laer into sound beer decisively proved the agency of Saocharobacillus
pastorianus in the production of "turning " in that beverage. It is, however,
incapable of developing or becoming injurious except when the percentage
content of hop extract (i.e. the hop resins inimical to bacteria) in the medium
is small. This influence of the hop resins was, however, not further investigated
by Van Laer.

As its generic name implies, Saccharobacillus paslorianus ferments sugars,
and especially saccharose, maltose, and dextrose, which it acts upon readily, but,
on the other hand, ferments lactose with difficulty. Saccharose is apparently
transformed without inversion, since the presence of invertin could not be
detected in the cultures. In media containing one of these sugars the bacillus
chiefly produces inactive lactic acid, in addition to ethyl-alcohol and a small
quantity of volatile acids (acetic and formic acids), the proportions varying with
the kind of sugar and the conditions of cultivation. Given a sufficiency of
sugar, the degree of acidity produced is then solely dependent on the composition
of the remainder of the medium ; in unhopped wort it amounts to as much as
1.26 grams (calculated to lactic acid) per 100 c.c., whilst in hopped wort it does
not exceed 0.27 gram. The development of the bacillus is not restricted by
alcohol unless more than 7 per cent, of this substance is present in the beer. It
thrives better in the warm, and consequently the malady is of frequent occur-
rence in summer in countries where the cellar accommodation is defective.
This explains the Flemish name " Zomerbier," applied to turned beer in general.
The organism cannot survive continuous exposure to 55°-6o° 0. for a short
time; consequently beer intended for export to tropical countries may be
protected against risk of " turning " by Pasteurisation.

§ 153.— White Beer, Lambic, Ginger-Beer.

A low percentage of lactic acid is met with even in the best beers. It is
derived partly from the malt itself, which contains on an average 0.05 per cent,
of this acid, but is chiefly produced during the mashing process, the amount then
developed being nevertheless small — ranging in normal beers between 0.05 and
0.2 per cent. The nature and amount of the acids produced during the malting
of barley, the kilning and mashing of the malt, and the boiling of the beer-wort,
have been investigated by E. PRIOR (I.). In addition to the rod-shaped species
already described, lactic ferments in the form of globular cells developing into
sheet colonies appear in the malt-mash. An acid-producing species of this class
was examined by P. LINDNER (I.), who named it Pediococcus atidi lactici. Its
diameter is 0.6-1.0 ft, and the optimum temperature is about 40° C., but the
organism is killed in two minutes by a temperature of 62° C., and it does not
thrive either in hopped wort or beer. — The spontaneous lactic fermentation
appearing under certain circumstances in malt-mashes has been investigated,
from a chemical point of view, by M. HAYBUCK (II.). The variety of the fission
fungi developing in these mashes is very considerable, the first in point of size
being the Sarcina maxima, described by P. LINDNER (II.), the individual cells of
the packet- colonies of this organism measuring 3-4 /i in diameter. Attempts
to obtain pure cultures of this, the largest species of sarcina, have hitherto proved
unsuccessful.

In many instances the appearance of a vigorous lactic fermentation in beer-

196 LACTIC ACID BACTERIA IN DISTILLING, ETC.

wort is regarded with favour and its development encouraged. This applies to
the so-called " Weissbier " (white beer). No careful bacteriological investigation
of the acidification process, which plays such an important part in the prepara-
tion of this refreshing beverage, has yet been made. Possibly Saccharobicilhis
pastorianus is concerned therein ; at present, however nothing definite can be
stated on this point.

A considerable amount of acidity is produced in the Belgian beers known as
Lambic, Faro, and Mars, beverages prepared by spontaneous fermentation with-
out any addition of yeast. The boiled and re-cooled wort is placed in barrels
which are only partly filled, the empty internal space communicating with the
external air by a small aperture. Sufficient yeast-cells to set up fermentation
are left adhering to the walls of the casks from the previous fermentation, so
that after a lapse of twenty-four hours an evolution of gas is already noticeable.
In addition to alcoholic ferinentaton, lactic, and subsequently also acetic, fermen-
tation sets in. L. v. D. HULLB and H. VAX LAER (I.) published in 1891 the results
of a chemical investigation of this matter. The more important of these are
tabulated below :

Age of the Lambic.

Alcohol per Cent.
by Weight.

Lactic Acid
per Cent.

Acetic Acid
per Cent.

10 months . . . . .
12 months . . . . " .
36 months

4.84
4.07

0.310
0.900

0.044
0. 121

o 098

47 months .....

5-24

0.939

0.336

The beverage is consumed after a storage period of three to five years, and, in
its matured condition, is known as " gueuse Iambic." The acidity then amounts

to about i per cent., and is
masked by an addition of
sugar immediately before

FIG. 52. — Section through Ginger-beer Plant.
The cells of Saccharomyces pyriformis are surrounded by the
cells of Bacterium vermiforme, the membranes of which are
very much thickened and swollen. Magn. 680. (After H.
M. Ward.)

mentation also occurs in the
case of ginger-beer (to which
reference has already been
made in § 64). The prepara-
tion of this foaming acid
beverage, which is largely
consumed in England in the
summer-time, is a very simple
matter. To a 10-20 percent,
solution of sugar are added
a few pieces of ginger and
a couple of granules of the ginger-beer plant the whole being then left
to stand uncovered. The liquid soon begins to ferment briskly, is bottled
at the end of twenty-four hours, and consumed within the next two days.
The so-called ginger-beer yeast was more closely examined by H. M.
WARD (II.). It consists of whitish translucent masses about the size of a hazel-
nut, and is a mixture (Fig. 52) of a yeast, Saccharomyces pyriformis, and a fission
fungus. The cell-walls of the latter organism are gelatinised in a manner with
which we shall become acquainted later on, more particularly in the case of cer-
tain filamentous bacteria : the greatly swollen outer layers of the cell-membrane

WHITE BEER, LAMBIC, GINGER-BEER 197

becoming detached, but only so far as to constitute an independent outer case or
sheath enveloping the cells. This jacket either surrounds the cell along its

FIG. 53. — Bacterium vermiforme.

a-h. The gradual evolution of the cells and chains into the worm form.

i-m. Development of a sheath increasing oil the one side ; the cell-membrane
thickens in certain directions only, and not equally all round. Magn. 680.

x-z. Dissolution of such unilaterally thickened cell-membranes into branched
forms, the cells being visible at the extremities of thj branches. Culture in
ginger-beer nutrient gelatin ; x, observed at 10 A.M. ; y, the same at 4 P.M., and
2, at 10 A.M. the following day. T..e parts indicated by x, xx, xxx, correspond.
Magu. 420. (After Ward.)

entire length or else only along one side thereof, and is in some cases absent
altogether. The breadth of the cell itself measures about 0.5 /*, and the length

1 98 LACTIC ACID BACTERIA IN DISTILLING, ETC.

varies between 0.5 and 50 /z. The thickness of the sheath is often ten times
greater than the diameter of the cell. When examined under the microscope,
this thick envelope with its comparatively thin enclosure resembles a worm, and
it is on this account that the name Bacterium vermiforme has been given to the
fungus.

The bacterium is shown in Fig. 53. It stands in symbiotic relation with the
Saccharomyces pyriformis, so that the development of the one is facilitated by
the presence and vital activity of the other. Ward also succeeded'in artificially
constructing the ginger-beer plant from its two components. The chief products
of this fermentation are carbonic acid and lactic acid, in addition to traces of
alcohol and acetic acid. The lumps of the plant are kept in a state of saltatory
motion by means of the carbonic acid gas, and increase considerably in size during
the fermentation. They are able to withstand desiccation, and shrink up on
drying to form a horny mass, in which condition they are stored for future use.
The origin of the ginger-beer plant is unknown.

CHAPTER XXVI.

THE LACTIC ACID BACTERIA IN THE PREPARATION
OF FODDER.

§ 154.— Brown Hay.

ONE of the processes wherein micro-organisms play an active part for the pre-
servation of juicy fodder, viz., that dealing with burnt hay, has already been
noticed in § 106; and we will now briefly sketch a second and more general
practice, viz., the preparation of brown hay. As in the former case, the warmth
necessary for driving off the water in the fodder has to be supplied by thermo-
genic bacteria. In addition to these, however, another series of organisms,
converting part of the carbohydrates into lactic acid, butyric acid, &c., comes
into play. The percentage of water in the green fodder employed for making
brown hay must be smaller than in that worked up into burnt hay. The
materials (grass, &c.) are built up into a round or square rick from 16 to 24 feet
in diameter, and 13 to 16 feet high, well trodden down, and thatched in thereof
to prevent the incursion of rain-water. At the end of about three days, spon-
taneous heating (" sweating ") will become manifest, and its progress can be
conveniently followed by means of a metal pipe laid in the stack and containing
a thermometer (provided with a couple of attached strings), which can be drawn
out as required, for the purpose of reading off the temperature prevailing in the
interior of the heap. In proportion as the temperature rises (generally up to
70° C., and frequently still higher), the mass begins to steam, and this goes on
for eight to fourteen days, a further four to eight weeks being allowed to pass
before the brown hay can be considered as finished. The product forms a firm,
dry mass, the colour of which, under normal conditions, is between pale and
dark brown, but is black when overheating has occurred. In point of cohesion
this brown hay is preferable to air-dried hay, being less brittle than the latter,
but tough and suitable for fodder. Its odour is aromatic, and recalls that of
freshly-baked bread or honeycomb. Comparative investigations into the chemical
changes produced in this method of preparation have been made by Dietrich,
Moser, Weiske, and others. The results obtained by Dietrich are given below
(see Table, p. 200), since they afford material for judging the process from the
Fermentation Physiologist's point of view. A parcel of aftermath was divided
into two portions, the one being worked up into withered (air-dried) hay, and
the other to brown hay ; and an average sample of each was subjected to analysis.
A comparison of the first and third lines of this table at once reveals the
high percentage of lactic acid and butyric acid in the brown hay, both of which
are entirely absent from air-dried hay. It will be evident, upon mature con-
sideration, that the production of these acids by bacterial activity does not occur
in the centre of the stack, since the temperatures (yo^go0 C.) prevailing there
are such as only the passive reproductive forms of these organisms are able to
withstand. To obtain a correct idea, we must picture the changes occurring
within the stack as proceeding in the following manner : the thermo-bacteria
develop in the centre of the mass and liberate heat, which radiates towards the
outside. Between this hot central layer and the external strata exposed to the

199

200

LACTIC ACID BACTERIA IN FODDER

Crude

Extractive

Protein.

Non-

Nitrog'enous

Portion

Matter.

Percentage
Composition of

Water.

Total.

Soluble
in
Water

Fat.

Woodv
Fibre.

Asli.

Citric
Acid.

F.actic
Acid.

Butyric
Acid.

Portion

( =

Total.

soinme

Amides.
&c.).

in
Water.

a. Air-dried hay

15.0

9-8

3-0

2-3

40.9

17-5

24.6

6.7

0.66

_

_

b. Brown hay .

2O. I

10.5

1.0

2.Q

21.9

9.0

28.1

7-3

6-97

2.23

c. The tetter)

calculated

to the same V

15.0

11. i

i.i

3-1

23.2

96

29.9

7.8

—

7.42

2.23

water - con- 1

tent as a. J

cool air lies a broad zone wherein the precise temperature (4o°-5o° C.) most
suitable for the development of the acid bacteria in question prevails. The meta-
bolic products from these organisms then gradually permeate the entire mass.
The chief material for these fermentations is afforded by carbohydrates (starch),
as may be seen from the foregoing table, according to which the air-dried hay
contains 40.9 per cent!, whilst the brown hay contains only 23.2 per cent, of
non-nitrogenous extractive matter (starch, sugar, &c.). Of the nitrogenous con-
stituents, those soluble in water, i.e. amides and kindred bodies — of which the
brown hay contained i.i per cent, and the air-dried hay 3.0 per cent. — are for
the most part consumed. The loss of matter attendant on the preparation of
brown hay is calculated by Dietrich as about 14 per cent.

Brown hay exhibits one advantage over both air-dried hay and burnt hay,
namely, that its preparation is much less dependent on the weather, a couple of
fine days sufficing for protecting the finished hay. These days can, however, be
selected at convenience, since the ricks of brown hay can be left untouched for
a long time without dread of spontaneous ignition. Hence this method is fre-
quently employed in rainy districts, e.g. the North Sea littoral and the Austrian
Alps. It is, however, inadvisable to resort to this practice where good air- dried
hay can be made from the green fodder at disposal, because the feeding experi-
ments performed by G. Kiihn and others, and reported by FR. ALBERT (I.) and
FR. FALKE (I.), concordantly demonstrate that the preparation of brown hay is
attended with a considerable loss (amounting to as much as 50 per cent, of the
total) of digestible protein substances.

§ 155.— Sweet Ensilage.

The preparation of brown hay is also partly dependent on the weather, in so
far that a certain amount of dryness in the material before stacking is essential.
Now, in many cases, it is either practically impossible or economically dis-
advantageous to remove from the green fodder even the small quantity of water
that must be got rid of in making brown hay. One instance of this kind is
afforded by the enormous quantities of beet leaves available for a few days only
in each year (during the ingathering of the beet crop), and another is the drying
of the de-sugared slices of beet, an operation impracticable in many places owing
to the lack of the necessary costly drying apparatus. In such, and many other
similar cases, putrefaction of the readily decomposable masses is prevented by
subjecting them to an acid fermentation without any previous drying. Formerly

SWEET ENSILAGE 201

this was effected exclusively in silos — whence the term ensilage, current for this
operation in England and France, is derived.

According to the composition of the raw materials, their water-content and
method of treatment, two classes of durable fodder are obtained, viz., green
pressed fodder and sour fodder. The main factor determining which of them
shall be produced is the height of the temperature attained, as the result of
spontaneous heating, in the mass. If this does not exceed 40° C., then the
butyric ferments develop along with the lactic acid bacteria, and a sour-smelling
product, known as sour fodder, results. More detailed particulars of this are
given in the next paragraph.

If, on the other hand, the thermogenic bacteria develop vigorously in the
heap, and thus cause the temperature to rise rapidly to 50° 0. and remain there
for some time, then the lactic acid bacteria develop by preference, overcome all
their competitors, and exert a practically undivided sway. In this case a
durable fodder is obtained, which is almost entirely free from volatile acids and
devoid of odour, or with a somewhat sweetish smell, on which account it is
known as sweet fodder ; though this name is hardly correct, owing to the
strongly lactic acid character of the product. A more suitable appellation has
latterly been bestowed on it, viz., green pressed fodder.

This process originated in England in 1885, under the name of sweet
ensilage, but through the explanatory treatise written by G. FRY (I.) became
known on the Continent, where it was at first styled Fry's ensilage. The
numerous investigations to which it was there subjected led to important
conclusions, both of a chemical and practical nature, which were fully reported
by FR. ALBERT (II.). Nevertheless, from the Fermentation Physiologist's point
of view, no advance has been made beyond the general information already
given by Fry. On account of this deficiency we are obliged to dismiss this
process (important though it is to agriculturists) with merely a very few
remarks.

As the name itself implies, green vegetable substances, such as waste beet
leaves, clover, green maize, &c., are used for the preparation of green pressed
fodder. Silos are unnecessary, the materials being stacked in the form of a
straw-thatched cottage, tightness of packing being an important feature. By
suitable means (e.g. horizontal beams with weighted ends, or similar' stack
presses) a continuous heavy pressure is exerted on the stack, the amount of
the pressure being a predominant factor influencing the degree of spontaneous
heating produced. Should the temperature not rise quickly enough, then the
pressure is moderated to admit air more freely to the thermogenic bacteria. On
the other hand, if the temperature rises immoderately (beyond 70° C.), then the
pressure is increased, the access of the air restricted, and the oxidising activity
of the said organisms consequently diminished. The state of the internal tem-
perature is observed by means of an ensilage thermometer designed by E.
Meissl, the scale of which projects from the side of the stack. In a word, the
stack is regulated in such a manner as to ensure the predominance of the lactic
acid bacteria, whereby, under normal conditions, a product of a green to olive-
green colour, and of an aromatic sweet flavour, is obtained. The structure of
the vegetable matters employed is still distinguishable. A sample of this fodder,
prepared from crimson clover, contained (according to Bbhmer) 71 per cent, of
water and about 0.36 per cent, of total acids, of which 0.27 per cent, was lactic
acid, and the remainder consisted of butyric acid, acetic acid, valeric acid, <fcc.
In the researches recorded by Fr. Albert the total acids (calculated to dry
matter) in a sample of green pressed fodder, prepared from meadow grass and
containing 68.4 per cent, of water, amounted to 2.49 per cent., of which 1.89 per
cent, was composed of non-volatile acids (lactic acid).

202 LACTIC ACID BACTERIA IN FODDER

With regard to the losses of matter inherent in this process, that consisting
of carbohydrates need not be further dilated upon, since it is evidently un-
avoidable, being intimately connected with the production of heat on the one
hand, and on the other with the formation of lactic acid. Greater importance
attaches to the modifications effected in the albuminoids during the process, and
these changes also afford a means for determining its value. The numerous
researches made on this point all tend to prove that the conversion of green
fodder into green pressed fodder is attended with a substantial loss of digestible
albuminoids, the amount of the loss being in direct proportion to the water-
content of the fresh material. The ferments consume a large amount of the
albuminoids initially present, and decompose them into amide compounds,
ammonia derivatives, and even ammonia — all substances of but little, if any,
use for the nutrition of animals which are to be killed for food. In the cases
reported by Albert, the amount of these matters eliminated ranged from 1 3 to 3 i
per cent, of the total nitrogenous matter (crude protein) present.

§ 156.— Sour Fodder

is prepared in pits some 40 to 80 inches in depth, and 80 to 120 inches wide, the
length depending on the quantity of fodder to be treated. The most important
raw materials are the exhausted slices of beet-root from sugar-factories, fodder-
beet, potatoes (previously steamed), frozen sugar-beet, chaffed maize stalks, &c.
The silo is tightly filled' with these and covered with a layer of chop (chaff),
surmounted with a thick stratum of soil, and over this again are laid boards,
weighted with sufficient stones to produce a pressure of about 1000 kilos, per
square metre (nearly 2000 Ibs. per square yard). Since practically no oxygen
can penetrate the interior of the compressed mass, the activity of the thermogenic
bacteria is very much impeded. Nevertheless, the temperature rises to some
extent, as a result of bacterial activity, but not to anything like the degree attained
in the case of green pressed fodder, and, in fact, generally remains below 35° C.
Observations on this point were made by K. Schatzmann in a silo of elliptical
ground plan, and having a capacity of 37 cubic metres (1307 cubic feet). On the
2nd day after filling, the internal temperature registered 26° 0., and attained on
the 1 6th day a maximum of 34° C. ; afterwards subsiding, so that on the 36th day
23° C., on the 56th day 19° 0., on the 86th day 12° C., and on the io6th day
8° C. were recorded. Under such conditions, the heat-loving lactic acid bac-
teria could not be expected to gain the upper hand ; and, as a matter of fact, a
number of highly divergent fermentative organisms take part in the production of
sour fodder, the percentage of volatile acids in the fodder being correspondingly
high, and the smell consequently sour. In this process the loss of matter is still
greater than was found in the case of green pressed fodder, and in an instance
examined by Julius Kiihn amounted to 23.4 per cent, of the total dry substance.
The loss is principally borne by the non-nitrogenous extractive matter (carbo-
hydrates, &c.), then by fermentable woody fibres, and finally by the albuminoids,
of which (in consequence of their conversion into amido-compounds) about 40
per cent, disappeared in the above-mentioned experiment (with soured green
maize). In a second instance (beet slices), the loss was 18-62 per cent. ; and in
a third (soured beet leaves tested by 0. Kellner) as much as 68 per cent.
Although these high proportions of loss in the souring process are regrettable,
it should not be forgotten that the fodder materials now in question would
be entirely wasted unless utilised by means of this process, whereas, when
so manipulated, a considerable proportion of their nutritive constituents can in
any case be preserved. This souring process can always be relied on to yield
comparatively good results where all other methods for preserving a given fodder

SOUR FODDER 203

are impracticable or disadvantageous. To this advantage must be added, in many
cases, an improvement in composition effected by the process, e.g. the reduction of
the percentage of (purgative) oxalic acid in beet-leaves, observed by 0. Kellner.
Numerous researches on this subject have been published, and the reader may
be referred for further information to the summary of these investigations which
appears in a useful treatise by JULIUS KUHN (I.), dealing with the matter from
the Agricultural Chemist's point of view.

Granting that the sacrifice of a certain portion of the given substance, in order
to retain the rest, is inherent in this process, it by no means follows that the
extent of the said sacrifice cannot be reduced. The great fluctuations exhibited
by the foregoing figures, referring to the amount of loss experienced, permit the
assumption that an excellent sour fodder can be prepared with a smaller pro-
portion of loss than the existing average. This opens up to the Fermentation
Physiologist a new and valuable field of work, still unexplored. The fermentative
nature of the souring process being acknowledged, it follows that the next object
in view is the attainment of a clear insight into the physiology of this fermenta-
tion ; which knowledge will then facilitate the cultivation of the ferments
combining maximum efficiency with minimum consumption of material, and
consequently capable of yielding the greatest proportion of best quality sour
fodder. Attention should be directed to the artificial inoculation of the silos with
selected ferments, since the resulting improvements are of great pecuniary value
to the agricultural interest. (The author estimates them as represented by an
annual sum — for Germany alone — of several millions of marks, i.e. shillings.)
These researches should also be extended so as to include the kindred process of
sauerkraut fermentation, the preparation of which important food-stuff, on lines
practised from time immemorial, formed the prototype for the production of sour
fodder. Little is known of the physiology of this method, the literature of the
subject being restricted to a couple of brief reports which ascribe the chief
agency in the process to the lactic acid bacteria.

CHAPTER XXVII.

THE PART PLAYED BY BACTERIA IN TANNING.

§ 157.— The Fermentation of the Plumping- Soak.

THE preservation of animal hides by simple drying is feasible only when they
are to be stored for some time an<l brought on the market in a dry state. To
accelerate this drying, the fleshy side is in many instances rubbed over with
arsenic or with common salt. A few investigations into the utility of the last-
named substance were made by F. HAENLEIN (I.). The brittle solidity and
fragility acquired by the hides in drying prevent their utilisation for practical
purposes, and the more so because putrefaction sets in directly they are wetted.
To remedy this defect is the object of tanning, i.e. the conversion of hides into
leather. Bacterial activity here comes into play at the outset of this operation,
micro-organisms being necessary even in the preliminary treatment known as
unhairing. As is well known, leather consists neither of the outer skin (epi-
dermis) nor of the next layer of mucous cells (mucous membrane), but of the
third layer (composed of closely interwoven cells), which, on this account, has
been termed the leather (or true) skin (corium). Several methods of exposing
this layer and simultaneously removing the hair exist, one of them being the
so-called sweating process. In this operation the cleansed, soft hides are
maintained at a moderate warmth in a chamber saturated with moisture — the
sweating pit. Putrefaction quickly sets in, but is only allowed to proceed so
far as to loosen the hairs and enable them to be scraped off, along with the
epidermis and mucous membrane. A second method of unhairing is by liming
or slackening, the hides being repeatedly steeped in an initially weak, but
progressively stronger, milk of lime. In the absence of investigation on the
subject, it is still uncertain whether an active part is played in this operation —
as is undoubtedly the case in the sweating process — by bacteria, certain species
of which, capable of resisting the action of the milk of lime, have been discovered
in the liquor by J. VON SCHRODER and W. SCHMITZ-DUMONT (I.). The result of
both operations is substantially the same, viz., the hair, epidermis, and mucous
membrane are loosened, and can then be removed.

In one respect, however, limed hides differ from those slackened by sweating.
In the former case, calcium carbonate has been deposited in the intercellular
spaces in the hide, which is thus rendered somewhat brittle and less pervious to
the tanning liquor. This carbonate precipitate is removed by steeping the hides
in a pickle or " bate " consisting principally of a mixture of bran, barley, groats,
and the excrement of various animals (dogs, fowls, pigeons, <fec.) in a state of acid
fermentation. The chief, if not the sole raison d'etre of this process, which has
been gradually developed through tentative experiments alone, has only been
brought to light in recent years. The first progressive step consisted in the
discovery, by AUG. FREUND (I.) in 1871, that lactic acid is the chief product of
the spontaneous acid fermentation of wheat bran. This explained the favourable
result of the bating of lime-slackened hides, the calcium carbonate being con-
verted into the soluble calcium lactate, and thus washed out of the hides.

Pickling, however, effects a second and remarkable alteration in the hide,

204

THE SOURING OF BARK LIQUOR 205

causing it to swell up (" plump ") to almost double its former thickness, thus
loosening the cellular tissue and facilitating the subsequent penetration of the
tannin. In order to make the most of this advantage, it is generally the custom
to pickle even the hides that have been unhaired by the sweating process, and
which consequently do not contain any lime that needs to be removed. The
object in this case is to plump the hides, i.e. cause them to swell up, and this
explains the term " plumping soak " applied to this acid liquor. This expansion
of the skin is due to the action of the gases liberated in the fermenting liquor.
The reactions occurring in this process are very diversified ; as might be
expected from the large number of bacterial species present in the bran itself
and in the added frecal matter. Of these organisms, such as attack the carbo-
hydrates develop most vigorously, these substances being present in large
amount.

J. T. WOOD and W. WILLCOX (I.) described one species of this kind and gave
it the name of Bacterium furfur is. The starch in the bran ishydrolised and con-
verted into glucoses by an enzyme, cerealin — isolated from bran extract by J. T.
WOOD (I.) — and these sugars are then acted upon by the bacteria in question,
organic acids and considerable quantities of gas being produced. In a sample
examined by WOOD (II.), i litre of the fermented bate contained 0.8 gram of
lactic acid, 0.2 gram of acetic acid, 0.03 gram of formic acid, and o.oi gram of
butyric acid. The disengaged gas was found, in different experiments, to consist
of 22-42 per cent, of C0g, 28-53 Per cent, of H, 24-26 per cent, of N, and 1-4
per cent, of 0, the percentage of carbon dioxide increasing as fermentation
progresses. This fermentation is attributed by Wood and Willcox to the said
bacterium, which they describe as short rods 0.7 p, long and 1.3/1 broad, united
to form chains, but not, so far as they could ascertain, producing endospores.
The cell-walls exhibit a tendency to swell up. The organism is incapable of
attacking solid or dissolved starch, and consequently can only come into play
when the cerealin has completed its diastatic action and converted the starch of
the bran, &c., into glucose.

These discoveries must be regarded as a preliminary step to be followed by
many others. The unappetising and (hygienically considered) objectionable use
of faeces is shown to be unnecessary ; and it would be advisable to endeavour to
artificially " sour " the plumping soak by means of a sufficient quantity of actively
fermenting leaven prepared from pure bran.

The next step in bacteriological investigation in this matter should be to
ascertain whether — as the figures above given lead one to suppose — pure lactic
fermentation is the best form of fermentation for this soak. It should be men-
tioned, moreover, that the removal of the lime from the slackened hides by the
aid of dilute mineral acids is impracticable, since it seriously impairs the quality
of the resulting leather.

§ 158.— The Souring1 of Bark Liquor.

For the actual tanning of the plumped hides there are, as is well known,
three different systems available, viz., alum- or white-tawing ; oil- tawing, or
shamoying ; and bark-tanning — the last of which will now be briefly considered
from a bacteriological standpoint. The chief tanning material employed in this
case is the bark of various trees (oak, pine, &c.), in addition to which gall-nuts,
myrobolams, sumach, &c., are also used.

Hides intended for sole-leather are placed in a tanpit in such a manner that
each hide is separated from its neighbour by a layer (a couple of fingers in
thickness) of coarsely-broken fresh bark, mixed with powdered gall-nuts, &c. The
empty corners and vacant spaces are packed with old spent tan, and the pit is then

206 THE PART PLAYED BY BACTERIA IN TANNING

filled up with water. At the end of eight or ten weeks the hides are taken out
and relaid in fresh strata of hark, and this operation is repeated (three or five
times) until the leather is impregnated with tan. There is no doubt at all but
that the activity of microbes is also manifested in the tanpit, but at present no
hypothesis can be formulated on this point, there being no scientific foundations
to build upon.

Our knowledge of the reactions occurring in the bark liqour is, however, in
a more satisfactory state. All the thinner hides, unsuitable for sole-leathers,
are steeped, not in the tanpit, but in an aqueous (cold-drawn) extract of tanning
substances, known as bark liquor or ooze (Fr.jusee, Ger. Lohbriihe). The actual
process of tanning, i.e. the combination of tannic acid and phlobaphene with the
fibres of the skin, does not concern us at present, this being a purely chemico-
physical operation. One circumstance, however, deserves brief mention here,
and that is the subsidiary phenomenon of the gradual souring of the bark liquor.
This change, well known to all tanners, was first chemically investigated by
H. BRACONNOT (I.), who, in 183 2, discovered in soured bark liquor an acid which
— as little was then known of the properties of lactic acid — he considered to
be a new, hitherto undiscovered acid, and therefore named it " acide nanceique,"
after the town of Nancy, where the discovery was made. This acid was, how-
ever, quickly recognised by L. Gmelin as lactic acid. J. WLADIKA (I.) in 1890
showed that volatile, as well as non-volatile, acids are present in old spent bark
liquors, the former consisting chiefly of acetic acid and the latter of lactic acid.
The reader will have no doubt that the reduction of these acids (which are not
present in the "sweet" liquor) is attributable to the activity of micro-organisms,
which are here present in abundance, HAENLEIN (II.) having found no less than
60,000 bacteria (along with a few budding fungi) in the liquor prepared from,
and corresponding to i mgrm. of, Silesian oak-bark. The acids are produced
from the saccharine constituents of the bark, a fact already evidenced by the
researches of B. KOHNSTEIN (I.) and J. VON SCHROEDER (I.), who showed that
in proportion as the sweet liquor becomes sour, so the percentage of these sugary
constituents decreases. In practical working their initial quantity is found to
be from 0.3 to 0.8 gram per litre.

A fission fungus named Bacillus corticalis, and recognised as an active partici-
pant in the souring of bark liquor, was discovered by HAENLEIN (III.) in large
quantities in sour pine-bark liquor, as also in the fresh pine-bark. This microbe
appears in the form of short rods 0.7-1 /* broad and one and a half to twice these
dimensions in length ; at the period of active reproduction the individual cells
are connected into many-jointed chains. The bacillus thrives on nutrient gela-
tin, nutrient agar-agar, and slices of potato, and requires but very little nutri-
ment. Being able to withstand desiccation, it remains alive in bark, where it is
found in large numbers. It has a great affinity for light, and grows more freely
when illuminated than in the dark, though it is still capable of performing its
vital functions under the latter condition. It thrives most vigorously at 30°-
40° C., and ceases to develop below 5° C. ; in habit it is facultatively anaerobic,
i.e. oxygen is not essential to its growth. Its most important property, so far as
we are now concerned, is its behaviour towards sugars, of which it acts upon
dextrose as well as saccharose and lactose, and produces, in addition to acids, a
considerable quantity of gas, composed (in one case investigated) of 95 per cent,
of hydrogen and 5 per cent, of carbon dioxide. Tannin is not attacked by this
microbe, a fact confirmed by the observation made by J. VON SCHROEDER and A.
BARTEL (I.), that the percentage of tannin in the liquors undergoes no alteration
on standing or during storage.

The acidity of tannin bark liquors amounts to 0.25 gram (reckoned as acetic
acid) per litre and is, as the previously mentioned researches of Wladika have

THE SOURING OF BARK LIQUOR 207

yhown, mainly due to lactic acid, the presence of which acid has a favourable
influence on the quality of the leather. According to J. PASSLER (I.) a leather
tanned with pure tannin handles poor and hard, but is rendered soft and supple
by the acid gradually formed in the bark liquor. It is therefore easy to under-
stand why the tanner likes to see his bark liquor turn sour, and even attempts
to favour and accelerate this state of things by mixing with the fresh liquor
some of an older, already soured batch ; thus unwittingly inoculating it with a
culture (in any case impure) of acid bacilli. Whether this microbe also acts in
other ways is at present unknown, but it is a subject worthy of investigation.
This applies particularly to the liberation of gas in bark liquors, which, according
to Haenlein's researches, amounts to 1-2 c.c. per gram of pine-bark. Important
researches on the dependence of the normal progress of this souring operation
on external conditions were made by F. ANDREASCH (I.).

Although Bacillus corticalis is by no means the only species found on pine-
bark, still the composition of the bark liquor is specially favourable to its
development. It is therefore probable that in the liquors prepared from other
(and especially tropical) tanning materials, other (but allied) species will be
found, not only because the bacterial flora of these materials (grown under
other conditions) is different, but because these latter differ in chemical com-
position from our indigenous tanning barks, &c., and consequently favour the
development of other species of bacteria. J. T. WOOD (III.) has described
the micro-organisms present in sumach infusions. — In conclusion, it may be
remarked that the process of tanning still presents a very profitable field of
research to the Technical Mycologist.

SECTION VII.

THE FOEMATION OF MUCUS AND ALLIED PHENOMENA
OF DECOMPOSITION.

CHAPTER XXVIII.

THE IMPORTANCE OF BACTERIA IN THE MANUFACTURE
OF SUGAR.

§ 159.— The Zoogrlcea of Leuconostoc Mesenterioides.

IN the manufacture of saccharose there are occasionally formed certain masses
of gelatinous mucus, for the most part merely small colourless or reddish agglo-
merations, resembling frog spawn in appearance, but, under special conditions,
increasing to masses exceeding a cubic foot in dimensions. Attention was first
drawn to them by C. SCHEIBLEK (I.), who, in 1874, subjected them to chemical
examination, the result^of which indicated the presence of dextran, a carbohydrate
first discovered by him in the plasma of unripe sugar-beet. Scheibler, in fact,
regarded this mucus as expressed beet plasma ; but this opinion (which was also
shared by E. FELTZ (I.)) was very soon opposed, P. JUBERT (I.) having shown,
a few months later, that these gelatinous lumps (known in France as " gomme
de sucrerie") continue to develop in sugar solutions. From this he concluded
that they are not extravasated beet plasma, but a ferment (a " plant," as he
expressly stated), whose reproductive power he succeeded in arresting by means
of carbolic acid. The microscopical examination of the mass, however, as first
made by F. MENDES and J. BOKSCHTSCHOFF (F.), led at the outset to no
satisfactory elucidation. For an accurate knowledge of the true state of the
case we are indebted to L. CIENKOWSKI (I.), who, in 1878, proved that the
mucous masses in question are composed of bacteria with extraordinarily
swollen and gelatinised cell-walls, which cause the individual organisms to
adhere together and form the aforesaid large masses, whose superficial con-
volutions frequently resemble those of the mesentery. Influenced by Billroth's
publication on Coccobacteria septica, and in view of the name Ascococcus
Billrothii given by COHN (II.) to a mucus-forming fission fungus, Cienkowski
called his microbe Ascococcus mesenteriotdes. The forms of cell which he detected
in these mucinous lumps he described as highly diversified : Coccus, Bacillus,
Vibrio.

In the same year VAN TIEGHEM (VII.) published a research on the organism
producing these mucinous masses, agreeing with his Russian colleague as to its
vegetable nature, though with respect to its position in the botanical system he
held other views. According to his observations, the development and repro-
duction of this microbe harmonised so well with those of the blue-green uni-
cellular algae of the genus Nostoc, that he felt obliged to call it '* white "
(chlorophyll-free) Nostoc, and bestowed on it the new generic name of Leuco-
nostoc. Contrary to the observations of Cienkowski, who ascribed a copious
polymorphism to this fission fungus, Van Tieghem could only discover globular

208

THE ZOOGLCEA OF LEUCONOSTOC MESENTERIOIDES 209

cells, as shown in Fig. 54 (which is made up from the drawings in the original
treatise, and is here reproduced on account of its historic interest). Since,
however, the researches of this French worker were not performed with pure
cultures, we cannot go more closely into the details of this illustration.

It was not until 1891 that a thoroughly satisfactory bacteriological investiga-
tion was made — by 0. LTESENBERG and W. ZOPF (I.)— on this organism. In the
interim, the same gelatinous
masses had also been observed
in Indian cane-sugar factories
by EL WINTER (I.). Adhering
to the existing nomenclature,
the two first-named workers
showed that Leuconostoc mesen-
terioides has only one morpho-
logical form, viz., a coccus of
0.8 to i.o/xin diameter. This
discovery in nowise detracts
from the accuracy of Cienkow-
ski's observations, for it must
be remembered that he did
not have pure cultures at his
disposal, but examined a
sample of the gelatinous mass,
which from its nature was
very likely to have become
infected by any number of
fission fungi during its re-
moval from the sugar-works
at Orlovetz to the laboratory

of Charkow.

mi i- . , , FIG. 54. — Leueonostoc mesenterioides.

The discoveries made by
Liesenberg and Zopf that this
microbe, when grown in or
upon nutrient media free from
cane- or grape-sugar, does not

develop a mucinous envelope, but grows in chains, and consequently assumes the
streptococcus form (Fig. 55), are entirely new. Such nutrient media are : Solid —
potato slices, peptoniserl meat-broth gelatin, milk gelatin, maltose gelatin, on all
of which it develops merely a thin pellicle without any formation of mucus ;
Liquid — milk and bouillon, in which merely a fine sediment of cells is deposited.
If, however, a small portion of such a culture of non-mucinous cells be trans-
ferred to a medium containing saccharose or dextrose, the formation of mucus
quickly ensues. Under these conditions there then develop (on slices of carrot,
in particular) large zooglcea, at first dry, like cartilage, but afterwards becom-
ing softer and resembling mesentery in appearance. Under the microscope it
appears as shown in Fig. 55. The cells, always globular, are invariably arranged
in pairs, i.e. as diplococci. The greatly swollen mucinous capsules of the indi-
vidual cells gradually coalesce and form agglomerations of constantly increasing
size, in which the diplococci are enclosed. Locomotion could not be detected in
any case.

• Not only was Cienkowski's work corrected by the researches of Liesenberg

and Zopf, but the same fate also befell some of the statements made by Van

Tieghem. The latter thought he had detected endospores in his Leuconostoc,

and asserted that in unfavourable media some of the cells increased in size, and

i o

1-8. Various stages of development of the Zoogloea formation.
9. Two chains of cocci, each exhibiting two enlarged (pre-
sumably sporogenic) members. Magn. about 500. (After
Van Tiegliem.)

210 BACTERIA IN THE MANUFACTURE OF SUGAR

formed spores 1.8-2.0 /* in diameter, the walls of which coincided with those of
the mother-cell. No. 9 of Fig. 54 shows two such cell-chains, each of which
exhibits two enlarged members wherein spore-formation has just commenced.
When transferred to a favourable medium, these spores were said to burst their
solid membrane and then reproduce themselves by fission. Liesenberg and
Zopf were, however, unable to discover such spores, and in any case their

presence would be unimportant, since
the organism already possesses in its
mucinous envelope an excellent means
of protection against adverse influ-
ences. Owing to this envelope it is
able — so Liesenberg and Zopf found
— to withstand three and a half years'
desiccation in the air, and to resist the
influence of dry heat at 100° C. for
over five minutes ; whereas the naked
modification, growing on sliced pota-
toes, succumbs after five minutes' ex-
posure to a temperature of 75° C.
Like other fungi, however, it is much
less able to resist moist heat (stearu,
heating in liquids), the envelope being
readily penetrable by moist warmth.
On warming a culture of the gela-
tinous form in a nutrient solution up
to 88° C. in forty-three minutes, and
keeping it at that temperature for
five minutes, all the cells were killed,
whereas a temperature of 86°-87°O.,
under otherwise identical conditions,
produced no injurious effect. The
naked variety showed itself even
somewhat more susceptible; never-
theless Leitconostoc mesenlerioides
must be classified among the heat-
resisting bacteria, by virtue of which
property it is enabled to appear in
the hot diffusion battery and juice
conduits of the sugar-factory. The
same faculty of resisting heat may

also be utilised in preparing a pure culture of the microbe : the sample (a
gelatinous lump in a solution of sugar) destined for this purpose being kept for
a quarter of an hour at 75° C., by which treatment most of the extraneous
germs (adherent to the mucus) are killed, leaving the Leuconostoc unhurt.

§ 160.— Physiology of Leuconostoc.

The mucinous envelope is soluble in zinc iodochloride, concentrated sulphuric
acid, strong caustic potash or soda, or in baryta water, but potassium iodide or
iodosulphuric acid produce no noticeable alteration. This fact by itself proves
that we have not in this case (as was supposed by Van Tieghem) to deal with
cellulose. A beautiful double staining can be produced by first treating the
cover-glass preparation with dahlia-violet, which stains the cocci alone, and then
immersing it in an aqueous solution of rosolic acid, which is absorbed by the

FIG. 55. — Lenconostoc mesenterioides.
, b. chains and bands of the non-capsuled variety,
potato culture ; c, e, cells with gelatinous capsule
in various stages of development. In d one pair
of cells is viewed in the direction of the line join-
ing their centres, and hence appears as a simple
cell, not as a diplococcus. The shading in c-e
merely indicates the configuration of the agglome-
rations, and not a stratification of the capsule.
Magn. about 1200. (After Liesenberg and Zopf.)

PHYSIOLOGY OF LEUCONOSTOC 211

mucinous envelope, the latter then surrounding the blued cells with a rose-red
halo. Preparations of this kind sometimes exhibit a scaly stratification of the
envelope, the explanation of which is clear : the outer layer of the cell membrane
swells up, detaches itself, and now encloses the cell on all sides, so that when
this process has been repeated several times the envelope continually increases
in size, whilst the dimensions of the bacterial cell itself remain unaltered. By
softly pressing the cover-glass of such a zoogloea preparation the cells can be
forced out of their mucinous envelopes.

As already remarked, the substance of which this envelope consists was
stated by Scheibler to be dextran, an opinion also shared (as the result of analy-
tical experiments) by P. DAEUMICHEX (I.). If, however, we consider the means
by which these chemists arrived at their discoveries, doubts will arise as to the
accuracy of their conclusions. In all attempts, made by macrochemical means,
to determine the composition of the vegetable cell membrane, the same difficulty
is encountered, viz., the solution and removal of the cell contents. In order to
attain their object, the reagents employed for this purpose must penetrate
through the cell wall, and since they come into contact with it whilst still in all
their pristine strength, they decompose it more or less effectually. When the
lixiviation of the cell contents is completed, then the product remaining behind
for ultimate analysis cannot be considered, in point of composition, as unaltered
cell-membrane substance, though its form may be still unchanged. This applies
to the case now under consideration. In order to obtain the substance of the
mucinous membrane in a pure condition Scheibler boiled the gelatinous mass
(freed from adherent sugar) with milk of lime, and found that only a small por-
tion was dissolved. This fact of itself bears evidence against the (chemical)
uniformity of the substance of the mucinous envelope of Leuconostoc. It is also
probable that even the dextran recovered from the lime extract is a decomposi-
tion product of a more readily hydrolised constituent of the said membrane.

The behaviour of this fission fungus towards sugars merits special considera-
tion. It has already been stated that the formation of mucus occurs only in
such nutrient media as contain grape- or cane-sugar, the other carbohydrates,
tested on this point by Liesenberg and Zopf, being found unsuitable. Leuco-
nostoc mesenterioides produces invertin, which then splits up the cane-sugar; so it
may be surmised that the development of the gelatinous membrane can only occur
in presence of grape-sugar (and perhaps also fructose !). This does not, however,
imply that lactose, maltose, and dextrin are unaffected by this fission fungus ;
on the contrary, it ferments them and forms lactic acid, a faint evolution of
gas being at the same time noticeable. The presence of a small quantity (3-5
per cent.) of calcium chloride in the nutrient medium favours the production of
mucus and the fermentative activity of the organism, the latter being brisker
when oxygen is excluded. The optimum temperature for the development of
this microbe being between 30° and 37° C., it is evident that in order to kill
this pest the juice in sugar- factories must be kept at higher temperatures.
Leuconostoc consumes a certain portion of the cane-sugar, and as it also produces
invertin, which forms invert sugar — the presence of which is well known to
seriously retard the crystallisation of the cane-sugar — another source of loss to
the sugar-manufacturer arises. Molasses naturally forms a highly suitable
nutrient medium for this microbe. The speed at which it increases therein is
reported (from practical experience) as follows by E. DURIN (I.) : A wooden vat,
previously used as a recipient for beet-juice, and the walls of which were covered
with a thin film of mucus (i.e. zoogloaa of Leuconostoc) was charged with 50 h.l.
(i 100 galls.) of neutral molasses. At the end of twelve hours the whole of this had
become converted into a mucinous coherent mass. This microbe also gives rise
to mischief in the refineries as well as in the raw-sugar works. F. STJIOHMEK (I.)

212 BACTERIA IN THE MANUFACTURE OF SUGAR

mentions such a gelatinous molasses derived from a colonial sugar-refinery,
and which yielded a pasty sediment consisting of the zoogloea of Leuconostoc
mesenterioides on dilution with water. It should be remembered, when deter-
mining the sugar-content of a molasses by polarisation, that the mucinous
envelope of Leuconostoc is optically active and deflects the beam of polarised
light three times as much as an equal weight of saccharose.

It may be mentioned as a curiosity that E. Durin — who regarded the chief
component of the mucinous masses of Leuconostoc as cellulose — took out a patent
in France (Feb. 14, -1876) for the "conversion of crystallisable sugar ( = cane-
sugar) into cellulose, and any uses (preparation of starch-sugar, dextrose, gun-
cotton, oxalic acid, &c.) to which this cellulose maybe applied" (Brevete sans
Garantie dn Gouvernement ! ).

A fission fungus ranking along with Levconostoc in so far as its importance
to the sugar industry is concerned was examined by A. KOCH and H. HOSAEUS (I.).

In a certain sugar-works the syrup de-
stined for working up into second pro-
duct was found to contain gelatinous
masses resembling the zooglcea of
Leuconostoc, but consisting of another
species of bacterium, shown in Fig. 56.
The special peculiarity of this microbe
is that the swelling of the membrane
is unusually great and extensive on
one longitudinal side only, so that a
long peduncular mucinous thread is
F,G.56.-Bacteri«mPediculatum. „$£&! formed in this direction.

A. Koch and H. ifosaeus.) was named Bacterium pedicidatum.

Unfortunately, it could not be ob-

tained as a pure culture. ]n respect of this peculiar unilateral gelatinisation
of the cell membrane it is not unique ; the Bacterium vermiforme (the chief
constituent of ginger-beer yeast, shown in Fig. 53), and also a fission fungus
(Nevskia ramosa) discovered by A. FAMINTZIN (I.) in aquarium water, having
similar characteristics. Moreover, in the algse, forming the neighbouring group
to the bacteria, and especially in the diatoms, many genera, e.g. Gomphonema,
exhibit well-developed and branched gelatinous stalks.

§ 161.— Mucinous Fermentation and Inversion.

The faculty of rendering sugar- juice mucinous is not restricted to the two
microbes just described, a number of other species being now known to be capable
of working similar injury. They, however, differ from the former in one cha-
racteristic, which, though unimportant for the practical man, is nevertheless
not without interest from a physiological point of view. The gelatinisation of
the nutrient media infested by the microbes described above must be charac-
terised as direct, since it is produced by the swollen cell-membranes of the
organisms themselves. Conversely the gelatin-forming property of the species
now to be described is an indirect one, it being here a question of the conversion
of sugar (outside the cell) into the mucinous matter which A. BECHAMP (II.)
proposed to call Viscose. In fact, we have to do with the actual production of
mucus, whereas the former case was one of zoogloea formation.

E. KRAMER (II.) in 1889 described a Bacillus viscosus sacchari which belongs
to this second group. The cells are rod-shaped, i /i thick and 2.5-4 /i long,
united into many-jointed chains ; neither locomotion nor endospore formation could

MUCINOUS FERMENTATION AND INVERSION 213

be detected. The organism thrives only on neutral or faintly alkaline nutrient
media, and in these it produces, in presence of cane-sugar, a mucus having the
elementary formula C6H1005. No swelling or gelatinisation of the cell membranes
occurs. The optimum temperature for the reaction is 22° C., but beet-sugar
juice will become changed to a viscid mass in one or two days at the ordinary
temperature.

FRITZ GLASER (I.) described — as Bacterium gelatinosum betce — a fission-fungus
discovered by him in mucinous beet-juice. Already by its active motility this
species differs from the others we have described ; and the same applies to several
other characteristics. It does not develop in neutral 10 per cent, molasses, unless
the medium has been previously qualified with a little of the precipitate thrown
down by alcohol from beet-juice — i.e. phosphates, &c., of alkaline earths extracted
from the molasses during the separation and saturation of the sugar -juice. The
chief products of the decomposition (preceded by inversion) of cane-sugar by this
organism are mucus and amyl alcohol. The former is identical in properties
with dextran, and is soluble in warm dilute acids and alkalies, but insoluble in
baryta water or milk of lime. An acid odour is evolved during this fermentation,
but no lactic acid is formed.

The number of species of bacteria capable of interfering with the normal
course of sugar manufacture is by no means exhausted with the examples
mentioned above, but owing to the paucity of observations on this point no
further reliable particulars can as yet be given. Consequently the subject
presents an admirable field for bacteriological research in order to elucidate the
causes and prescribe remedial measures for mucinous fermentation. It is well
known to sugar-makers that the percentage of invert sugar in molasses increases
during storage (sometimes for months) in the so-called reserves, and they are
also aware of the decomposition occurring in stored raw sugar and resulting in
the formation of invert sugar. Now the faculty of excreting an inverting
ferment is not very widespread among bacteria. For a comprehensive in-
vestigation on this point we are indebted to C. FERMI and G. MONTESANO (I.),
who examined about sixty (some of them pathogenic) species of bacteria, but
found only four, viz., Bacillus megatherium, Bacillus fluorescens liquefaciens, the
red Kiel bacillus, and Proteus vulgaris, capable of producing invertin in
saccharified bouillon. Experiments which have been made by A. HERZFELD
and U. PAETOW (I.), on the prevention (by hydrofluoric acid and alkali fluorides)
of inversion in molasses, lead to the hope that these antiseptics may prove
useful in many cases. Further researches on this subject are highly desirable. —
The nitric fermentation of molasses will be briefly mentioned in chapter xxx.

Sugar-juice and raw sugar are occasionally infested with higher fungi as well
as with bacteria. For instance, A. HERZFELD (I.) and A. B. FRANK (I.) report
the occurrence of a red pigmentary fungus in raw sugar. They found (in an
after-product) red lumps, about as large as hazel-nuts, which, under the
microscope, proved to be abundantly infested with a thread fungus, the proto-
plasm of which was stained by a red pigment, presumably generated by the
bacteria present in large numbers in the mass. The development of pigment
bacteria is also frequently noticeable in the saturation scum thrown out from the
sugar-works and spread over the fields, this scum being often found covered
with coloured (mostly red) patches, which are presumably zoogloea of Micrococcus
p>'odigiosus.

Large though the number of injurious fission fungi in sugar may be, it
is surpassed by the multitude of Eumycetes infesting the sugar-beet. These,
however, do not fall within our province, and readers who may be interested in
them are referred to the various text-books on plant diseases. The works
compiled by A. B. FBANK (II.) and P. SORAUER (II.) respectively, presuppose

214 BACTERIA IN THE MANUFACTURE OF SUGAR

a certain degree of (macroscopic) acquaintance with the individual maladies of
which they treat. On the other hand, the young sugar-technicist, who will, as
a rule, be mainly desirous of determining the nature of the disease brought
under his notice, is advised to study O. KIRCHXER'S (I.) " Handbuch der
Pflanzenkrankheiten " (" Handbook of Plant Diseases "). This work is admirably
supplemented by a good and cheap atlas (prepared by O. KIRCHNER and H.
BOLTSHAUSEN (I.) ) of coloured plates showing the chief diseases attacking
industrial plants. With the information thus gained, the learner will then be
able to resort with advantage to the two first-named standard works. A brief
review of the most important diseases set up in the sugar-beet by vegetable or
animal parasites has been written by A. STIFT (I. and II.), and particular
attention is devoted to Heterodera Schachtii (the cause of the so-called nematode
sickness) in a monograph by A. STRUBELL (I.),' as also in a useful work by
J. VANHA and J. STOKLASA (I.). Investigations on the influence of these worms
on the cellular activity of the beet, and on the resulting chemical changes
thereby induced, were made by J. STOKLASA (I.), and may now be mentioned.
At present we will merely refer briefly to the gummosis (Fr. yommose) of the
sugar-beet, a complaint first described by SORAUER (II.). The symptoms of this
disease are : extravasation of small drops of a gummy fluid from the unbroken
surface, and a gradual blackening of the vascular bundles and parenchyma of
the beet, from the tip of the root upwards. It is still uncertain whether the
bacteria so abundant in this gum should be regarded as the actual cause of the
disease or merely as harmless saprophytes.

CHAPTER XXIX.

ROPINESS IN MILK, WINE, BEER, AND OTHER LIQUIDS.

§ 162.— Ropy or Viscous Milk.

THE first attempt at a scientific study of this malady was made in 1847 by
GIRARDIN (I.), who hoped to elucidate it by chemical analysis, and sought the
cause in the defective composition of the fodder. This complaint may develop
to a variable extent in milk. In the worst cases the thickened liquid can be
drawn out to a thin thread a yard or so in length. J. LISTER (I.), in 1873, was
the first to reproduce this complaint by inoculation, and thus indicated the
probability of a living source of infection. To ascertain this by microscopic
examination was the task essayed by SCHMIDT-MUHLHEIM (I.) in 1882, who
found that ropy sour milk contained an unusually large number of cocci i /* in
diameter, frequently united as chains, but also in many cases isolated, and in
the latter case apparently endowed with motile powers. Although at that time
suitable methods of pure culture were no longer lacking, this observer made no
attempt to utilise them in his researches. This omission was, however, soon
remedied by E. DUCLAUX (IX.), who prepared pure cultures of two species of
bacteria from ropy milk, both of which belong, morphologically, to the so-called
capsule bacilli. The powerful lustre of the greatly swollen mucinous envelopes
surrounding these cells is the first thing to strike the assisted eye, on which
account the generic name, Actinobacter (lustrous bacterium, star bacterium),
was applied to both organisms. Under their influence the milk yields alcohol
and acetic acid.

To these two pests (known respectively as Actinobacter du lait visqueux and
A. polymorphus) a large number of others possessing similar powers have been
added by different observers ; e.g. a micrococcus discovered by HUEPPE (IV.) in
1884; the Bacillus mesentericus vulgalus, investigated by FLtiGGE (I.), and the
Bacillus pituitosi, a thick, slightly curved rod, discovered by LOEPFLER (III.).
Other allied species are : a streptococcus, described by HESS and BORGEAUD (I.),
and presumably identical with that observed by NOCARD and MOLLEREAU (I.) ;
and a bacillus, 1.2 /n broad and 2 p long, obtained by Schiitz from ropy milk,
and described by ST. VON RATZ (I.). In 1890, L. ADAMETZ (IV.) found in the
Liesing brook (which runs into the Danube in the south of Vienna) a capsule
bacillus, 0.7-1.2 p. long and 0.7 p broad, which he named Bacillus lactis viscosus,
and which is capable of turning both milk and cream ropy. It is fairly wide-
spread in nature, and was also detected by ADAMETZ (V.) in samples of milk
from the Sornthal (Switzerland). In addition to this, three other fission fungi
(named below) are found in Swiss soil, one of them being the Bacillus Guille-
beau c., which is not only dangerous to the cows (giving rise to inflammation of
the udder), but also produces various disturbances in the dairy by making the
milk " ropy " and the ripening cheese " blown." The facultatively anaerobic
Micrococcus Freudenreichii, 2 p. in diameter, discovered by A. GUILLEBEAU (I.),
is still more injurious to milk, since, whereas the other organisms just mentioned
act only at high temperatures — approaching blood-heat, and therefore easily
avoidable in practice — this coccus is active even at a moderate temperature, and

215

216 ROPINESS IN MILK, WINE, BEER, ETC.

turns milk ropy within five hours at 22° C. The optimum temperature of
development is two degrees lower, and the microbe is destroyed by an exposure
of two minutes to boiling heat. It has frequently been found in the district of
Berne, and often causes considerable damage.

Simultaneously with this last-named organism, a third microbe, also endowed
with the faculty of turning milk ropy, was introduced by Guillebeau under the
name of Bacterium Hessii. This species, which appears in the form of actively
motile rods, 3-5 p. long and 1.2 p. broad, is less injurious than the one just
described, since the ropiness it produces in milk disappears directly acidification
sets in.

The substantive cause of the mucinous condition may be of three kinds.
Either it is attributable to the swelling of the membrane of the bacteria in
question — as is apparently the case in those already alluded to as capsule bacilli,
e.g. Actinobacter, B. lactis viscosus, and also, according to the researches of
W. VIGNAL (I.), with B. mesentericus vulyatiis — or, secondly, the milk-sugar is
converted into a mucinous substance. This was asserted to be the case by
Storch for two species of bacteria discovered by him, and was proved by G.
LEICHMANN (III.) for a bacillus isolated from ropy inilk. This latter organism
acts on lactose, cane-sugar, maltose, galactose, levulose, and dextrose (but not
on mannite, arabin, or starch) in such a manner that mucus and lactic acid are
formed, together with a small quantity of ethyl alcohol. In the third place, the
ropy substance can also be produced from the casein of the milk. According to
H. WEIGMANN, this latter cause operates in the formation of the milk products
known as

§ 163.— Ropy Whey (Lang-e Wei) and Thick Milk (TsettemaBlk).

The Swiss dairymen discard ropy milk for cheese -making, being afraid of its
ca.using " nests," i.e. places within the cheese where the ripening proceeds
irregularly. They therefore devote particular attention to fumigating the stalls
out with burning sulphur, scouring the milk vessels with soda solution, &c., in
order to eradicate the evil as quickly as possible.

On the other hand, the Dutch look on the bright side of this evil, and even
derive benefit from it, the most palatable production of the Netherlands, viz.,
Edam cheese, being prepared with the aid of ropy whey (Dutch, Wei). The
first observations on and experiments with this ropy whey were made in the
" fifties" by a farmer (name unknown) of Assendelft, in Holland, but it did not
come into general use iu the manufacture of Edam cheese until 1887, when
Boekel recommended it most emphatically.

WEIGMANN (VII.) examined such whey, and found in it lai-ge quantities of
a fission fungus, which is mostly arranged in pairs, but frequently also in chains,
and bears the name of Streptococcus hollandicus. Sterile milk inoculated with
this organism becomes ropy and sour in twelve to fifteen hours at 25° C.

The same coccus was also found by Weigmann in the commercial products
known as Tcett&mcdk or Tcetmcdk (thick milk) in Norway, and Filmjolk (stringy
milk) in Finland and Sweden. This strongly sour, ropy, thick mass, the casein
of which is in the condition of fine flakes, is a highly prized article of nourish-
ment among the Scandinavian races, and is artificially prepared from normal
milk by either rubbing the interior of the milk-pails over with butter-wort
(Pinguiculavulgaris), called in Norway Tcettegrces, or by feeding this plant to the
milch-cows. The leaves are found to be infested with a fission fungus which
turns milk ropy, and is presumably identical with the above-named strepto-
coccus. As already remarked, the occurrence of ropiness in milk is usually
accompanied by acidification, whereby the development of numerous other

ROPINESS IN WINE 217

species of bacteria is prevented. This accounts for the circumstance that Tset-
maelk will keep for months without alteration if stored at a low temperature.

Herz was the first to record observations with regard to so-called soapy
milk, a term applied by him to milk that exhibits a taste of soap and lye, and
does not curdle, but only deposits a slimy sediment, even after prolonged
standing. The cream from this milk froths up very strongly when churned.
H. WEIGMANN and G. ZIRN (II.) had occasion, in 1893, to examine a milk of
this kind, and they succeeded in isolating therefrom a bacillus which is capable
of converting normal milk into the soapy condition, and is therefore termed
Bacillus lactis saponacvi. It was afterwards discovered that the organism
originated in the litter, which was in a damaged condition. When that was
withdrawn and the cows littered on sound straw, the milk no longer suffered
from this complaint.

§ 164.— Ropiness in Wine

was formerly attributed to a coagulation of the albuminoids, a hypothesis corrected
in 1856 by G. MULDER (II.), who traced the chief source of this malady to the
conversion of sugar into vegetable mucilage. Young white wines, in parti-
cular, fall victims to the disease, which in its incipient stage produces a faint
opalescence, followed by gradually increasing turbidity, until, finally, the liquid
becomes thick, and by degrees so viscid that it can be drawn out into threads a
yard or so in length, and can scarcely be poured out of the bottle. The flavour
is disagreeably slimy and insipid, though the odour (bouquet) is almost un-
altered. In France the malady is termed " Maladie de la graisse" or generally
" Vinfilant " or " Vin huileux," and the Italians style it " Vino Jilante." The
earliest microscopical studies on this point were made in 1861 by PASTEUR (XII.),
who found a very large number of fission fungi always present in ropy wine, and
also that by transferring a little of the liquid to sound wine of the same class
the disease was quickly communicated to the latter. He described two kinds of
cell form : small cocci united in chains (streptococci), and irregularly shaped cells
somewhat larger in diameter than those of yeast. The chief products of the
mucinous fermentation set up in wine by this mixture of organisms were found
to be gum, mannite, and carbon dioxide. Their ratio was represented by
Pasteur in the form of an equation as follows :

25Cj2H2.2Ou + 25H20 = izC^HaoOjo + 24C6H14O6 + I2C02 + I2H20.
Saccharose. Gum. Maunite.

These proportions were admittedly variable, but this was explained by
Pasteur by the supposition that the one species of ferment produces more
mannite, the other more gum ; and Monoyer, in 1862, attempted to represent
these reactions by splitting up the equation into two. Some observations on the
aforesaid streptococcus have also been published by E. DUCLAUX (X.).

The thoroughgoing microscopical investigations performed by J. NESSLER (II. )
showed that the streptococci described by Pasteur are frequently absent, or only
present in very small numbers, in ropy wine ; whereas, on the other hand,
the presence of certain unusually plentiful, extremely minute round bodies can
always be detected. Subsequently a few samples of ropy wines were examined
by E. KRAMER (II.), mainly with the object of obtaining pure cultures of the
organisms causing the malady, but this object has not yet been successfully
accomplished. By means of the dilution method approximately pure cultures
of such a fission fungus have been prepared ; and the name of Bacillus viscosus
vini has been given to the organism. It occurs in the form of rods, 0.6-0.8 /*
broad and 2-6 p, long, frequently united as many-jointed chains, and capable of
producing ropiness in white wines in the absence of air. A thorough mycological

218 ROPINESS IN MILK, WINE, BEER, ETC.

study of this malady has, however, still to be made. Neither the Bacillus
viscosus sacchari, mentioned in chapter xxviii., nor other similar cause of
mucinous fermentation, is capable of giving rise to ropiness in wine, since none
of them is able to develop in acid media.

One point is perfectly clear, viz., that the presence of sugar is a sine qua non
for the occurrence of the malady, since it forms the material from which the
mucus is produced. According to Nessler (an expert in the treatment of wines),
wines containing over 10 per cent, of alcohol are proof against ropiness.

With regard to the ropiness of cider — the most frequent malady to which
this beverage is subject — nothing reliable can at present be reported.

§ 165.— Ropiness in Infusions.

This was microscopically investigated as far back as 1834 by FR. KtTziNG
(I.), who ascertained that the lower orders of plants here in question are partly
algae and partly fungi, the schizomycetes being the most frequently found
members of the latter group. A few examples are given below.

It is well known that Infusum foliorum Digitalis very often becomes ropy, to
account for the occurrence of which divers hypotheses were formerly current.
Thus, for instance, it was asserted that the mucic acid in the leaves of digi-
talis exerts a coagulating influence on the pectin bodies also present therein.
W. BRAUTIGAM (I.) found in a ropy infusion of this kind a fission fungus, which
he named Micrococcus gelatinogenus, endowed with the property of gelatinising
vegetable infusions (e.g. Ipecacuanhas, Radix Althece, Senegce, Folia Farfane, and
especially Folia Digitalis), when mixed with sugar-cane, lactic acid being pro-
duced at the same time. The mucus is precipitable by alcohol. In nutrient
media devoid of sugar the micrococcus develops, but does not form mucus.

Of interest to the analytical chemist is the Bacterium yummosum, also
obtained by E. RITSERT (II.) from a ropy infusion of Digitalis. This organism
turns the nutrient medium ropy only when saccharose (but not dextrose or
lactose) is present, and can therefore be employed as a reagent for cane-sugar
to detect the latter in presence of large quantities of hexoses, e.g. in wine-must.
It will develop in highly concentrated solutions of this sugar, its growth not
being impeded until the concentration exceeds 60 per cent. The mucus produced
by this fission fungus has received the name of gummose, a term likely to lead
to error, since a somewhat widespread malady attacking the vine, the sugar-
beet, and other plants, has long borne the name of gummosis or gummose. This
mucus is distinguishable from dextran chiefly by being optically inactive. In
addition to mucus the organism produces an uninvestigated acid, and a compound
of unknown constitution, which deviates the plane of polarised light to the right
and reduces Fehling's solution. According to the conditions of cultivation, Bac-
terium gummosum appears as long or short rods, diplococcus or streptococcus,
the first forms being motile and producing endospores. The addition of acetate
of potash or soda or of yeast ash to the nutrient solution (e.g. sugar-beet juice,
&c.) is highly favourable to development and to the production of mucus.

The Bacillus gummosus, isolated by C. HAPP (I.) from a ropy infusion of
Digitalis, is characterised by its large size, the length being 5.0-7.5 /i, and the
breadth 0.6-2.0 /*. It exhibits an undulatory motion and forms endospores. In
cultures on slices of potato and beet the cells are globular, with a diameter of
0.7-0.8 (i, but when transferred to gelatin or agar-agar they quickly become
rod-shaped. Happ obtained from ropy Senega infusion a pure culture of
Micrococcus gummosus, the diameter of which is about 0.4 /t. A notable diffe-
rence exists between these two species with respect to their behaviour towards
sugars, the first-named being able to set up ropy fermentation only in presence

ROPINESS IN WORT AND BEER 219

of saccharose, whilst the Micrococcus also attacks maltose. The resulting mucus
(soluble in water, but insoluble in alcohol and ether), which has the elementary
formula CfiH1006, is, although the chief, not the sole product of this fermentation,
small amounts of mannite, butyric acid, lactic acid, and carbon dioxide being
also formed ; and a part of the saccharose is converted into glucose.

The so-called distilled waters (e.g. orange-flower water) often undergo
mucinous decomposition, some particulars of which have been reported by
L. VIRON (I.). As a remedy for this evil, P. Carles advised the shaking up of
the affected water with 2-3 grams of basic nitrate of bismuth per litre, and
filtering after standing. This is said to have answered particularly well with
orange-flower water. Ordinary distilled water is often rendered mucinous
by bacteria, especially when kept in wooden vessels ; A. GOLDBERG (I.) has
reported an instance of this kind.

A fission fungus, Bacterium gliscrogemim, 0.57-1.1 p. long and 0.4. /x broad,
has been isolated by P. MALERBA and G. SANNA-SALARIS (I.) from mucinous,
viscid urine (which often exhibits this property as soon as voided), and has
been recognised as the cause of this condition. According to a research of
MALERBA'S (I.), the mucus (gliscrin) thereby formed is nitrogenous.

It is well known that ink frequently becomes mucinous and viscid. M. HERY
(I.) investigated this matter and examined a bacterial species concerned therein.
As a preventive measure he recommends an addition of not less than 0.5 gram
of salicylic acid per litre of ink.

C. BOERSCH (I.) made an observation, interesting to the chemist, concerning
a fission fungus, Sarcinaflava, capable of producing ropiness in various liquids.
This organism attacks fumaric acid (in acid solutions), but, on the other hand,
leaves the isomeric maleic acid, COOH — CH=CH— COOH, untouched.

Eopiness in tan liquors is a phenomenon both well known and unwelcome to
the tanner, to whom it causes considerable damage and loss, since not only is the
liquor rendered worthless, but the hides steeped in it also suffer owing to the
masses of mucus adhering so firmly to the leather that great difficulty is expe-
rienced in getting them off again. This mucinous coating retards, or even entirely
prevents, the penetration of the tannin. Closer investigations regarding the
best means and methods of prevention would be valuable to this industry.

§ 166.— Ropiness in Wort and Beer.

PASTEUR (III.) was the first to study this phenomenon with the aid of the
microscope. He traced the cause of this complaint, which has many points in
common with ropiness in wine, to a fission fungus occurring abundantly in the
form of long chains in the affected liquids, and known by the name of Micro-
coccus viscosus. Morphologically, this organism greatly resembles a fission fungus
observed by J. BERSCH (II.) in a beer wort, which, instead of fermenting nor-
mally when pitched with yeast, became thick, oily, and finally viscid and ropy.

P. LINDNER (III.) in 1889 was the first to obtain a pure culture of a viscous
ferment. This was a pediococcus (not specifically named) occurring in large
numbers in ropy white beers, a class of beverage that is particularly liable to the
malady. The capacities of the microbe in question are restricted to, the pro-
duction of ropiness in white beer wort, it being unable to do so in hopped worts
and beers. Hence it is perfectly innocuous and unimportant, so far as true
brewing, in the narrow sense of the term, is concerned.

Other species appear in hopped beer. Two of these were found by H. VAN
LAER (II.) in a number of samples of ropy beer, from which they were isolated
to pure cultures, and named Bacillus viscosus I. and //. Both have several
identical characteristics, e.g. the form and dimensions of the cells, which are rod-

220 ROPINESS IN MILK, WINE, BEER, ETC.

shaped, 0.8 p. broad, 1.6-2.4 M l°ng> ana" mostly single, though not infrequently
joined in pairs.

In their behaviour towards beer-wort, however, they differ in a notable
manner. It is true that both of them produce ropiness, but not of the same
type. If B. v. I. is in action, then, in proportion as the viscidity of the liquid
increases, a number of mucinous, yellowish-white patches, terminating below in
branches, appear on the surface. In this way a coating of mucus is formed, the
surface of which is gradually covered with protuberances produced by bubbles of
the carbon dioxide liberated during this fermentation. With B. v. II., on the
contrary, this coating is absent ; moreover, the evolution of carbon dioxide is less
copious, and the ultimate degree of ropiness less pronounced than in the first
case. Whilst the malady is in progress, the colour of the wort changes to a
chicory-brown, and at the same time an odour develops, which cannot be more
closely defined, but which of itself suffices to reveal the presence of the com-
plaint. A further characteristic affording a means of distinguishing between
these two species of bacteria is their behaviour towards a sterilised solution of
3 grams of cane-sugar and i gram of peptone in 100 c.c. of water. This medium
is made viscid and ropy by B. v. I. alone, the second species producing nothing
more than a persistent turbidity, accompanied by the evolution of carbon dioxide.
Milk is altered by both species in the same manner as wort.

The fact that both these organisms also cause ropiness in nutrient solutions,
devoid of sugar and containing no organic matter beyond calcium lactate or
ammonium tartrate, is also interesting. As a matter of fact, a high content of
sugar is even injurious to the organisms. This discovery agrees with the ex-
perience gained in practice, that beers with a low attenuation (and therefore a
higher sugar-content) are comparatively seldom ropy. The proximate cause of
this alteration of the medium is a mucus excreted by the bacteria. In the
presence of sugar, carbon dioxide is liberated, arid presumably a small quantity
of another acid is also formed, since the acidity increases with the ropiness.
The mucus is not a uniform substance, but consists of at least two constituents,
one of which (insoluble in water) is characterised by its content of nitrogen.
This fact harmonises with the circumstance that the malady sets in earlier in
proportion as the nitrogen-content of the nutrient medium is greater. It also
explains the fact, noticed in practice, that worfcs rich in protein, peptones, and
the like, are those most readily becoming ropy. A higher content of acid (0.15
per cent, reckoned as lactic acid) restricts the development of both these species
of fission fungus ; but alcohol, even in the proportion of 6 per cent, by volume,
is powerless to injure them. In both cases growth proceeds at all temperatures
between 7° and 42° C., and is most vigorous at about 33° C.

A third viscous ferment, also discovered by Van Laer, differs from the other
two in its property of liquefying gelatinised meat- juice.

L. YANDAM (I.) obtained from ropy English beer pure cultures of a fourth
organism (Bacillus viscosus III.) in the form of rods 0.7 p. broad and 1.3-2.0 /u
long, mostly isolated, but frequently also forming bands of two or three cells.
So far as can be gathered from the particulars given, ropiness is produced, not
by any metabolic product excreted by the bacillus, but by the thickened cell
membrane of the organism. In other ways, too, this microbe differs from Van
Laer's bacilli. For instance, the development of the organism and the gelatini-
sation of the medium occur only in presence of sugar, and the degree of ropiness
is proportional to the amount of sugar eliminated. No evolution of gas could be
detected in wort cultures. The unrestricted access of air is essentially necessary
to the growth and activity of this bacillus. The organism is incapable of injuring
beer except when present in large numbers in the wort before the commencement
of primary fermentation.

THE SO-CALLED SARCINA TURBIDITY IN BEER 221

The number of organisms capable of rendering wort viscid is not exhausted
by the Schizomycetes already mentioned. In the second volume we shall become
acquainted with Dematium pullulans, a species of Eumycetes which is equally
capable of producing damage of this kind.

§ 167.— The So-called Sarcina Turbidity in Beer

will now be referred to, although no mucinous ferments are here in question.
Bottom-fermentation beer is required to be perfectly clear, and if it proves
defective in this particular it is considered poor or bad, according to the nature
of the turbidity. This may arise from several distinct causes: precipitated
albuminoids = gluten turbidity ; the presence of unsaccharified starch = starch
turbidity ; precipitated hop resins = hop dimness ; a high content of yeast
cells = yeast turbidity ; or, finally, strong infection with fission fungi = bacterial
turbidity. This latter, again, may be caused by different species of organisms,
a few of which (i.e. those producing turned and ropy beer) [have already been
mentioned, the turbidity in their case being merely a secondary phenomenon
attendant on another complaint. In the following lines, however, we will con-
fine ourselves to the turbidity caused by bacteria of the sarcina or pediococcus
form of growth. Very frequently these organisms (in enormous numbers) are
the only ones observable in samples of turbid beer.

The first observations on the subject were made by PASTEUR and J. BERSCH
(II.), and more minute researches were made by Julius Balcke, from whom
these organisms first received the name of Sarcina. Francke afterwards found
that this fission fungus always subdivides in two directions only (and not three),
and consequently forms sheet colonies. On this account FRANCKE (I.) in 1884
applied the new generic name of Pediococcus cerevisice to this microbe. Not-
withstanding this, it is still customary to term the malady under consideration
" sarcina turbidity " ; which is, moreover, partly correct, since true sarcina in
great numbers have also been found in turbid beers. The first successful
attempt to obtain a pure culture of such a pediococcus was made by P. LINDNER
(II.) in 1888. The Pediococcus cerevisice isolated by him from "sarcina turbid "
beer occurs as single cocci (0.9-1.5 p. diameter), diplococci, and tetrads. Still,
though it is undoubtedly the fact that this fission fungus occurs in large
numbers in such turbid beers, it by no means follows that the organism can be
positively assumed to be the cause of sarcina turbidity, attempts to grow it in
sterilised beer having proved unsuccessful. Moreover, as ANTON PETERSEN (I.),
E. CHR. HANSEN (V.), and ALFRED JORGENSEN (I.) have shown, a considerable
quantity of sarcina may be present in beer without any damage to the beverage
(turbidity or unpleasant flavour) resulting therefrom.

Further particulars given by them render it highly probable, however, that
" sarcina turbidity " is actually caused by fission fungi of the pediococcus and
sarcina groups, but that the mere presence of these organisms is not sufficient to
produce the malady, a special concurrent tendency thereto on the part of the
beer being essential. For the determination of the conditions under which the
''sarcina organisms" are capable of producing "sarcina sickness" in beer, we
are indebted to an instructive treatise by A. REICHARD (I.). He showed that
this turbidity occurs only when the secondary fermentation of the beer goes on
with vigour, and that, conversely, a similar degree of sarcina infection is
innocuous if the primary fermentation has been carried so far that only a weak
secondary fermentation ensue?. Reichard attributes this behaviour (confirmed
by searching experiments) to the avidity for oxygen (air-hunger) displayed by
the pediococci. It is only when the microbes are continually brought up to the
surface of the liquid by the bubbles of carbon dioxide given off during a brisk

222 ROPINESS IN MILK, WINE, BEER, ETC.

secondary fermentation that this avidity for oxygen can be satisfied and the
development of the organism proceed.

When no gas is liberated, and the pediococci consequently remain at the
bottom of the liquid, then no turbidity or unsatisfactory alteration of the flavour
or smell will occur. If, however, an infected beer be artificially brought into a
state of active secondary fermentation by priming (aufkrtiusen) with fermenting
wort, then sarcina turbidity will not be long in making its appearance. This
fact, determined by Reichard, indicates the necessity for caution in the employ-
ment of fermenting wort for priming beer. This practice, as is well known is
specially resorted to for livening up sluggish lager beers in the storage cask,
and is of itself unobjectionable. Care should, however, be taken to previously
ascertain that no large amount of sarcina is present in the cask. According to
the researches of REICHARD and RIEHL (I.) hops are very useful in combating
sarcina sickness. To prevent the appearance of the malady 30-40 grams of
hops per hectolitre of beer (or at the rate of 5 to 6 oz. per 100 galls.) should be
placed in the storage cask, and the latter then closed (bunged).

The injurious organisms in question either find their way into the wort in
the cooler, or — as stated by Balcke — may be transferred to the malt store on the
boots of a workman (floor-sweeper) who has previously been working on the
malting floor, where these organisms abound. It is, therefore, no wonder that
the thick worb is also rich in these organisms, and may consequently become the
source of acute troubles. The evil reputation of the thick wort and thick beer
is also easy to understand from a bacteriological standpoint. When such an
infected wort is fermented, then, of course, the yeast crop will be contaminated
with these injurious organisms and the malady will thus be perpetuated. To
purify such contaminated yeasts, S. VON HUTU (I.), in 1888, proposed an addition
of 5-7 grams of salicylic acid per hectolitre of beer (about i oz. per 100 galls.).
A second recipe of his, which was also approved by P. LINDNER (IV.) in 1895,
reads as follows: To each kilo. (2.2 Ibs.) of pulpy or liquid yeast take 6 grams
of tartaric acid dissolved in' water. After stirring them thoroughly together,
leave to stand for six to twelve hours, and then add the mixture to the wort in
the tun. The results of this treatment are said to be satisfactory.

It must, however, be expressly mentioned that this tartaric acid cure should
not be employed unless the yeast under treatment is either almost or entirely
free from wild yeasts, and is contaminated by sarcina alone. Otherwise it is best
to throw the batch away, since the tartaric acid treatment, by favouring the
development of the wild yeasts, would only make it worse than ever. This will
be referred to again in a subsequent section of vol. ii.

SECTION VIII.

DECOMPOSITIONS AND TRANSFORMATIONS OF ORGANIC
NITROGENOUS COMPOUNDS.

CHAPTER XXX.

THE PHENOMENA OF PUTREFACTION.

•

§ 168.— The Degradation of the Albuminoids.

IN § 15 of the Introduction it was stated that Liebig's differentiation between
fermentation and putrefaction is untenable, and that no sharply defined limit
between these terms exists. Enlarging the definition of the term fermentation
beyond its usual limits, we there defined this phenomenon as the transformation
of various chemical substances by the action of minute fungoid organisms.

Without prejudice to this general definition, we can nevertheless speak of
putrefaction in particular, limiting the application of this term to such fermen-
tations as chiefly effect the decomposition of albuminoid substances. Any further
attempt to analyse this more restricted term is at once frustrated by our igno-
rance of the constitution of the albuminoids themselves. The multiplicity of
contingencies here possible cannot be disregarded, and consequently no classifica-
tion according to the final products obtained is feasible. On the other hand, no
differentiation can be based on the composition of the bodies subjected to decom-
position, since we are here encountered by a question, hitherto unsolved by
chemists, viz., What are the albuminoids?

Obviously mycologists might postpone further researches on this point until
the necessary preliminaries have been performed by their chemical colleagues.
As a matter of fact, however, the opposite course has been adopted, and the
determination of the nature of the putrefaction products of albumen has not
only led to hypotheses regarding the composition of that substance, but will
probably also indicate the means whereby the nature and synthetic preparation
of these high-molecular nitrogen compounds can be established. Provided the
results obtained are of value to the chemist, and, though in a minor degree, to
the mycologist as well, the credit thus accruing to Fermentation Physiology is
not necessarily injured by the remark that, owing to the employment of indefinite
bacterial mixtures, these endeavours are not always free from objection from a
bacteriological point of view.

In future researches into albuminoid decomposition or putrefaction, it should
always be borne in mind that here also the co-operation and succession of various
organisms (i.e. symbiosis and metabiosis) will have to be taken into calculation.
Until this is done, mycological text-books will have nothing better to offer than
a varied collection of isolated observations, such as are given in the following
chapters and paragraphs.

It has long been observed that the natural decomposition of albumen yields
malodorous gases and vapours when proceeding in the absence of air. but that,
on the other hand, these attendant phenomena are wanting when air is allowed

223

224 THE PHENOMENA OF PUTREFACTION

froe access. Titular distinctions have been employed to express these differ-
ences, the natural inodorous decomposition of albuminoids being termed decay,
whilst the name of putrefaction has, in a narrowed sense, been applied to the
other set of phenomena. Formerly regarded from the chemical standpoint
alone, the fundamental physiological basis of this differentiation has now
been explained by the aid of mycological research as follows : Decay is the
result of aerobic microbial activity; putrefaction, of the energy of anaerobic
organisms. Of course both these processes may go on simultaneously in the
same substance, the outer surface, exposed to the air, decaying, Avhilst the
interior putrefies. This fact alone sufficiently proves how little value attaches
to researches wherein pure cultures are not employed. M. VON NENCKI (III.)
sought to explain the putrefactive decomposition of the albuminoids as a
process of hydration, and cited in support of this view the observation that
the products obtained are the same as those produced by the action of fusing
caustic potash.

The bad smell characteristic of putrefaction is often attributable to several

yCH^

compounds of the aromatic series. One of these is Indole, C6II/ /CH, which

\NH/

combines as an imide with nitrous acid to form the red nitroso-indole. This
property is utilised for the detection of indole in cultures. Since a great many
bacteria are capable of producing a small (though sufficient) quantity of nitrites
in ordinary nutrient media, this characteristic red coloration can be developed
(in presence of indole) by slightly acidifying the culture with sulphuric acid.
Of the pathogenic bacteria, Koch's Vibrio cholene asiaticcc was the first examined
for this reaction. This accounts for the current use of the term " cholera red
reaction," employed for this reaction by medical bacteriologists. /3-methyl indole
or skatole, which was first discovered in 1877 by L. BRIKGEE (II.) in human
faeces, is almost invariably produced during the putrefaction of albumen ; its
smell is. even more repulsive than that of indole. A closely allied derivative of
skatole, viz., /3-methyl indole acetic acid, was discovered by M. VON NENCKI
(IV.) among the putrefaction products occasioned by Jlacillus liquefaciens magntts
in the absence of air. Phenol was first recognised as a product of albumen
putrefaction by E. BAUMAKN (I.) in 1877, and orthocresol and paracresol by
E. BAUMANN and L. BRIEGEK (I.) in 1879. The capacity of a large number of
(mostly pathogenic) species of bacteria for producing the above-named substances
was investigated by A. LEWANDOWSKI (I.).

M. von Nencki and his pupils made a series of investigations on the products
of albuminoid putrefaction. Of their discoveries we will now briefly mention
those referring to leucine and tyrosine. These amido substances are secreted
by the pancreatic glands, and are almost always present in fresh faeces. They
are also produced, under certain condition^, in the putrefaction of various albu-
minoids. Now, according to NENCKI'S (V.) researches, leucine is further
decomposed by the activity of bacteria, the chief product being valeric acid,
along with carbon dioxide, hydrogen, and ammonia. The reaction is approxi-
mately expressed by the equation —

CH3— CH2— CH2— CH2— CH.Nttj— COOH + 2H20 =
Leucine.

(CH3)2 = CH— CH2— COOH + C02 + aH2 + NH3.
Valeric acid.

The decomposition of tyrosine may be effected in two different ways : in
presence of air — as was shown by E. BAUMANN (II.) — the NH2 group is separated,

THE DEGRADATION OF THE ALBUMINOIDS 225

and hydroparacumaric acid, of which tyrosine may be regarded as the amine
(alanine), is formed—

/OH /OH

C6H4< yields C6H4/

XCH2— CH.NH2— COOH \CH2— CH2— COOH.

Tyrosine. Hydroparacuuiaric acid.

When air is excluded, the results are, however, very different, indole, together
with carbon dioxide and hydrogen, being produced. This reaction is approxi-
mately represented by the equation —

/OH /HN\

C0H4( = C6H4<; \CH+C02 + HoO + H2
\CH2— CH.NH2— COOH \CH^

Tyrosine. Indole.

The evolution of sulphuretted hydrogen is a frequent accompaniment of
putrefaction. A large number of bacteria are endowed with the power of
liberating this gas, the production of which depends, however, not solely on the
species of ferment, but also on the composition of the nutrient medium, a circum-
stance which explains the contradictory results obtained by different workers.
Thus, for example, STAGNITTA-BALISTRERI (I.) denied that Bacillus subtilis,
Bacillus tetragenus, the so-called Wurzel bacillus, and others could form sul-
phuretted hydrogen ; but PETKI and MAASSEN (III.) then showed this contention
to be incorrect, and that, in presence of peptone, the gas in question is produced
by these microbes. In other cases, again, this product may be masked, e.g. by
combination with ammonia formed at the same time. A good deal of the sul-
phur present in the nutrient medium is utilised by the bacteria themselves for
structural purposes, the amount so consumed having been found by M. RUBNER
(I.) to be equivalent to 23-40 per cent, of the total sulphur in the medium. The
sulphur in organic combination is first occluded, a circumstance harmonising
with the well-known fact that the sulphur in albuminoids is very easily removed.
The more delicate processes leading finally to the evolution of sulphuretted
hydrogen, still remain unelucidated. PETRI and MAASSEN (IV.) are of opinion
that the bacteria liberate hydrogen, which in the nascent state then extracts
sulphur from the sulphur compounds and combines with it. They found that
very little of the gas in question is produced when nitrates are present in the
medium, but that these latter are thereby reduced to nitrites. With reference
to the fact (put forward to refute this explanation) that sulphuretted hydrogen
is liberated by aerobic bacteria in well " roused " (aerated) cultures, Petri and
Maassen showed that hydrogen is also liberated under this treatment, and that
consequently the presence of air favours the reducing action.

The faculty of producing sulphuretted hydrogen is very common among the
pathogenic bacteria, being absent in not a single one out of thirty-seven species
examined ; and in many of them — e.g. the bacilli of swine erysipelas — the inocu-
lated nutrient solution fairly bubbles from the quantity of gas liberated. A
convenient means of detecting and separating sulphuretted-hydrogen-generating
microbes from a mixture of bacteria by the aid of plate cultures is afforded by
the ferro-gelatin, recommended by A FROMME (I.) for this purpose ; i.e. a pepto-
nised meat-juice gelatin qualified by 3 per cent, of iron saccharate or tartrate.
In such nutrient media each colony of the sulphuretted hydrogen bacteria will
become surrounded by a black halo of FeS.

The conversion of sulphates into sulphides by bacterial agency is also a deci-
sive indication of reducing power. The conditions of vitality of a particularly
active species of fission fungus were investigated by BEYERINCK (II.), who named
the organism Spirillum desulfuricans. This strictly anaerobic microbe is utilised

226 THE PHENOMENA OF PUTREFACTION

in practice in so far that by skilfully encouraging its development pit-water very
rich in gypsum has been entirely freed from sulphates (CaS04 being converted
into CaS and FeS) and rendered suitable for various purposes, such as feed-water
for steam-boilers, &c. Further particulars on this matter will be found in the
treatise referred to. The sulphuretted hydrogen produced by the above-named
bacteria is consumed by a special group of fission fungi which will be more closely
considered in chapter xxxv.

Among the sulphurous products of albuminoid putrefaction mention must be
made of mercaptan (C2H..SH), which was first detected by M. VON NENCKI and
N. SIEBER (II.) in cultures of Bacillus liquefaciens magnus.

§ 169.— The Putrefactive Bacteria.

In the course of his investigations (frequently alluded to in previous para-
graphs) on the micro-organisms in putrescent liquids, Chr. Ehrenberg observed
a variety of forms and dimensions. The smallest of them bordered on the limits
(Lat. termo) of visibility, and was so minute as to be almost indistinguishable by
the aid of the optical instruments then available. On this account he, in 1830,
gave it the name of Bacterium termo, and subsequently, in 1838, expressed the
opinion that this species is identical with the Vibrio lineola already described by
O. F. Miiller. However, when FELIX DUJARDIN (I.), in 1841, undertook to
critically examine Ehrenberg's discoveries, and classified all the (infusorial) micro-
organisms devoid of visible organs of locomotion into the family Vibrionia, which
comprised the three genera Bacterium, Vibrio, and Spirillum, the old name of
Bacterium termo was re-applied to this organism. Dujardin also regarded this
" infusorium " as the smallest of all living creatures (le premier terme en quelque
sort de la serie animale), and described it as follows : " Form, cylindrical ; length,
2-3 p; thickness, 1.0-1.2 /n; frequently united in couples ; exhibiting a tremu-
lous movement," the latter being ascribed to alternate contractions and re-expan-
sions of the plasma. To these characteristics PERTY (I.) in 1852 added another,
viz., the grape-like form peculiar to the zooglcea of this microbe. One year later
COHN (V.) also described a like organism. Then when, towards the close of the
sixth decade of the century, Pasteur fully explained the theory of specific fer-
ments (originated by Kiitzing), and proved its accuracy by a series of examples,
of which lactic fermentation was the first, the inclination to regard putrefaction
as the work of a specific fission fungus gradually spread. Hence it was that
COHN (I.), in 1872, propounded the dictum that "putrefaction is a chemical
process excited by rod-bacteria" (Bacterium termo).

The more accurate (physiological) investigation of this process long remained
impossible owing to the lack of means for isolating and obtaining pure cultures
of its active organism. For this reason the results obtained by different investi-
gators (e.g. B. SANDERSON (I.) in 187:, and E. EIDAM (I.) in 1875) into the
physiological conditions of the so-called Bacterium termo are now only of historical
interest. On the introduction of plate-cultures into practical bacteriology, pure
cultures of the supposed Bacterium termo were soon obtained, and it was then
found that this term comprised a number of different species. ROSENBACH (II.),
in 1884, was the first to ascertain this fact, and described three distinct species
of decidedly putrefactive bacteria, which he named respectively Bacillus sapro-
genes /., //., and ///. Rosenbach undertook these researches from a medical
point of view, and consequently treated the morphological and physiological sides
of the question in a perfunctory manner. Nevertheless, he deserves the credit
of having finally banished the designation Bacterium termo from systematic
botany ; so that, though the name is still occasionally used, it has now no special
import, but merely serves as a convenient synonym for the term " putrefactive

THE PUTREFACTIVE BACTERIA 227

bacteria." In this general sense the term is used in Fig. 57. The figure itself
represents a species of bacterium (not more specifically identified) isolated from a
putrescent liquid.

G. HAUSER (I.) investigated this matter more thoroughly, and showed,
especially, that Bacterium terino, in the sense implied by Cohn, does not exist.
In 1885 he brought to our knowledge three putrefactive fission fungi, which are,
moreover, bacteriologically important from their indisputable polymorphism, a
peculiarity since recognised in many other species of bacteria, but at that time
much disputed. Hauser's discovery was welcomed by the supporters of this

B"IG. 57.— Bacterium tcrmo. FIG. 58. — Proteus vulgaris.

Cilia staining-. Magn. about One long1 rod and one short rod. Cilia staining.

1500. (After photograms by Magn. about 1500. (After photograms by Fraenkel

Fraenkel and Pfeiffer.) and Pfeiffer.)

theory, and the importance attached to it at the time was expressed in the name
given to the organisms, Hauser having chosen the generic name Proteus for these
extremely mutable Schizomycetes. A short description of their characteristics is
subjoined.

The cells of Proteus vulyarls are generally 0.9-1.2 p. in length, 0.4-0.6 Abroad,
and almost always occur in couples. In addition to these short rods, elongated
forms, very frequently attaining a length of 3.7 p., also occur. Some extremely
vigorous but very rare cells will measure 6 p. long by 0.9 p. broad. One of these
is shown Fig. 58. The large number of cilia indicates considerable locomotive
activity, and in fact this power is possessed by the various species of Proteus in
a high degree, manifesting itself both by a rapid forward movement and a con-
current (longitudinal) axial rotation. Hence, the coupled cells describe a kind
of double cone, the vertex of which is at their point of junction. In addition to
the above-named forms, gelatin cultures also yield spirilla, with two to four
convolutions; thread-cells, which may grow to a length of 100^1; and finally
" spirulina," or threads bent in the form of a bow, with ends twisted into a
queue. Under special circumstances involution forms are also produced : the
cells swell up in the shape of a pear, and resemble spermatozoa, dumb-bells, &c.,
in form.

Proteus mirabilis exhibits a very decided tendency for producing such involu-
tion forms. Globular or pear-shaped forms, 3-7 p, in diameter, are very frequently
developed in the cultures of this microbe, which also exhibits polymorphism in
a high degree, and in this particular greatly resembles the preceding species.
Here also we meet with short rods, long rods, spirilla, and thread-cells, rapidly
moving one through another in varied alternation. At the same time small
but unmistakable differences exist. Thus for example, these threads not unfre-
quently attain a length of 200 p., i.e. double the maximum size of the first-named
species.

Proteus Zenkeri differs from the two preceding species mainly in its inability
to liquefy gelatin, but resembles them in other particulars, though its cells are

228 THE PHENOMENA OF PUTREFACTION

generally smaller, the least of them being globular in form and 0.4 p. in diameter.
Short rods (0.8 p, long) joined in pairs are frequently encountered. These three
species are unique in the bacterial kingdom in point of motile power, which they
possess to such a high degree that a solid medium containing only 5 per cent, of
gelatin is unable to restrain them, and they make their way across it in all
directions. In order to stop this roving motion the gelatin content must be
increased to 10 per cent. This peculiarity is not only of physiological interest,
but is also decidedly important so far as practical bacteriology is concerned, in
that it indicates the futility of employing nutrient gelatin media containing less
than i o per cent, of gelatin for the preparation of pure plate cultures of Proteus
species. To complete the characterisation of these three species, it should be
mentioned that none of them forms endospores, and that their growth may be
arrested by depriving them of oxygen, though they do not necessarily die in
consequence. They will not thrive in mineral nutrient media, such as those of
Cohn and Nageli. When grown in albuminous media, they produce stinking
decomposition. A. BRODMEIER (I.) proved that in neutral or alkaline solutions
Proteus vulgaris is able to convert urea into ammonium carbonate. He thus
refuted the assertions of Leube to the contrary, and confirmed the discovery of
Schnitzler and Hofmeister.

No pretension can be made in the present work of giving a complete descrip-
tion of all known forms of putrefactive bacteria, and therefore the examples
already cited, being the species most frequently met with, must suffice. More-
over, we have already mentioned others of this class in previous paragraphs.
One of these, viz., the Bacterium Zopfii, discovered by KURTH (I.) in the
stomachs of fowls and shown in Fig. 31, is, according Czaplewski, identical with
Proteus Zenkeri. This note appears in an abstract of a work by CH. MOUGINET
(I.), who, also, minutely examined a number of putrefactive bacteria. HOLS-
CHEWNIKOFF (I.) described a fission fungus closely allied to Proteus vulgaris,
which, from its faculty of producing sulphurretted hydrogen, has been named
Proteus sulfureus.

Only one more species will be dealt with here, and that briefly, viz., Bacterium
coli commune, which is an invariable inhabitant of the alimentary canal of the
human subject (and of all the higher animals hitherto examined), and constitutes
the most important of the bacteria present in fseces. This parasite was first
described by TH. ESCHERICH (I.) as a slender short rod, 0.4 /* broad, the length
varying with the conditions of nutrition and cultivation, but mostly measuring
2-3 n, though occasionally it decreases to 0.5 /*. By some authors this fission
fungus is named Bacillus coli communis and Colon bacillus. Like the Proteus
species, it generally appears as double rods, but its movements are sluggish
and laboured. It does not liquefy gelatin. In media containing sugar it can
develop even in the absence of oxygen, and liberates a gas which — according to
FREMLIN (I.)— consists of two-thirds carbon dioxide and one-third hydrogen.
No development of endospores has hitherto been detected. In its manner of
growth in artificial media this organism agrees in many particulars with Bacillus
typhi abdominalis. Consequently they are extremely hard to differentiate, and
this makes the bacteriological examination of water a particularly difficult opera-
tion when the presence of typhus bacilli has to be quantitatively determined. A
further complication is imparted by the extreme sensitivity of B. coli commune
to modifications in the conditions of cultivation, and by its great tendency to
form varieties. For instance, a number of races of B. coli commune are nosv
known, which, under certain circumstances, are not merely saprophytic, but also
pathogenic. A more detailed treatment of this question would occupy too
much of our space, and besides, the matter is fully recorded in Tiemann-G'artner's
work on Water Analysis. A synopsis of the most important researches of

THE PUTREFACTIVE BACTERIA 229

Escherich, Kohler, Baginsky, Bischler, and others, on the methods of nutrition
of B. coli commune and its powers of decomposition, was prepared by M. IDE (I.)
in 1891. The facts brought to light since that date will be found in the several
yearly volumes of A. Koch's " Jahresbericht."

We will now briefly refer to the subject of intestinal putrefaction. Mention
has been made in a previous paragraph of the fundamental difference between
the processes of decomposition effected in the small intestine on the one hand and
in the colon on the other, in man. On issuing from the stomach — where, by
the action of the pepsin and hydrochloric acid secreted by the gastric glands, a
more or less extensive peptonisation of the digestive albuminoids in the food has
been effected — the pulpy food, now known as chyme, has a strongly acid reaction
(equivalent to 0.1-0.3 per cent, of hydrochloric acid). Immediately on its arrival
in the upper division of the alimentary canal (small intestine), it becomes mixed
with bile and pancreatic juice, under the influence of which the fat is emulsified
and the insoluble carbohydrates (starch) are hydrolysed. Both secretions have
an alkaline reaction, which, however, is not sufficiently strong to immediately
neutralise the acidity of the contents of the intestine. This slightly acid nutrient
medium, rich in sugar, offers a favourable field for the activity of the lactic acid
and allied bacteria introduced along with the food ; and, moreover, the acidity
restricts the development of the competitive putrefactive bacteria. In propor-
tion, however, as the contents of the intestine are forced onward and approach
the colon, the acid reaction is neutralised by the alkaline mucus secreted by the
intestinal glands. At the same time the composition of the mass has become
changed, since the products of the hydrolysis of starch, which have also to some
extent been converted by the aforesaid bacteria, have been absorbed into the
blood-vessels. Therefore in the contents of the colon it is the (undigested or
indigestible) albuminoids and biliary constituents which are decomposed by the
putrefactive bacteria now coming into action, and it is here that the malodorous
products (indole, skatole, volatile acids, sulphuretted hydrogen, &c.), to which the
intestinal contents (finally issuing from the rectum as faeces) owe their repulsive
smell, are produced.

The researches of MACFADYEN, NENCKI, and SIEBER (III.) revealed both the
actual course of the process just described, and the fact that, contrary to the
view expressed by Pasteur, the putrefaction occurring in the colon is not essen-
tial to digestion. The above-named workers performed their experiments on a
patient suffering from a strangulated hernia at the junction of the ileum and the
caecum. This portion of the intestine was removed by an operation, and the
subsequent surgical treatment necessitated the construction of an artificial
evacuatory passage (anus prceternaturalis) at the extremity of the small intestine,
until complete union of the severed portions was restored, an affair of six months'
duration. Meanwhile the contents of the intestine were discharged through this
artificial passage, and, though no digestive functions were performed by the colon,
the patient nevertheless kept in good health, and even increased in weight. This
will explain why Nencki regarded the development of antiseptic digestion as the
goal of the physiology of nutrition, i.e. digestion in which the putrefaction occur-
ring in the colon is either abolished, or at least reduced to a minimum, in order
to prevent the formation of decomposition products that are not only useless to
the body, but even troublesome and dangerous. As a matter of fact, GEORGE
NUTTALL and H. THIERFELDER (I.) recently afforded a convincing proof of
Nencki's theory by rearing some young porpoises, born by the aid of the Csesarean
operation, and nourished in a suitable sterilised chamber. On examination at
the close of the experiment, they were found perfectly healthy, though entirely
free from bacteria. Pasteur's assumption (which was also supported by Soxhlet
with reference to his incomplete process of milk sterilising) was thus shown

230 THE PHENOMENA OF PUTREFACTION

to be erroneous. A few observations on this point were also made by E.
DUCLAUX (XI.).

§ 170.— Proteolytic Enzymes.

All the fission fungi (with the few exceptions given in chapter xxxiii.)
require nitrogenous nutriment for the construction of their cells. Such of
these nitrogenous materials as are soluble in water, and therefore diffusible
through the cell-wall by osmosis, need not be referred to here. Mostly, how-
ever, the nutriment presented to the bacteria is insoluble in water, and this is
particularly the case with the protein albuminoids. To enable these latter to
supply the nitrogen required for the elaboration of the bacterial plasma they
must first be converted into soluble compounds, a task which is effected by the
proteolytic enzymes. So far no comprehensive study of these active bacterial
secretions has been made, and at present our knowledge is chiefly confined to
the enzymes dissolving gelatin and fibrin. A new classification of the bacteria
into two groups, the liquefactive and non-1 iquef active towards gelatin, according
to the presence or absence of a proteolytic enzyme, has obtained currency in
practical bacteriology since the introduction of the Koch system of plate-
cultures.

We are indebted to CL. FERMI (IT.) for the first extensive series of pure
culture investigations on this point. He proved that a gelatin-dissolving
enzyme is formed in cultures of the following species of Schizomyceles : —
Bacillus sublilis, B. anlhracis, B. megatherium, B. pyocyaneus, Vibrio choleras
asiaticce, Vibrio Finkler~ Prior, Micrococcus prodigiosus, M. ascoformis, M.
ramosus, spirilla from cheese, &c. Fibrin is dissolved as well as gelatin, but less
readily than the latter. Egg-albumen and coagulated blood-serum offer greater
resistance to these bacteria, thus indicating that pepsin is not present. Keasons
exist for assuming that the enzymes produced by the said microbes are not all of
the same kind, one conclusive indication being afforded by their behaviour under
different temperatures. Thus, for example, the proteolytic enzyme produced by
Micrococcus prodigiosus is rendered inactive (in solution) by a temperature of
55° C., that from B. pyocyaneut by 60° 0., that from B. anthracis by 65° C.,
and that from Vibrio F 'inkier-Prior not below 70° C. Similar differences of
behaviour are observed towards acids, bases, and poisons. A fundamental
difference exists between these enzymes and pepsin, since whereas the latter is
extremely sensitive towards alkalies, and is absolutely incapable of dissolving
albumin except in presence of free hydrochloric acid; the bacterial enzymes in
question act on fibrin in neutral or faintly alkaline solutions only, though they
will attack gelatin even when the liquid is slightly acid (0.5 per cent. HC1). On
this latter account they more nearly resemble trypsin, i.e. the enzyme secreted
by the gastric glands. None of the Schizomycetes under examination was found
eapable of producing an enzyme able (like pepsin) to dissolve fibrin in presence
of an acid. According to FERMI'S (HI.) results, the excretion of the proteolytic
enzyme occurs, as a rule, only when albumen is present in the nutrient medium.
Two only, of all the species examined by him, exhibited any variation in this
respect, viz., Micrococcus prodigiosus and B. pyocyaneus, which yielded a proteo-
lytic enzyme when cultivated in a mineral nutrient solution qualified with
glycerin or mannite.

It has long been known that antiseptics in small doses exert no injurious
influence on the action of enzymes. On this point some conclusive investigations
were published by FERMI and PERNOSSI (I.), and use is made of this property in
testing for the presence of a proteolytic enzyme in samples of liquids or bacterium
pultures, an easy metho4 proposed by FERMI (IV.) being employed, A so-

PROTEOLYTIC ENZYMES 231

called Thymol-gelatin is prepared in the following manner: — Water saturated
with thymol is qualified with 5—10 per cent, of purest gelatin, and after being
warmed on the water-bath is poured into test-tubes (10 c.c. in each). The
tubes are kept in a vertical position, and are ready for immediate use as soon as
the contents have set. The thymol present therein will prevent any develop-
ment of bacteria. A large stock of these tubes can be prepared, and the contents
preserved from desiccation by placing the (open) tubes, mouth downwards, in
a covered glass vessel containing a little distilled water. The liquid to be
examined is filtered to remove any solid particles. A few c.c. are then placed
in one of the thymol-gelatin tubes, and a little thymol is added to prevent the
development of any bacteria already present in the sample. The tube being
then left to stand at room temperature, the presence of any proteolytic enzyme
in the sample will be revealed in a few days by the liquefaction of an appreciable
stratum of the gelatin. To enable this change to be reliably ascertained a mark
is made on the tube at the time of filling, to denote the level of the gelatin.
The risk of the gelatin becoming dissolved by any large percentage of acid or
alkali present should be obviated by neutralising the sample before commencing
the experiment. Liquids containing substances such as tannin, glycerin, &c.,
capable of preventing or retarding the solution of the gelatin, are unsuitable
for use. This simple method may also be employed as an approximate
quantitative test for determining the relative strength of two solutions of a
proteolytic enzyme, since the amount of gelatin dissolved per unit of time under
identical conditions may be regarded as a measure of the concentration or
potency of the samples. If tubes of equal diameter are used, then this relation
is simply expressed by the height (thickness) of the two liquefied strata. Fermi
claims that his method is more reliable than those proposed (for the same
purpose) by Griinhagen, Griitzner, Briicke, and Schiitz, and which consist
chiefly in determining the amount of Jibrin dissolved by the sample under
certain definite conditions. As we have already mentioned that this latter
substance is attacked with greater difficulty than gelatin, it will be at once
evident that Fermi's method is the more delicate.

With regard to casease, i.e. the enzyme decomposing the casein of milk into
soluble products, the chief particulars have already been given in § 147. Many
bacterial species are, however, capable of dissolving this albuminoid without any
trace of casease being found in the cultures. One of these is the Bacterium
peptofaciens, isolated from milk by AL. BERNSTEIN (I.), which is particularly
active in converting casein into peptone and albumoses, a little (0.2 per cent.)
lactic acid being also formed. If, now, the milk be boiled after the bacterium
has been in action for a short time, the unconverted casein will be thrown down,
and, when filtered off, leaves behind a liquid which is rich in readily digestible
peptones, and has been named " galactone " by its inventor. The milk-sugar
present in this liquid may be fermented by the addition of suitable yeasts, and
then yields " galactone wine."

The bacteriological researches of the past few years have resulted in an
important modification of the opinions held regarding the so-called carnivorous
plants. According to earlier statements, the glands of the parts of the plant
acting as a snare secreted a dissolving albumen enzyme, which digested the
captured prey, i.e. converted its albuminoids into assimilable peptones, &c.
Hoppe-Seyler in 1876 threw doubts on the presence of this enzyme in Drosera
rotundifolia, and in 1889 N. TISCHUTKIN (I.) ascribed the phenomenon to
bacterial activity.

This observer ascertained that the juice collecting on the surface of the
leaves of Pinguicula is rendered inactive by painting the leaves over with
bactericidal media. The same conclusion was arrived at by R. DUBOIS (II.) in

232 THE PHENOMENA OF PUTREFACTION

1890, in his experiments on the contents of the urns of Nepenthes ; and two
years later the matter was again examined by TISCHUTKIN (II.) in the following
plants: — Drosera rolundifolia, L., D. Longifoli't, L., Dioncea muscipula, Ell.,
Nepenthes Mastersi, the results confirming the hypothesis expressed above, viz.,
that the digestion of the albuminoid bodies falling or introduced into the juice
excreted by these plants is exclusively due to the activity of bacteria settling in
the said liquid and there producing a proteolytic enzyme. According to an
analysis by Volker, the juice collecting in the cups of Nepenthes contains about
0.8-0.9 per cent, of dry matter, about 39 per cent, of which consists of malic
acid and 50 per cent, of potassium chloride, i.e. the two substances already
mentioned in § 41 as powerful bacterium stimulants. The juice in the unopened
young cups of Nepenthes contains neither proteolytic enzyme nor bacteria, the
latter falling out of the air into the liquid only after the cups are opened.
Ample opportunity is soon afforded for the exertion of their decomposing power
on the insects caught in these traps and prevented by special contrivances from
escaping. For the preparation of this nutrient material the organisms elaborate
enzymes, the proteolytic properties of which are utilised by the plant. These
so-called carnivorous plants consequently present a beautiful example of
symbiosis existing between higher plants and bacteria.

§ 171.— Ptomaines and Leucomaines.

The first step towards the elucidation of the regrettable fact that putrefying
albuminoids, when introduced into the blood-vessels of man or the higher
animals, set up violent reactions (sepsis, septiccemia), which may, under certain
circumstances, prove fatal, was made by P. L. PANUM (I.) in 1856, who proved
that putrescent albumen contains a poisonous fission product which cannot be
destroyed by boiling, treatment with alcohol, or similar methods, and is con-
sequently not an organised creature, but a chemical compound (known as
" extractive putrescent poison "). This discovery, which was tested and con-
firmed by M. HEMMER (I.) and F. SCHWENINGER (I.), is also of historical
importance in Pathological Bacteriology, since thenceforward medical views and
researches concerning the nature of the diseases engendered by bacteria pursued
two divergent paths : the one school holding these diseases to be toxic phenomena
produced by the poisonous metabolic products (toxins) of parasites growing
within the body, whilst the other regarded the vital activity of the organisms
themselves as the immediate cause of the malady. There is no occasion for us
to follow this conflict of opinions, which is still rife ; so we may confine our
attention to the efforts of Panum's successors in the narrower field of albuminoid
putrefaction. Among these E. BEIIGMANN (I.) and 0. SCHMIEDEBERG (I.) chiefly
deserve mention as being the first to obtain (1868) a poison of this group — by
precipitation as sulphate (the so-called sepsin sulphate) from putrescent beer-
yeast — in a crystalline form, and therefore available for closer chemical investi-
gation and characterisation. M. VON NENCKI (V.) was the first, in 1876, to
successfully prepare such a poison in the pure state, viz., the alkaloid collid'.ne
(isolated from putrid albumen), having the formula C8H,,N, and being (accord-
ing to its constitution) trimethyl pyridine, C5H,N. (CH3)3. Such alkaloids, are
also formed, as a matter of course, during the decomposition of the human
cadaver (Gr. ptoma), and on this account F. SELMI (II.) in 1878 gave the name
ptomaines to putrefaction alkaloids in general.

This newly discovered group was gradually enlarged, and now includes more
than fifty substances. Comparatively speaking, the majority of these new bodies
were discovered by L. BRIEGEH (III.), to whom we are also indebted for new
methods for the separation of these poisons from putrescent liquids. Of the

FPOMAINES AND LEUCOMAINES 233

ptomaines prepared by him, viz., choline, saprine (C.H16N2), putrescine (CUH12N2),
neuridine (C5H14N2), and cadaverine, peculiar interest attaches to the last-
named from its having been the first putrefaction alkaloid prepared by synthetic
methods. The first to accomplish this was Ladenburg, who determined its
formula as NH2.CH,— CH,— CH2— CH2— CH2.NH2,;.e., pentamethylene diamine.
Putrescine and cadaverine were detected by F. OBERMAYER and R. KERRY (I.) in
considerable quantities in the putrefaction of yeast. Choline (CH2.OH — CH2 —
N(CH3)3.OH) may be separated from lecithin, which forms an important con-
stituent of nerve and brain. By substituting hydroxyl for one of the hydrogen
atoms of the central CH, group, we obtain muscarine, CH,.OH — CH.OH — N.
(CH3)3.OH,' which O. SCHMIEDEBERG and E. HARNACK (I.) recognised as the
powerful poison of red agaric (Amanita muscaria), and to which must be ascribed
the intoxication resulting from the consumption of this fungus, or of the beverage
prepared therefrom, by the natives of Eastern Siberia. According to L. BRIEGER
(IV.) the same poison also results from the putrefaction of choline and certain
albuminoids, and it was also found in 1878 by Gantier in putrid fish. By
separating a hydrogen atom from the central CH, group in choline and the
hydroxyl adherent to the adjacent carbon, and combining these liberated equi-
valents to form water, we then have left behind neurine, CH2 = CH — N.(CH3)3.OH,
a vinyl derivative which may also be formed in the putrefaction of nerve tissue
and brain. According to the researches of P. JESERICH and F. NIEMANN (I.),
choline undergoes this conversion under the action of Bacteritmi coli commune.
Hydrocollidine, CsH,.lSr, is regularly produced during the putrefaction of the
flesh of horses and cattle, and is generally accompanied by the nearest homo-
logue of collidine, viz., parvoline, C9H13N. A more detailed characterisation of
these ptomaines must be omitted here, but the reader desiring instruction in this
particular will be able to obtain it from the concise monograph by F. JACQUEMART
(I.) Not every ptomaine is poisonous, — cadaverine, putrescine, and saprine
being devoid of this pix>perty.

The composition of tyrotoxicon, or cheese-poison, which was first discovered
by Y. VAUGHAN (1.), is still unknown, but from its chemical behaviour it appears
to consist principally of a diazo body (diazobenzene ?). It is formed (under con-
ditions still uninvestigated) in stored cheese by the action of bacteria, and when
eaten in such cheese produces symptoms of violent poisoning. A case of this
kind, in which fifty persons were simultaneously attacked, is recorded by Sen.
WALLACE (I.) The same poison is also occasionally formed in milk. Thus,
YAUGHAN (II.) reported an instance of eighteen persons being rendered ill by
eating vanilla ice, from which substance (chiefly composed of milk) crystals of
tyrotoxicon were obtained. L. DOKKUM (I.) extracted from a cheese recognised
as dangerous to health a ptomaine-like substance which he termed tyrotoxin,
but which is not identical with tyrotoxicon. In America such cases of cheese-
poisoning are more frequent than in Europe, Yaughan having enumerated
three hundred within two years.

It is not essentially necessary that the food should contain .ready-formed
ptomaines for symptoms of poisoning to appear. On the contrary, the ptomaines
may be formed in the body itself if the food contain bacteria capable of pro-
ducing them, and provided that the composition of the substances present in
the intestines is favourable at the moment. In such event the poisons are
called leucomaines, and most of the cases of so-called meat-poisoning are due
to this cause. Thus A. GARTNER (I.) reported a case wherein he succeeded in
identifying a fission fungus, Bacillus enteritidis, as the cause of the poison, and
the same microbe was discovered by J. KARLINSKI (II.) in a case of meat-
poisoning in Herzegovina, where sun-dried meat (" suche mieso ") is an ordinary
article of trade, and is frequently eaten raw by the natives. Many of the cases

234 THE PHENOMENA OF PUTREFACTION

of so-called fish-poisoning, i.e. illness produced by eating fish, also belong to this
category. On the other hand, these ill effects may also be brought about by
ptomaines produced during the storage of this (readily decomposable) food-stuff, a
remark which applies equally to the so-called sausage poisoning. Researches on
this point have been conducted by H. MAAS (I.). The poisonous decomposition
products developed by the activity of fission fungi in eggs, and also cases of
poisoning ensuing from the consumption of eggs so spoiled, have been investigated
by GLASMACHEE (I.), BONHOFF (II.), and GRIGORIEW (I.).

§ 172.— The Albuminous Poisons.

To attribute the poisonous effects of bacteria, in all cases, to the formation
of products of the ptomaine group would be incorrect. As a matter of fact,
the injury is frequently caused, not by these alkaloids at all, but by certain true
albuminoids, which, on account of their decomposing power, have been named
active albumen. We have to thank CHRISTMAS and HANKIN (I.) for the first
proof of this fact, though Pfliiger was cognisant of it as long ago as 1875. We
have already stated in § 82 that certain pathogenic fission fungi will develop on
nutrient media destitute of albumen and there elaborate poisons synthetically.

The fundamental differences between active albuminoids and ptomaines are
not confined to their production and composition, but extend also to their mode
of action : the former behaving like enzymes, and acting as a result of the
lability of their atoms, so that a small quantity of the active substance is able to
induce decomposition in a comparatively enormous mass of decomposable mate-
rial. On the other hand, the poisonous effects of the ptomaines depends on the
quantity coming into play, and increases therewith. As in the case of the
enzymes, the active albumen is completely deprived of its powers by moist heat
(100° C.), by which it is converted into non-poisonous passive albumen ; whereas
the ptomaines remain undecoinposed and undebilitated by the same treatment.
This fact is also of importance to the food-stuff chemist, since it will restrain him
from certifying a sample of suspected meat to be innocuous merely because a
negative result has been obtained with the current alkaloid reactions.

Many cases of meat poisoning are probably due to the presence and action
of active albumen. A fuller insight into this matter must first, however, be
gained by investigation. Thus we find it recorded by M. ARUSTAMOFF (I.) that
in the Lower Volga district the opinion 'prevails that only the consumption of
uncooked fish (salted sturgeon and salmon) is harmful. In view of the remarks
already made on the influence of heat on active albumen this observation becomes
intelligible. The danger resulting from the presence of living bacteria in in-
completely sterilised milk, and their developing in the intestines of the nursing
infant (see § 125), is probably in many cases due to active albumen formed by
the organisms. The author puts this interpretation on the results of the
experiments made by A. LUBBERT (I.) on this point.

As was first established by MITCHELL and REICHERT (I.) in 1886, it is to the
presence of such active albumen that the effects of snake-poison are due. More-
over, albuminous poisons are found in the normal blood of different animals, a
circumstance first established by A. Mosso (I.) in the case of Murcenidce, to which
family the common eel belongs. A list of fishes naturally containing poison has
been drawn up by J. POHL (I.) Poisonous albuminoids are likewise found in
various plants, e.g. abrin in the seeds of the paternoster pea (seeds of the wild
liquorice, Abrus precatorius), ricin in the seeds of fiicimis communic, and many
others.

The reaction between the animal body and bacteria is reciprocal. Just as
the latter are able to excrete noxious metabolic products, the effect of which on

LIBERATION OF NITROGEN, AND DE-NITRIFICATION 235

the infected animal body is manifested as disease, .so also the former can elaborate
substances having a poisonous effect on the parasitic micro-organisms. The
normal and continuous presence of such protective albuminoids, or alexines, as
they are called, in the blood, is the cause of the natural immunity enjoyed by
certain animals against certain pathogenic species .of bacteria. A closer con-
sideration of this matter would, however, be beyond the scope of the present
work, though it must be referred to, as throwing new light on the connection
between Bacteriology and Physiological Chemistry. Full information on the
subject of protective inoculation and serum therapeutics can be gathered from
the concise text-book prepared by HUEPPE (VI.), which at the same time pro-
vides an introduction to the study of Pathological Mycology. On this latter
subject P. BAUMGARTEN (I.) has written a reliable handbook which is hereby
recommended to food-stuff chemists and agriculturists.

§ 173.— The Liberation of Nitrogen, and De-nitrification.

The interest with which the farmer regards the decomposition of nitro-
genous substances, both in the manure heap and in the soil, always proceeds from
the same desire : to know what becomes of the nitrogen, and whether it is
retained in the soil.

The alterations suffered by nitrogenous manurial constituents derived from
urine will be described in chapters xxxii. and xxxvi., and at present we are
concerned merely with the putrefaction of the albuminoids, &c., evacuated in the
faeces.

In the first place, it must be remarked that the loss of nitrogen may occur,
not only as a result of its liberation in a free gaseous state, but also in conse-
quence of the volatilisation of ammonia produced by the action of micro-
organisms on the albuminoid matter of the manure. We are indebted to
E. MARCHAL (I.) for proving that the faculty of eliminating ammonia from
albuminoids is common to a great many fungi (both Schizomycetes and Eumycetes),
occurring in large numbers in the soil, and quite distinct from the Schizomycetes
effecting the conversion of urea. Among the fungi (widely distributed and
frequently discovered in the soil) examined and recognised by MARCHAL (II.) as
powerful ammonia- producers, may be mentioned in the Schizomycetes group : —
Bacillus mycoides, Fliigge ; B. fluorescens liquefaciens, FL; B . fluorescens putidus,
Fl. ; B. subtilis, B. arborescens, B. mesentericus vulgalus, Fl. ; B. mesentericus
ruber, Fl. ; B. janthinus, Zopf; Proteus vulgaris, H. ; Bacterium coli commune,
Sarcina lutea, Micrococcus roseus, Fl. ; M. flavus, Fl. ; M. candicans, Fl., &c. ;
and in the Eumycetes group : — Aspergilhis terricola, Penicillium glaucum, P.
cladosporioides, Mucor mucedo, M. racemosus, Botrytis cinerea, B. vtdgaris, Cephalo-
thecium roseuni, and others. The potency of the different species varies, the
largest quantity of ammonia (0.8 gram per litre of nutrient solution) being
produced by Bacillus mycoides. This last-named fission fungus, which was
minutely examined by Marchal, decomposes both albumen, leucine, and tyrosine,
but does not attack urea. The losses occasioned by the volatilisation of ammonia
produced in this manner may be very considerable, but will not be further
considered here. We will now turn to the liberation of uncombined nitrogen.

The first researches on this point were undertaken by JULES REISET (I.) in
1854 and 1855. He asserted that free nitrogen is always evolved during the
putrefaction of manure, whilst G. HUFNER (I.) arrived at the contrary opinion,
being unable to discover any liberation of free nitrogen when atmospheric air
or pure oxygen was led through the putrefying substances. The same result
was obtained by ALEXANDER EHRENBERG (I.), 0. KELLNER and T. YOSHII (I.), and
BR. TACKE (I.) ; and this view was also held by H. IMMENDORPF (II.) in 1893.

236 THE PHENOMENA OF PUTREFACTION

Although these discoveries may justify the conclusion that no free nitrogen
is disengaged during the putrefaction of albuminoids, it must not, however, be
assumed that the same also applies to the decomposition of manures in general
under natural conditions ; since, under these circumstances, very considerable
quantities of this element can be liberated and become lost to the soil. This
result is, however, due to the reduction of nitric salts, and not to the putrefaction
of albuminoids.

This de-nitrification in arable soil was first noticed by GOPPELSRODER (I.) in
1862, and was long regarded as a purely chemical process. The first reference
to the agency of bacteria in this decomposition was made by E. MEUSEL (I.) in
1875, and the earliest pure cultures of such organisms were obtained by
U. GAYON and G. DUPETIT (II.) in 1882. In succeeding years a large number
of species, all capable of reducing nitrates, was made known ; e.g. by W.
HERAEUS (I.) in 1886. Two years later P. FRANKLAND (II.) was able to
associate with the group in question 17 out of 32 species, and R. WAKINGTON (I.)
1 6 out of 25 species examined, among them being Bacillus ramosus, the so-called
" Wurzel-bacillus." All these reduce nitrates into nitrites, but these two
chemists do not say whether the latter substances in turn may be still further
reduced by the bacteria. For this reason we must revert to the labours of
GAYON and DUPETIT (III.), who made pure cultures of two bacterial species,
named Bacillus denitrificans a and /3, which exhibit a noteworthy difference in
their behaviour towards nitrates. Species a is the more energetic, decomposing
as much nitrate as is presented to its action, and reducing the same to nitric
oxide and free nitrogen. The /3 species, on the other hand, forms nitrites, and
ceases to act before the whole of the nitrate is destroyed, free nitrogen being the
only gaseous fermentation product. Quite distinct from these two species is the
Bacillus denitrificans, isolated from arable soil by E. GILTAY and J. H. ABERSON
(I.), which reduces the nitrates to free nitrogen in an almost quantitative degree.
When grown on nutrient gelatin the rods measure 0.5 p in breadth and 1.5-3 /*
in length, but in liquids they assume a somewhat more elongated form. Closely
allied to these three species is the Bacillus denitrificans II., discovered by R.
BURRI and A. STUTZER (II.) on old straw, but differing from them in that it
liberates as gas only some 80 per cent, of the nitrogen in the decomposed
nitrates, the remainder being elaborated into an organic compound (still
uninvestigated), which is precipitated in large flakes. The same observation
was made (though not with pure cultures) by E. BREAL (I.) in 1892. Like the
aforesaid three Schizomycetes, Bacillus denitrificans II. is anaerobic, and decom-
poses nitrates only when oxygen is excluded. Another (sporogenic) de-nitrifying
bacillus, isolated by J. SCHIROKIKH (I.) from horse-dung, may also be
mentioned.

The facultatively anaerobic Bacterium coli commune exhibits a peculiarity
worthy of special consideration. When kept in a nutrient solution by itself and
with exclusion of air, it reduces nitrates to the condition of nitrites ; but the
decomposition proceeds in quite a different manner when the organism is grown
in symbiosis with a second species of bacterium, invariably found in horse-dung
by both the above-named workers, and named Bacillus denilrificans I. In such
case, even when air is admitted, the nitrogen of the nitrate is set at liberty,
though neither species is able to produce the same effect by itself. Bacterium
coli commune can, however, be replaced by Bacillus typhi abdominalis. The
potassium or sodium present in the nitrates or nitrites is converted into a
hydroxide, which accumulates in the medium, and eventually arrests the vital
activity of the bacteria in question. For this reason not more than 5 or 6 grams
of saltpetre (potassium nitrate) can be fermented per litre. The fact that Bac-
terium coli commune in the absence of air (e.g. in the intestines) converts nitrates

LIBERATION OF NITROGEN, AND DE-NITRIFICATION 237

into the exceedingly poisonous nitrites is also of interest to Pathological
Mycology, but we cannot further discuss the matter here. The important point,
so far as we are now concerned, is, that the disengagement of free nitrogen from
nitric salts can go on even in the presence of air. The de-nitrification occurring
in stored manure and in arable soil appears to be a twofold process : the
anaerobic nitrate destroyers acting in the lower strata away from the air, whilst
the symbiotic activity of the Bactarium coli commune (so plentiful in animal
excreta) and the Bacillus denitrificans I. comes into play at the surface. From
this it is evident that the theory which assumes the possibility of preventing the
destruction of nitrates by thoroughly loosening, and consequently aerating the
soil, is of little value. — The bacteria in question are (for some unexplained
reason) present in enormous numbers in the excrement of various animals.
First in this respect is horse-dung, which has always been regarded by practical
men as a hot manure, a property which is explained by the foregoing observa-
tions. Consequently such manure should not be applied, especially when fresh,
to soil that has recently received a dressing of nitrate of soda ; otherwise a
serious loss of nitrogen will result. This injurious action is, however, not limited
merely to such fields as have been artificially manured with nitrate, since (as we
shall see in chapter xxxvi.) the ammonia salts in the soil are, under favourable
conditions, oxidised into nitrates by the activity of a special group of bacteria,
such nitrates then forming a welcome food for the organisms dealt with in the
present paragraph. That it is a question of more than insignificant quantities
will be evident from the discovery reported by PAUL WAGNER (I.) — a discovery
which led to the aforesaid researches of Burri and Stutzer — viz., that out of 100
parts by weight of nitrogen applied in the form of stall-manure to the soil, only
25 parts are, on an average, recovered in the crop, whilst the remaining 75 parts
are entirely lost. These figures do not fully represent the extent of the loss
occasioned in the soil and manures by the activity of the de-nitrifying bacteria,
and there still remains another phenomenon for consideration. We must recall
that the fission fungus known as Bacillus denitrificans (and probably also a number
of allied species not hitherto investigated) separates nitric oxide as well as nitro-
gen from nitrates. This oxide then escapes into the outer layers of the manure
heap or soil, where it is brought into contact with oxygen, and combines there-
with to form nitrogen trioxide —

2NO + 0 = N203.

This latter then reacts on the ammonia and ammonia derivatives (urea, &c.)
in the soil, in such a manner as to liberate both the nitrogen of the trioxide and
that of the ammonia as well —

N203 + 2NH3 = 2N2 + 3H20.

Consequently the nitrogen compounds insusceptible to the direct action of the
microbes in question are also included in the wasteful reaction set up. It was
on this account that the production of ammonia during the decomposition of
manure was casually referred to at the commencement of this paragraph. The
present is a fitting opportunity for referring to the statements of several workers
— e.g. H. B. GIBSON (I.) — who, like Reiset, thought they had observed a libera-
tion of nitrogen in their researches on putrefaction. Their results were all
obtained by the use of complex bacterial mixtures, and therefore cannot be
considered as reliable. In this case, also, those experiments alone are decisive in
which pure cultures have been employed.

By the activity of these bacteria an enormous quantity of combined nitrogen
is daily set at liberty in the soil. To replace this loss, and to restore the con-

238 - THE PHENOMENA OF PUTREFACTION

tinuity of the nitrogen cycle, is the task of a separate group of bacteria, which
will be dealt with in chapter xxxiii.

The reduction of nitric acid by bacteria does not always stop short at the
liberation of free nitrogen, but in many instances extends to the formation of
ammonia. Several investigations on this point were made by 0. LOEW (III.)>
but, unfortunately, not with pure cultures. He found that " ordinary putre-
factive bacteria," grown in a solution of i per cent, of peptone, 0.2 per cent, of
KN03, and 0.2 per cent, of K2HPO4, cause the potash and carbon dioxide to
combine, whereas the nitrogen of the nitric acid is converted into ammonium
carbonate. When 0.2 per cent, of ethyl alcohol is also present (in anaerobic
cultures) the acetate is formed instead of the carbonate.

What has already been detailed will explain the so-called nitric fermentation
of molasses. The cell sap of the sugar-beet contains a quantity — generally
small, but occasionally larger — of nitrates, principally potassium nitrate. This
is not separated during the saturation process, but remains in the mass in un-
diminished quantity, a portion crystallising out, and being then found in the raw
sugar from the centrifugal machine, whilst the rest remains in the mother liquor,
i.e. the separated syrup. If this syrup is then boiled up for the manufacture of
second product, and again passed through the centrifugal machine, the proportion
of nitrates in the mass will be still larger, — Pellet having found 1.9 per cent, of
KN03 in one sample examined by him. At this stage the molasses has a faintly
alkaline reaction, and is rich in organic and inorganic nutrient substances of
various kinds. Hence it is no wonder if bacteria rapidly develop therein.
Under special conditions the upper hand is gained by such organisms as reduce
potassium nitrate and eject its nitrogen in the form of NO, which compound, on
coming in contact with air, is oxidised into the dioxide N02. The latter hangs
as a dense red-brown vapour over the surface of the molasses, and the sugar-
maker then says his molasses is in a state of nitric fermentation. This pheno-
menon is of less frequent occurrence in the "reserves" in the sugar-factories
than in the dilute molasses of the molasses distilleries. Certainly, the activity
of these reducing bacteria can be arrested by souring, but this treatment liberates
organic acids inimical to the yeast. Bearing this in mind, Czeczetka proposed to
remedy the evil by boiling the molasses directly the malady is observed.
According to a report by DUBRUNFAUT (I.) in 1868, nitric fermentation was first
noticed by Tilloy at his distillery in Dijon, and was successfully suppressed by
him by boiling the molasses along with sulphuric acid. An explanation (charac-
teristic of the state of knowledge in the domain of Fermentation Physiology at
that time) of the favourable influence of this treatment was made in the same
year by J. REISET (II.), who stated that the NO or N02 formed during the so-
called nitric fermentation proceeds from the oxidation of ammonia in the
molasses, this being attacked only when present in combination with a weak acid,
whilst when in the form of sulphate it resists the action of oxygen ; consequently
the molasses treated in the manner adopted by Tilloy was exempt from this
decomposition. This view was left uncontradicted by BKCHAMP (III.), although
he had already ascribed de-nitrification to the agency of micro-organisms. A
closer investigation (embodying modern methods of working) of this nitric
fermentation of molasses is highly desirable. To be thoroughly satisfactory,
such research must trace the course followed by the potassium nitrate in the
juices of the sugar- works, and more narrowly examine the quantitative dependence
of the nitrate in molasses on the method of preparation employed, very little
being as yet known on these points.

The nitric decomposition in question is also of frequent occurrence in the
fermentation of tobacco in heaps. SCHLOSINO (III.) reported in 1868 on the
first observation of this phenomenon by Ch. Ray.

THE LOSS OF COLOUR IN WINE 239

§ 174.— The Loss of Colour (Umschlagen, Brechen) in Wine

was first examined chemically by G. MULDER (II.) in 1855. Of this complaint,
which is known in France as vin toume, and in Italy as vino girato, he gives the
following explanation : — " This alteration of wine consists in a decomposition
of the tartaric acid, but how this decomposition is induced is unknown. The
cream of tartar is converted into potassium carbonate, whereby the colour of red
wine is altered and becomes brown. The decomposition begins at the bottom of
the cask, and is hence undoubtedly a result of the decomposition of the organic
matter of wine-yeast, which contains a substance acting destructively on the
tartaric acid, and, in co-operation with air, oxidising it to carbon dioxide and
water. As the malady progresses, the alcohol is converted into acetic acid, and
a putrefactive fermentation ensues." The commencement of this malady, which
appears more frequently in red wines than in white ones, manifests itself by a
slight evolution of carbon dioxide, which preliminary symptom is known in
practice as " boiling away " (versieden). Tartaric acid is not the only substance
eliminated, glycerin also — according to the researches of P. CARLES (I.)— being
slowly decomposed. Simultaneously, the amount of volatile acids increases to an
unusual extent (up to 4 grams per litre), a fact observed by SCHULTZ (I.), and
afterwards confirmed by J. MACAGNO (I.).

Ten years after Mulder's observations, PASTEUR (XII.) undertook the task of
discovering the cause of this malady and proving that here also the activity of a
still unknown micro-organism was in question. He showed that in wines
affected with this complaint bacteria are always detectable in large numbers,
their length being 3-5 p., with a breadth of 1-1.5 /*• Greater probability was
imparted to this assumption by the observation made by SCHULTZ (I.), who, in
1877, succeeded in artificially imparting the malady to sound wine by inoculating
it with a small portion of a wine already infected. A closer study of the
organism could not at that time be made, owing to the lack of methods of pure
culture, a defect that, in this connection, was first overcome by E. KRAMER (I.)
with the organisms from a number of samples of Styrian and Croatian wines
affected with loss of colour. This malady, as is well known, is exceedingly
prevalent in southern countries, and causes great loss to the agricultural interest
every year. Kramer examined nine various species, all aerobic and liquefying
gelatin. The first seven of them he named Bacillus saprogenes vini I.-VII., and
the other two Jlficrococcus saprogenes vini I. and II. Details and experiments
to prove whether these species are capable of producing loss of colour in sound
wines are still wanting, and consequently the Schizomycetes in question possess a
merely morphological interest. The actively motile Bacillus saprogenes vini /,,
which is found in nearly every sample examined, is probably identical with
Pasteur's " Bacillus du vin tourne." It attains a breadth of i p and a length of
2.5-6 fi ; and bands composed of two or three cells are not. rare. Bacillus sapr.
v. III. and VI. form endospores, and the cells of Micrococcus saprogenes vini II.
have a diameter of 1-1.4 /*• A pure culture of a bacillus, which, however, was
recognised as innocuous, was obtained, from Italian wine suffering from loss of
colour, by J. GALEAZZI (I.) in 1894.

These remarks sum up all that has hitherto been discovered by fermentation
physiologists respecting the loss of colour in wines. Consequently, knowledge of
the subject is still only in a very early stage, and we can only hope that future
researches will succeed in affording us f urthe^enlightenment. This wine malady
is so diversified in its mode of development and so changeable in its course, that
we are obliged to ascribe it to a very fine example of metabiosis, i.e. that a single
bacterial species is insufficent to occasion the complaint, the successive action of

240 THE PHENOMENA OF PUTREFACTION

a number of species being essential. In fact, the number of decomposable
constituents in unaltered sound wine is so great as to preclude the possibility of
a single species effecting all the changes involved. Consequently, investigations
on this point will need to be carried out to a somewhat comprehensive scale.
Several purely chemical researches into the changes produced were maJe by
J. Konig, and abstracts of them are given in BABO and MACK'S (I.) " Handbuch
des Weinbaues " (Handbook of Viticulture). Similar researches should now be
made with pure cultures of bacteria isolated from wines that have lost their
colour, and such researches should also include the examination of the changes
produced by the different species of these organisms, in each of the most
important constituents of wine.

This malady is also known as the putrefactive fermentation or " decaying "
of wine, from the final condition attained by the liquid. Wines rich in albumen,
e.g. even the Hungarian red wines, according to M. PREYSS (I.), are found to
have a special tendency to loss of colour. In order to understand why southern
wines are so prone to this malady, it is necessary to recall the fact — already
mentioned in previous chapters, and first quantitatively investigated by
N". SIEBER (I.) — that putrefaction does not ensue in strongly acid liquids,
whereas these wines are poor in acid. FONSECA and CHIAROMONTE (I.) re-
commended the addition of citric acid to increase their power of resisting the
complaint. The destruction of the acids of wine must therefore precede its final
putrefactive fermentation; hence the primary object of research must be the
discovery of the changes produced in these acids. Here, again, everything still
remains to be done, since all the information at present available is derived
almost exclusively from experiments in which pure cultures were not employed.

According to the discoveries of PASTEUR (IX.) and A. FITZ (IV.), tartaric
acid (in the form of its calcium salt) can be decomposed by bacterial agency in
three ways : viz., either into propionic acid (along with a little acetic acid) ; to
butyric acid ; or, finally, to acetic acid, small quantities of ethyl alcohol, succinic
acid, and butyric acid being also produced. Malic acid also may yield very
different fermentation products, among which BECHAMP (IV.) mentions acetic
acid, propionic acid, butyric acid, carbon dioxide, and hydrogen. According to
the researches of A. FITZ (IV.), malic acid (combined with lime) may be split up
by different species of ferments in three different ways. In the first case,
succinic acid, acetic acid, and carbon dioxide are formed, the relative proportions
being approximately represented by the equation

3COOH— CH2— CH.OH— COOH = 2COOH— CH2— CH2— COOH +
CH3— COOH + 2C02 + HaO.

In a second case, propionic acid, acetic acid, and carbon dioxide may be found ;
or, thirdly, butyric acid may be the chief product, along with a small quantity of
carbon dioxide. With regard to succinic acid, BECHAMP (V.) asserts that this
also may be split up (by a bacterial mixture not more precisely specified) into
propionic acid and carbon dioxide, the following equation —

COOH— CH2— CH2— COOH = CH3— CILj— COOH + C02.

approximately expressing the reaction. The succeeding homologue of this acid,
viz., pyrotartaric acid, breaks up, under similar conditions, into carbon dioxide
and methane, according to the equation —

2COOH— CHjj— CH.CHg— COOH + aH20 = sCH4 + sC02.

According to the researches of Fitz, citric acid is converted, by an unspecified
bacterial mixture, into acetic acid and small quantities of ethyl alcohol and

THE LOSS OF COLOUR IN WINE 241

succinic acid. A series of experiments on the behaviour of fifty-two species of
bacteria towards twenty-one different organic acids was performed by A.
MAASSEN (I.), principally on medical grounds. One result of this research was
the discovery of a new characteristic — valuable in the bacteriological analysis of
water — for the differentiation of Bacillus typhi abdominalis from Bacterium coli
commune— viz., tricarballylic acid, COOH— CH2—CH.COOH—CH2— COOH,
which is attacked and partly destroyed by the first-named organism, but is left
altogether untouched by the second.

More minute investigations into the fermentation of the above-named organic
acids would be of value, not only in solving the preliminary questions involved in
the study of loss of colour in wine, but also in connection with the decrease in
the acidity of wines and fruit wines during storage, a phenomenon well known
in practice and one quantitatively examined by PAUL BEHREND (I.) and by
P. KULISCH (I.). This decrease — so long as it remains within narrow limits —
is looked upon with favour, as contributing to the rounding and improvement of
the flavour of the maturing wine. If, however, it proceeds too far and the
acidity falls too low, then a proportionate decrease in the power of the beverage
to withstand disease (especially loss of colour) ensues. This fermentation of the
acids is, as already stated, principally effected by fission fungi, on which point a
few particulars have been given by MULLER-THURGAU (V.). To a small extent
these acids are consumed by the yeast in the primary fermentation, so that the
quantity present in the young wine is less than in the must. Consequently, if
the total acidity in the former is found greater than that of the fresh grape-juice
and fruit-must, the excess is due to the carbonic acid held in solution.

Grapes from vines infested with mildew, whereby both development and
sugar formation are retarded, yield wine poor in alcohol and consequently of low
resisting power. Such wine frequently becomes diseased, and is then known in
France as vin mildiouse. Here again bacterial agency is at work, the rod-shaped
organisms forming many-jointed chains and reproducing so abundantly, that
they finally accumulate as a thick sediment. U. GAYON (II.) regards this malady
as identical with that causing the loss of colour, because he identified in vins
mildiouses the same volatile acids (acetic and propionic acids) as have been
discovered by others, e.g. E. DUCLAUX (XII.), in vins tournes.

The mannitic fermentation of wine, which presents a certain oppositeness of
character to the malady known as loss of colour, will be described now, because
otherwise no suitable occasion would arise. This complaint does not wait to
attack the finished wine, but even makes its appearance at the stage of primary
fermentation. If the surrounding temperature keeps above 30° 0., then
alcoholic fermentation is confined within narrow limits, and an opportunity is
thus afforded for the development of certain species of bacteria which convert
the sugar of the must into mannite. Of this hexatomic alcohol there will be
produced, according to circumstances, from i to 30 grams per litre of wine, in
addition to a little acetic acid. A knowledge of this fact is useful to the analytical
chemist as well. Attention was first drawn to the mannite content, of Algerian
wines in particular, by P. CARLES (II.) in 1891. Figs, as is well known, very
often contain considerable quantities of this alcohol ; hence Carles thought that
the presence of mannite in any wine indicated adulteration by fig-wine. How-
ever, as reported by J. BEHRENS (IV.)> the presence of mannite in reliably pure
natural wines (e.g. Bordeaux, Chateau- Yqu6m) had been proved a year previously
by Portes and Lafauric ; and very soon afterwards JEGON (I.) showed that in
wines of reliable purity, but imperfectly fermented, as much as eight grams of
mannite could be found per litre. L. Roos (I.) then proved that this result was
due to bacterial activity, a discovery confirmed by U. GAYON and E. DUBOURG (I.),
who isolated from such wine a pure culture of a non-motile short-rod fission

i Q

242 THE PHENOMENA OF PUTREFACTION

fungus, capable of converting sugar into mannite (up to 50 grams per litre).
In nutrient solutions devoid of sugar this species fails to develop, a circumstance
sufficient to distinguish it from the bacteria (presumably) causing the loss of
colour of wine. Moreover, these latter — as already observed by Mulder — attack
cream of tartar first of all, whilst the bacteria of mannitic fermentation leave
this salt completely untouched. The fact, now firmly established, that a high
temperature (36° 0. or over) favours the appearance of the last-named microbes,
explains the defective fermentation (familiar to Sicilian and Algerian wine-
growers) of wine must during the prevalence of the hot south -wine (sirocco or
simoom), the red wines, in particular, being greatly affected. According to G.
BASILE (I.), this wine disease is as frequent in Sicily as it is dreaded, and in some
years affects the greater part of the vintage.

The bacteria here coming into action can be destroyed by heating up to 60° C.,
a treatment impossible to apply hitherto on account of its fatal effect on yeast-
cells. However, by artificially inoculating (pitching) with strong, pure yeast, and
by cooling the mash down to 15°— 20° C., the liquid could be rapidly brought into a
state of alcoholic fermentation, which could be controlled by suitably regulating the
temperature. In this way the desired result would be ensured, and would amply
repay the increased outlay required. In this connection the experience gained by
M. RIETSCH and M. HERSELIN (I.) should also be borne in mind, viz., that the
iujurious influence of an excessively high temperature (36° 0.) can be reduced by
aerating the fermenting liquid.

In conclusion, it will be useful to remember that mannite is also formed
during the mucinous fermentation of sugar, and that this hexavalent alcohol is
also excreted as a metabolic product by certain E<umycetes, e.g. Penicillium
ylaucum.

CHAPTER XXXI.

THE FERMENTATION OF CHEESE, AND ALLIED DECOMPOSITIONS.

§ 175.— The Composition of Ripe Cheese.

THE conversion of the fresh curd into finished cheese is termed ripening. We
will, in the first place, consider this process from the purely chemical side. It
was explained in § 144 that fresh curd can be obtained from milk in two
different ways, either by precipitating with acids or by setting with rennet. In
both cases nitrogenous compounds are present in the coagulum, only in the acid
curd ("Quark") they consist of casein, and in the rennet curd (" Bruch ") of
paracasein. The cheeses obtained from the acid coagulum are, with the sole
exception of " Glarner Schabziger," of inferior quality, and only suitable for
early consumption. These have hitherto received but little attention from
fermentation physiologists ; consequently the following particulars are restricted
to cheeses obtained from rennet coagulum, which to emphasise the point once
more — contain only one nitrogenous compound, namely, paracasein.

In contrast to this uniformity stands the variety of the nitrogenous compounds
present in ripe cheese. The first observation on this point was made in 1818 by
J. L. PROUST (I.), who isolated leucine from ripe cheese. Some sixty years later
(1880) N. SIEBER (II.) detected the presence of tyrosine in Roquefort cheese.
Nevertheless, these discoveries, as also those of two French workers who will be
mentioned later, were of a casual nature, the first thorough attempt to follow the
ripening process in a quantitatively analytical manner being made in 1882 by
A. WEIDMANN (I.), whose results, especially as regards the qualitative composition
of the cheese, were admirably supplemented by a later (1888) research under-
taken by B. ROSE and E. SCHULZE (I.). These investigations were made in
Switzerland on Emmenthal cheese, in which the above-named chemists discovered
a considerable amount of leucine. On the other hand, comparatively little tyrosine
was found, and other amido-compounds, as well as bodies of the xanthine group,
were altogether lacking. The presence of ammonia, however, was readily proved,
different samples being found to contain from 0.16-0.44 Per cent., calculated to the
dry weight. The amount of nitrogen found in the form of ammonia, amido-acids,
and other compounds distinct from albumen and peptone, in three kinds of this
cheese, ranged between 1.22 and 1.48 per cent. ; i.e. about one-fifth of the total
quantity of this element present. Among the albuminoid constituents special
mention must be made of caseo-glutin, a body allied to the peptones, and one that
must be considered as among the chief products of the ripening process, since it
constitutes 20 per cent., and even more, of the total dry matter. In addition,
there was found (along with a small quantity of peptones) another albuminoid
body, recognised as paracasein. To the list of the constituents of ripe Emmenthal
cheese still another unit phenylamidopropionic acid, was added in 1887 by the
labours of F. BENECKE and E. SCHULZE (I.)

Several other kinds of cheese were included in the scope of these investigations.
In harmony with the similarity existing between the methods employed in their
preparation the qualitative composition of Spalen cheese was found to resemble
that of the Emmenthal product. Peptone was also detected in Gruyere, Vacherin,

243

244 THE FERMENTATION OF CHEESE

and Bellalay cheese, as also in Schabzig cheese ; the latter, however, differs from the
other kinds just named by not containing any ponderable quantity of caseo-glutin.

The comparative examination of the constitution of hard and soft cheeses was
undertaken by BONDZYNSKI (I.)

If cheese be allowed to become over-ripe, then the percentage of albuminoids
falls oft' still more. Thus, A. MAGGIORA (II.) found in a sample of over-ripe
Stracchino (Gorgonzola) cheese only one-seventh of the initial nitrogen in the form
of protein, the remaining six-sevenths being in the form of amido- and ammonia-
compounds.

We will now briefly consider the amount of fatty-matter in cheese. When
whole-milk is set for cheese, the whole of the fat passes into the coagulum, which
then contains almost as much fatty matter as albuminoids, the former constitu-
ting about 45 per cent, of the total dry matter of the curd. So far as the
observations hitherto made extend, it would appear that the fat suffers no great
alteration, whether of quality or quantity, during the ripening process. Bacterio-
logically exact investigations are, however, still wanting Reference has already
been made in § 120 to the influence exerted on the fat by light and air, and this
influence also makes itself felt during the ripening of the cheese. The saponifica-
tion of the glycerides mentioned in the said paragraph occurs in cheese to a still
greater extent than in butter. The course of this operation has been traced by
E. DUCLAUX (VI.), who found that, in one instance, about one-third of the
glycerin butyrate originally present was broken up into its two components.

The conversion of albumen into fat, the development of which question has
been reviewed by S. SOSKIN (I.) in a prize essay, is not only of the greatest
importance in the study of chemical alterations in the animal body, but also
comes under consideration in the ripening of cheese. BLONDEAU (I.), in 1864,
was the first to remark, in his researchs on Roquefort cheese, that in this process
fat was produced at the expense of albumen. The same has also lately ibeen
asserted by H. JACOBSTHAL (I.), but was denied by the majority of subsequent
workers, e.g. inter alia, BRASSIER (I.) in 1865, IS". SIEBER (II.) and 0. KELLNER (I.)
in 1880. NAGELI and 0. LOEW (I.), however, proved beyond doubt, in the case of
certain Eumycetes, that lower fungi are able to convert albumen into fat. The
above-cited researches of Weidmann show that no remarkable quantitative increase
of fat occurs in the ripening of cheese, but this does not disprove the possibility
of the formation of fat from albumen during the process. On the other hand,
G. Musso and A. Menozzi, on the basis of their researches on Stracchino cheese,
believe that such a formation of fat must be assumed to occur.

The probability of such a conversion of albumen into fat cannot be rejected if
we recall another process — very different, it is true, from an festhetic standpoint,
but, nevertheless, very similar from a chemical and bacteriological point of view
— namely, the formation of adipocere. Fatty concretions, which in many cases can
only have originated in albuminoids (muscular substance, &c.), are frequently
found in bodies which have undergone decomposition in-the grave. This question,
which chiefly concerns the medical profession, we need not dwell upon. Various
proofs will be found in a treatise on this subject by ERMANN (I.).

§ 176.— E. Duclaux' Studies on Cantal Cheese.

As ripenening progresses, the amount of the aforesaid amido-compounds
continually increases, whilst the paracasein concurrently decreases. This trans-
formation may be due to two causes: one entirely chemical, the other physiological.

The earliest worker who believed the ripening of cheese to be due to microbial
activity was FEED. COHN (II.), who, in 1875 — from tne researches by which he
controverted the hypotheses of Bastian on spontaneous generation — arrived at

BACTERIAL FLORA OF RIPENING CHEESE 245

the following conclusion : — " The ripening of cheese I hold to be a true fermen-
tation." This fermentation he ascribed to the organisms ("lab-bacilli") present
in the rennet liquid, and which he associated with the bacteria then grouped
under the name Bacillus suUilis, a general term not to be confounded with that
at present applied to an entirely distinct species. Cohn's decision was based
exclusively on the microscopical examination of rennet and cheese ; and the same
applies also to the statements of F. BENECKE (I.)

It was not until 1878, however, that attempts were made to obtain pure cultures
of the presumptive cause of the ripening of cheese, and to test the influence of
the organism. This was effected by E. DUCLAUX (VII. and XIII.) in his studies on
Cantal cheese, from which he isolated ten species of Schizomycetes and classified
these under the common generic name of Tyrothrix belonging to the large sub-
group of the so-called hay and potato bacilli. Out of these ten species, one,
T. virgula, being unable to grow in milk, will be omitted from further considera-
tion. Each of the remaining nine produces two classes of enzymes, one resembling
lab and coagulating milk, whilst the other, casease (§ 147), dissolves and splits up
the albumin thus precipitated. This proteolytic enzyme can be thrown down from
the bacterial cultures by means of alcohol. A particularly abundant yield is
obtained from Tyrothrix tenuis, an actively motile, sporogenic rod about 0.6 p.
broad and 3 p. long, and often growing in the form of filaments ; hence the name
Tyrothrix (= cheese-thread, cheese-hair). This species is aerobic, as are also
T. Jiliformis, T. distortus, T. geniculatus, T. turgidus, and T. scaber ; whilst T.
urocephalum, T. claviformis, and T. catenula are, on the other hand, anaerobic.
Cultures of Tyrothrix tennis obtained from Duclaux' laboratory were investigated
in 1895 by W. WINKLER (I.), who formed the opinion that this species can be
modified, by cultivation, into three varieties or races. This was, however,
contradicted by J. WITTLIN (I.) in 1896.

The metabolic products, e.g. leucine, tyrosine, and the ammonia salts of
acetic, valeric, and carbonic acids yielded by the Tyrothrix species are the
identical substances we have seen to be produced in the ripening of cheese.
This concordance necessarily strengthens Cohn's hypothesis, that the ripening of
cheese is effected by the vital activity of micro-organisms. On this point, how-
ever, Duclaux was unable to afford any certain proof. Later workers attempted
to .arrive by various ways at a solution of this highly important matter, and
mostly by endeavouring to ascertain whether the ripening of cheese could be
accomplished in the absence of any fermentative organisms. F. SCHAFFER and
ST. BONDZYNSKI (I.) showed that curd prepared from boiled milk does not ripen ;
and, according to FREUDENREICH (III.), the same applies equally to Pasteurised
milk. Moreover, L. ADAMETZ (VI.) found that neither does ripening occur
when bactericidal substances, such as thymol or creoline, are added to the fresh
curd; and the same result was attained by L. PAMMEL (I.) by the use of
hydrogen peroxide.

§ 177.— Changes in the Bacterial Flora of Ripening Cheese.

L. Adametz also attempted to ascertain the active cause of the ripening
process in a new way, namely, by tracing the quantitative and qualitative
alterations occurring in the bacterial content of the ripening curd. These
researches — made on a Sornthal (Switzerland) soft " household " cheese, in
addition to Emmenthal cheese — led to the following results: —

(i) The freshly precipitated curd, moulded in the press and freed from
excess of whey, contains between 90,000 and 140,000 bacteria per i gram, a
comparatively large number of these being able to liquefy gelatin, and conse-
quently excreting a peptic ferment.

246 THE FERMENTATION OF CHEESE

(2) During the period of ripening, the germ content gradually rose to
850,000 in Emmenthal cheese and up to 5,600,000 in the "household" cheese,
but only a small share in this increase fell to the liquefactive species ; since
whilst the quantitative ratio of these to the other (non-liquefactive) kinds was
in the fresh curd i : 40, only one colony of liquefactive bacteria was found in
150 to 1 80 in the gelatin plate cultures prepared from the ripe cheese.

The expectation of finding the liquefactive bacteria assume the upper hand
during the ripening process was thus dispelled, and the same result was attained
in a later research published by E. VON FREUDENEEICH (IV.) in 1894, according to
whom the number of lactic acid bacteria in cheese increases with the age of the
latter. Hence the chief, if not the sole, share in the ripening of Emmenthal
cheese must be ascribed to these lactic ferments. E. J. LLOYD (I.) also came to
the same opinion in his researches on the ripening of Cheddar cheese.

The reason why Duclaux, in his earlier investigations, arrived at a contrary
result will be readily understood when it is remembered that he prepared his
pure cultures exclusively by the dilution method, and therefore by the aid of
liquid nutrient media (bouillon in particular) ; since in this last-named liquid the
organisms of the genus Tyrothrix thrive exceedingly, whereas the lactic acid
bacteria grow badly, if at all, therein. Consequently mixed sowings in this
medium result in a preponderance of the liquefactive species described by
Duclaux. From the discovery made by FREUDENREICH and SCHAFFER (I.) that
the ripening of hard Swiss cheese also goes on uniformly throughout the mass
when air is excluded, it follows that the said lactic acid bacteria are (facultatively)
anaerobic.

The harmonious results of the researches of M. LANG and FREUDENREICH (I.)
with Swiss, and of E. MARCIIAL (III.) with Belgian (Limburg), samples show
that a principal part in the ripening of soft cheese is taken by O'idium lactis,
further particulars of which member of the Eumycetes group will be found in the
second volume. Various budding fungi also seem to aid in the process, but more
detailed information on this point is absent. According to the statements of
JUL. HENRICI (I.), Swiss cheeses are poor in yeast-like fungi and rich in bacteria ;
but the converse ratio has been shown to exist in American cheeses.

Odour is one of the chai-acteristics peculiar to individual kinds of cheese ; it
is but slightly developed in many, but is prominent in others. In isolated
instances it is produced by the mechanical incorporation of added flavouring
substances to the fresh cheese mass. This applies, for instance, to the already
mentioned " Glarner Schabziger " or herb-cheese (Krauter Kase), which owes its
characteristic aroma to the addition of dried Melilotus cterulea (blue or Swiss
melilot). The English " sage-cheese " and American " clover-cheese " may also
be mentioned as examples.

In many cases the odorous principle is, on the other hand, produced
spontaneously in ripening, i.e. by the activity of micro-organisms, of which
nothing is as yet difinitely known. L. PAMMEL (II.) discovered on cabbnge
leaves a Bacillus aromaticus which, when inoculated into fresh curd, produces
during the ripening process an aroma similar to that of "clover-cheese."

§ 178.— Pure Culture Ferments.

The results briefly recorded in the preceding paragraph must be regarded as
first steps inspiring us to further progress towards the goal of all methods
relating to the practical application of Fermentation Physiology, viz., the
establishment of control over the progress of fermentation. In the matter of
cheese-making the attainment of this desire is still remote, and Mycologists are
not yet able to recommend this or that particular microbe with any assurance of

NATTO AND MISO 247

success. On the contrary, practice has also in this respect taken the lead by
employing in special cases such additio»s as, without being pure cultures (in a
bacteriological sense), nevertheless contain a predominating proportion of the
organism most suitable for the object in view. One of the two classes of cheese
to which this applies is the Roquefort, the other being Edam cheese.

Any one examining for the first time the said French cheese (originally
prepared in the village of Roquefort (Dep. Aveyron), from unskimmed sheep's
milk) will notice the green growth of mould occupying all the cracks abundantly
intersecting this brittle, sharp-flavoured mass. This filamentous fungus, whose
presence is by no means a sign of unsound composition, has been shown to be
the organism known as Penicillium glaucum (described in vol. ii.), which settles
in the fissured cheese mass and there consumes the acid which has been groduced
by the lactic acid bacteria and is retarding the development of the albumin-
degrading organisms. The favourable influence of this thread fungus is so
indubitably established by experience, that the practice is now common to sow it
purposely in the fresh cheese mass. To this end bread is allowed to become
covered with a luxuriant growth of mould, and is then dried and ground, the
resulting powder (rich in mould spores) being then strewn between the separate
layers of the sliced curd. In order to favour the development of this aerobic
assistant, some 60 to 100 fine holes are pierced by a needle in each cylinder of
ripening cheese.

The coatings of mould appearing and tolerated in Gorgonzola, Brie, and
Stilton cheese seem to have a similar action. In other cheeses, again, such as
Emmenthal and Gouda cheese, the formation of a mould coating in the ripening
cheese is prevented as far as possible, since it would unfavourably influence their
specific flavour. For this purpose the surface of the cheese is repeatedly wiped
over with salt water or strewn with dry salt. A comparison of the surface of
Gouda cheese with that of Brie cheese will show the remarkable difference
between them.

In the above-mentioned instance a thread fungus is employed, whereas in the
case of Edam cheese a bacterium is mixed with the milk to be made into cheese.
This is the Streptococcus hollandicus, whose acquaintance we have already made
in § 163, as a microbe capable of making milk or whey ropy. It is employed by
adding 2 per cent, by volume of ropy whey to the milk to be set for cheese.

§ 179.— Natto and Miso.

The process of fermentation known as the ripening of cheese both improves
the flavour and increases the digestibility of the albuminoids by degrading them
into more readily assimilable products. On this account the ripening of cheese
might be termed a preliminary digestion of the casein.

Similar to fresh whole-milk curd in the nature and proportional ratio of its
chief constituents is the Soja bean, i.e. the seed which replaces a meat diet
among the natives of Eastern Asia. This bean was first introduced into Europe
at the Vienna Universal Exhibition in 1873, and was shortly afterwards more
minutely described by FR. HABERLANDT (I.). It contains 35-40 per cent, of
albuminoids and about 15 per cent, of fat; and, consequently, a dish of soja
beans prepared in the ordinary manner forms a heavy, tough food-stuff. How-
ever, it can bo made more attractive to the palate, and better suitable to the
stomach, by boiling the beans for five hours in salt water, then forming the mass
into balls from 4 to 18 ounces in weight, packing these in straw, and leaving
them in a warmed cellar for a few days. Under these conditions they undergo
a fermentation which loosens the cellular tissue, and effects a partial conversion
of the protein into amides, peptones, guanine, xanthine, and tyrosine. The

248 THE FERMENTATION OF CHEESE

mass is then sold (in Japan) under the name of natto. The nature of the
species of bacteria taking part in this fermentation has been studied by K. YABE(!.)
and 0. LOEW (IV.).

In the preparation of the second kind of vegetable cheese, viz., Miso, recourse
is had to a substance known as Koji (described in vol. ii.), which is added to the
boiled bean pulp before allowing the latter to ferment. Full particulars
respecting the production of this and several other Japanese articles of diet
(e.g. Shojou prepared from soja beans) have been published by 0. KELLNER (II.) ;
and a few details of the last-named sauce, also highly appreciated in England
under the name of soy or shoyn, have been furnished by A. BELOHOUBEK (I.).
For the preparation of Tofu and Nukamiso reference should be made to two
treatises by M. INOUYE (I. and II.) ; and H. C. PRINSEN-GEERLIGS (I.) has
reported, inter alia, on the preparation (by the aid of fungoid ferments) of other
dishes from soja beans in Chinese cookery, such as Taohu or bean-cheese, the
sauce Tao-yu, &c.

§ 180.— The Normal Pitting of Cheese.

The ripening process does not always progress satisfactorily, but very often
results in a defective, or spoiled, inferior, or quite unsaleable product. Thus
E. vox FREUDENREICH (V.) reports that about 40 per cent, of Emmenthal cheeses
ripen imperfectly. The pecuniary loss accruing to cheese-makers from this
cause is estimated to amount, in Switzerland alone, to about a quarter of a
million of francs (=£10,000) per annum.

The defects here in question are of various kinds. L. ADAMETZ (III.), in
his comprehensive monograph on the subject, enumerates the following : —

(1) Defects caused by the unfavourable constitution of the milk employed.

(2) Inflation ("blown cheese"). (3) Bitter cheese. (4) Discolorations.
(5) Poisonous cheese. Of these, the first named is beyond the scope of the
present work, the fourth has already been discussed in §§ 89, 95, and 98, and
the fifth in § 171. More frequent, however, than any of these is the malady
known as inflation, puffiness, or " blown " cheese (Fr. boursoiiflement ; Ger.
Bldheii), which will now be briefly mentioned.

In addition to the constituents detailed in § 144, the curd from sweet milk
contains a certain quantity of lactose. This is dissolved in the whey remaining
in the coagulum, and which cannot entirely be removed by pressing. Con-
sequently, sugar is always present, and is decomposed in various ways by the
organisms existing in the curd. Some of them, for example, form lactic acid ;
whilst others consume it and liberate an abundance of gas. This causes holes
(bubbles or eyes) in the mass of the ripening cheese, and these holes manifest
themselves as pittings in the cut suface of the ripe product. Both the dimensions
of these holes and the manner of their distribution throughout the mass are very
characteristically developed in individual classes of cheese. In order to render
this clear by examples, reference may be made to two main types, Emmenthal
cheese on the one hand, and Edam cheese on the other ; the former exhibiting
a few holes of large size, and the latter a greater number, but of small dimensions.
The appearance of small holes, regarded as indispensable in the said Dutch fatty
cheese, is looked upon as a defect in the Swiss cheese, which, if it contains many
holes of small size, is characterised as " Nissler," and considered as inferior.
Still, the other extreme of immoderately large cavities is also undesirable. This
last condition reveals itself, in the course of its development, by the bulging of
the surface of the ripening cheese, which, in particularly bad cases, is even split
open. This malady, known as inflation or puffiness, has already formed the
subject of several investigations, as a result of which both the cause of the

THE CAUSE OF PUFFY ("BLOWN") CHEESE 249

complaint and the means of preventing and combating it have been dis-
covered.

§ 181.— The Cause of Puffy (" Blown ") Cheese.

The naturally obvious hypothesis that pitting is due to the gas-producing
powers of microbes was experimentally confirmed by H. WEIGMANN (VIII.) in
1 890. Undoubted though it be that inflation is also a result of microbial activity,
it is, nevertheless, equally undecided whether the malady is caused by specific
ferments, or only differs from normal pitting in degree, i.e. arises from the same
cause.

Of the two possible methods of explanation here indicated, the first is
championed by ADAMETZ and FREUDENREICH (VI.) in particular. The latter in
1890 established, in the case of three species of bacteria — recognised as setting
up inflammation of the udder in cows, and named Bacillus Guillebeau, a, b, c,
after their discoverer — that when inoculated into fresh curd they produce
inflation and bad flavour. The fermentative activity and products of these
three microbes were minutely examined by A. MACFAYDEN (I.) and L. NENCKI
(I.). The gas evolved by B. G. c. is a mixture of carbon dioxide and hydrogen,
their relative proportions at the climax of fermentation being about 76 : 23,
but afterwards becoming so far modified that, at the close of fermentation, only
0.72 per cent, by volume of hydrogen is found in the gas. To these three
injurious organisms (pathogenic in cows and goats) a fourth was soon afterwards
added by FREUDENREICII (VII.), viz., Bacillus Schafferi (bacteriologically very
similar to Bacterium coli commune) which was first obtained as a pure culture
from "puffy" cheese, and subsequently also found in "Nissler" cheese.
Freudenreich consequently regarded this bacillus as the cause of both these
maladies in cheese, and explained its dual manner of working by stating that
puffiness is produced when the fresh cheese curd contains comparatively few
colonies of this bacillus, at considerable distances asunder, but of large size,
and therefore capable of liberating much gas; whereas, on the other hand,
" Nissler " cheese results when the microbe is distributed abundantly, as individual
cells, throughout the ripening mass. ADAMETZ (VII.) found in the milk and
cheese of a. Sornthal dairy a fission fungus which he named Micrococcus
Sornthalii, and which proved capable of causing puffiness in cheese. This
microbe is shown in Plate I. Fig. i.

The second method is founded on a different basis. Whereas Freudenreich
devoted his attention to the discovery of specific inflation ferments, FR. BAUMANN
(I.), on the other hand, endeavoured to ascertain the external conditions under
which a microbe producing the normal pitting of cheese would become the cause
of inflation. An example of this is afforded by the Bacillus diatrypeticus casei,
discovered by him. This organism, when sparsely inoculated in curd prepared
from Pasteurised milk, produces a "blind" cheese, i.e. one containing merely
a few holes ; but when it is added in large amount, it gives rise to puffiness.
This facultatively anaerobic fission fungus is a non-motile capsule bacillus
generally 1.5 p. in length and 0.7 p in breadth ; a photographic representation
is given in Plate I. Fig. 2. In media containing sugar it liberates a gas chiefly
consisting of carbon dioxide along with a not inconsiderable portion of hydrogen ;
and in addition to these products alcohol and lactic acid are formed.

If this bacillus be inoculated into fresh curd prepared from non-Pasteurised
milk, and consequently rich in a variety of bacteria, a struggle ensues between
them. In presence of a superior force of species that do not generate gas, the
B. diatrypeticus casei is suppressed, and " blind " cheese will result. On the
other hand, when the restrictive power of the other organisms is merely sufficient
to moderate, but not prevent, the development (and consequently gas-producing

250 THE FERMENTATION OF CHEESE

power) of our bacillus, then the pitting will be normal ; whilst, if finally these
adverse influences be almost entirely lacking, " puffy " cheese results. No
rejection of this explanation is implied by the mention of the error into which
its author has fallen in attributing to his bacillus alone the capacity of producing
pitting — the inaccuracy of which assumption has been noted, inter alia, by
ADAMETZ (VIII.).

§ 182.— Cheese-makers' Recipes.

In the light of Baumann's discovery, the reason for a number of (apparently
pedantic) rules current among cheese-makers both for the production and
subsequent treatment of the curd is made clear. The careful adherence to
certain temperatures during and after setting ; the time of exposure to their
influence ; the nature of the mechanical treatment, and even the extent of the
pressure applied to the moulded curd in the press, — all these conduce to the
result — the cause of which is certainly unknown to the operators — that a certain
bacterial species attains pre-eminence.

The researches published by FR. SCHAFFER (III.) and FREUDEXREICH (VIII.)
in 1895 tend t° elucidate the processes occurring during the after- warming of
the curd. As has already been indicated, the freshly precipitated curd is kept
for a short time at a certain constant temperature, the degree and duration of
exposure varying in different kinds of cheese. In the case of Emmenthal cheese,
Schaffer showed that a merely gentle after-warming of the curd results in a
quicker and more perfect ripening, so that the finished cheese contains a large
proportion of products formed by the decomposition of albumin. Freudenreich
investigated this condition from a bacteriological point of view, and explained
the fact already recorded in § 177 — viz., that the ripening of hard cheese is
almost exclusively brought about by bacteria, whilst that of soft cheese is chiefly
occasioned by higher fungi (budding fungi, oi'dium) — as due to the inferior
powers of resistance to high temperatures exhibited by the latter organisms.
Thus the cheese-maker's old rule that "curd for soft cheese should be only
gently warmed " is shown to be well founded.

Certain prescriptive methods of preparation for a large number of different
kinds of cheese have been gradually built up as the outcome of practical
experience. The possibility of such a result is a proof that the bacterial species
necessary in the ripening of cheese are present everywhere and at all times.
The exact observance of these recipes can, however, only continue to indefinitely
yield uniform results where the composition of the bacterial flora of the milk
set for cheese undergoes merely unimportant fluctuations. This is the case in
the Alpine dairies, where the grazing grounds seldom, if ever, receive any
application of manure from external sources, and consequently the same species
of bacteria are continually returned to the ground anew in the dung of the
grazing cattle. For this reason the cheese-dairying industry necessarily developed
first in the High Alps, since there the business is, in a bacteriological sense,
exposed to the minimum of danger. The case is different in the lowlands,
where the cows are fed with fodder of highly diversified origin, frequently
consisting of the residues of agricultural industries (grains, distillery residue,
grape skins, &c.). The bacterial flora of the dung of such animals will be liable
to frequent changes ; and since the bacterial content in the milk is for the most
part derived from the dung, it will be evident that the cheese-making process
will be affected by this change. Greater difficulties consequently attend the
pursuit of the industry in lowland districts, and much less reliance can be placed
on recipes. To explain this more clearly by an example, reference may be
made to an observation which chemists have been unable to explain, but which

BITTER MILK AND BITTER CHEESE 251

— regarded from a bacteriological standpoint — seems almost self-evident, viz.,
the difficulty experienced in the working up of milk at such times of the year
as a change is made from dry to green fodder, and vice versd. The bacterial
flora of fresh grass is of a much more diversified character than that on dry
hay ; only a few species remaining alive and capable of development in the
latter.

§ 183.— Counteracting1 Puffiness in Cheese.

The reader will now probably inquire whether any method exists whereby
milk that will produce puffy cheese may be recognised as dangerous before it
is worked up and rejected by the cheese-maker. This course will be advisable
when the gas-forming bacteria greatly preponderate, a condition ascertainable
by the so-called fermentation test. A sample of the milk to be examined is
kept in a fermentation flask (§ 126) for twelve hours at 40° 0., a conclusion
based on experience being then formed as to its suitability or the reverse,
according to the changes occurring during this period. Fuller particulars on
this point will be found in Adametz's monograph, as also in the highly com-
mendable text-book of W. FLEISCHMANN (I.).

As a means of preventing the malady, FREUDENREICH (IX.) recommends the
addition of 3 per cent, of common salt to the freshly precipitated curd, freed
from the main bulk of the whey. For restricting incipient puffiness, Adametz
counsels setting the cheese to cool, since the ferment is found, by experience, to
be violent and injurious solely at higher temperatures.

From the results of an investigation made by H. L. BOLLEY and C. M.
HALL (I.) it must be concluded that gas-forming bacteria are not present in milk
at the moment it leaves the udder. If this observation is confirmed by renewed
(highly, desirable) researches in other places and under different conditions, and
thus become a general law, it will indicate the means of preventing puffiness, viz.,
by taking care to keep the fresh-drawn milk free from dirt and dung, which are
the vehicles by which the gas-forming bacteria are introduced.

§ 184.— Bitter Milk and Bitter Cheese.

According to a rule based on experience, and observed by all housewives
skilled in cookery, boiled milk must be stored in uncovered vessels, otherwise
it is liable to turn bitter. The attention of Pasteur was also directed to this
matter in the course of his studies on spontaneous generation. We have already
seen, in a previous section, that the French investigator here made the important
discovery that, though the lactic acid bacteria are thus destroyed, the more highly
resistant spores of butyric acid bacteria can withstand such a brief exposure to
boiling heat. Now, since the majority of these latter are anaerobic, they can
then only manifest their activity provided the admission of oxygen is either
entirely prevented, or at least restricted, a condition ensured by covering the
milk-pan with a lid. There then gradually accumulates within the pan an
atmosphere of carbon dioxide, &c., produced by the vital activity of bacteria and
preventing the access of oxygen to the strongly fermenting milk. The existence
of this gaseous stratum can be detected by the sense of smell on carefully raising
the lid.

The bitter flavour developed in the milk was formerly ascribed— e.g. by
K. KRUEGER (II.) — to the chief product formed by these bacteria, viz., butyric
acid, until WEIGMANN (IX.) in 1890 showed that no bitter taste is produced in
milk by the addition of butyric acid. Like HUEPPE (VII.), he attributes the
bitter flavour to the peptone formed from the albuminoids in milk. Later re-
searches on this point have led, in the main, to the same results. Consequently

252 THE FERMENTATION OF CHEESE

the subject falls within the present section, which deals with the decomposition
of albumin.

Nageli was the first to attribute the development of bitter flavour in milk to
bacterial activity, and since then many attempts have been made to find and
prepare pure cultures of bacteria possessing such properties. Some of these are
capable of producing bitter principles both in milk and cheese, whilst others are
injurious solely to the former; but all liquefy gelatin, and consequently produce
a peptic ferment.

Of the first group two species are known up to the present, viz., (i) Tyrothrix
geniculattis — obtained as a pure culture by DUCLAUX (XIII.) from Cantal cheese,
and already noticed in § 176 — produces a bitter substance in both milk and soft
cheese. (2) Micrococcus casei amari was isolated to a pure culture by FREUDEN-
EEICH (X.) in 1894 from bitter, hard, Swiss cheese. This organism, which
measures about i /* in diameter, is endowed with the somewhat rare dual property
of forming lactic acid and liquefying gelatin. In milk and cheese — but not in
bouillon — it gives rise to a strongly bitter flavour, which Freudenreich only
partly ascribes to the peptone produced, since after the latter has been thrown
down by alcohol from milk cultures of the coccus, the filtered liquid leaves, on
evaporation, a bitter residue. Here the widespread experience of practical
cheese -makers, viz., that the bitter taste generally makes its appearance at the
stage of semi-ripeness, vanishing again as the cheese increases in age, may be
mentioned.

The capacity of developing a bitter flavour in milk alone is possessed by the
following organisms: — i. Weigmann described in his above-named treatise a
sporogenic bacillus, 1.5-1.8 /i long and 0.9-1.1 p. broad, which does not produce
gas, bub gives rise to a casein-dissolving enzyme and a volatile acid (differing
from butyric acid). 2. The micrococcus of bitter milk, of CONN (IV.), is aerobic
and non-motile, forms butyric acid, and develops a repulsive bitter flavour in
milk, cream, and butter. 3. M. BLEISCH (I.) obtained from milk, which had
become decomposed after sterilisation by the Neuhauss process, a pure culture
of a facultatively anaerobic motile bacillus, whose endogenous spores were
able to stand exposure ^ for six hours at a temperature of 100° 0. in milk.
When inoculated into s'terilised milk it produces a strongly bitter flavour, and
must, from its behavour towards casein, be grouped with Duclaux' Tyrothrix
species. 4. Bacillus ligiiefaciens lactis amari was found by FUEUDENREICH (X.)
in cream which had turned bitter spontaneously. The relative dimensions of
this motile bacillus vary considerably : the most usual measurements are 0.5 /u
for the breadth and 1.5 p for the length, but the latter may extend to 6 /u. It
iuduces coagulation in milk — which it also makes very bitter — but no formation
of acid takes place ; and it liquefies gelatin.

CHAPTER XXXII.

THE FERMENTATION OF UREA, URIC ACID, AND
HIPPURIC ACID.

§ 185.— Urea, the Final Product of Animal Metabolism.

THE natural cycle pursued by the elements normally present in the vegetable or
animal body is, with a few exceptions, very simple and easy to follow. Those
resisting the action of fire, and therefore found in the ash — viz., K20, Na20,
CaO, MgO, SiO,, S03, P,O5, Fe,O3— are taken up by the plant from the soil
(where they are generally present in sufficient quantity) and are returned thereto
in manures. Hydrogen and oxygen are, in the form of water, always plentifully
at hand. Carbon is absorbed from the air as carbon dioxide by the plant, and is
given up again by the animal in the same form.

The natural circulation of nitrogen is much more complex. This element,
which is indispensable for the construction of albuminoid substances, is an object
of solicitude not only to the farmer, who balances the incomings and outgoings
of his soil, but also to the bacteriologist, who carefully watches the changes of
form nitrogen undergoes during its passage from the plant through the body of
the animal and back to the earth, where it is again gradually enabled to renew
the cycle.

Only a portion of the nitrogen consumed by man and animals in the food —
chiefly in the form of albuminoids, but also as amido-compounds, &c. — and
transformed and again excreted, leaves the body vid the intestinal canal, i.e. in
the faeces. This portion consists, on the one hand, of indigestible or undigested
food constituents, and, on the other, of nitrogenous metabolic products ; such,
for example, as glycocholic acid and taurocholic acid from the bile ; leucine and
tyrosine from the gastric juices, &c. &c. The subsequent fate of these amido-
compounds passing into the excrement has already been dealt with in § 168.

The residual nitrogen takes another route in order to make its exit from the
body, namely, vid the kidneys, and is excreted in the urine. The most important
constituent of this substance is urea, but uric acid, hippuric acid, allantoin, &e.,
are also present, though in much smaller quantities. The qualitative content of
these bodies differs in the various kinds of animals, whilst quantitatively they
depend on the amount and composition of the food.

In human urine and that of the carnivorous mammalia, birds and reptiles —
which has an acid reaction owing to the presence of acid sodium sulphate — uric
acid is present to a greater extent than hippuric acid. The total amount voided
by a man of normal size is : —

Urea . 35-50 grams.

Uric acid Q-5--75 gram.

Hippuric acid . . . • . . .0.3 gram.

On the other hand, the urine of the herbivorous mammals and birds — which
has an alkaline reaction owing to its content of KHCO3 — exhibits a different
substantive ratio, uric acid being in very minute proportion (T^-gth per cent.),
whilst hippuric acid is comparatively plentiful, e.g. in cows' urine up to | per

254 THE FERMENTATION OF UREA

cent., in horses' urine up to 2 per cent, (in combination with lime). Urea
is present to the extent of 2-5 per cent, in cows' urine, and 3 per cent, in that
of the horse.

§ 186.— Urea Unassimilable by Higher Plants.

Assuming the total number of inhabitants in the world to be 1500 millions,
and estimating the diurnal excretion of urea at an average of only 25 grams per
individual, then there results a total daily production of 37,500 tons of urea by
the human race alone. This quantity in the solid state would occupy a space of
50,000 cubic metres (65,400 cubic yards), and contain 17,000 tons of combined
nitrogen. The excretions of the animal kingdom must be estimated at a much
higher figure.

The amount of nitrogen daily excreted in urine and passing into manure is
consequently enormous, and the question naturally arises : What becomes of it
afterwards ? Is urea (and also uric acid, <tc.) adapted to serve immediately and
directly for the nutrition of plants, and of cultivated plants in particular ?

Agricultural practice answers this query by a decided negative, knowing
from experience that manuring with fresh urine is at first either entirely useless
or actually injurious. No satisfactory explanation of this fact has yet been
discovered by scientific research. 0. KELLNER (III.), for example, sought it in
the circumstance that urea is not absorbed (retained) by the soil. We must
therefore turn away from research, and fall back on the fact that urea is
unsuitable for the nutrition of the higher plants ; and that consequently its
nitrogen, not being available for this purpose, is last. If this element is to
continue its cycle in the organic world, it must first be converted into other
forms and modes of combination ; and the question arises as to which of these
involves the least labour and smallest expenditure of energy.

It must be borne in mind that urea is a derivative of carbon dioxide on the
one hand and ammonia on the other — i.e. two compounds which are known to
be suitable for the nutrition of plants — and may be regarded as a condensation
product from these two atomic groups by dehydration. If their coherence can
be loosened, and the carbamide split up by hydrolysis to form carbon dioxide and
ammonia —

CO(NH2)a + 2HaO = C0a + (NH4)20,

this prevents the danger of the said quantity of urea remaining undecomposed
and accumulating, in consequence of which its nitrogen would be withheld from,
instead of restored to, the vegetable kingdom.

There is, however, no need to seek far for an instrument for this conversion,
Nature herself having already provided the implements for this work in the
micro-organisms known as urea bacteria. The next three paragraphs will be
devoted to the consideration of their character and capabilities ; a fourth dealing
with the decomposition of uric acid and hippuric acid.

§ 187.— Discovery of the Urea-Ferment by Pasteur.

The natural process of the decomposition of urea was discovered some two
years after the first successful attempt at the artificial preparation of this
substance. The well-known fact that urine, which is clear when first voided by
healthy individuals, becomes turbid on prolonged standing, while with increasing
age it turns more and more alkaline, and exhibits an increasing smell of
ammonia, found an explanation in 1830 at the hands of Dumas, who regarded
this modification (the ammoniacal fermentation of urine) as a conversion of urea

RESEARCHES OF P. MIQUEL 255

into ammonium carbonate by the absorption of water. This hydrolysis of urea
was considered as a purely chemical process, a readjustment of the atoms in the
molecule.

It was reserved for PASTEUR (I.) in 1862 to show the incorrectness of this
view. He discovered in fermented urine a micrococcus (0.8-1.0 /x in diameter,
and frequently united as diplococci, tetrads, and chains) which is capable of
inducing the change in question in sterilised urine. This organism was shortly
afterwards (in 1864) also described by VAN TIEGHEM (VIII.), and called
Bacterium urece, being subsequently named by Cohn Micrococcus urece.

The next researches on this important fermentation appeared in 1879, and
afforded proof that this capacity for converting urea into ammonium carbonate
is not restricted to one single species of microbe, but that, on the contrary,
Pasteur's micrococcus has competitors, not only in many bacteria, but also in a
few of the higher fungi. This work was performed by P. MIQUEL (V.), to
whom we owe most of our present knowledge of the fermentation of urea.
Before turning to his more recent labours on the subject, we will, however,
briefly review the endeavours made by his colleagues in the same direction.

ii. VON JAKSCH (I.) iu 1881 published a thoroughgoing investigation, the
morphological part of which was also instrumental in founding the theory of
bacterial pleomorphism ; the physiological results will be given in the next
paragraph. The urine-bacterium discovered by him throve best in a liquid
containing the following dissolved salts per litre of water : acid potassium
phosphate, 0.12 gram ; magnesium sulphate, 0.06 gram ; Seignette salt, 5 grams;
and uiea, 3 grams. This liquid is known in the literature of the subject as
Jaksch's nutrient solution.

LEUBE (I.) in 1885 added four new species of bacteria to the group of urea
ferments already known. One of them, called Bacterium urtce, appeared in the
form of plump rod?, 2 p. long and i /i broad, and of the remaining three, one
belongs to the sarcina group.

In contrast to the bacteria (forming solid colonies) mentioned above is the
urea-fermenting micrococcus discovered by FLUCCE (1.), and known from its
liquefying influence on gelatin as Micrococcus urece liquefaciens.

The report drawn up by C. LUNDSTROM (I.) and R. CAMBIER (I.) also made
known a few new species of urea-fermenting bacteria, and the same applies to a
research by R. BURRI, E. HERFELDT, and A. STUTZER (I.), which we will deal
with briefly below. We can now turn our attention to the above-mentioned
newer

§ 188.— Researches of P. Miquel (VI.).

This author isolated from air, soil, liquid manure, water, &c., some sixty
different species of bacteria, all of them capable of fermenting urea. Out of
these he selected seventeen as particularly worthy of interest, and has more
closely investigated and described them. Morphologically he distinguishes three
genera, Urobacillus, Urococcus, Urosarcina. In the further subdivision within
these three groups two piincipal factors of a chemico-physiological nature are
adopted as criteria, viz., the rapidity of fermentation, i.e. the amount of urea
fermented per unit of time ; and on the other hand, the fermentative power,
expressed by the maximum quantity of urea completely fermented by the
species in question per unit volume of nutrient solution. These two indications
acquire almost the character of mathematical constants for any determined

The most powerful as well as the most energetic is the Urobacillus Pasteurii,
so frequent in both natural and drainage waters. This organism ferments
3 grams of urea per hour in a 2 per cent, peptonised urea -bouillon, and

256 THE FERMENTATION OF UREA

completes its task even when the nutrient solution contains 140 grams of urea
per litre. Morphologically similar to this, but differing greatly in physiological
character, is the Urobacillus Freud enreichii, found with particular frequency in
the sweepings of the streets of Paris. This bacillus can only hydrolise 0.3 gram
of urea per hour, and cannot ferment a larger quantity than 45 grams per litre
of nutrient solution. It is an actively motile rod, 1.0-1.3 M Droad and of
variable length.

High degrees of speed and power of fermentation are not always found in
association. This is well exemplified by Urobacillus Schiitzenbergii, a rod i JLI
long and 0.5/1 broad, incapable of producing spores It was found by Miquel
both in natural and drainage waters, but never in the air. This species is very
energetic, i.e. transforms a large amount of urea per unit of time, but its activity
ceases as soon as the liquid has become somewhat enriched with ammonium
carbonate. That this is actually the cause of the cessation follows from the fact
that the fermentation proceeds further when this salt is removed by aerating the
medium.

To this injurious influence of ammonium carbonate — towards which different
degrees of susceptibility are exhibited by the various species — is due the rapid
dying of cultures of the bacteria under consideration, in liquids containing urea.
If it is desired to prolong their existence or to refresh debilitated cultures, they
must be transferred to nutrient media that are free from urea, and consequently
enable growth to proceed only at a slow rate, but, just for this reason, ensure a
longer life.

Even greater than their sensitiveness towards ammonium carbonate, of which
even the most delicate species can support a very considerable quantity, is the
susceptibility of the urea bacteria to the presence of free acid in the nutrient
medium. Burri and his collaborators ascertained (in their above-mentioned
researches) that 0.4 per cent, of sulphuric acid produces a fatal effect. This
observation can be practically utilised in the protection of stall manure from
early decomposition.

Into the remaining distinguishing characteristics of Miquel's urobacteria —
especially the degree of resistance to heat exhibited both by the vegetative forms
and the spores produced by all but the last-named species — we cannot enter
further. One common characteristic peculiar to the group must not, however,
be omitted from mention, and that is the aureole with which the colonies
surround themselves when grown on 2 per cent, urea-gelatin, and whereby they
are distinguishable from all other bacterial species, even at an early age, by
macroscopical examination or under a low power. The colonies are closely
surrounded, for a distance exceeding their diameter, with numerous biscuit-
shaped bodies, embedded in the gelatin and for the most part so close together
that they envelop the colony as in a cloud. These bodies are facetted crystals,
each composed of two combined globules, containing lime, carbonic acid, and
phosphoric acid, and formed by the reaction of ammonium carbonate (liberated
by the bacteria), on the salts of the alkaline earths contained in the medium.

Whether and how far the nitrogen in the urea contributes to the structure of
these bacteria is also an interesting point. Jaksch in his treatise asserted that
his bacillus preferentially takes up nitrogen from urea, other sources, such as
peptone, being less suitable ; but Miquel came to exactly the opposite conclusion,
the species examined by him greatly preferring peptone, or any similar
substance, before urea as a source of nitrogen.

Urobacteria are of very frequent occurrence in nature. For quantitative
determinations on this point we are indebted to Miquel, according to whom the
average number of such bacteria in the air of Paris was (in 1891) 151 germs per
cubic metre. The smallest number (90) was found in autumn and the highest

URASE 257

in spring (197) and summer (202). The relative content of these in natural
waters increases with the degree of impurity. A good example is afforded by
the Seine : before reaching Paris 103 urobacteria were found per 10,000
microbes, whereas a sample taken within the city limits gave 204, or twice as
many as before. According to Miquel, 1-2 per cent, of the bacteria present in
the soil, and 15 per cent, of those in cowhouse manure, are capable of hydrolys-
ing urea. It is therefore evident that Nature provides for the conversion of
urea into a more readily assimilable compound, which we have already found to
be essential.

The ammoniacal fermentation of the urine of herbivorous domestic animals
(horses, horned cattle) begins soon after its evacuation from the body. The
resulting ammonium carbonate partly volatilises, and thereby leads to a loss
of nitrogen, the extent of which — as A. MTJNTZ and A. GIRARD (I.) showed
in 1893 — was formerly under-estimated. Out of the many methods proposed
and attempted for the prevention of this loss and the combination of the
ammonia, a few (e.g. sulphuric acid) have proved unfit for application in the
stall, and others (such as peat litter, gypsum, kainit) insufficient, only a single
one being actually suitable : the superphosphate recommended by E. HEIDEN (I.)
in 1887, viz., the acid phosphate prepared by means of sulphuric acid, and
capable not only of chemically fixing ammonia, but also, by reason of its acid
reaction, preventing the inception of uric fermentation.

§ 189.— Urase.

In 1874 Museums (I.) expressed the opinion that the conversion of urea into
ammonium carbonate is merely an indirect result of bacterial activity, the
hydrolysis of the urea being effected not by the organisms themselves, but by the
enzyme they excrete. This enzyme was said to be particularly plentiful in, and
recoverable from, the urine of patients suffering from catarrh of the bladder, a
circumstance which would also account for the alkaline reaction of this urine
when in a freshly voided condition. This statement was investigated and con-
firmed by PASTEUR and JOUBERT (II.) in 1876. On the other hand, the attempts
made by LEUBE (I.) to separate the enzyme from the bacteria by filtration
through a clay cylinder failed. P. MIQUEL (VII.) then showed, in 1890, that
these conflicting results are due to the extreme decomposability of this enzyme,
which he proposed to name urase, and which, being very easily oxidised, ought
to be filtered in an atmosphere devoid of oxygen, a precaution neglected by
Leube. Urase decomposes in three to four hours at 50° C., and in a very few
minutes at 80° C. With regard to its chemical composition nothing is at present
known ; not even whether it is a single body or a mixture of several substances
(varying in constitution according to the conditions of fermentation). One
thing, however, Miquel placed beyond doubt, viz., its capacity to hydrolyse and
rapidly convert urea (in a sterilised solution) into ammonium carbonate. It is
therefore a true enzyme.

§ 190.— The Decomposition of Uric Acid and Hippuric Acid.

With regard to the disruption of the uric acid molecule by microbial agency,
F. and L. SESTINI (I.) instituted an investigation, according to which the
decomposition corresponds to the equation- —

,NH— C— NHX NH4,

II >C =0 + SH20 + 30 = 4 >C03 + C02

C-NH/ K'

^NH— C = 0

258 THE FERMENTATION OF UREA

Unfortunately, pure cultures were not employed in this research, which was
published in 1890, and the same defect attaches to an investigation made by
E. GERARD (I.) in 1896.

In a chemical sense, the statement just recorded was confirmed by the
treatise of Burri and his co-workers, mentioned in § 187. These workers also
included the decomposition of hippuric acid in the scope of their labours. Like
their Italian colleagues, however, they did not employ pure cultures of ferments,
but used " a drop of manure drainings" for inoculating the media. They found
that hippuric acid was not attacked per se, but only when in combination with
lime, the decomposition, moreover, being more difficult to effect than was the
case with uric acid or urea — which last named is the easiest of all to convei't into
ammonium carbonate. Both for the sake of completeness and also to show the
necessity for a more accurate investigation of the decomposition of hippuric acid,
we must refer to a remark made by Van Tieghem in his above-mentioned
treatise, namely, that his B. iirece is capable of splitting up hippuric acid into its
two components, glycocoll and benzoic acid, according to the equation —

CH2 . NH— CO CH0 . NHQ COOH

I I + H20 = | +|

COOH C6H5 COOH C6H5

Analytical data to prove that this decomposition actually goes on so smoothly
are, however, lacking.

So far as the destiny of hippuric acid in the soil is concerned, K. YOSHIMURA
(I.) has observed that its fermentation proceeds much more rapidly in the upper
layers than in the subsoil.

CHAPTER XXXIII.

THE FIXATION OF FREE NITROGEN BY BACTERIA.

§ 191.— Accumulators and Consumers of Nitrogen.

IF seeds of any of the leguminous plants, e.g. peas, lupins, clover, &c., be
sown in a soil containing all the food-stuff's (K20, P2O., &c.), except nitrogen,
necessary for the growth of plants, then, given sufficient moisture, germina-
tion will scon be observed. At the outset the young plant develops just as
well in the absence of nitrogen as if that substance were present in the soil :
it feeds upon the stores of nutrient substances (carbohydrates, albumen, fat,
<fec.) accumulated in the cotyledons or seed-leaves. When, however, this store
is exhausted, a complete cessation in the growth of the plant visibly ensues,
the leaves turning yellow and becoming partly dried up, and the whole plant
presenting a moribund appearance. The cause of this condition can only be
sought in the dearth of combined nitrogen that has now set in, since the
plant has all the other essential food-stuffs at its disposal. The condition
itself is consequently termed nitrogen-hunger. All other kinds of higher
plants hitherto examined suffer in the same way when grown under these
identical condition?. Differences, however, are noticeable in their subsequent
behaviour. If left unprovided with assimilable nitrogen, the representatives
of all other families of phanerogamic plants — apart from a few exceptions to
be enumerated later — persist in this state of starvation and finally die away.
Not so the Leguminosw. These will be observed to remain for some time —
a few days to three weeks, according to circumstances — in this debilitated
condition, but will then revive almost instantaneously, rapidly turning green,
throwing up thick, juicy stalks, embellishing themselves with luxuriant foli-
age, putting forth a large number of blooms, and producing a good crop of seed.
Such a plant may then overtake others that have been provided with nitro-
genous food (manure), and have not had to pass through the famine period ;
and may, finally, at harvest yield a quantity of haulm and seed as great,
and containing just as much nitrogen, as its more highly favoured fellows.
Hence the leguminosse, in contrast to (nearly) all the other cultivated plants
(cereals, such as wheat, oats, &c. ; hoed crops, such as beet, potatoes, &c. ; oil-
seeds, and so forth) that have been examined on this point, are characterised
by the capacity for growing and ripening in a soil perfectly devoid of nitro-
gen and without the application of nitrogenous manure. This circumstance
is so much the more remarkable since both the leaves and seed of pulse
contain an unusually large proportion of combined nitrogen, and are, in fact,
richer in this element than any other vegetable food-stuffs. This fact will be
best displayed by the subjoined table, giving the average figures, obtained
from a large number of analyses, of the percentage of nitrogen in the dry
matter of the seeds of —

Maize 1.8

Buckwheat . . . .1.9

Oats 1.9

Wheat 2.3

Peas 4.3

Beans 4.6

Lentils . . . • 4-7
Soja beans . . . .6.1

259

260 FIXATION OF FREE NITROGEN BY BACTERIA

From the large number of researches instituted on the assimilation of nitrogen
by plants, the following example, given by EMIL WOLFF (I.), may be selected,
and may easily be repeated on a small scale by the reader for his own information.
A number of zinc boxes were filled with 24 kilos. (53 Ibs.) of washed calcareous
river-sand destitute of nitrogen, the necessary mineral nutrient substances
(P20., SO3, K20, MgO) being then added and the seeds mentioned in the follow-
ing table sown. A certain number of these boxes contained 0.83 gram of nitrogen
apiece in the form of nitre. The total dry matter and total nitrogen in the crop
were determined, with the following results : —

Per Box.

Oats.

Buckwheat.

Rape.

Peas.

Vetches.

With-
out
X.

With

N.

With-
out
N.

With

N.

With-
out

N.

With
N.

With-
out

N-

With

N.

With-
out

N.

With
N.

Grms.

Grms.

Grms.

Grms.

Grms.

Grms.

Grms.

Grms.

Grms.

Grms.

Dry matter in the\
crop . . /

24

9i

12

44

13

50

352

330

250

241

Nitrogen therein

0.15

0.44

0.14

o.43

0.13

0.50

6.74

6.45

6.01

5.95

This table shows that, in the case of peas and vetches, a high yield containing
much nitrogen can be obtained, without the soil (initially destitute of nitrogen)
having received any application of this element in the form of manure. Such
manuring is consequently unnecessary to these plants.

The results of these experiments on a small scale are confirmed by the
observation of large farmers. Schultz of Lupitz, a land-owner in the Mark
(Brandenburg), states that he has gathered 227 kilos, of nitrogen per hectare
(about 200 Ibs. per acre) annually from his " lupin meadows," for fifteen years
without any nitrogenous manuring. Similar results are reported by Deherain
from experiments with sainfoin, by the cultivation of which from 205 to 237 kilos,
of (combined) nitrogen were obtained per hectare (180-208 Ibs. per acre).

For this reason agricultural chemists have termed the Leguminosw collectors
or accumulators of nitrogen, and have set them up as a class distinct from all
other cultivated plants, the latter being grouped under the name of nitrogen
consumers.

This does not, however, imply that the leguminous plants are averse to such
manuring; on the contrary, they readily absorb any nitrogenous food pi-esent in or
added to the soil, this food assisting the plant to tide over the stage of nitrogen-
hunger. So soon, however, as this period of stagnation is passed, they no longer
require nitrogenous manure, and the latter, when applied, does not influence the
size of the crop. Practical agriculturists utilise this observation by planting soils
which are poor in nitrogen with such nitrogen accumulators (especially lupins) as a
first crop, which is ploughed in when sufficiently developed. This constitutes the
practice known as green manuring. A field treated in this fashion will then
contain a much larger amount of nitrogen (in organic combination) than before,
and is rendered capable of properly developing other cultivated plants (cereals,
hoed crops, &c.), which, by reason of their high nitrogen requirements, would
otherwise have yielded only a miserable crop in such poor soils.

DISCOVERY OF THE LEGUMINOUS NODULES

261

§ 192.— The Discovery of the Leguminous Nodules.

The source of the free nitrogen accumulated by this class of plants must be
sought in the atmosphere alone.

Formerly the ammonia compounds of carbonic acid, nitrous acid, and nitric acid,
always present in the air (i.e. the rainfall), were considered as likely sources of
nitrogen. The quantities of the last-named acid brought down in the rain, in
temperate and tropical climates respectively, are given in the following table,
drawn up (partly from personal experience) by A. MtiNTZ and V. MARCANO (I.) : —

Liebfraueiiberg
in Elsass
(Boussingault).

Rotharuste'J
(Lawes and
Gilbert).

Caracas, in
Venezuela
(Milntz and
Afarcano).

Island of
Reunion
(Raimbault).

Mfr.

Mff.

Mg.

Mg.

HN03 per litre of)
rain- water . ./

o.iS

0.42

2.23

2.67

Amount of HN03"|

Kilos.

Kilos.

Kilos.

Kilos.1

thus brought into 1
the soil per hec- j

0-33

0.83

5-78

9-93

tare per annum . J

The researches and calculations made by HELLRIEGEL (I.), in particular,
showed, however, this addition of nitrogen is much too small to deserve all the
credit of the enrichment of the soil. Moreover, since the supply is delivered in
approximately legular quantity to all the fields in a given district, it would afford
no explanation of the fact that, of all these fields (without nitrogenous manure),
it is only just those that have been planted with leguminous crops that yield such
a surplus of nitrogenous matter.

Consequently, only one other feasible explanation remains, viz., that the
Leyuminosce possess the inherent property of absorbing nitrogen from the air, and
elaborating it into nitrogenous compounds (albumen, <fec.).

However compulsory this conclusion may be, its recognition by the majority
of vegetable physiologists and agricultural chemists was slow. Both classes
were under the influence of Boussingault, whose experiments (1837 to 1858) in
the cultivation of leguminous and other plants under bell-glasses in soils destitute
of nitrogen gave results — confirmed by the check experiments of Lawes and
Gilbert — which seemed to deny the absorption of free atmospheric nitrogen by
plants.

Shortly afterwards, however, attention was directed to the bodies now termed
leguminous nodules. These are lateral appendages or swellings on the roots (as
shown in Fig. 59), and occur both on the younger and older portions of same, the
former being the most thickly infested. In one and the same plant all the inter-
mediate stages of this formation, ranging from such as are just barely perceptible
to those as large as a pea or hazel-nut, can be found. Their form (Fig. 60)
differs in different species, observations on which point have been published by
A. TSCHIRCU (I.) and by LAWES and GILBERT (I.).

The earliest description of the leguminous nodules was given by MALPIGHI (I.)
in his book published as far back as 1687, and this observer referred to them as
galls, i.e. diseased excrescences, an opinion also shared by P. DE CANDOLLE (I.)

i i kilo per hectare = 0.89 Ib. per acre.

262 FIXATION OF FREE NITROGEN BY BACTERIA

in 1825. TJIEVIRANUS (I.), in 1853, was the first to regard these nodules as
normal growths, and thirteen years later they were studied by WOBOSIH (I.), who
made the (subsequently important) observation, that the formation contains
entirely closed cells filled with living bacteria. In the seventies ERIKSSON (I.)
and CORNU (I.) recognised these appendages as metamorphosed lateral roots of
perfectly unique structure.

When regarded in section (Fig. 61), a nodule of this kind is seen at the first
glance to consist of two different portions— a white or colourless external zone

FIG. 60.— Root-nodule of Vicia sativa.
mw. main root ; sw. lateral root ; the meaning of
the other letters is given in Figs. 61 aud 63.
Magn. 3. (After Jieyerinck.)

FIG. 59.— Root of Vicia Faba.
With young nodules on most of the lateral roots
and on the tap root. Somewhat reduced.
(After Strasburger.)

cl

FIG. 61.— Cross-section through a nodule of Vicia

saliva, cut along the line c in Fig. 60.
j)r. the primary integument with a few epidermal

bacteria (rb).

xl. the vascular bundles, each with a xylem fibre.
bact. thi; strongly developed bacteroidal tissue.

Magu. 10. (After Jieyerinck.)

and an interior layer, pale red in the young nodule, but afterwards greenish grey,
the line of demarcation between them being somewhat sharply denned, and the
outline indented like that of a blackberry. It is in the cells of this inner layer
that the bacteria now under consideration, and more fully described below, are
sheltered ; and the layer itself is known as the bacteroidal tissue. On account
of these enclosures the said root-nodules received from A. B. FRANK (III.) in
1879 the name of mycodomatia (i.e. fungus chambers), an expression that has,
however, been abandoned.

§ 193.— Formation and Functions of the Nodules.

The first to take under consideration the physiological importance of these
nodules was LACHMANN (I.), who in 1858 defined them as stores of albumen.

FORMATION AND FUNCTIONS OF THE NODULES 263

One-and-twenty years later FRANK (III.) showed that the formation of nodules
does not occur when the plants are grown in sterilised soil, thus proving that the
co-operation of microbes existing in the soil is a necessary factor. This result,
conjoined with Woronin's observations, led to the conclusion that the production
of the nodules is effected by soil bacteria. Frank's observation, and the con-
clusion deduced therefrom, were subsequently confirmed by H. M. WARD (V.),
who showed that the nodules are absent in water-cultures of Vicia Faba in
sterilised nutrient solutions, but, on the other hand, appear in large numbers if
chopped nodules, grown in the ordinary soil, be inserted amongst the root-hairs.
This discovery threw a little more light upon the manner in which the nodules
are produced, and increased the probability of the assumption that they result
from the activity of bacteria which gain access to the root, and there exert a
certain stimulance inducing a luxuriant cell-growth. A more intelligent inves-
tigation of the importance and mode of action of the nodules thus became possible,
and it was then remembered that the Leguminosce are precisely the plants found
capable of growing in soil destitute of nitrogen. Hence the obvious idea sprang
up that possibly these nodules should be regarded as organs facilitating the
absorption of uncombined nitrogen from the air.

It naturally follows that if this assumed faculty is actually possessed by these
growths, a direct connection between the formation of the nodules and the
development of the plant as a whole should be traceable, and this was accom-
plished by HELLRIEGEL (I.), in conjunction with WILFARTH, in the years 1884
to 1886. These workers, as a result of exhaustive investigation of plant-roots,
arrived at the conviction that the development of the root-nodules stands in
most intimate relation to the growth and assimilation of the whole plant. The
number of nodules per plant was found to be the greater in proportion as the
development of the latter was more perfect. Papilionaceous seeds (e.g. peas)
sown in boxes of sterilised soil devoid of nitrogen, and protected from subsequent
infection, perished after nitrogen-hunger set in, but, on the other hand, throve
and ripened when the boxes were supplied with a small quantity of an aqueous
extract from fertile soil. When the non-nitrogenous soil was watered with
a little of the said extract, and then sterilised and covered with a layer of
sterilised cotton-wool before planting the seeds, the result was identical with that
of the first experiment, i.e. the plants started well, arrived at the stage of
nitrogen-hunger after the unfolding of the sixth leaf, and then gradually fell
into a consumptive state and perished, barren. By these and many other experi-
ments Hellriegel arrived at the incontrovertible conclusion that the absorption
of atmospheric nitrogen by the Papilionacece is directly connected with the
development (or the presence and activity) of the so-called leguminous nodules,
which latter are produced solely by the action of certain bacteria on the roots.

Hellriegel confined his researches entirely to the Papilionacece, and left out
of consideration the other two families, viz., Ccesalpinacece and Himosacece, which
form with it the order Leguminosce. The formation of nodules in these two
families was subsequently investigated by D. MORCK (I.), with the result that
the faculty was discovered in each one of sixty-five species (from thirty-eight
genera) examined. These discoveries were supplemented by H. LECOMTE (I.),
who, in 1894, proved that nodules are formed in Arachis hypogcea, the earth-nut,
a fact already noted by Poiteau in 1852, but afterwards denied by Eriksson.
The nodule bacteria of the Soja bean (Sqja hispida) have been described by O.
KIRCHNER (II.). On the other hand, neither Morck nor FR. NOBBE (I.) was able
to discover the presence of root-nodules on Gleditschia ; and it is found impossible
to inoculate this plant by the nodule bacteria of other Leguminosce. Apart from
Leguminosce, the organs in question are found (so far as is at present known)
only on Alnus, Elceagnus angustifolius, IJippophae, and Podocarpus, all of which

264 FIXATION OF FREE NITROGEN BY BACTERIA

are able to thrive in soils destitute of nitrogen. The faculty of nodule formation
has also been ascribed to other plants. A. B. FRANK (IV.) goes the farthest in
this respect, and, indeed, assumes that all plants are in a position to take up
free nitrogen, an opinion also recently expressed by J. STOKLASA (II.). LIEB-
SCHER (I.) confines his opinion within narrower limits, but ascribes the power of
fixing free nitrogen to oats and mustard as well. The statements of Frank and
Liebscher have, however, been disproved by the searching criticisms of, e.g.
WILFARTH (I.), U. Kreusler, P. Wagner, F. Nobbe, and L. HILTNER (I.); and
J. H. AEBY (I.) has shown that mustard does not possess the faculty with which
it is credited by Liebscher. The same results were obtained by CH. E. COAXES
and W. R. DODSON (I.) in 1896, in their experiments on the cultivation of the
cotton plant (Gossypium).

§ 194.— The Nodule Bacteria.

The discoveries reported in the foregoing paragraph, and for which we have
chiefly to thank Hellriegel and Wilfarth, lead up to the question whether this
proved absorption of free nitrogen is effected in the root-nodule itself, or whether,
by the influence of this formation, the entire plant — particularly the foliage —
becomes capable of taking up this gas from the atmosphere and fixing it in
combination ?

This latter opinion, which was chiefly supported by A. B. Frank, has been
investigated by P. KOSSOWITSCH (I.), who was, however, unable to discern any
absorption of atmospheric nitrogen by the parts of the plants above ground.
Attention must, therefore, be concentrated on the root-nodules themselves ; but
before going into the question whether and in what manner the nitrogen is fixed
by them, it will be necessary to become more closely acquainted with the living
organisms they contain, viz., the nodule bacteria.

The discovery of these bacteria by Woronin in 1866 was not followed im-
mediately by their general recognition in scientific circles. For example, H.
DE VRIES (II.) in 1877 looked upon them as non-essential. Moreover, when
J. BRUNCHORST (I.), in 1885, examined them more closely, he came to quite a
different conclusion, and defined the supposed bacteria as organised albuminoids
collected in the interior of the nodule cells, and therefore termed them bacteroids
(on account of their external resemblance to bacteria). Hence the term bac-
teroidal tissue, applied to the internal portion of the nodules in which these
organisms appear in large numbers. Remarkably enough, Brunchorst's opinion
found favour in the eyes of A. B. FRANK (V.), although conflicting \vith his own
discoveries made in 1879. A. TSCHIRCH (I.) also ranged himself on the side of
Brunchorst.

A complete revolution of opinion took place in 1888, when BEYERINCK (XIV.)
indubitably established the fungoid nature of these supposed pseudo-bacteria,
by isolating them from the nodules, and cultivating them further in artificial
media. The pure culture obtained from the individual species of Papilionacecu
exhibited certain slight but undeniable differences, which, however, were not so
extensive as to make their discoverer feel justified in classifying the organisms as
separate species, so he defined them as varieties of a single species, for which he
proposed the name of Bacillus radicicola. The artificial formation of nodules
induced by inoculating the roots of Leguminosae with such pure cultures was
successfully attempted a year later by Prazrnowski, and will be noticed in § 195.
The bacillus in question develops either feebly or not at all on ordinary nutrient
gelatin, this substratum being too rich in nutrient materials. Beyerinck recom-
mends a decoction of the leaves of PapilionacecK, with the addition of 7 per cent,
of gelatin, \ per cent, of asparagin, and £ per cent, of cane-sugar. The requisite

THE NODULE BACTERIA 265

degree of acidity in the medium is represented by about 0.6 c.c. of normal acid
per 100 c.c. The plates coated with this nutrient medium are inoculated with
an infusion prepared by mixing a few c.c. of sterilised water with a small portion
of the contents of the bacteroidal tissue of a fresh, young nodule that has been
previously washed with water, then steeped a short time in alcohol, freed from
the latter by means of ether, and finally cut open with a sterilised knife. The
small quantity of inoculating liquid will be absorbed by the gelatin, leaving the
bacteria on the surface, where their growth progresses in the most favourable
manner. Here they develop to small mucinous colonies that do not liquefy the
gelatin.

Two forms of cells will be immediately noticeable in preparations made from
such a culture. Beyerinck distinguishes them as rods and rovers. The former
have a breadth of i p. and a length of 4 to 5 p, and wander eagerly towards the
edge of the cover-glass, where fresh oxygen obtains access. As veritable dwarfs
in comparison with these are the rovers, which are only 0.9 p. long and o. 18 /*
broad, and therefore belong to the smallest of known micro-organisms. Even the
Chambeiiand filter cannot restrain them, and they escape through its pores. As
the name implies, they are endowed with motile power, which is frequently so
strong that individual rovers are able to escape from the parent colony, make
their way across the gelatin plate, and found a daughter colony at a distance.
The rod cells are not invariably of the ordinary cylindrical shape ; on the contrary,
variously bulged or lobed forms appear in larger or smaller number according to
circumstances, and a forked branching, resembling the Greek y, is very frequent.
This peculiarity, be it remarked en passant, is also shared by other bacterial
species, e.g. the Pasteuria ramosa already mentioned in an earlier paragraph.
Bacillus radicicola does not produce an enzyme capable of dissolving gelatin,
starch, or cellulose, or of inverting saccharose ; neither has spore formation been
detected, a circumstance harmonising with the fact that a temperature of 6o°-
70° C. suffices to destroy this fission fungus. On the other hand, the organism
appears to support drought and frost without sustaining any injury.

A few words must be devoted to the varieties exhibited by the nodule
bacteria. Hellriegel established (though without employing pure cultures) that
the nodule bacteria of peas cannot develop nodules on lupins and Seradella
(Ornithopus sativus). This observation, which was challenged by A. B. Frank,
was confirmed by NOBBE, working with pure cultures, in conjunction with
SCHMID, HILTNER, and HOTTER (I.). Whether the species should be divided
merely into races or varieties, as advocated by the observers just named; or
whether we should here speak of different species in the sense used by naturalists,
and consequently express them by different specific names, as was attempted, e.g.
by A. SCHNEIDER (I.), is a point of remote importance. BEYERINCK (XV.) has
also become a convert to this view, having been unsuccessful in inducing the
formation of nodules in Vicia Faba by means of bacterial cultures from those of
Ornithopus. On this point an interesting discovery was made by NOBBE,
HILTNER, and SCHMID (I.), according to whom the bacteria from any given species
of Leguminosce produce the most plentiful development of root-nodules in the
shortest time when applied to other plants of the same species, the potency
diminishing in the case of plants of merely allied species, and finally becoming nil
when a greater specific difference exists between the original plant and the one
inoculated. The various separate species (or varieties) of the nodule bacteria
can, to a certain extent, therefore replace one another. Thus, those of the pea
are also efficacious for all the examined species of the genera Vicia and Phaseolus,
but, on the other hand, without effect on Robinia, Ornithopus, red clover,
kidney vetch, and other clovers. Those of Robinia form nodules only on Phaseolus
and a few species of the genus Trifolium. Finally, according to the researches

266

FIXATION OF FREE NITROGEN BY BACTERIA

of F. NOBBE, E. SCHMID, L. HiLTNER, and E. HOTTER (II.), the nodule bacteria of
Elasaynus differ greatly from those of Leguminosce.

These observations are also of importance to practical agriculture. Already,
for several years past, soils intended to be brought under cultivation (e.g. high
moorland soils) for the growth of nitrogen-collecting plants are previously
inoculated, i.e. strewn with a little earth from fields that have borne leguminous
plants for a long time, and are consequently rich in nodule-forming bacteria.
For this inoculation to have the desired result, it is necessary to use earth
containing the bacterial species most efficient for the kind of Leguminosce to be
afterwards grown. Practical experience on the importance of this consideration
is already available. Thus SALFELD (I.) has reported that a similar soil intended
for peas could not be rendered capable of yielding a crop unless strewn with a
little soil obtained from a good pea-field, soil from a lupin-field failing to produce
the desired efl'ect. A similar discovery was made by M. FLEISCHER (I.). This
treatment must, of course, be preceded by any improvement found necessary in
the chemical composition of the soil. Thus, for example, sour moorland must
previously be limed, in order to neutralise the acids preventing the development
of the nodule bacteria. Moreover, this operation must be performed with
discretion, an excessive addition of lime being avoided. Reference may be made

on this point to a communication by TACKE,
IMMENDORF, HESSENLAND, SCHUTTE and

'"\ ' / MlNSSEN (I.).

" W

§ 195.— The Bacteroids.

The bacteria in question are often met
with in air and water, and very frequently
in the soil. NOBBE, SCHMID, HILTNER, and
HOTTER (I.) made several quantitative bac-
teriological investigations on this point. The
bacteria pass from the soil into the roots of
buch plants as will admit them. The first
successful, artificial production of nodules
by the aid of pure cultures was made by
A. PRAZMOWSKI (IV.). This worker, in
view of the absence of the sporogenic
faculty in these organisms, changed the
name of Bacillus radicicola, bestowed on
them by Beyerinck, into Bacterium radici-
cola. According to his observations, this
fission fungus penetrates the epidermal cells
of the root-hairs, and there develops to a
colony which then surrounds itself with a
. , tough membrane. From this original posi-

Xuclear staining with i per cent, chromic acid ,1 i_ i. i j .cii j

and methyiene blue ; k. the nucleus with tion there branches out a lustrous sac, filled
a vacuole; ; /. the with bacteria, which turns towards the bark
cells and branches out amongst them. As
a result of this advance towards the centre

FIG. 62.— Section through the bacteroidal
tissue of Latbyrus sylvestris.

its nucieoius (lying i

°,f
central vacuole ; bact. the bacteroids.

(4/ter Beyerinck.) of the root-hair, the cells thereof are incited

to rapid increase and become densely

crowded, in consequence of which they assume a polygonal outline, and constitute

the bacteroidal tissue already mentioned (Fig. 62). The plasma of these cells,

with its fungoid enclosures, has been termed mycoplasma by A. B. FRANK (VI.).

Before tracing the career of the bacteria any further, a few explanatory

THE BACTEROIDS 267

words must be added concerning their mode of arrangement (already referred to
as a " sac ") at the time of penetration into the cells. The.-e branching bacterial
colonies, enveloped, as has been stated, in a membrane, were for a long time
misunderstood. Beyerinck at first considered them as the surplus matter from
the division of the nucleus of the nodule-cells (Kerntoimenfaden). By other
workers they were styled mucus threads, fungoid hyphte, plasmodial cords, &c.
Frank, for a long time, held them to be the mycelium of an independent higher
fungus, differing from the nodule bacteria and belonging to the genus Schinzia,
and consequently named them /Schinzia leguminosarum. Subsequently Frank,
by the new name infection threads, indicated their true nature, first recognised
by Prazmowski. Frank, moreover, in his studies on the immigration of the
nodule bacteria in the root, arrived at results differing in several points from
those of Prazmowski. We cannot, however, go further into this matter, which

FIG. 63.— Development of bacteria to bacteroids from the FIG, 64. — Keticulated band of bac-

meristem of a root-nodule ol Vicia sativa. Explanation teroids from the nodules of Vicia

in the text. Faba.

Magu. 700. (After Beyerinck.) Magn. 700. (After Beyerinck.)

is one more of botanical than mycological interest. He also discarded the name
bestowed on these root-dwelling organisms by Beyerinck in favour of the term
Rhizobium leguminosarum. The integument of the threads is not, as erroneously
opined by Frank, a product of the plasma of the nodule-cells, but is formed by
the union of the swollen membranes of the outer layers of the bacteria con-
stituting these filamentous colonies (or zooglcea). As was shown by A. KOCH (IV.)
and M. W. BEYERINCK (XVI.), it is stained blue by zinc iodo-chloride, and
therefore consists of a substance allied to cellulose. The structure and progress
of the infection threads in the nodule-cells can be readily recognised in sectional
preparations, stained by a solution of equal parts of fuchsine and methyl violet
in i per cent, acetic acid. This colours the plasmal contents and membrane of
the nodule-cells blue, the bacteria of the infection threads being stained red,
whilst the membrane of the latter remains uncoloured. It should be mentioned,
in conclusion, that these threads of capsuled bacterial colonies are but rarely
found in the nodules of lupins.

The bacteria which have gained access now develop in the cells of the
bacteroidal tissue, and, finally, under the influence of the surrounding protoplasm,
become modified into involution forms termed bacteroids, rich in albumen and no
longer capable of reproduction. This morphological change is represented in
Fig. 63, in an example taken from the nodule of Vicia satii-a shown in Fig. 60.

A section cut near the lower extremity meets the youngest meristem, where
only long rods (u) in a high state of development are to be seen. A little higher

268 FIXATION OF FREE NITROGEN BY BACTERIA

up, in older layers (v), the commencement of branching is already discernible?
and is found in a more forward state in a still higher position (x). Finally, in
a section across the cells of the fnternal tissue of the nodule in the direction y
(Fig. 60), none but variously shaped bacteroids (y} for the most part united to
form reticulated bands (shown in Fig. 64) occur. After attaining this condition
the bacteroids are soon dissolved by the surrounding cell plasma and disappear.
However, before this occurs, small globular vesicles of an unknown nature, which,
however, should not be regarded as endospores, not infrequently appear in the
interior of this formation. Little can as yet be said of the chemical composition
of the contents of these bacteroids. Micro-chemical reactions, however, indicate
that the greater part is composed of albumen. Certain enclosures are also
frequently observed, A. B. FRANK (VII.), for instance, having noticed such
bodies in the bacteroids of individual nodules of peas. He regarded them as
amylodextrin, the discovery leading him to the opinion that two kinds of nodules
develop on these Papilionacece : albumen nodules and amylodextrin nodules. A
subsequent investigation of this matter by H. MOLLER (I.) showed, however,
that these doubtful enclosures do not consist of carbohydrates, but of waxy
or fatty substances; that they are also to be found in the bacteroids of the
" albumen nodules " ; and that, moreover, there is but little probability of the
existence of dimorphism in the root-nodules of the pea, since these enclosures
are also occasionally met with in the bacteroids of the nodules of other Legu-
minosce (e.g. Trifoliunt repens).

Concurrently with the reproduction and transformation of the bacteria
marches the development of the nodule, which not only increases in size, but
also becomes richer in nitrogenous compounds. This gradual increase in the
percentage of combined nitrogen in the nodules and the relative proportion of
this substance there present, as compared with other parts of the root, were
quantitatively investigated by J. STOKLASA (II.). From his results a few
figures have been collected into the subjoined tables, which refer to yellow
lupins : —

Nitrogen Content in the Dry
Matter from : —

At Flowering'
Time.

At Fructifi-
cation.

In Fully
Ripe Fruit.

Koot-nodules
Roots free from nodules ....

Per Cent.
5-2
1.6

Per Cunt.

2.6

1.8

Per Cent.
1-7

1.4

That this nitrogen of the nodules was chiefly in the form of albumen is
revealed by the following table : —

In Nitrogen as : —

Percentage Content of the Dry
Substance of the Nodules.

Albumen.

Amides.

Asparagin.

Per Cent.

Per Cent.

Pur Cunt.

Flowering time

3-99

0-35

0-34

Ripened fruit

1-54

0.15

truces

The figures just given are surpassed in two analyses made by A. B. Frank,
according to which a few of the pea nodules examined by him contained 6.94 per
cent., and those from the dwarf bean 7.44 per cent, of nitrogen, corresponding —

CLOSTRIDIUM PASTEURIANUM 269

on the basis of the factor 6.25 — to an albumen content of 43.4 and 46.5 per cent.
As already remarked, the bacteroids — which, when they exhibit the aforesaid
vesicles, are frequently termed vesicular bacteroids — are finally dissolved by the
surrounding plasma, which is thus enriched with albuminoids, and is then diffused
through the plant. The cell contents of the (formerly reddish but now greenish
grey) bacteroidal tissue gradually vanish and are dispersed into other parts of
the plant, and the nodule consequently shrivels up. The commencement of this
process of evacuation of the cells is indicated by the appearance of a central
vacuole of gradually increasing size (Fig. 62).

Neither the transition of the nodule bacteria into bacteroids, nor the final
dissolution of the latter, goes on simultaneously in all parts of the individual
nodules, which, moreover, are themselves of different ages. Some of the bacteria
escape the converting influence of the cell plasma by remaining within the
protecting mucinous capsule (membrane) of the infection threads. Hence, it
happens that in the autumn large numbers of the bacteria are still present in
an active condition within the nodules. In the subsequent putrefaction of the
latter the organisms are set at liberty, pass the winter in the soil, and then act
again as nodule-formers in the following spring.

The bacteroid stage is not reached in every case, the plasma of the nodule
cells being unable in many instances to utilise the microbes. In such event it
swarms with bacteria alone, which then act solely as parasites towards their
host, and consequently the latter derives little or no benefit from the formation
of nodules. This phenomenon is termed by Beyerinck "an overgrowth of
bacteria." According to the observations of NOBBE and HILTNER (I.), it occurs
when the inoculation is performed with bacteria that have been grown on
artificial media for a long time.

§ 196.— Clostridium Pasteurianum.

The fact recorded in § 194, that absorption of nitrogen is not effected by
the superior (aerial) parts of the Leguminosce, led us to investigate the root
nodules more closely. We then observed that the possession of these appendages
enables the plant to grow well and ripen even in soils destitute of nitrogen,
and we furthermore learnt that the production of these nodules is directly
connected with the activity of certain special bacteria — the nodule bacteria.
However important and satisfactory this result may be to agricultural practice,
it still leaves unsolved the (from a theoretical standpoint) main question, " By
what is the free nitrogen fixed ? " Is this effected by the nodule bacteria
themselves, or do they merely exert a stimulative action on the plasma they
inhabit, which is thereby empowered or spurred to unwonted activity ?

The latter question cannot be answered by experimental means, since for
that purpose the chemical activity of the bacteria would need to be eliminated
and only their assumed stimulating action allowed to operate. However, the
wished-for decision may be expected from the experimental solution of the
previous question, and attempts may therefore be made to ascertain whether
the nodule bacteria are of themselves capable of fixing free nitrogen. On this
point BEYERINCK (XVII.) has conducted researches in nutrient solutions with
pure cultures of Bacillus radicicola, and in this manner ascertained that during
a period of two months an increase of 9-18 mgrms. of combined nitrogen
occurred per litre. Nevertheless he considers that this discovery leaves the
question still undecided, and consequently a repetition and extension of his
researches in this direction is highly desirable.

The exercise of the still imperfectly proved capacity of Bacillus radicicola
for fixing free nitrogen is opposed by a considerable obstacle consisting in the

2/o FIXATION OF FREE NITROGEN BY BACTERIA

difficult accessibility of the interior of the nodule (the chief seat of this fission
fungus) to the gas to be fixed. A. B. FRANK (VIII.) has ascertained that the
air passages (intercellular spaces) of the nodules lead only as far as the cambium
layer, but not into the bacteroidal tissue; consequently atmospheric nitrogen
cannot penetrate directly to this tissue. On this point R. BOUQUET (I.) has
expressed an opinion worthy of further investigation. According to him, it
is the water absorbed by the roots and exhaled through the leaves which
conducts and gives up free nitrogen in solution to the root cells, where it is then
combined by the plasma.

If, then, we must consider the question, whether the fixation of free
atmospheric nitrogen is effected within the nodules, to be still imperfectly solved,
it is, on the other hand, clearly proved that such an operation goes on in
(uncultivated) soil. This was first observed by M. BERTHELOT (I.) in 1885, and
he subsequently proved that this phenomenon is not occasioned by exclusively
chemical affinity, but is due to the activity of micro-organisms. Opinions on the
nature of the organisms were at first divided. TH; SCHLOESING, jun., and
EM. LAURENT (I.) ascribed the fixation of nitrogen to certain lower algse
(Conferva, Oscillaria, Nitzschia), and mosses (Bryum, Leptobryum). Doubt was
cast on this hypothesis by A. GAUTIER and R. DROUIN (I.), and P. KOSSOWITSCH
(II.) disproved it — so far as the algae are concerned — by the aid of pure
cultures. BERTHELOT (II.) then showed that the activity of fungi — both
Eumycetes (such as Aspergillus niger, Alternaria tenuis, <fec.) and Schizomycetes —
is in question.

For more precise investigations on this matter we have to thank S. Wixo-
GRADSKY (II.), who described a fission fungus belonging to the group of butyric
acid bacteria, and bearing the name of Clostridium Pasteurianum. This occurs
in the form of rods, 1.2 p. broad and 5 /j. long, each producing an endospore,
the cells thereby swelling up to the clostridium form, and storing up in their
interior (though not at both poles) substances that are stained a deep blue-blade
by iodine. The ripe spore escapes through the wall of the mother-cell in a
longitudinal direction. The great resemblance between this clostridium and
the butyric acid bacteria described in a previous section is not only morphological,
but also extends to the fermentative capacity. For example, Clostridium
Pasteurianum acts on sugar in such a manner that both volatile acids : butyric
acid and acetic acid (4 : i), and gases: carbon dioxide and hydrogen (60-75
per cent, of H. by volume) are formed. The importance to us of this fission
fungus on the present occasion consists in its behaviour towards nitrogen,
which gas it absorbs from the atmosphere, fixes it and employs it in the elaboration
of organic substances. The energy necessary thereto is supplied and liberated
by the decomposition of sugar ; consequently it is easy to understand that a
definite relation exists between the amounts of sugar fermented and of nitrogen
combined. This ratio was determined by Winogradsky as 2.5-3 mgrms. of
nitrogen to 1000 mgrms. of dextrose. This element (N.) when in a state of
combination is not only valueless to Clostridium Pasteurianum, but when
present in large quantity even injurious thereto. For the cultivation of the
microbe — which cannot be carried out on the ordinary nutrient media (gelatin,
bouillon) in use, though it grows on sliced potatoes — use is made, preferably,
of an aqueous solution containing i gram K3P04, 0.5 gram MgS04, 0.01-0.02 gram
NaCl,FeS04,MnSO4, a little CaCO3 (for fixing the acids), and 20-40 grams of
dextrose per litre. Clostridium Pasteurianum is strictly anaerobic, and in the
soil is therefore obliged to rely on the co-operation of aerobic fission fungi,
which remove the injurious oxygen from its sphere of influence, surround it
with an atmosphere of nitrogen, and as a reward for this service have the
opportunity of consuming the nitrogenous substances elaborated and excreted

CLOSTRIDIUM PASTEURIANUM 271

by the Clostridium. Winogradsky observed two species of such assistant
organisms, detailed mention of which can, however, be omitted, it being sufficient
for our purpose to have referred to this new case of symbiosis.

Whether the just-named faculty of fixing nitrogen is also possessed by the
organisms akin to Clostridium Pasteurianum, e.g. Prazmowski's Clostridium
butyricum, still remains undermined. WIXOGRADSKY (III.) examined fifteen
species of soil bacteria in this connection, with only negative results. In the
higher fungi K. PURIEWITSCH (I.), in an improved continuation of Berthelot's
researches, showed that both Aspergillus niger and Penicillium glaucum fix free
nitrogen. Their potency is, however, but slight, and is not to be compared
with that of Clostridium Pasteurianum, since it is not manifested in media
devoid of nitrogen. According to H. JUMELLE (I.), Spirillum luteum, also, is
capable of thriving in media free from nitrogen.

The proof of the fact that the fixation of free nitrogen occurs in the soil
affords a new possible explanation of the activity of the nodule bacteria of the
Leguminoscf, viz., that the actual absorption of the free nitrogen goes on outside
these root formations ; substances unassimilable by higher plants being formed,
and then converted into an assimilable form by the nodule bacteria. This
provisional interpretation, which will not encroach on future research, is not
without its analogies; one need only recall the Mycorhiza, discovered by Frank
on the rootlets of the majority of forest trees (Cupuliferce, Conifarcp), heaths
(A'/v'cYtcer*), &c., and in regard to which the most important discoveries made up
to the year 1888 will be found briefly reviewed in a treatise by F. BEXECKE (II.).

The great importance to General Physiology of the researches reported
above will be readily appreciated, since they have brought to our knowledge
organisms which dispense with combined nitrogen as a food-stuff, and are
consequently of the greatest importance in the economy of Nature, by revealing
the means for maintaining the circulation of nitrogen. Large quantities of
this element are daily liberated by the activity of both de-nitrifying and nitrifying
bacteria, and restored to the atmosphere, the result being that the stock of
nitrogenous compounds, so essential to the nutrition of all other plants and all
animals, becomes reduced. To compensate and reverse this loss is the task of
the nitrogen-fixing fungi, which, for this reason, must be regarded as the
benefactors and foster-mothers of all other living creatures.

SECTION IX.
OXIDISING FERMENTATIONS.

CHAPTER XXXIV.

THE IRON BACTERIA.

§ 197.— Morphology of the Genera Crenothrix and Cladothrix.

THE group of Schizomycetes known as thread bacteria can be divided into two
sub-groups. To the one of these belong the two genera (described in the follow-
ing chapter) which store up globules of free sulphur in their cells. The
appearance of these organisms is so characteristic that a skilled eye can detect
them with ease. On the other hand, the five genera of the second sub-group
lack this peculiarity. In three of these latter, viz., Streptothrix, Leptothrix, and
Cladothrix, the fission of the cell takes place in one direction only ; con-
sequently, rods are formed. In the other two, however, viz., Crenothrix and
Phragmidiothrix, the extremity of the thread is broken up, by division in all
three directions of space, into coccus-like cells. No perfectly satisfactory morpho-
logical description, or definite separation of these five genera into species, has
yet been drawn up on this basis. This defect — to which reference has also been
made by C. SAUVAGEAU and M. RADAIS (I.) — therefore restricts us to the con-
sideration of individual examples. A typical one is afforded by Crenothrix
polyspora. This thread bacterium was first described by F. COHN (XI.) in 1870,
and was subsequently also named Crenothrix Kiihniana. It is illustrated in
Fig. 65, which shows that the threads are sessile, each of them consisting of a
single row of short cells held together by a common tubular integument, called
a sheath. This latter is formed by the splitting of the cell membrane into two
layers, the outer one of each cell becoming merged into the adjacent membranes
above and below, and thus forming a uniform tube in which the cells can move
up and down. The threads are not cylindrical, but increase in diameter from
about half-way along their length towards the free end, so that the cross-sectional
diameter varies from 1.5 to 5 p. Moreover, as may be seen from the figure, the
various segments of the thread are of unequal length. As the cells multiply
rapidly, the upper members are forced out of the sheath, but are, for the most
part, already subdivided — by the development of partition walls in all three
directions of space — into small rounded cells, which thereupon make their
escape. The new cells vary in size according to the rapidity of this process of
subdivision, and are correspondingly distinguished by Zopf as micrococci (r),
and macrococci (q) (Fig. 65). Cohn proposed the terms microgonidia and
macrogonidia, because their method of formation bears a slight apparent re-
semblance to the endospores (known as gonidia) of certain Eumycetes, with
which we shall become acquainted in the second volume. The dimensions of these
cocci vary between i p and 6 /*. Their cell-walls swell up readily, and unite to
form zoogloea (f) up to i cm. in diameter. Under favourable conditions the
cocci then grow, by repeated subdivision and sheath-formation, into the thread

272

THE GENERA CRENOTHRIX AND CLADOTHRIX 273

forms already described. ZOPF (VI.) observed that in many cases this occurs
before they have quitted the sheath of the parent thread ; and in this manner
tufted forms, similar to that shown in Fig. 66, are produced. Otherwise, the
sheath is gradually emptied of
its contents, and then collapses
limply and withers up.

If we disregard the excep-
tions just named, wherein the
germination of the cocci pro-
ceeds within the parent thread,
and so causes it to present a
branched appearance, it may be
said that the genus Crenothrix
appears only in the form of
single, unbranched, filamentous
chains of cells. This charac-
teristic suffices to distinguish
this genus from that bearing
the name of Cladothrix, the
best-known species of which is
Cladothrix dichotoma. As this
name implies, we have here to
do with a forked thread, such
as shown in Fig. 67. This
false branching is produced in
the following manner : — One
of the rod-shaped joints of
the thread turns aside, and,
growing beyond its next higher
neighbouring cell, repeatedly
subdivides and forms a new >{. ^N

thread, on which a similar false
branching may also develop.
Hence there ensues a forma-
tion the internal structure of
which is represented diagram-
matically in Fig. 68. In many
species (not depicted here)
the sheath becomes greatly
thickened at the base, where
it attains a diameter many
times exceeding that of the
cells it encloses, but tapers oft'

gradually towards the free ex- n_2. macrococci, and r. micrococci splitting off. Magn. about
tremity. Cladothrix dichotoma 600.

al«n rliffpr.! from +>IA ahnvp a-e. reproduction of the cocci ;/. colony (zooglo3a) of cocci ;

also dittero trom the above- ^ ^^ uatura, gize . h game beginnin,, to gcrminate.

mentioned thread bacteria in Magn. 600. (After Zopf.)

another important particular,

viz., by the production of rod-shaped roving cells, called rod gonidia. which

develop at the extremity of the threads, and, after being initially embedded in

the swollen sheath (Fig. 69), are liberated, wander about, and finally settle down

to form new threads by subdivision and sheath-formation.

Allied to Cladothrix dichotoma — though not, as ZOPF (VII.) opined, belonging
to the morphological cycle of this organism — is Leptothrix ochracea, which was
i s

FIG. 65. — Crenothrix polyspora
thread forms of different diameter.

274 THE IRON BACTERIA

first described by KUtzing. A second sheath-forming thread bacterium, allied
to the genus Cladothrix, was also examined by him, and named Sphm-otilus natans.
It is still too imperfectly known to be dilated upon here, although ED. EIDAM (II.)

FIG. 66. — Crenothrix polyspora.

Germination of the cocci (g) within the sheath of the parent
thread. Magn. about 600. (After Zopf.)

also occupied himself with it. Associated with this colourless species is a second
(coloured) species, discovered by W. ZOPF (VIII.) in a Silesian river receiving
the drainage from a sugar-works. The cells of this, Sphcerotilus roseus, contain
a yellow and a red colouring-matter, which circumstance is of itself sufficient to
distinguish it from all other (colourless) species of thread bacteria hitherto
mentioned.

PHYSIOLOGY OF THE IRON BACTERIA 275

The genus Phragmidiothrix, one species of which — Ph. multiseptata — was
discovered by ENGLER (I.) in the so-called "dead ground" of the Bay of Kiel,
differs from all the foregoing in the absence of sheath formation.

§ 198.— Physiology of the Iron Bacteria.

It is not always possible to discern the structure of these thread bacteria
without some preliminary treatment, because in most cases the sheaths are

\
\

\

s I

H

N

V !'

\

\

y

FIG. 67.— Cladothrix dichotoma. FIG. 68.

Portion of a thread with several branching' forks. Diagram of the false branching of

Stained with fuchsine solution, and thus reveal- Cladothrix.

ing the articulation into long rods. Magn.
540. {After Zopf.)

surrounded and permeated by red-brown masses of ferric oxide. These deposits
and accumulations are characteristic of these plants, and facilitate their detection
and discovery. Since other fungi exposed to the same conditions do not
exhibit this peculiarity, Cohn formed the opinion that its occurrence is intimately
connected with the vital activity of the thread bacteria, the ferric oxide being
deposited in their sheathing in the same way that silica is accumulated in the
plates of the diatoms We are indebted to 8. WINOGRADSKY (IV.) for proving
the correctness of this view, and for refuting the opinion of Zopf that the deposi-
tion is purely mechanical ; and we have to thank the same observer for the more
intimate investigation of the process in question.

The species Crenothrix polyspora, Cladothrix dichotcwa, Leptothrix ochracea,&,c.,

276 THE IRON BACTERIA

occur in particular abundance in such waters as are rich in iron, not in the form
of oxide, but as the soluble bicarbonate of the protoxide, FeH,(C03),. Ferrugin-
ous springs, ascending from the deeper strata of the rocks, bring up this substance
in a ready-formed state ; and in the water of the
upper strata it is produced by the decomposition of
vegetable matter, the iron, both in this and in the
water itself, being converted during cellulose fermen-
tation into the hydrocarbonate. This compound is
then absorbed by osmosis into the bacterial cell, where
it is split up by the plasma and oxidised, according to

. ' ^r ^JC^K'

g « ,

2FeC03 + 3H20 + 0 = Fe2(OH)6 + 2C02

The ferric oxide is then stored up in the sheath, to
which it imparts a coloration, initially pale yellow but
gradually changing to dark brown. Freshly precipi-
FIG. 69. tated ferric hydroxide is, as we know, somewhat

Clatlothrix dichotoma. soluble in water, but afterwards gradually passes

Subdivision into roving rods at the into a condition in which it is only attackable by
extremity of a thread, s. the weakacids. This change can be traced in the young
rodse"vftif 'their5 (lat^ralTcllia Bacteria, the colouring-matter in the yellow sheath
(c). Magn. looo. (After A. being at first extractible by washing with water
Fischer.) Cilia staining. containing C02 in solution. Subsequently, however,

dilute hydrochloric acid must bo resorted to, and

at a still later stage even this solvent is powerless to extract the brown deposit.
A very fine and fast blue stain can be produced in young sheaths (the iron in
which is still soluble in acid) by exposing them to a mixture of hydrochloric acid
and yellow prussiate (potassium ferrocyanide), whereby the hydroxide is dissolved,
immediately converted into Berlin blue, and re-precipitated. In older threads
the deposits of ferric oxide increase to a thick incrustation, and entirely conceal
the structure of the cells.

Winogradsky discovered that these bacteria thrive only when ferrous carbonate
is available, and that growth is arrested directly the nutrient medium contains
no iron, or only iron in the condition of oxide. This fact entails the conclusion
that the life of these bacteria is mainly sustained by the energy liberated during
the oxidation of ferrous oxide to ferric oxide. Consequently, these organisms
rightly deserve their name of " iron bacteria." According to the discoveries of
H. MOLISCH (I.), iron can be replaced in this oxidation process by the chemically
allied metal manganese. These bacteria require but a very small quantity of
other nutrient materials, an addition of, e.g. a few thousandths of i per cent, of
sodium acetate to ferruginous water being entirely sufficient to bring them to
a state of perfect development. This inexigency is also indicated by the
observation, made by 0. ROSSLEE (I.), that Cladothrix polyspora can be grown
on bricks moistened with a little ferrous sulphate solution. In 1894 M. BtSGEN
(I.) succeeded in obtaining pure cultures of Cladothrix dichotoma on gelatin.

The decomposing power of these organisms is very great, the amount of
ferrous oxide oxidised by the cells being a high multiple of their own weight.
This high chemical energy on the one hand, and the inexacting demands in the
shape of food on the other, secure to these bacteria an important part in the
economy of Nature ; the enormous deposits of ferruginous ochre and bog-iron
ore, and probably certain manganese ores as well, being the result of the activity
of the iron bacteria.

Moreover, they make their presence evident not only in natural water basins,
but in all other places where water rich in iron is to be found in quantity.

PHYSIOLOGY OF THE IRON BACTERIA 277

Consequently, these organisms may develop into an- actual nuisance to water-
technicists by penetrating into the clarifying reservoirs and delivery pipes, and
there growing so vigorously as to completely obstruct the passage of the water,
and thus interrupt the service of distribution. Many towns deriving their
water-supply from a soil or river water rich in iron have suffered from this
nuisance ; Lille, for example, as reported by GIARD (III.), and Berlin, as
mentioned by W. Zopf in his (treatise already referred to. In the waterworks
at Lake Tegel, from which the greater part of Berlin derives its supply, these
bacteria (and especially the " well-pest," Crenothrix polyspora) flourished so
luxuriantly that they constituted more than one-half of the layer of sediment
(about forty inches in depth) gradually collecting at the bottom of the reservoirs.
One means of obviating this nuisance (although not practicable on a small scale)
is by freeing the water from its content of ferrous oxide, for which purpose
P. WOLTERING and A. SASSEN (I.) recommended a method (which is said to
answer) consisting in passing the water through coke towers where the ferrous
oxide is converted into ferric oxide, the latter being then removed by suitable
strainers.

Finally, Cladothrix odorifera merits brief consideration. Every one is
acquainted with the peculiar smell of the soil, more particularly when moist,
e.g. after a brief shower of rain. According to the researches M. BERTHELOT
and G. ANDRE (I.), this odour is due to a neutral organic compound, present in
the soil and volatilising at the same time as water vapour. The producer of
this (not yet precisely identified) compound has now been recognised by RULLMANN
(I.) in a new species of bacterium, viz., Cladothrix odorifera. It occurs along
with Cl. dichotoma in the soil, and, like the latter organism, can be cultivated
on nutrient gelatin ; but whereas the colonies of Cl. dichotoma are inodorous,
liquefactive, and turn the substratum brown in a short time (two days), those
of CL odorifera, on the other hand, retain their chalky white appearance and
evolve the aforesaid earthy smell. RULLMANN (II.) also found that this species
is capable of withstanding the influence of drought and poisons, being able to
bear exposure for twenty-four hours to a i : 1000 solution of corrosive sublimate.
Like its aforesaid congener, Cl. odorifera possesses considerable oxidising power,
though this is manifested by the transformation of ammonia into nitric acid,
and not by the conversion of ferrous into ferric oxide. This mode of action
is not peculiar to this organism alone, but is shared in a still higher degree by
a group of bacteria whose acquaintance we shall make in chapter xxxvi.

The iron bacteria are not the only Schizomycetes capable of liberating the
energy necessary for the maintenance of their existence from inorganic bodies.
In the next two chapters we shall make the acquaintance of fresh natural groups
and other processes similar to those described ; thus justifying the title of this
concluding section.

CHAPTER XXXV.

THE SULPHUR BACTERIA.

§ 199.— Morphology of the Genus Beggiatoa.

THE sulphur bacteria, so called on account of their peculiar properties, differ
both in structure and external appearance from the filamentous bacteria described
in the preceding chapter. They may be divided into two sub-groups, one of
which forms the species classified by Engelmann as purple bacteria, and already
noticed in chapter xiii. on account of their behaviour towards light. The other
sub-group of the sulphur bacteria, which assume the form of long threads, will
now be described.

It will be useful to preface this description with a few hints concerning arti-
ficial cultivation and reproduction for the purposes of investigation. The sulphur
bacteria are seldom absent in marsh water, although their number is frequently
so small as to elude the inquiring eye of the microscopist. In order to cause
them to increase, the conditions of the environment must be rendered favourable,
and with this object the simple method proposed by their careful observer, S.
WINOGRADSKY (V.), is employed. A few cuttings of the fresh root-stock of the
flowering rush, Butomus umbellatus (found in every pond, and by no means rare
on river banks), are placed, along with the adherent mud, in a deep vessel con-
taining 3-5 litres (about a gallon) of water, a couple of grams of gypsum being
added, and the whole left to stand uncovered at room temperature. After five
to seven days the liberation of sulphuretted hydrogen will already be noticeable,
the gas being disengaged by various species of fission fungi present in the mud
and acting on the gypsum. In this manner the ground is prepared for the
sulphur bacteria also present, and the latter then develop rapidly. At the end
of three to six weeks their presence can be ascertained by the aid of the micro-
scope, and they gradually increase to such an extent as to be recognisable by the
unassisted eye. Generally, this diversified mixture of sulphur bacteria is not
deficient in the red species as well, but the colourless long thread forms are most
plentiful.

Two genera were more closely investigated by Winogradsky. The one of
these bears the name of Beggiatoa, given by TREVISAN (I.) in 1842 in honour of
the Italian physician F. S. Beggiato of Vicenza, who, in 1838, published a com-
munication on the flora of the sulphur springs of the Euganean Hills, near Padua.
The species of this genus occur as actively motile cylindrical filaments, which
may attain a length of i c.m. and more. The breadth is always constant in each
separate species, and thus affords a means for differentiating between them.
Under favourable conditions of nutrition, and especially in presence of sul-
phuretted hydrogen, the interior of the individual threads (Fig. 70 a) is seen to
be well stocked with roundish, highly refractive granules, i.e. the sulphur
granules described later on. In this condition the transverse cell walls are
indiscernible or only detected with difficulty, as will be gathered from Fig. 70 c,
which shows the same thread after it has lost its enclosed sulphur granules by a
long sojourn in water devoid of sulphuretted hydrogen. Moreover, the length
of the cells varies in the different species. If this organism be deprived of the

278

MORPHOLOGY OF THE GENUS BEGGIATOA 279

said gas, which is indispensable to its continued existence, then the threads
begin to break up (Fig. 71), the contents — except a thin coating attached to the
walls — vanish, and they finally perish. No success has attended the search for
spore formation in the Beggiatoa. The most abundant species of this genus is
Beggiatoa alba, the threads of which are 2.8-2.9 (j. in thickness, whilst the length
of the individual members varies between 2.9 and 5.8 p, the shortest of them
being thus symmetrical. A second species, with a diameter of 1.6-1.7 /*, the
length of the separate cells being 4-8.5 /.i, has been named Beyglatoa media ; and

.

FIG. 71.— Beggiatoa alba.

Moribiind through lack of H2S. Thread falling
apart into its short members, which thereupon
assume a rounded form. Magn. 900. (After
Winogradsky.)

FIG. 70. — Beggiotoa alba.

The same portion of thread under different

conditions of existence.

«. in a medium rich in H2S ; the thread is densely
packed with sulphur granules ; b. after twenty-
four hours' sojourn in a liquid devoid of HgS ;
only a few sulphur granules remain ; c. at the
end of a further forty-eight hours ; sulphur
totally disappeared, transverse walls now visible,
contents of individual cells granulated. Magn.
900. (After W'inofjradsky.)

FIG. 72. — Terminal portion of ihreads of (x)
Beggiatoa media and (y) B. minima.
Magn. 900. (After Winogradsky.)

a third kind, whose diameter is only 0.8 p, is called Beggiatoa 'minima. Both
these species are shown in Fig. 72, magnified to the same extent as the first-
named species. In addition to these there is still a large number of species
whose threads vary in diameter between the above limits. Compared with all
these the Beggiatoa mirabilis noted by COHN (XII.), Warming, and Engler, but
not yet more minutely examined, the threads of which are said to attain a
breadth of 30 p, is gigantic. According to Winogradsky, the breadth of the cells
of any given species is — to emphasise this point once more — unalterable.

The growth of these Schizomycetes is very slow, a thread requiring at least
twenty-four hours to double its length. They are extremely susceptible, even
merely the grip of the forceps being fatal. For this reason they have to be
sucked up by means of a small tube, for purposes of examination, and protected
from the pressure of the cover-glass by introducing splinters of glass, &c., into
the liquid.

280 THE SULPHUR BACTERIA

§ 200.— The Species of the Genus Thiothrix,

which has been newly established by Winogradsky, differ from Beggiatoa by the
absence of free motility, they being sessile, i.e. attaching themselves at one '
extremity by means of a mucinous sucker to the walls of
the culture vessel, the cover-glass of the microscopical
preparation, to stones, remains of plants, and similar
quiescent substrata in the situations where they occur
naturally; whilst the other end extends into and grows
in the liquid. Such a one is shown in Fig. 73. In this
genus, also, the articulation of the threads is ordinarily
concealed by the abundant content of sulphur, but if the
latter be washed out with absolute alcohol and the cells
stained, e.g. with fuchsine, the transverse walls are plainly
revealed. The length of the joints gradually increases
towards the free end, as will be seen from the subjoined
measurements given by Winogradsky : — Length of joint
near the point of attachment, 4-8.5 p; at the apex,
FIG. 73.-Thiotrix nivea. S~I5 /*• However, there is no scarcity of considerably
. shorter cells. So far as the breadth of the threads is
Group of young threads , ,, . ... , ,. ,. .

with one end firmly at- concerned the above -conditions are reversed, the threads
tached to the substratum tapering off towards the free end, where, for example,

Z^wZ™£». their diameter is on]y '-5 * compared with 2.0 M at the
900. (After Winogradsky.) base. Consequently the cells are more slender towards
the tip.

A second characteristic point of difference from the genus previously described
is the appearance of a (merely slight) sheath, whereby the moribund members
are partly held together, whereas the Beggiatoa threads at this stage break into
short fragments and finally into separate cells.

A third characteristic of the genus Thiothrix is the dislocation (termed
conidia-formation by Winogradsky) of the uppermost joint of the thread. The
rod-shaped cell, thus loosened from the chain, crawls a short distance along the
solid substratum, then develops a mucinous sucker and grows into a new thread,
from which in turn conidia subsequently wander and settle in the vicinity, the
result being the formation of the whitish, tufted, thread colonies characteristic
of Thiothrix.

Here also the thickness of the threads constitutes a criterion for the classi-
fication of species. One of them, named by Winogradsky Thiothrix nivea, has a
diameter of 2-2.5 ** near the base, 1.7 p. in the middle, and 1.4-1.5 p, at the tip.
In a second species the diameter is almost uniformly i.o-i.i p throughout the
whole extent of the thread. It is known as Thiothrix tennis, and is probably
identical with a fission fungus discovered by ENGLER (I.), in the so-called " dead
ground " of the Bay of Kiel, and which he held to be a Beggiatoa and called by
the specific name B. alba var. universalis. The threads of a third species (Thio-
thrix tenuissima), from a sulphur spring at Adelboden (Switzerland), measure
only 0.4-0.5 p. in breadth. W. ZOPF (VII.) regarded the sessile sulphur bacteria
as belonging to the morphological cycle of the Beggiatoa, and named them
" sessile Beggiatoa," until Winogradsky proved that two distinct genera are here
in question.

As will be shown later on, the life of the sulphur bacteria is indissolubly
connected with the presence and availability of free oxygen. In the mode of
satisfying their needs in this respect the two genera differ. The Beggiatoa,
being endowed with the power of locomotion, can more readily accomplish this

NON-FILAMENTOUS SULPHUR BACTERIA 281

object by their ability to proceed at will to the surface of the liquid. Conse-
quently this species gains the upper hand in stagnant or quietly flowing waters,
in which they search about so eagerly that very little of the oxygen diffusing into
the water can reach the bottom where the Thiothrix species rest. The latter,
however, have the advantage in rapid running water, the loose Beggiatoa species
being washed away by the current. Jn either event, whitish mucinous masses
highly characteristic of sulphur springs accumulate in time — e.g. those of
Bareges in the French Pyrenees — and are known in France as baregine or
glairine.

§ 201.— Morphology of the Non-Filamentous Sulphur Bacteria.

Several red species of these organisms are already known to us (§ 91), viz.,
Chromatium (Monas} Okenii, Monas Warmingii, Spirillum rubrum., ^p. volutans,
Ophidomonas sanguined, Rhabdomonas rosea. These are again shown in Figs. 74 to
78. It was remarked in § 68 that Ray Lankester had assumed all these

FIG. 74.

Chromatium Okenii.
Magn. 600. (After F. Cohn.)

FIG. 75.

Rhabdomonas rosea.
Magn. 600. (After F. Colin.)

FIG. 76.

Monas Warmingii.
Magn. 600. (After F. Colin.)

FIG. 77.— Spirillum volutans.
Magn. 600. (After F. Cohn.)

FIG. 78. — Ophidoiuouas sanguine
Magn. 600. (After F. Cohn.)

organisms to be merely special forms of one species for which he proposed the
name Bacterium rubescens, The basis for this assumption was, however, a very
insufficient one, since it rested principally on the identity (which, moreover, was
not satisfactorily demonstrated) of the red colouring-matter, peculiar to these
organisms, and which received from Lankester the name bacterio-purpurin.
This investigator was supported in his views by Warming (in 1875), wno °n
his part classified a large number of the red sulphur bacteria examined by him
into a single species, viz., Bacterium sulfuratum. Zopf (in 1882) went still
farther than either by defining all these organisms as special forms of growth

282 THE SULPHUR BACTERIA

of a single species of thread bacterium, viz., Beggiatoa roseo-persicina, which,
under certain circumstances, was said to appear as long threads (Leptothrix),
and under others as fractions of such threads, viz., as Monas, Spirillum, &c.,
capable of developing once more into threads.

The re-investigation of these discoveries (which were not made with pure
cultures) by Winogradsky led to the refutation of this assumed variability of
form, and also to the discovery that the above-named red sulphur bacteria are
not capable of progressive development, i.e. of growing into thread form. Some
little doubt still prevails as to the existence of retrogressive development, i.e.
the dismemberment of short cells from filamentous red Beggiatoa species. In
contradiction of Winogradsky's statement that the filamentous sulphur bacteria
(Beggiatoa and Thiothrix) are invariably colourless, and consequently cannot
throw off coloured cells, W. ZOPP (VIII.), in a subsequent communication
(1895), reported the existence of red Beggiatoa species which become dismembered
into short (sulphur-bearing) cells. The question must consequently be considered
as requiring further investigation. The results will, however, be chiefly of
botanico-morphological interest, and will not affect either the firmly established
theory of the pleomorphism of the Schizomycetes, or touch the physiology of the
sulphur bacteria, which latter is the sole property meriting consideration, so
far as we are concerned. So long, however, as Winogradsky's discoveries remain
uncontroverted by any thoroughly reliable investigations, his deductions must
be allowed to stand, viz., that the sulphur bacteria are not pleomorphic — neither
the colourless, filamentous genera nor the non-filamentous red genera. The
Russian physiologist described a long series of species of the latter type, which,
as they are devoid of special physiological importance, we need not examine
more minutely. It will be sufficient to mention the chief forms.

The aforesaid purple bacteria are only a single sub-gr-oup comprising all those
sulphur bacteria whose living cells are free and capable of locomotion. It is
divided into three genera, Chromatium, Rhabdochromatium, and Thiospirillum.
Of these terms the penultimate one is synonymous with Cohn's Rhabdomonas,
whilst the last one comprises all red sulphur-bearing spirilla, and consequently
includes Ehrenberg's Ophidomonas. A contrast to this sub-group of free cells
is afforded by the species of red sulphur bacteria which are genet-ally united as
colonies. In the genera Thiocystis and Thiocapsa this union is effected by a
mucioous sheath, which is absent in Thiosarcina. In all three cases reproduction
occurs by fission in three directions, and the same behaviour is exhibited by the
genus Lamproci/stis, which principally differs from the other three in the
structure of its bag-shaped zoogloea, which is hollow internally, and consists
solely of a lattice-like network. A good representation of this species was given
by COHN (II.) who described it, along with other organisms, as Clathrocystis
roseo-persicina. The genus Thiopedia is characterised by the division of the
cells in two directions of space, and by the consequent flat colonies. In the
remaining species cell fission occurs in one direction only. The Ama;bobac(er
species are distinguished by an amoeboid movement ; those of Thiodlctyon owe
their name to the reticular conjugation of their spindle-shaped cells ; whilst
Thiopolycoccus forms zoogloea of closely crowded cocci. The genus Thiothece is
distinguishable from all other sulphur bacteria by its particularly thick gelatinous
sheath.

A few remarks with regard to the properties of bacterio-purpurin will be
opportune in this place. The difficulties in the way of preparing a quantity
sufficient for the performance of a chemical analysis have not even yet been
overcome ; consequently its chemical composition is still entirely undefined, and
we cannot yet say with certainty whether the colouring-matter is the same in
all red sulphur bacteria. This is, however, assumed to be the caee, on the

PHYSIOLOGY OF THE SULPHUR BACTERIA 283

ground of the concordant results yielded in separate instances by chemical
reactions, a few of which are now given. The pigment is insoluble in water or
ether, but is soluble in cold alcohol (as found by Winogradsky in contradiction of
Lankester's report). It is converted, by warming with water and by chloroform,
into a golden-brown compound, which is changed into brown by hot alcohol,
hydrochloric acid or acetic acid ; whereas ammonia or caustic potash produces
no visible change at first, but finally gradually develops a dirty shade of colour.
Concentrated sulphuric acid changes the red almost instantly into a deep blue ;
which afterwards gradually tones into a brownish green. This reaction resembles
that set up by the same acid with the lipochromes. Bacterio-purpurin is very
quickly destroyed by oxidising agents (e.g. dilute nitric acid or bromine water).
Iron and manganese appear to favour its production, a conclusion deduced from
the fact that the addition of the protosulphide of either of these rnetals to the
medium results in a much stronger coloration of the cells. The sensitiveness
of bacterio-purpurin to chemical influences explains the varied change of tone
produced in the colour in one and the same cell under different external
conditions, causing it to assume all shades, from pure violet to purple, peach-
blossom red, rose, orange, brown-red, and brown. With regard to the spectrum
of bacterio-purpurin, examined by Lankester, Warming, and Englemann, details
have already been given in § 92.

The classification of the non-filamentous sulphur bacteria, drawn up by
Winogradsky and briefly outlined above, received an important extension by
M. JEGUKOW'S (III.) discovery that, in addition to the red species already
described, certain colourless non-filamentous sulphur bacteria also occur in
Nature. Two of these he subjected to a closer physiological examination, which
will be referred to in the succeeding paragraph. The one of them, indicated as
species a, occurs as slightly curved motile rods, their breadth varying from 1.4
to 2.3 /JL, and the length between 4.5 and 9 /it. For the second species, known
as /3, the dimensions are 0.6-0.8 /z and 2.5-5 P- respectively.

§ 202.— Physiology of the Sulphur Bacteria.

The true nature of the rounded, highly refractive enclosures present in these
fission fungi, and attracting the eye of the microscopist, was first recognised by
Cramer, whose discoveries are noticed in a treatise by C. MULLER (I.). It was
shown in these experiments that these granules behaved exactly like sulphur in
presence of solvents, and they were therefore thought to consist of that element.
F. COHN'S (II.) extension of these investigations (which were confined to
Heggiatoa, and were confirmed by J. Mayer-Ahrens) to the red sulphur bacteria
as well, led to the same result : the granules appearing in these coloured
Schizomycetes, under certain — as yet undefined — conditions, are composed of
pure sulphur. The term granules applied to these forms is unsuitable, inasmuch
as they consist not of solid granular, but (as Winogradsky afterwards proved) of
oily, amorphoiis sulphur, the greater part of which is soluble in CS2. However,
when the enveloping cells are killed, the sulphur granules are gradually changed
into the crystalline modification of this element. If a few Beygiatoa threads
rich in these droplets be immersed in concentrated picric acid and left in water,
a number of very fine monoclinic prismatic plates and rhombic octahedra will
be found in the threads after a lapse of twenty-four hours, and it will at the
same time be noticeable that the growing crystals have penetrated the adjacent
cell walls.

F. Cohn was the first to investigate the origin of these internal constituents,
which occasionally fill the cell to such an extent as to exceed 90 per cent, of its
weight. Starting from the fact that the sulphur bacteria are only found in

284 THE SULPHUR BACTERIA

abundance in natural waters containing sulphuretted hydrogen, and are, on the
other hand, almost entirely lacking elsewhere in Nature, he came to the opinion
that this gas is produced by the reducing action of these fission fungi on the
sulphates in the water, and that they subsequently reoxidise the gas, sulphur
being left as a deposit in the cells. In forming this opinion he was chiefly
influenced by the result of an investigation made by LOTHAR MEYER (I.), who
kept a sample of sulphur-spring water (rich in Beggiatoa) from Landeck in
Silesia for four months in a stoppered flask, and found that at the end of that
time it contained five times as much H2S as at first. The same conclusion as
deduced by Conn was also arrived at by JE. PLAUCHU (I.), and by A. ETARD and
L. OLIVIER (I.). This hypothesis, which credited the sulphur bacteria with
both a reducing and an oxidising capacity, was first thoroughly investigated in
1886 by S. WINOGRADSKY (VI.), who showed that the sulphur bacteria consume
(instead of producing) sulphuretted hydrogen ; oxidising it and storing up the
separated sulphur in their cells. The amount of these enclosures in the cell is
larger or smaller according as this process can be carried on with a greater or
lesser degree of vigour. It depends, therefore, on external conditions, and
consequently cannot be relied on — as was done previously by various observers :
inter alia, Winter in Rabenhorst's " Kryptogamen Flora " and by Engler — as
a characteristic for the differentiation of species. The sulphur does not
permanently remain in the cells, but is oxidised by them to sulphuric acid, the
latter being then absorbed by the carbonates — usually CaH2(C03), — in the
water, and converted into sulphates.

If these Schizomycetes are deprived of sulphuretted hydrogen for a long time,
they consume their internal store of sulphur (which will be exhausted in twenty-
four to forty-eight hours), and then perish of hunger. This fact demonstrates
that the sulphur bacteria cannot permanently dispense with sulphuretted
hydrogen, but that this gas is actually their special (and almost exclusive) source
of energy. Sulphur, or rather its compound with hydrogen, plays the same part
towards these organisms as the carbohydrates do towards the majority of Schizo-
mycetes ; its combustion liberates the energy necessary to the maintenance of
their vitality. According to the observations of Winogradsky, the individual
threads of Beggiatoa daily consume from two to four times their own weight of
the gas. These Schizomycetes require but little other (organic) nutriment, and in
fact will not stand very much. This explains, on the one hand, their unusually
slow rate of growth in proportion to the amount of sulphur separated, and, on
the other, their inability to grow in the ordinary nutrient media employed in
bacteriology : e.g. on gelatin they perish in a very few minutes. Attempts to
grow them as pure cultures on a large scale have hitherto failed, and the
physiological facts determined concerning them have all had to be ascertained
very laboriously by cultivating single organisms in sulphur-water on microscope
slides.

The optimum, i.e. the maximum supportable, quantity of sulphuretted
hydrogen in the water is higher in the case of the red sulphur bacteria than
with the colourless, filamentous species. These latter require less, and in fact
die instantly if placed in water saturated with the gas, whereas the red kinds
will stand this degree of concentration very well. Consequently, under natural
conditions, these latter will gain the upper hand in such places where large
quantities of sulphuretted hydrogen are evolved, either as a result of the decom-
position of an abundance of organic matters (albumen) or by the powerful
reduction of sulphates. This is the case, for example, in the stagnant shallow
bays on the Danish Zeeland coast, and the same conditions obtain in the
Limanes so plentiful along the coast of the Black Sea (e.g. near Odessa). These
latter are shallow salt lakes, separated from the open sea merely by a low,

PHYSIOLOGY OF THE SULPHUR BACTERIA 285

narrow tongue of land. Their bottom is covered by a thick mud, which owes its
black colour to the FeS thrown down from the iron compounds in the water
(and in the plants rotting therein) by the sulphuretted hydrogen generated from
sulphates by the reducing action of bacteria investigated byE. BRUSILOWSKY (I.).
The red sulphur bacteria are but rarely found in mineral sulphur springs.
According to Cohn, they have been detected by Morren in the sulphur spring at
Ougree, on the Maas ; by Fontane and Jaly, in that at Sales, in the Pyrenees ;
by Meneghini, in that of the Euganean Hills, near Padua ; and by Cohn himself
in that of Tivoli, near Rome.

The existence of the sulphur bacteria is often a very hard one, because it
requires the simultaneous presence and availability of two gases which neutralise
one another and become converted into sulphur and water —

H2S + O = H20 + S.

So that actually the surface of liquids wherein H2S is produced in abundance by
the activity of reducing bacteria becomes coated with sulphur formed by purely
chemical means, in accordance with the foregoing equation. Now, in order that
the sulphur bacteria may be in a position to exert their powers of oxidation, it
becomes necessary for them to inhabit certain strata of the liquid between the
limits where the oxygen can gain access from above and sulphuretted hydrogen
reach them from below. If the liberation of the latter gas goes on briskly, this
level rises, and may ascend to the surface of the liquid ; otherwise it sinks and
approaches the bottom, where the sulphuretted hydrogen is generated. This
change of feeding-ground cannot, however, be followed by all species of sulphur
bacteria, since — just in the same way as has been explained with regard to
sulphuretted hydrogen — these organisms are adapted to a certain tension of
oxygen, which varies in the different species, i.e. they cannot stand the presence
of more than a certain quantity per unit of volume of the liquid. In the case
of oxygen, this tension is naturally greatest at the surface and smaller at greater
depths. It will be evident that even the fluctuations of atmospheric pressure
will suffice to produce a change in the predominating species of a diversified
mixture of sulphur bacteria in their natural haunts. The same applies to the
rate at which the sulphuretted hydrogen is disengaged.

For an instructive insight into these conditions we are indebted to the re-
searches of M. JEGUNOW (I.) on the colourless non-filamentous species referred to
at the close of the last paragraph. As already stated, the habitat of the sulphur
bacteria is in those strata of the liquid where the oxygen from above comes
into contact with the sulphuretted hydrogen from below. At this level the
organisms congregate to form an assemblage visible to the naked eye, and which
the above-named Russian physiologist termed the bacterial plane, the structure
of which he examined minutely. He artifically induced the processes going on
in the Limanes to repeat themselves — so far as necessary to the purpose in view
— on a small scale in the laboratory, by placing a certain quantity of the black
mud in suitable vessels containing water, and then leaving the whole to stand
uncovered. We will not go further into the matter of the rise and fall of the
bacterial plane as observed by him, because BEYERINCK (I.) had made similar
experiments two years earlier, and applied to the phenomenon a term (Bakterien-
Niveau) having the same significance as that used by Jegunow.

The discoveries made by JEGUNOW (II.) with regard to the construction of
this bacterial plane, in the case of the organisms now in question, must, however,
be considered as novel. When cultivated in higher and broader, but thinner,
strata of liquid, the plane assumes the form reproduced on a reduced scale in
Fig. 79, i.e. the bacteria do not form a simple plane, but become piled up in
places into tuft-like projections — each about 3-4 mm. long — four of these being

286 THE SULPHUR BACTERIA

shown (enlarged) in Fig. 80. The examination of these tufts by the aid of
a horizontal microscope shows that they are formed by the movement of the
individual bacteria, in a manner similar to the gushing of a spring ; they ascend
in the axis of the tuft, and then describe an arc in their return to the plane.
When inverted by the microscope this resemblance is still more striking ; so
that Jegunow has styled the planes " fountain planes." The velocity of the
individual cells he found to be 0.02 mm. per second. In tracing the chemical
activity of the bacteria, he made use of a simple and reliable reagent for sulphu-
retted hydrogen : a fine (woollen or similar) thread treated first with ferric

FIG. 80.— A portion of the Bacterial plane from the preceding Fig.
showing the arched construction of the plane itself, as well as
four of its fountains. Magn. n. (After Jegunow.)

FIG. 79. — Culture of Sulphur Bacteria from the Limanes (in a small vessel ; reduced scale).

The figures on the label give the dimensions, the thickness of the layer of liquid being 0.9 mm. The
bottom is occupied with black Limanes mud; above this is the liquid, the meuiscus'of which is
visible at the top of the Fig. ; and in between is the bacterial plane with five " fountains." (After
Jegunow. )

chloride and then with ammonia, both in such a very dilute condition that the
thread is stained merely a pale yellow. A glass weight is then attached to the
thread and let down into the liquid, whereupon the lower part of the thread, as
far as the summit of the tufts on the fountain plane, quickly turns black, from
the formation of FeS. From that point onwards, however, the colour gradually
changes to white. This experiment shows that in the summits of the tufts the
sulphuretted hydrogen arising from below is first oxidised to sulphur, and stored
up in the cell, which conveys it to a higher level (the actual plane), and there
oxidises it to sulphuric acid. This acid then dissolves the ferric oxide on the
upper part of the thread, which is consequently decolorised at this level. The
time occupied by the cells in making a single trip — and therefore the total

PHYSIOLOGY OF THE SULPHUR BACTERIA 287

period required for the conversion of H,S into SO3 and the expulsion of the
latter from the cell — was ascertained by Jegunow to be about five minutes.

The importance of the sulphur bacteria in the economy of Nature is unmis-
takable : in co-operation with the sulphate-reducing bacteria they ensure that
the sulphur cycle pursues an uninterrupted course, the element being taken up
by the higher plants in the condition of sulphates, and deposited in the cells in
the form of organic compounds, from which, in the course of putrefaction, it is
liberated as sulphuretted hydrogen, and is finally then reconverted into sulphates
by the sulphur bacteria and recommences its course through the higher plants.

CHAPTER XXXVI.

THE NITRIFYING BACTERIA.

§ 203.— The Recognition of Nitrification as a Physiological
Process.

THE nitrogen excreted from the animal body as urea has not, when converted
into ammonium carbonate (see chap, xxxii.), yet attained the form in which it is
usually taken up by plants. Although it is indubitable that plants in general
can obtain their requirements of nitrogen from the ammonia salts, it is never-
theless certain — both as a result of manuring experiments on the small scale and
also from the experience of agricultural practice — that the majority of cultivated
plants absorb the element in question more rapidly and abundantly when it is
offered them in the form of nitrates. In fact, for some of them, e.g. maize, buck-
wheat, and tobacco, JUL. LEHMANN (I.) puts forward the well-grounded assumption
that they derive their nitrogen exclusively from nitrates. Here again Nature
has made provision for the necessities of the case by converting into nitrates the
ammonia salts which — partly as a result of decomposition and partly as artificial
manures — find their way into the soil.

This process, long known and briefly termed nitrification, was defined in
1846 — on the basis of an experiment by J. DUMAS (II.) — as a purely chemical
process of oxidation. This observer regarded chalk as the intermediary facili-
tating the intimate combination of ammonia and atmospheric oxygen. Fifteen
years later this role of " go-between " was ascribed by MILLON (I.) to the porous
humic bodies in the soil — a view that still remained destitute of any convincing
proofs when revived in 1863 by BLONDEAU (II.).

Ten years later other opinions began to arise. The first adverse hypothesis
was expounded in 1873 by ALEX. MULLER (I.), but was not based on any
solid foundation, nor was it followed up any farther. Four years afterwards
SCHLOESING and MtNTZ (I.), relying on the results of their researches in this
direction, hazarded the opinion that the formation of nitre in the soil is due to
the vital activity of organised ferments (soil bacteria). In a subsequent com-
munication these two workers detailed some of the conditions requisite for the
inception and course of nitrification. The operation is almost stagnant below
5° C., but becomes apparent at 12° C., and attains its maximum at 37° C. As
the temperature rises still higher the reaction becomes weaker, and ceases
altogether at 55° C. It proceeds the more rapidly as the degree of moisture in
the soil increases, provided aeration is not thereby impeded. A faintly alkaline
reaction facilitates the progress of nitrification, which, moreover, may not always
result in the production of nitrates, but at times does not extend beyond the
formation of nitrites, especially at low temperatures (below 20° C.) and with a
restricted admission of air.

Both workers also endeavoured to obtain pure cultures of the organisms
under investigation. The result of their endeavours will not be judged too
harshly when the existing lack of any reliable method of pure cultivation at that
time is remembered. When the introduction of the Koch gelatin plate afforded
a new appliance for this purpose, it was pressed into the service now under

288

NITROSO-BACTERIA AND NITRO-BACTERIA 289

consideration by several workers, inter alia by ADAMETZ (IX.) and A. B. Frank ;
nevertheless, the result did not fulfil expectations. The last-named German
mycologist then contradicted the assumptions of the two French agricultural
chemists, and championed the views held by Dumas. To this revival of an old
hypothesis we owe the production of a comprehensive work by H. PLATH (I.),
which is commended to the attention of the reader not only on account of the
new discoveries it mentions, but also because the first part contains a collection —
rich in information for the chemist — of all the then known methods for the
production of nitric acid from ammonia by oxidation. In the second part of
this treatise it was stated, on the basis of new experiments, that completely
sterilised soil no longer possesses the faculty of converting ammonia into nitric
acid. It was furthermore shown that, when organisms are entirely excluded,
neither the soil as a whole, nor any one of its constituents, is capable of trans-
forming ammonia into nitric or nitrous acid by occluding atmospheric oxygen.
A re-examination of this work by H. LANDOLT (I.), who undertook the task in
consequence of an objection raised by A. B. FRANK (IX.), led to a complete con-
firmation of Plath's discovery on all points. It was thus ascertained (in 1888)
by the exclusion method that in the oxidation process now under our notice the
role of oxygen-carrier is played by living organisms, and that consequently
nitrification is a physiological process.

§ 204.— Nitroso-Bacteria and Nitro-Baeteria.

The discovery and closer investigation of these unknown organisms was
shortly afterwards effected by S. WINOGRADSKY (VII.). It is not surprising
that their preparation as pure cultures was so long delayed, when we remember
that these bacteria do not thrive on media rich in organic nutrient substances.
The above-named Russian physiologist successfully employed for this purpose
the gelatinous inorganic substance, viz., precipitated silica, recommended by
W. KtiHNE (I.). When prepared by precipitation from water-glass (alkali
silicate) by hydrochloric acid, and purified by dialysis, concentrated by boiling,
and then sterilised in the steamer, this silica forms a vitreous mucinous mass.
This is then incorporated with a sterilised solution of the sulphates of potash,
magnesia, ammonia, and carbonate of soda, inoculated with the bacterial sample.
These salts cause the silica to set, so that the germs in the sowing are fixed
separately, and thus may be kept apart, even when they have developed into
colonies. In this manner Winogradsky primarily succeeded in obtaining
cultures of assured purity, by means of which he was enabled to arrive at
conclusions unattainable by the fractional sowings and dilution method em-
ployed by previous workers, e.g. W. HERAEUS (I.), P. FRANKLAND (III.), and
R. WARINGTON (III.).

One of the weightiest of these results is the fact determined by WINO-
GRADSKY (VIII.) that the numerous species of the group of nitrifying bacteria
are classifiable into two sharply divided sub-groups : nitroso-bacteria and nitro-
bacteria.

The nitroso-bacteria oxidise ammonia to nitrous acid, in accordance with the
equation —

(NH4).p + 302 = N203 + 4H.20,

but no farther. For this reason nitrites are not altered by these bacteria,

On the other hand, the nitro-bacteria lack the facility of attacking ammonia,
but perform the task of converting nitrous acid into nitric acid, in accordance
with the equation —

I I

290 THE NITRIFYING BACTERIA

= O ^N = °

= 0

As is apparent from this equation, their powers differ from that of the
nitroso-bacteria, inasmuch as the latter convert the pentavalent nitrogen of
ammonia into the trivalent nitrogen of nitrous acid, whilst the nitro-bacteria
re-convert the element into the pentad condition.

It is evident that these oxidation processes can be effected only in the presence
of bases which take up the acids with which the ammonia was initially combined,
and also neutralise the resulting nitrous or nitric acid — thus protecting the
bacteria from injury from this source. This task is excellently performed in the
soil by calcium carbonate. The favourable influence exercised on the course of
nitrification by the presence of this salt is therefore readily explainable without
dragging in any hypothesis about the condensation of oxygen. Free alkali is
unsuitable here for the fixation of the acids, since the presence of this reagent
in quantity would be injurious to the bacteria. In artificial cultures calcium
carbonate can be replaced by magnesium carbonate, a practice adopted by
Winogradsky.

§ 205.— Nitrosomonas and Nitrosocoeeus.

Two main types of nitroso-bacteria can be differentiated in consequence of
the results of existing investigations. One of them (in several species) is found
in all the soils of the Old World (Europe, Asia, Africa) hitherto examined, and
is known as Nitrosomonas. The second is peculiar to the soil of the two remaining
continents, and has received the name of Nitrosocoeeus. The individual organisms
of the first-named type are each provided with a single cilium, and exhibit powers
of locomotion which are manifested at an eai-ly stage in the cultures, and cause
these to become decidedly opalescent. Subsequently the cells become quiescent
and collect as zoogloea, which rest in the form of greyish gelatinous clouds on the
carbonate at the bottom of the liquid. We will describe this (Nitrosomonas)
genus first.

Only a single species of nitroso-bacterium has been discovered in European
soils, viz., Nitrosomonas euro})cea. At the opalescent stage of the culture this
organism appears as briskly motile cells (fitted with a short flagellum) in the
shape of short rods 1.2-1.8 p. long and 0.9-1.0 p. broad. The cells of Nitroso-
monas javanica, cultivated from the soil of the Botanical Garden at Buitenzorg.
near Batavia, are globular, and only attain a diameter of 0.5-0.6 p, but their
flagellum is very long — as much as 30 p.. The Nitrosomonas japonica, found in
soil from Tokio, is — like the Nifrosomonas africana, isolated from samples of
soil from Tunis and from La Reghai'a, in Algeria — very similar to the European
species, only somewhat smaller.

Differing from these species are those of the genus Nilrosococcns, found in
South American and Australian soils. They do not form zooglcua, neither are
they ciliated. That obtained from Quito (Ecuador) is a coccus 1.5-1.7 ^ in
diameter. A similar species, except in point of size, is the Nitrosocoeeus brasili-
ensis, obtained as a pure culture from the soil of Campinas (Brazil), and attaining
a diameter of 2 p. ; and the species grown from Melbourne soil is undistinguish-
able from this latter. The nitroso-bacteria are, as observed by WINOGRADSKY (IX.),
very susceptible to desiccation, and consequently the amount of such organisms
in the soil decreases as drying progresses. They are almost entirely lacking in
the air.

ASSIMILATION IN THE DARK 291

§ 206.— The Nitro-Baeteria.

differ from the species already described, not only from a chemico-physiological,
but also from a morphological point of view, being smaller and more slender.
The cells are an elongated oval, mostly pear-shaped, 0.5 p. in length and 0.15-
0.25 fj, in breadth, and are therefore among the smallest of all known organisms.
In liquid cultures they develop and congregate to form a thin, mucinous skin
adhering firmly to the walls of the vessel. Compared with their powerful
oxidising action, the vegetative development of these organisms is astonishingly
slight. Spore formation has not been found either in these or in the nitroso-
bacteria ; and up to the present no subdivision of the genus Nilrobacler into
species has been made.

BURKI and STUTZER (III.) in 1895 obtained from Hanoverian soil a nitro-
bacterium which they assert will thrive both on nutrient gelatin and in bouillon,
but (so it is said) exhibits no nitrifying action in nutrient media of this kind, as
a rule, and, indeed, loses this power entirely, so that when re-transferred into
mineral nutrient solutions it does not attack the nitrites placed at its disposal.
A careful examination of such a culture, obtained direct from the above-named
chemists, was made in 1896 by S. WINOGRADSKY (X.), who showed that the
alleged pure culture contained, not only the nitro- bacterium, but also three other
species of (saprophytic) bacteria which thrive well in bouillon, a medium in which
the nitro-bacterium will not grow. Winogradsky's treatise is recommended
to the reader, more particularly because it mentions numerous contingencies
likely to arise in working, and render of no avail the trouble bestowed on the
nitrifying bacteria by the bacteriologist. Furthermore, he gives a new recipe
for a medium for the pure cultivation of nitro-bacteria, more convenient in use
than gelatinous silica, viz., nitrite agar-agar, i.e. a mineral solution containing
nitrites and qualified by 1.5 per cent, of agar-agar.

If the amount of nitrogen oxidised per unit of time be taken as the standard
for measuring the chemical energy of these organisms, then — as Winogradsky
ascertained by comparative investigations — the nitroso- bacteria will be found
the more active of the two. From this fact it is permissible to draw the further
deduction that the conversion of the trivalent nitrogen of nitrous acid into
pentavalent nitric nitrogen requires the expenditure of a greater amount of
internal force than is needed for the converse operation in the oxidation of
ammonia to nitrous acid.

Both nitroso- and nitro-bacteria are always present in the soil, the second
type of organism immediately oxidising the nitrous acid generated (from ammonia
salts) by the first. Whether nitrification begins already in the dung-heap, or
has its first inception in the field, is dependent on various circumstances. It
will proceed whenever a sufficient quantity of ammonia salts has been produced
by the fermentation of urea, provided air has ready access. Thus, H. IMMEN-
DORFF (III.) showed that in the outer layers of manure heaps (especially horse-
dung), the production of nitrous acid will set in briskly in a few days. There
are ample reasons why the formation of the easily lixi viable nitrates, which may,
moreover, expose the materials to wasteful reduction processes, should be pre-
vented in the manure heap. On this account endeavours should be made to
minimise the aeration of the manure by battening the heaps well down.

§ 207.— Assimilation in the Dark.

The incapacity (recorded in § 204) of the nitrifying bacteria to grow on
nutrient gelatin is, in the main, attributable to their general distaste for organic

292 THE NITRIFYING BACTERIA

nutriment, a peculiarity noted by MUNEO (I.) in 1886. The smaller the quantity
of organic food present, the more energetically do growth and oxidation proceed ;
and the latter effect is most powerful in solutions containing exclusively inorganic
matters. For nitroso- bacteria Winogradsky recommends a mixture of 2—2.5
grams of ammonia sulphate, 2 grams of common salt, and a sufficient quantity
of magnesium carbonate per litre of well-water. For mtro-bacteria the ammonia
salt is replaced by sodium nitrite.

When such a nutrient solution containing solely inorganic matters is inocu-
lated with a few nitroso- or nitro-bacteria, energetic oxidation occurs, accom-
panied— as was first brought into notice by W. HERAEUS (I.) in 1886 — by
a rapid reproduction of the bacteria. When development is concluded, and the
available quantity of ammonia or nitrite oxidised, then the bacterium crop grown
in this manner contains a certain quantity of organic matter, the carbon of
which has been exclusively derived from inorganic sources — in this case carbon
dioxide. The amount was ascertained by Winogradsky, by four quantitative
analyses, as 0.020-0.022 gram per 100 c.c. of liquid. Consequently the nitroso-
and nitro-bacteria are able to abtract from carbon dioxide, in the absence of light,
the carbon necessary for the construction of their cells, and are therefore able to
assimilate carbon dioxide in the dark.

Two sources of carbon dioxide are available to the nitrifying bacteria. One
of them is the carbonate present in the nutrient solution (or soil), and which is
also necessary for other reasons already given in § 204. According to Wino-
gradsky, this carbonate supplies carbon to the newly formed bacteria, which are
assumed to decompose it by means of the acids they produce, and then utilise the
carbon in the construction of new cells. He considers that the function of these
organisms is to liberate and restore into general circulation the carbon that, by
any means, has been converted into carbonates, and so withdrawn therefrom.
On the other hand, E. GODLEWSKI (I.) showed that it is chiefly from the atmo-
sphere that the carbon dioxide requisite for the construction of new cellular
substance is derived. He found that development did not occur in cultures con-
taining magnesium carbonate when only air free from carbon dioxide was
admitted. Now the atmosphere contains not only carbon dioxide, oxygen, and
water, but also ammonium carbonate, with which substances the nutrient require-
ments— ash constituents apart — of the nitrifying bacteria are satisfied. These
organisms will therefore be able to develop in places where there is nothing
present but bare rock, the cracks and fissures of which afford them a shelter
against the desiccating action of the winds. In fact, it was in such arid places
that A. MtJNTZ (1.) constantly found nitrifying bacteria. It can very easily be
shown that friable (" rotten ") stone, especially that from the Faulhorn, is thickly
impregnated with these organisms.

In order that the carbon of the carbon dioxide may be prepared for its
ultimate purpose, it must first be freed from the two attached atoms of oxygen.
In green plants the force requisite for this purpose is supplied by the thermal
power of the sun's rays ; but in the nitrifying bacteria, which also assimilate in
the dark, it is the energy liberated during the oxidation of nitrogen that effects
the dissociation of the carbon dioxide molecule. Consequently, the assimilation
of carbon is dependent on the oxidation of nitrogen, a fact quantitatively proved
by Winogradsky. According to this authority, about 35 mgrms. of nitrogen
are oxidised for each milligram of carbon assimilated, the atomic ratio being —

C : N = i : 30.

More accurate knowledge of the progress of this assimilation — especially
on the thermo-chemical side of the question — is at present lacking. F.
HUEPPE (VIII.) and O. LOKW (V.) constructed equations to represent the

WALL-SALTPETRE AND PLANTATION-SALTPETRE 293

changes occurring in the reaction, but these can merely be alluded to here.
Godlewski ascertained that by no means the whole of the ammoniacal nitrogen
eliminated during the nitrification is recovered as nitrous or nitric acid, but that
a portion is liberated in its elementary condition, and escapes from the solution
undergoing nitrification. It may be opined that this loss is not immediately
connected with the action of the nitrifying bacteria, but is only an associated
phenomenon produced by the reaction of the N2O3 on the still undecomposed
NH3, in accordance with the equation —

N203 + 2NH3 = 3H20 + 2N2.

The reason for this is that the nitrous acid liberated does not in every part of the
liquid come into immediate contact with the carbonate which would protect it
from the action of the ammonia.

§ 208.— Wall-Saltpetre and Plantation-Saltpetre.

The particulars already given of the life-conditions of the nitrifying bacteria
will explain the origin of wall-saltpetre, i.e. the corroding efflorescence of salt-
petre on masonry. This substance is a white snow-like mass, consisting princi-
pally of crystals of calcium nitrate, and occurring with particular frequency on
the walls of stables and closets. It is precisely in such places that the fission
fungi under discussion find an abundance of the food-stuffs they require : the
ammonia salt is supplied by the urea absorbed by and hydrated in the walls ;
calcium carbonate and a little alkali are present in the brickwork, and there is
no lack of the necessary oxygen. Consequently all the preliminary conditions
favouring the activity of the nitrifying bacteria introduced in dust, &c., are
fulfilled. However welcome this activity may be when restricted to the soil, it
is entirely undesirable in brickwork, the latter being gradually corroded and
rendered brittle by the calcium nitrate produced. Sprinkling the walls with
powerful antiseptics, such as antinonnin, may, however, afford a remedy. That
the phenomenon is really due to the nitrifying bacteria has been proved by the
researches of 0. HELM (II.) and G. TOLOMBI (II.).

A few words must also be devoted to the saltpetre plantations. Since the
discovery of the South American deposits of nitrate of soda, which substance
can be converted into saltpetre by treatment with potassium salts, the production
of plantation-saltpetre has decreased. It will, however, come to the front again
whenever the Chilian beds are exhausted. In fact, the production of saltpetre
for agricultural purposes by this method is even now worthy of consideration.
The quantity of Chili saltpetre imported by European countries is very con-
siderable, and large sums of money are annually disbursed to South America
which might be retained by producing the saltpetre at home. The accomplish-
ment of this project necessitates a searching investigation of all the conditions of
nitrification, in order to ascertain how the reaction may be suitably controlled.
The result would be that, instead of using expensive foreign nitrate, the ground
would be manured with cheap sulphate of ammonia, now formed as a waste
product in home gas-works and coke-factories, and put upon the market in con-
stantly increasing quantities. The consequent freedom from the hands of
Chilian speculators would be a great gain from the point of view of national
economy. Moreover, this method of manuring presents another advantage from
the standpoint of the agricultural economist. As is well known, the soil has no
power of fixing nitrates, a certain portion of the added saltpetre invariably — as
P. DEHERAIN (III. and IV.) and others have shown — escaping in the drainage-
water, so that more has to be added to the soil than is recovered in the crop.
This disadvantage does not attach to manuring with salts of ammonia, since they

294 THE NITRIFYING BACTERIA

are fixed by the soil and protected from wasteful lixiviation, the nitrifying
bacteria then oxidising the ammonia and supplying the plant with nitrates
according to its requirements.

So far as plantation-saltpetre is concerned, the external conditions favouring
the rapid formation of this compound have been gradually ascertained by means
of tentative experiments. A pyramidal heap, resting on an impervious clay
foundation, is prepared by mixing chalky soil with various kinds of organic
matter, and is frequently watered with liquid manure, an admixture of brush-
wood in the heap imparting porosity and facilitating aeration. The nitrates,
&c., formed in the interior appear — like wall-saltpetre—on the surface of the
mass, and gradually increase to form a crust which is richer in nitrates than the
interior of the heap. The crude lye obtained therefrom by lixiviation is treated
by adding a potassium salt in order to convert the nitrates of calcium, mag-
nesium, and sodium into potassium nitrate, the crude saltpetre thus produced
being then purified in refineries.

The elucidation of the optimum external conditions for influencing nitrification
has been attempted by numerous investigators, and a few of their results will
now be given. J. DUMONT and J. CROCHETELLE (II. and III.) found that the
chlorides of potassium injuriously affect nitrification, whereas the carbonates of
these metals, and also potassium sulphate, act beneficially. From what has
already been stated it will be evident that the merely faint (or altogether
inoperative) activity of the nitrifying bacteria in soils poor in calcium carbonate
(e.g. sour meadow-land) can be stimulated by the addition of the said
carbonate. On this point a few experiments have been made by J. DUMONT
and J. CROCHETELLE (I.). The kind of acid with which the ammonia is combined
must not be regarded as unimportant, Hueppe and Winogradsky having noticed
that — as afterwards shown by the special experiments of 0. LOEW (VI.) —
nitrifying bacteria do not attack ammonium formate at all, and that the oxalate
is acted upon only very imperfectly, and with great difficulty.

CHAPTER XXXVII.

ACETIC FERMENTATION.

§ 209.— Discovery of the Acetic Acid Bacteria.

IP beer, wine, or other similar alcoholic liquids, are left to stand exposed to the
air, they will, at the end a few days, become covered with a tough, mucinous
(usually smooth) skin or film. The alcohol gradually disappears, and, in
approximately the same ratio, the presence of acetic acid makes itself evident :
the beer, &c., is converted into vinegar. It has been known from the earliest
times that an unsoured sample of beer, wine, or the like can be quickly turned
into vinegar by the addition of a small quantity of such skin. This latter was
regarded as the carrier of the vinegar fermentation, and consequently received
the name of "mother of vinegar" (Fr. mere de vinaigre, Ger. JZssigmutter). The
first botanical investigation of this substance was made in 1822 by PERSOON (I.),
who described the organised skin developing on various liquids, and gave it the
general name of Mycoderma, i.e. mucinous skin or fungoid skin, but never
contemplated the existence of any direct connection between acetic fermentation
and the development of such a structure.

This was reserved for the German algologist FR. KtiTZiNG (I.). In his
treatise on this subject, published in 1837, he showed, without, apparently,
being acquainted with the labours of his predecessor — that the " mother of
vinegar " is constructed of a number of minute dot-like organisms (which we
now call bacteria), arranged together in the form of chains. These he classified
as algae, and named them Ulvina aceti, and asserted quite positively that alcohol
is converted into acetic acid by the vital activity of these organisms.

Kiitzing's results, however, attracted but little notice, because, two years
after their publication, LIEBIG (III.) appeared on the scene with his theory of
acetic fermentation (which will be described in a subsequent paragraph), in
which no mention was made of the potency of living organisms, but the " mother
of vinegar " was asserted to be a formation devoid of life : a structureless
precipitate of albuminous matter. Only one of the reasons put forward by the
German chemist in support of this view, which he stubbornly upheld, will be
mentioned here, and that merely as a curiosity. The Dutch chemist, G. MULDER
(III.), celebrated as a chemical expert on wine, subjected the "mother of
vinegar" to chemical analysis, and, because he failed to discover the presence
of any ash constituents, thought that it must be regarded as a compound of
protein and cellulose. Mulder's statement was refuted in 1852 by R. THOMSON
(I.), who showed that a sample (but by no means a pure culture) of "mother
of vinegar" contained 94.53 per cent, water, 5.134 per cent, organic matter, and
0.336 per cent. ash.

The diffusion of new light on this matter was reserved for PASTEUR (XIII.).
Taking up anew the question of the origin of acetic fermentation — examined by
Kittzing merely from the purely botanical side, and that only cursorily — he
controverted the opinions of the chemists, and proved, in 1864, that this
fermentation also is a physiological process, whose inception and maintenance
is bound up with the vital activity of minute fungoid organisms, to which he

295

296 ACETIC FERMENTATION

applied the specific name Mycoderma aceti, first employed by Thomson. Of
course, at that time, PASTEUR (XIV.) was not in a position to prepare or use
pure cultures, consequently the results of his experiments cannot now be credited
with more than the single value of having unimpeachably proved the dependence
of acetic fermentation on the vital activity of certain micro-organisms. Pasteur
did not determine to what group of living organisms Mycoderma aceti belongs,
the botanical, and especially the morphological side of the question concerning
him but little ; only, in one place in his treatise, he states that he cannot regard
the organism as a bacterium, as was done by Stack in 1863. Nevertheless, we,
at the present day, must agree with the opinion of the last-named : the cause
of acetic fermentation studied and described by Pasteur can only have been
a fission fungus.

The property of forming mucinous skins on the surface of liquids is not
peculiar to the acetic acid bacteria alone, but, on the contrary, is a very general
vital phenomenon among fungi. It is particularly noticeable among a group
(which will be considered in the second volume) of budding fungi, which have
been named, according to the nature of the medium in which they are found,
Mycoderma cerevisite, Mycoderma vini (Fr. fleur de la biere and fleur du vin
respectively). Pasteur denied that any of these skin-forming budding fungi
have the power of producing acetic acid, but the author refuted this opinion
by proving, in 1893, the existence of at least one such species endowed with this
faculty. More particulars concerning this will be given in one of the chapters
of the second volume. At present we are only concerned with the fact that
acetic fermentation is a vital manifestation not peculiar to fission fungi alone.

§ 210.— Morphology of the Acetic Acid Bacteria.

Strictly speaking, Pasteur's publication did not advance our knowledge of
the morphology of the organisms in question beyond the discoveries made by
Kiitzing ; and the case remained in statu quo for another fifteen years, until
taken up by Emil Christian Hansen, whose researches on the acetic acid bacteria
not only threw new light upon the organisms themselves, but were also— and
that in a dual sense — important to the subject of Fermentation Physiology
generally.

Until then the opinion was current that any given fermentation was carried
through by merely a single species of ferment. HANSEN (VI.), however, showed
in 1878 that, in the spontaneous souring of beer at least two different species
of bacteria can come into action, one of which he named Mycoderma aceti and
the other Mycoderma Pasteurianum, in honour of his predecessor. At the
suggestion of W. Zopf he afterwards changed these names to Bacterium aceti
and Bacterium Pasteurianum respectively. This important discovery was
subsequently extended, partly by HANSEN (VII.) himself — who afterwards
introduced into the literature of the subject a third species under the name
of Bacterium Kiitzingianum — and partly by A. J. BROWN (I.), W. PETERS (I.),
A. ZEIDLER (I.), WERMISCHEFF (I.) and the author. Of all these species, only
those described by Hansen have been thoroughly investigated- morphologically,
and for this reason they alone will be more closely considered in the following
lines.

When inoculated into lager-beer or the so-called " doppel-bier " — a Danish
high fermentation beer rich in extract and poor in alcohol — and kept at a
temperature of about 34° C., these three species — provided air is freely admitted
— will develop on the surface of the beer (which remains bright) to a pellicle
within twenty-four hours. In the case of B. aceti, this skin is moist and
mucinous, smooth and veined, but B. Pasteurianum is, on the other hand, dry,

MORPHOLOGY OF THE ACETIC ACID BACTERIA 297

and soon develops fine implications. That of B, Kiitzingianum resembles the
first species, but differs therefrom in raising itself high above the surface of the
liquid by gradually climbing up the walls of the vessel. Fresh differences make
their appearance when a small portion of the skin is examined under the
microscope. Whilst the cells of B. Kiitzingianum are, for the most part, single,
and are only rarely seen joined together as chains, those of the other two species
are seldom found as separate cells. The cells of B. aceti (Fig. 81) are somewhat
more slender, and frequently exhibit the sand-glass or figure 8 form (" en huit")
noticed by Pasteur. In B. Pasteurianum (Fig. 82) they are mostly rather longer
and considerably broader (plumper) than those of the other two species.

These bacterial pellicles are true zoogloea, i.e. the individual cells are attached

FIG. 81. — Bacterium aceti.

Cells from a freshly formed skin on
" doppel-bier."

Mag-n. 1000. (After Hanscn.)

FIG. 82. — Bacterium I'asteurianmn.

Cells from a freshly formed skin grown at 34° C.
on '-doppel-bier."

Magn. 1000. (After Honsen.)
FIG. 83. — Bacterium Kiitzing-ianum.

Cells from a freshly formed skin grown
at 34° C. on " doppel-bier."

Magn. 1000. (After Hansen~).

together by a mucinous envelope, formed by the swelling and mutual fusion of
the external layers of the cell membranes, in which the cells then become
embedded. In ordinary (unstained) preparations the presence of this envelope
is only deducible from the mutual cohesion of the cells ; it can, however, be
rendered visible by suitable treating and staining, e.g. by Loefflei-'s method.
Fig. 8 (p. 31) was drawn from a preparation of this kind.

The behaviour of the mucinous envelopes of these three species towards iodine
solution (iodine in water or alcohol or potassium iodide) is worthy of notice ; those
of B, Pasteurianum and B. Kiitzingianum are thereby stained blue, whilst that of
B. aceti remains unaltered. The point must be emphasised that it is the mucinous
envelopes and not the true cell membranes that are stained in this manner. The
cell plasma is in all three cases coloured yellow ; consequently the preparations
all exhibit yellow cells after the iodine treatment, these cells being embedded in
the case of B. aceti in a colourless matrix. In B. Pasteurianum and B. Kiitzingi-
anum this latter is blue, and to the unaided eye the appearance of the whole

298 ACETIC FERMENTATION

varies from green to bluish green, according to the proportion of the matrix.
It was this difference in the behaviour of the mucinous envelopes — which, how-
ever, is noticeable only in young and vigorous pellicles— that first directed the
attention of Hansen to the existence of two species of acetic acid bacteria. The
chemical composition of the envelope has not yet been determined, but that it is
not cellulose must be concluded from the negative results obtained from the tests
made with various reagents (iodosulphuric acid, zinc iodochloride) for that
substance. Already in this characteristic these three species differ from the
acetic acid bacterium introduced into the literature of the subject by A. J. Brown
under the name of Bacterium xylinum. The tough, leathery skin of zoogloea
(measuring as much as one inch in thickness), formed by this bacterium on the
surface of the nutrient solution, and generally known in England as the vinegar-
plant, consists principally of the extensively developed mucinous envelope of the
cells. If the contents be extracted by suitable means, a mass is left which
answers to the cellulose reactions (e.g. solubility in ammoniacal copper oxide)
and on ultimate analysis exhibits a composition agreeing with the formula
(C6HI005)H.

Moreover, the three Hansen species differ notably in the appearance and
development of their colonies, prepared by the transference of droplets (rich in
cells) from a pure culture grown at 25° C. on to solid nutrient media (wort
gelatin or doppel-bier gelatin). Those from B. aceti assume the form of exceed-
ingly pretty, many-rayed stars or rosettes ; those from B. Pasteurianum have an
almost perfectly smooth periphery (without dentations) and exhibit convolutions
of the surface resembling those of the brain ; whilst those of B. Kutzingi-
anum are readily recognisable by the absence of both the stellar form and
convolutions.

§ 211.— The Morphological Influence of Temperature.

Hansen's researches into the acetic acid bacteria also afford an important
support to the theory of bacterial pleomorphism, as will now be shown. The
cell forms described and illustrated in the previous paragraph are not the only
ones assumed by the fission fungi under consideration. On the contrary, the
pleomorphic variations are exceedingly plentiful, though they may all be grouped
under three main types, viz., chains of short rods (as already described), long
threads, and, finally, distended or bulged forms. The conditions ascertained by
HANSEN (VII.) as influencing the development of one of these forms, its gradual
conversion into the others, and, finally, its restoration to the original shape, will
now be briefly referred to. It must be premised that the minimum limit of
temperature at which development can proceed is for B. aceti, 4°-5° C. ; for
B. Pasteurianum, 5°-6° C. ; and for B. Kutzingianum, 6°-7° C., the maximum
being about 42° C., and the optimum temperature about 34° 0.

Cultures of Bacterium Pasteurianum on doppel-bier have shown that, at all
temperatures higher than 5° C. (but not greatly exceeding 34° C.), chains of
short rods develop, which, when grown at temperatures below 15° C., often
attain extraordinary dimensions, especially in the direction of the breadth. The
formation of chains proceeds most abundantly at about 34° C., the individual
short rods then having the ordinary form and being filled with firm, slightly
lustrous plasma.

If a small portion of such a skin, cultivated at 34° C., be transferred to a
fresh nutrient medium, and maintained at 4O°-4o.5° 0., a morphological altera-
tion of the cells (Fig. 84) occurs, and is already noticeable at the end of a few
hours. The short rods about 2 /j, long and i p. broad, of which the chains of the
seed were composed, begin to elongate, and at the end of eight to nine hours

FIG. 84. — Bacterium 1'asteuriamuu.

Morphological change from short rods to long threads. Culture on " doppel-hier " agar-agar in a
Bottcher chamber at about 40.5° C. «. chain of eight short rods ; a' -a'", the same after six, ten
and twenty hours ; b. chain of live short rods ; b'-b". the same after five and nine hours ; c and d.
after ten and twenty-one hours. Magu. 1000. {After Hansen.)

Fio. 85. — Bacterium Pasteuriauuin.

Long threads developed by twenty-four hours' cultivation at 40.5° C. on " doppel-bier.'
Magn. 1000. (After Hansen.)

300 ACETIC FERMENTATION

none but long rods are found, some of these being already disconnected, others
still retaining the chain form. The latter also finally become dismembered, so
that after a further four hours none but elongated cells, 40 p. and more in length,
are present. These now continue to grow, and in twenty -four hours from the
commencement of the experiment long threads (Fig. 85), some of them measuring
200 ft in length, are found exclusively.

A fresh modification of form sets in as soon as these long threads are exposed
to the original temperature of 34° C. — they begin to bulge. These forms (Fig. 86)

FIG. 86. — Bacterium Pasteurianum.

Conversion of long threads into swollen (bulged) forms and chains. Culture in " doppel-bier " at 34° C.
J. condition after four hours ; II. after five hours ; III. after seven hours. Magn. 1000. (After
Jiannen.)

can be already noticed at the end of four hours, and their number rapidly in-
creases from that time onward. At about the same time other portions of the
threads begin to break up into fragments, the disruption beginning indifferently
at either or both extremities, or in the middle of the thread, which is thus
modified into a chain of short rods, or one exhibiting both long rods, unaltered
portions of threads, and bulged articulations ; in short, great diversity prevails.
All intermediate stages of the last-named forms, between the very frequent
spindle cells on the one hand, and pear-shaped rounded forms on the other, are

FIG. 87. — Bacterium Pasteurianuin.

Conv ersion of long tlireads into chains of short rods. Culture on " doppel-bier agar-agar in a Bottcher
Chamber at 34° C. a. long serpentine thread at the commencement of the experiment ; a', the same
after five and a half hours ; a", the same after seven hours. The highly swollen central portion is
omitted in the drawing, b. long thread with' several lends; I', after four hours; b", after six
hours ; b"'. after nine hours. Only the central portion of the modified thread is shown. Magn.
icoo. (After Hamtn.)

302 ACETIC FERMENTATION

met with. Globular cells, measuring up to 10 p. in diameter, are also by no

means rare.

Finally,the threads become entirely dismembered into short rods (Fig. 87), even

the bulged cells undergoing this conversion, and leaving only the thickest portion

(Fig. 88) unchanged. This portion eventually, after the filamentous ends on

either side have broken up into short
rods, collapses in the surrounding liquid
and dissolves. An examination made
after the lapse of twenty-four hours then
reveals only chains of short rods. We
have thus induced a reversion to the
original forms of cell, and have there-
by learned the morphological influence of
temperature. Of course, neither the com-
position of the nutrient medium nor the
condition of the seed is a matter of in-
difference. Thus, for instance, if, instead
of sowing the young cells presupposed
in the foregoing demonstration, those
already forty-eight hours old are em-
ployed, then the conversion into threads
becomes very difficult. In the case of
lager- beer the development proceeds some-
what differently to that occurring in the
" doppel-bier " hitherto mentioned.

Bacterium aceti and B. Kiltzinyianiim

In a the pear-shaped swelling lias tapered behave very similarly under the circum-
ont into two thin threads, in & the lower stances now in question. A few small

of these has divided into short rods. In e the -\-cc • j. i 11

swelling has begun to disintegrate, a part of differences are, however, unmistakably
the plasmal cell contents escaping. In d the evident. Thus, for instance, in harmony
evacuation is complete only the thick cell wjth the plumper form of the short rods
wall being left. c. spindle-shaped swollen . n n . r . r ,11 1.1 c •, i
form, with two long threads undergoing in- « A Pasteunanum, the breadth of its long
cipient subdivision. Magn. 1000. {After threads is also greater, as will be evident
//a/wen<) on reference to Figs. 85 and 89 ; the

long threads of B. aceti are thinner, but

attain a greater length, viz., up to 500 ju. On the other hand, the long threads of
B. Kiitzingianum are considerably smaller than those of the other species.
Finally, it should be stated that branching of the long threads occasionally occurs.
A few of these comparatively rare forms are shown in Fig. 90. Pleomorphism
seems to be a general property of the acetic acid bacteria, since it was also found
by Hansen to prevail in four other species, including those discovered by Zeidler.
Like most of the other Schizomycetes, the acetic bacteria exhibit a preference
for darkness. Their development — as M. GIUNTI (I.) discovered — is restricted
(though not entirely prevented) by diffused daylight, as well as by direct exposure
to the sun; but according to the discoveries made by G. TOLOMEI (III.), this
result is due to the chemically active light rays alone. TOLOMEI (IV.) likewise
found that the discharge of strong electric sparks at a short distance above the
surface of the liquid also restricts development.

§ 212.— The Equation of Acetic Fermentation.

For a long time no clear perception was obtained of the mode of action of the
" mother of vinegar." True, it was known that the acetic acid is formed from the
alcohol present, and also that acidification does not occur when air is excluded,

FIG. 88. — Bacterium I'asteuriauuni.
Residue of swollen long threads after a sojourn of
one to two days in " doppel-bier " at 34° C.

THE EQUATION OF ACETIC FERMENTATION 303

but the reasons for these phenomena could not be given. The ABBE ROZIEK (I.)
concluded from his experiments that air is absorbed by the wine in process of
turning sour, but LAVOISIER (I.) afterwards showed that this is only true of one
of the constituents of the atmosphere, viz., oxygen. He stated that "acetic
fermentation is nothing more than the souring of wine, effected in-the open air
by absorption of oxygen." In 1821 Edmund Davy discovered platinum-black, a
substance which, when moistened with spirits of wine, becomes white-hot, the
formation of acetic acid being evidenced by the odour evolved. This observation
was followed up by DOBEREINER (I.), who found that, in this reaction, the alcohol

FIG. 8q.— Bacterium accti.

Long threads. Culture twenty -four hours old in < • doppel-bier " at 4O'-4o.5° C. In several places the
breadth of the threads is exaggerated. Magn. 1000. (After Hanscn.)

takes up oxygen — water and acetic acid, but no carbon dioxide, being formed.
By observing the volume of oxygen consumed by a weighed quantity of alcohol,
he arrived at the following equation for this oxidation process : —

C4H602 + 40 = C4H404 f- 2HO

which, translated from the symbolical language of equivalent formuke to that of
atomic formulae, reads as follows :

C2H6O + 02 = C2H402 + H./X

Hence, Dobereiner concluded that, for the production of acetic acid, only three
substances are required: alcohol, oxygen, and a body capable of absorbing and
condensing the latter, and thus bringing it into more intimate contact with the
first named, whereupon the reaction ensues.

The above experiment of Dobereiner's was taken by chemists as a starting-
point in attempts at elucidating the phenomenon of acetic fermentation. The
intermediary part played by the " mother of vinegar " in the souring of wine
was obvious, since it was well known that without this " mother " no conversion

304 ACETIC FERMENTATION

occurred. Nevertheless, more than one opinion was rife as to the mode of action
of this mucinous skin.

Berzelius, in 1829, on the basis of his theory of catalytic action, ascribed the
potency of this skin in acetic fermentation to the acetic acid " enclosed within
its pores." Ten years later, and two years after the appearance of Kiitzing's
work — which, being out of harmony with the spirit of the age, was consequently
disregarded — Liebig published his theory of acetic fermentation, in which the

" mother of vinegar " was classed along-
side platinum-black, their mode of
action being defined as identical and
of a purely chemical nature.

Owing to the endeavours of Pas-
teur, the theory promulgated by Kiit-
zing was experimentally shown to be
correct, and the true import of the
vinegar-mother once more recognised.
It would, however, be going too far to
also credit the French physiologist with
having recognised acetic fermentation
as a purely physiological process ; for
— remarkable as it may now appear
to us — Pasteur, with his followers,
stopped half-way and defined the vine-
gar fungus as " acting after the manner
of spongy platinum." He characterised
the skin-like zoogloea of the fission
fungus in question as " vegetations en-
dowed with the remarkable peculiarity
of retaining the oxygen of the air and
condensing it after the manner of
spongy platinum, by inducing the com-
bustion of alcohol and acetic acid." VV.
VON KNIERIEM and AD. MAYER (I.)
share theci'edit of having convincingly
proved, in 1873, that the oxidation of
alcohol by means of platinum-black
FIG oo-Bacturiiuniiceti cannot be classed along with the fer-

Filamentous cells of unusual form from cultures mentatk* setup by the "mother of
(several day sold) on wort and on" doppel- bier" Vinegar. Platinum - black OXldlSCS

at 39°-4i° c. Magn. 1000. (.(fter Hamm.) both concentrated and dilute alcohol,

whereas, according to the experience

of vinegar-makers, acetic fermentation cannot proceed in presence of more
than 14 per cent, of alcohol. Moreover, with regard to temperature, highly
impoi'tant differences — touching the very existence of \ the question — are
observed. Thus, whereas acetic fermentation proceeds most satisfactorily at
about 35° C. and is arrested altogether at 40° C., the energy of the oxidation
effected by platinum -black (starting at 35° C.) increases as the temperature
rises, and may become so violent that the alcohol ignites explosively and burns
away to water and carbon dioxide. Hence the composition of the (by no means
uniform) oxidation products thus formed differs greatly from those obtained
from acetic fermentation.

This latter process, whose purely physiological nature was placed beyond
doubt by these investigations, was examined more minutely by A. J. BROWN (III.)
in 1886. Meanwhile, Hansen's discovery of the existence of at least two species

PURE CULTURE FERMENTS IN VINEGAR 305

of acetic acid bacteria considerably enlarged the field of research, since thence-
forward " acetic fermentation " could no longer be spoken of without coupling
with it the name of the organism by which it was caused. The species forming
the subject of Brown's researches was obtained by him from sour (acetic) beer,
and was called Bacterium aceti, though not identical with Hansen's species
bearing the same name. Pasteur's discovery that the " acetic acid bacteria "
first convert alcohol into acetic acid, and then burn the latter to carbon dioxide
and water, was also made by Brown in connection with his B. aceti, but he did
not institute any closer examination (more particularly in connection with the
ratio of transformation) on this point, so that this theoretically and practically
most important question has still to be investigated.

Methyl alcohol, isobutyl alcohol, and amyl alcohol are not attacked by Brown's
B. aceti, but normal propyl alcohol is oxidised to propionic acid. If the nutrient
medium (yeast-water) contains dextrose but no alcohol, then gluconic acid is
formed, a fact already established by BOUTROUX (I.) in 1880, in connection with
another species of bacterium (of questionable purity). Saccharose, lactose, and
starch remain unaltered, but mannite is converted into Itevulose, which then
remains unchanged. Dulcite is unaffected, whilst glycol is converted into
gly collie acid. The behaviour of Bacterium xylinum is approximately the same
as in the organism just described. The extensive mucinous envelopes, consisting
of cellulose, are produced when the nutrient solution contains dextrose, kevulose,
or mannite ; whilst, on the other hand, cane-sugar and starch are useless for
this purpose,

We are indebted to G. BERTKAND (I.) for a beautiful experiment with a
fission fungus, not accurately identified, but presumably very closely allied to
Bacterium xylinum. Mountain-ash berries, i.e. the fruit of Sorbus aucuparia, S.
intermedia, and S. latifolia, contain, in addition to glucose, an alcohol isomeric with
mannite, viz., Sorbitol (C6H1406). If now the juice of these berries be subjected
to alcoholic fermentation (which sets in rapidly and spontaneously), the glucose
is decomposed, but not the sorbitol, this latter only being attacked when the
above-mentioned fission fungus obtains access into the fermented liquid, which
it does through the mediation of a small red fly (Drosophila funebris, Fabricius,
1). cettaris, Macquart), known to all fermentation technicists as the "vinegar-
fly." This insect haunts places where alcoholic juices (especially fermented fruit-
juices) are being stored and converted into vinegar, and there loads itself with
acetic acid bacteria, which it then transfers to other localities. The bacterium
introduced by these flies into the fermented juice of the mountain-ash berry
oxidises the hexavalent alcohol sorbitol to the ketose sorbin (also known as
sorbinose or sorbose), according to the equation —

C6H1406 + 0 = C6H1208 + H20.

This affords a convenient method for the production of sorbose.

"With regard to the fermentative capacity of B. aceti Hansen, and B, Pasteur-
ianum, the author, in 1895, published comparative investigations, showing that
a sowing of the first- named species on pale lager-beer is able to develop and
exert a powerful acidifying effect at 4°-4.5° C. ; whereas B. Pasteurianum is
unable to do this, or to reproduce itself at all, even at 4.5°-5° C.

§ 213.— Pure Culture Ferments in the Manufacture of Vinegar.

Searching investigations into the chemical activity of the different species of
acetic acid bacteria would be not only opportune in the interests of science, but
also highly important to the practice of the vinegar industry. In this business
i u

306 ACETIC FERMENTATION

the employment of selected pure culture ferments is not yet regarded as a funda-
mental rule, everything being still left to the mercy of chance.

As every reader will be aware, there are two different methods of making
vinegar. In one of them wine forms the raw material, this method being
known as the Orleans process, from having long been extensively carried on in
that locality. There (as elsewhere) the work is still performed in the same
manner as it was centuries ago, as follows : — A number of oaken casks, each of a
capacity of some 55 gallons, are arranged in rows in a chamber maintained at a
constant temperature of 1 8° to 22° C. In the upper part of the front end (head)
of each cask a circular aperture (a few cm. in width) is provided, through which
the cask can be filled or emptied, and which is generally kept closed, whilst near
it is a very small hole (vent) always left open for the admission of air. In
normal work each cask is about half full. Before setting a new cask in work,
it is scalded out several times with steam or hot water, in order to extract the
sap from the wood, and is then "soured" by impregnating it with good, boiling-
hot vinegar. About i hi. (22 galls.) of good clear vinegar and 2 1. (0.44 gall.)
of wine are then placed in the cask, another 3 1. of wine being added at the end
of eight days, 4 to 5 1. more after the lapse of another week, and so on until the
cask contains about 180-200 1. (40-44 galls.). Then, for the first time, vinegar
is drawn from the cask, and in such quantity that about 22 galls, are left
behind in the vessel. From that time the cask (" mother ") is in continuous
use, 10 litres (2.2 galls.) of vinegar being withdrawn every week and replaced
by an equal quantity of wine. The " mother " casks may remain in work during
six or eight years without interruption, but at the end of this period they will
, contain such a considerable accumulation of deposited yeast, tartar and mother
I of vinegar, as to necessitate their being emptied and cleansed. J A skin, known
as vinegar-flowers or mother of vinegar, and composed of acetic acid bacteria,
develops on the surface of the liquid, and the manner and luxuriance of its
growth enables the operator to judge the progress of the fermentation. How-
ever, at the outset the growth proceeds very slowly, since the wine employed
mostly contains but very few of these bacteria. Consequently an opportunity
is afforded for the development of rapid-growing injurious organisms, chiefly
certain budding fungi, which consume the acetic acid. The aerobic " vinegar
eels" also make their appearances To obviate this source of loss, PASTEUR (XV.),
in 1862, proposed that, instead of waiting until the acetic acid bacteria in the
wine had increased sufficiently to form a protective skin of " vinegar-flowers,"
the necessary ferment should be cultivated in small vessels, the skin thus
obtained being then carefully transferred in pieces of sufficient size, by the aid
of a wooden spatula, on to the surface of the wine to be soured, which was
i placed in shallow open vats.f This process was adopted by Breton-Lorion of
Orleans, in particular, and would be suitable for general application if the
presence of not more than one species of acetic ferment could be thereby ensured.
This, however, is not the case, and it is purely a matter of chance whether the
skin prepared by cultivation beforehand is composed of beneficial or injurious
organisms.) According to circumstances, there may be present several very
different species with divergent properties, faculties, conditions of vitality and
metabolic products. By reason of this uncertainty alone, the Pasteur method is
liable to produce very irregular results, and may, on occasion, actually give rise
to losses; and, as a matter of fact, it is just on this account that the method has
been abandoned both in France and Germany, where it was introduced by
E. WURM (I.). Up to the present it does not appear that any one has attempted
to work with really pure cultures.

In the second method actually employed for making vinegar, spirit is used
instead of wine. This method has been evolved from that originally prescribed

PURE CULTURE FERMENTS IN VINEGAR 307

by Hermann Boerhave, and has attained its present condition (since 1823) mainly
through the instrumentality of Karl Schiizenbach. The French term it the
" German method," but in Germany it is generally known as the " quick vinegar
method " (Schndlessig-Fabrikation). A detailed description cannot be given here,
but the gist of the process consists in slowly running the " goods " (i.e. spirit
diluted with vinegar) to be turned into vinegar over shavings or strips of beech-
wood contained in a closed vat (the vinegar-generator), so that the liquid presents
a large surface to the air, which is admitted through special ventilating holes
below and makes its escape at the top. That the fermentative activity of micro-
organisms also comes into play in this method can no longer be doubted since
the searching investigations of Pasteur, which were confirmed (on repetition) by
Mayer and Knieriem. Pasteur showed that no acidification takes place if the
alcohol be allowed to trickle over shavings destitute of fungi. He assumed that
the organism taking part in the quick vinegar process is the same as that forming
the superficial skin in the Orleans method, the fungus being supposed to settle
on the shavings in the vinegar -generator and convert the slowly running vinegar
goods into acetic acid. Up to the present no precise investigations on the bacteria
acting in this branch of industry have been made public. This highly necessitous
industry has, more perhaps than any other, to struggle against a variety of
difficulties ; the actual losses of alcohol are enoi'mous, and no one is able to offer
any reliable explanation of their cause. The introduction and intelligent use of
suitable pure culture ferments would be a great boon. How much still remains
to be done and ascertained in this instance can be estimated by a comparative
glance at the conduct of fermentation in the operation of brewing. Not least
among the advantages to be derived from such a method of working — which we
may hope will soon be elaborated — would be the possibility (not afforded by the
present method) of combating the " vinegar eels." With regard to these
objectionable parasites, it may be mentioned that detailed morphological and
physiological information concerning Anguillula aceti will be found both in
Czernat's monograph (excerpts from which are contained in Borgmann's trans-
lation of Pasteur's Etudes sur le Vinaiyre] and in a treatise by G. LINDNER (I.),
which latter work chiefly deals with the pathogenic potency of these worms. As
SADEBECK (I.) has found, these parasites are occasionally themselves infested and
killed by a fungoid parasite belonging to the group of Oomycetes (mentioned in
the second volume), and known as Pythium Anyuillulce aceti.

CHAPTER XXXVIII.

THE OXYDASES.

§ 214.— The Browning of Wines.

IN addition to the purely chemical absorption or fixation of oxygen (e.g. in the
conversion of SO., into S03), on the one hand, and the oxidation effected directly
by the vital activity of micro-organisms on the other, there is a third method of
transferring this gas, viz., by the action of enzymes, to which the name oxydases,
proposed for them by Weigert, may be generally applied. How many kinds of
oxydases exist is a matter for future research to determine. At the present
time the subject is merely in an incipient stage, though the commencement
made is a highly promising one, and has already led to the explanation of
several phenomena which only a short time back were regarded as extremely
puzzling.

One of these is the so-called " browning " of wines, known in France as
" la casse" "le cassage," or "cassure" (Ger. Rahn-Werden or Braunwerderi).
This occurs chiefly in white wines and was for a. long time classed along with the
malady known as loss of colour in (red) wines. In France attention, was first
directed to its distinct character by ARM. GAUTIER (I.) in 1878, but in Germany
this was known at an earlier date. The most important characteristic of " vin
casse " is the rapid change of colour undergone by the wine as soon as it is poured
out of the cask or bottle into an open glass, the colour of the upper layers
(exposed to the air) of the hitherto pale liquid becoming darker, and finally (in
the course of a few hours) becoming brown. This coloration also gradually pro-
gresses in the deeper layers, and, at the same time, the flavour becomes unpleasant
(air taste). Turbidity then ensues, but disappears in proportion as a fine dark
brown pulverulent sediment settles down. The liquid is now (three to four
hours after the commencement of the experiment) again bright, though darker
in shade than when newly drawn from the cask. The taste has also improved
again, without, however, being equal to what it was at first.

In view of the fact (indubitably proved by Nessler's experiments) that
this malady only occurs when air is admitted, it was regarded as an oxida-
tion process, without any more precise acceptable explanation being forthcoming.
After Gautier had presumed, and A. BOUFFARD (I.) in 1894 had denied, the
probability of bacterial activity in this phenomenon, it was shown by G.
GOUIKAND (I.) that we have here to do with the action of an enzyme which plays
the part of a carrier of oxygen. He isolated the same (though not in a pure
state) from browned (white and red) wines, and produced therewith the same
malady in previously sterilised sound wines. This enzyme must probably be re-
garded as acting by the absorption of atmospheric oxygen, which it then gives up
again, not only to the colouring-matter in the wine, but also to the tannin, and
thus converting them into insoluble dark-coloured compounds. It is to be hoped
that, ere long, this matter will have been made clear by further investigation.

It is found by experience that the wines obtained in wet autumns from
rotten grapes, as also those affected with " sweet-rot " (Edelfdule ; pourriture
noble), and such as are poor in acid, are subject to this malady with comparative

308

DISCOLORATION OF VEGETABLE JUICES 309

frequency. Nessler made searching investigations into the means of combating
this complaint in practical viticulture. The most important result obtained
was the discovery that the browning of wine can be prevented by thoroughly
fumigating the casks with 1-2 grams of sulphur per hectolitre — 22 galls, or
3.53 cubic feet — of cask-room before use, the malady being found, in Nessler's
experiments, not to ensue when the wine contained a minimum quantity of
0.003 per mil. of S02. According to the researches of Gouirand, this enzyme is
destroyed by a temperature of 80° C., 60° C. being apparently insufficiently
reliable for this purpose. MULLER-THURGAU (VI.) made the discovery, im-
portant in cellar management, that the tendency of wine to turn brown could
be prevented by Pasteurisation, i.e. keeping it for some time at a temperature
of 6o°-62° C., which it will endure without acquiring the so-called " boiled "
taste.

The acquisition of a more accurate characterisation of the enzyme, and the
consequent possibility of distinguishing it from other oxydases, is desirable, this
being a necessary preliminary to the elucidation of its origin. Possibly the
enzyme is not formed anterior to fermentation, but, on the other hand, its
presence in the grapes themselves and in the must is not absolutely precluded.
V. MARTINAND (I.) has actually found oxidising enzymes in wine-must on many
occasions. The elucidation of the conditions under which browning may be
caused in wines is a subject requiring further investigation, the question
whether the presence of special metabolic products is essential, or whether the
oxydase here concerned differs from those observed by Martinand, being still
unsolved. Moreover, it appears from the discoveries of this observer that, in
the maturing of wine', the alterations of flavour occurring — and which may be
accelerated by the influence of oxidising agents (ozone, the electric current) —
are, under natural conditions, brought about by the agency of oxydases which
still require closer identification. The same applies to the darkening of the
colour of wine during storage. According to G. TOLOMEI (V.), oxydases are
also produced by the wine-yeasts Saccharomyces apwidatus and Sacch. ellip-
soideiw.

§ 215.— The Rapid Discoloration of Fresh Vegetable Juices

is in many cases attributable to the action of oxydases. Technical interest
in the discoveries made on this point is chiefly centred in the researches of
G. BERTKAND (II.) on Japanese lacquer, that lustrous and extremely durable
varnish employed in Eastern Asia for coating wooden furniture and similar
articles. By making incisions in the bark of the indigenous Rhus vernicifera — a
tree of the family Anacardiacece and closely allied to the European garden-tree
Rhus cotinus (Venice sumach) — a juice is obtained which, on admixture with the
oil from Bignonia tomentosa and (for red lacquer) vermilion, yields the lacquer
in question. This juice resembles a thick pale cream and will keep unchanged
for a long time if stored in closed bottles, but quickly turns brown when air is
admitted, becoming covered in a few minutes with a tough black skin, and
finally hardening — this being, in fact, the property for which it is so highly
prized. That a process of oxidation is here in question cannot be doubted.
The constituent thus converted has been isolated by Bertrand under the name
of laccol, and recognised as a compound allied to the polyatomic phenols, and
capable of producing extremely violent reddening and inflammation of the skin
if applied in even very minute quantities. The juice also contains an oxydase,
named by Bertrand laccase, by the known oxygen-carrying powers of which the
laccol is rapidly converted into a hard, black oxy-compound, insoluble in water,
alcohol, &c. This product is not obtained in the absence of the enzyme, only a
resinous soluble grease, that remains sticky for a long time, being obtained under

3io THE OXYDASES

such circumstances, In addition to laccol, other polyatomic phenols (pyrogallol,
hydroquinone, &c.) and their acid derivatives (e.g. gallic acid and tannin) are
quickly oxidised by laccase in presence of air. According to the further dis-
coveries of BERTRAND (III.), the polyphenols containing at least two groups of
OH or NH2 (either in the ortho- or in the para-position) are also easily and
readily oxidised by this oxydase.

This interesting discovery gave an impulse to the elucidation of several other
phenomena interesting both to the food-stuff chemist and the agriculturist. It
is well known that the freshly broken or cut surfaces of raw apples rapidly
become discoloured on exposure to the air, at first turning reddish and then
becoming brown. This is the cause of the ugly colour of expressed apple-must.
Housewives skilled in cookery are aware that this alteration of colour does not
ensue if the cellular structure of this fruit is preserved unbroken until after the
apple has been boiled. L. LINDET (I.) in 1893 explained this discoloration as
resulting from the action of an enzyme, to which he subsequently gave the name
of laccase — without, however, implying the identity of this with the oxydase of
the lac-tree. The name of malase would probably be more suitable for this apple
enzyme. In the case of apple-juice also, oxygen is carried by the enzyme to the
tannin, and thus dark-coloured oxy-compounds are produced, which are pre-
cipitated on the cell walls as a fast, permanent dye. The spotting of sound
apples under the rind, the so-called brown spotting, is explainable in the same
manner. So long as the structure of the cell remains perfectly intact, the
atmospheric oxygen cannot obtain access to the enzyme (in the plasma) or to the
tannin. As soon, however, as by mechanical action (e.g. the dropping of the
apple from the tree, pressure in packing or transit, &c.) any of the cells become
ruptured, then an opportunity is afforded the oxygen to act on the now exposed
constituents of the plasma. If the rind of the fruit remains uninjured, the air
gains admission to the interior merely through the intracellular spaces alone, and,
in such event, will produce only a faint reaction and slight discoloration.
Whether, as assumed by Lindet, the enzyme and tannin are contained in separate
cells (i.e. distinct from each other), is a question still requiring more accurate
research on the part of the botanist to decide.

The darkening of beet-juice, or the rapid discoloration of the fresh slices of
beet in the sugar- works evidenced even when cutting-tools devoid of iron are
employed, is equally attributable to the action of an oxydase present in the sugar-
beet. This was discovered by G. BERTH AND (IV.), and received the name of
Tyrosinase, because it carries atmospheric oxygen to the tyrosine — well known
to be abundantly present in the cells of the sugar-beet — and thus produces the
discoloration in question. On the other hand, laccase has no effect on the said
amido-compound. Apart from this property, t}7rosinase is also characterised by
its greater susceptibility both to heat and chemical influences. It occurs in other
plants, e.g. the bulbs of the dahlia (Dahlia varidbilis). According to the
researches of G. BERTRAND (V.), oxidising enzymes are also found in other
plants, e.g. in the carrot ; the tubers of the potato (which, as is well known,
rapidly become discoloured when cut in an uncooked state) ; in the pear, quince,
and chestnut ; in the sprouts of asparagus, clover, lucerne, and rye-grass ; in
the leaves of the potato, sugar-beet, &c. For detecting this class of enzymes
Bertrand recommends the employment of guaiacum tincture, which produces
therewith a blue coloration when dabbed or poured on to the cut surface or juice
under examination. To isolate these enzymes the plant juice is mixed with
alcohol, the resulting precipitate being dissolved in a little water and filtered.
On pouring the filtrate into five volumes of alcohol, a precipitate consisting of the
desired enzyme will be formed.

The so-called rusting or tarnishing of many of the agarics, i.e. the rapid

THE BITTERING OF WINE 311

discoloration of freshly broken or cut surfaces in the body of the fungus, is well
known. The bluing of two of these, which he styled Boletus luridus and
Agaricus sanguineus (?), was explained by OH. SCHONBEIN (I.) in 1856, by stating
that these fungi contain a resinous substance soluble in alcohol (the above-
mentioned reaction with guaiacum tincture will be remembered !), and becoming
converted into a blue oxy-compound when brought into contact with ozone.
The formation of this latter from the oxygen of the air is accomplished by the
activity of another substance, also present in the fungi, and destructible by heat.
This active substance was subsequently (in 1895) proved by E. BOURQUELOT and
G. BERTRAND (I.) to be an oxydase, and was detected by them in 59 out of 107
species examined : e.g. in 18 species of the genera Russula and Lactarius,
10 species of the genus Boletus, 2 species of the genus Amanita, &c. According
to the researches of BOURQUELOT and BERTRAND (II.), the enzyme giving rise to
the bluing of Boletus cyanescens is similar to laccase ; but another oxydase,
causing the freshly fractured surfaces of Russula nigricans to first turn red and
finally become black, is certainly different.

G. TOLOMEI (VI.) discovered in ripe olives an oxydase which he called olease.
In many parts of Italy it is customary to allow the olives, before putting them
through the press, to undergo a spontaneous decomposition, which is chiefly
effected by this olease, but has not yet been sufiiciently investigated. This
enzyme also passes into the oil prepared at temperatures below 70° 0., and pre-
sumably continues to convey oxygen gradually thereto as well, oleic acid, acetic
acid, sebacic acid, &c., being formed.

Another phenomenon not yet accurately known (but possibly also attributable
to enzymatic activity) will now be considered, since otherwise no convenient
opportunity would offer, and that is

§ 216.— The Bittering of Wine.

This malady makes its appearance in many districts, such as the French Jura
(Burgundy wine), the Ahr valley (Rheinland), Voslau near Vienna, and Sicily
(Vino del Faro di Messina), with comparative frequency, and almost exclusively
affects red wines. The commencement of the disease is evidenced by a reduction
in the acid content, the wine becoming apparently sweeter again (French cellar-
masters say "le vin doucine"). By degrees the liquid turns paler, and is finally
decolorised completely, the colouring-matter being deposited as an insoluble
sediment or covering the walls of the bottle as with a skin. Concurrently, the
wine develops a strange odour and a bitter after-taste, which finally becomes so
strong as to render the liquid undrinkable. This malady first makes itself
apparent in the second or third year of storage, and oftentimes not before the
wine is bottled for maturing.

Respecting the cause of this incurable disease of wine, nothing reliable can
as yet be stated. PASTEUR (XVI.) attributed it to the activity of a rod-shaped
fission fungus, without, however, being able to throw any further light on the
matter. The bacteria found in large numbers in bitter wine are for the most
part covered with flakes and fragments of the precipitated brownish red colouring-
matter, and hence very often assume remarkable shapes. They may be freed
from these incrustations by the addition of a droplet of a solvent mixture of
alcohol and tartaric acid to the preparation. R. ADERHOLD (I.) unsuccessfully
attempted to prepare pure cultures of the organism suspected of causing this
malady, but PERRONCITO and MAGGIORA (I. ) were able to artificially induce the
complaint in sound wines by inoculating them with a bouillon culture of
microbes discovered in bitter wine ; the infection, however, succeeding only in
such samples as contained less than 8.5 per cent, of alcohol. The attempts at

312 THE OXYDASES

noculation made by E. Kramer with a bittered white wine from the province of
Kiistenland (Austria) did not prove satisfactory. At present, uncertainty pre-
vails not only with regard to the organism causing this complaint and the
external conditions influencing its development, but also as to the nature of the
bitter principle itself. The opinion expressed by Mulder, that citric ether is in
question, was refuted by C. NEDBAUER (I.), who proved that this (still uninvesti-
gated) bitter principle is a compound that is not volatilised by boiling the wine.
From experiments made by J. BERSCH (I.), it is permissible to conclude that the
tannin present is decomposed and consumed by the organisms here in question.
This observation would' suffice to explain the fact mentioned at the commence-
ment of this paragraph, that bittering is almost exclusively confined to red wines,
these containing, as is well known, a somewhat large amount of tannin absorbed
from the skins and kernels of the grape during the primary fermentation.

It may be useful to casually mention, in conclusion, that the bittering of
alcoholic beverages, beer in particular, may also be occasioned by higher fungi
(yeasts). Fuller particulars will be found in a subsequent chapter in the second
volume, dealing with Saccharomyces Pastorianus, and to which the reader is
hereby referred.

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