•

GIFT OF
Dr. Beckwith

SJIULUC i

LIBRA:;/

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AGRICULTURAL AND
INDUSTRIAL BACTERIOLOGY

AGRICULTURAL AND
INDUSTRIAL BACTERIOLOGY

BY

R. E. BUCHANAN

PROFESSOR OP BACTERIOLOGY IN THE IOWA STATE COLLEGE OP

AGRICULTURE AND THE MECHANIC ARTS, AND BACTERIOLOGIST OF THE

IOWA AGRICULTURAL EXPERIMENT STATION

D. APPLETON AND COMPANY
NEW YORK :: 1921 :: LONDON

BIOLOGY
LIBRARY

COPYRIGHT, 1921, BY

D. APPLETON AND COMPANY

PRINTED IN THE UNITED STATES OF AMERICA

PREFACE

AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY is based on
practical experience as a teacher and lecturer during the
last sixteen years, and has been written to supply a need
constantly felt during these years of practical work with
students of agriculture — the need for a book to include
those bacteriological topics fundamental in everyday life
on the farm and in the industries. The volume has also
been written for, textbook use and to serve as a reference
work for students and the many men and women interested
in agricultural and industrial problems.

Written along such lines, and for such a public, AGRICUL-
TURAL AND INDUSTRIAL BACTERIOLOGY contains no detailed
discussion of methods and technique, since it is not intended
to serve as a manual of laboratory practice. This volume
has been written to lay a satisfactory and practical founda-
tion on which may be based more advanced work in the
specialized applications of bacteriology in dairying, soil tech-
nology, forestry, plant pathology, and various industries
such as bottling, baking and canning.

In certain parts of the book it is assumed that the reader
possesses some knowledge of chemistry and biology ; but the
major portion of the volume requires no such background
of previous work and preparation.

R. E. BUCHANAN.

IOWA STATE COLLEGE.

792095

CONTENTS

SECTION I

MORPHOLOGY AND CLASSIFICATION OF
MICROORGANISMS

CHAPTER PAGE

I. THE DEVELOPMENT OF AGRICULTURAL AND INDUSTRIAL

BACTERIOLOGY 3

Development of the microscope — Controversy over the
theory of spontaneous generation — The Controversy
over the germ theory of fermentation — Controversy over
the theory of pleomorphism — The Controversy over
the germ theory of disease.

II. GROUPINGS AND GENERAL RELATIONSHIPS OP MICRO-
ORGANISMS 11

Differences between microscopic plants and animals —
Position of bacteria, yeasts, and molds in the plant king-
dom— Groups of Thallophyta showing position and
relationships of bacteria, yeasts, and molds — Thall-
ophyta— Differentiation of bacteria, yeasts, and molds —
The protozoa.

III. MORPHOLOGY AND CLASSIFICATION OF THE BACTERIA MOR-
PHOLOGY OF BACTERIA 15

Shape — Size — Multiplication of bacteria — All groupings of
bacteria — Structure of the bacterial cell and its appen-
dages— Spore formation — Conidia formation — Classifi-
cation of bacteria — Key to genera of bacteria of agricul-
tural and industrial significance — Outline of bacterial
classification— Order Eubacteriales— Key to families of the
Eubacteriales — Family Nitrobacteriacese — Acetobacter
— Nitrosomonas — Nitrobacter — Azotobacter — Rhizo-
bium — Family Bacillaceae — Bacillus — Clostridium —
Family Coccaceae — Neisseria — Streptococcus — Dip-
lococcus — Staphylococcus — Micrococcus — Sarcina —
Family Pseudomonadacese — Family Bacteriaceae — Ery-
throbacillus — Erwinia — Proteus — Bacterium — Pas-
teurella — Hemophilus — Lactobacillus — Family Spir-
illaceae — Vibrio — Spirillum — Order Actinomycetales
— Actinomyces — Mycobacterium — Corynebacterium —
Pf eitferella — Order Spirochaetales — Treponema .
vii

yjij CONTENTS

CHAPTER PAGE

IV. MORPHOLOGY AND CLASSIFICATION OF THE YEASTS 43

Size, shape, and grouping of yeast cells — Structure — Cell

contents — Vegetative multiplication of yeast cells —

Development of spores — The classification of yeasts —

f Relationships of true yeasts — Classification of the

yeasts — Saccharomyces — Schizosaccharomyces.

V. MORPHOLOGY AND CLASSIFICATION OF THE M'OLDS 50

Structure of mold hyphse — Structure of the mold cell —
Mold growth — Multiplication in molds — Sexual repro-
duction in molds — Asexual reproduction in molds —
Sporangium formation — Conidia — Classification of the
molds — Key for differentiation of common genera of
molds — Rhizopus — Mucor — Key to species of Mucor —
Oospora — Aspergillus — Key to Aspergillus Species —
Penicillium — Key to some common species of Penicil-
lium — Trichotheciuin — Hormodendrum and Clados-
porium — Alternaria .

SECTION II
METHODS OF STUDY

VI. CULTURAL METHODS 71

- Methods of sterilization — Sterilization by heat — Sterili-
zation by light — Sterilization by filtration — Culture
media — Nutrient substances used in preparation of
media — Solid media — Adjustment of the reaction of the
medium — Pure culture methods — Study of growth char-
acters.

VII. METHODS OF STUDYING PHYSIOLOGIC CHARACTERS 92

Determination of changes in hydrogen ion concentration —
Color changes of Clark and Lubs indicators — Deter-
mination of acid production — Determination of alcohol
production — Determination of aldehyde — Production
of acetyl methyl carbinol — Determination of oxygen re-
lationships— Determination of gas production — Deter-
mination of reduction changes — Determination of indol
— Digestion of starch and other insoluble carbohy-
drates— Digestion of proteins — Fixation of atmospheric
nitrogen — Oxidation of ammonia and of nitrites.

VIII. MICROSCOPIC METHODS 106

The oil immersion objective — Microscopic observation of
colonies and cultures — Unstained preparations — Stained
preparations — Spore stain — Study of acid fast bacteria —
Gram's stain — Staining of Flagella.

CONTENTS

SECTION III
PHISIOLOGY OF MICROORGANISM

CHAPTER PAGE

IX. EFFECT OF PHYSICAL ENVIRONMENT UPON MICROORGAN- •

ISMS ............................................. 115

Rates of growth and death — Effect of light — Effect of
heat — The minimum temperature — Optimum growth
temperature - - The maximum temperature — Growth
temperature range — Thermal death point — Effect of
electricity — Affect of X-rays and radium rays — Effect of
pressure — Effect of osmotic pressure — Effect of desicca-
tion.

X. PHYSICAL EFFECTS PRODUCED BY MICROORGANISMS ....... 127

Light produced by microorganisms — Production of heat —
Changes in viscosity — Effect on osmotic pressure.

XI. EFFECT OF CHEMICAL ENVIRONMENT UPON MICROORGAN-

ISMS ............................................. 131

Effect of chemicals on direction of movement — Effect of
chemicals on direction of growth — Effect of chemicals
on rapidity of growth — Effect of hydrogen ion concen-
tration on rapidity of growth — Effect of oxygen upon
rapidity of growth — Effect of kinds of nutrients present
— Effect of concentration of nutrients — Stimulating
effects of dilute poisons — Effect of autogenic substan-
ces — Effect of chemicals upon rate of death — Charac-
teristics of an ideal disinfectant — Effect of hydrogen
ion concentration upon death rates — Alkalinity or alka-
lies — Salts of the heavy metals — The non-electrolytes —
Oxidizing agents — Essential oils — Stimulation of disin-
fection — Autogenic products — Use in therapeutics.

XII. CHEMICAL CHANGES PRODUCED BY MICROORGANISMS

ENERGY RELATIONSHIPS ........................... 147

Sources of Energy — Sunlight as a source of energy to mi-
croorganisms — Oxidation as a source of energy for micro-
organisms — Methods of securing energy by oxidation —
Cycles of the elements — The nitrogen cycle — The car-
bon cycle — The sulphur cycle.

XIII. MECHANISM OF CHEMICAL CHANGES PRODUCED BY MICRO-

ORGANISMS — ENZYMES AND FERMENTATION ............ 157

Enzymes — The hydrolytic enzymes or hydrolases — The
splitting and oxidizing enzymes — Fermentation — The
origin of the products of fermentation.

CONTENTS

SECTION IV

BACTERIA IN TECHNICAL AGRICULTURE AND THE
INDUSTRIES

CHAPTER PAGE

XIV. RELATIONSHIPS OF MICROORGANISMS TO THE PRESERVA-
TION OF FOOD 173

Agencies which bring about deterioration of foods —
methods of preventing deterioration — Sterilization by
heat — Food preservation by pasteurization — Preserva-
tion by low temperatures — Preservation by chemicals —
Preservation of foods by drying.

XV. FERMENTATION OF CARBOHYDRATES — ALCOHOLIC FER-
MENTATION IN FOODS — COMMERCIAL PRODUCTION OF

ETHYL ALCOHOL — FUSEL OILS 183

Agencies active in alcoholic fermentation — Preliminary
preparation of carbohydrates for yeast fermentation —
Alcoholic fermentation of sugar solutions — Preparation
of distilled liquors — Manufacture of commercial alcohol
— Alcoholic fermentation in bread making — Alcohol
production in other foods — The commercial prepara-
tion of yeast — Development of fusel oils in alcoholic
fermentation.

XVI. LACTIC ACID FERMENTATION — THE GENERA LACTOBA-

CILLUS — STREPTOCOCCUS AND BACTERIUM 192

Organisms producing lactic acid — The genus Streptococ-
cus— The genus Lactobacillus — The genus Bacterium —
Technical utilization of lactic acid fermentation — Asso-
ciative action — Lactic acid bacteria in milk — Soured
milk beverages — Sour curd cheese — The use of starter —
Effects of pasteurization upon the souring of milk —
Secondary changes in soured milk — Lactic acid in cheese
manufacture — Lactic acid in food preservation —
Ensilage — Sauerkraut — Other fermented foods — Lactic
acid in yeast manufacture — Lactic acid in bread
making.

XVII. ACETIC, BUTYRIC, OXALIC AND CITRIC FERMENTATION —
THE GENERA ACETOBACTER, BACTERIUM, CLOSTRIDIUM,

ASPERGILLUS AND ClTROMYCES 209

Acetic fermentation — Types of acetic acid fermentation —
Groups of acetic bacteria — The genus Acetobacter -- Vine-
gar manufacture — Butyric fermentation — Organisms
producing butyric fermentation — Formation of other
volatile acids— Oxalic fermentation — Citric acid fer-
mentation.

CONTENTS

XI

CHAPTER PAGE

XVIII. FERMENTATION OF POLYSACCHARIDES AND FATS — THE

GUMS — HEMICELLULOSES AND CELLULOSES 220

Distribution — Fermentation — Fermentation of Pecgin, the
process of retting — Fermentation of starch and inulin —
Fermentation of fatty acids and glycerin.

XIX. DECOMPOSITION OP ORGANIC NITROGENUS COMPOUNDS —

RIPENING OF CHEESE AND OF MEAT 228

Types of organisms — The anaerobic, putrefactive bac-
teria — Aerobic, proteolytic bacteria — Proteolytic
changes in foods — The ripening of cheese — Types of
cheeses — Ripening of meat.

XX. BACTERIA OF THE SOIL — THE CYCLES OF THE ELEMENTS . . . 238
Importance of bacteria in the soil — Abundance and dis-
tribution of bacteria in the soil — Cycles of the elements —
The cycle of nitrogen in nature with special reference to
the soil — Outline — Proteolysis, ammonification or de-
aminization — Nitrification — Assimilation of nitrogen by
plants — Denitrification — Nitrogen fixation — The cycle
of carbon — Production of carbon dioxide in soil by mi-
croorganisms— The decomposition of insoluble carbohy-
drates in the carbonaceous matter in the soil — The
cycle of phosphorus — The cycle of sulphur — Organ-
isms oxidizing hydrogen sulphide and free sulphur — Or-
ganisms reducing sulphates to sulphides — The cycle of
iron.

SECTION V
MICROORGANISMS AND DISEASE

XXI. DISEASE, INFECTION, AND RESISTANCE 273

Disease—Contagious and noncontagious diseases —
Factors predisposing to disease — Heredity and dis-
ease— Immunity — Exhaustion theory of immunity —
Noxious retention theory — Pythogenic theory of
disease — Ehrlich's Humoral Theory of Immunity —
Metschnikoff's Theory of Phagocytosis — Kinds of
immunity — Factors which determine immunity —
Antigens and their corresponding antibodies — Toxins
and antitoxins — Sources of toxins — Origin of anti-
toxin— Manufacture of diphtheria antitoxin — Prep-
aration and standardization of toxins — Injection
of the toxin — Standardization of antitoxin serum —
Preparation and utilization of other antitoxins —
Agglutinins and pricipitins — Colloidal solutions and
suspensions — Agglutination — Protein Precipitation

xji CONTENTS

CHAPTER PAGE

— Cytolysins, including bacteriolysins and hemoly-
sins — Constitution of Cytolysins — Complement
fixation in the diagnosis of disease — Opsonins and
phagocytosis — Detention of opsonin — Opsonic index
— The presence of opsonins in antisera — Significance
of opsonins in immunity — Hypersusceptibility or
anaphylaxis — Examples of natural anaphylaxis —
Desensitization — Utilization of the anaphy lactic
shock in disease diagnosis.

XXII. MICROORGANISMS OF THE BODY IN HEALTH AND DIS-
EASE 305

Bacteria of the body — How bacteria gain admission to
the tissues of the body. Infection atria — How bac-
teria leave the body — The types of diseases produced
by microorganisms.

XXIII. NONSEPTIC INFLAMMATION AND SUPPURATION — THE

GENERA STREPTOCOCCUS AND STAPHYLOCOCCUS 309

Inflammation — Suppuration — Nonspecific inflamma-
tion — Streptococcus pyogenes — Synonym — Mor-
phology — Distribution — Culture — Physiology —
Pathogenesis — Focal infections — Immunity — Sta-
phylococcus aureus and Staphylococcus albus —
Synonyms — Morphology and staining characters —
Distribution — Cultural characters — Physiology —
Pathogenesis — Immunity .

XXIV. PNEUMONIA. MENINGITIS AND GONORRHEA — THE SPE-

CIFIC PYOGENIC Cocci — THE GENERA DIPLOCOCCUS

AND NEISSERIA ' 321

Diplococcus pneumonise — Synonyms — Distribution —
Morphology — Cultural characters — Physiology —
Pathogenesis — Bacteriological diagnosis — Immu-
nity — Neisseria gonorrhoeae — Synonyms — Mor-
phology — Culture — Physiology — Pathogenesis —
Bacteriological diagnosis • — Immunity — Neisseria
meningitidis — Synonyms — Morphology — Physi-
ology— Pathogenesis — Bacteriological diagnosis —
Immunity.

XXV. THE COLON-TYPHOID SERIES OF BACTERIA. THE GEN-
ERA BACTERIUM AND PROTEUS 331

The colon or colon aerogenes subgroup of bacteria —
Bacterium coli — Synonym — Distribution — Morphol-
ogy— Cultural characters — Physiology — Pathogene-
sis— Recognition — Intermediate or enteritidis sub-
group — Bacterium cholerae-suis — Synonyms —

CONTENTS

Xlll

CHAPTER

PAGE

Bacterium enteritidis — Synonym — Bacterium par-
atyphi and Bacterium schottmulleri — Synonyms —
. Bacterium pullorum — Typhoid-dysentery subgroup
— Bacterium typhi — Synonyms — ; Morphology —
Cultural characters — Physiology — Disease pro-
duced— Transmission — Recognition of the disease —
Prevention of disease — Bacterium dysenteriae —
Transmission — Bacterium abortus — Synonyms —
Morphology — Cultural characters — Pathogenesis —
Immunity.

XXVI. HEMORRHAGIC SEPTICEMIAS AND PLAGUE — THE GENUS

PASTEURELLA 345

The hemorrhagic septicemias — Pathology — Cultural
characters — Physiological characters — Pathogenic
characteristics — Pasteurella cholerse-gallinarum —
Synonyms — Pasteurella boviseptica — Synonyms —
Pasteurella suiseptica — Synonyms — Hemorrhagic
septicemias in other animals — Pasteurella pestis —
Synonyms.

XXVII. THE SPORE-BEARING RODS — THE GENERA BACILLUS
AND CLOSTRIDIUM. ANTHRAX, BLACKLEG, MALIGNANT
EDEMA, TETANUS, BOTULISM AND GASEOUS EDEMA 352
Bacillus anthracis — Synonym — Distribution — Mor-
phology — Culture — Physiology — Pathogenesis —
Immunity — Transmission — Clostridium tetani —
Synonyms — Morphology — Cultural characters —
Physiology — Pathogenesis — Immunity — Clostri-
dium chauvaei — Synonyms — Morphology — Cul-
tural characters — Physiology — Pathogeneses —
Immunity — Clostridium Welchii — Morphology
— Cultural characters — Physiology — Pathogenesis
— Immunity — Clostridium botulinum — Synonym
—Distribution — Morphology — Culture — Physio-
ology — Pathogenesis.

XXVIII. THE GROUP OF RAY BACTERIA, LUMPY JAW AND RE-
LATED INFECTIONS — THE GENUS ACTINOMYES 368

Actinomyes bovis — Synonyms — Morphology — Cul-
ture — Pathogenesis — Related organisms.

XXIX. THE ACID FAST GROUP — THE GENUS MYCOBACTER-
IUM — TUBERCULOSIS — PARATUBERCULAR DYSEN-
TERY AND LEPROSY 371

Mycobacterium tuberculosis — Synonyms — Morphol-
ogy— Cultural characters — Physiological characters
— Pathogenesis — Immunity — Diagnosis of the dis-

XJV CONTENTS

CHAPTER PAGE

ease — Transmission — Transmission of bovine tuber-
culosis to man — Mycobacterium paratuberculosis —
Synonyms — Morphology — Cultural characters —
Pathogenesis — Mycobacterium leprae — Synonyms .

XXX. DIPHTHERIA AND GLANDERS GROUP — THE GENERA

BACTERIUM AND PFEIFFERELLA 384

Corynebacterium — Corynebacterium diphtheriae —
Synonyms — Morphology — Cultural characters —
Physiological characters — Pathogenesis — Transmis-
sion— Genus Pfeifferella — Pfeifferella Mallei — Syn-
onyms — Morphology — Cultural characters — Phy-
siology — Pathogenesis — Immunity — Diagnosis —
Transmission.

XXXI. SPIRAL BACTERIA — ASIATIC CHOLERA — THE GENUS

VIBRIO 391

Vibrio cholerae — Synonyms — Morphology — Pathogen-
esis.

XXXII. PATHOGENIC FUNGI, MOLDS AND YEASTS — THE GEN-
ERA BLASTOMYES, ASPERGILLUS, SPOROTRICHUM —
TRICHOPHYTON, MICROSSPORUM, ACHORION, OID-

IUM 393

Pathogenic yeasts— The genus Blastomyces— Pathogenic
molds — Genus Sporotrichum — The genus Tricho-
phyton — The genus Microsporum — The genus Ach-
orion.

XXXIII. THE SPIROCHETE GROUP — THE GENERA TREPONEMA

AND LEPTOSPIRA, THE DISEASES SYPHILIS AND YEL-
LOW FEVER 397

Treponema pallida — Synonyms — Morphology —
Culture — Pathogenesis — Leptospira icteroides —
Pathogenesis.

XXXIV. BACTERIA WHICH CAUSE PLANT DISEASES — THE GEN-

ERA ERWINIA, PSEUDOMONAS AND LACTOBACILLUS. . . 401
Erwinia amylovora — Synonyms — Morphology —
Cultural characters — Pathogenesis — Erwinia solan-
acearum — Synonym — Morphology — Cultural char-
acters — Pathogenesis — Erwinia tracheiphila — Syn-
onym— Morphology — Erwinia carotovora — Syn-
onym — Morphology — Culture and physiology —
Pathogenesis — Erwinia melonis — Synonym —
Morphology, culture, and physiology — Pathogenesis
— Erwinia phy tophthora — Synonym — Lactoba cillus
teutlius — Synonym — Morphology — Cultural and

CONTENTS

XV

CHAPTER PAGE

physiological characters — Pathogenesis — Pseu-
domonas campestris — Synonym — Morphology —
Cultural and physiological characters — Pathogene-
sis — Pseudomonas tumefaciens — Synonym — Mor-
phology and culture, Pathogenesis — Pseudomonas
stewartii — Synonym — Pseudomonas juglandis —
Pseudomonas medicaginis.

XXXV. PROTOZOA CAUSING DISEASE — THE GENERA BABESIA,
PLASMODIUM, CENTAMOEBA, COCCIDIUM AND TRY-

PANOSOMA 409

Classification of the protozoa — Genus Trypanosoma —
Disease produced — The genus Entamoeba — The
genus Babesia — Babesia bigemina — The genus Plas-
modium — The genus Eimeria.

XXXVI. INFECTIOUS DISEASES WHOSE CAUSAL ORGANISMS ARE

NOT CERTAINLY KNOWN 418

Smallpox — Chicken pox — Measles — Scarlet fever — In-
fantile paralysis — Foot and mouth disease — Rabies
or hydrophobia — Hog cholera — Influenza — Swamp
fever or infectious anemia — Typhus fever.

SECTION VI
SANITARY BACTERIOLOGY

XXXVII. BACTERIOLOGY OP WATER AND SEWAGE — SEWAGE DIS-
POSAL 431

Economic importance of impure water — Methods of
determining purity of water — Methods of determin-
ing the potability of water — Presumptive tests —
Chemical studies of water to determine pollution —
Sanitary quality of various waters — Methods of
making impure water potable — Self-purification of
water — Coagulation and sedimentation — Chemical
treatment — Filtration — Sewage disposal — The rural
problem — The city problem.

XXXVIII. BACTERIOLOGY OF MILK 446

Fermentative and other changes in milk — Germicidal
stage — The development of lactic acid — Disappear-
ance of the acid — Decomposition of protein — Abnor-
mal milk fermentations — Bacterial infection of milk
— Udder infection — Bacteria from body surfaces —
Bacteria from the dust — Bacteria from the hands of
the milker — Bacteria from milking vessels — Disease
bacteria in milk — Classification of market milk —
Certified milk— Uninspected milk — Pasteurized milk.

INDEX.. . 457

ILLUSTRATIONS

FIO. PAQB

1. Shapes of bacteria 15

2 Involution forms 16

,.. Forms and groupings of the cocci 18

4. Capsules of various bacteria 19

5. Flagella of bacteria 21

6. Spores of bacteria 22

7. Acetobacter 31

8. Nitrosomonas 31

9. Nitrobacter 31

10. Azotobacter 32

11. Rhizobium 32

12. Bacillus 33

13. Clostridium 34

14. Neisseria 35

15. Streptococcus : 35

16. Diplococcus ' 36

17. Staphylococcus 36

18. Micrococcus 36

19. Sarcina 37

20. Pseudomonas 37

21. Erythrobacillus 37

22. Erwinia 38

23. Proteus 38

24. Bacterium 39

25. Pasteurella 39

26. Hemophilus 40

27. Lactobaeillus 40

28. Vibrio 40

29. Spirillum 40

30. Yeast cells 44

31. Multiplication of yeasts by budding 45

32. Development of spores in yeast 45

33. Types of hyphae in molds 51

34. Development of a zygospore in a Vucor 53

35. Formation of ascospores and asci in Aspergillus 54

36. Development of spores and sporangia of Mucor 55

37. Mold spores (conidia) 56

38. Types of spore production by molds 56

39. Rhizopus 59

40. Common species of Mucor 60

xvii

xviJi ILLUSTRATIONS

41. Species of Aspergillus 62

42. Aspergillus oryzae 63

43. Penicillium 64

44. Penicillium 65

45. Trichothecium 66

46. Hormodendrum and Cladosporium 67

47. Alternaria 67

48. The effective range of indicators most commonly used in

the bacteriological laboratory 95

49. Diagrammatic representation of the optics of the oil im-

mersion lens 106

50. Lactic acid bacteria 195

51. Acetobacter 211

52. Crystals of calcium oxalate in medium in which Aspergillus

is growing 218

53. Clostridium 224

54. Aspergillus oryzse 226

55. Outline of nitrogen cycle 240

56. Nitrosomonas 244

57. Rhizobium leguminosarum 251

58. Nodules on roots of a leguminous plant 254

59. Section of nodule of root of leguminous plant 255

60. Agglutination 291

61. Phagocytosis 297

62. Streptococcus pyogenes 311

63. Staphylococcus aureus 318

64. Diplococcus pneumonise 323

65. Neisseria meningitidis 329

66. Bacterium coli 334

67. Bacteria. , 337

SECTION I

MORPHOLOGY AND CLASSIFICATIONS
OF ORGANISMS

AGRICULTURAL AND
INDUSTRIAL BACTERIOLOGY

CHAPTER I

THE DEVELOPMENT OF AGRICULTURAL AND INDUSTRIAL
BACTERIOLOGY

BACTERIOLOGY is frequently defined as that branch of
science which has to do with bacteria, but in a broader
sense it may include consideration likewise of those yeasts,
molds, and protozoa which may be studied most conven-
iently and satisfactorily by the methods used in the study
of the true bacteria. Those who use the term bacteriology
in the narrower sense have adopted microbiology to desig-
nate that science which includes consideration of all forms
of microscopic life. In this volume the term bacteriology
will be used in the broader sense. The terms microbe,
microorganism, and germ are used popularly to designate
any one of these minute forms of life to be considered.
Of these terms, microorganism is probably most commonly
used, and is to be preferred. Agricultural and industrial
bacteriology may be defined, therefore, as that branch of
science which deals with microorganisms, that is, bacteria,
yeasts, molds and the protozoa, as they may affect agricul-
ture or the industries directly or indirectly.

Agricultural bacteriology draws its material from many
sources. It is necessary briefly to consider the size, shape,
and structure of microorganisms, that is to study mor-
phological bacteriology. The relationship of bacteria and

3

AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

« other microorganisms to each other must be considered
' from'; the "standpoint of systematic 'bacteriology. The re-
lationships between microorganisms and their environment,
such as the effect of heat and light upon their growth and
activities, and the manner in which microorganisms them-
selves bring about changes must also be considered. This is
the branch of science which may be termed physiological
bacteriology. The agriculturist is interested in microorgan-
isms present in foods, particularly in milk, and the changes
which are brought about in dairy products, that is, in dairy
bacteriology. The same is true of bacteria in the soil, or
soil bacteriology. He should acquaint himself with rela-
tionships of microorganisms to disease, particularly the
common diseases of animals ; in other words, with medical
or pathogenic bacteriology. He should know something of
the ways in which the body resists disease. He must, there-
fore, study some of the phases of immunology. Finally the
methods by which disease-producing microorganisms are
spread, and the methods by which such passage from one
individual to another may be prevented may be considered
under the heading of sanitary bacteriology.

Bacteriology as a science is of comparatively recent de-
velopment. It is probable that more has been accomplished,
more facts have been discovered relating to microorganisms,
since 1900 than in all previous time together. What is
true of bacteriology in general is even more true of agri-
cultural bacteriology. Practically all of our information
relative to this subject has been gained since 1880.

In the development of the science of bacteriology, five
factors have been of major importance, namely, the
development of the microscope; the controversy over the
theory of spontaneous generation; the controversy over the
germ theory of fermentation; the controversy over pleo-
morphism among microorganisms; and finally the contro-
versy over the germ theory of disease.

THE DEVELOPMENT 5

Development of the Microscope. — Microorganisms, and
bacteria in particular, are exceedingly minute. Individual
cells cannot be seen with the naked eye, and very few
observations can be made satisfactorily without the aid of
a compound microscope. It is apparent, therefore, that
little could be learned concerning bacteria until the micro-
scope had been highly perfected.

Apparently the first authentic record and description of
bacteria is that by a Dutch lens maker named van Leeu-
wenhoek. This investigator succeeded in so combining
lenses as to secure magnifications greater than those of his
predecessors. In a letter written in 1683 to the Royal
Society of London he commented at some length upon his
microscopic observations of tartar from teeth and of various
decaying materials. Accompanying his paper were draw-
ings of the minute organisms which he observed. These
were published in the proceedings of the society, and con-
stitute the first pictures of microorganisms. His observa-
tions and pictures are sufficiently accurate so that one
familiar with the microorganisms of the mouth can recog-
nize and identify at least1 some of the forms illustrated
with a high degree of probability.

During the next hundred years lenses were improved and
compound microscopes came into common use. In 1783
a Danish investigator, Miiller, published a study of micro-
organisms which were observed in various decaying mate-
rials and in water. He worked out the first scientific
classification of these lower forms of life. Undoubtedly
some of the organisms observed were bacteria, and it is
to Miiller that we owe certain of the common names used
in bacteriology, such as vibrio and bacillus.

During the next half century the microscope was still
further perfected. Ehrenberg in 1836 greatly elaborated
and improved the classification and observations of Miiller.
A few years later the principle of the oil immersion lens

6 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

was evolved, and finally in 1870 the substage condenser
developed by Abbe. Microscopes comparable in efficiency
to those in use now were unknown before this time. Cohn
in 1872 clearly presented the differences between the true
bacteria and the protozoa. He placed the bacteria with
the plants and worked out a fairly comprehensive and
satisfactory classification of genera and species. Since
that date advance has been comparatively rapid.

Controversy over the Theory of Spontaneous Genera-
tion.— Some of the earlier observers of bacteria and other
microorganisms expressed much curiosity as to their origin.
It was observed, for example, that if a decoction of vege-
tables or hay was exposed to the air for a time, the liquid
soon swarmed with bacteria. Apparently living things ex-
isted where there had been no observed living things be-
fore. Many investigators arrived at the conclusion that,
unlike higher forms of life, bacteria might originate de novo,
that is, without preexisting organisms of the same kind.
In fact, they concluded that when there existed a right mix-
ture of water and dead organic material, the right exposure
to air, and the right temperature, there was inherent ability
on the part of the organic material to change in part to
living cells. This point of view was opposed by other scien-
tists, particularly by Pasteur and Tyndall, who contended
that microorganisms must always come from preexisting
microorganisms of the same kind, and that there is no
proof of spontaneous generation as it concerns bacteria was
quite definitely demonstrated. As a result of the laboratory
studies on this problem the foundation was laid for modern
bacteriology, and many of the methods used in the labora-
tory to-day were first developed by those who took a lead-
ing part in the settlement of this dispute.

The Controversy over the Germ Theory of Fermenta-
tion,— Chemistry as a science is somewhat older than
bacteriology. Among the subjects which have interested

THE DEVELOPMENT 7

chemists from early days are the phenomena of fermenta-
tion, putrefaction, and decay. It has long been known,
for example, that if fruit juices are kept under the right
conditions fermentation will occur and alcohol (spirits of
wine) is formed. This was early regarded by the chemists
as purely a chemical transformation. During the first half
of the nineteenth century, however, it was repeatedly ob-
served that alcoholic fermentation was always accompanied
by the development of minute or microscopic cells which
were called yeasts. It was conjectured by those who ob-
served these living cells that they might be the cause of the
fermentation. Chemists opposed this conjecture and ar-
gued that these minute objects were the results and not the
cause of the fermentation. Here again it was necessary
to take the subject into the laboratory in order to decide
whether microorganisms caused fermentation or were the
result of fermentation. The great German chemist Liebig
argued for the latter, and Pasteur for the former concep-
tion. The controversy between these two schools of thought
waged for many years, but was at last settled conclusively
by the laboratory experiments of Pasteur. This investiga-
tor demonstrated beyond reasonable doubt that practically
all fermentation, putrefaction, and decay, are due to the
presence and growth of microorganisms, for the most part
bacteria, yeasts, and molds. In the course of the contro-
versy, however, many methods of laboratory work were de-
veloped, and many new species of microorganisms dis-
covered.

Controversy over the Theory of Pleomorphism. — It is
comparatively uncommon in nature to find single kinds or
species of bacteria growing by themselves apart from all
other species, or as the modern bacteriologist terms it "in
pure cultures." This fact was not recognized by some of
the earlier investigators, and led to a long argument or
controversy over the theory of pleomorphism. About 1870

8 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

a physician by the name of Billroth concluded that all bac-
teria were different growth forms of one single pleomorphic
species, which, when growing under different conditions
shows different cell forms. His suggestion was made be-
fore modern laboratory technic was well developed. Much
of the literature, particularly the medical literature, in the
two decades following the work of Billroth is greatly con-
fused because of adherence to this doctrine Here again
laboratory studies carried out in the most careful manner
were necessary to determine the right. They led to the
discovery of many new facts regarding microorganisms, and
showed quite conclusively that there are many distinct
species of bacteria, and that Billroth was mistaken in his
assumption that all were growth forms of a single pleo-
morphic species. It is true that microorganisms sometimes
vary in their shape, their appearance, and their size, when
grown under different conditions, and sometimes they can
be distinguished from each other only with difficulty.
Nevertheless there are many distinct kinds, and the
theory of pleomorphism in its original form has been dis-
proved.

The Controversy over the Germ Theory of Disease.—
The fact that bacteria can produce disease was first ade-
quately demonstrated and proved by Robert Koch in 1872.
Before this time there had been occasional conjectures that
microorganisms might have something to do with disease
but no real proof. Koch examined microscopically the
blood of cattle having the disease called anthrax, and ob-
served numerous rod-shaped organisms among the red blood
cells. These he succeeded in transferring to culture media
and in growing them free from every other kind of organ-
ism, that is, in pure culture. He found that he could culti-
vate them in this fashion indefinitely in the laboratory, and
that whenever they were introduced under the skin of sheep
or cattle the animal would invariably contract the disease

THE DEVELOPMENT 9

anthrax and usually succumb. Furthermore, he discovered
that in the bodies of animals artificially inoculated there
were present the same organisms which he introduced, and
that he could get pure cultures again from the tissues of the
animal. His proof of the causal relationship of organisms
to disease was quite adequate and complete.

Many investigators began studying other diseases and
soon bacteria were discovered which caused many of them.
These facts were of course in contradiction to the sup-
posed facts which had governed the treatment of disease
formerly, and there was much opposition to the acceptance
of the statement that bacteria can cause disease. The proof
finally became overwhelming, and the whole development of
medicine was modified in consequence. The importance
of bacteria as causes of disease led to a large amount of
study, and gave the needed incentive to the rapid develop-
ment of the science of bacteriology, and to the modern con-
ception that diseases are of two classes; the so-called in-
fectious diseases, which are caused by microorganisms; and
the non-infectious diseases, which are not so caused. The
development of the germ theory of disease led also to a
study of the means by which the animal* body resists dis-
ease, that is, to the development of immunology. It re-
sulted also in a study of the means by which disease-pro-
ducing microorganisms travel from one person to another,
that is, to the development of sanitary bacteriology.

During the growth and development of the science of
bacteriology, and in the laboratory studies necessitated by
the various controversies noted, many facts of interest and
importance to agriculture were discovered. The knowledge
that microorganisms cause fermentation and disease led to
a study of foods, particularly of milk and milk products,
and to the development of dairy bacteriology. A study of
bacteria in their relation to the nitrogen of the soil, and the
discovery of bacteria living in the roots of leguminous

10 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

plants such as clover and alfalfa, laid the foundation for
the growth of soil bacteriology.

It is apparent from the preceding discussion that the ag-
ricultural and technical bacteriologist finds his material in
many different fields. Agricultural bacteriology, in other
words, cannot be considered as a distinct science, but rather
as a compendium of material taken from the various dis-
tinct branches of bacteriology, and having agricultural
application.

CHAPTER II

GROUPINGS AND GENERAL RELATIONSHIPS OF
MICROORGANISMS

IT has already been noted that four distinct groups of
microorganisms are to be considered in agricultural bac-
teriology, namely, the bacteria, the yeasts, the molds, and a
few of the protozoa. It is important that the differences
separating these groups from each other should be recog-
nized, and that the position of all in their relationship to
other plants and animals should be understood.

Three of these groups, the bacteria, the yeasts, and the
molds, are placed in the plant kingdom, the protozoa in the
animal kingdom. It is necessary, therefore, first to differ-
entiate microscopic animals from plants.

Differences between Microscopic Plants and Animals. —
Bacteria are probably the simplest 'living plants and proto-
zoa the simplest living animals. Both belong near the bot-
tom in the evolutionary scheme. It is natural, therefore,
that many resemblances are to be found between the lower
plants and the lower animals. Some investigators, in fact,
group all of the unicellular plants and animals together
under the name Protista. The differentiation between bac-
teria and protozoa is rendered particularly difficult be-
cause there are a few forms which apparently are inter-
mediate, and which are grouped by some investigators with
the bacteria and by others with the protozoa. Neverthe-
less, most forms in the two groups are not difficult of differ-
entiation. Plant cells usually possess a firm and well-de-
fined cell wall, while the protozoa, at least during their
active development, usually do not. The bacteria, in gen-

It

12 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

eral, have the attributes of plant cells. They multiply by
transverse fission, while many of the protozoa multiply by
fission which is either longitudinal or at least not at right
angles to the longest axis of the cells. Bacteria are never
amoeboid in their movements as are protozoa of many types.
Furthermore, many intergrading forms exist between the
true bacteria and that group of plants known as the blue-
green algae. No careful student of bacterial morphology
can escape the conclusion that the relationships are prob-
ably closest to this group of plants, although a few of the
bacteria show close resemblances and relationships to cer-
tain of the mold fungi.

Both from the standpoint of structure and of connecting
forms, the bacteria on the whole show closer relationship to
the plants than to the animals or protozoa.

Confusion sometimes arises because of the fact that many
of the bacteria are actively motile. It should be recognized
that the ability to swim about is not a characteristic pe-
culiar either to plants or to animals. Many forms which
are definitely known to be plants possess this power, the
sperm cells, for "example, of even some of the higher forms
of plants such as mosses, ferns and cycads, can move inde-
pendently. A misunderstanding of this fact led to some
confusion in early studies of bacteria, for it is only within
the last half century that their true relationships have been
understood, and they have been placed definitely with the
plants.

Position of Bacteria, Yeasts, and Molds in the Plant
Kingdom. — Botanists generally recognize four great sub-
divisions of plants, the seed plants or Spermatophyta, the
fern plants or Pteridophyta, the moss plants or Bryophyta,
and the thallus plants or Thallophyta. The bacteria,
yeasts, and molds all belong in this last group.

The Thallophyta are separated from other plants in that
they do not develop a complex plant body with differenti-

GROUPINGS AND GENERAL RELATIONSHIPS 13

ation into roots, stem and leaves. Sometimes the entire
plant consists of a single cell, that is, is unicellular. In
other cases it may consist of a chain of cells united to form
more complex structures, though this is never the case with
bacteria, yeasts, or molds. The group Thallophyta has
three important subdivisions. The first is that of the algae,
forms containing green coloring matter or chlorophyll.
The second group is that of the fungi which differ from the
algae in that they do not possess chlorophyll. It includes
such forms as yeasts, molds, puffballs, mushrooms, mil-
dews, etc. The third subgroup is that of the Schizophyta
or fission plants in which multiplication is of some other
type than that of simple cell fission. One of the subdivi-
sions of this group is that of the Schizomycetes or bacteria.
In the following scheme of classification an attempt is made
to show the various important subgroups of the Thallo-
phyta. Those subgroups which contain organisms to be
studied in bacteriology are designated by bold-faced
type.

GROUPS OF THALLOPHYTA SHOWING POSITION AND RELATION-
SHIPS OF BACTERIA, YEASTS, AND MOLDS

Thallophyta. — Simple plants, never differentiated into
roots, stems and leaves.

A. Unicellular plants, multiplying by cell fission only.

1. Without chlorophyll. Bacteria or Schizomycetes.

2. With chlorophyll. Blue-green algae or Schizo-

phyta.

B. Unicellular or multicellular. Multiplying by means

other than simple cell fission.

1. Without chlorophyll, the fungi, including the

yeasts, molds, mildews, smuts, rusts, puffballs,
mushrooms, etc.

2. With chlorophyll, including the seaweeds, pond

scums, water silks, etc.

14 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Differentiation of Bacteria, Yeasts, and Molds.— The

bacteria and yeasts are readily differentiated from molds in
that they are always unicellular while the molds, in gen-
eral, are multicellular. Bacteria always multiply by fis-
sion, that is, the cells divide at right angles to their longest
axis. Yeasts, in general, multiply by budding, although a
few yeasts are known to multiply by fission. When bac-
teria produce spores, usually but one spore is found in a
single cell, while with yeasts there is usually more than
one. This is always true in those yeasts which multiply by
fission. There are also some minute structural differences
which may usually be observed between yeasts and bac-
teria. The yeast' cell shows a definite nucleus while the
presence of a definite nucleus is not so clearly established
for bacteria. In most cases the yeast cells are decidedly
larger than those of bacteria.

Botanists do not recognize a distinct group which they
term molds. Organisms belonging to this group, which is
convenient for the purposes of the bacteriologist, are really
members of different groups of fungi. They all resemble
each other, however, in forming more or less cottony or vel-
vety masses of fungus threads from which spore stalks in
various shapes and spores in various groupings are pro-
duced. A few of the molds show forms which intergrade
with the bacteria. Multiplication in molds is usually by
means of spores which are produced in large numbers by
single plants.

The Protozoa. — The protozoa are the one-celled animal
forms existing in a great variety of shapes, sizes, and meth-
ods of life in soil, in water, and occasionally producing dis-
eases in man and animals. The group is a very large one.
From the standpoint of agricultural bacteriology we are
interested in the group because of the possible importance
of some forms in soils and the undoubted significance of
others in the production of diseases.

CHAPTER III

MORPHOLOGY AND CLASSIFICATION OF THE BACTERIA

MORPHOLOGY OF BACTERIA

Shape. — Bacterial cells are usually one of four shapes,
straight rods, curved rods, spheres, or filaments. A
straight rod is termed a bacillus; a curved rod a spirillum;
a spherical cell a coccus; the filamentous forms are some-
times termed trichobacteria.

Bacteria not infrequently develop cells which are not of
the normal size and shape. In some cases these are prob-

FIG. 1. — SHAPES OF BACTERIA. A. Spherical bacteria (cocci). B.
Rod-shaped bacteria (bacilli). C. Spiral bacteria (spirilla).

ably degenerate types, cells produced as a result of growing
under abnormal or unfavorable conditions. In other cases
they may represent special developmental phases of the life
history of the organism. In general, such unusual cells are
termed involution forms. Some authors insist that this
term should be used only for those which represent de-
generate types and not those which represent phases in the
normal life cycle.

15

16 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Size. — Bacteria are the smallest plants. The microscope,
and usually the compound microscope, is necessary for the
study of the individual cells. It is, therefore, convenient
to use the microscopic unit of measurement. The micron
(abbreviated /*) is defined as one one-thousandth of a milli-
meter, or one ten-thousandth of a centimeter, or approxi-
mately one twenty-five-thousandth of an inch. Most bac-
teria are 0.5 to 5/x in diameter, and from 0.5 to 10/x in
length. Probably the average bacterial cell does not have
a volume much greater than one cubic micron. The mi-
nuteness of these organisms is emphasized when it is re-
called that there are one trillion cubic microns in a cubic

flo
o.o

Ox c/^

~ c? /) Qs

FIG. 2. — INVOLUTION FORMS, a. Normal shapes, b. Involution
forms. 1. Acetobacter. 2. Mycobacterium. 3. Rhizobium,

centimeter, therefore, approximately this number of bac-
teria of average size would be required to fill a space of one
cubic centimeter. The specific gravity of the cell is usually
only slightly greater than that of water. It would require,
therefore, nearly a trillion of such bacteria to weigh a gram.
When water is heavily polluted it may sometimes contain
one million bacteria per cubic centimeter. This number
of bacteria, of average size, would not occupy more than
one millionth of the space within that cubic centimeter.

CLASSIFICATION OF THE BACTERIA 17

Multiplication of Bacteria.— Bacteria multiply by a
process of simple fission. A rod elongates to about double
its original length and splits in the middle to form two in-
dividuals. Spherical cells frequently elongate somewhat
before they divide.

Bacteria in many cases may multiply with considerable
rapidity. Certain of the common bacteria, for example,
when placed under the most favorable conditions for
growth, that is, with an abundance of food, moisture, the
right temperature, and all conditions optimum, may grow
to their full size and divide once every twenty minutes.
Probably most of the more active bacteria can grow to their
full size and divide to form two individuals within one-
half hour. If it is assumed that this process continues for
two days the number of bacteria will be 2no, a number
having some twenty-eight figures. A rough calculation of
the weight of such a bacterial mass will show it to be more
than a trillion tons. Obviously no such bacterial masses
can ever ber formed, inasmuch as the optimum conditions
for growth cannot long continue. The principal factors
which limit the rapidity of growth are the disappearance
of the available food material, and the production of sub-
stances by the bacteria which are more or less harmful to
their own development. This rapidity of multiplication,
however, explains why fermentative changes, such as the
souring of milk, may take place so rapidly. A compara-
tively small number of bacteria introduced into a bottle
of milk and kept at a suitable temperature will cause it to
become sour within a few hours.

Cell Groupings of Bacteria. — Bacterial cells do not al-
ways become detached from each other immediately follow-
ing cell division. In many cases the presence of capsules
or of mucilaginous or gummy walls may cause the cells to
cling together. It is obvious that rod-shaped bacteria, in-
asmuch as they can divide or multiply only by splitting

18 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

crosswise, must occur eith'er as isolated cells or in chains.
Such an organism is sometimes termed a streptobacillus.
Spiral bacteria very rarely occur in groups though occasion-
ally they are found in chains.

It has been stated that bacteria always divide at right
angles to the longest axis of the cell. Spherical cells, or
cocci, have no longest axis. There is, therefore, a priori no
reason why they cannot divide in any plane. Some spheri-
cal cells show no regularity in the manner in which they
divide. If such cells cling together they will form an irreg-
ular or grape-clusterlike mass. Such an organism is termed
a staphylococcus. Some cocci show a decided tendency to re-
main united in pairs. An organism of this type is termed
a diplococcus. When spherical cells divide persistently
in parallel planes a chain of cocci will be produced. Such

FIG. 3. — FORMS AND GROUPINGS OF THF cocci. 1. Single or isolated
cells. 2. Irregular masses. 3. Pairs. 4. Chainp. 5. Plates. 6.
Packets or cubes.

an organism is termed a streptococcus. A few kinds of
cocci have been described which divide alternately in two
planes forming first pairs, and then squares of cells. An
organism of this type may be termed a pediococcus. Still
others divide in three planes, each plane at right angles. to
the other two. This will result in the formation first of
pairs, then of squares, then of cubes of bacteria. Such a
cubical collection of bacteria is termed a sarcina.

It should be noted that the names given above are not the
scientific names of genera, but are common -descriptive
terms. It will be found later that some of these same words

CLASSIFICATION OF THE BACTERIA 19

are used also as generic names. When used in a simple
descriptive sense they are not capitalized. When used as
the names of genera they are capitalized and are generally
written in italics.

Structure of the Bacterial Cell and Its Appendages. —
The bacterial cell is a comparatively simple structure. It
possesses a definite cell wall. This may be surrounded by
various layers of gelatinous material or by membranes,
termed capsules or sheaths. It contains the protoplasm or
living cell material and various other cell inclusions, and
it may be motile by means of flagella.

The Cell Wall. — Each bacterial cell is surrounded by a
relatively firm membrane or cell wall. Its composition ap-
parently is not uniform among all bacteria. In a few cases
the cell wall will give the microchemical reaction charac-

FIG. 4. — CAPSULES OF VARIOUS BACTERIA. 1. Capsulated diplcrccfccus.
2. Capsulated streptococcus. 3. Capsulated bacilli. 3a, Bacilli
with asymmetric capsules.

teristic of cellulose, indicating a close relationship in com-
position to the cell walls of higher plants. Most bacteria
are supposed to have cell walls which are somewhat chiti-
nous, that is, they are nitrogenous in nature. Chitin is
closely related to the carbohydrates. When hydrolyzed it
breaks down into glucosamine and acetic acid. The cell wall
generally is thin and layers cannot be differentiated.

Cell Capsules and Sheaths. — Some species of bacteria
secrete large amounts of gelatinous material about the cell.
In some cases this dissolves relatively rapidly in the medium
in which the organism is growing, sometimes making it

20 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

slimy or gelatinous in consistency. In other cases this
material remains attached to the cell, forming a layer of
greater or less thickness. This may be regarded as the
outer portion of a greatly swollen cell wall. Such a mass
is termed a capsule. This capsule is usually composed of
gum or gumlike material. Occasionally it is nitrogenous in
nature.

A few filamentous or rod-shaped bacteria growing in
water surround themselves by a relatively firm membrane,
a tube in which the filaments or chain of cells lie. This tube
is termed a sheath. In same species this sheath becomes
impregnated with iron.

Protoplasm and Cell Inclusions. — The protoplasm or
living material within the bacterial cell is usually not clearly
differentiated as in the high.er plants into nucleus and cyto-
plasm. It is probable that in most cases nuclear material,
that is, chromatin, is scattered more or less throughout the
protoplasm. Some bacteria apparently have a primitive
type of nucleus.

The outermost portion of the protoplasm, that is, that
portion lying next to the cell wall, is differentiated as a
membrane termed the ectoplast. This ectoplast apparently
functions in determining what can enter and what can leave
the cell.

Bacterial cells may contain inclusions of many kinds.
Most common are granules which stain relatively deeply
with some of the ordinary laboratory dyes such as methylene
blue. Because their color reactions res'emble chromatin they
are termed metachromatic granules. In some species of
bacteria vacuoles are developed. These apparently are
spaces in the protoplasm filled with cell sap but not with
living material. Each vacuole is surrounded by a mem-
brane of the same general type as that surrounding the
entire mass of protoplasm. Granules of carbohydrate are
observable in some bacteria. The exact composition is not

CLASSIFICATION OF THE BACTERIA 21

well understood, but in some cases it is apparent that the
reserve carbohydrate food material resembles glycogen, in
others it is perhaps more closely related to starch. Gran-
ules which give the starch color reaction with iodine have
been termed granulose. Certain bacteria which live in
water containing hydrogen sulphide show sulphur granules
in the protoplasm. It is possible that in some cases oil drops
may be detected.

Flagella. — A very few species of cocci, many species of
bacilli, practically all species of spirilla, and none of the
filamentous bacteria, possess organs of locomotion termed
flagella. These are exceedingly slender, so slender in fact
that they are observed under the microscope usually only
by the most careful staining methods. They may be re-

FIG. 5. — FLAGELLA OF BACTERIA. A. Non-flagellate or atrichous bac-
teria. B. Bacteria with single polar flogellum, monotriehous.
C. Bacteria with clusters of polar flagella, lopotrichous. D. Bac-
teria with flagella on all sides, peritrichous.

garded as differentiated protoplasmic organs projecting
through the cell wall to the outside. They are relatively
long, sometimes many times the length of the cell which
produces them. By means of a spiral or corkscrew motion
these flagella propel the organism.

Those species of bacteria which do not possess flagella are
said to be atrichous. Some species possess a single flagellum
at one end of the cell. Such organisms are termed mono-
triehous. Organisms having a tuft of flagella at one end
are termed lophotrichous. Those bacteria which have fla-
gella distributed over the entire surface of the cell are said

22 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

to be peritrichous. Some investigators recognize another
group termed amphtirich&U8f that is, cells having flagella
at both ends. Such organisms, however, are in reality the
same as those having flagella at one end only. When a
monotrichous or lophotrichous motile cell is about to divide,
flagella frequently develop at the other end so that each
daughter cell, following the division of the mother cell, will
be motile. The flagella ordinarily point in the direction in
which the organism is swimming, that is, they pull the
organism about rather than push it.

Spore Formation. — A spore may be defined as a cell or
group of cells set apart specifically for purposes of repro-
duction. Usually, though not invariably, spores are more
resistant to unfavorable conditions than the cells which
produce them.

FIG. 6. — SPORES OF BACTERIA. 1. Spores centrally located, cells not
swollen. 2. Spores central, cells swollen. 3. Spores polar, cells
swollen. 4. Spores polar, cells not swolllen.

Certain kinds of bacteria may produce spores on the
inside of the cells. With the exception of two or three
little known species a bacterial cell produces only a single
spore. Because of their location bacterial spores are some-
times termed endospores. It is apparent that inasmuch as
a single spore forms within a cell, spore production among
bacteria does not involve multiplication or increase in
numbers,

None of the common species of cocci or spirilla produce
spores, but some species of the rod-shaped organisms or
bacilli do.

The first evidence of spore formation in the bacterial cell
is usually the appearance of certain granules. Gradually

CLASSIFICATION OF THE BACTERIA 23

the protoplasm at the point where the spore is to form
becomes more dense and finally surrounds itself by a definite
membrane or membranes. A mother cell containing a spore
is sometimes termed a sporangium. The spore differs from
the cell in which it is produced usually by beiri£ somewhat
smaller, by having a much firmer membrane, and by its
lower water content. It is usually well fitted to survive
unfavorable conditions for a considerable period of time.
In most cases the spore mother cell wall eventually breaks
away leaving the spore free.

Usually each species of spore-producing bacillus has its
own characteristic shape, size, and position for the spore.
These spores may be located either at the center or at or
near the end of a cell. In some species in which the spore
is located near the equator the mother cell wall bulges,
making the cell spindle-shaped. Such an organism is some-
times termed a clostridium. In other cases the spore does
not cause such cell enlargement. A few species of bacteria
produce spores at the end of the cell, causing considerable
enlargement. Such a drumstick-shaped cell is sometimes
termed a plectridium.

When spores come under favorable conditions for de-
velopment they will germinate, producing rods similar to
those from which they originated. These rods will then
go on multiplying by fission until stimulated in some way
themselves to produce spores. The stimulation to spore
production is not necessarily unfavorable conditions for
growth as is ordinarily understood. In other words, al-
though spores undoubtedly serve the purpose of tiding
bacteria over unfavorable conditions, the organism does not
need to be subjected to these unfavorable conditions before
producing spores. It appears to be quite as much a part of
the regular life cycle of some bacteria to produce spores as
it is a part of the regular life cycle of the corn plant to
produce an ear of corn.

24 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Conidia Formation. — Certain of the filamentous or so-
called higher bacteria sometimes divide by numerous cross
walls and split into small cells which function as spores.
These are termed conidia. They are probably not com-
monly found among the cocci, bacilli, or spirilla.

CLASSIFICATION OF BACTERIA

The true bacteria belong to the class SMzomycetes.
They may be characterized as minute, one-celled, chloro-
phyll free, colorless, rarely violet-red or pigmented plants,
which typically multiply by dividing in one, two, or three
directions of space. The cells thus formed are usually
spherical, cylindrical, comma-shaped, spiral or filamentous,
and are often united into filamentous, flat, or cubical aggre-
gates. Filamentous species are often surrounded by a com-
mon sheath. The cell plasma is generally homogenous with-
out a definite nucleus. Sexual reproduction is absent. In
some species reproductive bodies are produced, either endo-
spores or conidia. The cells in some species are motile by
means of flagella.

The class Schizomycetes may be subdivided into six dis-
tinct orders. Of these, four are not of great agricultural
significance. Two, however, are of sufficient interest to
require discussion. The four orders not further discussed
are the slime bacteria (belonging to the order Myxobac-
teriales), the sulphur bacteria (belonging to the order
ThMacteriales,) the iron bacteria (belonging to the order
Chlamydobacteriales) and the spirochetes (Spirochaetales) .
The two orders of agricultural significance are the filamen-
tous or branching bacteria (belonging to the order Actino-
mycetales) and the true bacteria (order E 'ub act en 'ales).

Orders in botany are divided into families, families into
genera, and genera into species. Each kind of bacterium
constitutes a species. The related species are grouped
together into genera, and related genera into families. For

CLASSIFICATION OF THE BACTERIA 25

example, there is one type of rod-shaped organism which
produces typhoid fever, another type somewhat closely
related to it which causes dysentery. These two species are
brought together in the genus Bacterium. This genus,
with several other genera of bacteria, are united into the
family Bacteriaccae, and this family again united with
others to form the order Eubacteriales.

The rules which govern the giving of scientific names to
plants and animals should be recognized also in bacteriology.
To every kind of organism a name should be given con-
sisting of two words : the first of these a proper name, al-
ways capitalized, the name of the genus; the second name
is the name of the species. This latter name is never capi-
talized unless it is derived from a proper noun. All genus
and species names are in Latin. The name of the organism
which causes typhoid fever is Bacterium typhosum. Fre-
quently the name of the man who first described the organ-
ism under the particular name accepted is given also, thus,
Bacterium coli Escherich, indicates that Escherich first ap-
plied this name to the organism called the colon bacillus.
Sometimes investigators have named species but have placed
them in the wrong genus. The man who first recognized
the species may have his name written in parentheses be-
tween the name of the species and the name of the individ-
ual who first used the right combination, for example, an
organism sometimes associated with pus production in
animals is Pseudomonas aeruginosa (Schroeter) Migula.
This indicates that Schroeter first used the name aeruginosa
but that Migula placed this species in the genus Pseudo-
monas.

The species name of an organism should ordinarily con-
sist of a single word. Some bacteriologists have not fol-
lowed this rule and have given complex Latin names to
organisms on the theory that a name should be a descrip-
tion, for example, one species was termed Bacillus saccharo-

26 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

butyricus fluorescens liquefaciens immobilis. The author
was indicating that he was dealing with a rod-shaped organ-
ism which fermented sucrose by the formation of butyric
acid, produced a fluorescent pigment in artificial media,
liquefied gelatin, and was nonmotile. It is quite as unnec-
essary to use complex names of this type as it is to have
the Christian name applied to an individual necessarily
descriptive of that individual.

Many microorganisms have received common names.
These should not be confused with the true scientific names
even though they somewhat resemble them, for example,
the organism which causes epidemic pneumonia is termed
the pneumococcus. Its true scientific name is Diplococcus
pneumoniae.

Many of the genera of bacteria belonging to the two
orders to be studied ar,e not of sufficient economic import-
ance to warrant discussion in this connection. Only genera
which contain species that bring about noteworthy changes
are included in the discussion.

First there is presented an artificial key for the differen-
tiation of the economic genera. This is followed by the
classification of genera and families recommended by the
Committee on Nomenclature of the Society of American
Bacteriologists.

It will be noted in the key and in the description of
genera and other groups that it is sometimes necessary to
use physiological rather than morphological criteria for
differentiation. For example, Gram's staining method
(discussed in a later chapter) results in coloring some
organisms and leaving other organisms unstained. Some
organisms can produce acid or acid and gas from cer-
tain carbohydrates, other forms cannot. This fact is of
great value in separating related groups. Occasionally
the type of growth upon culture media may be of im-
portance.

CLASSIFICATION OF THE BACTERIA 27

KEY TO GENERA OF BACTERIA OF AGRICULTURAL AND INDUS-
TRIAL SIGNIFICANCE

A. Typically never filamentous nor producing a
mycelium.
B. Cells spherical.

C. Cells occurring typically in chains.
D. Cells forming gelatinous masses in su-
gar media, saprophytic Leuconostoc.

DD. Parasitic. Cells not forming zoo-

gloeal masses Streptococcus.

CC. Cells not in chains.
D. Cells typically in pairs.
E. Cells Gram-negative. Uusually coffee-
bean-shaped Neisseria.

EE. Cells Gram-positive. Usually lan-
ceolate Diplococcus.

DD. Cells not in pairs.

E. Typically parasitic. Cells in irregu-
lar groups Staphylococcus.

EE. Typically saprophytic.

F. Cells arranged in regular packets. Sarcina.
FF. Cells not arranged in packets, not
regularly grouped.

G. Pigment usually yellow Micrococcus.

GG. Pigment red Rhodococcus.

BB. Cells elongate, not spherical.
C. Rods not spiral or curved.

D. Cells acid fast Mycobacterium.

DD. Cells not a<-id fast.
E. Endospores present.

F. Aerobic Bacillus.

FF. Anaerobic Clostridium.

EE. Endospores absent.

F. Not growing on ordinary organic
media, oxidizing ammonia or ni-
trites.

G. Oxidizing ammonia Xitrosomonas.

GG. Oxidizing nitrous acid Kitrobacter.

FF. Not securing growth energy by ox-
idation of ammonia or nitrites.
G, Oxidizing carbon compounds aero-
bically, saprophytic.

H. Oxidizing alcohol to acetic acid. Acetobacter.
HH. Oxidbing carbohvdrates.
Fixing atmospheric nitrogen.

I. Cells relatively large. Free

living soil bacteria. Azotobacter.

II. Cells smaller. Symbiotic in

roots of leguminous plants. Rhizobium.

28 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

GG. Not securing growth energy ex-
clusively by the aerobic oxidation
of carbon compounds.

I. Usually producing a fluores-
cent pigment. Usually motile

by polar flagella Pseudomonas.

II. Without fluorescent pigment.
Motile or nonmotile, if the for-
mer, flagella peritrichous.

J. Rods of irregular shape,
sometimes clubbed or branched.
Nonmotile animal parasites.
K. Gram-positive rods, fre-
quently granular or clubbed. Corynebacterium.
KK. Gram-negative, rods some-
times elongated or branched.

Not granular Pfeifferella.

JJ. Rods relatively regular, and

staining evenly or bipolar.

Motile or nonmotile.

K. Usually showing bipolar

staining. Weak power of

fermentation Pasteurella.

KK. Bipolar staining absent,
is L. Growing only or best in

presence of hemoglobin. Hemophilus. .
LL. Not requiring hemoglo-
bin.

M. Saprophytic red or
violet forms.

N. Pigment red Erythrobacillus.

NN. Pigment violet. . . Chromobacterium.
MM. Without red or vio-
let pigment.
N. Gram-positive lactic

acid bacteria Lactobacillus.

NN. Gram-negative.
O. Producing plant

diseases Erwinia.

00. Not producing
plant diseases.
P. Rods not uni-
form. Ferment
sucrose not lac-
tose Proteus.

PP. Rods uniform.
Never ferment
sucrose except
that lactose is
also fermented. Bacterium.
CC. Rods curved.

D. Short, comma-shaped Vibrio.

DD. Longer, spiral Spirillum.

CLASSIFICATION OF THE BACTERIA 29

OUTLINE OF BACTERIAL CLASSIFICATION

It was noted above that two only of the five orders of
bacteria will be discussed : the order of the true bacteria or
Eubacter idles, and the order of the thread bacteria or Acti-
nomycetales.

ORDER EUBACTERIALES

This order contains the forms usually termed the true
bacteria, that is, those which are considered least differen-
tiated and least specialized. They do not require hydrogen
sulphide or other sulphur compounds in abundance, and
the cells do not contain either sulphur granules or bacterio-
purpurin. The cells may be motile by means of flagella or
nonmotile, but they are never notably flexuous. Cell multi-
plication is always by transverse fission. Some of the gen-
era, particularly among the rod-shaped types, produce endo-
spores. The cells are usually minute and spherical, rod-
shaped, or spiral in shape, not commonly producing true
filaments, and never branching except in the so-called invo-
lution forms. This order includes most of the common bac-
teria. Altogether seven families belonging to order Eubac-
teriales are recognized. The following key may be used for
the differentiation of the families :

KEY TO FAMILIES OF THE EUBACTERIALES

A. Securing growth energy by direct oxida-
tion of carbon, hydrogen or nitrogen or
their compounds. Aerobic. Cells rod-
shaped or occasionally spherical I. Nitrobacteriaceae.

AA. Not securing growth energy solely as
in preceding.

B. Cells producing endospores II. Bacillaceae.

BB. Cells not producing endospores.

C. Cells spherical

CC. Cells not spherical. ^ III. Coccaceae.

D. Cells rod-shaped.

30 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

E. When motile, with polar fla-
gella, frequently producing a

fluorescent or a yellow pigment. IV. Pseudomonadaceae.
EE. Not as E. Fla.gella when pre-
sent, peritrichous V. Bacteriaceae.

DD. Cells bent or spiral VI. Spirillaceae.

I. FAMILY NITROBACTERIACEAE

Bacteria belonging to this family are usually rod-shaped,
occasionally spherical. In some genera the cells are motile
and in others nonmotile. In a few genera there is a de-
cided tendency toward the formation of threadlike or
branched involution forms. Endospores ;are never pro-
duced. These bacteria are grouped together primarily
because they must grow in the presence of free oxygen, and
secure their growth energy by the direct oxidation of car-
bon, or hydrogen, or simple compounds of these elements
and of nitrogen. None produce disease, and with the ex-
ception of the genus which lives in the nodules on the roots
of leguminous plants, none are parasitic.

The genera of sufficient agricultural interest to be dis-
cussed are Acetobacter, Nitrosomonas, Nitrobacter, Azoto-
bacter and Rhizobium. In addition to these genera three
others 1 have been described.

Acetobacter. — In this genus the organisms are rod-
shaped, frequently in chains, and usually nonmotile. The
bacteria grow on the surface of alcoholic solutions, securing
the energy for their growth usually by the oxidation of
alcohol to acetic acid, or occasionally by the oxidation of
sugars to simpler compounds. Involution forms are not
infrequent, occurring as filamentous, club-shaped, and even
branched cells. Organisms belonging to this genus con-
stitute the so-called mother of vinegar which forms over

1 The genus Hydrogenomonas secures its energy by the oxidation
of free hydrogen, Methanomonas by the oxidation of methane,
Carboxydomonas by the oxidation of carbon monoxide.

CLASSIFICATION OF THE BACTERIA 31

the surface of cider or wine in the process of vinegar
manufacture.

FIG. 7. — ACETOBACTER. A. Normal cells. B. Involution forms.

Nitrosomonas.1 — Bacteria belonging to this genus have
cells either rod-shaped or spherical, motile or nonmotile.
These organisms are soil forms securing their growth
energy by the oxidation of ammonia to nitrous acid. They
do not require organic compounds for growth. They are

V

a

FIG. 8. — NITBOSOMONAS. .A. FIG. 9.— NITBOBACTEB.

Non-motile spherical type.
B. Motile, rod-shaped type.

responsible in the soil for initiating the changes later to be
discussed under the heading of * * nitrification. ' '

Nitrobacter. — The organisms belonging to this genus are
rod-shaped, nonmotile, and do not require the presence of
organic matter for growth. They are commonly present
in the soil and secure the energy for growth by the oxida-
tion of nitrites to nitrates. They complete the process of

i This genus includes the organisms sometimes described as
\itrosococcu8.

32 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

"nitrification." This genus, together with the preceding
genus, are of considerable importance in determination of
soil fertility.

Azotobacter.1 — Organisms belonging to this genus are
relatively large rods, sometimes spherical, and occasionally
almost yeastlike in appearance. They are typically soil
forms securing their growth energy by the oxidation of
carbonaceous material, and of unusual importance because
of their ability to fix atmospheric nitrogen for their own

FIG. 10. — AZOTOBACTER. FIG. 11. — RHizoBiUM. A. Species mo-
tile by peritrichic flagella. 1.
Young motile cells. 2. Involu-
tion forms. B. Species motile
by means of polar fla2,ella. 1 .
Young cells. 2. Involution
forms.

use, thus increasing the nitrogen content of soils. They
do not live upon the roots of leguminous plants, but free
in the soil. In the laboratory their ability to use atmos-
pheric nitrogen may be shown by growing them in solutions
containing carbohydrates but deficient in nitrogen com-
pounds.

Rhizobium. — These organisms are minute rods, motile
when young, by means of flagella which may be either polar
or peritrichous. The cells may occur free in the soil, but
usually are present in the characteristic nodules on the

i Other names sometimes used for Azotobacter are Parachromatium,
and Azotomonas.

CLASSIFICATION OF THE BACTERIA 33

roots of leguminous plants. By use of the energy secured
from the oxidation of carbon compounds, usually sugars,
they are capable of fixing atmospheric nitrogen. The
species found generally upon the roots of leguminous plants
is Rhizobium leguminosarum. x

It is capable of fixing atmospheric nitrogen when grown
in the laboratory in solutions containing carbohydrates.

II. FAMILY BACILLACEAE

The organisms belonging to this group are always rod-
shaped, all produce endospores, and the flagella when pres-
ent are peritrichous. For the most part they are able to
decompose complex organic compounds, and are active in
producing decay in nature. Two genera are included in
this family ; the genus Bacillus, including those forms which
require free oxygen for their development; and Clostri-
dium, including those forms which do not grow in the
presence of free oxygen.

Bacillus. — The organisms belonging to this genus are all

FIG. 12.— BACILLUS. A. Motile species. 1. Vegetative cells. 2.
Speculating cells. 3. Free spores. B. Nonmotile species. 1.
Vegetative cells in capsules. 2. Left. Noncapsulated vege-
tative cells. Right. Sporulating cells. 3. Free spores.

rod-shaped, sometimes occurring in chains. Under the
right conditions these bacteria are among the most active

i Many other names have been applied to this organism. It is
frequently discussed in literature as the Bacillus radicicola.

34

AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

in nature, producing decomposition of organic materials.
One species produces the disease anthrax in animals and
man, and one or two species produce the diseases termed
foul brood of bee.

Clostridium. — The organisms of this genus are all rod-
shaped, producing endospores, but not growing in the pres-
ence of free atmospheric oxygen. Frequently the rods are
swollen at time of spore production, producing spindle-
shaped or club-shaped cells. Some of the bacteria of this
group are among the most active of the putrefactive forms,

FIG. 13. — CLOSTRIDIUM. A. Motile species. 1. Vegetative cells.
Sporulating, swollen cells, spores equatorial. B. Motile species.
1. Vegetative cells. 2. Sporulating cells, spores, polar, cell
swollen. 3. Free spores. C. Nonmotile species. 1. Vegetative
cells. 2. Sporulating cells, cells not swollen.

particularly in the production of malodorous compounds
such as butyric acid. Other species are capable of produc-
ing disease, especially when introduced into wounds.
Among the diseases produced are gaseous gangrene in man,
malignant edema, blackleg, and bradsot in animals, and
tetanus in man and animals.

III. FAMILY COCCACEAE

The organisms belonging to this family have cells usually
spherical when free, though during division they may be

CLASSIFICATION OF THE BACTERIA 35

somewhat elliptical. If the cells remain in contact after
division they are frequently flattened at the surface of
contact. They may form chains, packets or irregular
masses. Very few cocci are motile; none of the motile
species are of economic importance. Spores are not pro-
duced. Most of the genera of this family are parasitic,
many species are disease-producing. A few are of im-
portance because of the fermentative changes which they
bring about. The most important genera are Neisseria,
Streptococcus, Diplococcus, Staphylococcus, Micrococcus,
Sarcina.1

Neisseria. — The organisms of the genus are strict para-
sites, usually growing rather poorly in artificial culture
media. The cells usually occur in pairs, flattened at the
proximal sides, usually coffee-bean-shaped, and Gram-neg-
ative. Two of the diseases produced by organisms of this

FIG. 14. — NEISSERIA. FIG. 15. — STREPTOCOCCUS.

group are of importance, namely, cerebrospinal meningitis,
and gonorrhea.

Streptococcus. — This genus includes those spherical bac-
teria whose cells occur normally in chairs. For the most
part the cells are Gram-positive. Certain of the species
are important in that they bring about lactic acid fermenta-

i Other genera are: Leuconostoc, forming large gelatinous masses
in sugar solutions; and Rhodococcus, including the cocci which
produce a red pigment.

36 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

tion in milk. Others are capable of producing diseases.
Certain species, for example, are commonly associated with
tonsillitis, rheumatism, with the disease strangles in the
horse, wound infection, and pus production.

Diplococcus. — The organisms belonging to this genus are
strict parasites, Gram-positive, occurring in pairs, the cells
usually somewhat pointed. The most important species is
the organism usually associated with pneumonia.

00

o

FIG. 16. — DIPLOCOCCUS. Flo. 17. — STAPHYLOCOCCUS

Capsulated.

Staphylococcus. — In this genus the spherical cells are
united in more or less irregular masses. They are Gram-
positive, and usually parasitic. They are commonly asso-
ciated with pus production, wound infection, the develop-
ment of boils, abcesses, and similar conditions in the bodies
of man and animals.

_ Micrococcus. — The organisms of this

genus are usually not parasitic, but are
present commonly in water and soil.
They are frequently Gram-negative,
occasionally motile, and when grown in

the laboratory usually develop a yellow
FIG. 18 — MICROCOCCUS. . * _

pigment. There are no species of con-
siderable economic importance.

Sarcina. — Organisms belonging to this genus have their
spherical cells usually massed in regular packets, consisting
of cubes of eight cells each. When grown in the labora-
tory pigment is frequently produced, usually yellow in
color.

CLASSIFICATION OF THE BACTERIA 37

IV. FAMILY PSEUDOMONADACEAE

This family contains a single genus, Pseudomonas. The
cells are rod-shaped, and usually motile by means of polar
flagella. Frequently a fluorescent green or brown pigment
is produced in culture media. In other species an insolu-

tt
* I

FIG. 19. — SARCINA. FIG. 20. — PSEUDOMONAS.

ble yellow pigment is formed. Among those producing a
green fluorescent pigment is the Pseudomonas aeruginosa,
an organism sometimes found in wounds. Many of the yel-
low and a few white species produce diseases in plants.

V. FAMILY BACTERIACEAE

This is the largest of the families of bac-
teria. It includes those rod-shaped bacteria
whose cells are usually regular in shape, do
not produce endospores, and when motile do

FlG 21 EB. n°t nave polar flagella only. In most genera

YTHBOBACLLLUS the cells are gram-negative. Fluorescent
pigment is not produced. The important genera are
Erythrobacillus, Erwinia, Proteus, Bacterium, Pasteurella,
Hemophilus, and Lactobacilhts.

Erythrobacillus. — The organisms of this group are small
aerobic bacteria, producing a red or pink coloring matter,
sometimes yellow or orange. Some species are motile.
The most important species is Erythrobacillus prodigiosus.

38 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

This is found to cause red spots in bread and other carbo-
hydrate foods.1

Erwinia. 2 — The organisms belonging to this genus all
produce disease in plants. Their growth in the laboratory
is usually whitish. They form acids in certain carbohy-
drates, but as a rule no gas. One of the most important
species is Erwinia amylovora, the organism causing pear
blight.

Proteus. — The organisms belonging to this genus are
highly pleomorphic rods, frequently producing filaments
and curved cells as involution forms. They are Gram-
negative and actively motile. Upon suitable culture media
they form amoeboid colonies. The organisms ferment glu-
cose and sucrose but not lactose with the formation of acid

FIG. 22. — ERWINIA. FIG. 23. — PROTEUS.

and gas. Bacteria belonging to this group are among the
most important causes of putrefaction and decay. They
have been occasionally found associated with disease.

Bacterium, — The organisms belonging to this genus are
Gram-negative rods, frequently motile, and easily cultiva-
ble. Most species ferment certain carbohydrates with the
formation of acid and frequently of gas. They are typi-
cally intestinal parasites in man and higher animals. Some

1 Another genus belonging to this family is Chromobacterium in
which the cells produce a violet or blue pigment.

2 This genus is variously incorporated by writers in plant path-
ology with the genus 'Bacterium or the genus Bacillus.

CLASSIFICATION OF THE BACTERIA 39

species are not infrequent in the soil. A few species are
pathogenic, including the organisms causing typhoid fever,
paratyphoid fever, dysentery, and certain types of food
poisoning. One species, the Bacterium coli, is frequently
used as an index for the determination of the presence of
sewage in water.

Pasteurella. — The organisms of this genus are all rod-
shaped cells, Gram-negative, and show bipolar staining.
They have very slight powers of fermentation. Members
of this genus produce many diseases in man and animals,
including the bubonic plague of man and the hemorrhagic
septicemias of animals, such as fowl cholera and related
diseases in swine, sheep, cattle and horses.

Hemophilus. — The organisms belonging to this genus are
minute, parasitic, Gram-negative rods, which grow in the

FIG. 24. — BACTERIUM. FIG. 25. — PASTEURELLA

laboratory only in special media, preferably containing
hemoglobin. The most important species is Hemophilus
influenzae, the organism which has been supposed to cause
influenza and which is quite certainly associated with many
cases of the disease.

Lactobacillus.— The cells are rod-shaped, often long and
relatively slender, Gram-positive, and nonmotile. Some
species have very short cells and are almost coccuslike. No
endospores are developed. Acid, particularly lactic acid,
is usually produced in considerable quantities from carbo-
hydrates. Many of the species prefer to grow in the ab-

40 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

sence of oxygen, although they are not strictly anaerobic.
Included in this genus are many bacteria important in the
souring of milk, the formation of lactic acid in silage, sauer
kraut, etc.

FIG. 26.— HEMOPHILUS. FIG. 27.— LACTOBACILLUS.

VI. FAMILY SPIRILLACEAE

The organisms that belong to this group are curved
rods. Two genera are included, namely, Vibrio and Spiril-
lum.

Vibrio. — This genus includes short curved rods, motile
by means of one, two or three polar flagella. Many are
intestinal parasites and some are capable of causing disease.
The most important organism is the Vibrio cholerae pro-
ducing Asiatic cholera in man.

Spirillum. — Organisms belonging to this group are
longer, spiral cells, with a tuft of relatively long flagella
at the tip. They are not uncommon in water, and partic-

FIG. 28. — VIBRIO. FIG. 29. — SPIRILLUM.

ularly in water that contains decaying organic matter.
None of the species are of very much economic importance.

CLASSIFICATION OF THE BACTERIA 41

ORDER ACTINOMYCETALES

The organisms belonging to this order usually have the
cells somewhat elongated, frequently filamentous, and with
a decided tendency to the formation of branches. In some
genera a definite branched mycelium is developed. The
cells frequently show swelling, or are clubbed or irregular
in shape. Endospores are not produced, although conidia
are formed Ly some genera. Many of the genera are Gram-
positive and all are nonmotile. Some species are parasitic
in animals or in plants. Most of the species are aerobic.
The genera belonging to this group are Actinomyces, Myco-
bacterium, Corynebacterium, and Pfeifferella. x

Actinomyces. — The organisms belonging to this group
form fine threads (or mycelium,) which break up into
short segments which function as spores or conidia. Some
of the species are parasitic, one, the Actinomyces bovis, pro-
ducing lumpy jaw in cattle. Many other species are widely
distributed in the soil, apparently growing upon decaying
roots and similar organic matter.

Mycobacterium. — These organisms are slender rods
which are stained with difficulty, but when once stained are
resistant to decolorization by acids, that is, they are termed
acid fast. The cells are sometimes swollen, showing club
shapes or forked forms, occasionally the cells are branched.
They are nonmotile and Gram-positive. Some of the
species are present in the soil, a few are pathogenic to man
and animals. The most important of the diseases caused
are tuberculosis and leprosy in man, and paratubercular
dysentery in cattle.

1 Other genera are Actinobacillus, one species producing the disease
called actinohacillosis in cattle; Leptotrichia found in the mouth;
Erysipelothrix producing disease in man and animals, particularly
the disease swine erysipelas; and Fusiformis, including certain mouth
and throat forms, some of them associated with diseases of the throat
and the mouth.

42 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Corynebacterium. — The cells are slender, often slightly
curved rods with a tendency toward the formation of clubs,
and containing granules which give the cells when stained
a barred or irregular appearance. They are not acid fast
but are Gram-positive and aerobic. Most species are para-
sites, the most important being Cory neb act eriumi diph-
theriae, the cause of diphtheria in man.

Pfeifferella. — These organisms are nonmotile rods, slen-
der, and Gram-negative, staining poorly, sometimes form-
ing threads, and showing a tendency toward branching.
When grown upon potato in the laboratory they develop a
characteristic honeylike growth. The most important
species is Pfeifferella mallei, the organism which causes the
disease glanders in the horse.

ORDER SPIROCHAETALES

The organisms belonging to this group in many respects
resemble the protozoa, and may be regarded as intermediate
between the true bacteria and the protozoa. They are all
more or less curved rods, frequently very slender. Many
species are motile but there has been no definite demon-
stration of flagella. They multiply either by longitudinal
or transverse fission. It is probable that in some cases
they have a relatively complex life history . Several genera
have been described, the most important being Treponema.

Treponema. — The organisms^ belonging to this genus are
exceedingly slender spiral rods, motile by means of flexuous
bending of the body. The most important species is Tre-
ponema pallida, the cause of the disease syphilis in man.

CHAPTER IV
MORPHOLOGY AND CLASSIFICATION OF THE YEASTS

Size, Shape, and Grouping of Yeast Cells. — Yeasts are
usually somewhat larger than bacteria. This usually ren-
ders comparatively easy differentiation from the bacteria.
The yeast cell is usually spherical, ovoid, or ellipsoid in
shape, occasionally it is considerably elongated or even
cylindrical. In most species there is very little of the reg-
ularity of grouping which is characteristic of many types
of bacteria. The cells occur in masses or occasionally in
chains or filaments. Yeasts are typically unicellular
plants, and can by this means be differentiated from the
molds, although in a few species the chains of cells (fila-
ments) approach in appearance the hyphae of the true
mold.

STRUCTURE

The cell wall is relatively thin in young yeast cells but
may be considerably thickened in old. Occasionally the
cells are embedded in gelatinous masses. The chemical
composition of the cell wall has not been clearly estab-
lished. It is frequently said to consist of yeast cellulose,
although not identical in composition with the cellulose of
the cell walls of higher plants. The organism practically
never produces definite capsules and is never motile, that
is, flagella are not produced.

Cell Contents. — The contents of the cell are somewhat
more clearly differentiated than is the case with the bac-
teria. Furthermore, one may note marked differences in
the appearance of cell contents of young cells and older

43

44 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

cells. The young cell has a very thin cell wall, protoplasm
which is relatively homogenous, and a relatively large
nucleus. The latter is easily demonstrated by the use of
appropriate stains. As the cells increase in size, vacuoles
begin to appear in the protoplasm; both in these vacuoles
and in the protoplasm are formed granules which stain
intensely with methylene blue, the so-called metachromatic
granules. Eventually still larger vacuoles develop, which
in some cases are crowded full of granules giving the yel-
low glycogen reaction with .iodine. Still later the glycogen
may disappear but the relatively large vacuoles persist. In
an old cell the cytoplasm and nucleus may constitute a
relatively small proportion of the cell contents.

By means of suitable reagents it is possible to demon-

FIG. 30. — YEAST CELLS. A. Vegetative cell. n. Nucleus, v. Vacuole.
B. Yeast cell filled with glycogen.

strate the presence of an outer layer of the protoplasm, the
ectoplast, corresponding in function to that found within
the bacteria. It undoubtedly acts as a semipermeable
membrane in the determination of substances which may
leave and enter the cell.

Vegetative Multiplication of Yeast Cells.— Most of the
common yeasts multiply vegetatively by a process of bud-
ding. A few species belonging to a single genus (Schizo-
saccharomyces) multiply by fission, in a manner analogous
to that already described for bacteria.

Those yeasts which multiply by budding show first a
minute protuberance on one side of the cell. The nucleus
of the mother cell goes to a point near the opening between

CLASSIFICATION OF THE YEAST

45

the cell and the bud, elongates in the direction of this open-
ing and eventually pinches off a nucleus which migrates
into the bud. This rapidly increases in size and the open-
ing between the two cells soon closes. In a very young
bud the cell wall is either absent or very thin, but soon

FIG. 31. — MULTIPLICATION OF YEASTS BY BUDDING.

develops, probably in all cases becoming evident before
separation from the mother cell occurs.

Development of Spores.— All species of true yeasts are
capable of producing spores under suitable conditions.
These conditions differ with the species. In most species
the spores are not developed in the ordinary cultural media
or by the usual methods. With many of the more common
yeasts of economic importance sporulation may be induced
by placing young vigorous cells under such conditions that
they will have an abundance of oxygen, plenty of moisture,
and the right temperature. This is most frequently accom-

FIG. 32. — DEVELOPMENT OF SPORES IN YEAST.

plished by preparing small, flat gypsum blocks (plaster of
Paris). These are placed in deep Petri dishes, water

46 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

poured into the Petri dish until it reaches half the height
of the block, when the yeast mass to be studied is spread
over the surface. This is placed in a thermostat at the
right temperature, usually about 25° C. The cells will
usually develop spores in from twenty-four to forty-eight
hours. Some species of yeast which form films over the
surface of their nutrient solutions may develop the spores
in such films.

Yeast spores, like the spores of bacteria, are always
formed inside the mother cell, but differ in that usually
more than one spore is formed within a single cell.

The first evidence of sporulation in yeasts is the division
of the nucleus first into two, then into four, six, or eight
nuclei, by a process of mitosis. Each one of the nuclei then
surrounds itself by a bit of protoplasm and finally by a cell
wall or membrane. The spores grow in size by absorption
of the cytoplasm from the mother cell until they come to
occupy practically the entire interior. The number of
divisions of the nucleus determines the number of spores
which will develop. This is usually fairly constant in a
given species, although variations may be found. Yeasts
are known which typically produce one, two, four and eight
spores to a cell.

The mother cell with its spores shows such close rela-
tionship to the spore-bearing sacs produced by certain of
the fungi that it is usually given the name ascus, and the
spores are known as ascospores.

The spores vary not only in number but in shape and
size as well. Some are spherical, some elliptical, others are
hat-shaped, or even elongate. Some have a single mem-
brane and others apparently a double membrane.

Sexual reproduction of a primitive type may be observed
in some species of yeasts, though not with the most common
types. In a few species cells which are about to form
spores fuse in pairs, the nuclei also fuse and then divide

CLASSIFICATION OF THE YEASTS 47

to form the number of nuclei characteristic of the species,
each nucleus becoming a spore. In some forms the cells
which fuse are of the same size; in others they differ. In
one the spores fuse in pairs during the process of germi-
nation and the young yeast cells are produced by budding.
Occasionally yeast cells surround themselves with an
unusually heavy membrane and pass into a resting state.
Such cells are termed chlamydospores.

THE CLASSIFICATION OP YEASTS

Relationships of True Yeasts. — The development of asci
and ascospores by yeasts show that their relationship is
primarily to the group of fungi termed ascus-fungi or
Ascomycetes. Yeasts are sometimes conveniently sepa-
rated into true yeasts and false yeasts. The former are
those which have already been described. To the latter
belong certain forms which have every appearance of being
the vegetative stages of true yeasts, but which do not pro-
duce spores, and are frequently not active in causing alco-
holic fermentation. An organism of the latter type is
sometimes termed a torula.

Classification of the Yeasts. — Botanists recognize a dozen
or more different genera of yeasts. Only two are of suffi-
cient importance to be considered here. The first of these
is the genus Saccharomyces, which includes those common
yeasts which multiply by process of budding, and Schizo-
saccharomyces, in which the cells multiply by a process of
fission.

Saccharomyces. — To this genus belong most of the yeasts
of economic importance, including those which are used in
wine making, in manufacture of beer and commercial
alcohol, and in bread making. Many different species have
been described and several different methods of classifica-
tion have been used.

One common method of differentiating yeasts is based

48 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

upon the type of fermentation. Some yeasts settle rela-
tively rapidly to the bottom of the fermenting liquid and
do not form a heavy scum or pellicle over the surface.
They are termed bottom yeasts. Certain of the wine and
cider yeasts are of this type. In other species growth is
usually characterized by the development of a foamy scum
on the surface. Such are termed top yeasts. Interme-
diate forms, however, are known.

Many of the great brewing districts in Europe have
developed special strains of yeasts which are used for manu-
facture of particular types of beverages. Such yeasts have
been named after the town, locality, or establishment in
which they originated. For example, one may read of
Saaz yeasts, of Carlsberg bottom yeasts, etc.

Among the earlier investigators an attempt was made
to separate the various species of yeasts on the basis of
shape. The ordinary brewers' or bread yeast, known as
Saccharomyces cerevisiae, was described as oval or nearly
spherical ; wine yeasts were found usually to be ellipsoid
or elongate, and were termed Saccharomyces ellipsoideus.
The name Saccharomyces pastorianus was given to those
which produced long or cylindric cells. The conditions
under which yeasts are grown, however, influence to such
a marked degree the shape and size of the cells that this
method of differentiation is frequently difficult to use.

The most satisfactory methods for differentiation of
yeast species are based upon such morphological differences
as may be observed microscopically, the development of
spores, the ability of yeasts to ferment particular sugars,
to grow in the presence or absence of air, and to produce
definite quantities of alcohol. It is customary to recognize
six groups of yeasts, the groupings being determined by
the carbohydrates which may be fermented by the forma-
tion of alcohol and carbon dioxide. The following table
gives the characteristics of these groups :

CLASSIFICATION OF THE YEASTS

49

Production of Alcohol and Gas from

Number of group

Dextrose

Saccharose

Maltose

Lactose

1

+

+

_l_

2

-f-

-f-

—

—

3

-j-

—

-}-

—

4

-f-

—

—

—

5

-|-

-|-

6

—

—

• —

—

Schizosaccharomyces. — As was noted above, the organ-
isms belonging to this genus multiply by fission rather than
by budding. The cells when about to produce spores fuse
in pairs and within the resulting cell eight spores are
usually developed. Several species have been described.
One is the active fermentative agent in the production of
an African beverage made from millet.

CHAPTER V
MORPHOLOGY AND CLASSIFICATION OF THE MOLDS

STRUCTURALLY the molds differ from the bacteria and the
yeasts in that they are multicellular, that is, the plant body
is more complex than that of bacteria or yeasts, and is made
up of numerous cells. Furthermore, the cells which make
up the plant body of a mold are not all uniform in size,
shape or function. Some are differentiated for purposes
of securing nutrients, others may be differentiated for pur-
poses of reproduction.

It should be emphasized that the molds as considered
here do not constitute a group recognized as such by the
botanist. All belong to the great class Fungi, but they
are scattered among several distinct subgroups.

The plant body of the mold is made up of a mass of
threads, usually branched. This whole mass of threads
taken collectively, that is, the entire plant body, is termed
the mycelium. The individual threads are called hyphae.
In most molds the hyphae are of two principal types, those
which 'are differentiated for the purpose of producing
spores, called the fertile hyphae, and those which serve to
secure nutrients for growth, termed the vegetative hyphae.

Structure of Mold Hyphae. — Two kinds of hyphae may
be recognized among the molds. In the first the cells of
the hyphae are separated from each other by definite cell
walls, that is, the threads consist of definite cells arranged
in chains, that is, cross walls are formed in the hyphae. A
cross wall is termed a septum, and the hyphae are said to
be septate. In the second group of molds the hyphae do
not usually show cross walls except during the process of

50

MORPHOLOGY OF THE MOLDS 51

sporulation. The hyphae are more or less branched, con-
tinuous tubes. Such a mycelium is said to be nonseptate.
In general those molds which show a separate mycelium
have a single nucleus with the cytoplasm in each section
of the hyphae. In those molds which are nonseptate
numerous nuclei are to be found scattered throughout the
protoplasm. A cell is sometimes defined as a nucleus sur-
rounded by a bit of protoplasm, hence it is not incorrect
to speak of the nonseptate molds as being multicellular.

a-

FIG. 33. — TYPES OF HYPHAE ix MOLDS, a. Nonseptate hypha. Note
numerous nuclei and absence of cross walls or septa, b. Septate
hypha. Note single nucleus in each cell.

Structure of the Mold Cell.— The cell of the mold does
not differ in many respects from that of the yeast. The
cell wall consists in some instances of a substance closely
resembling cellulose, in others it may contain callose and
pectose. It is sometimes markedly thickened. This is par-
ticularly true of the outer walls of spores and of resting
cells. Such are sometimes cutinized. Such cells may occa-
sionally show ridges, thickenings, spines, knobs, or other
protuberances.

The nuclei may ordinarily be readily differentiated from
the remainder of the cytoplasm. In the young cell the
cytoplasm is relatively dense. In the older cells it becomes

52 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

vacuolated-, and in cells which have reached maturity fre-
quently the protoplasm containing the nucleus occupies
but a small portion of the cell volume, being reduced to a
comparatively thin layer just inside the cell wall. Gran-
ules of various kinds, including metachromatic granules,
globules of fat and of glycogen may develop. Apparently
these products may function as reserve food materials.

Mold Growth. — Most molds increase in size by growth at
the tip of the hyphae. Any cells lying back of the tip that
start to grow develop into branches. Such growth is
termed apical. A few molds are known in which all cells
may continue to grow in length and divide. Such growth
is termed intercalary. In apical growth the tip cell only
divides, while in intercalary growth cells throughout the
filament may divide.

Multiplication in Molds. — Practically all molds multiply
by means of specialized cells termed spores. In many in-
stances these spores are numerous and well adapted to carry
the organism over unfavorable conditions and to facilitate
its distribution.

A single species of mold may produce several kinds of
spores. A few molds have relatively complex life histories,
producing a different type of spore at each stage in de-
velopment. In general the spores developed belong to
two types, those which are asexual in origin and those which
are sexual. The asexual spore may be defined as one which
is not the result of the fusion of two sex cells or gametes.

Practically all of the common types of molds produce
asexual spores, some several kinds. A smaller number of
molds produce also sexual spores. From the standpoint of
the botanist who is seeking to determine true interrelation-
ships, the sexual- spores are of great interest. From the
standpoint, however, of the practical worker it is possible
to differentiate the molds satisfactorily by observing the
types of asexual spores produced.

MORPHOLOGY OF THE MOLDS 53

Sexual Reproduction in Molds. — Molds having nonsep-
tate mycelium produce a type of sexual spore termed a
zygospore. Those molds with septate mycelium, which pro-
duce sexual spores, develop ascospores.

The process of zygospore formation can most readily be
understood by reference to the accompanying figure. Two
filaments lying near each other send out branches which
approach each other and finally touch. The cell walls sepa-
rating the two tips are absorbed, resulting in union of
the protoplasm of the two cells. Cell walls are then
formed, separating the fused mass of protoplasm from the
parent hyphae. The nuclei of the two masses of proto-

3

FIG. 34. — DEVELOPMENT OF A ZYGOSPORE IN A MUCOE.

plasm apparently fuse in pairs. The resultant spore or
cell enlarges markedly in size, develops a very thick, usually
brownish cell wall, frequently marked by spines or by
irregular thickenings. Under favorable conditions this
zygospore germinates and develops a new mold.

The development of ascospores is somewhat more complex
than that of zygospores. Two cells, frequently of some-
what different size and shape, develop from the same or
adjoining threads. The cells coil together and the tips of
the cells fuse. The cell resulting from this fusion begins
to grow, branching more or less, and becomes surrounded
in most cases by a mass of hyphae, which develop from
adjacent hyphal threads. The branches of the fertilized

54 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

cell develop into sacs. In each one of these sacs, or asci,
two or more spores, usually eight, form. The structure
developed surrounding the spore sacs is termed a perithe-
cium. This is usually globular in shape and frequently
brightly colored. Comparatively few of the molds usually
encountered in the laboratory produce these perithecia.
An exception is to be found in certain common species of
Aspergillus, which readily develop orange or bright yellow
perithecia under suitable conditions.

Asexual Reproduction in Molds. — The asexual spores of
molds may be developed free or they may be enclosed in
special spore cases termed sporangia. Many of the molds,

FIG. 35. — FORMATION OF ASCOSPORES AND ASCI IN ASPERILLUS. 1, 2,
3. Stages in the development of a peritheeium. 4. A single ascus
from interior of the peritheeium. (Adapted from De Bary. )

with nonseptate hyphae produce these sporangia, but they
are never developed by the molds with septate hyphae.

Sporangium Formation. — The development of the spo-
rangium may be illustrated by the genus Mucor. The first
evidence of spore formation is the appearance of a fertile
hypha produced at right angles to the surface on which the
mold is growing. This becomes enlarged at the end, in-
creases in length, and finally consists of a relatively long
thread with a much swollen tip. The interior of the latter
is filled with protoplasm containing numerous nuclei. A
special wall then forms extending from the tip of the thread
up into the swollen apex. The nuclei surround themselves
by bits of protoplasm and develop around these, in turn,

MORPHOLOGY OF THE MOLDS

55

definite cell walls forming spores. The spore case is
termed a sporangium. The structure reaching to the in-
terior is a columella, and the stalk upon which the sporan-
gium is borne a sporangiophore. In some species the
sporangiophore may branch, each branch bearing a terminal
sporangium. When ripe the sporangium walls break and
spores are scattered.

Conidia. — Asexual spores of molds not bearing sporangia
are frequently termed conidia. Asexual spores may be
developed on specialized branches or by changes occurring in
the vegetative cells of the mycelium.

FIG. 36. — DEVELOPMENT OF SPORES AND SPORANGIA OF MUCOR. 1.
Young sporangium with numerous nuclei. 2. Segmentation of
protoplasm. 3. Segmentation of protoplasm and separation of
spores. 4. Mature sporangium. (Adapted from Blakeslee.)

Chlamydospores are asexual spores having a relatively
thick cell wall. -O'idia are developed when the vegetative , /
mycelium breaks up into spores without any differentia-
tion of special spore-bearing threads. In most molds a
definite fertile thread termed the conidiophore is developed
to produce the conidia. The conidiophore may be of many
shapes and types, sometimes simple, sometimes branched,
and sometimes club-shaped.

Conidia borne on the tip of branches may be developed
in one of two ways. In some species of molds the tip of
the conidiophore pinches off spores one after another; if
they stick together a chain is formed in which the spore

56

AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

nearest the mother cell is the youngest. In other molds
spore formation results from the budding out of a cell (not
a pinching off) from the terminal cell. The spore then
grows to its full size, and from its tip another spore buds
out. A continuation of this process also results in the for-
mation of a chain of spores. In this type of spore forma-
tion, however, the chains are sometimes branched, that is,

FIG. 37. — MOLD SPORES (CONIDIA). 1. Various types of unicellular
spores. 2. Types of spores with two cells. 3. Multicellular
spores.

more than qne spore may bud from a single cell. The
terminal cell of the chain is always the youngest.

The conidia developed by molds are of many shapes and

FIG. 38. — TYPES OF SPORE PRODUCTION BY MOLDS. 1. Chlamydospores
of Mucor. 2. Conidia production in MullerieUa, no conidio-
phores produced, the conidia arising laterally directly from the
mycelium. 3. Urospora showing simple conidiopliores. 4. Con-
idiophore of Aspergillus. 5. Conidiophore of Penicillium.
(After Brefeld.)

sizes. The simplest type of conidium is that which is sin-
gle-celled and oval or spherical in shape. Other single-

CLASSIFICATION OF THE MOLDS 57

celled spores may be elongate or even spindle-shaped, cylin-
drical, or club-shaped. Two-celled spores occur in many
species of molds. Others produce elongate spores, having
several or many cross walls. The spores of some molds are
many-celled, the walls being not only transverse but longi-
tudinal or irregularly disposed. The spores of some molds
branch, in others they are curved, sometimes being coiled
like a spring.

Spores are usually adapted to wind distribution, being
caught up by air currents and carried to considerable dis-
tances. They are, therefore, commonly present in the air
and are constantly observed in the laboratory. When they
are brought under favorable conditions for growth, they
germinate, developing a mycelium.

CLASSIFICATION OF THE MOLDS

"It has already been noted that the term mold as used in
bacteriology is not recognized in this exact sense by the
botanist. The molds are plants belonging to several dif-
ferent groups of fungi. They are all alike, however, in
possessing a much-branched mycelium and growing as a
velvety or cottony mass upon ordinary culture media, or
upon decaying materials.

Many different kinds of molds are known, in fact, more
than a hundred different genera have been described. Of
these, however, comparatively few are common, and the
student should readily recognize nine tenths of the molds
with which he may come in contact by comparatively sim-
ple microscopic examination. In many cases the genus to
which the mold belongs can be determined by its general
appearance. Some of these common genera have many
species. In the following account a description will be
Driven of the commonest of the genera together with notes
on methods of differentiating some of the species.

58 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

The following key based upon asexual spore production
differentiates nine genera of molds :

KEY FOR DIFFERENTIATION OF COMMON GENERA OF MOLDS

A. Spores borne in sporangia. Mycelium non-
septate.
B. Sporangiophores arising in clusters from

the nodes of runners or stolons 1. Rhizopus.

BB. Sporangiophores arising singly, without

stolons 2. Mucor.

AA, Spores (conidia) never borne in sporangia.
B. Neither conidia nor hyphae smoky or
dark in color.
C. Conidia one-celled.

D. No distinct conidiophores. Conidia

formed as oidia 3. Oospora.

DD. Conidia on distinct conidiophores.
E. Conidiophores enlarged or club-
shaped at tip 4. Aspergillus.

EE. Conidiophores branched at tip 5. Penicillium.

CC. Conidia two-celled, pear-shaped 6. Trichothecium.

BB. Either conidia or hyphae smoky or dark,
or both.

C. Conidia one-celled, in branched chain. . . 7. Hormodendrum.
CC. Conidia more than one-celled.

D. Like Hormodendrum, but condidia two-
celled when old 8. Cladosporium.

DD. Spores many-celled, club-shaped, in
chains 9. Alternaria.

Rhizopus. — Molds belonging to this genus are frequently
termed the black or bread molds. They are among the
most common, occurring upon bread, decaying vegetables,
and similar material. When young they are cottony and
white, as they grow older they become grayish because of
the development of -numerous dark sporangia. The vege-
tative mycelium spreads through the medium upon which
the organism is growing. When ready to produce spores,
long slender threads are thrown out which grow until the
tip touches some solid material. For example, in the labo-
ratory the tip may touch the glass side of the dish in which
the organism is growing. From this tip are sent out clus-
ters of dark brown, short branches, termed rhizoids. These
act as holdfasts, they are not roots in any sense. The

CLASSIFICATION OF THE MOLDS 59

hypha, called a stolon, grows on from this cluster of rhizoids
until it again attaches itself at some other point. From
each cluster of rhizoids there arises a group of sporangio-
phores which grow into the air, developing at the tip the
large dark brown or black sporangia. These sporangia are
readily recognized under the microscope. When mounted
in a drop of water the ripe sporangium usually collapses,
the outer wall disappearing and the spores go free. The
columella in this form is partly adherent at its base to the
outer wall of the sporangium. Usually when the sporan-
gium collapses it also collapses, the lower side invaginates,

FIG. 39. — RIIIZOPUS. 1. Growth habit of the mold, showing stolons,
rhizoids, sporangiophores and sporangia. 2, 3. Columella.

and together with the stalk or sj)orangiophore gives rise to
a structure having the appearance of a small toadstool or
umbrella.

)^Some species of Rhizopus are used commercially in the
transformation of starchy materials into sugar, precedent
to the fermentation of the sugar into alcohol in the com-
mercial manufacture of this material.

The most common of the species of Rhizopus is Rhizopus
nigricans, the common black bread mold. The species
used in hydrolysis of starch are Rhizopus oryzae and R.
japonicus. V

Mucor. — The genus Mucor is not as abundant as Rhizopus
but is found under the same general conditions. Usually
the mold is not quite so coarse. It does not develop stolons.

60

AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Its pporangiophores are sometimes branched, do not arise in
clusters, and no rhizoids are developed. In the sporangium
the columella is not attached to the sporangium wall, but
may be spherical in shape, pear-shaped, and in one species
pointed or covered with spines. The various species of

FIG. 40. — COMMON SPECIES OF MUCOR. a. Sporangiophore showing
branching and grouping of sporangia, b. Longitudinal section
through sporangium, c. Spores, d. Columella. 1. Mucor hie-
malis. 2. Mucor piriformis. 3. Mucor muceflo. 4. Mucor plum-
beus. 5 Mucor alternans. (1, 2, 3, after Wehmer. 4, 5, after
Gray.)

Mucor are separated from each other on the basis of
the size,, shape, and color of the sporangium, size and
shape of columella, and the branching of the sporangio-
phores.

Some eleven species of Mucor have been described and are
comparatively common. They may be differentiated from
each other by use of the key given in the footnote.1

i KEY TO SPECIES OF MUCOR
A. Sporangiophores rarely or never branched.

B. Columella spherical Mucor hiemalis.

BB. Columella pear-shaped (piriform) M. piriformis.

BBB. Columella elongate to ellipsoidal M. nwcedo.

AA. Sporangiophores usually* branched.

B. Columella usually knobbed or spiny near tip. . M. plumbeus.
BB. Columella not roughened at tip.

C. Rarely fruiting at room temperatures, im-
portant in commercial alcohol manufac-
ture, rapidly saccharifying starch M. rouxU,

CC. Readily fruiting at room temperature,
not commercial types.

D. Sporangiophores with definite main
stem and secondary lateral branches,
racemose.

E. Columella ovoid M. racemosus.

EE. Columella spherical

CLASSIFICATION OF THE MOLDS 61

>ne of the species of Mucor, Mucor rouxii, like the species
of Ehizopus above mentioned, has been used in the sacchari-
fication of starch. Some of the species produce heavy-
walled resting cells or chlamydospores in the vegetative
mycelium. This is particularly true of Mucor racemosus.

Oospora. — This genus is sometimes known as Oidium.
Tne most common species is Oospora lactis, a mold very com-
mon upon sour milk and cheese; in fact, it is to this mold
that many types of cheese owe in part their characteristic
flavor and aroma. The mycelium of this fungus grows very
largely below tfnTsurface of the medium on which it is de-
veloping, and later splits up by transverse fission into nu-
merous spores or oidia. These spores frequently do not
stand out upon the surface of the medium but are developed
below. No distinct conidiophores are formed. The myce-
lium shows a characteristic type of branching. Instead of
having main shoots with side branches, the terminal cells
usually divide and two branches of equal size develop from
tijx_ This type of branching is termed dichotomous.

Aspergillus. — The spores of Aspergillus are relatively
common in the air. The organism grows on all sorts of
decaying vegetation, on moldy corn, grain, bread, etc. In
color it is usually greenish, yellow, orange, black or brown.
The mold has generally a velvety or powdery appearance.
The hyphae are much branched and septate. The fertile
hyphae grow out into the air, the tip becoming enlarged
or club-shaped. From this enlarged tip are developed
numerous small branches called sterigmata. These usually

F. Sporangium gray, yellow M. erectus.

FF. Sporangium black M. fragilis.

DD. Branches of sporangiophore nearly

equal, .cymose.

E. Sporangia borne irregularly M. ambiguus.

EE. Sporangia in two rows, alter-
nating.

F. Spores spherical to short ellip-
soidal M . circinelloides.

FF. Spores longer, ellipsoidal M. alternans.

62

AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

cover the entire end and frequently the entire swollen por-
tion of the conidiophore, giving it something of the appear-
ance of a war club covered with spikes. These sterigmata
in some species branch into groups of branches, and in other
species they give rise directly to the spores. The spores
are pinched off in chains from the tips of the primary or
secondary sterigmata. In some species the spores radiate
in all directions from the head, in others the chains cling
together giving rise to a long column of spores like a plume.
Many species of Aspergillus have been described. A few
of them are of considerable economic importance. The

FIG. 41. — SPECIES OF ASPERGILLUS. a. View of conidiophore and
condia b. Longitudinal section through conidiophore to show
attachment and arrangement of sterigmata and conidia. c.
•Conidia. 1. Aspergillus fumigatus. 2. A. niger. 3. A. glaucus.
(Adapted from Wehmer.)

species known as Aspergillus fumigatus, for example, may
produce a type of pneumonia in birds when it is inhaled,
and it may also be pathogenic for other animals. Asper-
gillus niger has been much studied because of its ability to
produce a great variety of fermentative changes. The com-
monest is probably Aspergillus glaucus, a green species not
uncommon on canned fruits, jams, moldy grains, silage,
and in similar locations. It frequently produces abundant
yellow perithecia containing asci and ascospores. Asper-
gillus oryzae has been used in the manufacture of industrial
alcohol, for the conversion of starchy material into sugar
preliminary to the alcoholic fermentation, j

CLASSIFICATION OF THE MOLDS

63

The key given in the footnote will enable the student to
separate some of the more common species.1

FlG. 42. ASPERGILLUS OBYZAE.

i KEY TO ASPERGILLUS SPECIES
A. Spores white or nearly so.

B. With unbranched sterigmata Aspergillus candidus.

BB. With branched sterigmata A. albus.

AA. Spores colored.

B. Spores green, grayish, bluish or yellow-
ish-green,
r. With unbranched sterigmata.

D. Producing perithecia readily. ^

E. Perithecium naked, not embedded. A. glaucus.
EE. Perithecium embedded.

F. Tip of conidiophores only slightly
swollen, club-shaped* sterigmata
along sides for a considerable

distance A. clavatus.

FF. Tip of conidiophore hemi-
spherical, sterigmata produced

only from terminal portion A. fumigatus.

DD. Not producing perithecia (so far
as known).
E. Tip of conidiophore, very large,

••longates 80-100X500-800/A A. giganteus.

EE. Tip of conidiophore smaller,
spherical, or hemispherical.

F. Conidiophore rough, warty A. flaws.

FF. Conidiophore smoother A. oryzae.

CC. With branched sterigmata.

D. Mycelium rusty brown A. versicolor.

DD. Mycelium not so.

64

AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

ble Aspergilli.

Penicillium. — Certain species of this genus probably are
the most common of all molds. They are variously colored,
usually grayish-green or bluish-green. They occur upon
the surfaces of decaying vegetable matter such as silage,
oranges, lemons, bread, jams, jellies, etc. The spores are
relatively common in the air and are usually met with in
the laboratory. In many respects these organisms resem-
They differ primarily in the manner in
which the spores are produced. The erect
conidiophores branch at the tip, a group of
two, three, or four branches usually being
formed. These continue parallel to each
other and usually again branch. This may
occur a second or a third time. The
terminal branches pinch off chains of spores
which may reach a considerable length.
Microscopically the conidiophore with its
branches, tip and long chains of spores re-
sembles a brush, hence the name Penicillium
(a little brush).

Certain of the species of Penicillium are
of importance in the ripening of cheese^
particularly Roquefort, in which Penicil-
lium roquefortii is present, and Camem-
bert, in which Penicillium camembertii occurs. The char-

E. Tip of conidiophore club-shaped,
sterigmata both lateral and termi-
nal A. psreudoclavatus.

EE. Tip of conidiophore hemispheri-
cal, sterigmata terminal A. nidulans.

BB. Spores black or dark brown.

€. With unbranched sterigmata A. calyptratus.

CC. With branched sterigmata A. niger.

BBB. Spores, yellowish-brown, yellow, brown,
or reddish.

D. With unbranched sterigmata, coffee-
brown spores A. wentii.

DD. With branched sterigmata, yellow-
brown spores A. ochraceus.

FIG. 43. — PENI-
CILLIUM.

CLASSIFICATION OF THE MOLDS

65

acteristic flavor and consistency of these cheeses is due in
large measure to the growth of these Penicillia.

Some hundreds of species of Penicittium have been de-
scribed. They are comparatively difficult to identify
because of the variation in the appearance of the same mold

FIG. 44. — PENICILLIUM.
camembcrtii.

1. Penicillium roquefortii. 2. Penicillium

when grown under different conditions. A few species may
frequently be determined from the food or substratum on
which they are growing. The following key to such species
has been adapted from Thorn.1

i U. S. Dept. Agriculture, Bureau of Animal Industry, Bull. 118.

KEY TO SOME COMMON SPECIES OF PENICILLIUM
A. Growing on cheese.
B. Camembert or Brie.

C. Floccose colonies, white to gray-
green Penicillium camembertii.

CC. Powdery colonies, yellowish-
white P. brevicaule var. glabrum.

CCC. Forming yellow-brown areas,

spores rough P. breviccvule.

BB. Roquefort, forming green streaks

inside cheese P. roquefortii.

AA. Growing on citrus fruits (lemons,
oranges).

B. Mold colonies blue-green P. italicum.

BB. Mold colonies olive-green P. digitatum.

66

AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Trichothecium. — A common pink mold occurring particu-
larly on decaying fruit such as apples is Trichothecium
roseum. In some cases it may cause considerable damage
due to the development of rot. The spores are not un-
common and sometimes the mold makes its appearance
on laboratory media. The mold itself is white in color,
sends up numerous unbranched conidiophores which pro-
duce a cluster of two-celled pear-shaped spores at the tip.
The spores do not occur in chains. The terminal cell of the
spore is larger than the basal cell.

0

FIG. 45. — TRICHOTHECIUM.

Hormodendrum and Cladosporium. — These molds are
abundant upon decaying paper, straw, and similar mate-
rials. They usually produce irregular dark or sooty
patches of various sizes. The mycelium and spores are both
smoky or fuscous in color. The conidiophores are usually
well differentiated, branching more or less at the tip and
producing chains of spores which likewise branch. These
two molds belong to the group in which the terminal spore
is the youngest in the chain. The most common species is
the Cladosporium herbarum. The mycelium frequently
produces many cells of irregular shape and size. The coni-

AAA. Growing on pomaceoua fruits
(apples, pears). Blue-green colonies
forming coremia P. expansum.

CLASSIFICATION OF THE MOLDS

67

dia of Hormodendrum are one-celled, while those of Clados-
porium, at least in the old and mature colonies, are in part
two-celled.

Alternaria. — In this genus the spores are large and club-
shaped, occurring in chains, the youngest spore at the tip.

e

FIG. 46. — HORMODEXDBUM AT*D CLADOSPOEiuM. A. Hormodendrum.
B. Cladosporium.

The mycelium is usually also brownish or fuscou^ in color.
The spores are many-celled and muriform. The species
Alternaria tennis is abundant in moldy grains, in the soil,
and the cells are frequently found in laboratory air. *i

FIG. 47.— ALTERNABIA.

SECTION II
METHODS OF STUDY

CHAPTER VI

CULTURAL METHODS

WHENEVER practicable it is customary to study cultures
of bacteria when grown under laboratory conditions. Many
species of bacteria, as they occur in nature, are usually
found growing together, that is, in mixed cultures. They
are widely distributed, making it somewhat difficult to
maintain the bacteria in pure cultures after they are once
obtained. For an adequate laboratory study of cultural
characters it is necessary first to sterilize suitable contain-
ers, usually glassware, second to prepare a suitable medium
or nutrient mixture upon which the organisms will grow,
third to secure the organism to be studied fti pure culture,
that is, free from admixture with all other kinds of bacteria,
and fourth to study the characteristic growth and appear-
ance upon the culture media.

METHODS OF STERILIZATION

Sterilization may be defined as that process whereby
materials may be rendered entirely free from living micro-
organisms. Bacteria are so widely distributed in nature
that it is usually taken for granted that they are present in
or upon practically all the materials which are used in the
laboratory. Practically the only source of material natu-
rally sterile is from the normal tissues of animals or plants.
It is, for example, possible so to bleed an animal as to secure
sterile blood serum without any process of sterilization. In
most cases some artificial method of removing the bacteria
is used.

71

72 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Organisms may be removed or destroyed, that is. mate-
rials may be sterilized, either by physical or by chemical
means. The process of chemical sterilization will be dis-
cussed later under the heading of action of disinfectants
and antiseptics. The physical processes of sterilization are
the ones most frequently used in preparation of material
for the growth of microorganisms. The physical agencies
most important in sterilization are heat, light, and filtra-
tion.

Sterilization by Heat. — Bacteria, yeasts, and molds, like
all higher forms of life, are killed if they are subjected to a
temperature sufficiently high, and for a sufficient length of
time. To sterilize by heat implies subjecting the material
to be sterilized to such a temperature and for such a period
of time as will quite certainly destroy all microorganisms
which are present. The methods commonly used are ex-
posure to the direct flame, to hot air, to streaming steam,
and to steam under pressure.

Platinum needles and other small metallic (or occasion-
ally glass) objects used in the laboratory are usually steril-
ized by placing them directly in the Bunsen flame. A
needle, for example, may be quickly raised to a red or
white heat. Momentary exposure, under these conditions
will, of course, destroy all organisms which are present.
Incineration is also the method most commonly used in the
disposal of the bodies of animals that have died of infec-
tious diseases, and for the destruction of materials of little
value which have been contaminated with disease-producing
microorganisms.

Dry heat is commonly used for sterilization of the labora-
tory glassware such as test tubes, flasks, and pipettes. The
so-called hot air oven is ordinarily used for this purpose.
Materials to be sterilized are subjected to a temperature of
165° to 175° C. for an hour or more. The long exposure is
in part necessary to make sure that the heat has penetrated

CULTURAL METHODS 73

to all parts of the material to be sterilized. The cotton
which is used for closing the openings of bottles and flasks
is usually slightly browned by this exposure. Care should
be used that the temperature does not reach so high a point
that the cotton is charred.

Many objects which will not withstand heating at high
temperatures or in a dry atmosphere may be sterilized by
long continued or repeated boiling in water, or exposure to
streaming steam, that is, to steam at atmospheric pressure.
The latter is usually accomplished by the use of an instru-
ment termed an Arnold sterilizer which supplies a con-
tinuous flow of live steam to the box in which the materials
to be sterilized are placed. In such sterilization it is fre-
quently customary to heat for fifteen minutes on each of
two or three consecutive days. In other cases the material
is heated for a longer period at one time. •

Sterilization is most efficiently effected by the use of
steam under pressure. Certain types of media cannot be
sterilized in this fashion because they may be decomposed,
but most materials used in the laboratory in media, can
withstand a temperature of 110° to 122° C. secured by
steam pressure of eight to fifteen pounds per square inch.
The apparatus commonly used is some form of an auto-
clave. In the use of the autoclave care should be exercised
that all of the air has been driven out by the entering
steam before it is tightly closed, otherwise the temperature
will not be as high as would be assumed from the reading on
the pressure gauge of the instrument. Ten to twenty min-
utes exposure at fifteen pounds pressure will destroy any
living organism. Where large bulk of material is exposed
or where the material is of such a nature that penetration
of steam is slow a longer period of time must be used.

Sterilization by Light. — Sunlight destroys microorgan-
isms rapidly. The ultra-violet rays of the spectrum are
particularly potent. It is possible quite efficiently and sat-

74 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

isfactorily to destroy the bacteria present in transparent
fluids, such as water, by exposure to the light from some
sources having high ultra-violet and violet intensity. This
is most effectively accomplished by the use of the Cooper-
Hewitt mercury vapor lamp in a quartz tube. Such
apparatus has been used on a commercial scale for the
purification and sterilization of water.

Sterilization by Filtration. — Experience has shown that
the larger bacteria, at least, are held back when liquids
containing them are filtered through porcelain filters
(bougies, or candles). These filters in many degrees of
fineness (porosity) may be purchased upon the market in
many shapes and sizes. Filters are particularly adapted
to the sterilization of media which would be decomposed or
injured by heat. Many of the materials sterilized by filtra-
tion are more or less viscous, such as blood serum, and in
consequence do not pass through the filter readily. The
rapidity of filtration can be greatly increased by exhausting
the air from the flask into which the material is being
filtered, the air pressure forcing the material through the
pores of the filter. Filters have also been devised whereby
it is possible to apply pressure directly to the fluid, forcing
it through the pores of the filter with comparative rapidity.

CULTURE MEDIA

The material or substratum upon which an organism is
grown is called a medium or substrate. If the organism is
to be successfully cultivated it must contain all of the
materials essential to its growth. The various species of
bacteria, yeasts, molds, and protozoa show wide differences
in the nutrient materials which they require. Some forms
require inorganic material only; others require a mixture
of inorganic and organic substances; and some will grow
only in the presence of the most complex proteins or similar
materials, under conditions simulating those which exist in

CULTURAL METHODS 75

the animal body. It is apparent, therefore, that media of
many different kinds will be found useful in the laboratory.

Several steps are common to the preparation of all kinds
of media. First, there must be the right mixture of the
necessary nutrients. Second, these must be present in the
right concentration in water. Third, certain substances
may or may not be added to cause the medium to be solid or
liquid as required. Fourth, the reaction must be adjusted
when necessary. Fifth, the medium should be placed in
suitable containers. Sixth, it should be sterilized.

Nutrient Substances Used in Preparation of Media. — A
few microorganisms, some of them of considerable economic
importance, do not require any organic material for food,
that is, they are able by oxidation of certain inorganic sub-
stances to secure the energy necessary for them to build up
complex organic substances from inorganic. In preparing
media for such organisms, therefore, it is necessary to use
certain inorganic salts. In most instances some salt of nitro-
gen, either ammonia or nitrous acid, is necessary, likewise
some source from which the carbon can be secured, usually
a carbonate or carbon dioxide. Most organisms are stimu-
lated in growth by the presence of phosphate. As an exam-
ple of such a simple nutrient medium may be cited one
useful in cultivating from the soil the bacteria capable of
oxidizing ammonia to nitrous acid. Such a medium con-
tains ammonium sulphate, calcium or magnesium carbonate,
usually a little phosphate and traces of certain other salts,
but no organic matter.

Most of the common bacteria and those usually cultivated
in the laboratory require more or less organic material as
food. Some forms will utilize inorganic nitrogen providing
they have organic carbon compounds. They will, for exam-
ple, take nitrogen from ammonia or from nitrates providing
carbohydrates are furnished. Some may even utilize free
nitrogen from the air. Other bacteria require organic

76 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

nitrogen compounds in the form of amino acids, peptids,
peptones, and in a few instances apparently even more
complex organic compounds.

Media are sometimes divided into synthetic and nonsyn-
thetic. A synthetic medium is one in which the exact
chemical composition of each of the components is known.
A nonsynthetic medium is one in which the exact chemical
composition is not fully understood. Some kinds of bac-
teria can be studied best by the use of the simple synthetic
media, but suitable ingredients for such media are not
readily available for many other kinds of bacteria.

Synthetic media usually contain as a basis an aqueous
solution of certain salts, among them potassium phosphate
and sodium chloride. One of the most commonly used is
Uschinsky 's solution :

Water, distilled 1000 c.c.

Asparagin 4 grams

Ammonium lactate 6 grams

K2HPO4 2 grams

NaCl 5 grams

Media of this type have proved very useful in studying the
products of fermentation of microorganisms.

Were it possible to secure pure amino acids and peptids
in commercial quantities, suitable synthetic media for many
of the pathogenic bacteria could be devised which would be
of real value in their study and differentiation.

The basis for most laboratory media is nutrient broth or
bouillon. It contains either meat infusion or meat extract,
peptone, usually salt, and water. Meat infusion is pre-
pared by soaking 500 grams of lean minced beef in water
in the ice chest for twenty-four hours, filtering the liquid
through cheesecloth -by means of a meat press, and making
up the filtrate to one liter. Meat extract broth is prepared
by using (in place of the meat infusion) about three grams
of commercial beef extract to a liter of water.

Many makes of peptone are upon the market. Peptone

CULTURAL METHODS 77

is prepared by the action of the enzyme pepsin of the gastric
juice upon proteins. Peptones contain mixtures in varying
proportions of albumoses, peptids, and amino acids. Those
in which digestion is most complete, that is, those which
have the highest percentage of amino acid are frequently
most useful in stimulating bacterial growth. However,
they cannot be used for certain purposes. In the manu-
facture of diphtheria toxin, for example, apparently the
more complex peptones and albumoses are required. This
variation in composition of peptones from different manu-
facturers and from different lots should be borne in mind
in making comparisons in the growth of bacteria grown on
media containing these peptones.

Sugars and glycerin are frequently added to broth or
bouillon in studying the fermentative activities of many
species of bacteria, that is, in the study both of acid and
of gas production.

Dunham's solution or peptone water is a solution of pep-
tone in water without the addition of meat infusion or beef
extract. When the peptone used is of the right composi-
tion, that is, when it contains the amino acid tryptophane
it may be used for testing the ability of microorganisms to
produce the compound indol.

Milk serves as a satisfactory medium for the growth of
many species of bacteria. Usually the fresh separated milk
is used ; whole milk may be kept in an ice chest for twenty-
four hours and the milk pipetted from below the cream
layer.

Sterile defibrinated blood or blood serum alone, mixed
with sterile broth, are frequently used in media, particu-
larly when it is desired to grow certain of the pathogenic
or disease-producing bacteria. Inasmuch as it is not prac-
ticable to sterilize serum without coagulation after removal
from the body, precautions are taken to insure sterility and

78 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

to prevent contamination at the time the blood is drawn.
It is then allowed to stand until it clots when the clear
serum is pipetted off or it is shaken with glass beads or
whipped with a glass rod until defibrinated.

Beer-wort — is probably the most satisfactory medium
for cultivation of molds and yeasts. The unhopped wort
may be secured from breweries or it may be prepared by
soaking malt in warm water. The starch of the malt is
rapidly changed into the sugar maltose and certain of the
protein constituents likewise pass into solution.

Solid media — Solid media may be divided into three
types, media which are permanently firm or solid, media
which are at first liquid and later solidified and in which
the condition is irreversible, and media which may be liquid
or solid at will, that is, solid media which are liquefiable.

To the first group of natural solid media belong materials
such as potatoes and other vegetables and the meats. Pota-
toes when sliced and sterilized prove to be a suitable medium
for the growth of many kinds of bacteria.

Blood sera, or mixtures of blood serum with broth, may
be solidified by exposure to heat, and constitute satisfactory
media for the growth of many of the pathogenic bacteria.
Silicic acid in pure colloidal solutions may be caused to
form a gel by the addition of certain salts. This consti- -
tutes, when mixed with these salts, a satisfactory solid
medium for the growth of certain of the soil bacteria which
develop best in the absence of organic compounds.

The liquefiable solid media are prepared usually by the
addition either of gelatin or of agar-agar to a liquid
medium.

Gelatin is manufactured by boiling bones, joints, tendons,
and similar animal tissues, concentrating the extract, and
cooling and drying. When dissolved in water it has the
property of solidifying or gelatinizing when cool, and of
being liquid when heated. Chemically gelatin is closely

CULTURAL METHODS 79

related to proteins. Usually from ten to fifteen per cent
of gelatin is required in order to cause a medium to solidify
when cool. It is not adapted to the solidification of media
used for the growth of organisms requiring temperatures
higher than average room temperature. Bacteria, for ex-
ample, which grow only at the temperature of the body
cannot well be tested on gelatin as this medium would be
usually liquid at this temperature.

Agar-agar is a complex carbohydrate prepared from cer-
tain kinds of seaweed. Its solutions resemble those of
gelatin in their power of gelatinizing when cool. From
one to three per cent is ordinarily used in preparing solid
media. It may be noted that the melting temperature and
the temperature at which the agar medium solidifies are not
identical. A tube of bouillon agar, for example, must be
brought nearly to the boiling point before the agar liquefies,
but may be cooled to a temperature of forty-two to forty-
five degrees before it begins to solidify. The agar-agar is
not attacked by microorganisms generally, two or three
species only are known which can dissolve or digest it.
Gelatin, on the other hand, is readily attacked by many
species of bacteria, and one of the differential characters
noted is the ability of certain forms to digest or liquefy the
gelatin.

By the addition of agar or gelatin practically any of the
ordinary liquid media may be solidified.

Many other types of media and variations in the media
thus far mentioned will be noted later in the discussion of
the growth requirements of the specific kinds of bacteria.

ADJUSTMENT OF THE REACTION OP THE MEDIUM

The reaction of a medium, that is, its relative acidity or
alkalinity, may be designated in one of two ways: first, by
the amount of normal acid or normal alkali required to
bring one hundred cubic centimeters of the medium to the

80 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

neutral point of some particular indicator, or second, by
the designation of the true hydrogen ion concentration or,
conversely, of the true hydroxyl ion concentration of the
medium. While the first method is still the more commonly
used, the second method is in most instances preferable.

Bacteria, yeasts, and molds are usually quite sensitive to
the presence of an excess of acid or alkali. Some grow best
in a medium which is strictly neutral, others prefer one
which is somewhat on the acid side of neutrality, still others
on the alkaline side of neutrality. Careful adjustment of
reactions is, therefore, necessary in many cases. Some
organisms, for example, will not grow unless the medium
has almost exactly the same reaction as does the blood or
the tissues of the body in which they are accustomed to grow,

The chemist tells us that acidity is determined by the
presence of free hydrogen ions, alkalinity is determined by
the presence of free hydroxyl ions, and that a solution is
truly neutral when equal numbers of hydrogen and
hydroxyl ions are present in a given volume. Pure dis-
tilled water is neutral. This does not mean that it has no
free hydrogen ions and no free hydroxyl ions, but that when
molecules of water dissociate they break up into equal
numbers of each ion. The chemist has discovered, further-
more, that pure water (and therefore any neutral solution
of materials in water) contains approximately one ten-
millionth of a gram of hydrogen ions to the liter. This may
also be written 10~7 grams hydrogen ions per liter, or still
more conveniently expressed in terms of normality of hy-
drogen ions. A normal solution of hydrogen ions is one
which contains one gram of hydrogen ions per liter. A
neutral solution, therefore, is one which has a concentration
of hydrogen ions of 10~7 normal. It was noted above that
in a neutral solution there is the same number of hydroxyl
ions as hydrogen ions. The hydroxyl ion concentration
must, therefore, be also 10~7 normal.

CULTURAL METHODS 81

The physical chemist has also discovered that the product
of the normality of hydrogen ions by the normality of
hydroxyl ions is a constant number. What this number is
may be determined by multiplying 10~7 (normality of
hydrogen ions in a neutral solution) by 10~7 (normality of
hydroxyl ions in a neutral solution) giving 10~14. In other
words, the product of the normality of the hydrogen ion
concentration of a solution by the normality of the hydroxyl
ion concentration must always be approximately 10~14. If
we know either the hydroxyl or hydrogen ion concentration
we can at once determine the concentration of the other.
For example, 4f we are dealing with a solution having an
hydrogen ion concentration of 10~5 normal its hydroxyl
ion concentration must be 10"9 normal. It is thus possible
to arrange a scale to designate the acidity of any solution
in terms of its hydrogen ion concentration as follows :

100 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 10-9 10-10 10-11
10-1210-13 10-14.

On this scale it will be noted that the larger the numerical
value of the exponent, the smaller the hydrogen ion con-
centration. It will be recalled that 10~7 represents
neutrality. Numbers to the right of 10~7 represent increas-
ing values of alkalinity or decreasing hydrogen ion concen-
tration. Numbers toward the left represent increasing
acidity. Inasmuch as this method of statement is somewhat
cumbersome, it has been suggested by Sorenson that the ex-
ponents be used to indicate the scale, using positive signs
instead of negative. This gives the scale :

0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14.

Each of these numbers is termed the pH of a solution. A
solution having pH of 0, for example, would have a nor-
mality of hydrogen ion concentration of 10° normal or 1.
One having a pH value of 7 would have a hydrogen ion
concentration of 10~7 normal, that is, it would be neutral.
It is evident, therefore, that the smaller the number in

82 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

this scale, the higher the hydrogen ion concentration, that
is, the greater the acidity ; and the larger the number, the
greater the alkalinity.

Indicators are chemical substances which are of one color
in a certain range of pH values or hydrogen ion concen-
trations, and of another color in other ranges. The indi-
cator most commonly used is litmus, which has a lilac color
at true neutrality, that is, at a pH value of 7. At a pH
value of 8, that is, in a more alkaline solution, it is blue.
At a pH value of 5, that is, in a stronger concentration of
hydrogen ions, it is red. Other indicators change color at
other pH values. For example, phenolphthalein is color-
less in all hydrogen ion concentrations heaving a pH value
less than 8.2 ; in more alkaline solutions, it is red. Many
other indicators are known which change color at other
points in the hydrogen ion scale. This change of color is
not instantaneous. In adding alkali, for example, to an
acid solution containing litmus, there is not an instantane-
ous transformation of red into blue. By the use of stand-
ards whose hydrogen ion concentration is known it is possi-
ble to determine approximately the hydrogen ion concen-
tration of any material which it is desired to test, by the
use of the intensity of color of appropriate indicators.

Most bacteria grow in a hydrogen ion concentration of
10~7 to 10~8, that is, in solutions having a pH value between
7 and 8. The pH value of blood is usually about 7.35
This indicates, therefore, the hydrogen ion concentration
most useful in cultivating many species of pathogenic bac-
teria. Inasmuch as most media are on the acid side of true
neutrality it is necessary to add alkali, usually potassium
or sodium hydrate, until test shows that the hydrogen ion
concentration has become satisfactory.

The hydrogen ion concentration of medium or solution
depends not only upon the actual concentration and kind
of the acid but upon the concentration of substances which

CULTURAL METHODS 83

are termed buffers. A buffer is any substance in a solution
which tends to prevent rapid changes in hydrogen ion con-
centration upon the additions of alkalies or acids. Certain
salts, particularly the phosphates, and many organic sub-
stances, particularly the amino acids and peptones, act in
this manner. For example, the addition of a small amount
of an acid to distilled water will give a marked change in
the hydrogen ion concentration, but the same amount of
acid added to solutions containing considerable amounts of
buffers may result in very slight differences in hydrogen ion
concentration. It is evident that since microorganisms are
affected far more by differences in hydrogen ion concentra-
tion than they are by the total amount of acid present,
heavily buffered media are preferred for their growth.

It is noted above that media are sometimes standardized
by determining the amount of acid or alkali required to
bring a hundred cubic centimeters to the neutral point of
some indicator. The one usually chosen in bacteriology is
phenolphthalein. A solution is said to be — 1, for example,
when it will require one cubic centimeter of a normal solu-
tion cf acid to bring it to the neutral point of phenolph-
thalein. A heavily buffered medium, such as ordinary pep-
tone broth, having a reaction of +1 or +1.5, usually has a
pH value between 7 and 8. Direct determination of hydro-
gen ion concentration is preferable to titration in adjusting
the reaction of the medium.1

When a medium has been finally adjusted in its reaction,
that is, when the right amount of alkali or acid has been
added to give the desired hydrogen ion concentration, it is
frequently necessary to boil or heat the medium to precipi-
tate any materials not soluble in boiling water at the new
hydrogen ion concentration. This is followed by filtration.
If this step is omitted the medium will ordinarily be cloudy
or full of sediment.

i For methods of adjusting reactions see laboratory manual.

84 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

After being placed in suitable containers the medium is
then ready for sterilization. The method used will depend
upon the character of the medium. Most media are steril-
ized by heating in the autoclave at one atmosphere pressure
for from ten to fifteen minutes. Care must be used in the
sterilization of sugar media, however, because at certain
hydrogen ion concentrations they are rapidly decomposed,
the disaccharides in particular being hydrolyzed to mono-
saccharides. Some media are sterilized by exposure either
for one long period or for short periods on successive days,
to streaming steam. Still other media, particularly those
containing blood serum, are solidified by heating at a tem-
perature of from 75° to 80° C., and sterilized by exposure to
these temperatures on several successive days. In some
cases it is necessary to sterilize the ingredients of the
medium separately arid mix them in 'the right proportions
after sterilization.

PURE CULTURE METHODS

It has previously been noted that it is exceptional in
nature to find a single kind of microorganism growing by
itself, that is, free from all other kinds. This sometimes
occurs with disease-producing bacteria in the blood or tis-
sues, and under some exceptional conditions of environment
it may be possible for only one kind of germ to grow. It
is evident, therefore, that if bacteria are to be studied they
usually must first be separated from each other and grown
in pure cultures. A mixed or impure culture is one in
which several kinds of organisms are growing together.

It is possible sometimes to secure pure cultures by direct
transfer. For example, it is usually possible to get a pure
culture of a mold such as Mucor by touching a sterile needle
to the sporangia. A few spores will adhere and these may
be scattered over the surface of the culture medium upon
which the organism is to be grown. If the medium is suit-
able some at least of the young mold plants developing will

CULTURAL. METHODS 85

be free from contamination, that is, they will be pure. It
is much more difficult to secure pure cultures of bacteria
in this mechanical fashion. It may be accomplished, how-
ever, when the necessity arises, by the use of Barber's
capillary pipette. This is an instrument consisting of
an extremely slender capillary glass tube so arranged with
a thumb screw that water can be forced in and out. The
bacteria to be separated are placed in a drop of water
and examined under the microscope. By careful manipula-
tion the germ desired may be drawn into the capillary
pipette and blown out again into a drop of water free
from bacteria. Then it may be examined to be sure that
there are no other bacteria present, when it may be trans-
ferred to the medium upon which it is to be cultivated.
This method is cumbersome and the technic is difficult.
It has proved valuable for certain special studies but is
not in common use.

Perhaps the most common method of securing pure
cultures of bacteria is by plating. The mixture of bac-
teria to be separated is placed in liquid nutrient agar or
nutrient gelatin, that is, in one of the liquefiable culture
media. When shaken thoroughly so that the bacteria are
well distributed, this is poured into a sterile glass Petri
dish and covered with a sterile glass cover. The separated
bacteria are soon fixed in position by the hardening of
the culture medium. They can no longer move about, but
their growth is not interfered with. In consequence they
develop rapidly and in the course of a few hours to a few
days produce a mass of bacteria large enough so that
it can be easily seen with the naked eye. The mass of
organisms is termed a colony. If the number of bacteria in-
troduced into the original medium is not too large and
they are properly mixed through the medium the colonies
will be well separated from each other. Theoretically, at
least, each organism gives rise to a separate colony, prac-

86 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

tically, bacteria sometimes stick together, and a colony
will occasionally consist of more than one kind of organism.
In the majority of instances, however, each colony con-
sists of a pure culture.

A method closely related to the one just described for
purification of bacterial cultures is that of streaking. A
solid medium such as agar is prepared and the bacteria
to be separated smeared back and forth over its surface
in an effort to separate the cells from each other. When
successfully done the individual species may be separated
in colonies and pure cultures secured.

It is sometimes possible to use as a medium for plating
or for streaking some material which will not allow of the
growth of any organisms but the particular one desired.
Media of this type are termed differential media. For ex-
ample, ox bile is sometimes used to eliminate bacteria which
are not of intestinal origin. Some kinds of pathogenic
organisms may be secured in pure culture by the device
of animal inoculation. If, for example, it is desired to se-
cure a pure culture of the Mycobacterium tuberculosis, the
organism causing tuberculosis in cattle, from a sample of
milk, this may be accomplished most readily by inoculat-
ing some of the milk containing the organism into a suit-
able animal, particularly, perhaps best, into a guinea pig.
Within a few weeks the animal may be killed and the or-
ganism will be found growing in pure culture in the lymph
glands.

When once colonies consisting of one kind of organism
only have been secured, these may be transferred to other
tubes of media by means of the platinum loop or the
platinum wire. By successive transfers, it is possible to
keep most kinds of bacteria growing indefinitely in the
laboratory.

Before any important study of an organism is under-
taken it is customary to plate or in some other way deter-

CULTURAL METHODS 87

mine whether or not it is still pure. Inasmuch as micro-
organisms are common in the air there is always a chance
that some of the air forms may gain entrance when tubes
of media are opened. Many serious errors have been
made by students and investigators because they were
dealing not with pure cultures but with mixtures of va-
rious species.

STUDY OF GROWTH CHARACTERS

It is relatively difficult to tell many kinds of microorgan-
isms apart, consequently every observable point of differ-
ence should be noted. Wherever possible bacteria are dif-
ferentiated by observation of size, shape, motility, spore
production, and other morphologic characters. However,
these frequently fail, and species must be differentiated
from each other by their appearance when grown upon
culture media, and by differences in their behavior, that is,
in their physiologic characteristics.

For the purpose of arranging in logical order the most
important of the morphologic, cultural, and physiologic
characteristics of an organism, a committee of the Society
of American Bacteriologists has evolved a descriptive chart.
A- copy of this chart is included on pages 88 to 90. First
on the chart is the name of the the organism, the correct
genus and species to be given together with the authority,
when this can be determined. Next, it will be noted, a
statement is inserted concerning invigoration of culture.
Bacteria that have been grown for some time on unsuitable
laboratory media sometimes need rejuvenation, that is,
cultivation upon some unusually suitable medium for a
period in order that they may regain their vigor.

The cultures to be studied are usually slant or streak
cultures on solid media, stab cultures and plate cultures,
in gelatin or agar, or cultures in a liquid medium such as
broth or milk.

88

AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

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CULTURAL METHODS 91

On the second page of the descriptive chart will be
found the characteristics to be observed in the various
cultures, and some of the more common terms used to
designate these.

CHAPTER VII

METHODS OF STUDYING PHYSIOLOGIC CHARACTERS

MANY species of microorganisms are differentiated from
each other on the basis of differences in their physiologic
characters. For example, certain closely related disease-
producing bacteria are differentiated because of their abil-
ity to produce acid or gas from certain sugars. A study of
these characters teaches something of the parts which these
microorganisms play in nature in bringing about chemical
changes. Only a few of the most important of these physi-
ologic reactions and characters are here discussed, many
others will be noted in subsequent chapters. Those here
given are the ones most frequently used in the differentia-
tion of species.

A study of physiologic characters of bacteria is largely
a study of specific chemical changes brought about by
these microorganisms. The characters most frequently used
in the laboratory are changes in hydrogen ion concentration,
determination of acid production, alcohol production, oxy-
gen relationship, gas production, reduction changes, pro-
duction of indol, digestion of starch and other insoluble car-
bohydrates, digestion of proteins, nitrogen fixation, oxida-
tion of ammonia to nitrites, and oxidation of nitrites to
nitrates.

Determination of Changes in Hydrogen Ion Concentra-
tion.— Many bacteria when grown in nutrient solutions,
particularly those containing carbohydrates, bring about
marked changes in hydrogen ion concentration. The
amount of such change accomplished by a given organism
depends upon several factors, the most important being
the following: first, the amount of acid produced, second

92

METHODS OF STUDYING PH-YSIOljOGIC CHARACTERS 93

the kind of acid produced, third, the amount of buffer
present in the nutrient medium, fourth, the amount of
alkali (that is, the concentration of hydroxyl ions) pro-
duced at the same time. Alkali may be developed either
by the production of ammonia or by the transformation
of the salt of a strong acid to the salt of a relatively weak
or little dissociated acid. For example, certain bacteria
may transform sodium citrate into sodium carbonate, the
latter being decidedly alkaline in its reaction.

Two methods are in common use for detecting changes in
hydrogen ion concentration. The first is by a determina-
tion of the electric conductivity. The second is a colori-
metric method. For usual laboratory routine the colori-
metric method is the simpler and is the only one which will
be discussed. In this method it is customary to add a
suitable indicator either to the solution to be tested or
to a portion of this solution diluted somewhat with dis-
tilled water. The color secured is then compared with the
color produced by similar addition of indicator to standard
solutions whose hydrogen ion concentration is known. The
indicators most used in the bacteriology laboratory for this
purpose are those developed by Clark and Lubs. In Table
I the name of each of these indicators is given followed by
the color in its acid range, next the color in its alkaline
range, and finally the range of pH values through which it
changes color.

It is apparent that an approximate idea can be secured
of the change in hydrogen ion concentration by using dif-
ferent indicators and determining which gives an acid
color and which an alkaline color. For example, if it is
found that phenol red gives a yellow reaction the medium
is acid and must be below the pH value of 6.8. If methyl
red, on the other hand, gives an alkaline color it must have
a pH value above 6. The exact pH value can be deter-
mined then by the use of brom thymol blue, comparing the

94 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

COLOR CHANGES OF CLARK AND LUBS INDICATORS

Indicators

Full acid
color

Full alkaline
color

Sensitive range.
The indicator
changes from the
acid color to the
alkaline color be-
tween the follow-
ing PH values

Thymol blue
(Acid range)

Red

Yellow

1.2-3.8

Brom phenol
blue

Yellow

Blue

3.0-4.6

Methyl red

Red

Yellow

4.4-6.0

Brom cresol
purple

Yellow

Purple

5.2-6.8

Brom thymol
blue

Yellow

Blue

6.0-7.6

Phenol red

Yellow

Red

6.8-8.4

Cresol red

Yellow

Red

7.2-8.8

Thymol blue
(Alkaline
range )

Yellow

Blue

8.0-9.6

Phenolph-
thalein

Colorless

Red

8.0-9.8

Cresolph-
thalein

Colorless

Red

8.2-9.8

intensity of the color change from yellow to blue with stan-
dard solutions whose pH values are known.

In figure 48 is given diagrammatically a scale of pH
values from 1 to 14. This scale is designated to the left.
To the right are designated the pH values of certain solu-
tions and of media commonly used in the laboratory. The
heavy portions of the lines designating the indicators show
those particular ranges in which the color changes are most
rapid. It should be remembered that a decrease of 1 in
pH value multiplies the hydrogen ion concentration by
ten. A pH of 0 means normal concentration of hydrogen
ions, and a pH of 14 a normal solution of hydroxyl ions.

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96 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

For discussion of the methods of preparing standard
solutions and colors for accurate determinations for hy-
drogen ion concentrations a suitable laboratory manual
should be consulted.

Determination of Acid Production. — What has been
stated above concerning determination of hydrogen ion
concentration will indicate the method of determining
whether or not acid has actually been produced by an
organism. It should be noted, however, that the deter-
mination of hydrogen ion concentration is not a determina-
tion of the total amount of acid produced. This can be
determined only by a comparative titration. It is custo-
mary to titrate to a definite tint a sample of the sterile
medium retained as a check, using phenolphthalein as an
indicator. For this purpose it is usual to place 5 cc. of the
sterile material mixed with 45 cc. of distilled water in a
porcelain evaporating dish, add a drop or two of alcoholic
solution of phenolphthalein and add twentieth normal
(N/20) alkali until a definite pink color has been estab-
lished. This is kept as a standard, and the amount of
alkali required noted. A similar titration is made using
the medium in which the organism to be studied has been
grown. This is treated in exactly the same fashion and
the twentieth normal alkali added until the same tint
has been secured as with the check. The differences be-
tween the amounts of alkali required for bringing to the
same tint of red will vary directly with the amount of acid
produced. This- may be calculated in grams per liter if
the kind of acid formed is known.

This method of acid determination is not absolutely ac-
curate inasmuch as there may be alkalies developed simul-
talneously with formation of acid. There is no simple
method of correcting for this possible error.

In some cases it is desirable to determine the kind of
acid which has been produced. Simple chemical tests for

METHODS OF STUDYING PHYSIOLOGIC CHARACTERS 97

the various organic acids have not in most instances been
developed. Something of their nature, however, may be
determined by separating them into volatile and nonvola-
tile. If a solution containing an acid is acidified with
sulphuric acid and the materials distilled, certain acids,
particularly acetic, propionic and butyric will pass over,
while other acids, particularly lactic, will not. This makes
it possible by titration of the distillate to determine the
relative proportion of volatile and nonvolatile acid.

Determination of Alcohol Production. — Yeasts produce
considerable quantities of ethyl alcohol, and smaller amounts
are formed as a result of the growth of a few bacteria and
molds. Occasionally other alcohols may be developed such
as amyl, butyl and propyl. With yeasts alcoholic fermen-
tation is accompanied by the production of carbon dioxide.
The presence of ethyl alcohol may be detected by placing a
few cubic centimeters of the material to be tested in a
suitable container such as a test tube, and adding a small
crystal of iodine and several cubic centimeters of a strong
solution of sodium hydrate. If alcohol is present heat-
ing this material over a Bunsen flame will give a distinct
odor of iodoform. The amount of alcohol developed may
be determined by distillation and test of the specific grav-
ity of the distillate.

Determination of Aldehyde.— Certain microorganisms,
particularly when growing in carbohydrate solutions, pro-
duce aldehyde in sufficient quantities to give a distinctive
reaction. The detection of the presence of aldehyde is
best accomplished by the use of a fuchsin indicator. If a
solution of basic fuchsin is decolorized by the addition of
sodium sulphite (or better suphurous acid) until the color
has just disappeared or until the material is of a very
light pink color, and added to a solution in which aldehyde
has developed, the color of the fuchsin is restored. This
is the principle made use of in the so-called Endo medium.

98 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Certain bacteria when growing upon this medium form red
colonies because the fuchsin turns red as a result of the
development of aldehyde and acid. Other species of bac-
teria grown upon this medium do not change the color
at all.

Production of Acetyl Methyl Carbinol. — This compound
is produced by certain bacteria growing in the presence of
carbohydrates. It is recognized by the addition of strong
alkali such as sodium hydrate or potassium hydrate. When
allowed to stand for a few hours an eosin pink or red color
will develop, particularly near the surface, providing there
is some peptone present. This is frequently called the
Voges-Proskauer reaction, after the men who first noted
it.

Determination of Oxygen Relationships. — Bacteria are
frequently divided into two groups, those which require
free oxygen of the air for their development and those which
will grow without. The first are termed aerobes, the second
anaerobes. Those bacteria which can grow either in the
presence or the absence of free oxygen are termed faculta-
tive anaerobes, and those which will not grow in the pres-
ence of free oxygen are termed obligate anaerobes.

It is frequently necessary in the laboratory to determine
accurately the oxygen preferences of a microorganism.
The bacteria which will grow only upon the surface of a
medium, but never grow in the closed arm of a fermenta-
tion tube under any conditions, are the obligate aerobes.
For the most part these are organisms which are actual
oxidizers, changing carbonaceous materials to carbon di-
oxide and water, for example. The facultative anaerobes
are those which will grow in contact with air, but, at least
under certain conditions, will grow in the absence of free
atmospheric oxygen. Most of the facultative organisms
are obligate aerobes on certain media and facultative on
others. Many bacteria, for example, which usually require

METHODS OF STUDYING PHYSIOLOGIC CHARACTERS 99

atmospheric oxygen can grow in its absence providing ni-
trates are present to furnish an available supply of oxygen
though not in the free form. Other bacteria will grow in
the absence of oxygen providing they have suitable carbo-
hydrates. For example, the organism known as Bacterium
coli will grow only in the open arm of a fermentation tube
if sugar is absent, but in the presence of a suitable sugar
such as dextrose it grows both in the open and in the
closed arm.

The obligate anaerobes are those which will grow only
in the absence of free oxygen or are definitely injured by
its presence. It is evident that special cultural conditions
are necessary for their study.

A few bacteria have been termed mi^roaerophiles be-
cause they tend to grow in a definite concentration of oxy-
gen. When mixed with melted agar in a test tube and the
agar allowed to solidify, they will grow in a definite zone
some distance below the surface of the medium.

Determination of Gas Production. — Bacteria which
require free oxygen for their development frequently pro-
duce considerable quantities of carbon dioxide. Inasmuch
as they are growing in contact with air, however, this gas
diffuses into the air and does not collect as gas bubbles in
the medium. When it is desired to study gas production
by such bacteria it is necessary to grow them in a closed
vessel to which air enters through a small opening and
leaves through a tube which then passes through a suitable
absorbing agent, for example, strong sodium hydrate, which
will collect the carbon dioxide. The amount of gas may
be calculated by the increased weight of the sodium hy-
drate.

Most of the bacteria producing considerable amounts of
gas in laboratory media are anaerobes or facultative anae-
robes. The gases developed depend somewhat on the com-
position of the medium, and also upon the character of the

100 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

organism. Carbon dioxide, hydrogen, nitrogen, and meth-
ane are the most common gases formed.

As a test for the ability of an organism to produce gas
we may inoculate a liquid culture of agar or gelatin contain-
ing a suitable carbohydrate. The development of gas will
cause the formation of numerous bubbles. A qualitative
determination of the kinds of gas developed may be made
by use of a fermentation tube containing a suitable liquid
carbohydrate medium. The closed arm is entirely filled
with the medium. Organisms grown in the closed arm
will produce gas which will accumulate in the upper part
of this closed arm. The amount produced may be
readily estimated by noting the proportionate part of the
length of the tube displaced by gas. A qualitative test may
be made by filling the open arm with normal sodium hy-
drate, closing the open arm, shaking the gas into the open
arm, returning the gas to the closed arm, and removing the
cork. The liquid will rise to replace any carbon dioxide
which has been produced. Yeasts and some bacteria pro-
duce carbon dioxide only. With these the liquid will rise
practically to the top of the tube after the absorption of
the gas. Many bacteria produce both hydrogen and carbon
dioxide. The hydrogen may be measured after the absorp-
tion of the carbon dioxide by the alkali. When drawn into
the open arm, mixed with air, and brought near to a flame,
it will explode. A few microorganisms growing in the
presence of nitrates produce considerable amounts of free
nitrogen gas.

Methane is produced by a few bacteria, but not by organ-
isms most commonly grown in the laboratory.

Accurate studies of the comparative amounts of carbon
dioxide and hydrogen produced by bacteria have proved
useful in some cases in differentiating them. Such studies
can be made only by the use of an apparatus filled with
medium and containing no gas, sealed to prevent the escape

METHODS OF STUDYING PHYSIOLOGIC CH^IjACTERS 101

of gas until the material is subjected t

As the result of such a chemical test, the ratio of carbon

dioxide to hydrogen may be determined.

Determination of Reduction Changes. — It has already
been noted that certain facultative and other bacteria
growing in the absence of free oxygen, may take oxygen
from certain compounds, reducing nitrates to nitrites, sul-
phates to sulphides, and decolorizing certain pigments, such
as litmus or methylene blue. The facts are sometimes of
advantage in the differentation of bacteria.

Reduction of Nitrates to Nitrites — The transformation of
nitrates into nitrites may take place in a number of differ-
ent ways as the result of growth of microorganisms. The
test is, therefore, not particularly reliable and is not empha-
sized by bacteriologists as it was formerly. A peptone
broth containing usually two-tenths per cent of potassium
nitrate may be inoculated with the organism to be studied.
After standing for four days the nitrite test may be per-
formed by adding two cubic centimeters of each of the
following solutions:

(a) 5N Acetic acid, 1000 cc.

Sulphanilic acid 8 grams

(b) 5N Acetic acid 1000 cc.

Alpha amido napthalene 5 grams

If nitrite is present a reddish or rose color will develop. It
is always necessary to test an uninoculated tube at the same
time as a check to be sure that no nitrites were initially
present in the medium used.

Reduction of Sulphates to Sulphides — Some organisms
reduce sulphates to sulphides under anaerobic conditions.
Media may be prepared containing either lead acetate or
iron chloride. The production of sulphides from sulphates
will be evident by the blackening of the medium. It should
be recognized, however, that sulphides may also originate
during the decomposition of organic sulphur compounds.

102 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

In studying ,the reduction of sulphates to sulphides, there-
fore, a medium should be chosen which does not contain
any organic sulphur. Hydrogen sulphide may be recog-
nized by heating the medium and exposing lead acetate
paper to the vapor. The presence of hydrogen sulphide
will be shown by the darkening or blackening of this paper.

Reduction of Pigments — Certain pigments, such as litmus
and methylene blue, may be partially or completely decolor-
ized by the growth of organisms under anaerobic conditions.
This fact is made use of in litmus milk. Many bacteria will
completely reduce the litmus to its leuco base. The ability
of microorganisms in the presence of organic matter to
reduce methylene blue has been made the basis for a test
to determine the amount of putrescible organic matter
present in solution. Raw sewage, for example, if shaken
up with methylene blue and corked tightly in a bottle, »vill
soon become decolorized. The amount of pollution in the
sewage or amount of organic material present in the sewage
is a function of the length of time which it will take the
methylene to become decolorized under standard condi-
tions. The time which passes before the blue color com-
pletely disappears is usually regarded as inversely propor-
tional to the amount of organic matter present which may
be decomposed readily.

Determination of Indol. — The compound indol is pro-
duced by certain species of bacteria from the amino acid
tryptophane. This tryptophane in turn is a product of the
hydrolysis of certain proteins. Some organisms are capa-
ble of producing indol from proteins, that is, they are
capable of hydrolyzing the proteins, breaking them down
into their component amino acids, including tryptophane,
and then decomposing tryptophane with the development
of indol. Most of the indol-producing bacteria, however,
are incapable of attacking the proteins and must have
tryptophane present before they can produce indol. The

METHODS OF STUDYING PHYSIOLOGIC CHARACTERS 1Q3

various peptones on the market differ considerably in their
content in amino acid and tryptophane. For demonstra-
tion of this property a peptone relatively high in trypto-
phane should be chosen.

The development of indol from tryptophane is markedly
influenced by the presence of other substances. Most bac-
teria, for example, which can produce indol from trypto-
phane will not bring about this change in the presence of
carbohydrates.

Indol has a characteristic, disagreeable, fecal odor.
Many different chemical tests have been suggested for its
detection. The simplest of these is the nitroso-indol test,
which is carried out by adding a cubic centimeter of a
0.1 per cent solution of sodium nitrite together with a few
drops of strong acid such as sulphuric to the medium. The
nitrite is decomposed by the sulphuric acid, freeing nitrous
acid. This in turn combines with indol to form nitroso-
indol which has a bright red color. For other laboratory
tests for indol, and for methods of detection in very small
quantities the student should consult a laboratory manual.

Some bacteria are capable, of producing the closely
related compounds skatol and phenol from tryptophane.

Digestion of Starch and Other Insoluble Carbohydrates.
— Many bacteria when grown upon potato or in agar or
other media containing starch hydrolize the starch or fer-
ment it more or less completely. The ability to bring about
this change is termed the diastatic power of the microor-
ganism. It may be recognized by using an agar containing
enough starch to make it somewhat opaque. The organism
when grown upon this medium will produce a clear area
immediately about the colony. This may be emphasized by
the addition of a solution of iodine, when the undigested or
unfermented starch will turn blue, leaving a colorless zone
about the colony. Not all bacteria hydrolize the starch
with the production of sugars.

104 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Other insoluble carbohydrates may be substituted for
starch in culture media. Finely powdered cellulose or
hemicellulose, for example, may be used in agar to test the
ability of microorganisms to digest these substances.

Digestion of Proteins. — Many microorganisms have the
power of liquefying gelatin due to the production of an
enzyme called gelatinase. This is best observed in stab
cultures in test tubes. A few bacteria possess the power
of liquefying coagulated proteins, such as blood serum.
Some bacteria are able to digest casein in milk bringing
about a peptonization of this material. This latter phe-
nomenon may also be demonstrated by the use of a casein
agar, that is, nutrient agar to which casein has been added.
This medium is somewhat opaque in appearance. Those
organisms which digest the casein will clear the medium for
a certain distance around the individual colonies.

Fixation of Atmospheric Nitrogen. — Usually bacteria
which grow luxuriantly in a nutrient solution containing no
compounds of nitrogen are those which are capable of
securing their needed nitrogen from the atmosphere.
Quantitative tests for amount of nitrogen fixation can be
made only by chemical analyses of the culture medium
when the organism is well grown. In soil bacteriology it
is frequently customary to use the soil itself as a culture
medium, applying a suitable carbohydrate to supply growth
energy to the nitrogen fixing bacteria. Comparative analy-
ses of soils which have received a carbohydrate with others
kept under the same conditions but without this amend-
ment will indicate the amount of nitrogen fixed.

Oxidation of Ammonia and of Nitrites. — Certain soil
bacteria are capable of oxidizing ammonia to nitrites.
When they are grown in a suitable solution containing am-
monium sulphate without organic nitrogen, the develop-
ment of the nitrite may be observed by using the test above
indicated for the detection of nitrites.

METHODS OF STUDYING PHYSIOLOGIC CHARACTERS 1Q5

Other soil bacteria are capable of oxidizing nitrites to
nitrates. Observation of this change is frequently made
in soil cultures. Chemical analyses may be made at inter-
vals to show the development of this compound.

CHAPTER VIII

MICROSCOPIC METHODS

THE fact that bacteria, yeasts, and molds are so minute
necessitates the constant use of the microscope in the in-
vestigation of the size, shape, and arrangement of cells of
bacteria, the form of yeast cells, and the method of spore
production and general morphology of the molds. For

FIG. 49. — DIAGRAMMATIC REPRESENTATION OF THE OPTICS OF THE OIL
IMMERSION LENS.

many purposes the lower powers of the compound micro-
scope may be used, but in a critical study of the individual
bacterial cells it is customary to employ the highest power,
or the homogeneous oil immersion objective.

The Oil Immersion Objective. — Even a superficial exam-
ination of the various objectives used on compound micro-

106

MICROSCOPIC METHODS 107

scopes will show that in general the higher the power, that
is, the greater the magnification, the smaller is the opening
through which the light must pass at the lower end of the
objective. It is evident that with the higher powers the
amount of light which may enter the lens is considerably
reduced. It has been found in practice that the interposi-
tion of a drop of oil between the lens and the object to be
examined make a far more satisfactory definition, the object
being seen much more clearly. Inasmuch as oil must al-
ways be used with the highest power lens, and should never
be used with other lenses, a brief explanation of the reasons
for its use will be given.

The diagram in figure 49 should be consulted. Let C
represent the glass cover slip over the object to be examined,
and L the lens through which light must pass to the eye
of the observer. The diagram shows a drop of oil, H, lying
to the left of the line NB. This oil is usually oil of cedar
wood, having the same refractive index as the glass used
in the slide. By means of the mirror and the substage con-
denser the rays of light are focused upon the object which
is to be examined. These rays of light may be represented
by AB and A'B. Eays of light which are normal, that is,
perpendicular to the surface of C, such as the ray BN, will
pass straight through without refraction in glass, air, or
oil. A ray such as AB, however, upon entering the denser
medium, glass, will be refracted in the path BD. Here it
enters the air and is again refracted in a direction parallel
to the incident ray, that is, in the direction of DE. If,
however, a drop of oil replaces the air this second refrac-
tion would not occur. This is illustrated by following the
ray A'B which is refracted in the glass as BD'. Upon
entering the oil it continues in the same direction as the ray
BF. It is evident that all rays lying between A'B and BN
and N'N will enter the lens L when oil is present, but that
only a portion will enter if air is between the lens and the

108 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

cover glass. Evidently the light entering the lens is more
concentrated when oil is used. This results in a more
brilliantly illuminated field and more satisfactory observa-
tion.

Microscopic Observation of Colonies and Cultures. —
Bacteria, yeasts, or molds, growing on agar or gelatin plates
are advantageously observed under the low power of the
microscope, either by inverting the plate and examining the
colony through the medium, or by removing the cover and
observing directly. This is particularly advantageous in
studying molds. Frequently the arrangement of spores on
conidiophores and the manner in which conidiophores
branch can be better determined in this fashion than by
mounting material under a cover glass. Observation of
bacterial colonies under the lower power will frequently
show structural peculiarities which may be useful in dif-
ferentiating the related species from each other.

Unstained Preparations. — Microorganisms are not infre-
quently examined in the living condition by placing them in
a drop of suitable liquid such as physiological salt solu-
tion, water, or broth on a slide and covering them with a
cover glass. Such preparations are made for the purpose
of observing the appearance of the living organism, also to
determine motility, and in some cases to study method and
rate of growth. It is sometimes difficult to mount molds in
this manner without numerous air bubbles interfering with
satisfactory observation. This difficulty may be obviated
by a drop of alcohol followed by a drop of water.

Most microorganisms (bacteria in particular) are com-
paratively transparent and can be seen under the micro-
scope unstained only because their refractive index is dif-
ferent from that of water. Considerable care is, therefore,
frequently necessary in the adjustment of the light, by
shifting the mirror and the iris diaphragm of the substage,
or of the Abbe condenser.

MICROSCOPIC METHODS 109

Stained Preparations. — It is comparatively difficult to
observe unstained bacteria satisfactorily. The details of
the morphology of the molds and of the yeasts are also
better indicated in stained preparation. Some of the mor-
phologic characters cannot be seen at all except by the use
of an appropriate stain.

The stains commonly used in the bacteriological labora-
tory are certain of the so-called aniline dyes. These are
usually divided into two groups, the basic dyes, and the
acid dyes. In staining the cells of higher plants and ani-
mals it will usually be observed that the basic dyes stain
such objects as the chromatin material in the nucleus, while
the acid dyes more commonly stain the cytoplasm of the
cell. Most bacteria stain best by the use of the basic dyes.
Among these are gentian violet, methylene blue, and basic
fuchsin. For special purposes, and for use as contrast
stains, acid dyes are sometimes used, particularly Bismarck
brown, eosin, and safranine. These stains are usually em-
ployed in aqueous solutions. Methods of preparation of
the particular stains used may be found in laboratory
manuals.

Substances not in themselves true dyes are sometimes of
considerable significance in staining. When cells are
treated with certain compounds such as aniline oil, iodine,
or iron tannate, the ability of certain parts of the cell or
certain cell structures to take up stain may be greatly
increased. Substances 'thus used to increase the staining
power of an organism are called mordants. For example,
the flagella of bacteria are in general so slender and so
difficult to stain tha*, they are not to be seen in ordinary
preparations. However, when the bacteria are suitably
mordanted with iron tannate the flagella may then take up
the stain in sufficient amount as to make them readily
visible.

In the preparation of any stained mount the following

110 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

steps are usually necessary: spreading, drying, fixing,
staining, washing, and mounting. A small amount of the
organism to be studied, usually bacteria or yeasts, may be
mixed in a drop of water on a clean cover «glass and spread
over the surface in a uniform layer. The glass should be
sufficiently clean and free from grease or oil so that there
will be no tendency for the water to round up in the form
of a drop. If the organism to be studied has been growing
in a liquid medium, such as broth, the water may not be
necessary, but it may be spread directly in its nutrient
medium upon the cover glass. After spreading, the mate-
rial is allowed to dry in the air, or it may be gently heated,
using care that the organisms are not overheated. The
preparation is then fixed by passing it rapidly two or three
times through the flame of the Bunsen burner. This hard-
ens the protoplasm of the organisms and causes them to
stick firmly to the glass, so firmly in fact that they may
be washed vigorously without being loosened. A drop of
the stain to be used is then spread over the surface,
allowed to act for the desired length of time, then washed
off by means of the tap. The cover glass may then be
dried and inverted over a drop of water or a drop of
balsam on a glass slide and is then ready for microscopic
observation.

In many laboratories the cover glass is dispensed with,
the mount being made directly upon the glass slip or slide.
When dried after staining, the immersion oil is placed
directly upon the mount and examination made without
the interposition of the cover glass.

For staining particular parts of the cell, and for demon-
strating peculiarities of staining reactions, ^certain special
methods have been devised. Of these, four are of sufficient
importance to deserve comment in this connection. They
are the spore stain, the stain for acid fast bacteria, the Gram
stain, and the stain for flagella.

MICROSCOPIC METHODS 111

Spore Stain. — The spores of yeasts and of bacteria are
not readily penetrated by stains, but when they are stained
they are somewhat more resistent to decolorization than the
vegetative cells ; this,makes possible a staining method which
will show* spores of one color and vegetative cells of another.
The material to be examined is spread and fixed in the
manner already described. It is then flooded with some
powerful stain, usually carbol fuchsin. The mount is then
heated until the carbol fuchsin steams for a period of five
minutes. Care must be used to replace stain with fresh
as rapidly as it evaporates so that the mount does not be-
come dry over any part. The excess stain is washed off
with water, then with dilute (usually 5 per cent) acetic
acid, until the mount is light pink in color. Usually a few
seconds will be sufficient. It is then thoroughly washed in
water to remove all traces of acid. This procedure leaves
the spores red but the vegetative rods colorless. The latter
are stained by adding some Loeffler's methylene blue.
When this has acted for a few seconds the mount is
washed, dried, and examined. The spores will ordinarily
appear red and vegetative cells blue. With the yeasts the
spores will appear red and the asci blue.

Study of Acid Fast Bacteria.— A few species of bacteria,
some of them of considerable economic importance, such
as the organism causing tuberculosis, are said to be acid fast
or acid proof. Like bacterial spores they resist the pene-
tration of stains, but when once the stain has penetrated
they are not easily decolorized, in fact, they will withstand
the decolorizing action of strong acids or alcohol for a
considerable length of time. The method of procedure is
essentially the same as in staining spores, except that as a
decolorizing agent a strong solution of acid, usually sul-
phuric acid, is used. Other bacteria or materials present
may be counterstained by methylene blue. The acid fast
bacteria appear as red rods in a blue field.

112 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Gram's Stain. — Gram's staining method is one very fre-
quently used for the differentiation of bacteria. It was
originally used for the demonstration of bacteria in tis-
sues. Essentially it consists in staining bacteria with one
of the aniline dyes, usually gentian violet or thionin, add-
ing a solution of iodine as a mordant, then decolorizing
with alcohol. Bacteria may be divided into two groups,
those which are Gram-positive, that is, retain the color, and
those which are Gram-negative.

Staining* of Flagella. — It was noted above that the flagella
of bacteria are usually so slender and so difficult to stain
that they are not seen in the ordinary stained preparation.
Many methods of mordanting the flagella so that they will
stain more readily have been suggested and used in prac-
tice. One of the most common methods is to use iron tan-
nate as a mordant, followed by carbol fuchsin as a stain.
It is important in making such preparation that only young
actively motile cells be used. It is best not to employ
cultures more than twelve to eighteen hours old.

SECTION III
PHYSIOLOGY OF MICROORGANISMS

CHAPTER IX

EFFECT OF PHYSICAL ENVIRONMENT UPON
MICROORGANISMS

BACTERIA as well as all other microorganisms are con*
stantly being subjected to physical and chemical environ-
ment which bring about increases or decreases- in the rates
of growth, or increases and decreases in the rates of death,
and various alterations in morphology, physiology and cul-
tural characters. Before discussing these effects it is neces-
sary to note the laws which govern rates of bacterial
multiplication and rates of death.

RATES OF GROWTH AND DEATH

With large plants and animals it is comparatively easy
to determine rates of growth by weighing at suitable inter-
vals. With organisms like bacteria and yeasts, however, it
is usually best to count the number of living cells present
after varying lengths of time. It has already been noted
that bacteria multiply by fission, that is, each moth'er cell
divides into two daughter cells. The length of time which
elapses between consecutive cell divisions, that is, the length
of time that is required for a single cell to grow to its full
size, and divide to form two individuals, is called the gen-
eration time. It is apparent that the shorter the genera-
tion time the more rapidly are the bacteria multiplying.
We can, therefore, judge of the effect of various physical
or chemical influences upon the growth of an organism by
noting any variations in the length of the generation time
which are produced. It is usually not convenient actually
to watch the microorganisms under the microscope and to

115

116 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

determine by means of a stop watch the length of time
required for cell division or the length of the generation
time. Methods of counting, however, have been devised so
that we may know the number of living bacteria present in
a cubic centimeter of liquid at the beginning of any definite
period of time and the number at the end of that period.
From this it is possible to calculate the length of the gen-
eration time as follows : let n be the number of cell divisions,
that is, the number of generations which develop during a
given time, which we may represent by t. If we start with
one organism we shall have at the end of one generation
period two organisms, and at the end of the second genera-
tion period four organisms, and at the end of third period
eight organisms. It is apparent, therefore, that the number
of organisms originating from a single cell will be at the end
of the first period 21, at the end of the second 22, at the end
of third period 23, and at the end of the nih period 2n.
If instead of beginning with a single organism we start
with any number, which we may represent by B, the number
at the end of the nih generation period will be

B 2n

If we let the number of bacteria after time t be represented
by b we have the equation

b=B 2n

If this equation is solved for n it will be found that

_ log b— log B

~lo^~

It is evident that the number of generations which will
develop in time t will be equal to the total time divided by
the generation time, that is,

n=g

EFFECT OF PHYSICAL ENVIRONMENT 117

or conversely

g=L

By use of these formulae for determining the value of g in
any growing culture of microorganisms we may detect the
effect of changes in environment. Within certain limits,
increasing the temperature at which the culture is kept will
increase the rate of growth, that is, g will diminish, and
the smaller g is, the more favorable are the conditions.
Conversely, the larger g is, the more unfavorable are the
conditions.

When organisms are placed under sufficiently unfavor-
able conditions they cease to multiply and begin to die in-
stead. In most of the cases which have been carefully
studied the bacteria die off in accordance with a definite
law, which may be stated as follows : with a given kind of
organism under uniform conditions, the number of bacteria
present in a culture will always be reduced by one-half in
equal periods of time, that is, no matter how many bacteria
there are at the beginning of a definite period of time, one-
half that number will always be alive at the end of the
proper interval. For example, suppose that two cultures,
one containing a million bacteria and the other a thousand
bacteria are subjected to* the same unfavorable conditions.
It is found that at the end of a definite period of time, say
ten minutes, the bacteria in the less concentrated suspension
average five hundred. It will be found that in the same
perod of time the other culture has also been halved, that is,
there are 500,000 bacteria left. Another way of stating is
this : during each equal interval of time a definite percent-
age of those bacteria living at the beginning of the period
will be killed. If we wish to compare unfavorable condi-
tions in their effect upon the death of microorganisms we
may compare the length of time required to reduce the

118 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

numbers of bacteria by a definite percentage, say one half.
If at one temperature, for example, half of the bacteria are
killed in ten minutes, and at another temperature one half
are killed in five minutes, it is evident that the second
temperature is far more destructive than the first. It will
be noted that the time required to kill half the bacteria
is mathematically the converse of the generation time.

In summary, it may be emphasized that all effects of
environment upon microorganisms may be manifested in
growing cultures by changes in the length of the generation
time, changes in morphology, and in the physiologic and
cultural reactions. Likewise the effect of environment upon
the rate of death of bacteria may be noted by comparing
the length of time necessary to kill a definite percentage of
the microorganisms present.

In the present chapter the effect of the physical environ-
ment will be discussed. The most important of the physical
agencies are light, heat, electricity, the radiations from
Kontgen tubes and from radium, pressure, osmotic pressure
and desiccation.

EFFECT OF LIGHT

Light affects organisms in several ways. Certain species
of bacteria, particularly those which contain a purple color-
ing matter known as bacteriopurpurin, are attracted by
light ; when motile they swim in the direction of the light
source. When growing, for example, in a suitable medium
illuminated from one side, they will move toward the
brighter side and collect on the surface of the glass. The
effect of any agency upon the direction of movement of an
organism is termed a taxy. The power of light to attract
is termed phototaxis. Most of the bacteria, however, do
not grow well in the presence of light ; in fact, light exerts
a destructive influence.

Light in general interferes with the growth of micro-

EFFECT OF PHYSICAL ENVIRONMENT 119

organisms, and if too intense stops their growth and accel-
erates the death rate.

Light sometimes affects the direction of growth of micro-
organisms, particularly molds. The fertile hyphae, that is,
the conidiophores or sporangiophores of certain species of
molds tend to bend in the direction of light. An effect of
any external agency upon the direction of growth is called
a tropism; this effect produced by light is therefore termed
phototropism.

Living protoplasm in general is more or less injuriously
affected by light. Too intense light applied to the human
skin, for example, will cause sunburn. Green plants accus-
tomed to growing in the shade will likewise be sunburned
when exposed to direct sunlight. Certain light rays have
the power of causing coagulation of proteins. | It is not
surprising, therefore, that microorganisms wTSch do not
require light for their development are particularly sensi-
tive to it, and may be rapidly destroyed by certain rays.

It will be recalled that sunlight, that is, white light, may
be divided into various rays of different wave lengths. At
one end of the visible spectrum are the reds, at the other
end the blue and the violet. Beyond the violet with wave
lengths still shorter is the so-called ultraviolet. ; In general
the rays of light in the blue and violet and in the ultra-
violet are particularly destructive to bacteria. It may be
observed that these are the same rays which also affect most
intensely the photographic plate.

Commercial ;use of ultra-violet light in sterilization has
been attempted. The most satisfactory source of such light
is the mercury vapor arc lamp, or so-called Cooper-Hewitt
lamp. This is a quartz, not glass (glass is opaque to ultra-
violet light) cylinder from which most of the air has been
exhausted and which contains a small amount of mercury.
This mercury is vaporized and an arc passes from one end
to the other, causing the entire tube to glow. The light

120 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

given off is largely blue, violet and ultra-violet. Relatively
short exposures of living cells to this light will kill them.

EFFECT OF HEAT

^ For every organism there exists a group of temperature
relationships. The lowest temperature at which any appre-
ciable growth will develop is called an organism 's minimum
temperature. That temperature at which it grows most
rapidly is its optimum. The highest temperature to which
it will grow is its maximum. The number of degrees dif-
ference between the maximum and minimum growth tem-
peratures is termed the growth temperature range. An
additional temperature relationship has been termed the
thermal death point.

The Minimum Temperature. — As the medium in which
an organism is growing is cooled, in general the generation
time increases until it reaches a point where there is prac-
tically no multiplication ; below this temperature there will
be no growth and the bacteria will die off more or less
rapidly, depending upon circumstances. If other condi-
tions are constant, the colder the microorganisms, the less
slowly do they die. Dried bacteria, for example, may be
exposed to the temperature of liquid air without being
killed.

The minimum growth temperature varies greatly with
different bacteria. Some microoganisms can multiply at
temperatures below 0° C. Such multiplication, for exam-
ple, has been observed in butter and in brine in cold storage.

Optimum Growth Temperature. — As the temperature of
a culture is increased from the minimum the generation
time of the microorganisms will decrease in length, that is,
the bacteria will multiply more and more rapidly. This
continues with increase in temperature to a certain point ;
heating beyond this will then again increase the generation
time, that is, slow down the growth rate. That tempera-

EFFECT OF PHYSICAL ENVIRONMENT 121

ture at which the generation time is the least or that
temperature at which the organisms are multiplying most
rapidly is the optimum.

Bacteria are sometimes divided into several groups using
the optimum temperature as the basis for classification.
Those bacteria which grow best at relatively low tempera-
tures, such as the temperature of the ice box, are termed
psychrophilic. Such bacteria are found in cold water, or
in lakes, the arctic regions, etc. Not many of them are of
economic importance. Those bacteria which prefer to grow
at moderate temperatures, such as room temperature or
blood temperature, are termed mesophilic. The mesophilic
bacteria may be again subdivided into those which grow
best at temperatures of 20-25°, that is, at ordinary room
temperature, and those which grow best at blood heat at
37.5°. Among the organisms belonging to the latter group
are those which are the disease producers in man and ani-
mals. A few bacteria are known which have optimum tem-
peratures decidedly above blood heat, that is, above 37.5°
Some species of bacteria will grow only at temperatures of
45° to 60°. They are termed thermophilic. Such organ-
isms are not uncommon in the soil, and are of significance in
food canning.

The Maximum Temperature. — As the temperature is in-
creased above the optimum, the generation time of bacteria
increases, that is, the rate of growth decreases, until at last
it comes practically to a standstill. The highest tempera-
ture at which there is appreciable growth is termed the
maximum. Here again much variation may be found
among microorganisms. Certain of the psychrophilic forms
will not grow at temperatures above that of the room.
Many of the pathogenic bacteria have maximum tempera-
tures between 40° and 45° C. Certain of the thermophilic
bacteria will continue to grow at comparatively high tem-
peratures, even 65° to 70°, that is, they will grow at a

122 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

temperature which will cause slow coagulation or cooking
of egg white.

Growth Temperature Range. — Some bacteria have a very
wide growth temperature range ; for example, the Bacterium
coli will grow slowly at both 12° and 45° C., giving a growth
temperature range of at least 33°. Other bacteria, such as
some of the pathogenic forms, may have maximum temper-
ature at 40° and minimum temperature at 35°, giving a
growth temperature range of only 5°. It is apparent that
for such bacteria the temperature at which they are grown
must be carefully controlled if successful results are to be
secured.

Thermal Death Point. — While the term thermal death
point is commonly used in literature, it is not entirely satis-
factory to express the particular idea. A thermal death
point is sometimes defined as that temperature which in a
given length of time will kill a particular organism.; We
have already seen that all the organisms in a culture do not
die instantaneously upon being subjected to unfavorable
conditions, but that under a definite set of conditions, there
will be a definite rate of death. Theoretically it would be
better to designate the rate of death under certain standard
conditions at a definite temperature. The rate at which
bacteria die off at a given temperature is influenced by
several factors. Bacteria which produce spores are fre-
quently said to have two thermal death points, one for the
vegetative rod and another for the spores. The latter are
much more difficult to kill, that is, they die off less rapidly
under unfavorable conditions than do the vegetative cells.
The hydrogen ion concentration of the medium also is
important. The higher the hydrogen ion concentration or
the higher the hydroxyl ion concentration, the more rapidly
will bacteria, die off at a given temperature. The presence
or absence of moisture" may also be important. Bacteria
that are not killed by drying are much more resistant in the

EFFECT OF PHYSICAL ENVIRONMENT 123

dried condition than the same organisms when moist. It
has already been noted that steam is more effective in
sterilization than is dry heat. If the thermal death point
is to be determined it should be defined as that temperature
which in a given period of time will destroy all of a definite
number of bacteria, noting particularly the composition of
the medium and its hydrogen ion concentration. Another
method of determination is to give under definite conditions
the length of time required at ar definite temperature to
reduce the number of bacteria by one half.

EFFECT OF ELECTRICITY

Bacteria are usually little affected directly by the pas-
sage of electricity, (However, the passage of the electric
current may cause electrolysis and the consequent forma-
tion of substances which act as chemical disinfectants. '
When a current of sufficient strength is passed through a
solution containing sodium chloride, for example, chlorine
gas appears at one electrode and alkali at the other. The
free chlorine and hypochlorites which are formed act as
powerful disinfectants. This method has been utilized to
some extent in the sterilization of sewage and water.

EFFECT OF X-RAYS AND RADIUM RAYS

The rays given off by the Rontgen tube and by radium
have been studied in their bactericidal effect, but they
have not proved to be sufficiently powerful to be of eco-
nomic importance.

EFFECT OF PRESSURE

Bacteria are in general resistant to relatively high pres-
sures. In one series of experiments a pressure of three
thousand atmospheres (an atmosphere is about 15 pounds
per square inch) was shown not to kill the Bacterium
typhi, Bacterium coli, or many other species of bacteria. A

124 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

pressure of six thousand atmospheres continued for four-
teen hours was found to destroy all non-spore-forming
organisms. The spores of bacteria are not uniformly and
regularly killed even at pressures of twelve thousand atmos-
pheres. The pressures required to kill bacteria are so
enormous that they have not been found practicable in
the sterilization of food materials. In general bacteria that
are killed by high pressure are found to be difficult to
stain, only shadows being discernible. Gram-positive bac-
teria in general lose their specific staining reaction.

EFFECT OF OSMOTIC PRESSURE

| It will be recalled that the protoplasm of all cells is
bounded by a semipermeable membrane termed the ecto-
plast. Some substances in solution can pass through this,
others cannot. Water usually penetrates it readily.
Whenever the concentration of solutes on the interior is such
that osmotic pressure is greater on the inside of the cell
than on the outside, water passes in and the cell becomes
turgid. This is the normal condition of cells. When a
cell is plunged into a concentrated solution of some sub-
stance which will not pass through ectoplast, the water
passes from the inside of the cell to the exterior, the pro-
toplasm tends to shrink, and the cell is said to have under-
gone plasmolysis. On the other hand, when an organism
has become accustomed to growing in concentrated solutions,
such as syrups, and is then dropped into distilled water
the pressure on the interior of the cell is much greater than
on the exterior. The cell will then tend to swell and per-
haps burst. This condition is termed plasmoptysis. This
latter phenomenon may be readily observed by placing a
drop of distilled water over the tips of the hyphae at the
margin of a mold colony growing upon beer-wort agar.
The distilled water suddenly diminishes this concentration
locally, the pressure on the interior of the cell being such

EFFECT OF PHYSICAL ENVIRONMENT 125

that the tip is forced open and the protoplasm is
squeezed out much as tooth paste may be forced out from
a collapsible tube.

By suitably increasing the concentration of solutes the
point may be reached beyond which an organism cannot
adapt itself. Such a concentrated solution then acts as a
preservative. This is the important factor in preserving
such foods as syrups, jellies, and jams.

Solutions which have equal osmotic pressures are said
to be isotonic. Solutions are isotonic for a cell when they
will cause neither shrinking nor swelling of the cell con-
tents. The most common of the isotonic solutions used in
the laboratory is the so-called physiological salt solution,
containing usually 0.85 per cent sodium chloride. This
concentration of sodium chloride is such that it is isotonic
with the red blood cells of the blood. It is also isotonic
with the blood serum, consequently red blood corpuscles
dropped into such solution neither shrink nor swell.

EFFECT OF DESICCATION

Most yeasts and bacteria grow best in the presence of an
abundance of moisture. Complete desiccation, that is,
drying, will cause many kinds of bacteria to die off rela-
tively rapidly. Other species, particularly the spore
formers, are more resistant to the drying and they die off
much more slowly. Some bacteria that do not produce
spores and many of the yeasts are also relatively resistant
to drying.

The reasons why drying should be so destructive to cer-
tain kinds of bacteria are not easily determined. Undoubt-
edly in some cases the death of the cell is due to the
unusual concentration of solutes about the organisms when
it is drying, and to the consequent increase in the osmotic
pressure. It has been found that certain microorganisms
if frozen in a vacuum and dried quickly, are not killed.

126 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Evidently the freezing prevents the concentration of the
solute with increased osmotic pressure. Drying, of course,
will prevent any growth of microorganisms, so that this
method is used extensively in the preservation of foods.

CHAPTER X

PHYSICAL EFFECTS PRODUCED BY MICROORGANISMS

PRACTICALLY all of the chemical changes brought about
by microorganisms result in physical changes becoming
apparent in the medium in which the organisms have
been growing. There are four of these changes, however,
distinctly physical in nature that are worthy of notice.
These are: the production of light, the development of
heat, changes in viscosity of medium, and changes in
osmotic pressure.

LIGHT PRODUCTION BY MICROORGANISMS

The property of producing light, that is, of being pho-
togenic or phosphorescent, is one which is common to plants
and animals of a great many different types. A number
of species of bacteria have been described as photogenic.
Practically all of them have been found in sea water or
upon food materials, such as salt fish, taken from the sea.
These bacteria may be grown readily upon suitable culture
media, particularly media containing proper amounts of
salts. When grown upon an agar slant, for example, dis-
tinct light may be observed when the culture is examined in
a dark room. Sufficient light may be given off so that the
organisms may be photographed by their own light. The
bacteria which are found to be phosphorescent or photo-
genic are all .ae'robes, growing well only in the presence of
oxygen and not producing light in its absence. The phe-
nomenon of photogenesis in bacteria has not been adequately
studied, but there is no reason to suppose that it differs
materially from the same phenomenon observed in higher

127

128 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

plants and animals. In these forms it has been found
possible to extract by suitable methods from the phos-
phorescent organs, two substances which when mixed will
cause a solution to glow. The light developed is rather
yellowish-green in color. When these organisms occur in
great numbers, as they may occasionally in sea water, they
may be the cause of diffused phosphorescence. They
sometimes produce luminescence on decaying fish that have
been thrown up by the waves on the seashore.

PRODUCTION OF HEAT

Microorganisms in general secure their energy for de-
velopment and growth by oxidative processes. In most
instances some of the energy secured is released in the form
of heat. Such heat production may be studied by use of
Dewar flasks, which are especially designed to conserve
heat and prevent its radiation. By this use it is usually
possible to measure the amount of heat developed by vari-
ous fermenting materials, such as by yeast in cane sugar,
or by bacteria in the souring of milk. The latter phenome-
non has been studied by Hill (1911). This author found
that in a little less than twenty-four hours the bacteria
produced approximately one and three-tenths calories of
heat per gram of milk, during the process of souring.

Bacteria in nature sometimes produce appreciable in-
creases in temperature where there is opportunity for
oxidation, and the heat is not rapidly radiated. Loosely
piled manure, for example, or damp straw will heat, the
temperature rising in some cases to 60° or 70° C. The fact
that heat is gradually given off during the process of
decomposition is made use of by the market gardeners in
the preparation of hot beds. The amount of heat given
off by the manure used is sufficient to raise appreciably the
temperature under the glass frame. Loosely packed ensi-
lage may also show some rise in temperature, and frequently

PRODUCED BY MICROORGANISMS 129

there is a very marked increase within a few feet of the
top of the silo where air may have access to the material.
The heat of oxidation is also made use of in certain methods
of curing hay in which it is allowed to heat in piles, and is
then spread and dried rapidly. It is possible by this means
to secure a palatable product in countries where the cli-
mate is too moist to dry the hay in the ordinary manner.

CHANGES IN VISCOSITY

Some organisms may markedly increase, others decrease
the viscosity of solutions in which they are growing, as will
be discussed in greater detail in a later chapter. Many
species of bacteria are known to produce gums. The accu-
mulations of these gummy or mucilaginous materials in
the solution in which the organism is growing will increase
materially its viscosity. For example, certain bacteria
growing in milk may cause it to become ropy or stringy.
It is possible that in some cases the increase in viscosity is
due to partial digestion or hydrolysis of protein material.
Under certain conditions bread may become viscous or ropy
as the result of the growth of certain putrefactive bacteria.
Apparently the most marked change in this case is the
transformation of the gluten.

Conversely some bacteria are markedly capable of reduc-
ing the viscosity of the medium in which they are grown.
Certain bacteria digest gelatin and thus decrease its vis-
cosity. Other organisms are able to soften starch paste, or
to liquefy coagulated blood serum; a few species can even
digest agar-agar.

EFFECT ON OSMOTIC PRESSURE

The osmotic pressure of any solution varies directly with
the total number of ions and molecules dissolved. Conse-
quently when complex molecules are broken down into
simple molecules there is a resultant increase in the osmotic

130 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

pressure, for example, when proteins are broken 'down
through peptones to amico acids, there is marked increase
in osmotic pressure. When starch, which has almost no
osmotic pressure, is broken down into dextrins, maltose, and
finally to dextrose there is relatively an enormous increase
in osmotic pressure. Bacteria when growing in the tissues
in the body may bring about changes which increase the
osmotic pressure and tend in consequence to abstract water
from the tissues.

Conversely, when simple molecules are combined with the
more complex there is a resultant decrease in the osmotic
pressure. Certain bacteria polymerize sugars into poly-
saccharides, the latter having practically no osmotic pres-
sure.

CHAPTER XI

EFFECT OF CHEMICAL ENVIEONMENT UPON
MICROORGANISMS

THERE are four distinct and relatively important
methods by which the chemical environment may influ-
ence microorganisms: motile bacteria may be influenced
in their direction of movement, molds and filamentous
bacteria may be influenced in their direction of growth,
all types of microorganisms may be influenced as to their
rapidity of growth, and as to their rate of death under
unfavorable conditions.

EFFECT OF CHEMICALS ON DIRECTION OF MOVEMENT

It has previously been stated (Chapter IX) that the
effect of any external agency upon the direction of move-
ment of an organism is termed a taxy. When the agent
influencing such direction of movement is a chemical, the
phenomenon is termed chemotaxis.

Chemotaxis may be most readily demonstrated by
.immersing the tip of a fine capillary glass tube partly
filled with a solution of beef extract or peptone, in a drop
of water containing numerous motile bacteria. Within
a short time most of the bacteria in the drop will be
found moving about the tip of the tube, and many will
have entered. The chemical in solution has been passing,
by diffusion, out through the water. The bacteria appar-
ently tend to swim in the direction of the greater con-
centration. Such a phenomenon of attraction may be
termed positive chemotaxis. Some chemicals may exert the
reverse influence, that is negative chemotaxis. In general

131

132 AGEICULTUKAL AND INDUSTRIAL BACTERIOLOGY

bacteria are repulsed by solutions containing alcohol or
strong acids.

The white blood corpuscles of the blood are attracted
under certain conditions to the invading bacteria. The
cells have some power of independent motion. This posi-
tive chemotaxis toward bacteria is one of the most im-
portant items in the list of body defenses against disease.

Some bacteria are markedly attracted by oxygen, a phe-
nomenon termed aerotaxis. If a drop of sewage swarming
with bacteria be mounted under a cover glass on a glass
slide, using care to include a few small air bubbles,
within a short period of time the microorganisms, both
bacteria and protozoa, will be found to arrange them-
selves in quite regular concentric lines about these
bubbles. Apparently each motile species influenced by
oxygen concentration seeks to remain within that zone
where the oxygen concentration is most favorable. This fact
has been put to use by bacteriologists in isolating certain
species of bacteria. For example, the organism which causes
Asiatic cholera may be detected in f eces by adding a small
amount of the stool to broth. The many kinds of bac-
teria present will multiply but the cholera vibrio is
attracted most by oxygen and will be present, therefore,
in relatively much larger numbers in the surface layer.
An examination of a drop from the surface will show the
vibrios to be numerous if the test is positive.

EFFECT OF CHEMICALS ON DIRECTION OF GROWTH

The effect of an external agency upon the direction of
growth has already been defined as a tropism. The effect of
a chemical is correspondingly termed a chemotropism.
That type of chemotropism most frequently observed is the
attraction or repulsion exerted by water, termed liydrotrop-
ism. Most species of mold show this to a marked degree.
Those hyphas which are differentiated for spore production,

EFFECT OF CHEMICAL ENVIRONMENT 133

that is, the fertile hyphae, show in general a negative hydro-
tropism. This is the reason why the conidiophores and spor-
angiophores of the various molds generally rise at right
angles to the surface of the medium upon which the organ-
ism is growing. As a result the spores are developed in an
environment relatively dry, and the conditions are, there-
fore, much more favorable for their distribution by cur-
rents of air. It will be noted that hydrotropism is much
more important in controlling the direction of growth
of molds than it is in higher plants. For the latter,
gravity is frequently the agency most active in deter-
mining growth direction.

EFFECT OF CHEMICALS ON RAPIDITY OF GROWTH

Various chemicals exert a marked influence upon the
rapidity of growth of microorganisms, that is, upon the
length of the average generation time. Among the more
important of these may be mentioned the concentration
of hydrogen ions, the concentration of oxygen, the con-
centration of nutrient substances, the kinds of nutrient
substances present, the stimulating effect of dilute
poisons, and the effect of substances of autogenic origin
not belonging to any of the preceding groups.

Effect of Hydrogen Ion Concentration on Rapidity of
Growth. — For every organism there exists that concentra-
tion of hydrogen ions in the medium which is most
favorable for development, that is, the optimum; likewise
a maximum concentration beyond which it cannot grow;
and a minimum concentration, that is, a concentration of
hydroxyl ions likewise in which it cannot develop. The
growth limits vary widely with 'different species. Most
species of bacteria prefer to grow in hydrogen ion concen-
trations represented by the PH values 7 to 8. Yeasts and
certain species of molds prefer solutions which are some-
what more acid. It has been found by laboratory tests that

134 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

moderate changes in hydrogen ion concentration do not
markedly change the rate at which bacteria grow except
near the limits. Practically the optimum is a relatively
wide range. It has previously been emphasized that in the
preparation of certain types of media great care is neces-
sary to insure the correct hydrogen ion concentration;
otherwise growth will not occur.

Effect of Oxygen upon Rapidity of Growth. — Appar-
ently for each kind of organism under a definite set of
conditions or in a particular culture medium, there exists a
certain concentration of oxygen which is most favorable to
growth. This may be termed the optimum. For most bac-
teria there likewise exists a point in diminished oxygen
pressure where the concentration is not sufficient to allow
of growth. This may be termed the minimum. For certain
bacteria there likewise exists a maximum concentration, and
the organisms will not grow in a medium containing more
than this amount. Bacteria which require a relatively high
concentration of oxygen, usually about that of the atmos-
phere, are said to be aerobic. For such bacteria the opti-
mum and the minimum are particularly significant. These
bacteria will grow usually only on the surface of media or
in the surface layers. When inoculated into a fermentation
tube, for example, they will grow only in the open arm
near the surface of the medium. At the other extreme there
are bacteria whose optimum condition apparently is
approximately complete absence of free atmospheric oxy-
gen. Many of these will not develop at all in an atmos-
pheric concentration. Such organisms are termed obligate
anaerobes. For these anaerobic bacteria the optimum and
the maximum concentrations of oxygen are most important.
Lying between these two extremes are certain bacteria
termed micro-aerophiles, which grow best in a concentration
of oxygen something less than that of the atmosphere, but

EFFECT OF CHEMICAL ENVIKONMENT 135

do not grow in the entire absence of oxygen. In a suitable
medium such organisms will develop in a definite layer
some distance below the surface. Still other bacteria are
termed facultative. These organisms apparently will grow
either in the presence of atmospheric oxygen or in its
absence. In practically every instance, however, they grow
in the absence of oxygen only when there is nitrate or some
sugar or other available carbohydrate present. The Bacterium
coli, for example, will grow only in the open arm of the fer-
mentation tube in broth, but when sugar is added it will
grow equally well in the closed arm. It is possible that the
oxygen relationships of some microorganisms are more
complex than indicated. It is stated that the organism of
contagious abortion in cattle has two oxygen optimums, it
will grow either in decreased oxygen concentration or in
greatly increased oxygen concentration, but not well in the
presence of atmospheric concentration except after long
continued cultivation.

The fact that many organisms will not grow in the
presence of free oxygen makes it necessary to use special
methods of cultivation in the laboratory. Media in which
anaerobic bacteria are to be cultivated may -be boiled
just before use in order to drive out the oxygen. After
inoculation they may be covered by a layer of oil or paraffin,
or the air may be displaced in closed tubes by some other
gas. Most frequently hydrogen is used or oxygen is
absorbed by use of some reagent such as sodium pyrogal-
late, leaving an atmosphere of nitrogen. In some cases
a vacuum may be used satisfactorily.

Effect of Kinds of Nutrients Present. — By careful ex-
perimentation it is usually possible to find that combina-
tion of nutrient materials which will allow of the most
rapid development of a particular microorganism. The
substances needed and the proportions vary greatly with

136 AGEICULTUEAL AND INDUSTKIAL BACTERIOLOGY

the microorganism. Some forms can utilize inorganic
materials exclusively, others require the most complex
of organic compounds for their best growth.

Effect of Concentration of Nutrients. — For each nutri-
ent there must be an optimum concentration at which
the microorganisms will grow most rapidly. An increase
in concentration beyond this point will more or less
rapidly cut down the rate of growth, that is increase the
length of the generation time.

Stimulating Effects of Dilute Poisons. — Substances
which are ordinarily regarded as distinctly poisonous to
microorganisms, may when sufficiently dilute exert a stim-
ulating effect. Certain of the aniline dyes, for example,
when present in sufficient concentration prevent all
growth of microorganisms, but when greatly diluted they
may increase the rate of growth.

Effect of Autogenic Substances. — Practically all micro-
organisms more or less rapidly produce substances
inimical to their own development. In some instances the
number of bacteria which develop in a culture medium
is limited by the lack of some particular food constituent.
More frequently in the laboratory it is limited by the
accumulation of these toxic substances. Just what these
are is not known in most cases. Apparently in some
instances they are destroyed by heat and may be removed
by filtration through porcelain filters. As these substances
accumulate, the rate of development decreases, the gene-
ration time increases, until finally the bacteria are dying
off more rapidly than they multiply.

EFFECT OF CHEMICALS UPON RATE OF DEATH

In any culture or mass of bacteria in which there is no
multiplication the cells are dying. The death rate may
be very small, but the bacteria are, nevertheless, decreas-
ing in number.

EFFECT OF CHEMICAL ENVIEONMENT 137

An antiseptic is any substance which will inhibit the
growth of microorganisms. It is, of course, true that if
growth is prevented the cells will die off more or less
rapidly. A substance which causes comparatively rapid
death is termed a germicide. It will be apparent that the
difference between antiseptic and germicide is quantitative
and not qualitative. Both kill bacteria, antiseptics slowly,
germicides rapidly.

The term disinfectant is for practical purposes synon-
ymous with germicide. It is usually used with reference to
disease-producing or pathogenic bacteria.

A preservative is an antiseptic substance which is added
to foods to prevent the growth of harmful microorganisms.
The term fumigant is sometimes applied to a gaseous disin-
fectant. A deodorant is a substance which will mask or
destroy obnoxious odors ; it is not necessarily a fumigant or
a disinfectant. A substance which will kill bacteria is said
to exert a bactericidal action.

For convenience in discussion, substances capable of
destroying bacteria may be divided into three groups : first
those which will kill bacteria free in nature ; second, those
which may be injected into or absorbed by the body of man
or animal and will destroy microorganisms growing there;
third, those substances capable of killing microorganisms
which are produced by animals and are present in the blood
serum.

The germicidal agents commonly used in the destruction
of microorganisms may be grouped under the headings of
electrolytes, including the acids, alkalies, and salts; the
nonelectrolytes, including the alcohols, aldehydes, ances-
thetics, the phenol derivatives, the essential oils; and lastly
the oxidizing agents, including oxygen, ozone, chlorine, and
similar materials.

Characteristics of an Ideal Disinfectant.— Certain char-
acteristics should be possessed by an ideal disinfectant. To

138 AGRICULTURAL AND INDUSTKIAL BACTERIOLOGY

the extent that a particular disinfectant measures up in its
characteristics to those of the ideal disinfectant, it is valu-
able for general use. The important characteristics of dis-
infectants are as follows :

"""I. High Germicidal Power. — The ability of a particular
disinfectant to kill_ microorganisms is usually compared
with that of phenol. In making such comparisons it is cus-
tomary to use the Bacterium typhosum, the cause of typhoid
fever, as the test organism. / 'A series of test tubes are pre-
pared containing varying dilutions respectively of phenol
and of disinfectant to be tested. Equal numbers of Bac-
terium typhosum are then introduced into each tube,/ At in-
tervals of two and one-half minutes samples are taken from
each tube and transferred to a nutrient medium suitable
for growth. The transfers are continued for fifteen minutes.
The strength of phenol which is required to kill the organ-
isms, that is, so that no growth is secured when a loopful is
transferred to sterile broth after two and one-half minutes
exposure, is determined, likewise the strength of the
disinfectant to be tested which will produce the same
results. The ratio between the dilutions of the disinfec-
tant to be tested and the phenol is determined and the
number recorded. The ratio between the concentration
of the phenol and of the test disinfectant required to
kill in fifteen minutes is also determined. These two
ratios are averaged. For example, if phenol kills in two
and one-half minutes in a strength of one to one hun-
dred, and the disinfectant to be tested in one to five hun-
dred, the first ratio would be five. If in fifteen minutes
phenol kills in one to two hundred and the disinfectant
to be tested in one to twelve hundred the ratio would be
six. The average of the two would be five and one-half.
Most commercial disinfectants, particularly the coal tar
products, are sold upon the basis of their plienol co-
efficient.

EFFECT OF CHEMICAL ENVIRONMENT 139

2. Stability.— JThe disinfectant, to be most valuable,
should be relatively stable in the presence of organic
matt crT Some of the most powerful of the disinfectants
combine with organic matter forming insoluble com-
pounds and pass out of solution relatively completely. The
strength of the disinfectant may be thereby rapidly de-
er* used .to a point where it no longer destroys micro-
organisms.

3. Homogeneity. — -Disinfectants should be homogeneous
in composition. Substances which may be bought in pure
condition or in crystalline form such as mercuric chloride,
are ideal in this respect. / Many of the commercial disin-
fectants, particularly those prepared from coal tars, may
vary considerably in their composition from time to time,
and consequently_in their germicidal value.

4. Solubility.— !-The ideal disinfectant is one which will
dissolve in all proportions in water? /

5. Nontoxic to Higher Life.— (-An ideal disinfectant would
be one which is nonpoisonous fb man and animals. Obvi-
ously disinfectants which will kill one kind of cell and
not injure another are difficult to find. Most of the valu-
able disinfectants are more or less injurious to tissues.
Certain disinfectants, however, may be injected into the
blood, exerting a more harmful influence upon microor-
ganisms than upon tissues of the body, destroying the
former without seriously injuring the latter. Such, for
example, is the salvarsan used in the treatment of syphilis.

6. Noncorrosive. — Inasmuch as disinfectants must fre-
quently be used in contact with metal surfaces it is highly
desirable that they should not attack metals, injure
fabrics, leave stains, or bleach color.

7. Penet ration.— (Disinfectants differ decidedly in their
power to penetrate materials to be sterilized. An ideal
disinfectant is one which penetrates rapidly and effici-
ent lyj

140 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

8. Economy. -{The ideal disinfectant should be low in
cost.'T Certain valuable disinfectants, because they are so
expensive, can be used only in limited quantities and for
special purposes. The high cost of salts of silver, for
example, limit the use of silver almost entirely to medi-
cine. If the entire water supply of a city is to be steri-
lized, obviously a disinfectant must be chosen which is
relatively inexpensive.

9. Power to Remove Dirt and Grease. — This is related to
power of penetration. A film of oil or grease over the
surface of certain materials may wholly prevent the
action of many disinfectants. Those which have power
to dissolve or remove grease and other kinds of dirt are
naturally more efficient.

10. Deodorizing Power. — A disinfectant which can com-
bine with and destroy malodorous substances is prefer-
able to one which does not interfere with them.

Effect of Hydrogen Ion Concentration upon Death
Rates. — The addition of acid to any nonalkaline solution
containing bacteria (with the consequent increase in hydro-
gen ion concentration) increases markedly the rate of
death.

Bacteria which grow well in a relatively high hydrogen
ion concentration are termed acidophiles. The ability of
these organisms to grow in the presence of high concentra-
tions of acid is sometimes utilized in their isolation.

Acids are most commonly used as preservatives. The
most important are the acetic (usually in the form of vine-
gar) and the lactic acids, the latter the preservative agent
in sour milk, sauerkraut, and silage.

Strong acids are those which ionize most completely.
Weak acids are those which ionize poorly. Lactic and
acetic acids are relatively weak acids, and must be present
in relatively large amounts in order to secure a high con-
centration of hydrogen ions. For example, tenth normal

EFFECT OF CHEMICAL ENVIRONMENT

hydrochloric acid contains about ten times as many hydro-
gen ions as normal acetic acid.

In a few acids either the anion or undissociated acid
molecule may also exert a disinfecting action. In some
this action is much more important than that of the hydro-
gen ion; for example, benzoic acid is a comparatively
weak acid and yet it has relatively high antiseptic or
disinfecting value. This is likewise true of salicylic acid
and related compounds.

Alkalinity or Alkalies.— iHigh alkalinity, that is, high
concentration of hydroxyl ions likewise exerts a destruc-
tive action upon microorganisms. Lime owes its value as
a disinfectant to this fact. Unslaked lime (calcium
oxide) in the presence of water becomes calcium hydrate
and when used in strong solutions, as in whitewash, it has
a marked disinfecting value. It is a valuable disinfectant
to mix with stools or body excreta in order to destroy,
disease-producing bacteria. It should be noted, however,
that upon exposure to air calcium hydrate is gradually
converted into calcium carbonate which is no longer ger-
micidal.

Some bacteria will grow in relatively alkaline solutions.
For example, the organism causing Asiatic cholera, the
Vibrio cholera, will grow in much higher hydroxyl ion con-
centration, that is, in more alkaline solutions, than will
most other species of bacteria. Use is made of this fact
in isolating this organism from feces, the medium being
made so alkaline as to inhibit the growth of most other
microorganisms rendering the securing of this organism
in pure culture comparatively easy.

Salts of the Heavy Metals.— frhe soluble salts of the
heavy metals are almost without exception more or less toxic
to bacteria. In practically all cases their efficiency as disin-
fectants depends upon the degree of ionization. Salts of
silver, mercury, and copper are of particular importance.

142 AGEICULTUEAL AND INDUSTRIAL BACTERIOLOGY

Silver salts, particularly silver nitrate, possess high
germicidal power but are relatively unstable, especially in
the presence of light and organic matter. Colloidal solu-
tions of metallic silver are extensively used in medicine, the
high cost of silver preventing their extensive use for other
purposes.

The soluble salts of mercury are high in disinfecting
power. This is particularly true of mercuric chloride and
mercuric iodide. Mercuric chloride in solutions of one to
one thousand destroys microorganisms rapidly. The soluble
salts of copper, particularly copper sulphate, are also high
in disinfecting value. It has found extensive use in the
purification of water for domestic use. Copper combines
very largely with the organic material present and settles
out in insoluble form so that very little copper appears in
the water when drawn from the tap. Within recent years,
however, its use in water supplies has been very largely
[ displaced by chlorine. Copper apparently has a particularly
ihigh power of destroying algae, that is, it is an algicide.
Solutions of copper sulphate as dilute as one part of copper
to a million parts of water are effective for such purposes.
Copper is the most important constituent of most fungi-
cides, as Bordeaux mixture.

The Non-electrolytes. — Of the alcohols, ethyl alcohol
(C2H5OH) is most commonly used. Its power of disinfec-
tion, however, is usually greatly overestimated. It is most
effective in a concentration of 70 per cent. Either higher or
lower concentrations will fall off rapidly in disinfecting
power.

Formaldehyde (HCHO) is the most commonly used
aldehyde and is most efficient as a disinfectant. It is
usually sold on the market as formalin, the commercial or
trade name for a 40 per cent solution of formaldehyde in
water. Formalin of standard strength should not be below
37 per cent formaldehyde gas. Formaldehyde may be used

EFFECT OF CHEMICAL ENVIRONMENT 143

either in solution or as a gas. It finds extensive use as a
fungicide in the destruction of smut spores on certain
grains. For the destruction of bacteria it is most commonly
used as a fumigant. The formaldehyde gas may be gen-
erated either by boiling solutions of the gas, that is, by boil-
ing formalin, or from some of the polymers of formalde-
hyde, such as paraform. Formaldehyde gas is most efficient
in the presence of moisture. Vessels containing formalin
may be heated directly over a flame, the formalin may be
sprinkled upon sheets and hung about in the room to be
fumigated, or the formalin may be poured over a heap of
crystals of potassium permanganate in a container that will
not be corroded. In the last-named process the heat pro-
duced by the oxidation of a portion of the formaldehyde by
the permanganate vaporizes the remainder.

Phenol and the closely related compounds are among the
most satisfactory of disinfectants for general use. Phenol
(CGH5OH) in a 5 per cent solution will destroy most non-
spore-producing bacteria in a relatively short time. Some
bacterial spores, however, are extremely resistant. The
cresols or methyl phenols (C0H4CH3OH) are the chief con-
stituents of many of the so-called coal tar disinfectants sold
on the market. The cresols are not as soluble in water
as is phenol. A saturated solution contains about 2y2 per
cent. They are even more effective than is phenol. Fre-
quently acids or alkalies are added to the phenols and
cresols to increase their disinfecting power. Benzoic
acid (Cf)H5COOH) and sodium benzoate (C6H5COONa),
salicylic acid (CCH4OHCOOH), and sodium salicylate
(C0H4OHCOONa) have been used frequently as preserva-
tives in foods, usually in concentrations of not more than 0.2
per cent. Salicylates are generally forbidden in foods under
our pure food laws, but benzoates are permitted providing
the amount is fairly stated on the .label. Some of the
so-called canning powders sometimes used by housewives in

144 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

preserving vegetables are various mixtures of benzoates and
salicylates. Their use is unnecessary and objectionable.
The preservative action of the creosotes, the principal dis-
infective agency of smoke in the preservation of meat, is
closely related to that of phenol.

Many of the members of the series of aniline dyes have
decided antiseptic and disinfecting powers. They show
decided differences, however, in their effect on different
kinds or species of bacteria. For example, it has been found
possible quite completely to inhibit the growth of most of
the Gram-positive organisms by the presence of gentian
violet, the Gram-negative forms growing well in its pres-
ence. The so-called Endo medium is a mixture of basic
fuchsin and sulphites. It quite completely inhibits the
growth of most organisms other than those belonging to the
colon typhoid group of bacteria, that is to the genus Bac-
terium. Malachite green and other dyes have been used in
differential media, inhibiting growth of some kinds of bac-
teria and not interfering with the development of others.
I Oxidizing Agents.— The most important of the oxidiz-
ing agents which have been used for sterilization are
oxygen in the nascent condition, chlorine, the hypochlorites,
and potassium permanganate. Ozone (03) has frequently
been heralded as an excellent fumigant. Various types of
so-called t i ozonizers ' ' and "ozonaters" have been placed
upon the market. While concentrated ozone undoubtedly
has some destructive effect upon microorganisms, its pres-
ence in the air in considerable quantities is probably
decidedly more injurious to human beings than it is to the
microorganisms which may be present. Satisfactory and
reliable methods of utilizing ozone have not been worked
out.

Calcium hypochlorite and various other hypochlorites are
used extensively in the purification of water. In the pres-
ence of organic matter these break up, releasing free chlor-

EFFECT OF CHEMICAL ENVIRONMENT 145

ine. Eventually the chlorine goes into combination with
various bases and disappears in this form. The hypochlor-
ites are therefore disinfectants which act for a short period
of time only, after which their potency disappears. This
makes them exceptionally valuable for certain purposes,
but unreliable for others. The spore-producing bacteria are
relatively resistant to sterilization with hypochlorites.
Alkaline solutions of hypochlorites have also been used for
wound disinfection. Such, for example, is the Dakin's
solution used extensively in the sterilization of badly
infected war wounds. Potassium permanganate by its
direct oxidizing activity will completely destroy micro-
organisms in a comparatively short period of time.

Essential Oils. — The essential oils of many spices and
other plants have considerable antiseptic power. Among
them may be mentioned oil of cloves, oil of cinnamon, oil of
mint and especially oil of eucalyptus.

Stimulation of Disinfection. — The disinfecting action of
disinfectant in some cases may be materially increased by
the addition of other substances. For example, the addition
of alcohol to a solution of phenol materially increases the
rapidity with which it destroys microorganisms. Hydrogen
ions frequently increase the speed of disinfection. On the
other hand disinfecting action may be materially reduced
by the addition of certain substances. Sodium chloride, for
example, when added to mercuric chloride, decreases ioniza-
tion and correspondingly the rate at which microorganisms
are destroyed.

Autogenic Products. — Substances produced by the
growth of microorganisms in culture media frequently not
only eventually stop their growth, but determine, in part at
least, the rate at which they die off. In short, bacteria are
usually eventually killed by products of their own. growth.
In some cases this is due to the development of acids or
alkalies, but in many cases the substances produced are not

146 AGEICULTUKAL AND INDUSTKIAL BACTEKIOLOGY

well known. Some of them are easily destroyed by heat.
For example, Bacterium coli when grown upon a culture
medium renders that medium unfit for future growth of
the same organism until it has been heated. Heating
apparently destroys the toxicity of the growth product.

Use in Therapeutics. — Antiseptics or disinfectants
which may be used in or upon the human body are, of
course, much to be desired. Such substances must have a
much higher destructive action upon microorganisms than
upon the tissues of the body with which they come in con-
tact. Within the past two decades persistent effort has been
made to find substances which may be injected into the body
to kill certain kinds of microorganisms without injuring the
body tissues. Such studies have led to the discovery of sub-
stances such as arsphenamine (salvarsan), the so-called
specific for syphilis. Certain of the aniline dyes and related
chlorine compounds have been found useful in wound dis-
infection.

In a later chapter the ability of the body to produce sub-
stances harmful to microorganisms will be discussed. It
will be found that in the blood serum substances toxic to
bacteria may be developed in sufficient quantities entirely
to prevent their growth in the body.

CHAPTER XII

CHEMICAL CHANGES PRODUCED BY MICROORGANISMS
ENERGY RELATIONSHIPS

MICROORGANISMS, like all other forms of life, as long as
they are living and growing require a constant supply of
energy. This is used by the organism in various ways.
Energy is required in some cases for manufacture of food,
in all cases for the building up of complex compounds
such as protoplasm. Energy is necessary for growth and
for movement. Many of the chemical changes brought
about by microorganisms are the result of their methods
of securing energy.

Microorganisms show great variation in the manner
in which they secure their growth energy, and in the
manner in which it is utilized. Some microorganisms
require organic food, others are capable of manufacturing
their own food. Some live under aerobic conditions,
others under anaerobic conditions. A knowledge of the
sources of energy available to microorganisms and of the
ways in which they make use of this energy is therefore
necessary.

Sources of Energy. — Apparently the common, source of
< lu-rgy made use of by all forms of life is oxidation. An
additional source of energy utilized by some forms of life
is sunlight. Most species of higher plants possess the
green coloring material chlorophyll which enables them
to make use of the energy of the sun's rays for the pur-
pose of manufacturing their food from inorganic com-
pounds, primarily from carbon dioxide and water with
the formation of carbohydrates, particularly starch.

147

148 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Sunlight as a Source of Energy to Microorganisms.— It

is somewhat doubtful whether any group of bacteria,
yeasts, or molds, are capable of utilizing the energy of
light for manufacture of foods from inorganic compounds.
The possible exceptions are to be found in that group of
bacteria termed the Thiobacteriales (sulphur bacteria).
Certain of these species contain two coloring materials,
bacteriopurpurin, a purple coloring substance, and bac-
teriochlorin, a green coloring substance. The organisms
possessing these pigments apparently prefer to live in the
presence of light and are attracted toward the more bril-
liantly illuminated side of the vessel in which they are
growing, that is, they show positive phototaxis. That
they actually make use of the sunlight, however, as a
source of energy for the manufacture of foods from simple
inorganic substances has not been definitely proved.

Oxidation as a Source of Energy for Microorganisms. —
All bacteria, yeasts, and molds secure energy by the
oxidation of compounds, either organic or inorganic. For
convenience in discussion they may be divided into three
groups. 1. The prototrophic bacteria secure energy by the
oxidation of inorganic elements and compounds and
utilize the energy thus secured in building up their own
foods. Such organisms are totally independent of ex-
traneous organic food substances. 2. The metatrophic
organisms apparently require organic sources of carbon
and secure growth energy usually by oxidation. They
may, however, utilize inorganic compounds of nitrogen.
3. The paratropkic bacteria usually require both nitrogen
and carbon in the form of organic compounds. For the
most part they are parasitic bacteria and many of them
are disease producers.

The Prototrophic Bacteria. — These are the organisms
which by means of the oxidation of purely inorganic sub-
stances are able to make use of the same or other inor-

CHEMICAL CHANGES 149

ganic substances in building up their own food. They
are not the ones most commonly studied and found in the
laboratory, however. Certain bacteria in the soil are
known to oxidize ammonia into nitrous acid, using the
energy thus secured for building up their own food and
protoplasm. Still other bacteria oxidize nitrites into
nitrates making similar use of the energy secured. Water
in springs containing a considerable amount of hydrogen
sulphide usually abound with so-called sulphur bacteria
which can oxidize hydrogen sulphide to free sulphur and
finally to sulphuric acid, utilizing the large amount of
energy thus secured for their own growth. These sulphur
bacteria also occur in the soil and are doubtless effective
in oxidizing the hydrogen sulphide developed during the
decomposition of organic substances. Bacteria have been
discovered also which can oxidize hydrogen gas, and
others which can oxidize carbon monoxide, thus utilizing
them as sources of growth energy. Certain water bac-
teria oxidize ferrous iron to ferric iron, and manganous
manganese to manganic manganese.

It is probable that in all cases organisms belonging to
the prototrophic group use a considerable proportion of
the energy secured in building up carbon compounds from
carbon dioxide and water. The fact that they are inde-
pendent of organic materials and of sunlight makes them
apparently among the most primitive of living organisms.

MetatropJiic Bacteria. — To this group belong the yeasts,
the molds, and the most of the bacteria. These organisms
require organic compounds of carbon, and by oxidation of
these compounds they are able to secure growth energy.
They also use these compounds in building up their proto-
plasm. They are in many cases able to utilize inorganic
sources of nitrogen. Some forms, for example, can fix
atmospheric or free gaseous nitrogen. As a result of the
energy secured by the oxidation of carbohydrates many

150 AGEICULTUEAL AND INDUSTRIAL BACTEEIOLOGY

forms among the bacteria, yeasts, and molds, can utilize
nitrogen in the form of ammonia or of nitrates. Most of
the bacteria producing fermentation, decay, and putre-
faction belong here.

Paratrophic Bacteria. — These bacteria require particular
compounds of carbon and usually of nitrogen for their
development. Most of them are parasitic, living upon
the tissues of plants and animals. When grown in the
laboratory care must be used that the right amino acids,
vitamines, and carbon compounds are supplied. This
group is important because of the disease-producing
organisms which belong to it.

Methods of Securing Energy by Oxidation.— It has
already been emphasized that microorganisms secure
growth energy by oxidation. This may occur as a result
of direct oxidation of carbon or other compounds or ele-
ments by atmospheric oxygen, the oxidation of one com-
pound and simultaneous reduction of another, and by
intramolecular oxidation.

Many of the aerobic bacteria secure their growth energy
by direct oxidation of inorganic or organic elements
and compounds with atmospheric oxygen. As an example
of this type of transformation may be cited the oxidation
of ethyl alcohol into acetic acid by the vinegar bacteria
of the genus Acetobacter. The change may be illustrated
by the equation :

C2H5OH + 02 = CH3COOH + H20
Alcohol Acetic acid

The same bacteria are also capable of oxidizing acetic
acid to carbon dioxide and water. This may be illustrated
by the following equation:

CH3COOH + 202 = 2C02 + 2H20

In these two steps the alcohol has been completely oxi-
dized and the maximum possible amount of energy is
secured from the alcohol.

CHEMICAL CHANGES 151

Many species of bacteria, particularly the forms known
as facultative anaerobes oxidize carbon compounds in the
absence of atmospheric nitrogen, providing some other
compound containing oxygen which may be reduced is
readily available. For example, nitrates when introduced
into broth enable many bacteria that would otherwise be
aerobic to live under anaerobic conditions. These reduce
the nitrates to nitrites, utilizing the oxygen thus secured
for the oxidation of carbon compounds. It is evident that
the energy yield secured by this process is not as high as
by direct oxidation with atmospheric oxygen.

Many organisms are able to grow in the absence of free
atmospheric oxygen because of their ability to bring about
intramolecular rearrangement of atoms or intramolecular
oxidation of carbon in other compounds. Certain bac-
teria, for example, directly or indirectly are able to trans-
form the sugar dextrose into lactic acid. This may be
represented by the equation

C6H1206 = 2C3H603

Dextrose Lactic acid

If both dextrose and lactic acid are studied by the
methods of the physical chemist it will be found that the
number of calories of heat secured by the complete oxida-
tion of one gram of dextrose is greater than that secured
by the complete oxidation of the same weight of lactic
acid. In other words, inasmuch as energy is indestruct-
ible, when sugar is changed into lactic acid an intramole-
cular oxidation has occurred with the liberation of a cer-
tain amount of energy. It is apparent that this method
is least efficient of all, that is, much larger quantities
of materials are needed for the development of a given
amount of energy by organisms belonging to the third
group than by those belonging to the first. The complete
oxidation of a gram of glucose gives 3.76 large calories
of heat. Similar oxidation of a gram of lactic acid will

152 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

give 3.6 large calories of heat. The energy secured by the
transformation of the glucose into lactic acid is repre-
sented by the difference between these two or .16 calorie.
In other words more than twenty-three times as much
energy may be secured by the complete oxidation of a
given amount of glucose than is secured by the changing
of this amount of glucose into lactic acid.

A gram molecule of ethyl alcohol yields 325.7 large
calories. A gram molecule of acetic acid yields 209.4
large calories. By the transformation of a gram molecule
of ethyl alcohol into acetic acid there is released the dif-
ference, or 116.3 large calories.

CYCLES OP THE ELEMENTS

The fact that many organisms are constantly oxidizing
certain compounds and reducing others, or oxidizing com-
pounds containing one element under certain conditions
and reducing under others, makes possible the study of a
cycle of changes which these elements undergo. Three
of the elements are of sufficient importance from an agri-
cultural point of view, and are sufficiently changed by
microorganisms, as to warrant their discussion. These
elements are nitrogen, carbon, and sulphur.

The Nitrogen Cycle. — The cycle of changes brought
about by microorganisms in nitrogen compounds in nature
can best be followed by reference to the diagram on
page 240. Complex nitrogen compounds are constantly
being broken down by a process of hydrolysis into am-
monia, the ammonia oxidized to nitrates, the nitrates
taken up by higher plants, converted into ammo acids,
and built up into proteins, thus completing the cycle.
Microorganisms bringing about changes of economic sig-
nificance will be discussed in much greater detail under
later chapters.

Proteolysis and Ammonification. — It is perhaps most con-

CHEMICAL CHANGES 153

venient to commence consideration of the nitrogen cycle
with one of the more complex organic nitrogenous com-
pounds. Such compounds, for example, are the proteins
of plants and animals. These undergo decomposition
after the death of the plant or the animal, the more or
less insoluble compounds being k converted into soluble
forms by a process of hydrolysis. This transformation is
termed proteolysis. Finally the nitrogen appears in the
form of ammonia, so that from this point of view the full
process may be termed one of ammonification. The first-
step in the process apparently is the breaking down of
the proteins into the proteoses, differing somewhat in the
size of the molecule though still very large but showing
greater solubility. The proteins are in turn transformed
into peptones, still very complex compounds. The pep-
tones in turn are split into peptids and the peptids finally
into alpha amino acids. Some eighteen or twenty dif-
ferent amino acids have been detected among the decom-
position products of proteins. Certain species of bacteria
and other microorganisms help to bring about the process
termed deaminization, in which nitrogen is given off in form
of ammonia.

A considerable proportion of the nitrogen of animal
bodies is excreted in the form of urea or in some species
of animals as uric acid. These compounds are likewise
attacked by bacteria and are broken down into ammonia.
Eventually, therefore, practically all organic nitrogenous
compounds under the influence of bacterial action liberate
their nitrogen in the form of ammonia. The ammonia thus
produced in soil, for example, is readily available to many
species of plants and to various microorganisms. Most
of the ammonia, however, is rapidly converted by other
bacteria by a process termed nitrification.

Nitrification. — Certain species of bacteria present in soil
oxidize ammonia to nitrous acid and to nitrites. Others

154 AGEICULTUKAL AND INDUSTEIAL BACTERIOLOGY

convert the nitrites into nitrates. The first of these
processes is termed nitrosation, the second nitratation, and
the two together nitrification. All of these terms are mis-
nomers. Instead of being nitrification the process is in
reality an oxidation.

Denitrification. — It has been previously noted that micro-
organisms living in the presence of organic matter and of
nitrates, in the absence of free atmospheric nitrogen, may
reduce the nitrates to nitrites making use of the oxygen thus
secured for oxidizing carbon compounds. Other microorgan-
isms carry the change still farther, breaking down the nitrites
with the formation of free nitrogen gas. This latter process,
like the former, takes place only in the absence of free
atmospheric nitrogen and is not very common in soil. How-
ever, it constitutes in a sense a leak in the nitrogen cycle,
the nitrogen passing off into the free gaseous nitrogen of
the air.

Nitrogen Assimilation. — The green plants as well as many
microorganisms living in the soil take up nitrogen either in
the form of ammonia or of nitrates, and build it up into
complex organic compounds. This process is the reverse of
that which we have just been considering. It is the build-
ing up of proteins from inorganic nitrogen. Animal pro-
teins are in all cases derived directly or indirectly from
plant proteins. This completes the cycle of nitrogen.

Nitrogen Fixation. — The fact that there is a leak in the
nitrogen cycle, and that nitrogen may also be lost from
nitrogenous compounds in the process of burning makes it
evident that there must be methods whereby nitrogen can
be returned from the atmosphere to the nitrogen cycle.
This is in large part accomplished by certain bacteria which,
as a result of the energy secured by the oxidation of carbon
and other compounds, are able to take up free atmospheric
nitrogen, combine it with other elements, and work it up
into their own protoplasm. Such bacteria in some cases live

CHEMICAL CHANGES 155

free in the soil. In other cases they live in the root nodules
of leguminous plants.

The Carbon Cycle. — All plants and animals are con-
stantly producing carbon dioxide during the process of
metabolism. A part, at least, of the growth energy of every
living cell is secured by the oxidation of carbon compounds,
and carbon dioxide is eliminated. However, there are a few
microorganisms and the whole group of green plants which
are capable of synthesizing carbon dioxide and water into
complex organic compounds, particularly carbohydrates
and proteins (together with ammonia). We have constantly,
therefore, in action the two processes, formation of carbon
dioxide from complex organic compounds, a process of
oxidation, and the formation of organic compounds from
carbon dioxide, a process of reduction. Microorganisms are
constantly producing considerable quantities of carbon
dioxide in the decomposition of organic material. A few,
however, have been noted, such as those which oxidize
ammonia to nitrites, which apparently can make use of
carbon dioxide in the synthesis of their own food. The
carbon cycle is therefore comparatively simple, consisting of
this alternate oxidation and reduction.

The Sulphur Cycle. — Hydrogen sulphide apparently
originates in nature in two ways : first, as the result of the
decomposition of organic compounds containing sulphur by
microorganisms with the formation of free H2S ; and second,
the reduction of sulphates under anaerobic conditions by
microorganisms with the formation of free H2S. Hydrogen
sulphide is also found in water of certain springs.

The ordinary putrefactive and decay-producing bacteria,
for the most part, are capable of liberating hydrogen
sulphide from the complex protein compounds in which it
is found. A much smaller number of species are capable
of changing sulphates to sulphides under suitable condi-
tions. In some cases the decomposition of sulphates to

156 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

sulphides may give rise to such large amounts of hydrogen
sulphide as to constitute a nuisance. This is one of the
difficulties found, for example, in the operation of certain
types of septic tanks in sewage disposal. Where cities have
a water supply which is unusually high in sulphates, its
mixture with organic material and retention under ana-
erobic conditions is apt to lead to the formation of undesir-
able amounts of hydrogen sulphide.

Certain bacteria already noted, particularly forms be-
longing to the group of so-called sulphur bacteria, are able
to oxidize hydrogen sulphide to free sulphur, and free sul-
phur to sulphuric acid or to sulphates.

The sulphur cycle is completed by the assimilation of
sulphates by higher plants and the building up of the
sulphur into more complex organic compounds. As with
the nitrogen and the carbon cycles we have constant
changes, oxidation occurring in the presence of free atmos-
pheric oxygen, reduction in the absence of atmospheric oxy-
gen and assimilation by growing plants. Some authors,
particularly in soil bacteriology, have used the term sulpho-
fication in a sense analogous to nitrification, and desulpho-
fication for the reduction process, analogous to denitrifica-
tion.

CHAPTER XIII

MECHANISM OF CHEMICAL CHANGES PEODUCED BY
MICROORGANISMS— ENZYMES AND FERMENTATION

THE bacteria, yeasts and molds must secure all of the
materials useful to the cell by a process of absorption or of
os nosis. All organisms grow only in contact with water.
The substances to be used must be dissolved in the water
outside the cell and diffuse through to the interior. There
is nothing corresponding to a mouth through which solid
particles may enter. It is apparent, therefore, that micro-
organisms must bring about changes in substances outside
the cells in order either that they be made soluble in
water, thus capable of diffusing into the cell, or so changed
that even though soluble they may diffuse into the cell and
be utilized. 'Further, the cell must make use of materials
which diffuse to the interior in various ways. Sometimes
these must be broken down into simpler compounds. Usually
they must be oxidized directly or indirectly and the energy
thus secured utilized for growth purposes. Certain of the
compounds introduced in some instances may be reduced.
Carbon dioxide and water, for example, diffusing into the
cell may be combined to form carbohydrates.

The fact that microorganisms can bring about changes in
the chemical nature of compounds led many years ago to
their designation as organized ferments. They were con-
trasted with so-called body secretions such as the gastric
juice, which could also bring about fermentative changes and
digestion, and which were termed unorganized ferments.
Later it was discovered that many microorganisms excreted
substances which brought about changes outside the cell

157

158 AGEICULTUEAL AND INDUSTRIAL BACTERIOLOGY

body in just the same manner that the juices secreted by the
glands of the stomach act outside the stomach walls, that
is, away from the cells which produce these substances. In
other words, it soon became evident that the so-called
organized ferments acted at least in part through the
agency of unorganized ferments which were excreted.
However, it was not found possible to find unorganized fer-
ments excreted from the cell which would bring about all
of the chemical changes produced by microorganisms.
Finally it was discovered that certain of these substances
capable of bringing about chemical changes were constancy
retained inside the cell and usually worked there. For
example, it was found that yeast was incapable of pro-
ducing alcohol and carbon dioxide from sugar outside
the yeast cell; however, by suitable methods it was found
possible to press out the yeast contents, filter them entirely
free from living cells, and demonstrate that this material
could bring about alcoholic fermentation. It was shown, in
other words, that the changes brought about inside the cell
are brought about by agencies similar to those which may be
excreted. Finally the term enzyme was introduced to indi-
cate any substance produced by living cells capable of
bringing about chemical change. Instead of classifying
ferments into organized and unorganized, differentiation
was made between enzymes that act outside the cell, that
is, that are extracellular, and those that act inside the cell,
that is, that are intracellular. It is quite possible that all of
the chemical changes observed produced by microorganisms
are the direct or indirect result of enzyme action. It is,
therefore, important that the nature of the enzymes pro-
duced and the reactions which they bring about should be
understood.

ENZYMES

Many substances are known in nature which can bring
about chemical changes in other compounds without becom-

MECHANISM OF CHEMICAL CHANGES 159

ing themselves a part of the final product, or without being
used up in the process of being used. For example, a
platinum sponge thrust into a jet of hydrogen will become
hot, finally glow and ignite the hydrogen. It has the
capacity of adsorbing upon its surfaces the hydrogen and
oxygen, and these are brought into such intimate contact
that they unite to form water, giving off heat. The platinum
apparently does not take part in the chemical change ; cer-
tainly it does not become a part of the final product, water.
Substances which can act in this fashion, that is, can bring (
about chemical changes without themselves becoming part
of the final product and that are not used up by being used,
are produced by many cells, probably by all cells. Such
substances are termed enzymes. Enzymes, therefore, may
be_jsomewhat inadequately defined as organic catalysts."

Enzymes are undoubtedly extremely complex organic
compounds. They are relatively unstable, that is, they are
easily destroyed by unfavorable conditions. High tem-
peratures, excessive acidity or alkalinity, the presence of
certain chemicals, and light, all tend to destroy them. There
is, therefore, in any enzyme solution an inevitable deteriora-
tion.

(It will be noted that the main function of enzymes is
apparently to bring about chemical changes. Methods of
isolating and identifying the enzymes themselves have not
proved successful in most cases at least. The exact chemical
composition of none of the enzymes is as yet known. In
fact there seems to be no chemical test which will enable
one to recognize the presence of enzymes other than that of
allowing the enzyme to act under suitable conditions, and
noting the changes brought about, the amount of enzyme
present being deduced from the amount of change pro-
duced.

(The action brought about by an enzyme is probably in
general reversible-, that is, an enzyme brings about a change

160 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

in a compound until the new compound formed is in
equilibrium with the unchanged compound. In other words,
enzymes not only can tear down, they are not only analytic,
but they are synthetic as well, being capable of building up
complex compounds from the simpler. For example, the
enzyme maltase, when brought in contact with maltose in
solution in water, changes most of this maltose into glucose.
Finally an equilibrium is established, there being present a
small amount of maltose and a large proportion of glucose.
Conversely when maltase is placed in a solution containing
glucose it will build up a small amount of maltose (iso-
maltose) until again the solutions are in equilibrium.
Undoubtedly in the body and in the cells of plants and
animals this synthetic action is quite as important as the
analytic.

1 For convenience in study the enzymes may be divided
into three principal groups. The first includes those whose
function, from the standpoint of the cell, is primarily
digestive. These enzymes, for the most part at least, act
by a process of hydrolysis, that is, one or more molecules of
water are incorporated into the molecule which is to be
changed, which then breaks down into two or more simpler
molecules. Most of the extracellular enzymes are of this
nature. They are collectively known as hydrolases. The
second group includes those enzymes that bring about
changes primarily oxidative in nature. The changes which
they bring about usually yield energy to the cell. They may, in
a sense, be termed the respiratory enzymes. In some cases they
function by the addition of atmospheric oxygen or oxygen
derived from other compounds easily reduced to the com-
pound, usually a carbon compound, to be changed. Such
enzymes are generally termed the oxidases. Others bring
about a rearrangement of atoms within the molecule, an
intramolecular oxidation, and the molecule splits into two
or more new molecules. Such enzymes are frequently

MECHANISM OF CHEMICAL CHANGES 161

termed the splitting enzymes or zymases. Third, still other
enzymes are primarily concerned in reduction processes. It
is evident that wherever food material is built up from
simple inorganic substances that there must be reduction
taking place. The building up of sugar, for example, from
carbon dioxide and water implies the reduction of carbonic
acid (H2C03) to formaldehyde (HCHO), and the poly-
merization of this product into sugar. Such enzymes are
reductases.

The hydrolytic enzymes are both extracellular and intra-
cellular. Most of the oxidizing, splitting, and reducing
enzymes are intracellular.

; From the standpoint of agricultural or economic sig-
nificance, the hydrolytic and oxidizing enzymes are by far
the most important. The hydrolytic and oxidatlve enzymes
produced by microorganisms will therefore be discussed.

The Hydrolytic Enzymes or Hydrolases. — The hydro-
lases, that is, the enzymes which bring about splitting of
organic compounds by the addition of water, are for con-
venience divided into three groups, the carbohydrases, the
proteases or amidases, and the esterases or lipases. The
first group, the carbohydrases, bring about hydrolytic
decomposition of carbohydrates and related compounds.
The proteases hydrolyze the proteins and their various
decomposition products to amino acids. The esterases or
lipases hydrolyze the esters and particularly the glycerine
esters or fats. Enzymes belonging to all of the groups are
known among microorganisms.

Carbohydrases. — Enzymes capable of hydrolyzing the
various complex carbohydrates down to the simple sugars
are known. The principal sub-groups of carbohydrases are
the cytases and cellulases which attack the celluloses and
hemicelluloses, the pectinases attacking pectin, the diastases
and inulases attacking starch and inulin, and the invertases,
which hydrolyze the disaccharides to simple sugars.

162 AGRICULTURAL AND INDUSTRIAL BACTEKIOLOGY

The celluloses, hemicelluloses and vegetable gums are
carbohydrates having very large molecules made up by the
polymerization of the simple sugar radicles. Inasmuch as
cellulose and hemicellulose and closely related compounds
constitute a large proportion of the bodies of the higher
plants, it is evident that were it not for the decomposition
brought about by microorganisms, there would be large
accumulations of such materials in soil. Many species of
molds and bacteria are known which can decompose cel-
lulose, breaking it down into simpler compounds. The
enzymes capable of bringing about these changes are
usually termed cytases or cellulases. A few organisms are
also known which can decompose vegetable gums which are
closely related chemically to the hemicelluloses. The devel-
opment of cytase by microorganisms may be demonstrated
by mixing finely pulverized cellulose or hemicellulose with
suitable melted agar, and pouring into a Petri dish. Bac-
teria or molds capable of digesting this carbohydrate will
be visible because of the clear ring surrounding the colony.
By the action of the cytase undoubtedly considerable quan-
tities of carbohydrate food are released to other soil bacteria,
not capable of attacking the cellulose directly.

Pectinases are enzymes capable of dissolving or digesting
the carbohydratelike substance called pectin. This is present
in most plants, constituting the layer between the cellulose
walls of adjoining cells. In morphologic botany it is called
the middle lamella. It may be termed the glue which holds
plant cells together. "Certain microorganisms, particu-
larly among the molds and the bacteria, secrete pectinase
which dissolves this middle lamella and frees the cells from
each other. From an agricultural point of view this is par-
ticularly important in the retting of flax and hemp, in
which molds or bacteria dissolve out the pectins which bind
the linen 'fibers and hemp fibers together; it is important
also with certain of the disease-producing bacteria, such as

MECHANISM OF CHEMICAL CHANGES 163

the organism causing cabbage black rot, which softens the
middle lamella over large areas of the leaf causing the cells
eventually to die and decompose.

Among the higher plants the most common reserve carbo-
hydrate materials stored up are starch and inulin. Starch
is a polymer of dextrose, inulin a polymer of levulose.
Starch is attacked or hydrolyzed by the enzyme amylase
(diastase) which changes it into dextrin and finally into
the sugar maltose (C^H^On). This enzyme is produced
by sprouting seeds, is present in most leaves and growing
tissues of plants, and is formed by many species of bacteria
and molds, though, so far as is known, not by any of the
yeasts. The property of amylase production or starch
digestion may be demonstrated with molds or bacteria by
the use of starch plates, that is, by plates poured from agar
containing dissolved or suspended starch. Those organisms
which digest the starch will change its appearance from
milky or opaque to transparent and clear. The ability to
saccharify or liquefy starch possessed by certain molds has
been made use of in industries to prepare starchy materials
for alcoholic fermentation by yeasts. These will be dis-
cussed later. It should be noted that not all organisms
which attack starch and break it down into simple com-
pounds, change it into the intermediate simple sugars. For
example, the Bacterium aerogenes, although it is able to
produce gas and acid in starch solutions, does not develop
the enzyme amylase.

The carbohydrate inulin is very common as a reserve food
product in the plants belonging to the sunflower or com-
posite family. The sunflower plant itself, chicory, dande-y
lion and dahlia all contain considerable amounts of this car-
bohydrate. The enzyme inulase converts (by hydrolysis)
inulin into levulose. Certain molds are known which pro-
duce inulase. Whether or not this is also produced by
bacteria has not been adequately demonstrated. There are

164 AGEICULTUEAL AND INDUSTRIAL BACTERIOLOGY

bacteria known which will ferment inulin but whether the
inulin is attacked directly or first broken down into levulose
has not been proved.

Three of the common disaccharides are frequently used in
the study of microorganisms. These are sucrose or cane
sugar, maltose or malt sugar, and lactose or milk sugar.
These are hydrolyzed by the enzymes sucrose, maltase and
lactase respectively. The reaction may be indicated as
follows :

Ci2H22°ii + H2° + invertase — C6H1206 + C6H12O6 + invertase
sucrose sucrase dextrose levulose sucrase

or or or or

maltose maltase dextrose dextrose maltase

or or or or

lactose lactase dextrose galactose lactase

Many species of molds and yeasts are known which produce
one or more of these enzymes. Common bread yeast, for
example, produces the enzymes sucrase and maltase but not
lactase. Certain of the lactic yeasts, however, are able to
produce lactase as well. These enzymes are collectively
known as invertases, and the process of hydrolysis of a
disaccharide is termed an inversion. It should here be
remarked as under starch fermentation, that many bacteria
are capable of attacking disaccharides without hydrolyzing
them into the simple sugars; that is, the fact that a micro-
organism can ferment sucrose does not indicate that it
necessarily produces the enzyme sucrase, or that dextrose
and levulose are intermediate products in the fermentation.
Proteases and Amidases. — In the discussion of the nitro-
gen cycle it was noted that proteins are exceedingly complex
nitrogenous organic compounds built up by the polymeriza-
tion of alpha amino acids. The hydrolysis of these com-
pounds is brought about by a group of enzymes termed pro-
teases and amidases. Those most common are the pepsins,
the trypsins, and the erepsins. The pepsins are enzymes

MECHANISM OF CHEMICAL CHANGES 165

capable of attacking proteins, breaking them, down into
peptones, and perhaps in some eases into polypeptids and
peptids. They require an acid medium. Trypsins are
enzymes capable of attacking proteins, breaking them down
through the stage of peptones into the peptids, the amino
acids, and some cases even to ammonia. They require an
alkaline medium. The erepsins resemble trypsins in their
end products, but attack only compounds somewhat less
complex than the proteins. They will attack, for example,
the proteoses and the peptones. The general types of reac-
tion brought about may be illustrated by the hydrolysis of
a dipeptid into amino acid.

R.CH-COOH R.CH.COOH

NH NH2

H20

R.CH-CO R.CH-COOH

NH2 NH2

The amino acids may be regarded as the building stones
out of which proteins are constructed. The cement which
binds them together is the abstraction of water. Enzymes
which attack proteins insert water and the compounds fall
apart on the lines of cleavage.

Microorganisms are known to produce various types of
proteases. One of these most commonly observed in the
laboratory is gelatinase, the enzyme capable of liquefying
gelatin. Bacteria have been described which will liquefy
egg white and digest blood serum. These changes are some-
times noted as cultural characteristics of microorganisms
studied. Many microorganisms, too, are able to digest the
casein of milk in which they are grown, either with or with-
out the preliminary coagulating action of the so-called lab
enzyme or rennet.

Esterases and Lipases. — Fats, that is, the glycerine esters
of the fatty acids, are hydrolyzed by lipases into the corre-

166 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

spending fatty acids and glycerine. For example, certain
microorganisms live in butter and produce lipases which
may decompose stearin in accordance with the following
reaction :

(C^COOaCsHs + 3H20 + lipase
Stearin

= 3C17H35COOH + C3H5 (OH), + lipase
Stearic acid Glycerine

Lipases have been described for certain species of molds but
they are not very common among the bacteria. They may
be demonstrated by preparing an emulsion of a suitable fat,
such as butter fat, or one of the vegetable oils, in agar.
Microorganisms grown upon the surface of such a medium
will show ability to hydrolyze the fat by the clearing of the
medium immediately surrounding the colony.

The Splitting and Oxidizing Enzymes. — The most im-
portant of the so-called splitting enzymes are the zymase
of yeast, and the lactacidase of the lactic acid bacteria. The
enzyme zymase in the presence of phosphoric acid is capable
of transforming the monosaccharide sugars such as dextrose,
into alcohol and carbon dioxide. There probably are one or
more intermediate steps in the process, but the transforma-
tion may be illustrated by the equation :

C6H1206 = 2C2H5OH + 2C02
Dextrose Alcohol

This enzyme is characteristic of most species of yeasts ; it is
present also in a few of the molds and probably in some
species of bacteria. The enzyme is intracellular. Lactaci-
dase likewise transforms sugars, including the monosac-
charides, with the end product lactic acid. Here again there
are probably several intermediate steps in the process, but
the ultimate transformation may be indicated by the equa-
tion:

C6H1206 = 2CH3CHOHCOOH
Dextrose Lactic acid

MECHANISM OF CHEMICAL CHANGES 167

This reaction is brought about within the cells of certain
species of bacteria, and probably to a less degree by certain
species of yeasts and molds. The enzyme lactacidase, how-
ever, has apparently never been secured outside the bac-
terial cell.

Oxidizing enzymes which combine organic compounds
with oxygen from the air are comparatively common in
microorganisms. Probably most of them are intracellular.
But little detailed work has been done upon them. Much
more is known concerning the oxidases developed by higher
forms of life, particularly the higher plants. One of the
most striking of the oxidative changes is that produced by
members of the genus Acetobacter in the oxidation of alcohol
to ''acetic acid. This may be illustrated by the following
equation :

C2H5OH + 02 = CH3COOH + H2O
Alcohol Acetic acid

Many color reactions are the result of the presence of
oxidases. This is particularly true among higher forms.
The bruised surfaces of many fruits, for example, are
known to change color because of the action of oxidases in
the cells which are exposed to the air. It is quite possible,
though by no means demonstrated, that the oxidizing
activity of organisms belonging to the genera Nitrosomonas
and Nitrobacter is due to the presence of enzymes of this
group. Probably all cells contain oxidizing enzymes capable
of releasing energy from the food, particularly from carbo-
hydrate substances.

FERMENTATION

Fermentation may be very broadly denned as any
chemical change brought about directly by microorganisms
or indirectly as the result of the action of enzymes or other
substances produced by them. It is sometimes somewhat
more narrowly defined to include only chemical changes

168 AGRICULTURAL AND INDUSTRIAL BACTEEIOLOGY

brought about in carbohydrates. The term putrefaction is
sometimes applied to decomposition, particularly of protein
materials, occurring under more or less anaerobic condi-
tions, usually with the development of malodorous sub-
stances. Decay is similar decomposition occurring usually
under aerobic conditions without noteworthy development
of malodorous compounds.

Practically all fermentation, putrefaction, and decay in
nature occur as the result of the activity of microorganisms,
that is, of the bacteria, the yeasts and the molds.

The Origin of the Products of Fermentation. — The prod-
ucts developed during the process of fermentation may be
grouped under four headings, using the origin as a basis for
the classification.

1. The Analytic Action of Extracellular Enzymes. — The
digestive changes brought about by extracellular enzymes
break down compounds on the exterior of the cell into
simpler compounds. These are usually formed greatly in
excess of the immediate needs of the microorganisms. Such,
for example, is the transformation of starch into maltose by
certain molds, or the digestion of casein or gelatin by bac-
teria.

2. The Analytic Action of Intracellular Enzymes. — Many
of the intracellular enzymes are active in bringing about
changes in those food substances which diffuse into the cell.
Such, for example, is the origin of alcohol and carbon di-
oxide in yeasts, and of lactic acid in the lactic acid bac-
terium. It may be noted furthermore that the activity of
the analytic enzymes contained within a cell do not cease
necessarily with the death of that cell. Such enzymes as
continue to act after the death of the cell and bring about
more or less decomposition of the cell substance itself are
termed autolytic. In an old culture in broth in which most
of the organisms have died, the cells will be found more or
less autolyzed; and chemical test will reveal substances

MECHANISM OF CHEMICAL CHANGES 169

present in the medium which have dissolved out from the
bacterial cell and which are not present in rapidly growing
young cultures. Sometimes these products resulting from
autolysis or self-digestion are poisonous in nature.

3. The Synthetic Action of Enzymes. — It will be recalled
that not only are enzymes analytic in their action, but they
are synthetic as well. Many times complex compounds are
produced from simple compounds as a result of bacterial
activity. Such, for example, are the gums and the slimes of
various species of bacteria. The organism producing ropy
milk, for example, polymerizes the sugars present into much
more complex gums, usually galactans. The toxins and the
enzymes themselves are likewise the result of synthetic
action.

4. Secondary Action of Growth Products. — Some of the
ultimate products of the action of microorganisms are
secondary, that is, products formed within a cell may upon
excretion bring about changes in other substances wholly
outside of the cell. For example, certain bacteria growing
in milk containing calcium carbonate would produce lactic
acid, this, upon diffusing from the cell, would form calcium
lactate, with the evolution of carbon dioxide. It can scarcely
be said that the carbon dioxide is developed as a result of
the direct action of the microorganism, but that it is the
result of a secondary change. Some of these secondary
reactions are of considerable importance in the soil. The
insoluble phosphates, for example, are in part at least
made soluble and available to the plant roots through the
action of secondary products of the growth of bacteria. Not
infrequently in fermenting materials gaseous hydrogen is
developed. This as nascent hydrogen may exert a powerful
reducing action upon various substances present. For
example, levulose when fermented by certain bacteria is
transformed in part into mannitol, that is, the sugar is
changed into the corresponding polyatomic alcohol.

SECTION IV

BACTERIA IN TECHNICAL AGRICULTURE AND
THE INDUSTRIES

CHAPTER XIV

KELATIONSHIPS OF MICROORGANISMS TO THE
PRESERVATION OF FOOD

Agencies which Bring about Deterioration of Foods. —

The agencies which bring about deterioration in foods and
food products may be grouped under two general headings :
the intrinsic, including those which are normally present
in the food itself and have not been derived from outside
influences ; and second, those which may be termed extrinsic,
that is, those which enter the food at some time during the
process of preparation or ripening. To the first group
belong the so-called autolytic enzymes. In certain fatty
foods, for example, there may be hydrolyzing enzymes
which will bring about rancidity. In fruits and certain
vegetables the enzymes which normally bring about ripen-
ing may bring about a condition of overripeness. Certain
of the oxidizing enzymes, such as those present in fruits,
may produce discoloration. Of the extrinsic agencies
capable of bringing about food deterioration, there are the
bacteria, the yeasts and the molds together with the
enzymes which they produce.

In general the bacteria thrive best in nitrogenous foods
containing considerable proportion of water and not too
much acid; they grow best in a medium which is nearly
neutral. The yeasts, for the most part, grow best in a
medium rich in carbohydrates, particularly in the ferment-
able sugars, and preferably in the presence of some acid.
Like the bacteria, they prefer a considerable amount of
moisture. The molds will grow in media somewhat drier
and of almost any type. Certain molds will grow on foods

173

174 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

that are slightly acid as well as upon those which are neu-
tral or slightly alkaline.

Methods of Preventing Deterioration. — In order to pre-
vent deterioration food must be kept so that there can be
no appreciable activity of autolytic enzymes and no growth
of bacteria, yeasts, or molds. This may be accomplished by
heating to a temperature and for a time sufficient to destroy
all microorganisms and enzymes present, or by the process
termed pasteurization, whereby those organisms capable of
bringing about detrimental changes are destroyed without
necessarily making the material sterile; by holding the
foods at temperatures so low that undesirable changes can-
not be brought about; by the presence of chemicals which
will act as preservatives or antiseptics; and finally by the
elimination of water to such a degree that microorganisms
cannot grow and enzymes cannot act.

Sterilization by Heat. — If food materials are heated to
such a temperature and for such a time as will destroy all
of the living microorganisms present, and so sealed that
microorganisms from the environment cannot come in con-
tact, they may be preserved indefinitely. The process most
used for this purpose is that commonly termed canning, in
which the food material either before or after sterilization
is hermetically sealed in containers. Meat, fruits, vege-
tables, and in general those foods not seriously damaged by
heat and which cannot readily be preserved by drying, are
utilized.

In any method of canning it is necessary that the food be
properly prepared, placed in suitable containers, the air
exhausted and the food hermetically sealed, and sterilized.
We are here concerned primarily with the factors which
determine the time and the temperature necessary for
sterilization.

The first of the factors determining the time necessary for
sterilization is the initial infection, its extent, and the kind.

FOOD PRESEEVATION 175

It has been previously learned that bacteria are not killed
off instantly by heat, but that the temperature determines
the rate of death or rate at which the microorganisms die
off. Other things being equal, the larger the initial number
of microorganisms to be destroyed the longer will be the
time necessary for their destruction. It is also apparent
that the kinds of organisms present may have some influ-
ence. The spore-producing bacteria are more difficult to
destroy than the nonsporulating types. A process com-
monly used preliminary to canning is termed blanching.
The food products, usually vegetables or fruits, are dipped
first into hot water, then into cold water. This process
shrinks the tissues somewhat, makes them somewhat firmer,
and tends to set the color, but also washes off many of the
bacteria which were present, thus reducing the initial infec-
tion and possibly thereby somewhat decreasing the time
necessary for sterilization. It is sometimes stated, further-
more, that bacteria are more easily destroyed when first
heated, then cooled (or chilled) and reheated. Several tests
on this point have failed to show any marked effect of this
1 ' cold shock."

The second factor in determining time of sterilization is
the size of the food container or can. It is evident that the
center of any can is that portion which last reaches the
desired temperature. The larger the container the longer
will it take this central portion to assume the desired tem-
perature.

The third factor is the ease with which heat may pene-
trate to the center of the canned material. Where the
particles of food are solid and are surrounded by water or
thin syrup, convection currents are at once set up in the
water and the interior heats relatively quickly by a process
analogous to that used in a hot water system in the heating
of a home. If the material lying between the food particles
is viscous or gelatinous, or there is not much, if any, free

176 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

water, convection does not occur, and heat passes to the
center much more slowly and only by conduction. Cans of
corn heat more slowly at the center than cherries. Sub-
stances like pumpkin paste and spinach heat most slowly
of all. With the latter the rate of heat conduction is
practically the same as the rate of heat conduction through
water.

Fourth, the hydrogen ion concentration, that is, the
actual acidity of the food material, is also important. It is
common experience that foods such as peaches or apples are
much more easily sterilized by heat than vegetables such as
peas, beans and corn. The presence of acid greatly increases
the death rate of microorganisms at high temperatures.

Fifth, in some commercial canneries devices (agitators)
are employed to keep the canned foods continually in
motion during sterilization. This induces constant mixing
of the food material and increases markedly the rate at
which the heating will occur. Agitation acts in very much
the same fashion as convection currents in water. The
method of cooling and the time required will also determine
to some extent the efficiency of sterilization.

Sixth, the temperature which must be employed is of
importance. Home canning is ordinarily accomplished by
the use of boiling water, and 100° c. is the highest tem-
perature available. By the use of pressure cookers or in
commercial establishments by the use of large autoclaves or
pressure cookers, higher temperatures may be employed and
the length of time required materially decreased.

Several methods having their counterpart in commercial
processes have been utilized in the home canning of foods,
important among them being the intermitten process of
sterilization and the cold pack process. In intermittent
sterilization it is customary to heat the food to be preserved
in its container for a certain length of time, frequently an
hour, on each of two, three or more successive days. The

FOOD PRESEKVATION 177

theory is that the first heating will destroy all except the
spores of the bacteria, these will germinate during the next
twenty-four hours and be destroyed as vegetative cells upon
the next heating or quite certainly upon the third. In the
cold pack process the food material is heated in boiling
water or flowing steam at approximately 100° c. for such
time as will make certain the destruction of all microorgan-
isms, or at least those which might bring about deteriora-
tion.

Not all canned foods, even though they may keep per-
fectly well, are completely sterilized. It has been deter-
mined as the result of many studies within the last few
years that the microorganisms which may bring about
deterioration in canned foods are: first, those which may
enter the food as the result of defective sealing; second,
those aerobic spore-producing bacteria which normally are
unable to grow in canned foods because of the complete
exclusion of air and which may find conditions suitable for
growth if air is admitted due to defective sealing; third,
anaerobic spore-producing bacteria capable of growing at
normal temperature occasionally escape sterilization and
produce deleterious changes, frequently accompanied by
evolution of gas and the development of malodorous and
bad tasting compounds, and sometimes poisons or toxins
(such as Clostridium botulinum) • and lastly, certain of the
most resistant of the spore-forming bacteria belonging to
the group of thermophiles. These spores of thermophilic
bacteria will not germinate unless the canned food is held
at a high temperature for some time. When canned foods
are not adequately cooled in a commercial cannery and are
stacked away in a warehouse, they may retain their heat
for days or even weeks and conditions be particularly
good for the development of the thermophiles. The same
may happen when canned goods are stored in hot climates.

178 AGRICULTUEAL AND INDUSTRIAL BACTERIOLOGY

Food Preservation by Pasteurization. — Pasteurization is
that process of food preservation in which the food is heated
to a temperature sufficient to destroy certain types of
undesirable bacteria, but not necessarily to destroy all living
microorganisms present. The process was first used with
wines and beers in which it was desirable to destroy certain
nonspore-producing bacteria. Heating such materials to
high temperatures would injuriously affect their flavor. By
careful tests the time and temperature sufficient to destroy
the undesirable organisms may be determined without in-
juring the flavor. The process has in recent times come to
be used most extensively with milk. In the modern milk
pasteurizing plants, however, the effort is made to heat the
milk to that temperature and for a sufficient length of time
to kill all disease-producing germs. Other kinds of organ-
isms may not be destroyed. Milk, or more particularly
cream, may also be pasteurized to destroy microorganisms
which may give undesirable flavors or aroma to the cream
products, particularly butter. It is evident that pasteuriza-
tion of milk is primarily a sanitary measure rather than a
means for increasing its keeping qualities.

Preservation by Low Temperatures. — The rate at which
microorganisms grow and at which enzymes act decreases
with decrease in temperature. Articles of food which may
be frozen without damage may be preserved almost in-
definitely, although even in frozen foods, such as poultry
and fish, there is a slow deterioration, probably due to the
action of enzymes. Other foods may be kept at low tem-
peratures above the freezing point. Such, for example, are
eggs and various other foods. It should be noted that at
low temperatures the types of organisms which may grow
may differ from those which develop at higher temperatures
and the changes brought about will not be the same.

Preservation by Chemicals. — Some foods naturally con-
tain substances which are preservative, for example, many

FOOD PRESERVATION 179

of the spices contain essential oils in sufficient quantities
to have a decided preservative action. It is a well-known fact
in the household that certain acid fruits and vegetables,
such as stalks of rhubarb and the fruit of cranberries and
gooseberries, may be preserved for considerable lengths of
time in water without any sterilization.

Another class of foods is preserved as a result of chemicals
produced during the process of fermentation. Without
exception these are foods relatively high in sugar. Sauer-
kraut, for example, is preserved as the result of develop-
ment of lactic and some acetic acid by bacterial growth. As
long as it is kept from the air, deterioration will not take
place after the acid has been developed. Dill pickles are
prepared in a somewhat similar fashion. The process of
preparation of silage from corn constitutes another ex-
ample.

In some cases chemicals are added directly to foods in
order to preserve them. The preservative may be some
organic food acid such as acetic. Vinegar is used for
pickling meats, fruits, and vegetables. Sodium benzoate in
small quantities, although not advocated, is nevertheless
permitted by our pure food laws, providing the quantities
added are plainly indicated. Still other chemicals are used,
such as spices and the essential oils. Certain preservatives
are in general forbidden by our pure food laws. Such, for
example, are salicylic acid and the salicylates and formalde-
hyde.

Preservation of Foods by Drying. — The amount of
water which must be removed from any food material in
order to prevent the growth of microorganisms will depend
upon several factors, most important being the amount of
soluble materials present in the food, the kinds of solutes
present, the distribution of the water, the kinds of organ-
isms or enzymes present, and the method of desiccation.

The amount of material in solution in the water in a food

180 AGRICULTUKAL AND INDUSTRIAL BACTERIOLOGY

determines osmotic pressure, and this is an important factor
in determining the ability of microorganisms to grow. Most
microorganisms cannot develop in concentrated sugar solu-
tions. Because of their relatively high sugar content, many
fruits are in consequence readily preserved by drying, and
may contain a much higher percentage of water than could
other carbohydrate foods, such as flour, without deteriora-
tion.

The amount of moisture that must be removed may also
in part be determined by the concentration of acids present.
Essential oils are also important in some instances, although
\the drying process may occasionally remove some of these.
The malic acid of the apple is a factor of no inconsiderable
importance in preserving dried apples.

The amount of water available for the growth of micro-
organisms is not always the amount which is revealed upon
comparison of the moist and dry weight of ^he food
material. Many foods hold their water tenaciously, not
yielding it readily to microorganisms. Then, too, in some
instances the water is not equally distributed throughout
the food material. Butter, for example, retains water in the
form of small droplets. Butter, therefore, containing 15
per cent of moisture may afford much better moisture con-
ditions for microorganisms' growth and yield up moisture
more readily than some other food containing 30 per cent.
When the butter fat is completely freed from water it does
not change readily. During the process of drying, further-
more, external layers of the food may be changed so as to
render the penetration of microorganisms difficult. For
example, meats which are dried or partially dried soon come
to have the external layers saturated with fats and oils, con-
stituting a waterproof exterior through which microorgan-
isms cannot readily penetrate.

The kinds of organisms present or the nature of the
enzymes may also determine the amount of moisture which

KELATIONSHIP OF MICROORGANISMS 181

must be removed in order to make food keep. In general
bacteria seem to require somewhat more available moisture
than do the molds. Yeasts and molds grow fairly well in
solutions that are somewhat acid, the yeasts requiring also
sugar for their development.

The methods used in the process of desiccation also
influence the keeping qualities. Certain fruits, for example,
are generally sulphured, that is, exposed to the fumes of
S02 or sulphurous acid in order to bleach them. This
effectually destroys many microorganisms which are pres-
ent. Other foods may be exposed to the direct rays of the
sun during the process of drying and the microorganisms
present destroyed.

Many methods are used for the drying of foods. Drying
is expedited by the use of high temperatures, by the use of
air which is relatively dry, by increasing the rapidity of air
circulation by blowing or fanning, and by the use of a par-
tial vacuum.

Dried foods may be divided into groups, using as a basis
of classification the proportion of carbohydrates, fats, or
proteins, present.

The carbohydrate foods such as grains, and the flours and
meals produced from them usually are sufficiently dried
during the process of ripening and manufacture so that
they will keep indefinitely. Fats and oils usually keep well
providing they are completely freed from water. Proteins
are usually somewhat more difficult to dry and to preserve.

Many starchy products produced by the baker and the
manufacturer, such as macaroni, vermicelli, yeast cake,
crackers, biscuits, etc., are dried for preservation. Methods
have also been developed for the rapid desiccation of vege-
tables and fruits.

Many dried fruits may contain as much as 30 per cent of
moisture and yet not spoil. Syrups, molasses, sorghum,
jams, jellies, preserves, etc., are readily preserved because

182 AGEICULTUKAL AND INDUSTRIAL BACTERIOLOGY

of the concentration of sugar present. Milk powder is
prepared by several processes, among them the forcing of
milk in the form of fine spray into a heated compartment
from which the air has been partially exhausted. As it falls
to the bottom it is transformed into a fine, dry powder
which keeps well. Jerked meat, smoked meat, fish, and
beef extract are preserved by the removal of water. Eggs
are sometimes dried, and find extensive use among bakers.

CHAPTER XV

FERMENTATION OF CAEBOHYDRATES— ALCOHOLIC FER-
MENTATION IN FOODS— COMMERCIAL PRODUCTION OF
ETHYL ALCHOL— FUSEL OILS

ONE of the commonest and most important of the changes
brought about in carbohydrates by microorganisms is that
known as alcoholic fermentation, that is, the transformation
of monosaccharides into ethyl alcohol and carbon dioxide.
This type of fermentation is important in the preparation
of certain beverages, in the manufacture of distilled liquors,
and particularly in the preparation of commercial alcohol,
bread and other foods. Inasmuch as yeasts are the most
active of the organisms in bringing about alcoholic fermenta-
tion, consideration of the preparation of commercial yeasts
is of importance.

Agencies Active in Alcoholic Fermentation. — A few
bacteria are known which may produce alcohol in the
process of fermentation. The amounts formed, however,
are small, both actually and .relatively to the amounts of
other fermentative products. Certain species of molds, par-
ticularly when grown in sugar solutions under anaerobic
conditions, can produce somewhat larger amounts of alco-
hol. From a practical point of view, however, all alcoholic
fermentation of importance is brought about by members of
the yeast genus Saccharomyczs.

The transformation of sugar into alcohol and carbon
dioxide may be represented by the equation :

C.HUO« = 2C2H5OH + 2CO,
Dextrose Ethyl alcohol

The intracellular enzyme zymase has been found capable

183

184 AGEICULTUEAL AND INDUSTRIAL BACTEEIOLOGY

of bringing about this change. It may be prepared from
yeast cells by grinding them with fine sand and diatomace-
ous earth until the cell bodies have been ruptured, then
squeezing them by means of a hydraulic press to force out
the cell juices. Under suitable conditions these cell juices
containing zymase will produce transformation of sugar
into alcohol and carbon dioxide. Apparently zymase will
not act except in the presence of phosphates or phosphoric
acid. Probably there is first brought about a union of the
hexose sugar and the phosphoric acid to form hexose phos-
phate, and this is then decomposed by the zymase with the
formation of alcohol and carbon dioxide.

Preliminary Preparation of Carbohydrates for Yeast
Fermentation. — It has already been emphasized that
yeasts are able to act directly upon monosaccharide sugars
only with the production of carbon dioxide and alcohol.
Carbohydrates in nature frequently exist in more complex
forms, as disaccharides and polysaccharides. These must
first be transformed into the monosaccharides before they
can be fermented by the yeast,

The extracellular digesting or hydrolyzing powers of
yeasts show marked differences. None are known which are
able to attack the complex polysaccharides, such as starches
and cellulose. Most species, however, are able to produce
the enzyme maltase which hydrolyzes maltose to two mole-
cules of dextrose. Likewise most species produce the
enzyme sucrase which hydrolyzes sucrose or cane sugar into
dextrose and levulose, and a few species are also known
which produce the enzyme lactase, hydrolyzing lactose or
milk sugar into dextrose and galactose. It is apparent,
therefore, that no preliminary treatment is necessary for
the fermentation of cane sugar or malt sugar by yeast, and
that some yeasts can also ferment lactose or milk sugar.

In manufacture of alcohol generally the polysaccharides,
particularly starch and cellulose, constitute the cheapest

FERMENTATION OF CARBOHYDRATES 185

and most easily available form of carbohydrate. Inasmuch
as these cannot be attacked directly by yeasts, if they are to
be used in alcoholic fermentation they must be hydrolyzed
into sugars which can be directly attacked. Several methods
have been worked out for this purpose.

When starch or cellulose are heated in the presence of
acids and water they are more or less- rapidly broken down
into dextrins and finally into dextrose. Some attempts have
been made upon a commercial scale to hydrolyze the cel-
lulose of sawdust and wood to dextrose, utilizing" the sugar
formed for yeast fermentation. The commercial manu-
facture of glucose from starch is based upon the acid
hydrolysis.

A much more convenient method of preparing sugar from
starch, preliminary to alcoholic fermentation, is by the use
of malt. Malt is usually prepared from barley. This is
steeped or soaked in warm water for twenty-four hours,
drained and kept exposed to the air, thoroughly stirred and
moist, until it germinates. Various mechanical devices are
used for this purpose. In some cases large revolving drums
containing the moist barley are used. In other cases large
tanks with mechanical stirrers are employed, and in still
others the malt is spread out on the floor and turned over
with shovels, by workmen. An examination of a barley
grain, or the grain of any grass, will show that the young
plant or germ lies on one side near the base of the starch or
endosperm of the seed. The major portion of this germ is
made up of a comparatively large shield-shaped organ
called the scutellum. When the seed is soaked and started
to germinate, certain cells of this scutellum form consider-
able quantities of the enzymes amylase or diastase. As the
plant begins to grow this diastase diffuses out into the
endosperm, transforming the starch into soluble sugars,
which may be absorbed by the growing plantlet and used as
food. The maltster has learned to recognize that stage in

186 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

the sprouting of the barley grain in which there has devel-
oped the maximum amount of diastase together with the
minimum amount of transformation of starch. The sprouts
are then broken off and the grain dried. This is termed the
malt and is usually ground before use. The malt contains
not only enough amylase or diastase to transform the starch
present but much greater quantities as well. When ground
malt is mixed with warm water the diastase goes into solu-
tion and rapidly transforms the starch present into the
sugar maltose. If malt is added to any solution of boiled
starch, there will be rapid transformation of practically all
the starch present into maltose. For example, corn meal,
rice meal, or boiled potatoes, when mixed with malt, soon
dissolve and become sweet, due to the development of the
malt sugar. Malt is the most common of the agencies
employed for the saccharification of starch, preliminary to
alcoholic fermentation, or when such transformation is
required for other purposes.

In the commercial manufacture of alcohol for industrial
purposes, cheaper methods for saccharifying starch have
been discovered. Certain species of molds, particularly
certain members of the genera Aspergillus and Rhizopus,
are known to secrete large quantities of the enzyme amylase.
These molds may be grown in the laboratory upon suitable
sugar media, and the spores collected in large quantities.
The material to be saccharified is usually boiled to soften it.
Most frequently starch from rice, potatoes, or corn, is
employed for this purpose. When sufficiently cooled, the
spores of the Aspergillus or Rhizopus are introduced and
mixed through thoroughly. Frequently air is bubbled
through the mass to facilitate rapid growth of the mold
mycelium. This soon penetrates to all portions of the mass
and at once begins to excrete the enzyme amylase. Within
a few hours or days, the whole mass of starch has been
liquefied and transformed into sugar. Yeast can then be

FERMENTATION OF CARBOHYDRATES 187

added and the sugar fermented to alcohol and carbon
dioxide.

Alcoholic Fermentation of Sugar Solutions. — The juices
of many fruits contain sufficient quantities of sugar, usually
dextrose, levulose, sometimes cane sugar, so that they fur-
nish a natural medium for the growth of yeasts. In fact,
yeasts in nature apparently grow for the most part upon
the surfaces of bruised and diseased fruits. They are
probably transported from one fruit to another by the
agency of flies and various sucking insects. It is apparent,
therefore, that when juice is pressed from fruits it usually
contains a considerable inoculation of yeasts. The juices of
fruits likewise usually contain considerable quantities of
acids, particularly malic, citric, and tartaric. The hydrogen
ion concentration in consequence is so high that most species
of bacteria are inhibited from growth. This acidity is some-
times intensified by the wine manufacturer by the so-called
process of sulphuring. In this process fumes of S02 are
introduced, or compounds readily yielding S02 are added.
The yeasts most commonly present on the surfaces of fruits,
and which develop in their juices, belong to the group
frequently termed Saccharomyces ellipsoideus because of
the ellipsoid form characteristic of the cells.

Wines are prepared from the juice of the grape, cider
from the juice of the apple, and perry from the juice of the
pear. These may in turn be utilized for'the manufacture of
vinegar. It should be noted that -afeoholifc fermentation is
essentially an anaerobic process. If any of these juices are
left exposed to the air, the acetic bacteria will ordinarily
begin to grow, transforming the product into vinegar. The
juice of the century plant or agave in Mexico is fermented
to produce pulque, and in certain tropical countries the
juices of certain palms are fermented for the production of
palm urine.

As previously noted, most of the common yeasts do not

188 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

possess the power to ferment lactose. A few species are
known, however, which do have the power of developing
lactase, therefore they are capable of fermenting milk.
Alcoholic milk beverages have been prepared in many coun-
tries. The alcoholic fermentation in this case is usually
accompanied by the formation of lactic acid as well, the
product being a type of sour milk with a low percentage of
alcohol.

Beer and ale are prepared by soaking malt in water to
allow the action of the diastase upon the starch, transform-
ing it into the sugar maltose. Certain proteolytic enzymes
at the same time dissolve portions of the protein content of
the malt. The solution is termed beerwort. To this, both in
order to control undesirable fermentation and to impart a
desirable flavor, an extract of hops is added. This hopped
wort is then placed in large fermenting vats and yeast
added, usually in pure culture. Fermentation proceeds
until the desired concentration of alcohol has been secured.
When fermentation is complete, the beer is clarified and put
in bottles or kegs for distribution. The character of the
product depends in large measure upon the kind of yeast
used, particularly upon the use of bottom or top yeast.

Preparation of Distilled Liquors. — By a process of dis-
tillation the percentage of alcohol present in the final
product may be greatly increased. Rum is prepared by
fermenting molasses, the resulting alcoholic solution being
distilled and flavored. Brandy is prepared by the prepara-
tion of wine or cider. Whisky is prepared by the inocula-
tion of a mash made of malt and either ground grains or
potatoes, which is then inoculated with yeasts. When the
alcohol content has reached a sufficient degree of concentra-
tion and the fermentation is complete, the alcohol is distilled
over and the product aged in suitable containers.

Manufacture of Commercial Alcohol. — The primary
requisite for the manufacture of alcohol on a commercial

FERMENTATION OF CARBOHYDRATES 189

scale, is a cheap source of carbohydrate. Attempts have
been made to hydrolyze the cellulose of wood and other
materials, and this is being carried on on a commercial scale
in some of the southern states. Usually, however, potatoes
or cornstarch constitute the cheapest available carbo-
hydrate. This is hydrolyzed either by the use of malt or of
certain of the molds already described. It is then inoculated
with yeast and when fermentation is complete, the alcohol
distilled over.

Alcoholic Fermentation in Bread Making. — Many dif-
ferent leavening agents have been utilized in the prepara-
tion of bread. When wheat flour and water are mixed in
proper proportions, the gluten of the flour forms a sticky
or pasty mass, taking up most of the water. This is made
porous by the introduction of minute gas bubbles, usually
of carbon dioxide. This gas is, generally, on a commercial
scale, generated by the growth of yeasts. The yeast used in
bread making is of the same general type as that employed
in the manufacture of beer. It will be recalled that yeast
does not have the power to produce diastase, therefore fer-
mentation induced by the yeast cannot take place until
sugar is supplied. Flour contains small quantities of dias-
tase and when mixed with water there is sufficient formation
of sugar to allow of some growth of yeast. In commercial
bread manufacturing, however, there is frequently added
sugar, either maltose, dextrose, or sucrose, to increase the
rapidity of the yeast development. Frequently also various
chemicals are added to act as yeast stimulants. Certain
salts of ammonia and of phosphoric acid increase materially
the rapidity of yeast growth, and the production of alcohol
and carbon dioxide. When, as the result of the growth of
the yeast and proper kneading and mixing, the gas bubbles
of carbon dioxide have been sufficiently distributed through-
out the dough, it may be molded into form and baked. This
process drives off most of the alcohol, coagulates the protein

190 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

or gluten, and increases at the same time the size of the gas
bubbles.

In some cases bacteria have been substituted for yeast.
Certain organisms belonging to the genus Bacterium are
known to be able to split starch with the formation of car-
bon dioxide and hydrogen. "Salt-rising bread" is made
by utilizing microorganisms of this type.

In order to prevent rapid deterioration of the bread after
baking it is necessary that a certain amount of acid be pres-
ent in the dough. This is usually formed as a result of the
growth of certain lactic acid bacteria. Occasionally the acid
is added by bakers as such. Proper observance of the
acidity improves the texture of the loaf and renders impos-
sible the development of ropy or stringy bread.

Alcohol Production in Other Foods. — More or less alco-
holic fermentation may be incident to manufacture by fer-
mentation of certain articles of food for man and domestic
animals. During the process of fermentation of sauerkraut
and in the preparation of ensilage more or less alcohol may
be formed. Usually, however, the lactic bacteria outgrow
the yeast, and alcoholic fermentation is secondary.

The Commercial Preparation of Yeast. — Originally it
was customary for the housewife or the baker to carry along
his own culture of yeast. Such a culture was ordinarily
prepared by inoculating a mixture of potato water, sugar,
hops, salt, and sometimes other ingredients, with a little of
a preceding batch of the material. The culture consisted
of a mixture of alcohol-forming yeasts and lactic acid bac-
teria. Such a home-made culture is, in the United States,
usually termed a starter. In Great Britain it is usually
called a barm.

In the commercial manufacture of yeast, it is necessary to
grow large quantities of the yeast plants, free them from
the material in which they have been growing, and press
them into cakes. This is usually aecomplishe/1 by seeding a

FERMENTATION OF CARBOHYDRATES 191

sugar solution with a suitable type of yeast and small quan-
tities of lactic acid bacteria. Usually air is bubbled through
the fermenting mixture. The yeasts multiply rapidly.
They are removed from the liquid in which they are grow-
ing, either by sedimentation or by centrifugation in a
machine somewhat resembling a milk separator. The yeast
cells are then washed, again separated and pressed into
cakes. In the so-called compressed yeast a comparatively
small amount of starch will be present. In the dried yeast
cakes the yeast is mixed with corn meal, or similar absorb-
ent material, pressed into cakes and dried.

Development of Fusel Oils in Alcoholic Fermentation.—
Important by-products in alcoholic fermentation are the
fusel oils, consisting primarily of isoamyl alcohol. Formerly
it was supposed that these were derived from the sugar, but
more recent study has shown these to be derived -from the
amino acids developed from the digestion of proteins. Iso-
amyl alcohol is developed from isoleucine.

CHAPTER XVI

LACTIC ACID FEEMENTATION— THE GENEEA LACTOBACIL-
LUS, STKEPTOCOCCUS AND BACTERIUM

LACTIC acid is produced in nature commonly as the result
of the action of various microorganisms upon carbo-
hydrates; it is also found in small quantities in tissues of
the body as a result of muscular activity. Lactic acid fer-
mentation is the most common change observed in milk, and
fermentation of this type is most important in the develop-
ment of the acid so necessary in the preservation of certain
foodstuffs such as silage, sauerkraut, pickles and other fer-
mented foods.

The exact chemistry of the formation of lactic acid has
not been adequately explained. It is generally assumed to
be due to the activity of an intracellular enzyme, the so-
called lactacidase. The transformation does not require the
presence of free oxygen, that is, it is essentially anaerobic.
The change is one brought about by the organism as a
means of securing energy. It is quite possible that the
chemical transformation of sugar into lactic acid occurs in
several steps. Disregarding the intermediate products,
however, the reaction may be written :

C6H1206 = 2CH3 • CHOH • COOH
Glucose Lactic acid

It will be noted that the lactic acid molecule contains
one asymmetric carbon atom. There are, therefore, two
lactic acid forms, a levo acid and a dextro acid, the former
turning the plane of polarized light to the left, the latter to
the right. The kind of lactic acid developed in a particular

192

LACTIC ACID FERMENTATION 193

fermentative process apparently depends very largely upon
the kind of organisms bringing about the change, the carbo-
hydrate fermented and the temperature. Very frequently
the acid formed is of the inactive type, that is, it consists of
an equal mixture of the two acids, levo and dextro.

Lactic acid is the principal fermentative product of many
kinds of bacteria belonging to the genera Streptococcus and
Lactobacillus. Bacteria belonging to other groups, however,
may sometimes produce small amounts of lactic acid asso-
ciated with larger amounts of succinic, acetic, butyric or
propionic acids.

ORGANISMS PRODUCING LACTIC ACID

The organisms producing lactic acid primarily belong
for the most part to the genera Streptococcus and Lacto-
bacillus. Organisms producing smaller amounts of lactic
acid and larger amounts of volatile acids, proteolytic fer-
ments, etc., in general belong to the genera Bacterium and
Staphylococcus.

The Genus Streptococcus. — There is much discussion in
the literature relating to the souring of milk as to whether
lactic acid production in milk is usually due to the presence
of streptococci or of short rods occurring in chains. This
has given rise to considerable confusion in names. It seems,
however, that the organisms frequently alluded to in liter-
ature as Bacillus lactis acidi or Bacterium lactis acidi are
for the most part, at least, streptococci and may be included
under the species name, Streptococcus lacticus.

Another source of confusion in discussions of the strepto-
cocci as lactic acid producers has been the fact that the first
species of Streptococcus studied, S. pyogenes, as well as many
other species since described, are found associated with dis-
ease in man and animals. This led at one time to recommenda-
tion for the condemnation of milk which was found upon
microscopic examination to show chains of cocci. It seems to

194 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

be well established that at least one, perhaps several, species
of nonpathogenic streptococci are the most common organ-
isms instrumental in bringing about lactic acid fermenta-
tion of milk.

Streptococcus lacticus has spherical cells sometimes
slightly elongated, occurring in chains of greater or less
length. It is Gram-positive, does not produce spores and is
nonmotile. Apparently there are some strains, perhaps dis-
tinct varieties, capable of producing capsules. Certain of
these capsulated types are responsible occasionally for the
development of ropiness in milk, and for the sliminess
experienced in certain cultures or starters. Streptococcus
lacticus grows well, but never luxuriantly, on many of the
laboratory media. Its growth is greatly increased in amount
by the presence of suitable sugars which it may ferment.
Media prepared from whey are frequently found to be use-
ful. Lactose agar plates poured from souring milk will
show the organisms developing as small pin-point colonies,
frequently lying somewhat below the surface of the medium.
If litmus or some other suitable indicator is present, the
medium surrounding the colony will be noted to have
become intensely acid. Upon agar slants, the colonies are
usually more or less separated or discrete, at first scarcely
visible, and at last dewdroplike in appearance. In ordinary
broth the organism does not grow well unless sugar is
present. Usually the medium clears rapidly by sedimenta-
tion. Gelatin is not liquefied.

The common sugars, dextrose, sucrose and lactose, are all
fermented with the production of lactic acid but never with
the formation of gas. There is some confusion in the liter-
ature as to the type of acid produced, but pure cultures in
certain sugars, at least, frequently produce the dextro type-
only. When grown in milk sufficient acid is usually formed
from the lactose (.5 to 1.25 per cent) so that coagulation or
development of an acid curd occurs. The curd so formed is

LACTIC ACID FERMENTATION 195

usually smooth, free from gas bubbles, has a pleasant acid
flavor and with little or no tendency to shrink and expel the
whey. Its optimum growth temperature, apparently, is
about blood heat, but it grows well (particularly in milk) at
room temperatures and slowly at the temperature of the ice
chest. Apparently there are strains of this organism which
can resist the ordinary temperatures used in pasteurization.
This is evidenced by the fact that milk which has been pas-
teurized in commercial plants usually retains sufficient num-
bers of these organisms in the living condition so that it
sours.

The Grenus Lactobacillus. — The bacteria of this genus
are all Gram-positive, nonspore-producing rods developing
commonly in milk and various fermented foodstuffs. A
few species have been reported from the alimentary tract
of man and animals. The group is sometimes known as the
group of high acid bacteria because certain of the species
will grow in a medium having a higher hydrogen ion con-
centration than will permit the growth of most bacteria,
even of the Streptococci. Some of the species are relatively

a bed

FIG. 50. — LACTIC ACID BACTERIA. A. Streptococcus lacticus. B. Lac-
tobacillus bulgaricus. G. Lactobacillus lactus acidi. D. Bacterium
aero genes.

short rods. Most of them, however, are decidedly elongated,
sometimes almost filamentous. The shortest rod forms
apparently intergrade with the Streptococci; it is accord-
ingly sometimes difficult to differentiate between Strepto-
coccus lacticus and the Lactobacillus lactis acidi.

One of the best known species belonging to this group is
the Lactobacillus bulgaricus. The organism was first

196 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

described by Metschnikoff who, when traveling through
southern Russia and the Balkan countries, was much im-
pressed by the large quantities of fermented milk and sour
milk beverages used by the peasant classes as food. He also
reached the conclusion that the peasants of this region were
particularly long lived. He attributed this longevity to the
large use of soured milk in the diet. He made a study of
the favorite milk beverages and succeeded in isolating the
organism chiefly responsible for the lactic acid formation.
Inasmuch as it was isolated from the Bulgarian soured milk,
it was given the specific name bulgaricus. The organism is
a relatively large rod, sometimes filamentous. The cells are
usually .5 to I//, in diameter, and 2 to 3/x in length. In old
cultures, however, some of the rods may be many microns in
length. While the organism is definitely Gram-positive in
young cultures, smears made from old cultures may show a
mixture of Gram-positive and Gram-negative rods. Single
cells may sometimes show a portion retaining the Gram
stain and another portion decolorized and taking the con-
trast stain. This organism does not grow very readily upon
the ordinary culture media, although its development in
milk at the right temperature is abundant. The colonies
produced in agar resemble a tiny mass of wool. The thread-
shaped rods will be seen to penetrate the medium in all
directions. A stab culture in whey agar or lactose agar
gives a fir-tree appearance. Lactobacillus bulgaricus grows
best at temperatures above blood heat, the optimum being
42° to 45°. Growth is slow at room temperatures. The total
amount of acid formed is usually somewhat greater than
produced by the Streptococcus lacticus, sometimes as much
as four per cent being observed. Inasmuch as growth
evidently continues in these relatively strong solutions of
acid, the organism is termed acid tolerant or acidopkilous.
The curd produced in milk is usually somewhat slimy,
smooth, and does not tend to contract and expel the whey.

LACTIC ACID FERMENTATION 197

The organism grows best under anaerobic conditions, better,
therefore, below the surface of the medium than at the top.

Lactobacillus caucasicus and certain related species have
been isolated from cheese and are probably important in the
ripening process. Still other forms have been isolated from
fermenting silage, sauerkraut, and other fermented foods.
Many of them will grow at moderate temperatures or room
temperatures and' a few of them apparently possess the
power of producing gas, particularly carbon dioxide.

Bacteria belonging to the genera Bacterium, and Staphy-
lococcus are also capable of producing some acid in milk.
Members of the former genus are important in development
of lactic acid in certain foodstuffs.

The Genus Bacterium. — The organisms belonging to this
group are all Gram-negative nonspore-forming rods, which
develop readily upon the ordinary culture media, and are
usually either aerobic or facultative. The most important
forms are Bacterium aerogenes from milk and several
species associated with the fermentation of sauerkraut and
of silage. The organisms of .this group, when present in
milk, are usually termed the abnormal and undesirable
lactic acid bacteria. Some of them, however, may be desir-
able in the fermentation of other food products. Most of
the bacteria belonging to this group, particularly those of
importance in milk and the dairy industry, have the power
of producing gas from sugars. They develop also certain of
the volatile acids in addition to the formation of lactic acid.

Bacterium aerogenes grows well on ordinary culture
media. The colonies on agar or gelatin are much larger and
more inclined to be slimy than those of the organisms
belonging to the genera Streptococcus or Lactobacillus.
For the most part they grow best at blood heat. Most of
them also develop well at lower temperatures. When suit-
able carbohydrates which they can ferment are present, the
organisms will develop anaerobically. In the absence of

198 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

such sugars they are aerobic. Dextrose, sucrose, maltose,
and lactose are all fermented with the development of acid
and gas. A total of one to one and one-quarter per cent of
acid may develop in milk, but as mentioned above, it is
only in part lactic acjd, acetic and other volatile acids being
present. Usually the lactic acid formed is levorotatory.
The gas developed is a mixture of hydrogen and carbon
dioxide. When grown in milk the curd produced is usually
torn by gas bubbles and shrinks, expelling a considerable
proportion of the whey. When present in milk in unusually
large quantities, such organisms may interfere with the pro-
duction of the best quality of cheese.

TECHNICAL UTILIZATION OP LACTIC ACID
FERMENTATION

Associative Action. — It has frequently been observed,
particularly in the development of bacteria in milk, that
mixed cultures are able to bring about changes which pure
cultures of the constituent organisms could not produce.
In some cases there is an increased production of acid. In
other cases the type of the change in milk is altered materi-
ally. It will be noted below that several of these associative
actions, particularly those among the desirable bacteria,
bring about changes of considerable economic importance.
Inasmuch as bacteria rarely occur in pure culture in nature,
the spontaneous changes brought about in milk and the
various fermented foods must, therefore, in general be the
result of associative action.

LACTIC ACID BACTERIA IN MILK

Milk when freshly drawn, practically always contains
some lactic acid bacteria, usually streptococci and lacto-
bacilli. These may have come in part from the udder, from
the coat of the cow and very largely, usually, from the milk
utensils. When the milk is allowed to stand, particularly in

LACTIC ACID FERMENTATION 199

a warm place, they multiply very rapidly and usually over-
grow other kinds of bacteria which may be present. Sugar
is rapidly transformed into lactic acid. The total number
of the lactic acid bacteria developing will usually run into
the hundreds of millions per cubic centimeter. Eventually
sufficient lactic acid develops to curdle the milk and
effectually stops the growth of the bacteria. Streptococcus
lacticus is usually inhibited first. Certain species of lacto-
bacilli may go on growing, producing larger quantities of
lactic acid, after the inhibition of the streptococci.

When members of the genus Bacterium, particularly
Bacterium aerogenes, are present in sufficient quantities to
modify the course of fermentation, the curd developed, as
noted above, is usually broken and tends to shrink.

Soured Milk Beverages. — An artificial buttermilk (fre-
quently sold at soda fountains) is prepared by inoculating
milk with a previously soured sample of milk or with a pure
culture of lactic acid bacteria. In most cases the resultant
souring is produced by an organism of the Streptococcus
lacticus type. When the milk has curdled it is beaten or
churned, so that the casein becomes finely divided. In some
respects it is preferable to the true buttermilk as a beverage
because of the greater uniformity in flavor and texture
secured.

Milk sherbets (as lacto) are frequently prepared from
milk soured in this manner, flavored with fruit juices, and
frozen.

In some cases, the organisms used for souring the milk
belong to the genus Lactobacillus, usually Lactobacillus
bulgaricus. As noted above, milk soured by this organism
is somewhat more slimy and has a smoother curd than milk
soured with Streptococcus lacticus or related organisms.
The vogue of "the so-called Bulgarian soured milk owes its
origin to the work of Metschnikoff. This observer came to
the conclusion that the consumption of soured milk bev-

200 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

erages of this type is decidedly conducive to longevity be-
cause it induced the development of a lactic acid flora in
the large intestine, and there was, in consequence, little or
no development and consequent absorption of poisonous
putrefactive substances from the colon. Acting upon this
suggestion pharmaceutical houses have prepared tablets
consisting usually of mixtures of milk sugar and dried
cultures of Lactobacillus bulgaricus. More recent work
seems to indicate that while soured milk of this type is an
excellent food, nevertheless, the unusual health-promoting
qualities ascribed to it by Metschnikoff have scarcely been
confirmed by the facts as they have developed. Bulgarian
soured milk undoubtedly has a place in the dietary of
invalids and convalescents.

Soured milk beverages have been used apparently since
ancient times practically all around the shore of the Medi-
terranean both in Africa and Europe, about the Black Sea
and the Balkan countries, in Greece, in southern Russia, the
Caucasus and parts of India. The methods of manufacture
differ materially with the locality. In some cases the milk
is subjected to a preliminary heating and is then inoculated
by introducing a small amount of a previously fermented
batch of milk. It is then kept in a comparatively warm
place. This encourages the growth of bacteria belonging to
the group of lactobacilli, particularly Lactobacittus ~bul-
garicus. A soured milk of good flavor and comparatively
high acid content is thereby secured.

In c'ertain districts dried kernels, termed kefir grains, are
introduced into the milk which is to be fermented. When
examined microscopically these kefir grains are found to be
made up of gelatinous masses of bacteria with yeast cells
embedded throughout. When introduced into milk a com-
bined lactic acid and alcoholic fermentation occurs. The
resultant beverage is somewhat acid and effervescent, con-
taining a low percentage of alcohol. It should be observed

LACTIC ACID FERMENTATION 201

that the yeasts present are those capable of producing the
enzyme lactase, therefore, of fermenting lactose.

Sour Curd Cheese. — When milk that has been soured
is heated, the curd contracts, the whey is expelled and may
readily be drained away. The curd may then be worked or
molded, or mixed with butter or various flavoring materials,
and converted into a sour milk cheese usually termed Dutch
cheese or cottage cheese. This type of cheese is not usually
subjected to a ripening process.

The Use of Starter.— While cream may be churned into
butter without any preliminary fermentation and while
, butter from this sweet cream is preferred in certain locali-
ties and for certain purposes, nevertheless, most of the
butter sold in the United States is churned from cream
which has been allowed to ripen, that is, it has been allowed
to undergo a process of acid fermentation. The flavor and
aroma of the butter will depend very largely upon the type
- of organisms which have been growing in the cream. The
butter fat absorbs considerable amounts of dissolved sub-
stances present in the fermenting cream, it is, therefore,
highly desirable that these should be of the proper char-
acter. If batches of cream are allowed to ripen spontane-
ously without the addition of any starter, there is apt to be
a marked lack of uniformity in the product. A much better
and more uniform result is secured by inoculating the
cream which is to be ripened with a considerable amount of
desirable lactic acid bacteria. Such organisms are sold
under the name of commercial starters. It is generally
assumed that they are pure cultures, or practically pure
cultures, of Streptococcus lacticus or some very closely
related organism. This is introduced into pasteurized milk
and allowed to grow until the milk has reached a satisfac-
tory state of acidity. With this a larger bulk of milk is
inoculated and finally the whole mass used as a starter for
the cream which is to be churned. A heavy inoculation with

202 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

the desirable microorganisms and their growth products
usually overwhelms undesirable types of bacteria and leads
to the development of a satisfactory product.

Recent studies have made it evident that the changes
brought about in a satisfactory starter are not the results
simply of the growth of Streptococcus lacticus. Another
organism closely related to it must ordinarily grow with it.
Cream which has been ripened by means of a pure culture
of the ordinary Streptococcus lacticus does not produce but-
ter with as satisfactory a flavor and aroma as that produced
by cream which has been inoculated with a mixture of these
organisms. The ripening produced by a starter is, there-
fore, an excellent example of associative action. The true
Streptococcues lacticus apparently produces very little
change in milk or in cream other than the development of
the pure lactic acid from lactose. The associative organism,
also a coccus (either Streptococcus citrovorus or Strepto-
coccus paracitrovorus) can by its own growth bring about
very little change in milk. When grown in the presence of
Streptococcus lacticus, however, the activity of this organ-
ism is greatly stimulated and small amounts of volatile
acids are developed. It is to the absorption of these and
perhaps of other growth products that the butter owes its
characteristic aroma and flavor.

EFFECTS OF PASTEURIZATION UPON THE SOURING OF MILK

Some of the earlier opponents of pasteurized milk urged
that the process of pasteurization, if properly performed in
a manner to eliminate all disease-producing bacteria in the
milk, would likewise kill all of the lactic acid bacteria, and
that the only organisms remaining alive would be spore-
producing bacilli and clostridia. These would then begin
to grow, after the milk had cooled, and bring about putre-
factive changes. However, the work of Ayers and his
associates in the Dairy Division of the Bureau of Animal

LACTIC ACID FERMENTATION 203

Industry seemed to indicate that commercially pasteurized
milk practically always contains resistant strains of
Streptococcus lacticus which will bring about normal sour-
ing, and that putrefactive changes in pasteurized milk are
little if any more common than in milk which has not been
pasteurized.

Secondary Changes in Soured Milk. — When soured milk
is allowed to stand, lactic acid formation eventually ceases
and very little change occurs in the milk for a considerable
period of time unless air is allowed access to it. This has
been taken advantage of in certain districts in northern
Europe to preserve meat for a time in soured milk.

In certain of the Scandinavian countries, particularly in
Norway, milk is kept from the winter months for use in the
home during the summer months while the cattle are away
in the mountain pastures. The milk is first heated and then
stored in casks after inoculation with a suitable starter.
The organisms present are lactic acid bacteria and a species
capable of producing a considerable degree of viscosity.
This ropy milk will keep for a considerable period of time
if air is excluded.

When air gains access to soured milk, certain aerobic
forms, particularly certain molds such as O'idium lactis,(
begin to grow upon the surface. These organisms rapidly
oxidize the lactic acid to carbon dioxide and water, thus
decreasing the acidity of the solution. At the same time
certain of the molds produce proteolytic ferments which
bring about partial or complete digestion of the casein.
When the acidity of the solution has been sufficiently
decreased, certain of the aerobic and anaerobic spore-
producing bacteria find conditions favorable for develop-
ment and bring about proteolytic or even putrefactive
changes.

Lactic Acid in Cheese Manufacture. — Rennet curd
cheeses are in part ripened through the activity of lactic

204 AGEICULTURAL AND INDUSTRIAL BACTERIOLOGY

acid bacteria. Usually rennet acts rather more rapidly and
more satisfactorily upon milk in which a slight amount of
lactic acid has developed. Bacteria of the group Strepto-
coccus lacticus and particularly Lactobacillus caucasicus
and related species rapidly transform any residual lactose
in cheese into lactic acid. The other changes incident to
cheese ripening will be discussed in another connection.

LACTIC ACID IN FOOD PRESERVATION

Ensilage. — Ensilage or silage is prepared by chopping
or shredding various green foods and packing them com-
pactly into an air-tight structure termed a silo. In general
the food plants most used for a silo are those which contain
considerable quantities of sugar when green and of starch
when mature; such, for example, are corn and sorghum.
Other fodder plants may be ensiled but unless the content
of sugar or starch is sufficiently high, abnormal or putre-
factive changes may occur. This may be obviated by mix-
ing with corn or with sorghum. Such, for example, would
be silage made from mixtures of Indian corn and soy beans
or cow peas.

The most essential change occurring in the ensilage is
the transformation of a part of the carbohydrates present
into lactic acid. Carbon dioxide, acetic acid, traces of
butyric and other acids and sometimes alcohol may be
formed. The lactic acid accumulates in quantities sufficient
to prevent the development of putrefactive or other unde-
sirable microorganisms, providing conditions are satisfac-
tory.

For proper fermentation in the preparation of silage it
is necessary to have, first, material properly prepared by
shredding; second, the ensilage packed tightly into an air-
tight container or silo so as to leave as little air space as is
practicable; third, the presence of an optimum amount of
moisture, and fourth, the presence of suitable bacteria.

LACTIC ACID FEEMENTATION 205

Most of the difficulties in the preparation of satisfactory
ensilage are due to defects of one of the first three factors.
The suitable lactic acid bacteria are usually present. How-
ever, certain investigators have found increased uniformity
of results when the silage is inoculated with suitable mix-
tures of lactic acid bacteria at the time of preparation.

Usually the materials placed in the silo are green, that is,
they contain many living cells. Among the first changes
which occur, therefore, in the silo are those due to the
activity of these living cells and of the enzymes present in
them. Certain of the earlier investigators of silage con-
cluded that these changes were by far the most important.
More recent investigations, however, have shown that the
bacteria are responsible for most of the fermentative
changes. Yeasts, which are usually present, may bring
about some alcoholic fermentation. The lactic acid bacteria,
however, usually overgrow and outgrow the other organisms
present and produce sufficient lactic acid to inhibit their
development completely. Eventually the growth of the
lactic acid bacteria is likewise stopped, and the food
material or silage will then usually keep almost indefinitely,
providing air does not gain access.

A considerable rise in temperature may take place in the
silo during the process of fermentation. If this increase
is excessive it usually indicates that there is not sufficient
moisture present and the material has not been tightly
enough packed. The lactic acid fermentation does not give
rise to quantities of heat comparable with the amount
developed during oxidative changes occurring due to the
growth of molds and bacteria in the presence of atmos-
pheric oxygen. The surface layers of silage usually become
quite warm, for air can penetrate to the depth of several
feet. These, therefore, can be preserved only by covering
the surface carefully with something which will exclude the
air.

206 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

If air gains access to the silage, molds will soon develop
and oxidize the lactic acid to carbon dioxide and water, and
putrefactive changes may then be brought about. Moldy
silage, of course, has lost much of its feeding value and is
dangerous particularly for horses.

The organisms responsible for bringing about the fer-
mentation are members of the genera Bacterium and
Lactobacillus for the most part. Organisms closely resem-
bling if not identical with Lactobacillus bulgaricus and
Bacterium aerogenes are usually abundant. The character-
istic odor of good silage is due to the presence of certain
volatile acids and to the formation of esters with the traces
of alcohol present.

Carbon dioxide may be produced in considerable amounts
during the fermentation of the silage. Care should be used,
therefore, in entering a silo which is partly filled with
silage. It may be recalled that carbon dioxide is heavier
than air and that there are numerous cases of asphyxiation
on record where care has not been used to see that the silo
is free from this gas in injurious quantities. The danger
would be particularly great in a silo where the process of
filling has been interrupted and men enter the silo a few
days later to tramp down new silage as it enters.

The fact that the fermentation is essentially anaerobic
and that there are organisms present, such as members of
the genus Bacterium, which produce not only carbon
dioxide but hydrogen as well, leads to strong reducing
action. Considerable amounts of sugar are transformed
into the polyatomic alcohols. Fructose especially is con-
verted into mannitol (C6H1406).

Sauerkraut. — Sauerkraut is prepared by shredding or
slicing cabbage, mixing with salt and pressing firmly into
casks or vats. The sugary juices of the cabbage leaves
together with the salt soon form a liquid which covers the
mass. Lactic acid fermentation, brought about largely by

LACTIC ACID FEKMENTATION 207

microorganisms belonging to the genus Bacterium, perhaps
secondarily by organisms of the genus Lactouacillus, ensues.
Gas is given off in considerable quantities. Among the
gases developed are considerable quantities of those con-
taining sulphur, particularly hydrogen sulphide. It may
be noted that cabbage contains appreciable amounts of
glucosides containing sulphur. These are largely broken
down during the process of fermentation. Finally the lactic
acid content of the brine rises to a point which will inhibit
the growth of all microorganisms which are not aerobic.
Sauerkraut thus prepared will keep for a considerable
length of time if not exposed to atmospheric oxygen. When
so exposed, however, it rapidly molds, the lactic acid is
oxidized and decay occurs.

Other Fermented Foods. — Sweet corn, beets, and some
other foods are occasionally preserved in a manner similar
to sauerkraut. When mixed with a suitable amount of salt
and pressed firmly into jars or barrels, lactic acid fermenta-
tion will occur and effectually preserve the food material as
long as it is not in contact with the air.

Lactic Acid in Yeast Manufacture. — A medium some-
what acid is usually required in the production of commer-
cial yeast in order to prevent the development of undesir-
able microorganisms. Certain bacteria, members of the
genus Lactobacillus, are usually introduced into the vats
preliminary to the growth of yeasts in the commercial
manufacture of compressed yeast or dried yeast cakes.

Lactic Acid in Bread Making. — The bacteria present in
yeast cakes and in flour usually bring about the develop-
ment of some lactic acid during the leavening process in
the bread. The texture of the loaf and its flavor are in part
dependent upon the development of the lactic acid. Fur-
thermore, the keeping quality of the bread is directly
related to it. If sufficient lactic acid does not form during
the leavening process the bread is very apt to become ropy

208 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

when kept in a warm place after baking. Commercial
bakers not infrequently add lactic acid to the dough to
insure its presence in sufficient quantities.

So-called " salt-rising ' ' and certain types of "sour
dough7' bread are leavened by the action of organisms
belonging to the genus Bacterium. They produce small
quantities of lactic acid and considerable quantities of gas,
particularly carbon dioxide and hydrogen. " They thus
replace yeast in forming the gas bubbles essential to the
leavening of the loaf.

CHAPTER XVII

ACETIC, BUTYRIC, OXALIC AND CITRIC FERMENTATION—
THE GENERA ACETOBACTER, BACTERIUM, CLOSTRID-
IUM, ASPERGILLUS AND CITROMYCES.

MANY different acids have been described as produced by
microorganisms. Among the most important in this list are
formic, acetic, propionic, butyric, valerianic, succinic, lactic,
oxalic, and citric. Of these, lactic acid has already been
discussed. Acetic, butyric, oxalic and citric acids are of
considerable economic importance.

ACETIC FERMENTATION

Many microorganisms are known which are capable of
producing acetic acid. Comparatively few of these, how-
ever, are of economic significance. Acetic acid production
is most important in the manufacture of vinegar for use as
a condiment, although large quantities are produced by
bacterial fermentation for the manufacture of ingredients
(particularly amyl acetate) used in making certain ex-
plosives. Small quantities of acetic acid are also produced
in many fermented foods such as sauerkraut, ensilage, and
fermented pickles.

Types of Acetic Acid Fermentation. — Bacteria produc-
ing acetic acid may be divided into two groups; those
which bring about the change under aerobic conditions,
oxidizing alcohol to acetic acid; and those which bring
about fermentation of sugars under anaerobic conditions,
acetic acid being one of several of the products.

Alcoholic solutions, such as wine, beer or cider, when
allowed to stand open to the air soon become covered with a
growth popularly termed mother of vinegar. This is com-

209

210 AGKICULTUEAL AND INDUSTEIAL BACTERIOLOGY

posed largely of microorganisms which rapidly oxidize the
alcohol to acetic acid. The enzyme produced by the organ-
isms capable of bringing about this change has not been
isolated, but it probably may be regarded as an intracellular
enzyme belonging to the general group of the oxidases.
The reaction may be indicated as follows :

C2H5OH + 02 = CH3COOH + H20

The bacteria which bring about the change under ana-
erobic conditions usually produce considerable quantities
of butyric, lactic, succinic or other acids simultaneously
with the formation of acetic acid. The changes are rela-
tively complex and the process of the fermentation is not
well understood ; it cannot, therefore, readily be represented
by a simple chemical equation.

Groups of Acetic Bacteria. — As previously noted, the
bacteria producing acetic fermentation are either aerobic
or facultative and anaerobic. Of the former group the
organism Acetobacter aceti may be taken as a type. Of the
latter group, organisms belonging to the genera Bacterium
and Clostridium are worthy of mention.

The Genus Acetobacter.— To this genus of bacteria
belong most of the forms capable of oxidizing ethyl alcohol
to acetic acid under aerobic conditions. Many species of
Acetobacter have been described but all are apparently
closely related. They differ from each other slightly in
morphology, in their ability to produce large or small
amounts of acid and to attack various sugars. The "mother
of vinegar " which forms on the surface of wine, cider or
other alcoholic solutions when allowed to stand, consists for
the most part of a tangled mass of bacteria belonging to this
genus together with yeast cells instrumental in bringing
about the preliminary alcoholic fermentation. All of the
bacteria belonging to this genus are rod-shaped, usually
relatively long and occur in chains. They are usually non-

ACETIC FERMENTATION 211

motile and do not produce spores. For the most part, they
grow readily upon suitable culture media and are obligate
aerobes. Some produce such small quantities of acid that
they are of no economic significance. Others produce the
acid rapidly and in large quantities. Different species have
been described from wines, from cider, from beer and from
the so-called quick process of vinegar manufacture. The
cells for the most part produce considerable amounts of
slime and tend to form films on the surface of the medium.
Involution forms, that is, cells showing unusual shapes such

Pis. 51. — ACTETOBACTER. Normal rods and involution forms.

as clubs, branched forms, spheres, etc., are very common,
particularly in the older films.

Acetobacter aceti. — This organism may usually be dif-
ferentiated from related acetic bacteria by the fact that it
produces a surface pellicle upon the alcoholic solution which
can be easily broken up ; that is, the development of slime
is not so evident as in some other species. The slime from
this organism is not stained by the use of a solution of
iodine or of potassium iodide. The organism has usually
been isolated from the surface of souring beer.

Another organism which has been described from beer
vinegar is the Acetobacter pasteurianum. Some authors
have also described it as one of the important agencies in
the production of wine and cider vinegar. It develops as a
membrane on the surface of the liquid, at first appearing

212 AGEICULTURAL AND INDUSTRIAL BACTERIOLOGY

moist, later dry, and finally wrinkled, while the medium
below the surface remains quite clear. The bacteria occur
in chains, although greatly elongated filaments may be
observed. This organism produces a much larger amount of
slime than the Acetobacter aceti, and the slime turns blue
when potassium iodide or iodine is added. The involution
forms are particularly abundant. Upon beer-wort and
beer- wort gelatin the organism grows readily. It has a high
power of oxidizing alcohols to corresponding acids, ethyl
alcohol being rapidly transformed to acetic acid, propyl
alcohol to propionic acid, and dextrose to gluconic acid

(CH2OH(CHOH)4COOH).

Acetobacter orleanense is regarded as one of the most
common causes of acetic fermentation in fruit juices such
as the wines and ciders. It differs only in minor morpho-
logical and physiological characters from species of the
Acetobacter previously described. It can produce acid
from many sugars but best where it can oxidize alcohol to
acetic acid. It may be noted that this organism, as well as
other species of Aceiobacter may continue the action beyond
the point of complete oxidation to acetic acid, and may also,
when the alcohol supply has been exhausted, oxidize the
acetic acid to carbon dioxide and water. This makes it
necessary to pasteurize or to seal vinegar from the air when
it has been prepared, otherwise the same organisms which
produced it will destroy it or cause it to "lose its strength."

The Acetobacter schutzenbachii may be taken as a repre-
sentative of the species which have been described in the
so-called quick process of vinegar manufacture. While it
grows readily upon ordinary culture media, it does not
produce a heavy film upon the surface of liquids. It is,
therefore, not an organism which would ordinarily bring
about the transformation of cider or wine into vinegar by
the slow process requiring film formation. The bacterial

ACETIC FERMENTATION 213

cells are usually oval, sometimes elongated or pickle-shaped.
Osually they occur in pairs though sometimes they are
united into chains of different lengths. This organism
develops rapidly upon the shavings used in the quick vine-
gar process and is very active in the oxidation of alcohol to
acetic acid. As much as 11.5 per cent of acetic acid has
been produced in alcoholic solutions by organisms of this
type.

Vinegar Manufacture. — Vinegar may be defined as a
solution of acetic acid (usually from 4 to 8 per cent,
preferably about 5 to 6 per cent) developed by the fer-
mentation of alcoholic solutions of various types. The
origin of vinegar is ordinarily indicated by such phrases
as "cider vinegar," "wine vinegar, " "malt vinegar,"
"honey vinegar,' etc. The manufacture of vinegar will be
discussed under the headings of manufacture in the home
and on the farm and commercial manufacture.

Home Manufacture of Vinegar. — The materials most
commonly used for the home manufacture of vinegar are
cider or other fruit juices, honey, and artificial sugar solu-
tions. The first step in the process is always an alcoholic
fermentation, the transformation of the sugar, at least in
large part, into alcohol and carbon dioxide. This change
may be initiated by the introduction of yeast, but with the
fruit juices this usually is not necessary, the native wild
yeasts present being sufficiently numerous and active. For
the manufacture of a good quality of vinegar it is necessary
to have first, a sufficient exposure of surface of the liquid to
the air to allow the abundant development of the surface
film of acetic bacteria; second, the rapid transformation of
the alcohol into acetic acid due to the presence of desirable
acetic bacteria; third, maintenance of the material at a
suitable temperature, and finally, some method of stopping
the process upon the formation of the maximum amount of
acetic acid and before microorganisms have transformed

214 AGEICULTUEAL AND INDUSTKIAL BACTERIOLOGY

any considerable proportion of the acetic acid into carbon
dioxide and water, thus weakening the vinegar. For this
purpose casks or barrels may be partly filled with cider or
wine (preferably not more than one-half to two-thirds full
at the most), placed upon their sides and an opening left
for ingress of air. This opening should be closed with a
layer of cheesecloth or gauze to prevent insects from gain-
ing access. The process may be considerably hastened by
adding some good vinegar or some "mother of vinegar,"
or best of all a culture of the most suitable acetic bacteria.

Washings from honey extractors and improperly ripened
honey may be manufactured into vinegar of very fair
quality by the same process.

Many housewives in recent years liave manufactured
vinegar in small quantities in the home by making use of
what have come to be known popularly as "vinegar bees/'
These consist of dried, brownish, gelatinous lumps, varying
from the size of a pinhead to a bean. When examined
microscopically they are found to consist of a gelatinous
mass of bacteria with many yeasts. When dropped into a
solution of sugar, the yeasts at first multiply rapidly and
bring about alcoholic fermentation. The bacteria soon
begin to develop, produce the characteristic acetic film, and
transform the alcoholic solution into vinegar. Homemade
vinegar of this type is often prepared by the use of fruit
juices, syrups that have been used in preserving fruits,
from molasses, or even from cane sugar or honey. Some
samples of vinegar of fair quality are prepared in this way,
though usually the vinegar is not as good as that secured
by the fermentation of cider. In most instances the acid
content is relatively low, rarely reaching 4 or 5 per cent.
It should not be used, therefore, for preservation of food
materials requiring a high percentage of acid.

Commercial Manufacture of Vinegar. — The best commer-
cial vinegars are manufactured from cider and wine,

ACETIC FEEMENTATION 215

though vinegar of good quality may be secured from beer-
wort or malt extract. Much vinegar, however, is manu-
factured by the alcoholic fermentation of molasses or cheap
syrups. In some cases a mixture of acetic acid secured
from the destructive distillation of wood diluted with water
and colored with caramel is sold as vinegar. This is gen-
erally prohibited by law as fraudulent.

One of the chief aims in commercial manufacture of vine-
gar is the quickening of the process. One of the earlier
methods, and one still practiced, is the so-called Orleans
method, resembling in many respects the method of vinegar
manufacture already described as suitable for the home and
farm. A cask is partly filled with a mixture of wine and
good vinegar. It is allowed to stand until a satisfactory
scum or membrane has formed over the surface and until
the entire contents have been converted into vinegar. A
definite amount of the vinegar, usually a gallon or more, is
withdrawn and an equal amount of wine added, care being
taken not to break the membrane covering the surface of the
liquid. After the process is well started a portion of the
contents of the barrel may be withdrawn daily or at other
suitable intervals and equal amounts of wine added. The
method has certain disadvantages. The membrane is apt to
be broken and portions will settle to the bottom where the
organisms composing it cease growing and decompose, add-
ing to sediment and producing undesirable flavors.

Pasteur and his collaborators developed a so-called vat
method of vinegar manufacture in which a suitable mixture
of wine and good vinegar was poured into a relatively
shallow vat and a light wooden lattice floated upon the sur-
face. The membrane, consisting of acetic bacteria, soon
developed and remained supported by the wooden frame.
As soon as the material had been converted into vinegar a
portion was withdrawn and an equal amount of unfer-
mented material added. This withdrawal can be made

216 AGEICULTUEAL AND INDUSTRIAL BACTERIOLOGY

daily and a constant supply of vinegar thus secured. The
process has the advantage of providing abundant surface
area with consequent more rapid fermentation and it like-
wise preserves the acidifying membrane intact.

It may be readily deduced that the larger the relative
area of the surface of the liquid exposed to the air, and the
more rapid and thorough the aeration, the more rapid,
under given conditions, will be the transformation of
alcohol into acetic acid. This has led to the manufacture
and development of various devices for hastening the manu-
facture of vinegar by exposing the liquid in very thin layers
to the action of organisms in the presence of air, layers
much thinner than those present in the Pasteur vat method.
The device probably most used at the present time is the
so-called quick or German method of vinegar manufacture.
The apparatus consists essentially of a tall cask or wooden
cylinder filled with beech-wood shavings. Beech-wood is
chosen in preference to other woods because it contributes
but little to the flavor of water coming in contact with it.
The shavings rest upon a false bottom bored full of holes.
An automatic sprinkling device is installed above the top
of the shavings. These shavings are first inoculated with
suitable bacteria, usually organisms of the type of Aceto-
bacter schutzenbachii. This may be accomplished by
sprinkling the beech-wood shavings with a good quality of
vinegar previously manufactured by the process or pure
cultures may be used. The alcoholic solution to be fer-
mented is sprayed or sprinkled over the top of the shavings
and trickles down over their surfaces to the bottom and
finally into a suitable receptacle. The surface area of the
shavings is relatively large. The liquid flows down over
them in very thin layers. The microorganisms find favor-
able conditions for growth inasmuch as air is constantly
passing in at the bottom, up through the shavings and out
at the top. A constant circulation of air is easily main-

BUTYRIC FERMENTATION 217

tained because some heat is generated during the process of
fermentation, giving rise to convection currents. The bac-
teria soon cover the surfaces of the shavings with a con-
tinuous film. Conditions are optimum, therefore, for the
rapid oxidation of alcohol to acetic acid. After the
apparatus is in working order and the shavings thoroughly
inoculated and the rate of flow properly adjusted, the
alcoholic solution may run in at the top in a continuous
stream and vinegar be drawn off at the bottom.

BUTYRIC FERMENTATION

The development of butyric acid (C3H7COOH) in fer-
mentation is usually regarded as an undesirable change,
although in some operations such as retting of flax and
hemp, it may be desirable and even essential.

Butyric acid is produced in several distinct ways in
various fermentative processes. Certain anaerobic bacteria
ferment sugars (and in some cases polysaccharides) with
the formation of butyric acid and gas, usually carbon
dioxide and hydrogen. If the intermediate steps are dis-
regarded the reaction may be indicated as follows :

C6H120<; = C3H7COOH + 2C02 + 2H

Lactic acid or the lactates may also be fermented by these
organisms. The reaction in this case may be represented as :

2CH3-CHOH-COOH = C3H7COOH + 2C02 + 2H2

In some cases glycerin is fermented with the development
of butyric acid. The acid may also originate as a result of
the hydrolysis of the fat butyrin.

Organisms Producing Butyric Fermentation. — Most of
the bacteria responsible for butyric fermentation of carbo-
hydrates belong to the genus Clostridium; in fact, probably
most of the species of this genus produce more or less

218 AGEICULTUKAL AND INDUSTRIAL BACTERIOLOGY

butyric acid. Among these are the organisms which pro-
duce tetanus or lockjaw, blackleg in cattle, gaseous gan-
grene and malignant edema. Certain of the bacteria most
important in bringing about putrefaction, such as Clos-
tridium putrificum, are likewise important. The organism
most commonly associated with butyric fermentation is
Clostridium butyricum. It is a rod-shaped, Gram-positive,
motile rod, producing spores, the cell during sporulation
becoming swollen or spindle-shaped. It can be grown in
ordinary culture media under anaerobic conditions. In the

FIG. 52. — CRYSTALS OF CALCIUM OXALATE IN MEDIUM IN WHICH
Aspergillus is GROWING

presence of sugars, particularly of dextrose, abundant gas
formation and development of butyric acid occurs. The
process is soon halted, however, by the increasing acidity of
the solution unless some means is taken to neutralize the
developing acid by the presence of some base, such as
calcium carbonate.

The Clostridium pectinovorum has been repeatedly
isolated from hemp during the process of retting by the wet
method. This organism has markedly developed the power
to dissolve the pectin from the middle lamella joining the
cells and bundles of bast fibers together in the stems of
plants. Pure cultures have been used to some extent in
facilitating the process.

OXALIC AND CITRIC FERMENTATION 219

Formation of Other Volatile Acids.— Certain bacteria
are capable of producing formic, propioiiic and valerianic
acids from carbohydrates. These are less frequently met
with and are not as important, therefore, as those bringing
about acetic and butyric fermentations.

OXALIC FERMENTATION

Certain species of molds are known which are capable of
producing oxalic acid from sugars. The most important of
these belong to the genera Aspergillus and Pemcillium.
The process is essentially aerobic. The presence of the
oxalic acid is easily demonstrated by growing the organisms
in a medium containing some soluble salts of calcium. The
insoluble calcium oxalate is precipitated. The crystals of
this salt usually may be readily observed in an agar plate
culture of such a mold.

CITRIC ACID FERMENTATION

Certain species of molds belonging to the genera Citro-
myces and the closely related genus Penieillium have been
found to possess the power of transforming sugar into citric
acid. The acid is produced in sufficient quantities so that
patents upon this method of preparation have been taken
out by commercial firms in Europe. Most of the citric acid
on the market, however, comes from native fruits such as
the orange and lemon and not from citric fermentation.

CHAPTER XVIII

FEKMENTAJTION OF POLYSACCHAKIDES AND FATS

THE GUMS — HEMICELLULOSES AND CELLULOSES

Characteristics. — The gums, hemicelluloses and cellu-
loses are the most complex of the carbohydrates known to
occur in nature. They all have much the same empirical
formula, in most cases represented by (C6H1005)n. They
differ in their action toward various reagents. The gums
will swell in water and are soluble in hot water. The hemi-
celluloses may be softened and even put into solution by hot
water. Celluloses, however, are soluble only in strong
sulphuric acid and in ammoniacal copper sulphate. All
may be broken down by hydrolysis with hot acids through
a series of intermediate compounds to simple sugars having
the formula C6H1206, or in a few cases to pentoses,

(C5HJ005).

Distribution. — The gums occur commonly in nature as
products of plant growth. The agar-agar used in the bac-
teriological laboratory is a gum derived from various mem-
bers of the group of red or purple seaweeds. The gum
tragacanth and the gum arabic of commerce are examples of
gums produced by higher plants. Gums may frequently be
observed upon certain trees, such as the cherry. The hemi-
celluloses are relatively abundant in certain seeds. The
date seed, for example, consists for the most part of com-
pounds belonging to this group. The true celluloses make
up most of the cell wall of higher plants.

Fermentation. — It is evident from even casual observa-
tion in nature that cellulose and plant bodies rapidly decay
and disappear, particularly when incorporated into the soil.

220

FERMENTATION OF POLYSACCHARIDES 221

TJiis change is brought about by the activity of microorgan-
isms of many types. Some species apparently work best
under anaerobic conditions. Others are essentially aerobic.

Several species of bacteria belonging to the genus Clos-
tridium have been described as capable of bringing about
anaerobic decomposition of cellulose. It is a matter of com-
mon observation that when dead plant tissues decompose
under anaerobic conditions, as in the mud at the bottom of
a pond, carbon dioxide, methane (CHJ and hydrogen gas
are evolved. It is found that during this process of decom-
position there are developed compounds having higher per-
centages of carbon until, under the right conditions, prac-
tically the entire organic remains consist of carbon and
hydrocarbon. This is the kind of change occurring in the
process #f peat formation. The plant roots, mosses and
remains of stems buried under water gradually decompose,
but residues high in carbon are left. It is probable that
coal has originated in the same manner. Probably a por-
tion at least of the humus in the soil is developed by the
same general process, although aerobic bacteria probably
play a considerable part in its manufacture.

A distinct group of organisms, either anaerobic or facul-
tative forms, are of importance in bringing about cellulose
digestion in the alimentary tract of herbivorous animals.
The horse and the cow, for example, eat large quantities of
cellulose. A study of the digestion occurring in these ani-
mals reveals that a part, at least, of this cellulose is digested
and utilized. Careful search, however, has failed to show
that herbivorous animals in general are capable of pro-
ducing any enzyme which will digest cellulose. It is quite
probable that microorganisms present in the paunch of
ruminants and in the large intestine of the horse are
responsible for much, if not all, of this fermentation and
digestion. Many efforts have been made to isolate the
organisms responsible, and although some species capable of

222 AGEICULTUEAL AND INDUSTRIAL BACTERIOLOGY

digesting cellulose slowly have been isolated, apparently the
most important types have not thus far been found.

Many species of bacteria have been isolated capable of
decomposing cellulose under aerobic conditions. Agar plate
cultures containing finely granulated precipitate of cellu-
lose, constitute a suitable substrate for the growth of such
organisms. The bacteria produce an enzyme, cytase, which
dissolves the cellulose in the area immediately about the
colony and this area then becomes clear. Certain species
belonging to the genus Actinomyces are also believed to be
of importance in the soil. They are very common upon
decaying plant roots, and are abundant in and on the
decomposing straw of manure piles. Certain molds, among
them many species of Penicillium, are known which actively
digest cellulose under aerobic conditions.

The products resulting from the action of microorganisms
upon cellulose have not been adequately investigated.
Whether or not the cellulose is actually broken down into
sugar, for example, before it is utilized by the bacteria or
transformed further, is not known. It seems fairly certain,
however, that in some cases, at least, the intermediate prod-
ucts of the decomposition of the cellulose are utilized more
or less directly and sugar is not formed at all.

Organisms capable of bringing about cellulose fermenta-
tion are of great importance in nature, particularly in the
soil. By the changes they bring about they make available
sources of energy to many other types of soil bacteria.
Their functions will be noted later in connection with soil
bacteriology.

Fermentation of Pectin, the Process of Retting. —
Pectin is a gumlike compound closely related to the carbo-
hydrates, occurring generally in plant tissues. In con-
stitutes the major portion of the binding material which
goes to make up the structure known as the middle lamella.
A careful study of a cross-section of any plant tissue will

FERMENTATION OF POLYSACCHARIDES 223

show that each individiual cell has its own separate cell wall
made up of cellulose, and that there is a binding material
between the two cellulose walls holding the cells together in
the form of a tissue. In some plants this middle lamella is
relatively thick. In other cases it is thin and can be
demonstrated only with some difficulty. Microorganisms
of several kinds are known which are capable of producing
the enzyme pectinase which softens and dissolves this pectin,
thus allowing cells or groups of cells to fall apart.

Bacteria capable of producing pectinase are of consider-
able importance in certain plant diseases. The disease soft
rot or black rot of cabbage may be used as an illustration.
In this disease the bacteria gain entrance to the tissues of
the leaf through the \vater pores or through insect injuries.
They begin to grow in the intracellular spaces, producing
the enzyme pectinase which diffuses between the cell walls,
softens and removes the pectin, and the cells no longer are
joined together into a tissue. Microscopic examination of
rotting or rotten wood, for example, will show that the
cells are no longer joined together and they may be readily
separated from each other.

A practical utilization of this property of pectinase
formation by microorganisms is to be observed in the
process known as retting. The long cellulose bast fibers of
the flax plant and of the hemp plant are used largely in
textile industries in the manufacture of linen and hemp
articles. In the mature plants the bundles of bast fibers are
so closely united to other plant tissues that it is not prac-
ticable to remove them and clean them by mechanical
means. In order to soften the middle lamella the plants
are subjected to the process known as retting. It may be
noted that this is the old English form of the word rotting.
Literally the plants are rotted for a given length of time.
The process of retting may be either anaerobic or aerobic in
nature. In the former the bundles of flax or hemp are

224 AGEICULTUEAL AND INDUSTRIAL BACTERIOLOGY

bound and sunk in water of a pond or stream. Certain ana-
erobic bacteria closely related to the clostridia already
noted as bringing about cellulose fermentation find con-
ditions favorable for growth under these conditions and
produce pectinase, dissolving and digesting the binding
elements between the bundles of bast fibers. Several species
have been described as instrumental in bringing about this
cnange. The ones most commonly noted are Clostridium
asterosporum and Clostridium pectinovorum. "When the

FIG. 53. — CLOSTRIDIUMS. 1. Clostridium butyricum. 2. Clostridium
pectinovorum.

process has continued sufficiently long and before these
microorganisms attack the cellulose, the bundles are re-
moved, dried and are ready for the extraction of the bast
fibers which can now be readily accomplished by mechanical
means. In the aerobic process the flax or hemp is allowed
to lie on the surface of the ground exposed to the dew and
rain. Under these conditions certain molds and possibly
certain bacteria as well, grow through the stems and
gradually bring about the same kind of change as in water
retting. It may be noted that pure cultures of anaerobic
retting bacteria have been prepared and their use has
proved successful in an experimental way.

FERMENTATION OF STARCH AND INULIN
Starch and inulin are two of the most commonly formed
polysaccharides stored as reserve food material by plants.

FEKMENTATION OF POLYSACCHARIDES 225

Starch appears in plant tissues and plant cells in the form
of starch grains. These are insoluble in cold water. The
starch has the empirical formula (CGH1005)n. Inulin on
the other hand is soluble in cold water, does not form gran-
ules in plant tissues, but is widely distributed in the great
family of plants known as the composites. It is abundant,
for example, in the sunflower, the dahlia, the dandelion and
chicory. It has the same empirical formula as starch.
Upon hydrolysis starch breaks down through a series of
dextrins to maltose and finally to dextrose. Inulin, on the
other hand, breaks down through a series of intermediate
compounds to fructose (levulose).

Starch and inulin may be fermented in two principal
ways. Some microorganisms hydrolyze the complex carbo-
hydrates into simpler compounds, finally forming sugar.
They may or may not utilize the sugar thus formed, though
usually they do. Other microorganisms break down starch
but utilize the intermediate products, changing them into
acids, gases, Acetone and other compounds. In this process
sugar apparently is never formed or at least, if formed, is
so evanescent that its presence is not detected.

Microorganisms capable of bringing about hydrolysis of
starch are of considerable economic importance. It has
already been noted that certain fungi may be utilized com-
mercially in the transformation of starch into sugar pre-
liminary to the manufacture of alcohol in the so-called
amylomyces process. Apparently the first utilization of
fungi for saccharification of starch was in oriental coun-
tries, probably in China. The Chinese use this method in
the preparation of a medium for the growth of yeasts in the
manufacture of Chinese whisky. Rice is first softened by
boiling, mixed with chopped rice straw, and made into
cakes. These cakes are exposed to the air and allowed to
mold. The molds most frequently occurring are certain
species of Aspergillus, particularly Aspergillus oryzce, and
several species of Mucor and Rhizopus. When the mold is

226 AGRICULTUKAL AND INDUSTRIAL BACTERIOLOGY

well developed the cakes are dried and kept for future use.
In the manufacture of the rice whisky, rice is boiled until
soft, cooled, and inoculated with these moldy rice cakes.
The spores are thoroughly stirred into the mixture. They
soon germinate and the mycelium produced develops abun-
dance of amylase. The starch is rapidly converted by this
means into sugar and the alcoholic fermentation resulting
from yeast introduction may go forward.

Microorganisms capable of digesting or decomposing

FlG. 54. ASPERGILLUS ORYZAE.

starch and inulin without the formation of sugars are com-
paratively common. Many of the bacteria of the alimentary
tract and of the soil can bring about these changes. Some
of the products formed are the higher alcohols, such as amyl
alcohol, and the various simpler acids of the fatty acid
series, such as acetic, butyric and valerianic. The ability
or lack of ability of various organisms to attack starch or
inulin is of great importance in the differentiation of
species in the bacteriological laboratory.

FERMENTATION OF FATTY ACIDS AND GLYCERIN
Fats are present in practically all plant and animal cells,
sometimes occurring in considerable quantities. Fats and
oils constitute a large proportion of the food of man and

FERMENTATION OF POLYSACCHARIDES 227

animals, consequently changes brought about by micro-
organisms are of considerable economic importance.

Fats are glycerides of the fatty acids. Among the com-
moner fats are palmitin (C15H31COO)3C3H5, and olein
(C17H33COO)3C3H5.

The fermentation of fats is usually primarily an hydroly-
sis of the fat into its constituent fatty acids and glycerin.
This process may be illustrated by the following reaction:

(C17H35COO)3C3H5 + 3H20 - C3H5 (OH)8 + 3C17H35COOH
Stearin Glycerin Stearin acid

Microorganisms may then attack either the glycerin or the
fatty acid, bringing about secondary fermentation.

The list of bacteria capable of bringing about fat hydrol-
ysis is not very extensive. Many species of molds, however,
are known which can bring about this change. It may
readily be observed by growing the organism to be studied
upon an agar plate, in which the agar contains an emulsion
of the particular fat to be studied. The development of the
characteristic enzyme, lipase, will give rise to a transparent
area immediately surrounding the colony of the lipolytic
organism.

Very few microorganisms are known which can directly
digest or ferment fatty acids with long carbon chains. A
considerable number of molds and bacteria, however, are
able to utilize the volatile acids of the fatty acid series. It
has already been noted that molds and bacteria may oxidize
acetic acid, butyric acid and their salts.

Many species of bacteria are capable of fermenting
glycerin. This may be regarded as a simple carbohydrate.
It is one of the substances frequently used in the laboratory
for differentiating the fermentative powers of microorgan-
isms and for consequent characterization of species.

CHAPTER XIX

DECOMPOSITION OF ORGANIC NITROGENOUS COMPOUNDS
—RIPENING OF CHEESE AND OF MEAT

ALL plant and animal tissues and cells contain proteins,
and frequently other complex nitrogenous compounds. They
probably are the most essential of the constituents of proto-
plasm. They contain the elements carbon, hydrogen, oxy-
gen, nitrogen, frequently sulphur or phosphorus, and some-
times iron. They may be characterized chemically as ex-
ceedingly complex nitrogenous compounds, which, when
hydrolyzed, break down into a large proportion of alpha-
amino acids. Conversely it may be stated that proteins are
built up by combinations of various amino acids.

The general formula for the alpha-ammo acids is
RCH NH2 COOH. Some nineteen or twenty distinct alpha-
amino acids have been found by chemists in their studies of
the hydrolysis of protein, that is to say, the R in the gen-
eralized formula may represent some nineteen or twenty
different groupings. The manner in which the proteins are
built up may be illustrated by the use of glycocoll, one of
the simplest of the amino acids, as an example. Glycocoll
is an amino-acetic acid, CH2NH2COOH. It has been found
possible by chemical means to cause two molecules of an
alpha-amino acid to unite with each other. It may be noted
that these acids contain both an acid (carboxyl or COOH)
and a basic (NH2) group. The NH2 (or basic portion) of
the molecule may be caused to combine with the carboxyl
radical of another. Disregarding the intervening steps

228

DECOMPOSITION OF ORGANIC COMPOUND 229

necessary in bringing about this combination, the reaction
may be written :

CH2NH2COOH + CH2NH2COOH

= NH2CH2CONHCH2COOH + H20

By the abstraction of a molecule of water, then, it is possible
to cause alpha-amino acids to unite with each other. The
resulting compound is called a peptid. An examination of
the structure of the peptid shows it also to be an amino
acid, that is, it contains an NH2 and a carboxyl radical. It
is, therefore, possible to duplicate the preceding reaction
and cause peptids to unite with each other. If two dipeptids
unite, for example, the resulting compound will be made up
of four alpha-amino acid radicals joined together. This
process may be continued indefinitely. As many as sixteen
of these alpha-amino-acid radicals have been caused to join
to each other in laboratory studies. A complex compound
of this type is termed a polypeptid. In nature, undoubtedly,
poly peptids unite to form peptones, peptones to form pro-
teoses and proteoses finally to form proteins.

Microorganisms in bringing about changes may attack
the proteins by adding water to the molecule and causing
them to break down into simpler proteoses. The proteoses
in turn may be hydrolyzed into peptones and these through
polypeptids and peptids and finally to the alpha-amino
acids. Such a change may be regarded as a digestive action
on the part of the microorganisms. Such changes are due
to the development of pepsin and trypsinlike ferments
which act externally to the cell of the microorganism, that
is, this is an example of the analytic action of an extra-
cellular enzyme. The amino acids thus formed as the result
of the activity of microorganisms may themselves be
attacked in various ways. One of the most important of
these is the process termed deaminization. Certain micro-
organisms have the power of removing the amino group,

230 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

leaving the corresponding acid behind. The amino group
is thus converted into ammonia and the process from the
standpoint of the soil bacteriologists is frequently termed
ammonification. As an example of this change may be
given the transformation of glycocoll into acetic acid and
ammonia. The reaction may be written as follows :

CH3NH3COOH + H2 = CH3COOH + NH3
Glycocoll Acetic acid Ammonia

It will be noted that this transformation requires the
presence of nascent hydrogen. The change is one, therefore,
which is essentially anaerobic. Microorganisms which grow
in the presence of oxygen, that is, under aerobic conditions,
may in some cases bring about deaminization by oxidation.
In this case a fatty acid containing one less carbon atom in
the chain is usually formed. The reaction may be indicated
as follows:

CH3CHNH?COOH + 02 = CH2COOH + NH3 + C02
Alanin Acetic Acid

It is evident, therefore, that either under aerobic or ana-
erobic conditions microorganisms may free ammonia from
amino acids. This is important, because ammonia may be
utilized directly, or after conversion into nitrates, by
higher plants as nutrient material.

Certain microorganisms when growing under anaerobic
conditions may attack amino acids eliminating carbon
dioxide, thereby changing the compound from an amino
acid to an amine. This process is termed decarboxlyation.
It may be illustrated by the following reaction :

CH3CHNH2COOH = CH3CH2NH2 + C02
Alanin Ethylamine

Amines produced as a result of the action of microorgan-
isms are frequently termed ptomaines. Some of the amines

DECOMPOSITION OF ORGANIC COMPOUND 231

thus developed are malodorous. A few are distinctly poison-
ous. For example, the histamin formed by the decarboxyla-
tion of the amino acid called histidin is extremely poisonous.
Among the ptomaines (amines) most commonly produced
by microorganisms is trimethylamin (CH3)3N. This gives
a characteristic odor to herring brine and decaying fish. It
is produced in pure cultures by certain bacteria. It is not
highly poisonous. Other ptomaines which have been iso-
lated are putrescin (tetramethylenediamin, NH2C4H8NH2)
and cadaverin (pentamethylenediamin, NH2C5H10NH2) and
neurin (CH3)3C2H3NOH. The latter is very poisonous. It
should be noted that apparently food poisoning is but
rarely due to the presence of ptomaines. Most of the cases
of ptomaine poisoning which have been recorded in the
literature or which are popularly supposed to be of this
type are in reality either infections with specific bacteria
or are the result of eating foods containing true toxins or
endotoxins produced by the specific bacteria, and are not
primarily the result of a decomposed protein.

Certain microorganisms, particularly yeasts, change
amino acids by hydrolysis to a primary alcohol, at the same
time bringing about decarboxylation and deaminization.
For example :

CH3CHNH2COOH + H20 = CH3CH2OH -f NH2 + C02
Alanin Ethyl alcohol

One of the common products in alcoholic fermentation is
amyl alcohol or so-called fusel oil. This is produced by
yeasts from isoleucin. The reaction may be illustrated as
follows :

C5H10NH2COOH + H20 = CBHnOH + NH3 + C02
Isoleucin Amyl alcohol

The amyl alcohol thus formed finds extensive use in the
industries, particularly in the form of amyl acetate.

232 AGEICULTUEAL AND INDUSTEIAL BACTEEIOLOGY

TYPES OF ORGANISMS

A comparatively small number of species of bacteria are
known which can attack pure protein in the absence of other
nutrients. A much larger proportion are able to decompose
proteoses, peptones and amino acids.

The microorganisms responsible for bringing about
decomposition of protein may be grouped under the two
headings, anaerobic and aerobic. The former produce most
of the malodorous compounds ordinarily associated with
putrefaction. The latter are responsible for decomposition
in which such compounds are oxidized, that is, they bring
about the change ordinarily termed decay.

The Anaerobic, Putrefactive Bacteria. — Certain mem-
bers of this group of microorganisms are capable of causing
disease. Such are the forms developing in blackleg and
malignant edema in animals, and the gaseous gangrene in
man. The saprophytic bacteria belonging to the group are
comparatively numerous. The most important species is
Clostridium putrificum. This organism is abundant in
nature in the soil. Together with several closely related
species it probably is the most common cause of putrefaction
of meat and similar proteins. The organism itself is a
slender rod, single or in chains. Its spores are developed in
the ends of the rods, causing them to be swollen and assum-
ing the shape of drumsticks. Microscopically the organism
has much the same appearance as Clostridium tetani, the
cause of tetanus. It grows readily in ordinary culture
media. It is capable of developing in a mixture of egg
white and water into which has been inoculated a little soil,
cheese or fecal material. "When growing under these condi-
tions an intense putrefactive odor develops, probably due
largely to the formation of hydrogen sulphide and possibly
of methyl mercaptan (CH3SH). Butyric acid and am-
monia are also formed. This is one of the few bacteria

DECOMPOSITION OF OEGANIC COMPOUND 233

capable of bringing about decomposition of such native
proteins as albumin or fibrin under anaerobic conditions
without the presence of any carbohydrates. The bacteria
may usually be seen readily in stained mounts taken from
putrefying material.

The Aerobic, Proteolytic Bacteria. — Various bacteria
belonging to the genera Bacillus, Proteus and Pseudomonas,
together with certain molds are able to bring about decom-
position of proteins under aerobic conditions. The organ-
isms belonging to the genus Bacillus have usually been
regarded as among the most important of the bacteria in the
soil in bringing about decomposition of nitrogenous com-
pounds. The recent work of Conn and others seems to
throw some doubt upon their importance in the soil, inas-
much as apparently they are usually present in the form of
spores. "When grown in the laboratory, however, most
species of the genus Bacillus are capable of digesting gela-
tin and frequently proteins such as albumin or egg white
are digested as well. Typical of this group is the Bacillus
mycoides. This organism is a comparatively large rod,
usually occurring in long chains and, when grown upon
culture media, producing rootlike extensions from the sides
of the colonies. The spores are formed readily, are
equatorial in position and do not cause an enlargement of
the cell. It grows readily upon ordinary culture media.
Milk, blood serum and gelatin are all liquefied. The organ-
ism is particularly active in the development of am-
monia.

Members of the genus Pseudomonas, such as Pseudomonas
fluorescens, and members of the genus Proteus, such as
Proteus vulgaris, are probably also important in bringing
about ammonification.

Certain molds, particularly members of the genus Peni-
cillium, are active in producing hydrolysis in certain pro-
teins, particularly the, proteins of milk. It will be noted

234 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

later that they are active in bringing about the changes
incident to the ripening of certain kinds of cheese.

PROTEOLYTIC CHANGES IN FOODS

The Ripening of Cheese. — Cheese is a product prepared
from milk by causing a coagulation or curdling of the
simple milk protein and a more or less complete removal of
the liquid portion or whey. Many different kinds of cheeses
are prepared. Probably in all more than one hundred dif-
ferent varieties have been described. There are several
factors which contribute to determine the character of a
cheese. They may be enumerated as follows:

1. The kind of milk. — Cheese is prepared in various
countries from the milk of the cow, goat, sheep and mare.
These various milks differ somewhat in chemical composi-
tion and consequently cause variation in the composition of
the cheese produced.

2. The method of causing curdling. — Milk may be curdled
either by the development or addition of acid or by the
addition of the enzyme rennet. The curds produced by
these two methods are not identical.

3. The proportion of whey expelled. — The amount of
moisture left in the cheese is determined by the amount or
proportion of the whey squeezed out. Cheeses are some-
times classified as moist and dry.

4. The size of the cheese prepared. — Small cheeses give
opportunity for microorganisms growing upon the surface
in the presence of oxygen to exert an influence on the
character and flavor of the cheese. This does not occur in
the large cheeses.

5. Amount of salt and condiment. — Various materials
are used to flavor cheeses.

6. The temperature of ripening. — The temperature at
which the cheese is held during the ripening process may

DECOMPOSITION OF ORGANIC COMPOUND 235

make a marked difference in the texture and flavor of the
product.

7. Time of ripening. — Some cheeses require a very much
longer period of time for their complete ripening than do
others.

8. The organisms present and active in the ripening
process. — The kinds of microorganisms present and active
will depend very largely upon the factors already enumer-
ated. Perhaps it may be emphasized that all the preceding
conditions are attempts to vary the conditions under which
microorganisms will be active and consequently vary the
type and proportion of products developed. In some cases
particular microorganisms will be active and consequently
vary the type and proportion of products developed. In
other cases, certain microorganisms are intentionally intro-
duced. Usually they are present in the material and there
is no need of special inoculation.

Types of Cheeses. — Cheeses may be differentiated into
acid curd cheeses and rennet curd cheeses; the latter, in
turn, into the hard cheeses and soft cheeses.

Acid Curd Cheeses. — Acid curd cheeses are prepared by
allowing the milk, usually whole milk, to sour spontaneously
or after the introduction of a suitable starter. When the
acidity has reached a suitable point the material is heated,
whereupon the whey separates out. The curd is then
made into balls or cakes and sometimes mixed with cream
or butter before use. The so-called Dutch cheeses and
cottage cheeses prepared in the household are of this type.
They are usually not ripened before they are used. By a
special process, sour curd cheeses are ripened in some of the
Scandanavian countries and a peculiar type of cheese pro-
duced. Ordinarily acid curd cheeses are not held for very
long periods of time, inasmuch as the yeasts and molds soon
make the cheese unfit for use.

Rennet Curd Cheeses. — Of the many types of rennet curd

236 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

cheeses produced, three only may be mentioned as illustrat-
ing various processes of ripening and of manufacture.
These are Cheddar cheese, an example of a hard cheese, and.
Camembert and Roquefort, examples of soft cheeses.

Ordinary Cheddar cheese is prepared from milk of good
quality, care being used that gas-producing bacteria of the
Bacterium aerogenes or Bacterium coli type are not present
in sufficient numbers to cause gas bubbles to develop in the
ripening cheese. The milk is allowed to stand until a small
amount of acid, usually about .2 per cent has been devel-
oped. The rennet is then added, whereupon the milk
quickly curdles. The curd is broken up by means of knives
and gradually shrinks until most of the whey is drained off.
The whey is finally removed quite completely by pressing
and the curd is formed into large cakes. The consistency
of the casein gradually changes until it becomes rubbery
and unites readily to form a coherent mass. The cheese is
then placed in the curing room until the ripening process
has been completed. The ripening is probably due to the
combined action of proteolytic enzymes originally present in
the milk and introduced with the rennet, and the proteo-
lytic and fermentative action of certain bacteria which are
present. Most of the lactose is soon transformed into lactic
acid. This acid in large part combines with the casein and
furnishes suitable conditions for activity of the pepsinizing
or proteolytic enzymes. The proteins are attacked and par-
tially hydrolyzed, that is, the casein becomes more and more
soluble in water. Flavoring substances of various types are
produced. It is probable that these* are in part ammonia
and related compounds, in part volatile acids, and in part
the ethyl esters of various fatty .acids.

Roquefort cheese was originally made from sheep's milk
but milk of the cow is also used to some extent. This is a
characteristic soft cheese in which opportunity has been
afforded for the growth of a specific mold, Pencillium

DECOMPOSITION OF OEGANIC COMPOUND 237

roquefortii, throughout the mass. Air is supplied by means
of holes punched through the cheese by stiff wires. The
cheese is originally inoculated in some cases by the addition
of moldy bread crumbs to the curd.

Camembert cheese is also a soft cheese in which a much
larger proportion of whey is retained than in Cheddar
cheese. The cheese is molded into a small mass and kept in
a .suitable curing room. Certain molds soon cover the
entire surface of the cheese. The most important of these
are the Oospora lactis and various species of Pencillium,
particularly Pencillium camembertii. These molds, par-
ticularly the latter, produce a proteolytic enzyme which
gradually diffuses toward the interior of the cheese. There
is, therefore, a progressive softening of the cheese mass from
the exterior to the interior, and the cheese is considered
completely ripe when the softening has extended to the
center. The mold itself usually does not penetrate very
deeply into the cheese mass and is removed before the
cheese is consumed.

Ripening of Meat. — The so-called ripening process occur-
ring in meat is not brought about by the activity of micro-
organisms, although there are certain types of sausages in
which some bacterial and mold action is important in con-
tributing to the flavor. The ripening of meat is for the
most part due to the activity of the autolytic proteolytic
enzymes present in the meat itself.

CHAPTER XX
BACTEKIA OF THE SOIL— THE CYCLES OF THE ELEMENTS

Importance of Bacteria in the Soil. — Were it not for
the presence and activities of the bacteria in the soil the
growth of higher plants would soon become impossible. By
their activity the soil bacteria are constantly making avail-
able for the higher plants the various substances necessary
for their growth. Growing crops need, among . other ele-
ments, carbon, hydrogen, oxygen, nitrogen, sulphur, phos-
phorus, iron and calcium. Bacteria play only a minor role
in making available the carbon, hydrogen and oxygen, but
the other elements are practically all of them made avail-
able in suitable compounds by the action of the bacteria.

Abundance and Distribution of Bacteria in the Soil. —
No entirely satisfactory method has been worked out for
determining the actual numbers of bacteria of all kinds in
a sample of soil. By plating and dilution methods, it is
possible to secure approximate counts of many kinds of
microorganisms, but some forms do not grow readily upon
ordinary culture media and may be overgrown or lost
entirely. The actual numbers which may be enumerated
vary from a few thousand to many million per gram of soil.
Of the microorganisms present the most abundant usually
are bacteria, next come molds, yeasts and the protozoa. The
species which are present, particularly in their comparative
proportion of individuals, differ greatly, depending upon
climatic conditions and the character of the soil. They are
most abundant near the surface, in general, and decrease
rapidly with increase in depth. In some soils which are

238

BACTERIA OF THE SOIL 239

uniform to a great depth, as in certain of the semiarid soils
of the western United States, bacteria are moderately
abundant to considerable depths.

Cycles of the Elements. — Bacteria, yeasts and molds in
the soil are of primary importance because of the chemical
changes which they are able to bring about. The changes
which have been enumerated above make available elements
necessary for plant nutrition. Complex compounds are
constantly being broken down into simpler compounds and
simple compounds built up again into more complex forms.
These successive transformations and changes in complexity
of the various important elements may be usually grouped
together under the heading of the term " cycle. " The
elements which are important from this point of view are
nitrogen, sulphur, phosphorus, iron, carbon and calcium.

THE CYCLE OF NITROGEN IN NATURE WITH SPECIAL
REFERENCE TO THE SOIL

Outline. — The transformations which nitrogen may
undergo are relatively numerous. Microorganisms play a
large part in bringing about these changes. Simple nitrogen
compounds are assimilated by higher plants and built into
complex compounds. These, after the death of the plant, or
of the animal which has eaten the plant, are returned to the
soil and broken down into simpler compounds. As the
starting point in the discussion of the cycle of nitrogen one
may take the more complex protein. Many microorganisms
may attack this, bringing about the change termed proteoly-
sis. Eventually the nitrogen appears in the form of am-
monia and the process termed ammonification is complete.
Various microorganisms in the soil are capable of oxidizing
ammonia-producing nitrities. Still other bacteria oxidize
the nitrites to nitrates. These are assimilated by the
green plant, gradually built up into compounds more
and more complex until finally plant proteins are de-

240 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

veloped. Under certain conditions microorganisms may
attack nitrates, removing oxygen and changing them to
nitrites. Nitrites may also be attacked or reduced and
free nitrogen given off. These processes are usually termed
electrification. Certain microorganisms are likewise capable
of taking up atmospheric nitrogen and converting it to
their own use, or in some cases turning it over to higher
plants, particularly the legumes. This process is termed
nitrogen fixation. For convenience in discussion it is

Plant Rr&i n>>

FIG. 55. — OUTLINE OF NITROGEN CYCLE. Heavy lines indicate changes
produced by bacteria.

desirable to take up these various subdivisions or sections
of the nitrogen cycle one after the other.

Proteolysis, Ammonification or Deaminization. — The
most complex of the nitrogen compounds are the proteins.
They have already been denned as complex organic com-
pounds made up of carbon, hydrogen, oxygen and nitrogen,
sometimes including also phosphorus, iron or sulphur, and
which yield, upon hydrolysis, a large portion of alpha-
amino acid (RCHNH2COOH). It has also been shown that
it is possible for the chemist to join chains of amino acid
radicles indefinitely, the cement which joins them together
being the abstraction of water. When proteins are decom-
posed as the result of the action of digestive enzymes or the

BACTERIA OF THE SOIL 241

action of bacteria, a process the converse of that just dis-
cussed occurs. Water is inserted into the complex molecule
which then breaks apart at this point of cleavage forming
two molecules somewhat less complex. Bacteria which are
capable of attacking proteins and breaking them down in
this fashion by the introduction of water, that is, by hydrol-
ysis, are said to be proteolytic. Such organisms are com-
paratively common in the soil. Many of the spore-pro-
ducing bacteria, such as Bacillus subtilis and Bacillus
mycoides are of this character. The same kind of a change
may be brought about by some of the species of cocci and by
species of Actinomyces. The proteoses and peptones pro-
duced by these organisms are attacked by a still greater
variety of forms and broken down into simpler and simpler
compounds, eventually with the formation of considerable
amounts of alpha-amino acids. These amino acids, as they
develop, may be attacked by species of bacteria and molds in
the soil. One of the most common changes is that known as
ammonification, or more properly deaminization. Ammonia
is split off. In most instances apparently the microorgan-
ism is not primarily concerned with the ammonia which is
thus released, but has removed the amino group in order
that it may utilize the remainder of the acid as food.

It is evident that not all of the nitrogen compounds which
gain access to the soil are in the form of plant and animal
protein. A considerable part, for example, of the nitrogen
in barnyard manure may be in the form of urea and liip-
puric acid. Many species of spore-producing bacteria in
particular are capable of attacking urea, bringing about
deaminization or ammonification in accordance with the
following reaction:

(NH2)2CO + 2H20 = (NH4)2C03

Other nitrogenous compounds which may be added to the
soil such as hippuric acid, calcium cyanamide, etc., are

242 AGBICULTUKAL AND INDUSTRIAL BACTERIOLOGY

deaminized in an analogous fashion. In general, it may be
noted that practically any organic compound of nitrogen is
eventually broken down by soil microorganisms with the
formation of ammonia.

Well-aerated soils show little tendency for ammonia to
accumulate. As rapidly as it is formed it is changed by the
nitrifying bacteria, but under anaerobic conditions, such as
exist in the mud in the bottom of a pond or in water-logged
soil, the nitrogen remains more or less indefinitely in the
form of ammonia. Not infrequently the fact that the am-
monia is usually oxidized in arable soils and the nitrogen
available to the roots of green plants, therefore, is generally
nitrate, has led to the erroneous assumption that green
plants cannot take up ammonia through their roots. In
general, this is a fallacy. In fact, many kinds of plants,
particularly those which live with their roots in wet or
water-logged soils or in mud, secure practically all of their
nitrogen in the form of ammonia. However, ammonia is
relatively volatile and would be readily lost from the soil
and manure were it not for the fact that it is, in the pres-
ence of oxygen, readily oxidized to nitrites and nitrates.

Inasmuch as the rapidity with which the microorganisms
in a given soil can bring about complete proteolysis and
ammonification of nitrogenous manures added is one of
the indices to soil fertility, methods have been devised for
testing out this soil power in the laboratory. The test is
carried out by adding a- given amount of a particular
nitrogenous material, such as casein, blood meal or other
organic nitrogenous compound, to a given weight of the soil
to be tested, mixing thoroughly, placing in suitable vessels,
usually tumblers, adding water to the optimum and incu-
bating. At the end of a given period of time these samples
of soil together with similar samples to which no manure
has been added are analyzed for their ammonia content.

Until recent years it has generally been thought that the

BACTERIA OF THE SOIL 243

spore-producing bacilli in the soil were the most important
of the ammonifying bacteria. It is undoubtedly true that
in the laboratory in culture media these microorganisms are
capable of digesting proteins quite readily. Careful studies
of soils, however, seem to indicate that most of these species
are present except under unusually favorable conditions, in
the form of spores and that they are numerous in the soil
not so much because they are rapidly multiplying or are
present in the vegetative stage, but because there is a grad-
ual accumulation of highly resistant spores.. The bacteria
apparently, which are somewhat more important in bring-
ing about ammonification, are members of the genus
Pseudomonas, particularly forms of the type Pseudomonas
fluorescens. These bacteria can likewise bring about rapid
ammonification in laboratory media.

It is probably a very unusual condition for soils to be
markedly deficient in microorganisms capable of bringing
about ammonification. The bacteria are probably most
important in ordinary arable soil, although in some acid
soils, in leaf mold and in similar locations, it is probable
that molds are more important.

Though the microorganisms in the soil usually are pro-
ducing ammonia from complex nitrogenous compounds
greatly in excess of their own needs, nevertheless, it must be
remembered that some of the ammonia is assimilated by
them. That is, some of the nitrogen of the soil becomes
locked up in the cells of the bacteria, yeasts and molds
present. Chemical analysis of the soil, therefore, will
always show a small portion at least of the nitrogen in the
soil in organic form. This may later be released by the
death and decomposition of the bacterial or mold cells.

Nitrification. — The term "nitrification" as applied to
the transformation of ammonia into nitrites and finally into
nitrates is a misnomer chemically. The process is actually
one of oxidation. The name, however, has become so firmly

244 AGEICULTUEAL AND INDUSTEIAL BACTERIOLOGY

fixed in discussions of soil bacteriology that it probably
cannot be replaced by a better one.

The process of nitrification as it occurs in soils usually
takes place in two distinct steps; first, the oxidation of
ammonia to nitrous acid or nitrites, a process which may
be termed nitrosation, and second, an oxidation of the
nitrous acid or nitrites to nitrates, a process termed nitra-
tation.

The process of nitrosation is brought about by a peculiar
group of prototrophic bacteria. All of them belong to the
genus Nitrosomonas. Some are short rods and motile.
Others are spherical and nonmotile. They do not grow at

FIG. 56. — NITROSOMONAS. A. Nonmotile spherical series. B. Motile
rod-shaped form.

all readily in the laboratory upon the commonly used media
and may be isolated and studied only by the use of special
methods. They require for their growth the presence of
ammonia and free atmospheric oxygen. For the most
part they do not grow well in the presence of an
abundance of organic matter, particularly of amino acids
and peptone. They are found in the laboratory not to
require any organic matter whatever for their development.
Apparently they oxidize the ammonia in accordance with
the following equation :

2NH3 + 302 = 2HN02 + 2H20

and secure by this means energy for their growth and for
the manufacture of food. They are capable of securing

BACTERIA OF THE SOIL 245

carbon from carbonates and building this up into complex
compounds.

The organisms belonging to this genus of bacteria may
be isolated in the laboratory by placing a small amount of
soil in a solution containing ammonium sulphate, carbonates
and salts (not nitrogenous), such as phosphates, necessary
for the growth of bacteria. A clouding of the medium will
eventually develop and a microscopic examination show the
characteristic bacteria to be present in abundance. Chem-
ical tests will also show that a portion, if not all, of the
ammonia has been transformed into nitrite. It may be
emphasized that organisms such as these must be among the
most primitive of living things. They are capable of living
wholly upon inorganic sources of materials and they do not
even require sunlight for securing energy for manufacture
of their food. They may be grown in pure culture in media
such as agar or silica jelly containing ammonium sulphate
and suitable salts.

It may further be emphasized that these microorganisms
do not oxidize the ammonia to nitrite in order to make use
of the nitrite formed, but rather that they may secure
energy from the oxidation. The nitrites then constitute a
waste product of the bacterial metabolism. Very rarely is
there any tendency for the nitrites to accumulate in
appreciable quantities in any soil. The conditions which
favor the growth of the Nitrosomonas will also favor the
growth of organisms which can transform the nitrites to
nitrates. Whenever nitrites are present in abundance in
soils, it may be taken as evidence that they have formed, not
as a result of the oxidation of ammonia, but as a result of
anaerobic denitrification, to be discussed later. Nitrites
are not utilized by higher plants as a source of nitrogen. In
fact, they are in general injurious to plants, particularly to
plant roots.

Nitrites as rapidly as they form in soil are usually

246 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

oxidized into nitrates by bacteria belonging to the genus
Nitrobacter. These are small, nonmotile rods present in
most arable and fertile soils. Like Nitrosomonas they do
not require the presence of organic material for their
development. They are likewise prototrophic and secure
their growth energy by the oxidation of the nitrites to
nitrates in accordance with the following reaction:

2HNT02 + 02 = 2HN03

Nitrates, therefore, tend to accumulate in the soil. Under
certain conditions they may be leached out and concentrated
in other places. It is claimed, for example, that the great
nitrate deposits of certain arid regions, as in Chile, are due
to the concentration brought about in this way. Nitrates
are easily taken up by the roots of most higher plants.
Consequently it is in this form that nitrogen is assimilated
by most growing crops.

Nitrobacter may be readily cultivated in the laboratory
by preparing a solution containing potassium nitrite, car-
bonates, phosphates and other salts necessary for growth,
but containing no organic material. When such a solution
is inoculated with a bit of soil a clouding will soon develop
and the nitrites will be found to be changed to nitrates. The
oxidation may be materially increased by suitable aeration.
The ability of a particular soil to transform nitrites into
nitrates may be tested by a manner similar to that already
noted for ammonification, but in general it is not differen-
tiated from the process of nitrification as a whole.

It has already been noted that the process of nitrification,
or oxidation of ammonia, starts with the ammonia and ends
with nitrates. The ability of soil to bring about this entire
change of nitrification may be studied by adding ammonium
sulphate or some other compound containing available
ammonia to a suitable soil sample in the laboratory, adding
the optimum amount of water and analyzing after a suit-

BACTERIA OF THE SOIL 247

able period for the content in nitrates. Determination of
the ammonifying and nitrifying power of soils is among
the more valuable determinations made in the laboratory in
an effort to estimate soil fertility.

Assimilation of Nitrogen by Plants. — All plants which
live in the soil, the bacteria, the yeasts, the molds, the
higher fungi, many alg®, the mosses, the ferns, and the
seed plants all require nitrogen for their growth. The
process of taking up nitrogen and utilizing it in metabolism,
particularly in the building up of protoplasm, is termed
nitrogen assimilation.

It was previously noted that bacteria of different kinds
utilize many different sources for their nitrogen supply. A
few species are capable of taking it directly from the air.
Many more can utilize amino acids, ammonia or nitrates.
Nitrogen taken up by bacteria and built into protoplasm is
locked up, of course, in a form unavailable to higher plants.
Molds and yeasts resemble bacteria in their ability to use
various compounds of nitrogen in their metabolism. It is
not strange, therefore, that in fertile soils an appreciable
amount of the total nitrogen contained in the soil should be
found in the cells of the yeasts, molds, bacteria and other
fungi present.

Some higher plants have the ability to assimilate amino
acids, but these latter are present usually in very small
quantities and only at intervals in most soils. Many species
likewise can utilize ammonia, and most species can utilize
nitrates. Nitrites in general are poisonous to plant roots.
The nitrogen upon entering the plant is rapidly built up
into more complex nitrogenous compounds; first, probably
into amino acids, then into peptids, then finally into the
complex plant proteins. Inasmuch as animal proteins are
in all cases derived directly or indirectly from plant pro-
teins this may be said to complete the nitrogen cycle. There
are, however, several transformations of nitrogen not in-

248 AGBICULTUEAL AND INDUSTRIAL BACTERIOLOGY

eluded in the main cycle which are of great importance
from an economic point of view. These are denitrification
and nitrogen fixation.

Denitrification. — Denitrification is a term applied to the
reduction of nitrates to nitrites and to the process of
evolution of free atmospheric nitrogen from either nitrates
or nitrites by the activity of microorganisms. Like the
term nitrification it is a misnomer, as the process is essen-
tially a reduction.

Nitrates are transformed to nitrites by certain kinds of
bacteria when placed under anaerobic conditions and in the
presence of certain easily oxidizable substances. For
example, in the presence of organic material and the
absence of free oxygen, certain microorganisms remove
oxygen from the nitrate molecule, reducing it to nitrite, and
use the oxygen thus obtained for oxidizing carbon com-
pounds. A reaction of this type may be illustrated as fol-
lows:

C6H1206 + 12HN03 = 6C02 + 6H20 + 12HN02

As a result of this action energy is secured for growth. The
reduction of the nitrate requires less energy than is secured
by the oxidation of the carbon compounds, hence there is a
net gain in energy to the microorganism.

Many bacteria are known which bring about this trans-
formation. Most of them are termed facultative, as they
will grow well under normal aerobic conditions, but do not
grow in the absence of atmospheric oxygen unless an ade-
quate supply of this element can be secured from sources
such as nitrates. The reduction of nitrates to nitrites is
often observed in the laboratory and is a valuable means of
differentiation of bacteria from each other.

Certain bacteria, the true denitrifiers, are capable of
bringing about a reduction of nitrites with the evolution
of free nitrogen gas. The chemistry of this transformation

BACTERIA OF THE SOIL 249

has not been adequately worked out. The reaction itself,
however, may be illustrated as follows:

C6H1206 + 8HN02 = 6C02 + 10H20 + 4N2

It is probable that in many cases the evolution of
elementary nitrogen is due to the simultaneous presence of
nitrites and amino acid. In general an amino acid and
nitrous acid react with each as follows :

R • CO • COOH + HN02 = R • CHOH • COOH + H20 + N2

NH2

To what extent denitrification can occur in the absence of
amino compounds is not well understood. This latter
reaction explains the small amount of nitrogen gas fre-
quently found accompanying carbon dioxide or hydrogen in
fermentation tests of bacteria of the colon typhoid group.
Many of these forms apparently can transform nitrates into
nitrites but cannot directly attack the nitrite molecule. If
amino acids are present, however, there may be an evolution
of nitrogen gas.

Denitrification is negligible in its results in most well-
aerated soils. It may prove to be of importance, however,
in certain soils containing considerable quantities of nitrate
which have become water-logged. The free oxygen soon
disappears and the nitrate may be transformed into nitrite
by the bacteria present. It is possible that in some cases
the amount of nitrites formed in this fashion may be suf-
ficient to exert a toxic action upon plant roots. It has been
found, for example, that the addition of nitrate as a fer-
tilizer to rice is not good practice but that ammonia salts
are much to be preferred. Apparently, this is because the
roots of the rice grow in a water-logged soil containing little
free oxygen. Nitrates may be reduced to nitrites and ren-
dered not only unavailable to the plant roots, but actually
toxic. Ammonia salts, on the other hand, are not trans-

250 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

formed and may be taken up and utilized directly by the
plant.

Nitrogen Fixation. — A study of the cycle of nitrogen
makes it evident that the amounts of living material upon
the earth, that is, the number of plants and animals, must
depend quite directly upon the amount of nitrogen in the
nitrogen cycle. There are several ways in which nitrogen
may be lost. First, there is the occasional process of
denitrification and evolution of free nitrogen gas. Then
explosives give off free nitrogen when fired. Fuel, when
consumed, gives off some free nitrogen. It is evident that
unless there is some way in which this nitrogen can be
replaced in compounds, the number of living creatures on
earth must gradually diminish. Until methods of fixation
of atmospheric nitrogen were discovered this fact was a
matter of grave concern to chemists. Laboratory studies
have shown that nitrogen may be fixed, that is, atmospheric
nitrogen may be combined with the other elements, by the
use of high temperatures or the electric current. It is noted
that there is some such combination of nitrogen during the
electric discharges of a thunder storm. The amount of
nitrogen fixed in this way, however, is very small in com-
parison to the amount fixed by the activity of microorgan-
isms, particularly of bacteria and perhaps of some of the
molds.

Nitrogen as an element is exceedingly inert. The nitro-
gen molecule is apparently in a stable condition and in
order to force it to combine with other elements there is
usually necessary an expenditure of energy. This is evident
in the laboratory, for, as has been stated, high temperatures
or the electric current must be utilized. These forces are
not available to microorganisms. It is necessary, therefore,
in all cases that some easily oxidizable food material be
present so that energy may be secured for fixation. In most
cases the nitrogen-fixing bacteria utilize carbohydrates.

BACTERIA OF THE SOIL 251

The microorganisms capable of fixing atmospheric nitro-
gen are in part symbiotic, that is, living upon or in the roots
of higher plants, and nonsymbiotic, that is, living free in
the soil. Under each of these headings bacteria and fungi
are to be considered.

Nitrogen Fixation ~by Symbiotic Bacteria. — The bacteria
which live in the roots of plants and are capable of fixing
atmospheric nitrogen belong to the genus Rhizobium.
Whether or not more than one species should be recognized
is somewhat uncertain. There is some evidence that there

FIG. 57. — RHIZOBIUM LEQUMINOSARUM. A. Peritrichous type. B. Mo-
notrichous type. 1. Motile rods. 2. Non-motile or involution
forms.

are several distinct species on the roots of distantly related
leguminous plants. For the present discussion, however,
all may be regarded as belonging to a single species termed
Rhizobium leguminosarum.1

This organism is apparently widely distributed in soil.
Whether or not it multiplies actively in soil is not very
definitely proved. It is usually found associated with the
roots of leguminous plants, causing the formation of
nodules, sometimes termed "tubercles," upon them. No
species of leguminous plant is known which, under its native
conditions, does not have these nodules appearing upon the

1 This organism is variously known in the literature as Bacillus
radicicola and Pseudomonas radicicola.

252 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

roots. The family Leguminosce is a relatively large one.
Some forms are trees, some are shrubs, and many are
herbaceous. They are found in abundance from the tropics
to the arctic region, and from the low plain to the limits of
vegetation upon the mountain. Some species develop best
in very moist soil, others under relatively arid conditions.
Some species prefer soils which are distinctly acid. Most
species prefer a neutral to a somewhat alkaline soil. It is
evident, therefore, that the Rhizobium leguminosarum is
practically world-wide in its distribution. Apparently the
only conditions under which it does not grow are those
under which legumes will not grow.

Rhizobium leguminosarum may be readily cultivated in
the laboratory from the nodules of legumes. It grows best
on neutral media containing suitable sugars and phosphate.
Peptone is not necessary. The colonies which develop upon
sugar agar are usually somewhat slimy, sometimes almost
clear and colorless. In sugar broth the medium becomes
clouded and with some strains considerable amounts of gum
are produced. This may be precipitated by the addition of
alcohol. The bacteria grow readily upon a nitrogen-free
medium and are able to fix appreciable quantities of atmos-
pheric nitrogen. In determining the relative nitrogen-
fixing power of various types of these bacteria, it is cus-
tomary to compare the number of milligrams of atmospheric
nitrogen fixed per gram of sugar (usually glucose) used.
Usually Rhizobium leguminosarum is able to fix from one
to five milligrams of nitrogen per gram of glucose utilized.
The organism is aerobic, completely oxidizing sugar with
the development of carbon dioxide and water. There is,
therefore, an abundant evolution of gas, but as the oxidation
can take place only near the surface of the medium and in
the presence of an abundance of oxygen, gas bubbles are not
observed and no gas is formed in the closed arm of the
fermentation tube.

BACTERIA OF THE SOIL 253

The young bacteria are motile, Gram-negative rods.
There is some question as to the distribution of flagella.
Some authors have claimed that a single polar flagellum is
to be demonstrated, others that there are several flagella
distributed over the surface of the cell. This may be due
to the fact that in reality several species of bacteria have
been studied, or because the cultures examined were of dif-
ferent ages. In many media the older organisms become
considerably enlarged and more or less vacuolate. Fre-
quently they are decidedly irregular. Branched forms and
forms showing irregularities in staining are common. The
appearance frequently leads to the inference that multi-
plication is by a process of budding and not by normal
transverse fission.

While there are some points not well understood, in
general the method of root infection has been fairly well
worked out. The bacteria in the soil apparently come in
contact with the root hairs. It will be recalled that root
hairs develop just back of the root caps of the young grow-
ing roots and are somewhat evanescent. Older roots do not
show them. In some way this species of organism finds a
way to penetrate the wall of the root hair. Apparently it
digests its way through. Once inside the root hair it begins
multiplying relatively rapidly and produces a gelatinous
strand which was formerly regarded as a fungous filament.
In fact, this organism was originally described as a root-
dwelling fungus. The strand of bacteria increases in length
until it comes in contact with the cell wall toward the
interior of the root. Here a buttonlike enlargement in the
strand may take place. Eventually the bacteria make their
way through the cell wall and invade the next cell. Infec-
tion of the root has now been established.

The presence of the organisms in the cells of the roots
leads to an increased growth. It is probable that some
substance excreted by the bacteria possesses the property

254 AGKICULTURAL AND INDUSTRIAL BACTERIOLOGY

of stimulating the meristematic cells to increased rapidity
of development. In consequence a small tumor or mass of

FIG. 58. — NODULES ON ROOTS OF A LEGUMINOUS PLANT.

cells develops rapidly and protrudes from the main portion
of the root as a nodule. This nodule may be regarded as a
modified rootlet.

BACTERIA OF THE SOIL 255

The nodules of different legumes show some differences
in structure. In general, however, there is an epidermal
layer with a portion of the interior consisting of large cells
more or less compactly filled with bacteria. Scattered
among these cells there may be uninfected cells, in some
cases packed with starch granules. Fibrovascular bundles
leave the main root and pass from base to tip of the nodule

FIG. 59. — SECTION OF NODULE OF ROOT OF LEGUMINOUS PLANT. Cells
containing bacteria shaded black.

just underneath the epidermis. It is interesting to note
that in these bundles the arrangement of the conduction
tissues, that is, of the phloem and xylem, is the reverse to
that found in the normal root. The nodules in some species
of legumes grow rapidly to full size and then stop growing.
There is, in such nodules, no permanent meristematic
tissue. In others, a group of growing cells remain active
near the tip of the nodule. This meristematic mass con-

256 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

tinues growing and may branch from time to time. Soon
plants, in consequence, show forked nodules sometimes
resembling masses of coral. Certain well-differentiated
areas apparently are always set apart for the growth of the
microorganisms. The nuclei of the invaded cells eventually
undergo degeneration. Stained preparations made from
nodules show that in the older cells at least, most of the
bacteria have assumed involution shapes resembling in
many respects those found in culture media. These were
termed bacteroids by the earlier investigators before their
true bacterial relationship was understood. The morphology
of the organism seems to vary somewhat with the age of the
nodule and the species of legume.

Comparative studies of legumes grown in soils which
have been sterilized and freed from Rhizobium legumino-
sarum, consequently having no nodules upon the roots, with
plants having nodules prove conclusively that the organism
fixes atmospheric nitrogen and in some manner passes this
on in combined form to the plant, that is, to the legume.
Legumes are, therefore, not dependent merely upon soil
nitrogen, but by the aid of the bacteria can utilize the
atmospheric nitrogen for their development. There seems
to be no reasonable doubt but that the energy required for
fixation of the nitrogen by the bacteria is furnished by the
plant through carbohydrates, probably sugars, which are
supplied. In just what manner the legume secures the
nitrogen through the help of the bacteria, is somewhat less
certain. It has been assumed by some writers that the
bacteria fix the nitrogen and use it in building up their own
protoplasm, and that only after the death of the bacterial
cell and its digestion, either as a result of autolysis or as a
result of the secretion of enzymes in the nodules by the
plant, the digested products may be absorbed by the
legume. Other investigators have concluded that a part, at
least, of the nitrogen fixed is excreted by the bacteria in the

BACTEKIA OF THE SOIL 257

form of some soluble compound and that the plant can
benefit by the presence of the bacteria without destroying
them.

It already has been noted that there probably should be
recognized more than one species of the genus Rhizobium.
This is evidenced particularly when attempts are made to
inoculate legumes of one kind growing in sterile sand with
the organisms originating from the root of some other
species. In general leguminous species of plants which are
closely related botanically may be cross-inoculated readily.
For example, the various species of the genus Medicago, to
which the alfalfa belongs, readily cross-inoculate. Further-
more, bacteria from the roots of sweet clover belonging to
the closely related genus Melilotus also cross-inoculate
readily with alfalfa. Forms, however, so distantly related
as soy beans, lupine, and the clovers seem quite impossible
of cross-inoculation. It is entirely probable that the organ-
isms producing the nodules on these three plants belong to
different species.

It 'is evident from the preceding discussion that although
bacteria capable of causing root nodules to form on the roots
of leguminous plants are widely distributed and present in
most soils, it is nevertheless true that in many soils those
particular forms, possibly species, capable of inoculating
the roots of certain legumes may be absent. Care must be
used, however, to differentiate betweei* the lack of organ-
isms capable of inoculating roots and physical or chemical
soil conditions unfavorable to the growth of plants. Many
of the clovers, for example, prefer to grow in a soil con-
taining a considerable quantity of lime, one which is, there-
fore, neutral or somewhat alkaline in reaction. With such
plants, lack of a good stand may be quite as apt to be due
to wrong soil conditions as to the lack of the proper inocu-
lating organism. Various methods of artificial introduction
of organisms capable of inoculating the roots of certain

258 AGEICULTUKAL AND INDUSTRIAL BACTERIOLOGY

species of legumes have been suggested. Soil from a field in
which a particular legume has been successfully grown is
often used for the inoculation of other fields, sometimes
being spread at the same time that the seeds of the legume
are sown. This method has the obvious disadvantage of
great bulk and the chance of introducing undesirable weeds.
The second method commonly practiced is that of the use
of pure cultures. Some of the agricultural experiment
stations in the United States and Canada, as well as certain
commercial firms, supply these regularly, usually in the
form of a liquid culture. It is customary to supply a cul-
ture of the organism isolated from the nodules of the par-
ticular legume which it is desired to inoculate. In some
cases it is necessary for the organism to be increased. A
culture medium containing phosphate, sugar, and water
may be inoculated with the culture of the organism it is
desired to grow, and within a few hours the numbers of
bacteria will greatly increase. Inoculation may be effected
by moistening the seeds which are to be sown with such a
culture or in some cases by mixing with dry soil and spread-
ing this upon the ground to be inoculated.

Much has been written in bacteriological literature con-
cerning the possibility of exalting or increasing the nodule-
producing power of particular strains of Rhizobium
leguminosarum. This increased power of nodule formation
has been termed virulence. It is claimed by certain manu-
facturers and by some authors that it is possible by growing
in certain kinds of culture media, and by subjecting thq
organisms to suitable environment, to increase or decrease
this virulence. Adequate scientific demonstrations of such
variations in virulence have apparently not been made. It
seems that quite as wide variations in inoculating power
may be found among various cultures isolated from differ-
ent plants of the same legume species.

Inoculation of soil for growing bacteria has been widely

BACTERIA OF THE SOIL 259

practiced. When once the bacteria are thoroughly inocu-
lated through the soil, that is, after there have been one or
more successful crops of legumes grown on a particular
area, the organisms will persist for a considerable period
of time even though the particular legume is not present.

Nitrogen Fixation by Symbiotic Fungi. — Many kinds of
higher plants are found to have growing in or upon their
roots species of fungi apparently intimately connected with
the metabolism, life history and successful development of
the plant. The plants which show this characteristic are in
many cases inhabitants of swamps and bogs, others are the
so-called epiphytes or air plants. In addition, however,
many species are known to possess these fungi upon their
roots, though they grow under normal soil • conditions.
Fungi growing in or upon plant roots are generally termed
mycorrhiza. In some species the fungi occur inside the root,
frequently giving rise to swellings, nodules, tuber forma-
tion, etc. In other cases the fungi grow over the surface of
the root, penetration, if any, being relatively shallow. The
former are termed endotrophic and the latter ectotrophic.

Among the endotrophic mycorrhizas are some which grow
in the roots of orchids. All members of this great plant
family (Orchidacece) show a certain zone in the roots just
inside the epidermis given over to hyphae of a fungus. It is
found that seeds will die soon after germination, if this
fungus is not present and functioning. The roots of the
Russian olive tree, furthermore, and its close relatives, such
as the shepherd berry, have nodules on their roots much
resembling the nodules of legumes, but harboring an endo-
trophic fungus. The same is true of the roots of the alder
and the New Jersey tea of our prairies. Apparently these
nodules are normal to the plant and the organisms growing
in them are in some respects beneficial. In certain species
of flowering plants which have lost the power to produce
chlorophyl, such as the Dutchman's pipe, apparently the

260 AGEICULTUEAL AND INDUSTRIAL BACTERIOLOGY

task of taking up plant food from the soil and elaborating
it for the use of the plant is largely given over to organ-
isms growing in the tuberous or nodular roots. Sufficient
work has not been done with most of these plants to prove
conclusively that the primary function of the mold is to fix
atmospheric nitrogen. There is some evidence pointing to
this as a fact for certain forms such as the alder, while
experiments upon New Jersey tea did not lead to the same
conclusion. Apparently there is a condition of symbiosis.
The exact benefit reaped by the plant has not been deter-
mined with certainty.

The roots of many trees, and particularly plants growing
in bogs and swampy places, are covered with the mycelium
of a fungus or so-called ectotrophic mycorrhiza. In certain
groups of plants, such as those belonging to the genus Vac-
cinium, which includes the huckleberries, blueberries and
cranberries, practically no growth will occur, at least nor-
mal development will not take place unless the fungi are
present upon the roots. In other cases it is apparently
true that the mycorrhiza is largely parasitic and more harm
is done than good. Certain of the toadstools and mush-
rooms which appear in woods are of this character and the
hyphse may be traced from the base of the fruiting body to
the tip of the tree roots.

In summary, it may be concluded that many plants not
belonging to the group of legumes depend, in part at least,
upon the activity of molds growing in their roots or in root
nodules, for their best development. It is not at all improb-
able that in some cases nitrogen is fixed and furnished to
the growing plant by these organisms, but adequate proof
has not been brought forward in most cases.

Nitrogen Fixation by Nonsymbiotic Bacteria. — It has been
abundantly demonstrated that there are several, probably
many, kinds of bacteria living in the soil not in symbiosis
with higher plants, which are capable, under certain condi-

BACTERIA OF THE SOIL 261

tions at least, of fixing atmospheric nitrogen and utilizing
it for their own metabolism. Some species of these are ana-
erobic, most are aerobic. As noted above with Rhizobium,
it is customary to rate the rapidity and amount of nitrogen
fixation by comparing the number of milligrams of nitrogen
fixed with the number of grams of sugar utilized as energy,
for these organisms are just as dependent as are the sym-
biotic type upon some easily oxidizable substance, such as
carbohydrates, from which they may secure energy. The
group of organisms which has been most studied and is
probably, on the whole, most important in such nitrogen
fixation, is the one whose members belong to the genus
Azotobacter.

Azotobacter. — The genus Azotobacter includes those soil
organisms which are aerobic, secure their growth energy
almost entirely by the oxidation of carbohydrate material
and which are capable of fixing atmospheric nitrogen. The
cells usually are somewhat larger than those of other soil
bacteria. Under favorable conditions in the laboratory in
suitable media, they may appear almost yeastlike. They
are usually spherical, ellipsoid, or in some cases elongate.
Some species are motile, usually by means of polar flagella.
Others are nonmotile. Some species possess the power of
producing a diffusible pigment, greenish or brown in color.
Species belonging to this genus are widely distributed in
nature and about ten have been described in the literature.

The species first described, and one of those most com-
monly met with is Azotobacter chrodcoccum. This and
another species known as Azotobacter Beyerincki are prob-
ably the most widely distributed in soils. They grow
readily upon laboratory media containing phosphate and
certain other inorganic salts plus some easily available
source of carbon, particularly sugars and higher alcohols.
They do not develop in media or in soils having an acid
reaction. One species of Azotobacter, A. Vinelandii, de-

262 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

scribed by Lipman in New Jersey, has been found able, in
the laboratory, to fix from fifteen to twenty milligrams of
nitrogen per gram of sugar or alcohol (such as mannitol)
used. The amount of nitrogen fixed is also influenced by
other constituents of the medium. It has been shown by
certain authors that the addition of soil extract, particularly
of humates, exerts a decided stimulating action. It has been
found that under certain conditions species of Azotobacter
growing together with algas are capable of fixing consider-
able quantities of nitrogen, apparently there existing sym-
biotic relationship between the two types of organisms.

Efforts have been made to utilize cultures of Azotobacter.
Most fertile soils contain bacteria of this type in abundance.
The addition of such organisms, therefore, is superfluous in
such soils and useless in soils that are not adapted to their
growth.

Organisms belonging to this group are not of such im-
mediate value to the farmer as are those growing in sym-
biosis with leguminous plants. Nevertheless, in the long
run they are probably responsible for the accumulation and
storing up of large quantities of nitrogen in the soil. It
should be noted, however, that there is no evidence that
these bacteria take up free nitrogen and turn it over to
plants growing in the soil. It is probable that they utilize
the nitrogen in their own metabolism and it is only after the
death and decay of the Azotobacter cells that the nitrogen
becomes available to higher plants. Inasmuch as these
bacteria can utilize sugars, and salts of various organic
acids as sources of energy in the laboratory, it is probable
that they secure their energy from the same sources in the
soil. Their development, therefore, is dependent upon an
abundant supply of such materials.

Anaerobic Nitrogen-fixing Bacteria. — Several species of
anaerobic bacteria have been described as capable of fixing
atmospheric nitrogen. The most important of these is

BACTERIA OF THE SOIL 263

Clostridium pasteurianum. While this type of fixation may
be shown in the laboratory it is not probable that it is of
any great economic significance. The amounts of nitrogen
fixed are not large and the utilization of carbohydrate as a
source of energy must be much greater under anaerobic
than under aerobic conditions. Such organisms are, there-
fore, far less efficient nitrogen-fixing machines than are the
aerobic species such as Azotobacter.

Nitrogen Fixation by Nonsymbiotic Molds. — Considerable
study has been devoted to the question of whether or not
molds not living in symbiosis with plant roots are capable
in the soil of fixing atmospheric nitrogen. There is some
diversity of opinion, but on the whole it may be said that it
has not been definitely proved that molds do possess this
capacity. However, under certain soil conditions, such as
in the leaf mold occurring in forests, molds are undoubtedly
very important in breaking down organic compounds and
it is not impossible that some species may be found capable
of fixing atmospheric nitrogen, adding thereby to the fer-
tility of forest soils. Such has not been definitely proved.

THE CYCLE OF CARBON

The common forms in which carbon is found in nature
are as the element, (that is as carbon), as carbon dioxide,
and as relatively complex carbon compounds, the so-called
organic compounds. The cycle of carbon, therefore, is not
as complex as that of nitrogen, and many species of organ-
isms are known which can bring about transformations of
carbon from one compound to the next.

Carbon dioxide, as it exists in the atmosphere and in solu-
tion in soil water, is taken up by green plants and these, by
utilizing the energy from the sun's rays, are capable of
forcing this into combination with hydrogen, producing
formaldehyde, and then polymerizing this compound into
carbohydrates, first into sugars and then into starch. Some

264 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

bacteria are apparently also capable of taking up atmos-
pheric carbon dioxide or carbon dioxide in solution and
utilizing the energy secured by oxidation of inorganic com-
pounds to assimilate the carbon dioxide into their own
protoplasm. Such organisms have already been noted
among the so-called nitrifiers, the energy secured by oxida-
tion of ammonia, for example, being in part used for the
reduction of carbon dioxide and the production of organic
carbon compounds.

Microorganisms in general (as well as animals) possess
the ability to break down complex compounds of carbon,
securing growth energy by their oxidation, and giving off
carbon dioxide continuously. In the main, therefore, the
carbon cycle consists in manufacture of carbon compounds
by green plants and their oxidation not only by these plants
but also by animals and microorganisms.

Under certain anaerobic conditions, microorganisms may
attack carbon compounds, evolving hydrogen and carbon
dioxide. In general the carbohydrates have the empirical
formula CnH^nOn- ^ *s apparent, therefore, that if free
hydrogen gas is given off together with carbon dioxide that
there will be a residuum of carbon. Apparently changes
of this kind have been responsible for the formation of peat
and of coal.

Production of Carbon Dioxide in Soil by Microorgan-
isms.— Practically all of the aerobic bacteria and many of
the anaerobic bacteria produce carbon dioxide in the decom-
position of carbonaceous material. It is, therefore, constantly
being produced in the soil, and is of considerable significance
there. A large proportion of the carbon dioxide evolved
escapes in the air, some portions are dissolved in the soil
water and in part may be taken up by plant roots. It is
evident that the soil solution must, under most conditions,
be practically a saturated solution of carbon dioxide. This
exerts a marked solvent action upon many of the minerals

BACTERIA OF THE SOIL 265

present in the soil and undoubtedly is one of the important
factors in bringing about the solution of materials necessary
for the growth of higher plants.

The Decomposition of Insoluble Carbohydrates in the
Carbonaceous Matter in the Soil. — A considerable pro-
portion of the carbonaceous material added to the soil is in
the form of cellulose and closely related compounds. This
is not readily attacked by most kinds of bacteria, but there
exist in the soil certain specialized forms which decompose
cellulose readily. Members of the genus Actinomyces are
particularly active in this respect, causing rapid digestion
of cellulose. Certain molfts are also active, particularly in
acid soils. Then, too, a considerable number of species of
bacteria have also been isolated from the soil, capable of
bringing about cellulose digestion. They belong to a
variety of genera. They may be recognized in the labora-
tory by their ability to grow upon an agar containing finely
divided cellulose and to produce a clearing of the medium.
By the destruction of cellulose in the soil, decomposition
products such as the simple soluble carbohydrates and
organic acids, are produced which may be utilized by many
other soil forms.

The changing organic matter present in soil is usually
termed collectively "the soil humus." Most plant residues
added to soils are comparatively high in carbon and poorer
in nitrogen. In the process of decomposition carbon, in the
form of carbon dioxide, is given off more rapidly than is
nitrogen. In consequence, there is a tendency for the
humus, as it becomes older, to show a smaller ratio of nitro-
gen to carbon. This carbonaceous material present in soil
is of significance from many points of view. It ameliorates
the physical condition of the soil and furnishes directly and
indirectly from the products of its decomposition food
material for many of the nitrogen-fixing and other soil
bacteria. It is made up in part, of course, of the bodies of

266 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

bacteria and fungi themselves. Its color probably is in part
determined by the pigments produced by the bacteria and
the molds.

THE CYCLE OF PHOSPHORUS

Phosphates are quite essential to the development of most
higher plants. They are usually present in soil in the form
of tricalcium phosphate which is quite insoluble. It must
be converted into soluble phosphate to be taken up by plant
roots and combined into organic compounds such as the
phosphoproteins, nucleic acid, etc. The solution of the
insoluble phosphate in soil is probably in large part the
result of the action of organic acid and the carbon dioxide
produced as the result of the growth of microorganisms and
of the formation of nitric acid from ammonia. There seems
to be good evidence that rapid nitrification in the soil brings
about a considerable solution of the insoluble phosphates,
rendering them available for plant growth.

Phosphates also have a relationship to the activity of soil
microorganisms in their direct stimulating effect upon
growth. Many species of bacteria bring about fermentative
changes much more rapidly in the presence of phosphates
than in their absence. This is most certainly true with the
bacteria which are capable of fixing atmospheric nitrogen.

THE CYCLE OF SULPHUR

Sulphur in nature is found in its free form, as sulphates,
sulphides and in organic compounds, particularly in cer-
tain classes of proteins and in certain essential oils, such
as mustard oil. Microorganisms are active in bringing
about the various transformations of sulphur from one
condition to another. Protein compounds are broken down
into amino acids, particularly the amino acid cystein, and
from this is formed hydrogen sulphide. This may be
oxidized to sulphates. Sulphates are taken up by the plant

BACTERIA OF THE SOIL 267

roots and built up again into protein. Under anaerobic
conditions sulphates may be changed into sulphides. Cer-
tain bacteria are also known which can produce free sul-
phur, and others which can oxidize free sulphur to sul-
phates.

Org-anisms Oxidizing Hydrogen Sulphide and Free Sul-
phur.— A large group containing many families and numer-
ous genera of the so-called purple or sulphur bacteria (thio-
bactcria) are known which evidently utilize hydrogen
sulphide, very largely in their metabolism. Many springs
and ground waters contain considerable amounts of sul-
phides which, upon aeration, give off free hydrogen
sulphide. Bacteria belonging to this group are active in
bringing about oxidation in such waters. Among the most
common forms are members of the genera Beggiatoa and
Thiothrix. These organisms, by the oxidation o5 hydrogen
sulphide, produce granules of free sulphur inside of the
cells. When examined microscopically these free sulphur
granules will be noted as glistening globules. If such bac-
teria are dried upon a slide and a small amount of a sulphur
solvent, such as carbon disulphide is added and allowed to
evaporate, sulphur crystals may be detected. These bacteria
also possess the power of oxidizing the free sulphur to sul-
phates. It is not probable that these organisms are par-
ticularly significant in the soil as hydrogen sulphide is
rather readily oxidized to sulphates by chemical means and
by various catalytic agents in the soil. The species of bac-
teria which may bear some relationship to this phenomenon
in the soil are not well known. The process has been some-
times termed sulphofication.

Bacteria belonging to this group may be of some signifi-
cance in the destruction of concrete structures such as
cement tile through the formation of sulphuric acid and
consequent disintegration.

Sulphates are quite as essential to plant growth as are

268 AGEICULTUBAL AND INDUSTEIAL BACTEEIOLOGY

phosphates. Most soils are somewhat higher in sulphates
than in phosphates and consequently additions of sulphates
are not as frequently required for soils as are the phos-
phates. In some cases, however, they are deficient.

Organisms Reducing Sulphates to Sulphides. — Under
anaerobic conditions, in the presence of organic matter,
many species of microorganisms are known which will
change sulphates to sulphides, in many cases releasing free
hydrogen sulphide. This power particularly is possessed
by many of the soil bacteria and by forms belonging to the
genus Vibrio, particularly the species Vibrio desulphuri-
cans.

Bacteria of this type may be of significance in soils which
are water-logged and which contain an abundance of sul-
phates and organic matter. Hydrogen sulphide is quite
poisonous to many plant roots. The reduction of the
sulphates to sulphides, therefore, will make conditions par-
ticularly unfavorable for the development of many kinds of
plants. Processes such as the reduction of sulphates to sul-
phides and of nitrates to nitrites are among the agencies
which are important in causing plants such as corn to turn
yellow when the soil in which they are growing becomes
water-logged. These organisms are also important in
sewage disposal. When organic matter, such as sewage, is
mixed with a city water supply high in sulphates, such as
magnesium sulphate, the reduction to sulphides and the
formation of hydrogen sulphide may be so great as to
create a nuisance. Special methods of sewage disposal have
been adopted in some cases in which the sewage is more
thoroughly aerated than is necessary with sewage which is
lower in sulphates.

THE CYCLE OF IRON

Iron in nature occurs in organic form and in the inor-
ganic form in the oxidized and reduced condition. Micro-

BACTERIA OF THE SOIL 269

organisms are known which oxidize the reduced iron to the
ferric condition, and other organisms which, under ana-
erobic conditions in the presence of organic matter will
reduce ferric iron to ferrous iron. The latter are of some
practical significance as the ferrous iron compounds may
be somewhat poisonous to plant roots.

Bacteria oxidizing ferrous to ferric iron are of consider-
able importance in the deposition of iron ore. It is prob-
able that most of the great iron ore beds, consisting of iron
oxide and iron carbonate, have been formed through the
action of microorganisms. Bacteria belonging to this group
for the most part are forms which oxidize the soluble salts
of ferrous iron to an insoluble ferric condition and deposit
them in the sheaths of the filaments. In the city water sup-
plies they may accumulate in quantities such as to form
reddish or yellowish deposits in the pipes, the so-called
deposit of iron rust.

SECTION V

MICROORGANISMS AND DISEASE

CHAPTER XXI

DISEASE, INFECTION, AND RESISTANCE

Disease. — Disease may be defined as any abnormality
in form or functioning of the body or of any of its parts.
Thus denned the term includes many conditions frequently
not thought of as disease. It includes, for example, condi-
tions usually termed traumata, that is, the changes resulting
from mechanical injury, such as a blow, a bruise or a cut.
In addition to the traumatic diseases there are so-called
dietary or deficiency diseases. These are known both in
plants and in animals. A lack of sufficient iron, for ex-
ample, will, in certain plants, result in a whitening of the
leaves, that is, in a decrease in the amount of chlorophyl
present. In man and animals diseases sometimes result
from an improper balance among the principal food con-
stituents: fats, proteins and carbohydrates. Still more
frequently occur those diseases due to the lack of certain
so-called accessory substances or vitamines. Such, for
example, are the diseases scurvy, beriberi and probably
pellagra. Still another group of diseases is due to the
improper functioning of certain glands of the body such as
goitre and diabetes. There may be also hereditary defects
such as color blindness or congenital defects such as hare
lip or clubfoot. All the conditions enumerated may be
grouped together as noninfectious diseases.

An infectious disease is one caused by some microorgan-
ism. It is evident, therefore, that the primary grouping of
diseases may be made into infectious and noninfectious.

273

274 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

The student in bacteriology is interested in the former and
in the bacteria, yeasts, molds and protozoa which may pro-
duce them.

Among the infectious diseases of man the following are
important: pneumonia, tuberculosis, boils, abscesses and
bone infections, cerebrospinal meningitis, gonorrhea, Malta
fever, anthrax, gaseous gangrene, tetanus, bubonic plague,
glanders, paratyphoid, typhoid, dysentery, diphtheria, in-
fluenza, smallpox, chicken pox, measles, hydrophobia,
mumps, Asiatic cholera, syphilis, malaria, yellow fever,
trench fever, typhus fever.

Among the infectious diseases of domestic animals and
birds may be noted the following: strangles or distemper,
glanders, infectious abortion, fistula, poll-evil, wound infec-
tion, tetanus, hemorrhagic septicemia, pseudotuberculosis,
dourine, horse sickness, swamp fever, pox and pneumonia in
the horse ; wound infection, mastitis, pneumonia, milk fever,
blackleg, malignant edema, hemorrhagic septicemia, infec-
tious abortion, pseudotuberculosis, lumpy jaw, surra, Texas
fever, pleuropneumonia, foot and mouth disease, rinderpest,
cowpox and anthrax in cattle; hog cholera, swine plague,
swine erysipelas, hemorrhagic septicemia, anthrax, tuber-
culosis, foot and mouth disease in the hog; anthrax, pneu-
monia, Malta fever, pseudotuberculosis, sheep pox, in goats
and sheep; fowl cholera, fowl plague, fowl typhoid, white
diarrhea of chicks, roup, tuberculosis, spirochastosis, asper-
gillosis, epithelioma contagiosum in domestic fowls; and
rabies and dog distemper in the dog.

Infectious diseases among plants may be differentiated
into those caused by fungi and those caused by bacteria. A
large number of these latter have been described under the
name of bacterioses. Among the more common may be
noted the fire blight of the apple and pear, the black rot of
cabbage, crown gall of apple and other fruits and various
blights, galls, tumors, leaf spots and wilts of various plants.

DISEASE, INFECTION, AND RESISTANCE 275

Certain infectious diseases of insects are also of economic
importance. The best known of these are the flacherie of
the silk worm, American foul brood and European foul
brood of bees.

Contagious and Noncontagious Diseases. — A contagious
disease is one which is more or less readily transmissible
from one individual to another by direct or indirect con-
tact. A noncontagious disease is one that is not thus trans-
mitted. In practice the term contagious is usually modi-
fied. We speak of certain diseases as being highly con-
tagious, of others as being moderately or slightly contagious.
At the other end of the series we have diseases which are
noncontagious. Particular notice may be drawn to the
fact that the terms infectious and contagious are frequently
confused in popular literature and discussion. It will be
noted that infectious refers to cause, contagious to method
of spread.

Factors Predisposing to Disease. — The resistance of an
individual to a disease undoubtedly varies from time to
time. It is probable that unusual fatigue, hunger, unusual
chilling or excessive heating of the body may increase the
susceptibility of the individual. Certain diseases are more
apt to attack children, others more frequently attack adults.
There are also diseases characterized usually as diseases of
old age.

Heredity and Disease. — To be inherited, in the strict
sense of the term, a disease must be of a character such that
it can be transmitted indefinitely from one generation to
the next. Certain defects, such as color blindness, may be
transmitted in this fashion, but this is not true of any of
the infectious diseases. Animals may at birth occasionally
be infected with certain diseases acquired by them from the
mother before birth. Such diseases are, however, not in-
herited in the strict sense of the term.

276 AGRICULTURAL AND INDUSTRIAL BACTEEIOLOGY

, IMMUNITY

Immunity may be defined as resistance to disease. Its
converse is susceptibility. The general fact that individuals
vary greatly in their ability to resist disease has of course
been known since history began. Many explanations were
suggested to explain the reason for an individual becoming
immune to a disease after having once been attacked by it,
as by smallpox, for example. Some five of these theories
are worthy of note. Three of the earlier theories have been
quite generally discarded as imperfect or not tenable. Two
of the others, however, have played a very large part in the
development of modern views.

Exhaustion Theory of Immunity. — When it was recog-
nized that bacteria and other microorganisms sometimes
cause disease it was suggested that an individual became
immune to the disease caused by such an organism due to
the using up or abstraction from his body of some of the
material essential for the growth of the organism. As long
as this deficiency existed bacteria of that type could not
grow in the body. It was necessary to discard this concep-
tion, however, when it was shown that the blood, for
example, of individuals entirely immune to certain diseases
contained all of the nutrients necessary for the active
growth of their causal organisms.

Noxious Retention Theory. — When bacteria are culti-
vated in the laboratory, it is always noted that they grow
most rapidly in the early stages of development. As time
progresses the growth becomes slower and slower and finally
ceases. It was suggested early that this stopping of growth
in culture media was due to the development of substances
injurious to the organism. Bacteria growing in milk, for
example, produce lactic acid in sufficient quantities event-
ually to stop the growth of the organism producing the acid.
Keasoning by analogy it was then suggested that bacteria

DISEASE, INFECTION, AND RESISTANCE 277

growing in the body and producing certain diseases might
develop there substances not injurious to the health of the
individual, but which would prevent further growth of the
microorganism in question. Studies of the blood, however,
soon revealed this suggestion to be fallacious. Immunity
apparently is not due to the retention of noxious substances
produced by the microorganisms causing the disease.

Pythogenic Theory of Disease. — It was also suggested
that filth and decaying organic material might be the cause
of disease. A half century ago it was believed demon-
strably true that malaria was due to the inhalation of the
miasms arising from the decomposition of organic matter in
swamps. Murchison, in England, contended that filth and
accumulated dirt, particularly decaying organic matter,
produced many types of fever. When the germ theory of
disease was suggested, the pythogenic theory was modified
and it was contended that pathogenic or disease-producing
microorganisms might be generated in such decaying heaps
of organic substances, gain access to the air, and produce
disease as a result of inhalation. It was even contended that
disease germs might originate spontaneously in such ma-
terial. While we now know that disease may sometimes be
spread as a result of the accumulation or presence of filth
and dirt, nevertheless, it has been quite definitely proved
that these factors do not stand in the causal relationship
to disease which was suggested by the proponents of the
pythogenic theory.

Ehrlich's Humoral Theory of Immunity. — As a result
of detailed studies Ehrlich came to the conclusion that
immunity is due to the development by the body tissues of
substances which will in some way antagonize the invading
microorganisms or neutralize their poisonous and deleteri-
ous products. The substances thus produced in the tissues
he termed antibodies. An individual, therefore, becomes
immune or is naturally immune to disease because of the

278 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

sufficient presence of the appropriate type of antibody in
the blood or in the body tissues. This theory has apparently
stood the test and there is little reason to doubt that im-
munity in many diseases, probably in most, is due to the
presence or to the development of antibodies which can
specifically antagonize the invading microorganism.

Metschnikoff's Theory of Phagocytosis. — As a result
of the microscopic examination of the blood of animals
which had been injected with various kinds of bacteria,
Metschnikoff came to the conclusion that certain body cells,
particularly certain types of white blood corpuscles, were
capable of engulfing and destroying invading microorgan-
isms. He concluded that immunity was due to the power of
these cells to destroy bacteria. An individual who became
immune to disease had, so to speak, trained his phagocytic
body cells to destroy the particular type of organism in ques-
tion. Such an individual, therefore, would be immune to the
disease as long as this function on the part of the white
blood cells persisted. It will be shown later that Metschni-
koff 's theory contained much of value. Undoubtedly the
white blood cells are important in preventing disease, and
any adequate explanation of immunity must take into con-
sideration their activity. However, it has been found that
the white blood cells by themselves usually are relatively
incapable of destroying a microorganism but must act in
conjunction with certain antibodies present in the blood
stream. It will be noted, therefore, that Ehrlich's theories
and Metschnikoff's theories have been finally united and
together constitute the basis for the modern science of im-
munology.

Kinds of Immunity. — Common observation teaches that
man is immune to many diseases which attack animals. For
example, man does not contract hog cholera or dog dis-
temper. Most diseases of plants are not transmissible to
animals or vice versa. Many diseases of man cannot be

DISEASE, INFECTION, AND EESISTANCE 279

transmitted to lower animals. Within a certain species,
furthermore, certain individuals are found from birth to be
much more resistant than other individuals to a particular
disease. All of these are examples of natural, that is, con-
genital, immunity.

An immunity, on the other hand, which develops after
birth is said to be acquired. It may develop as the result of
any one of a considerable variety of conditions. The body
itself may take an active part in the development of immune
substances, that is, of antibodies. Such an immunity is
termed an active immunity. Such, for example, is the
immunity which develops as a result of having had small-
pox or diphtheria. Immunity of this type, furthermore,
results from vaccination, that is, inoculation with dead or
attenuated cultures of microorganisms causing the disease,
against which it is desired to immunize. The blood serum
of an actively immunized animal comes to contain antibodies
specific against the disease.

The blood serum of an actively immunized animal may
be injected into an animal which has not been immunized
and such an individual may acquire, as a result, a consider-
able degree of immunity. In this case, the tissues of the
individual into which the serum is inoculated or injected
do not play any part in the manufacture of the antibodies,
that is, the body is passive. This type of immunity, there-
fore, is termed passive immunity. The immunity conferred
as a result of the injection of antidiphtheritic serum is of
this type, likewise the immunity conferred by the injection
of anti-hog cholera serum.

An acquired immunity may be either permanent or tem-
porary. In general an active, acquired immunity is some-
what more permanent than a passive acquired immunity.
For example, hogs which are passively immunized against
hog cholera by the so-called single treatment, that is, by
the injection of an anti-hog cholera serum, acquire an im-

280 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

munity which is distinctly temporary. The animal is pro-
tected for only a few weeks. By the use of the vaccine,
however, the animal may be caused to develop an active
immunity which will last for a much longer perior of time,
probably for years in many cases.

Factors Which Determine Immunity. — The factors which
determine the resistance against disease in plants and ani-
mals may be grouped under two general headings: those
which are of importance in what may be termed general
resistance, and those of importance in specific resistance
against particular diseases.

In animals general resistance is due in large part to the
coverings of the body and to the membranes lining its
respiratory and digestive tract. The skin is an effectual
barrier to the entrance of microorganisms generally. The
mucous membrane of the nose and respiratory tract con-
stantly throw off mucus upon which dust and microorgan-
isms inhaled generally catch, and are pushed up toward the
body openings into the throat and nose by the cilia of the
epithelial cells. In other words, a cleansing stream of mucus
is constantly rising from the lungs and prevents the accu-
mulation of dust particles in this organ. In the alimentary
tract the digestive juices, particularly the strongly acid
gastric juice, destroy many microorganisms. The sub-
cutaneous tissues, particularly the layers of fascia, lying
parallel to and just under the skin in many parts of the
body and separating muscles and subdivisions of tissues
from each other, are important agencies in preventing the
distribution of microorganisms through the body.

In plants the general resistance is usually due to the
presence of bark or cuticularized layers covering the living
tissues. Many plants also contain substances, such as acids,
tannins, glucosides, etc., which prevent the development
within them of most kinds of microorganisms.

Specific resistance in animals, as already noted, depends

DISEASE, INFECTION, AND RESISTANCE 281

upon the development, in the tissues of the animal, of
antagonizing substances termed antibodies. Any substance
which, when injected into the tissues or when present in
the tissues, causes these tissues to develop an antibody is
termed antigen. A consideration of specific immunity,
therefore, is primarily a discussion of the various substances
or antigens ivhich can incite the tissues of the body to the
development of immunizing substances and a study likewise
of these immunizing substances or antibodies.

The following table lists the more important of the anti-
gens and their corresponding antibodies, which will be dis-
cussed on the succeeding pages :

ANTIGENS AND THEIR CORRESPONDING ANTIBODIES

ANTIGENS ANTIBODIES

Toxin Antitoxin

Bacterial cell (agglutinogen) Agglutinin

Soluble protein (precipitogen) Precipitin

Cells foreign to body: Cytolysin

Bacteria Bacteriolysin

Red blood cell Hemolysin

Bacterial cell Opsonin

Certain kinds of bacteria capable of producing disease are
known to produce poisons termed toxins. The body devel-
ops immunity against such by the production of antitoxins.
When certain kinds of bacteria or other cells gain access to
the body, the latter may sometimes produce substances which
will cause these bacteria to clump together or agglutinate.
These are termed agglutinins. Foreign proteins, that is pro-
teins not native to the body of the animal into which they
are injected or coming from another species of animal or
plant, will cause an individual injected to develop sub-
stances in the blood serum which will precipitate the corre-
sponding protein. Such antibodies are termed precipitins.
The presence of foreign cells (such as bacteria) may cause
the tissues to develop substances which will digest or dis-

282 AGEICULTUEAL AND INDUSTRIAL BACTERIOLOGY

solve the particular organism in question. A person who
has completely recovered from typhoid fever, for example,
will generally contain in his blood serum substances which
will dissolve or digest typhoid bacilli. Such substances or
antibodies are termed b act erioly sins. Sometimes another
type of antibody may be developed against bacterial or
other cells. These substances function by making the bac-
teria more attractive apparently to the white blood cells.
In other words, they enable the white blood cells to destroy
the microorganism. They are termed opsonins. Our con-
sideration of the factors underlying immunity, therefore,
must include a brief consideration of these various anti-
bodies and their corresponding antigens.

TOXINS AND ANTITOXINS

A toxin may be defined as an organic poison, that is, a
poison produced by living cells of plants or animals and
having certain characteristics of which the following are
most important:

1. Toxins are very easily destroyed (labile). Particularly
are they thermolabile, that is, easily destroyed by heat.
They deteriorate rapidly when kept in the light and in the
presence of moisture.

2. When toxins are injected into suitable animals in
amounts smaller than sufficient to cause death (sublethal
doses) these animals will respond by the development in the
blood serum of antitoxins or substances which will neutral-
ize the toxins. No poison, therefore, is a true toxin which
will not cause animals to produce antitoxin to neu-
tralize it.

It will be noted that the above definition does not include
all organic poisons. The immunity resulting from the
development of antitoxins in the body must be carefully
differentiated from so-called drug habituation. Repeated
injection of morphine (an organic poison), for example,

DISEASE, INFECTION, AND RESISTANCE 283

will cause the body to become accustomed to its presence
and to endure relatively large amounts of the material.
This resistance, however, is not due to the presence or for-
mation of substances which definitely neutralize the activity
of the morphine.

The exact chemical constitution of none of the toxins has
thus far been determined. Generally when injected into
an animal they require a period of incubation before the
results of the poisoning action are manifest. In general
they injure the body probably by combining chemically
with certain of the body cells or tissues. The toxin pro-
duced, for example, by the bacillus causing tetanus or lock-
jaw combines with the central nerve cells, injuring them
and producing thereby the symptoms of the disease.

Sources of Toxins. — A relatively small number of the
disease-producing bacteria develop toxin. The most im-
portant of these are the bacteria which produce the diseases
diphtheria, tetanus and botulism (one type of food poison-
ing). The organisms causing blackleg in cattle and gaseous
gangrene in man and certain types of dysentery bacilli
develop toxins likewise. It is evident, inasmuch as
toxins are produced by only few of the pathogenic bacteria,
that antitoxins for immunizing against or curing such
diseases must be limited in number. There is no probability,
for example, that an antitoxin can ever be secured for a
disease such as tuberculosis in which there is no evidence of
any toxin production by the bacteria.

These toxins are excretions of the bacterial cells. Certain
other plant cells are also known which can produce toxins.
The juice of the castor oil bean seed, the inner bark of the
black locust and the seed of the jequirity bean, all contain
toxins. Certain poisonous animals are also toxin producers.
Snake venom, for example, contains one or more toxins.
The stings of many insects, fish and other animals are
injurious because of the presence of true toxins.

284 AGKICULTUKAL AND INDUSTRIAL BACTEEIOLOGY

Origin of Antitoxin. — It has already been noted that
when a toxin is injected in sublethal doses into a suitable
animal, this animal will react eventually by the develop-
ment in its blood serum of substances (called antitoxins)
capable of neutralizing toxin of the type injected. Experi-
ence has shown that these antitoxins are developed not only
in quantities sufficient to neutralize the amount of toxin
injected, but quantities greatly in excess of this. It was
also noted that toxins poison because of their ability to unite
chemically with certain cells of the body and injure them.
According to the Ehrlichian conception, when these toxins
are not combined with the cells in amount sufficient to cause
death of the cells, these cells are stimulated to an excessive
production of antitoxins which are thrown off into the
blood serum and by combining there with the toxin prevent
its union with the cells. It may be emphasized that anti-
toxins are specific, that is, each kind of toxin causes the
development in the animal's body only of its own particular
type of antitoxin and this antitoxin will neutralize only the
type of toxin which caused its production.

Manufacture of Diphtheria Antitoxin. — The blood
serum from an animal which has been actively immunized
by repeated injections of the toxin of the diphtheria bacillus
may be used for the prevention or treatment of diphtheria
in man. An account of the method of preparation will
serve to show the general principles governing manufacture
of antitoxins. Production of antitoxins of other types dif-
fers from that described for diphtheria only in details.

Preparation and Standardization of Toxins. — Inasmuch
as the diphtheria toxin is an excretion of the diptheria
bacillus it can best be prepared by growing the bacteria in
broth and when the toxin formation has reached the maxi-
mum the bacteria may be filtered off by means of a porcelain
filter. The broth is usually placed in large flat-bottomed
flasks, the layer of broth being comparatively thin. The

DISEASE, INFECTION, AND EESISTANCE 285

diphtheria . bacteria grow as a film over the surface and
within a few days produce the maximum amount of toxin.
Some substance, such as cresol, which will kill the diph-
theria bacilli without injuring the toxin, is added and the
broth filtered through a suitable porcelain filter. This broth
containing the toxin is now ready for standardization. In-
asmuch as no chemical test has been devised for the detec-
tion or estimation of a toxin it is necessary to standardize
by injection of the material into a suitable animal. The
animal of choice is the guinea pig. The unit of toxicity is
determined by injecting varying amounts of the toxic broth
into a series of guinea pigs weighing about 250 grams each.
Those animals which die in just three days have received a
minimum lethal (fatal) dose of the toxin. This unit of
toxicity is usually abbreviated as the M. L. D. dose. A
broth having a satisfactory content of toxin frequently will
show one thousand or more M. L. D. per cubic centimeter,
that is, each c.c. will contain enough poison to kill one
thousand guinea pigs weighing 250 grams each.

Injection of the Toxin. — Several species of animals may
be used in the production of diphtheria antitoxin. The
horse has proved to be the most suitable from the commer-
cial standpoint because it will yield a large quantity of
serum at one bleeding and usually will develop an antitoxin
of relatively high potency. The horses used must first be
examined to make sure that they are free from disease and
in good physical condition. They are then injected sub-
cutaneously with a small amount of diphtheria toxin. This
may cause some local swelling and fever. This soon disap-
pears, however, and the animal may then receive a second
dose of the toxin. At intervals larger and larger doses of
toxin are injected until it is evident that the animal has
reached a high degree of immunity and the blood contains
considerable quantities of antitoxin. A small sample of
blood is then withdrawn from the horse and tested to deter-

286 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

mine its antitoxin content. The horse is bled from the
right jugular vein by means of a sterile trocar and tube into
a sterile glass vessel. Usually about one liter of blood may
be secured per one hundred pounds weight of horse. This
amount can be withdrawn from the animal without serious
injury. After a period for recuperation the animal may
then be injected several times more with diphtheria toxin
and again bled. Successive bleedings from the same animal
may thus be secured over a period sometimes of several
years. The blood is allowed to clot, that is, the fibrinogen
of the plasma is transformed into fibrin which catches the
blood cells as in a net and by its contraction squeezes out the
blood serum from the clot. This clear, straw-colored blood
serum is found upon examination to contain the antitoxin.
Standardization of Antitoxin Serum. — Inasmuch as
there is great variation in the relative antitoxic content of
the serum secured from different horses it is necessary to
standardize each sample. For this purpose, the antitoxin
content is compared with a sample of standard antitoxin,
secured in the United States from the Hygienic Laboratory
of the Public Health Service in Washington, D. C. The
standard serum secured from this source is diluted so that
each cubic centimeter contains one immunity or antitoxic
unit. It is evident that the antitoxin which has been
manufactured cannot be compared directly with the stand-
ard antitoxin but only by use of titration of each of these
against a toxin. The manufacturer, therefore, uses a suit-
able sample of toxic broth. This is first titrated against the
standard antitoxin secured. To each one of a series
of tubes one antitoxic unit of the standard serum is
added. To each of these tubes, then, graduated amounts
of the toxic broth are likewise added; to the first
tube a small amount, to the next a larger amount and
so on to the last. Each tube is then made up to the
same volume with sterile physiological salt solution and the

DISEASE, INFECTION, AND RESISTANCE 287

contents thoroughly mixed. The content of each tube is
then injected into a guinea pig weighing 250 grams. It is
probable that the guinea pig receiving the injection of the
contents of the first tube will survive, inasmuch as the
amount of antitoxin is more than sufficient to neutralize all,
of the toxin. At the other extreme, however, it is probable
that the guinea pig will die, inasmuch as the toxin present
is more than completely sufficient to neutralize the anti-
toxin. It is evident, then, that the guinea pigs at one end
of the series will live and those at the other end will die.
"Where these two series come together is an approximation
of the neutralization point. The guinea pig, in other words,
is used as an indicator in the titration of the toxin against
the standard antitoxin. It is necessary arbitrarily to select
the point of neutrality. That amount of toxin which when
mixed with one immunity unit of the standard antitoxin
and injected into a 250 gram guinea pig will kill that guinea
pig in just four days is termed an L -f- dose. This amount
of the toxic broth is now placed in each of a series of test
tubes. That is, each test tube contains one L -f- dose of the
now standardized toxin. To the series of tubes is then added
graduated amounts of the antitoxin which has been manu-
factured and which it is desired to standardize. The contents
of these tubes are in turn injected into suitable guinea pigs.
Those animals receiving sufficient antitoxin completely to
neutralize the toxin injected will live. Those receiving an
excess of the toxin will die. Here again an animal may be
chosen which dies in just four days. Inasmuch as it received
an L + dose of toxin and yet died in just four days it must
have also received one immunity unit of antitoxin.

The entire process may be compared to the standardiza-
tion of an unknown alkali by use of a known alkali and
unknown acid. The acid is first standardized against the
known alkali, and then used for the standardization of the
unknown alkali.

288 AGKICULTUEAL AND INDUSTEIAL BACTEEIOLOGY

The standardized antitoxin is now filtered through por-
celain filters and placed in suitable small containers. It
may be used in prevention or curing of the disease diph-
theria by injection subcutaneously by means of a hypo-
dermic needle.

Preparation and Utilization of Other Antitoxins. —
Antitoxins may be prepared in a somewhat similar manner
for the prevention and treatment of tetanus or lockjaw,
certain types of gaseous edema and for botulism. Anti-
toxins have also been prepared for the toxins of the venoms
of various snakes. i

AGGLUTININS AND PRECIPITINS

Two men working independently, Gruber and Widal, dis-
covered almost simultaneously that when certain kinds of
bacteria were present in the body, as in certain diseases
such as typhoid, the blood serum came to contain some sub-
stance which would cause agglutination or flocculation of
bacteria of this particular type. For example, when a drop
of blood serum from a person who has typhoid fever is
placed in a broth culture of typhoid bacilli and allowed to
stand for a time, the bacteria gradually lose their power
of motion, begin to clump together in masses which grow in
size until finally they settle out, and the medium which was
originally cloudy and turbid becomes clear with a flocculent
sediment. Serum from normal individuals usually does not
show this power. The substances developed in the blood
capable of flocculating bacteria in this fashion were termed
agglutinins.

Somewhat later the independent observation was made
that whenever foreign proteins of any kind were injected
into an animal, eventually the blood of that animal acquired
the property of precipitating the corresponding or homol-
ogous protein. For example, if a dilute solution of egg white
be injected into the blood stream of a rabbit at suitable

DISEASE, INFECTION, AND RESISTANCE 289

intervals, and after a time the rabbit bled, the blood allowed
to coagulate and the serum secured, this serum, when added
to a dilute solution of egg white will cause the medium to
become opaque or cloudy and a flocculent precipitate will
settle out. This will occur, however, only when the blood
serum is mixed with the particular protein injected in the
first instance. The substance produced in the blood serum
is termed a precipitin.

Colloidal Solutions and Suspensions. — It was not at first
recognized that the phenomena of agglutination and pre-
cipitation were fundamentally the same. Substances in
extreme state of division or fineness are comparatively
coarse colloids, or rather, their suspension constitutes a
colloidal suspension. The protein molecule, such as that
of egg white, apparently is large enough, or exists in aggre-
gates which are large enough, to behave as suspended par-
ticles rather than as substances in true solution. Experi-
ence has shown that a great variety of substances added to
particular proteins will cause precipitation, as in the so-
called salting out process. A saturated solution, for
example, of ammonium sulphate added to many proteins
will cause them to go out of solution, or precipitate. The
same is true with bacterial suspensions. A saturated solu-
tion of ammonium sulphate will also cause typhoid bacilli
to flocculate out of suspension. This ability of certain salts
to cause flocculation or agglutination is particularly well
shown by mixing fine silt or clay with water. The solution
will remain turbid almost indefinitely, that is, the particles
will settle out very slowly indeed unless some salt, such as
alum be added. When this is added, however, the clay
particles bunch together, flocculate and settle to the bottom
rapidly.

The resemblance between the phenomena of agglutination
and precipitation by means of blood serum and the agglu-
tination or flocculation by means of salts escaped notice for

290 - AGEICULTUEAL AND INDUSTKIAL BACTERIOLOGY

many years. It has now been shown definitely, however,
that the blood serum which is capable of causing flocculation
of a particular kind of bacterium does this by sensitizing
the bacteria to the action of certain salts. For example,
common salt, that is, sodium chloride, in dilute solution does
not cause flocculation or agglutination of typhoid bacteria.
A dilute solution of serum from a typhoid patient with
typhoid bacilli in distilled water will not cause agglutina-
tion. When, however, typhoid serum, bacteria and common
salt are all present, flocculation or agglutination does
occur. It can be readily shown that the agglutinin of the
serum combines with the bacteria and makes these bacteria
sensitive to the action of salt. Apparently the same explan-
ation holds for precipitation as with proteins when a
specific antiserum is used.

Agglutination. — Agglutinins are usually highly specific,
that is, an agglutinin capable of causing clumping of one
kind of microorganism will not cause clumping of other
species. There are same exceptions to this rule, however.
In some cases closely related bacteria will cause antibodies to
develop which will cause some agglutination of the other
species. In many instances, however, the reaction is so
specific that it can be used readily in the diagnosis of
disease. For example, the most common method of recog-
nizing typhoid fever in the earlier stages of the disease is
by the so-called Widal test (named after the man who first
described typhoid agglutination). The test may be either
microscopic or macroscopic. If the former, some of the
diluted serum from the patient suspected of having typhoid
is mixed with a drop of typhoid broth culture and examined
under the microscope. If the serum contains agglutinin,
that is, if the patient has typhoid, the bacteria will at first
be observed to move about rapidly, finally their motion
decreases, they begin to clump together and in a few min-
utes to half an hour comparatively few, if any motile bac-

DISEASE, INFECTION, AND RESISTANCE

291

teria are to be seen, the cells being arranged in large clumps.
In the macroscopic test blood serum is added to a broth
culture of typhoid bacilli or to a suspension of the typhoid
bacilli in a physiological salt solution. When allowed to
stand for a suitable length of time, the bacteria will floc-
culate out leaving a clear, supernatant liquid and a floc-
culent sediment.

Agglutinins apparently are not produced in quantities
sufficient for assistance in recognition of many of the com-
mon diseases. The agglutination test is used most fre-

FIG. 60. — AGGLUTINATION. Left: Unagglutinated bacteria. Bight:
Clumped or agglutinated bacteria.

quently in typhoid, paratyphoid, dysentery, glanders, and
for the differentiation of the various types of bacteria
capable of causing pneumonia and meningitis.

Protein Precipitation. — Like the agglutinins the pre-
cipitins are highly specific. Precipitins developed for one
kind of protein will usually not cause precipitation of other
types. This property of precipitin formation upon the
introduction of foreign protein has been used for the dif-
ferentiation of proteins in several instances. By the injec-
tion of a particular protein into a suitable animal it is
entirely possible to secure from that animal a serum which
can thereafter be used as a specific test for the protein
injected. This method is used in practice in the differen-

292 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

tiation of horse flesh and beef in countries where both are
used. If some of the juice from known horse flesh is
injected into a rabbit a serum can be secured which will
always precipitate the juice from horse flesh. Similarly
rabbit serum may be secured specific for other kinds of
meat. To carry out the test some of the juice may be
pressed from the sample of meat suspected of being horse
flesh, treated with some of the serum from the immunized
rabbit and the identification made by the absence or pres-
ence of a clouding or precipitate. In a somewhat similar
manner the origin of blood stains has been determined. It
is possible to secure an antiserum which is a specific precipi-
tin for the blood serum of man or of any of the animals.
The various flours (such as wheat and buckwheat) contain-
ing proteins may be separated from each other by means of
such a test. The presence or absence of eggs in pastries may
also be recognized. In some cases precipitin tests may be
used in the recognition of disease. The blood serum of a
patient having typhoid fever, for example, will not only
agglutinate the typhoid germ but will also cause precipita-
tion in broth in which typhoid bacilli have been grown and
from which they have been filtered off. In some cases, as in
anthrax, the tissues suspected of coming from animals hav-
ing this disease may be extracted with physiological salt
solution and brought in contact with serum from an animal
which has been immunized against the particular disease.
The development of a precipitate shows the presence of the
specific organism in the tissue under investigation.

CYTOLYSINS, INCLUDING BACTERIOLYSINS AND HEMOLYSINS

It was first noted by Pfeiffer that the blood serum of
animals may sometimes possess the property of dissolving
or digesting bacteria. This observation was first made with
the organism which causes Asiatic cholera, the Vibrio
cholerce. It was found that when these organisms were

DISEASE, INFECTION, AND RESISTANCE 293

injected into the peritoneal cavity of a suitable animal,
samples withdrawn at intervals and stained preparations
made for microscopic observation, the bacteria were found
at first to stain sharply and definitely, later to stain
indefinitely, then as granules, and at last completely to
disappear. It was then found possible to immunize animals
against various disease-producing organisms and to study in
the blood serum of such immunized animals the substances
capable of destroying the organism. Such substances were
named bacteriolysins. Later it was found that the injection
of other kinds of foreign cells would lead to the develop-
ment of corresponding antibodies. For example, if red
blood corpuscles (carefully washed to remove serum) of a
guinea pig be injected into the blood of a rabbit, the latter
will finally acquire the property of dissolving or digesting
the red blood cells of the guinea pig. This phenomenon may
be observed in a test tube. If a suspension of guinea pig
corpuscles in physiological salt solution be made and a drop
of blood serum from a rabbit which has been immunized
against such corpuscles be added to it, the opaque red sus-
pension will change in the course of a few minutes or half
an hour to a transparent, red solution. Such an antibody
capable of bringing about solution of red blood cells is
termed an hemolysin. Other types of cells were also found
to be capable of inciting the development of lytic substances.
Antibodies of this general type capable of dissolving cells
are termed cytolysins.

Constitution of Cytolysins. — Cytolysins are thermola-
bile, that is, when exposed to a temperature of 56° c. or
above for a half hour, they lose their power to destroy
cells. However, this power to destroy cells may be com-
pletely restored by adding to the antiserum some serum
from normal blood, that is, serum from an animal which
has never been immunized. It is evident, therefore, that a
cytohjsin is in reality made up of two substances; one pro-

294 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

duced as a result of the immunization and present only in
the blood of immunized animals; the other a normal con-
stituent of blood serum common to the blood of most, if not
all, species of animals. The substance produced as a result
of the immunization, that is, the specific antibody, has been
termed the amboceptor. The substance present normally
in the blood serum of most animals is termed the comple-
ment. Cytolysin, therefore, is amboceptor plus comple-
ment.

Experiment has shown that amboceptors are highly
specific. They unite only with the kinds of cells instru-
mental in causing their production. It may be shown
experimentally that whenever amboceptor comes in contact
with its specific antigen, the two unite. There is, however,
no visible change of any kind. If complement is then
added, it in turn unites with the antigen and brings about
the hemolysis. The amboceptor, in other words, is a spe-
cific substance which sensitizes bacteria or other cells to the
action of a universally present complement. Thi& may be
shown by mixing a heated antiserum, that is, a serum con-
taining cytolysin in which the complement has been
destroyed by heat (leaving the amboceptor only) with suit-
able cells, allowing them to stand in contact for a time, then
washing repeatedly (by means of the centrifuge) in physio-
logical salt solution. Cells treated in this fashion
brought into contact with serum containing complements
will be rapidly destroyed. Neither the heated serum alone,
containing amboceptor, nor the normal serum alone, con-
taining complement, is able to destroy the cells, but when
the cells are sensitized by the presence of amboceptors, the
complement is capable of destroying them.

It is apparent that immunity to many diseases is due, at
least in part, to the production of these specific amboceptors
in the blood. Such amboceptors, for example, may be
demonstrated in the blood serum of a person who has recov-

DISEASE, INFECTION, AND EESISTANCE 295

ered from typhoid fever. It is possible also to produce
antisera containing specific bacteriolysins in the horse, for
example, and to use such antisera in the treatment or pre-
vention of disease. The serum used in the treatment of the
disease cerebrospinal meningitis usually contains consider-
able amounts of bacteriolysin specific for the meningococcus,
the organism which causes this disease.

Complement Fixation in the Diagnosis of Disease.— In
certain diseases amboceptors specific for the causal micro-
organism are formed early. Their development does not
necessarily mean the recovery or effective immunization of
the patient. This is particularly true in certain chronic
infections. For example, the disease glanders in the horse
may and frequently does assume a chronic form which is
not at all readily recognized by clinical examination.
Whether or not the animal has the disease, however, may be
determined by examining a sample of the blood for the
presence or absence of the amboceptors specific for the
glanders bacillus. If they are present it is usually consid-
ered as conclusive evidence that the animal has the disease
glanders. This test, that is, hunting for specific ambo-
ceptors in the blood of suspected individuals, is termed the
complement fixation test. Two groups of substances are
necessary. The first is that group required for the specific
reaction, and second, a group used as an indicator for the
recognition of the reaction.

The materials of the first group are as follows :

1. Suspension of specific organisms such as glanders
bacillus (the antigen).

2. Serum from the animal, such as the horse, suspected
of having the disease. — If the disease is present this serum
will contain amboceptor and complement. The latter is
removed, however, before the test by heating. A positive
test will mean the presence of an amboceptor specific for
the glanders bacillus.

296 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

3. Blood serum containing complement. — This is usually
obtained from the guinea pig. It is carefully titrated
before use in order to determine the amount of complement
present.

Suitable amounts of numbers one, two and three, that is,
of the antigen, serum containing amboceptor, and serum
containing complement are mixed. The amboceptor should
at once unite with the specific organism, such as the
glanders bacillus, and then the complement unite with this
sensitized antigen. Theoretically one might expect to
determine the presence of amboceptor then by examining
such material microscopically. Practically, however, this
does not prove to be feasible. The changes brought about
by the complement in the bacteria may be in some cases so
slight that they cannot be observed microscopically. It will
be noted, however, that if amboceptor is present the comple-
ment will be used up or fixed. If amboceptor is not present
the complement will not be fixed. To determine then,
whether or not the amboceptor is present, some reagent is
desirable which may be added to this mixture to determine
whether or not the complement has disappeared. This
reagent (the second group of materials) is prepared from
the following:

4. Red blood cells of some suitable animal such as sheep.
— These should be washed carefully and suspended in
physiological salt solution.

5. Serum from rabbit which has been immunized by
repeated injection of sheep red blood corpuscles. — This is
heated to destroy any complement present. It should con-
tain amboceptors specific for sheep red blood cells.

Number four and five are mixed. The amboceptor unites
with the red blood cells, sensitizing them to complement.
These sensitized cells may now be used as an indicator in
solutions for the presence or absence of complement. This
mixture of 4 and 5 is added to the first mixture, containing

DISEASE, INFECTION, AND RESISTANCE

297

1, 2, and 3. If no hemolysis occurs it is evident that the
complement has been used up, therefore, that there is
amboceptor present in the patient 's serum and the disease is
diagnosed as glanders. On the other hand, when the mix-
ture of 4 and 5 is added to 1, 2 and 3 hemolysis will occur
if the complement has not been fixed. This will occur if
amboceptor is absent. The diagnosis then would be
negative.

A somewhat similar method, usually termed the Wasser-
mann test, is commonly used in the diagnosis of syphilis in

FIG. 61. — PHAGOCYTOSES. 1. Of bacilli. 2. Of cocci.

man. While theoretically many disease-producing organ-
isms cause the development of amboceptors, their presence
in the body is usually not used as a means of diagnosis with
the exception of the diseases glanders, syphilis, Malta fever
and gonorrhea.

OPSONINS AND PHAGOCYTOSIS

It has already been noted (page 278) that Metschnikoff
first called attention to the fact that certain body cells,
particularly certain types of white blood corpuscles are

298 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

capable of engulfing and destroying foreign cells such as
bacteria which may gain access to the body. He conceived
immunity, therefore, as being the process of training the
white blood cells to engulf and destroy invading micro-
organisms. Later two English investigators, Wright and
Douglas, showed that the white blood cells by themselves
usually do not possess the ability to engulf and destroy
microorganisms, but they require the presence of a serum
constituent of antibody to which the name opsonin has been
given. They showed that immunity in many diseases is due
to the development of these opsonins which so change the
bacteria that they become attractive or positively chemo-
tactic for the white blood corpuscles.

Detection of Opsonin.— The fact that white blood cor-
puscles have the power of destroying bacteria may be shown
by making injections of suitable bacteria into the blood
stream of an animal, removing samples of blood at intervals
and preparing stained mounts. By the use of an appro-
priate dye, the cytoplasm of the white blood corpuscles will
stain very lightly, the nucleus of the cell deeply and any
bacteria either on the inside or outside of the cell will also
stain deeply. It is possible by microscopic observation in
this fashion to count the number of bacteria which have
been engulfed by a white blood cell. Study will show
that the bacteria in the blood will at first be free, later and
in larger proportions, they will be found inside of the
white blood cells. Finally they will begin to break to pieces,
become granular and finally completely disappear.

Similar observations may be made outside of the body.
For this purpose it is necessary to secure suspensions of
white blood corpuscles, bacteria and the serum to be tested.
The white blood cells are secured by centrifuging blood
which has been prevented from coagulating by the addition
of sodium citrate. The red blood cells, having a higher
specific gravity, settle to the bottom. The serum remains

DISEASE, INFECTION, AND RESISTANCE 299

on top. The white blood corpuscles are intermediate and
will appear in large numbers in the surface layers of the
sediment. The serum may be then pipetted away, the white
blood cell layer removed by means of a small spoon or
spatula, resuspended in physiological salt solution and
centrifuged once or twice again to wash away all of the
serum. The cells are then suspended in physiological salt
solution for study.

A suspension of the bacteria to be studied is also prepared
in physiological salt solution. A suitable dilution of the
serum to be tested is also prepared. By means of a capillary
pipette a small amount of the suspension of white blood
cells, and equal amounts of the bacteria suspension and of
the serum to be tested are drawn up, then blown out upon
a glass slide and mixed. They are then drawn again into
the pipette, the end sealed and placed in an incubator at
37 y2° c. for fifteen minutes. The mixture is then blown
again onto a glass slide, spread, fixed and stained. In
general there will be a direct relationship between the
amount of opsonin specific for the organism used present in
the serum and the number of bacteria engulfed on the
average by white blood corpuscles. If bacteria and white
blood cells alone are mixed without serum containing
opsonin, very few if any of the bacteria will be engulfed.

Opsonic Index. — It is possible to compare the opsonin
content of one individual and that of another by determina-
tion of what is termed the opsonic index. This is a number
which represents roughly the ratio between the amount of
opsonin contained in the two bloods. When white blood
corpuscles, bacteria and serum from a patient, for example,
are tested and the average number of bacteria engulfed per
white blood cell determined, and the same process carried
out with the same bacteria, white blood cells and serum from
a normal individual, the ratio of the average number of
bacteria engulfed as the result of the use of patient serum

300 AGRICULTURAL AND INDUSTRIAL BACTEEIOLOGY

with that of those engulfed when using normal serum, is
termed the opsonic index. For example, if patient serum
on the average should cause the white blood cells to engulf
an average of ten bacteria, and the normal serum cause the
white blood cells to engulf five bacteria, the opsonic index
would be the ratio of ten to five.

The Presence of Opsonins in Antisera. — Certain types
of antisera are prepared by injection of suitable animals
with dead or living cultures of bacteria in an effort to secure
a high concentration of opsonin specific for the disease in
the blood of such an animal. Such sera are sometimes used
in the treatment of disease. Most of the so-called anti-
bacterial (not antitoxic) sera used in disease treatment con-
tain opsonins and usually cytolysins as well.

Significance of Opsonins in Immunity. — It is probable
that the body owes more of its resistance to disease to the
presence of opsonin than to the presence of any other single
antibody. Development of opsonin may be stimulated by
the use of vaccines. A vaccine may be defined as a dead or
attenuated culture of an organism injected into an animal
or individual for the purpose of causing it to develop an
active immunity. The immune substances formed are prob-
ably in large part cytolysin and opsonin. In some cases
the term vaccine is restricted to living or attenuated bac-
teria, and for dead cultures injected in a similar fashion the
word bacterin has been coined.

It is possible, following vaccination, to note increased
resistance by studying the opsonic index of the vaccinated
individual.

HYPERSUSCEPTIBILITY OR ANAPHYLAXIS

In certain respects the phenomenon of anaphylaxis or
hypersusceptibility is the converse of immunity. It is,
nevertheless, very closely related to the latter phenomenon
and should be discussed, in consequence, at this point.

DISEASE, INFECTION, AND EESISTANCE 3Q1

It has long been known that certain substances, par-
ticularly certain proteins, are poisonous to certain indi-
viduals. There are some persons, for example, who cannot
eat eggs without being poisoned thereby. The specific
phenomenon, however, was not carefully investigated until
after certain accidental observations were made upon the
conduct of guinea pigs. In the study of the standardization
of antitoxin attempts were made to use guinea pigs a second
time as test animals, that is, animals which had received
doses of antitoxin and toxin and had recovered were later
tested out to determine whether or not they might be
utilized a second time for standardization. It developed,
however, that almost invariably a second injection of anti-
toxin proved fatal. This led to a study of the effect of
injecting pure proteins into guinea pigs. It was found
whenever a dilute solution of a protein is injected under the
skin of a guinea pig or introduced into the blood stream, the ,
animal then allowed to remain without subsequent injection
for a period of two weeks or more, that a second injection
made after the end of this period resulted in the develop-
ment of a certain reaction which has been termed the ana-
phylactic shock. For example, a guinea pig may be
injected with a small quantity of egg white. It will show
no untoward reactions, will remain apparently healthy and
vigorous, but when injected intraperitoneally or intra-
venously two weeks or more later with a similar solution of
egg white there will be a rapid development of severe symp-
toms. The animal will show difficulty in respiration. It
will urinate and defecate rapidly. It will usually scratch
its nose with its front paws, thus showing evidence of diffi-
culty in respiration. It may finally develop convulsions,
fall over on its side and die within a few minutes after
injection. What was, upon the first injection, a nonpoison-
ous substance is found upon a second injection to become a
deadly poison for the guinea pig. An animal which has

302 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

been thus sensitized to the protein is said to be in a condi-
tion of hypersusceptibility or anaphylaxis.

The anaphylactic reaction is very specific. A guinea pig
injected with one kind of protein does not become sensi-
tized thereby against other kinds of protein.

Other species of animals than the guinea pig may be
similarly sensitized by the parenteral introduction of pro-
tein, that is by an injection or introduction of protein by
some means other than through the normal channel of the
alimentary tract. The symptoms developed as a result of
the second injection into these other animals may differ
materially from those exhibited by the guinea pig.

Examples of Natural Anaphylaxis. — It was noted above
that occasionally individuals may develop sensitiveness to
the presence of particular proteins without any intentional
preliminary sensitization. Such, for example, is the sensi-
tization of individuals to the pollen of certain plants. In
the United States in many localities the rag weed pollen
during the months of August and September causes intense
irritation of the mucous membrane of the respiratory tract
of those individuals said to have the disease hay fever. Just
how such individuals became sensitized, it is difficult to
ascertain.

Certain individuals become sensitized to the proteins
specific for particular animals. There are persons, for
example, that cannot be in the vicinity of a horse or inhale
the scurf from the skin of the horse without being subjected
to an attack of asthma. In fact many types of asthma are
undoubtedly due to the inhalation of particles of dust con-
taining proteins or other substances to which the individual
has in some way become sensitized. Individuals who have
thus become sensitized to proteins will show the character-
istic reaction in a variety of ways. If an extract of rag
weed pollen be rubbed vigorously into the skin of a person
subject to hay fever caused by this particular type of

DISEASE, INFECTION, AND RESISTANCE 3Q3

pollen, a marked inflammation and swelling at this point
will be noted. It will not have this effect if rubbed into the
skin of a normal individual. In a similar way an extract
from the hair or skin of the horse rubbed into the skin of a
person subject to horse asthma will cause a decided local
reaction.

Occasionally individuals are found who are subject to
anaphylactic shock when injected with horse serum. Care
must be used, therefore, in injecting diphtheria antitoxin
and other antisera to make sure that the individual is not
markedly anaphylactic toward this protein. In some in-
stances the injection has been known to cause a temporary
illness and is not infrequently followed by the development
of a rash.

Desensitization. — If a guinea pig be sensitized by the
injection of protein, and two weeks or more later a second
injection of the same protein be made in a dose too small to
cause fatal results, yet large enough to evoke some of the
symptoms of the anaphylactic shock, the animal will for a
time be temporarily desensitized. That is, after the ana-
phylactic shock has been evinced a third injection of the
same protein, unless much larger, will not produce the
anaphylactic symptoms. Use has been made of this prin-
ciple in desensitizing individuals sensitive to particular
proteins.

Utilization of the Anaphylactic Shock in Disease Diag-
nosis.— When certain bacteria, particularly organisms
causing chronic infections such as glanders or tuberculosis,
gain access to the body they apparently act in the same
manner as a sensitizing dose of a protein. That is, as a
result of their growth in the body the individual animal
becomes highly sensitized to the organisms and the products
of their growth. This fact is made use of in the recognition
or diagnosis of such diseases. For example, a cow which has
been infected with tuberculosis becomes hypersensitive to

304 AGEICULTUEAL AND INDUSTRIAL BACTERIOLOGY

the products of growth of the tubercle bacillus. Conse-
quently it is possible by growing the tubercle bacilli in the
laboratory in broth, killing the organisms by heat, filtering
and concentrating the product to develop what is practically
a glycerin extract of tubercle bacilli which, when injected
into animals suspected of having tuberculosis, will deter-
mine the presence or absence of the disease by the reaction
produced. If the injection is made subcutaneously, there
will generally develop in from twelve to sixteen hours after
injection a fever leading to a rise of one or more degrees in
the temperature. If the injection is made into the skin
there may be considerable local swelling developed. A
purified tuberculin brought into contact with the mucous
membrane covering the eye will cause decided inflammation.
The material thus prepared for diagnosing tuberculosis has
been termed tuberculin. A similar material prepared from
glanders bacilli termed mallein has been extensively used in
the diagnosis of glanders in the horse.

CHAPTER XXII
MICROORGANISMS OF THE BODY IN HEALTH AND DISEASE

Bacteria of the Body. — Many of the bacteria found
associated with the bodies of plants and animals are sapro-
phytic and are found in these locations by accident. Soil
and water contain great numbers of living organisms and
many are associated with dust particles in the air. Such
organisms, except as they may lead to some confusion in
studies of organisms normal to the body, are of no par-
ticular importance from the standpoint of health or dis-
ease.

The bodies of animals and many plants harbor what may
be termed a normal flora. Many of the organisms compos-
ing this flora are commensals, that is, they live upon the
waste products of the host and do not in any way cause
disease. Some are even decidedly useful. It is probable,
for example, that the digestion of cellulose in the alimen-
tary tract of ruminants and herbivorous animals in general
is due, at least in part, to the activity of certain kinds of
bacteria. Still other organisms are true parasites, living in
or on the tissues of the body. In a few instances these are
known to be helpful. The bacteria, for example, which live
in the roots of leguminous plants are truly parasitic, yet
they are on the whole helpful and not injurious to the plant.

Many of the bacteria present in the body are capable of
producing disease under certain conditions. Some kinds of.
organisms have very low degree of virulence or attacking
power and under normal conditions are quite incapable of
destroying or injuring living tissues. Other forms of bac-
teria which occasionally gain admission to the body are

305

306 AGKICULTURAL AND INDUSTRIAL BACTERIOLOGY

highly virulent and in many cases can overcome resistance
readily.

Certain species of cocci, particularly organisms belonging
to the genus Staphylococcus, are abundant upon the skin.
These bacteria, together with certain species of Strepto-
coccus, are found frequently in the mouth and intestines.
In the alimentary tract, particularly in the colon, a con-
siderable number of species of bacteria grow normally.
Most important and most common in this group is the
organism known as s the Bacterium coli. Under certain
abnormal conditions the bacterial flora of the intestines may
contain a great variety of other kinds of bacteria.

How Bacteria Gain Admission to the Tissues of the
Body, Infection Atria. — Microorganisms capable of caus-
ing disease gain admission to the body in a variety of ways.
In some cases they enter as a result of traumata, that is,
through direct mechanical injury to the skin or mucous
membranes. Bacteria, for example, frequently gain admis-
sion through wounds, giving rise to inflammation, pus
production and sometimes generalized infection. A special-
ized type of traumatic infection is to be found in the bites
of certain insects. The mosquito, for example, when
infected from the blood of a person having malaria may
later inject the organisms capable of causing this disease
into another individual. In a few cases organisms may
apparently gain access to the body through the unbroken
skin or mucous membrane and produce disease. Certain of
the molds, for example, capable of causing diseases such as
ringworm and favus apparently do not require any injury
-to the skin in order to begin their growth. A very frequent
channel of infection is the alimentary tract. Many diseases
are contracted as a result of swallowing the causal micro-
organisms. Typhoid fever is a disease of this type. In
certain cases the respiratory tract constitutes the infection
atrium. One may inhale, for example, the organism
capable of causing pneumonic plague or other respiratory

MICEOOEGANISMS OF THE BODY 307

diseases. Finally, certain deseases are transmitted usually
through the genito-urinary tract. Such, for example, are
certain of the venereal diseases, particularly gonorrhea and
syphilis in man and dourine in the horse.

How Bacteria Leave the Body. — Inasmuch as most dis-
ease-producing bacteria do not multiply outside the body
of the man or animal in which they live or produce disease,
it is important to know the channels through which these
microorganisms may leave the body. In some cases the
organisms are found in the pus or exudates from the
wounds or surface lesions of the body. In other cases they
may be found in mucous secretions of the nose and respira-
tory tract; in still other cases in the saliva. The organism
causing diphtheria, for example, is usually present in the
throat and mouth. In many diseases the causal microorgan-
isms are excreted with the feces. This is particularly true
of the so-called intestinal diseases such as typhoid, para-
typhoid, Asiatic cholera and dysentery. Occasionally the
organisms may be excreted in the urine. This occurs occa-
sionally in typhoid fever. Insects are sometimes responsible
for withdrawal of organisms from the body when they suck
up the blood. This occurs in yellow fever, malaria and some
other diseases.

Bacteria causing plant diseases frequently are not
released from the injured tissues until after the death and
disintegration of the tissue. In other cases exudates may
form and the organisms escape to surface. In still other
cases apparently the disease organism is transmitted by
sucking insects as in the curly leaf of the sugar beet.

The Types of Diseases Produced by Microorganisms. —
In the animal body the microorganisms sometimes invade
the blood stream, growing in the blood in all parts of the
body. Diseases of this type are termed bacteremias. A
bacteremia caused by entrance into the blood of an organism
usually responsible for pus production and inflammation
(such as Streptococcus or Staphylococcus) is frequently

308 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

termed a septicemia. Some authors use the term septicemia
and bacteremia as entirely synonymous. A toxemia is a
disease such as diphtheria or tetanus in which the causal
microorganism remains more or less localized in or on the
tissue where it produces a poison or toxin which enters the
circulation and causes injury to the various body tissues. In
some diseases the microorganisms tend to remain localized
and produce marked inflammation without any generalized
symptoms. Infections of this type are sometimes referred
to as phlogistic. A sapremia is a disease caused by the
absorption of poisonous putrefactive products from decay-
ing or necrotic tissue. For example, the retention of the
placenta or afterbirth in a cow after parturition and its
subsequent decay or decomposition in the uterus may lead
to the development of a sapremia.

Diseases are sometimes named after the causal organism.
A disease caused by a Bacterium, for example, might be
termed a bacteriosis, one caused by a Spirillum a spirillosis,
and one caused by a Spirochceta a spirochcetosis.

Diseases of plants produced by bacteria are sometimes
known as galls or tumors, as for example, crown gall of the
the apple ; blights, such as pear blight ; leaf spots ; rots, such
as the blackrot of cabbage; and wilts, such as the wilt of
cucumber or sweet corn.

The infectious diseases, that is, the diseases caused by
microorganisms, may be divided into two general groups;
those in which the causal agency is definitely known, and
those in which it is not definitely described. Under the
first heading we have diseases which may be caused by
bacteria, by yeasts, by molds and by protozoa. Under the
second heading there are diseases caused by filterable
viruses, that is, microorganisms apparently so small that,
at least in certain phases of their life history, they can pass
through the porcelain filters. In still other cases the causal
organism has completely eluded the search of the bacteriol-
ogist thus far.

CHAPTER XXIII

NONSPECIFIC INFLAMMATION AND SUPPURATION— THE
GENERA STREPTOCOCCUS AND STAPHYLOCOCCUS

Inflammation. — Inflammation is an abnormal condition
of a tissue usually characterized by excessive redness, by
pain, by swelling and usually by fever. The redness is due
to the engorgement of the capillaries with blood, the swell-
ing to the extrusion from the blood vessels of blood serum
or plasma and the pain or pressure upon nerve endings.
Inflammation may be produced in a variety of ways in the
bodies of man and animals. It sometimes results from
physical injuries. A burn or excessive exposure of the skin
to bright light will lead to inflammation. The injection of
certain chemicals will likewise product inflammation. There
are also many species of bacteria which when growing in the
tissues of the body will bring about inflammation. These
bacteria probably bring about the irritation necessary to
cause inflammation because of their injurious effect upon
the cells either directly or through the formation of various
poisonous substances.

Inflammation may be regarded as a protective device on
the part of the body. When microorganisms gain access to
tissues and give rise to inflammation, the changes incident
to this process may prevent them from spreading to other
parts of the body. The manner in which this is accom-
plished will be discussed below.

Suppuration. — Suppuration or pus production occurs
when certain organisms are growing in tissues and causing
localized inflammation. For example, when the so-called
pyogenic or pus-producing bacteria in some way penetrate

309

310 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

the skin frequently they find conditions favorable for devel-
opment. They have optimum moisture, optimum food con-
ditions and optimum temperature for their rapid growth.
They can be stopped only by specific defensive devices on
the part of the body. As a result of their growth and of the
irritation of tissues due to the resultant injury, the blood
capillaries expand and become engorged. Serum is poured
out into the tissues and passes into the wound. This serum
ordinarily contains antibodies, particularly opsonins. As a
result of the union of opsonin with the bacteria, the white
blood cells are also attracted to the area involved. These
white blood cells have the power of leaving the blood stream,
passing out through the capillary walls, and soon come to
lie in great numbers around the injured area. They are
so tightly packed together in many cases that they consti-
tute what is termed a pyogenic membrane, capable in most
instances of effectually preventing further spread of the
microorganisms through the tissues. The opsonized bacteria
are then more or less rapidly destroyed by the white blood
corpuscles. The blood serum and the white blood cells
together with some bacteria and disintegrated tissue cells
constitute pus, and a wound producing pus is said to sup-
purate. Eventually the microoganisms are usually disposed
of and healing occurs as the result of the development of
new tissue. It is evident that suppuration is one manifesta-
tion of an inflammation.

Nonspecific Inflammation. — As noted above, many kinds
of bacteria are capable of producing inflammation. In
some cases these bacteria are capable of causing the so-
called specific diseases, such as the typhoid bacillus, the
pneumococcus (producing pneumonia), or the bacillus of
tuberculosis. In other cases inflammations are produced
by what are termed the nonspecific, pyogenic cocci, that is
microorganisms usually not responsible for the so-called
specific infectious diseases, but capable of causing a great

NONSPECIFIC INFLAMMATION 311

variety of inflammations, the type depending upon the con-
ditions governing infection and the degree of resistance of
the individual.

An inflammation "of any organ of the body is usually
named by adding "itis" to the root of the name of the
organ concerned. Examples of such inflammation are ton-
silitis (inflammation of the tonsils) ; appendicitis (inflam-
mation of the appendix), etc.

Three organisms are most commonly responsible for the
nonspecific inflammations. These are the Streptococcus

• •

FIG. 62. — STREPTOCOCCUS PYOGENES.

pyogenes, the Staphylococcus aureus, and the Staphylococ-
cus albus. Many other species have been described but are
much less important. They differ only in minor respects
from the organisms mentioned. These forms, therefore, are
the only ones which will be discussed.

STREPTOCOCCUS PYOGENES

>

Synonym. — Streptococcus erysipelatis.

This organism probably should be regarded as a group of
closely related species rather than as a single species. The
classification of the various species, however, has not been
satisfactorily worked out and the differences in disease pro-

312 AGKICULTUEAL AND INDUSTRIAL BACTERIOLOGY

auction or pathogenicity need not be emphasized here. It
may be noted that Streptococcus pyogenes is morpho-
logically very much like the Streptococcus Iwticus usually
associated with the souring of milk.

Morphology. — Streptococcus pyogenes is a coccus about
one micron in diameter, practically always occurring in
chains, sometimes short and sometimes long. It is non-
motile, does not produce spores, is easily stained and is
Gram-positive. Some types produce capsules, although
most of the more highly pathogenic forms are capsule-free.

Distribution. — Non virulent strains of Streptococcus
pyogenes are quite common upon the surface of the skin
and particularly in the mouth and on the mucous mem-
brane. Virulent strains have been isolated from a great
variety of inflammatory conditions.

Culture. — This organism grows fairly readily upon most
of the ordinary culture media but not particularly
luxuriantly. An agar slant culture, for example, develops
in the form of minute colonies, rarely larger than a pin
head, at first transparent and almost dewdroplike; later
they may become somewhat more opaque. In general they
grow particularly well upon coagulated blood serum, but
not upon potato. In broth the medium is sometimes clouded
as a result of the formation of numerous short chains, in
other cases it remains clear, the organism evidently growing
in the form of long tangled threads as a sediment in the
bottom of the tube.

Physiology. — This organism is aerobic and facultative
anaerobic. It grows best at blood heat but growth will
usually take place also at room temperature. No pigment
is produced and no coagulating or proteolytic enzyme. No
gas is produced from any of the sugars, though acid is
formed by most strains from many sugars, particularly
from dextrose and lactose. Efforts, in part successful, have
been made to classify the various strains of streptococci,

NONSPECIFIC INFLAMMATION 313

using acid production in various sugars as a basis for this
classification.

Pathogenesis. — Various strains of Streptococcus pyo-
genes show very great differences in their ability to produce
infection. Some strains are rleatively nonvirulent, others
are highly virulent. Some when inoculated into the body
tend to grow best in the blood stream, others grow in the
joints or in some particular organ of the body. In general
the disease produced is a more or less diffused inflammation.
In general infections with Streptococcus pyogenes are
somewhat more serious than those with staphylococci.
Apparently it is somewhat more difficult for the body to rid
itself of these organisms than of the other nonspecific pyog-
enic types.

Focal Infections. — Streptococcus pyogenes when grow-
ing in the body is apt to develop in some particular tissue
or organ producing in it either an acute or chronic inflam-
mation. As will be noted below, organisms from such
chronic inflammations not infrequently pass to other parts
of the body, being carried there by the blood stream. A
few of the more important focal infections will be noted
briefly.

Streptococci not infrequently produce chronic inflam-
mation of the tonsils (tonsilitis). From these they may
spread to neighboring lymph glands causing their enlarge-
ment or inflammation, sometimes resulting in the condition
known as Quinsy. Chronic tonsilitis (as is true in chronic
inflammation of any part of the body) usually leads to the
formation of more or less cicatricial or scar tissue, the ton-
sils becoming hardened, and local areas of inflammation per-
sist. Occasionally the organisms pass to the middle ear,
probably by way of the Eustachian tube, causing the con-
dition known as otitis media. From this location the organ-
ism may also occasionally pass to the interior of the mastoid
bone (or rather the mastoid process of the temporal bone)

314 AGEICULTUKAL AND INDUSTRIAL BACTERIOLOGY

lying just behind the ear, causing the condition known as
mastoiditis. The latter condition is particularly dangerous
because of the possibility eventually of the organism passing
through the bony inner wall of the mastoid, gaining access
to the brain cavity and there causing inflammation of the
covering membranes of the brain and spinal cord or
meningitis. Surgical intervention is sometimes necessary.

Organisms of this type may also cause ulcer ation of teeth
and in improperly filled teeth the organisms may pass
through the root, gain access to the gums and cause chronic
inflammation (or pyorrhea) about the roots of the teeth, in
some cases leading to a more diffused inflammation usually
termed gingivitis or inflammation of the gum. The mucous
membranes lining the nose may also be inflamed by organ-
isms belonging to this group, giving rise to the condition
known as rhinitis or more commonly as a cold. From these
membranes the organism sometimes passes by means of the
connecting canals into the sinuses of the head, that is, into
those cavities in the bones lined by mucous membranes.
Among the more important of these are the frontal sinuses.
This condition is known as a sinusitis. Inflammation of the
udder sometimes occurs in cattle as a result of infection
with organisms of this general type, producing the condi-
tion known as mastitis or garget. Erysipelas in man appar-
ently is due to the growth of this organism in subcutaneous
tissues producing extensive or general inflammation.

In some cases a focal infection with a highly virulent
organism may lead to the passage of the organism to the
blood stream and the development of septicemia (or "blood
poisoning"). Certain types of pneumonia, particularly
many following influenza, are due to infection of the lungs
from highly virulent strains of streptococci. Streptococcus
infection is not uncommon in lower animals. Such, for
example, is the so-called navel ill of the colt. The organism
enters through the umbilicus soon after birth. Wound

NONSPECIFIC INFLAMMATION 315

infection and suppuration in both man and animals are
sometimes caused by streptococci, although in most cases
staphylococci are more abundant.

As a result of focal infections in various parts of the
body, more particularly in tonsils, about the teeth, and in
the sinuses, Streptococcus pyogenes may gain entrance to
lymph channels or the blood stream and localize in other
parts of the body. Most cases of articular rheumatism, for
example, are of this nature, the organism localizing and
growing in the membranes about the joints. Another
secondary effect following tonsilitis and similar infections
is endocarditis, that is, inflammation of the lining mem-
branes of the heart, including the membrane which covers
the various heart valves. In a certain proportion of cases
the latter leads to a deposition of fibrin upon the heart
valves, later the development of scar tissue and the warping
of the heart valves out of shape, resulting in leakage
through the valves. In most cases the organisms are finally
eliminated but the defective heart valve, of course, cannot
be repaired. This is one of the most common causes of heart
trouble. Income cases the fibrin collecting upon the heart
valves is considerable in amount. It may form smajl cauli-
flowerlike masses. Portions of this material occasionally
break away and pass into the blood stream. Such a particle
is termed t an embolus and when it reaches a blood vessel
too small for its passage it may obstruct the flow of blood.
When this occurs in an end artery it may entirely cut off
the blood supply from that portion of the body. If this
should occur in the brain, for example, it would lead to
paralysis.

Immunity. — Resistance to infection by streptococci
seems to be due principally to the activity of opsonins and
white blood cells. The problem is rendered complex by the
fact that there are many strains, varieties or perhaps even
species of streptococci and the opsonins or immune bodies

316 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

for each are so specific that they will not immunize against
other strains. Immunization against streptococcus infec-
tions by the use of vaccine, consisting of suspensions of
killed streptococci, has been frequently attempted. While
some favorable results have been reported, the results on the
whole do not appear to be particularly encouraging unless
great care is taken that the organisms injected are the ones
against which immunity is actually desired. So-called
autogenic vaccines have been used with some degree of suc-
cess in chronic infections. In chronic erysipelas, for ex-
ample, there are reports of favorable results secured by
injecting dead cultures of the type of organism causing the
infection in the particular individual to be protected.
Streptococci are common constituents of many of the so-
called influenza vaccines, inasmuch as the streptococci have
been found in many instances to produce the post-influenza
pneumonias.

Vaccines against streptococcus infections have been pre-
pared in a variety of ways. Todd made such a vaccine by
first growing the streptococci on blood serum for twenty-
four hours, and inoculating from these cultures into large
flasks containing ten per cent serum broth. These were
incubated for one month. Six per cent of sterile glycerin
is added and this material concentrated at 60° C. for two
days over unslaked lime. It is evident that the thick paste
thus secured is made up of the products of autolysis of
the bacteria, as well as the growth products and unchanged
serum broth. It is diluted before use as a vaccine. Other
investigators have prepared bacterial extracts by shaking
broth cultures either with urea or galactose in twenty-five
per cent solution for several days. The high concentration
of the solution tends to destroy the organisms, and the
shaking to disintegrate them. The vaccine is thereby
rendered sterile without heat, and the antigen, therefore,
is not altered.

NONSPECIFIC INFLAMMATION 317

In some cases the streptococci are grown in broth in
large quantities, and separated by centrifugation. An
apparatus working on the principle of a cream separator
has been devised and used for this purpose.

A hemolytic toxin, streptolysin, has been demonstrated
for some strains of Streptococcus pyogenes, and for this
an antitoxin has been produced. However the antitoxin
has not been shown to have any curative or prophylactic
effect. It is not probable that the disease-producing power
of the organism can be accounted for on the basis of toxin
produced. So-called endotoxins are developed both by
virulent and by non-virulent strains. Bacteriolysins are
probably not important. Immunity is largely to be ascribed
to the activity of opsonins.

Antisera prepared from horses by injecting first with
dead and then with living cultures of streptococci until a
high degree of immunity has been obtained, have been
claimed to be successful by some users. The results have
not, however, in general proved encouraging. In general
it may be stated that no very satisfactory method of im-
munizing against Streptococcus pyogenes has been devel-
oped.

~

STAPHYLOCOCCUS AUBEUS AND STAPHYLOCOCCUS ALBUS

Synonyms. — Staphylococcus pyogenes aureus and Staphy-
lococcus pyogenes albus, Micrococcus aureus and Micrococ-
cus albus, Aurococcus aureus and Albococcus albus.

Morphology and Staining Characters. — These organisms
are considered together inasmuch as they are found in the
same general situations, are very closely related, differing
from each other only in minor characteristics such as pig-
ment production. These bacteria are spherical, occurring in
irregular masses like grape clusters. The cells are usually
0.7/X-0.9/X in diameter, occasionally larger. They are non-

318 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

motile, do not produce spores, stain readily with the
ordinary aniline dyes and are Gram-positive.

Distribution. — The staphylococci are quite common
upon the skin and hair in man and animals, likewise in the
nose and mouth and occasionally in the feces. They are not
uncommon in water, especially when this has been contam-
inated with sewage. They are present in many cases of
local infection accompanied by pus production.

Cultural Characters. — These bacteria may frequently
be secured in pure culture directly from the pus of suppurat-
ing wounds, boils or abscesses. The bacteria grow readily
upon the ordinary culture media. In gelatin a slow lique-
faction occurs. The two species may be differentiated from
each other in general by the color produced upon media.
"When grown upon potato, for example, Staphylococcus
aureus produces a bright orange pigment while the Staphy-
lococcus albus is white. Growth in milk is followed by the
production of some acid and ultimate digestion of the curd.

Physiology. — Small amounts of acid are produced from
some sugars, but no gas. Nitrates are reduced to nitrites.
Enzymes capable of digesting casein and gelatin have been
demonstrated.

Pathogenesis. — The staphylococci in general produce
infections such as abscesses, carbuncles, boils, acne, fur-
uncles in man, wound infection, poll-evil and fistula in the
horse, and similar lesions in other animals. Inasmuch as
these bacteria are normally present upon the surface of the
skin, they generally invade the tissues whenever there is a
break in the skin, that is, through any injury. They are
therefore, the common causes of wound infection or sup-
puration in wounds. A boil results from the entrance of
organisms of this group under the skin, where they grow
(providing the individual is not sufficiently immune) and
destroy a certain amount of tissue. A reaction or inflam-
matory condition is set up immediately about the injured

NONSPECIFIC INFLAMMATION 319

portion. The pyogenic membrane previously described is
formed and the* bacteria are prevented from spreading from
the initial source of infection. The portion of the tissue
killed by the bacteria gradually softens and is discharged
through the surface, that is, the boil is said to "come to a
head/' The purulent discharge which follows is a mixture
of white blood cells, serum, bacteria and disintegrated
tissue. When the bacteria have been cleansed from the

*

FIG. 63. — STAPHYLOCOCCUS AUREUS.

injured area, cicatricial tissue or scar tissue grows into the
wound, replacing the tissue destroyed by the microorganism.

A somewhat similar condition, frequently becoming
chronic, is the fistula on the withers or shoulders of a horse.
The organisms have gained access through the skin as a
result of a bruise or injury, grow between the muscles and
layers of fascia, producing severe inflammation. In chronic
fistula the area is more or less surrounded by a growth of
scar tissue. Poll-evil is a somewhat similar affection on the
top of the poll or head of the horse.

Immunity. — As in infection with Streptococcus, immu-
nity, when developed, is probably due to the presence of
sufficient quantities of opsonin. The popular conception
that boils are beneficial because they drain impurities from
the blood is fallacious. A person is subject to boils primarily

320 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

because of a deficiency in immunizing substances, probably
in large part opsonin. Occasionally apparently the presence
of bacteria in a wound or in a boil is not sufficient to incite
the individual to the production of immune substances or
antibodies such as opsonin. In such instances, boils may
recur at frequent intervals and the individual is said to
suffer from furunculosis. Under such conditions it has been
found possible to use successfully vaccines prepared from
the organism causing the trouble in the particular individ-
ual. These are prepared by growing the bacteria on slanted
agar cultures, suspending in physiological salt solution,
destroying by heat. Subcutaneous injection of such a vac-
cine tends to incite the tissues to the production of suitable
antibodies.

CHAPTER XXIV

PNEUMONIA, MENINGITIS AND GONORRHEA— THE SPE-
CIFIC PYOGENIC COCCI— THE GENERA DIPLOCOCCUS
AND NEISSERIA

THE organisms belonging to the genera Diplococcus and
Xeisseria include for the most part pathogenic, or at least
parasitic bacteria, spherical in shape, whose cells occur
generally in pairs.

The organisms may be differentiated by use of the Gram 's
stain. The members of the genus Diplococcus are Gram-
positive, those of the Neisseria Gram-negative. The organ-
isms belonging in this group which are pathogenic and of
economic importance are three in number: the Diplococcus
pneumonice, causing pneumonia ; the Neisseria meningitidis,
causing infectious cerebrospinal meningitis in man, and the
Neisseria gonorrhoea, causing »the disease gonorrhea in man.

These organisms include the most common forms of the
so-called specific pyogenic cocci, that is, those cocci re-
sponsible for inflammatory changes in connection with
specific diseases.

DIPLOCOCCUS PNEUMONLE

Synonyms. — Streptococcus pneumonice, Diplococcus lan-
ceolatus.

The organism is usually termed the pneumococcus.-
The term pneumonia literally designates an inflammation
of the lungs. This may be caused by any one of several
species of bacteria. In the great majority of cases the
pneumococcus is the cause and is ordinarily spoken of or
referred to as the cause of specific infectious pneumonia.

321

322 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Distribution. — Several distinct types, races or varieties
of pneumococcus have been described. Some of these are
not uncommon in the normal mouth and throat. Others
are usually associated with disease production or are pres-
ent in the mouths and throats of those associated with those
who are diseased. Within the same type there are appar-
ently considerable variations in virulence. Probably in no
disease do we have a better illustration of the fact that pro-
duction of disease is due to lack of immunity on the part of
the individual attacked or to high virulence on the part of
the attacking organism. In other words, the ability to pro-
duce disease always depends upon the relative virulence of
the organism and the resisting power of the individual.

Morphology. — The Diplococcus pneumonia usually oc-
curs in pairs, more rarely in chains with few elements. The
cells are sometimes spherical, but are most frequently some-
what flattened at the point of contact and with the opposite
side somewhat elongated and pointed. This lance-shaped
appearance has given rise to the name Diplococcus lanceo-
latus. Capsules may usually be readily demonstrated in
sputum or in body fluids. The organism stains readily and
is Gram-positive.

Cultural Characters. — The pneumococcus is most readily
isolated upon blood agar, that is, agar which has been
melted and has had added to it a small amount of fresh
blood which has been drawn under aseptic precautions to
insure its sterility. If culture is desired from sputum
where other organisms are apt to be present, it is usually
necessary first to inoculate a suitable laboratory animal,
such as a mouse. The colonies are never luxuriant, the
organisms developing as discrete, transparent, dewdroplike
colonies upon the surface. Broth is clouded and milk may
be somewhat acidified.

Physiology. — The pneumococcus grows but little or not
at all at temperatures lower than blood heat. Acids are

PNEUMONIA, MENINGITIS AND GONORRHEA 323

produced from certain carbohydrates. Cultures lose their
viability relatively quickly. When the organism is sus-
pended in broth or physiological salt solution it readily
undergoes autolysis or self-digestion.

Pathogenesis. — The pneumococcus was first described
as an organism present in sputum in healthy individuals,
which when injected into a mouse, produced a quickly fatal
type of septicemia. Later it was found quite consistently
associated with acute infectious pneumonia in man and in
certain animals, particularly the horse. In pneumonia the
organisms are present in the lung tissue causing severe local
inflammation. The blood vessels become engorged and

FIG. 64. — DIPLOCOCCUS PNEUMONIAE.

blood plasma is poured out into the air sacs or alveoli. The
fibrinogen is converted into fibrin and the alveoli become
filled with fibrin clots. Lung tissue which has been changed
in this fashion is no longer spongy and capable of floating
upon water, but is dense and when placed in water sinks to
the bottom. Such lung tissue is said to have undergone
hepatization, that is, it has become liverlike in consistency.
In some cases there is more or less diffused bleeding into the
tissues and they become red. Such a condition is known as
red hepatization. When white blood cells are present in
great numbers, the lungs are said to have undergone gray
hepatization. The organisms may also gain entrance to the

324 AGEICULTUEAL AND INDUSTKIAL BACTERIOLOGY

blood stream, causing septicemia. In fact, septicemia is
apparently, in many cases at least, the immediate cause of
death from pneumonia. When the body defenses gain the
upper hand, the bacteria may be eliminated and the lung
tissues undergo resolution. The clotted contents of the
alveoli undergo partial or complete autolytic digestion and
the material passes to the mouth or is resorbed into the
blood.

Bacteriological Diagnosis. — The Diplococcus pneumonia
is usually recognized in sputum, when stained by Gram's
method, as a capsulated diplococcus, Gram-positive. Inas-
much as several distinct types of pneumococci have been
described, each capable of causing pneumonia in man and
perhaps in animals, it is sometimes necessary to differentiate
the types by the use of the agglutination test. Antisera,
capable of conferring immunity and having curative prop-
erties, have been prepared for certain types of pneumococci,
but not for others. It is of advantage, therefore, for a
physician to know what type of pneumococcus is causing the
disease in a particular case. This may be ascertained by
securing a pure culture of the organism causing the par-
ticular infection and testing it against known antisera con-
taining agglutinins. The pneumococci are usually divided
into four types. Types 1, 2 and 3 are most commonly pres-
ent in diseases and are relatively distinct and easily recog-
nized. Type 4 includes forms not included in types 1, 2
and 3. The test is frequently carried out as follows : a bit
of sputum from the patient is injected intraperitoneally
into a mouse. At the end of a few hours the animal is
killed and the peritoneal cavity washed out with physio-
logical salt solution. The pneumococci will have multiplied
rapidly and by this means a milky suspension of the organ-
ism may be secured in relatively pure culture. Portions
of this suspension are then tested against sera known to
agglutinate respectively types 1, 2 and 3.

PNEUMONIA, MENINGITIS AND GONORRHEA 325

Immunity. — The problem of immunization against pneu-
monia is complicated by the variety of types of pneumococci
and other organisms causing the disease. It is also compli-
cated by the marked variation which pneumococci appar-
ently may show in virulence and by the great differences
which may occur in resistance or immunity to the disease.
Specific antisera have been prepared for certain types by
injecting horses or other suitable animals, first with killed
and finally with living cultures of pneumococci. The use
of a potent antiserum against the right type of microorgan-
ism has proved markedly advantageous. Difficulty in typ-
ing organisms and in the preparation of potent antisera has
interfered with its widespread use.

Pneumococci together with streptococci have generally
been included in considerable numbers in the vaccines used
to prevent influenza or rather to prevent the development
of secondary complications following attacks of influenza.
The evidence concerning the value of these vaccines is very
conflicting.

Immunity against pneumonia apparently is short-lived.
While a person convalescing from the disease may show
evidence of a high degree of acquired immunity, this usually
does not persist for a very long period.

NEISSERIA GONORRHOLE
Synonyms. — Microeoccus gonorrh&ce, Diplococcus gonor-

The causal name usually applied is gonococcus, occa-
sionally diplococcus of Neisser.

The disease produced is gonorrhea in man, not transmis-
sible to animals.

Morphology. — Stained mounts of gonorrheal pus show
the Gram-negative, coffee-bean-shaped diplococci, lying gen-
erally in pairs inside the polymorphonuclear leucocytes.
In culture there is little or no tendency to chain formation,

326 AGKICULTURAL AND INDUSTRIAL BACTEEIOLOGY

the cells being arranged in irregular masses or in pairs.
The gonococcus stains readily with the ordinary aniline
dyes. The fact that it is Gram-negative facilitates differen-
tial diagnosis between infections with gonococcus and the
other pus-producing bacteria.

Culture. — The gonococcus is usually secured by smear-
ing serum agar in plates with gonorrheal pus. The colonies
which develop resemble somewhat those of streptococci,
being small, well-separated and transparent. Care must be
used to maintain a medium that has a moist surface and the
right hydrogen ion concentration, best that of the blood.
After a few transfers the organism develops more luxuri-
antly on artificial media.

Physiology. — The gonococcus is aerobic, easily de-
stroyed by drying, and in the laboratory rapidly undergoes
autolysis, making it necessary to transfer cultures every two
or three days when they are kept at blood heat. The organ-
ism remains viable in culture media for a somewhat longer
period of time in the ice chest.

Pathogenesis. — Gonorrheal infection of the membranes
of the eye sometimes occurs at birth, leading to the develop-
ment of the so-called gonorrheal ophthalmia, or ophthalmia
neonatorum. In some localities physicians and midwives
are. required to drop solutions containing suitable silver
salts into the eyes of newborn children to prevent the devel-
opment of this disease.

Gonorrhea is usually regarded as the most important of
the so-called venereal diseases. In the male it is first an
acute inflammation of the urethra which extends in many
cases to the prostate gland. Chronic inflammation of this
gland may lead to the formation of scar tissue and a
decrease in the size of the urethral tube, producing the so-
called stricture. In the early stages the disease is charac-
terized by considerable quantities of purulent discharge.
"When it becomes chronic, as it may in prostatic infection,

PNEUMONIA, MENINGITIS AND GONORRHEA 327

the discharge changes to a more or less viscous serum. The
gonococci occasionally invade the urinary bladder, pro-
ducing an acute cystitis. They may also pass along the vas
deferens to the seminal vesicles and finally to the epi-
didymis, causing epididymitis and orchitis. The disease is
peculiarly difficult to treat because after the first occurrence
of urethritis, the organism frequently invades the neighbor-
ing glands, such as the prostate, and local applications of
disinfectants do not reach them. In a considerable per-
centage of cases, therefore, the disease becomes chronic. In
metastatic infection from cases showing prostatitis or vesi-
culitis, the organisms may localize in the joints producing
gonorrheal rheumatism. More rarely there may be involve-
ment of the heart valves.

In the female gonorrhea is usually primarily a vaginitis
followed by involvement of the uterus and not infrequently
of the Fallopian tubes. The acute inflammation here may
give rise to the condition known as "pus tubes " (pyosalpin-
gitis) in which there is danger of the tubes bursting and
producing peritonitis. The urethra is frequently also
involved in the disease.

Because of its wide distribution, the variety of the symp-
toms and the seriousness of the after effects, such as rheu-
matism, sterility, pyosalpingitis requiring surgical inter-
vention, etc., the disease probably is to be regarded as the
most important of the venereal infections.

Bacteriological Diagnosis. — As noted above, the dis-
ease may frequently be recognized by the identification of
the Gram-negative, coffee-bean-shaped diplococci present in
the pus. The complement fixation test has also been used
to detect the presence of the organism where there are no
apparent or evident symptoms of the disease, particularly
in determining when a cure has been effected.

Immunity. — Gonorrhea is a disease from which spon-
taneous cure occurs but slowly, the individual frequently

328 AGBICULTUEAL AND INDUSTRIAL BACTERIOLOGY

retaining the organisms for months or for years. Appar-
ently in some cases the organisms lie dormant or latent and
produce symptoms not sufficiently acute to be noticed for
long periods of time. Under certain conditions the disease
may flare up again.

No antisera have been prepared which give relief or
effect a cure in this disease. It has been claimed that vac-
cines have been used with some success in immunizing those
who have the disease in chronic form.

NEISSERIA MENINGITIDIS

Synonyms. — Diplococcus meningitidis f Diploeoccus intru-
cellularis, Micrococcus meningitidis. The organism is fre-
quently known by its casual name, meningococcus.

The disease produced, acute contagious cerebrospinal
meningitis, is characteristic of man, although it may be
transmitted by experimental inoculations to some animals,
particularly to the monkey.

Morphology. — The meningococcus in the body exudates
usually appears as a diplococcus, or occasionally in groups
of four. In culture media, the organism is usually about IM
in diameter and commonly occurs in pairs, rarely in short
chains. Capsules are not produced. The organisms stain
readily with the usual aniline dyes and are Gram-negative.
Inasmuch as there are other organisms, such as streptococci
and the pneumococcus which occasionally cause meningitis,
the latter fact is important in differential diagnosis.

The organism may frequently be secured in pure culture
by making a lumbar puncture with a sterile hypodermic
needle and transferring directly to a suitable medium. The
medium must be carefully prepared, have the correct acid-
ity and usually it is necessary, upon first culture at least, to
add blood serum. Upon this medium white discrete colonies
develop. The organism undergoes autolysis or self-digestion
so readily that frequent transfers must be made to keep it

PNEUMONIA, MENINGITIS AND GONOKRHEA 329

viable except under the most favorable of conditions.
Growth does not occur readily upon most of the ordinary
culture media.

Physiology. — The meningococcus is killed readily by
drying. Small amounts of acid are produced from dex-
trose ; milk is not changed and gelatin is not digested.

Pathogenesis. — The meningococcus apparently gains
access to the body through the respiratory tract. It finds in
many individuals a suitable site for growth in the naso-
pharynx, that is, the back portion of the nasal cavity just
above the pharynx. From this point in a rather uncertain
proportion of individuals the organism finds its way, per-

FIG. 65. — NEISSERIA MENINGITIDIS.

haps through the lymph channels or through the blood
stream, to the central nervous system, where it grows upon
and in the meninges (the membranes which cover the brain
and the spinal cord). This is followed by the usual evi-
dences of inflammation. The clear cerebrospinal fluid
becomes clouded with white blood cells, that is, it becomes
more or less purulent. A mount of this material secured
by means of a lumbar puncture, that is, by the insertion of
a hypodermic needle between the lumbar vertebrae into the
spinal cavity, shows more or less numerous Gram-negative
diplococci frequently lying within the white blood cor-
puscles. The disease is not infrequently accompanied by a
septicemia, that is, the meningococci gain access to the blood
stream and grow rather generally through the body.

330 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Bacteriological Diagnosis.— It has already been noted
that the disease produced by the meningococcus is differen-
tiated from the other types of meningitis by identifying
the organisms by means of lumbar puncture. They may
usually be identified in a Gram-stained preparation. They
may also be grown upon suitable culture media.

At least two relatively distinct types of meningococci
have been described, some investigators insisting that there
are even more, perhaps four. This is of importance because
antisera prepared against one type does not appear to be
particularly efficacious against another type. It is advan-
tageous in some instances to determine the type of meningo-
coccus causing the disease in the particular individual.
Pure cultures may be secured and tested by the agglutina-
tion reaction against serum specific for each of the meningo-
coccus types.

Immunity. — The problem of immunization against
meningococcus is complicated, as is the case with the
pneumococcus, by the fact that there are two or more dis-
tinct varieties. Antisera containing antibodies specific for
the disease have been used with a marked degree of success.
The antisera are prepared by injecting dead and, later, liv-
ing cultures of meningococci into a suitable animal, such as
the horse. When a high degree of immunity has been
secured the horse is bled, the blood serum secured and fil-
tered. This is usually injected intraspinally, that is
cerebrospinal fluid is removed and a sufficient amount of
the antiserum introduced. This comes in immediate con-
tact with the microorganisms growing on the meninges.

CHAPTER XXV

THE COLON-TYPHOID SEKIES OF BACTERIA
THE GENERA BACTERIUM AND PROTEUS

This group of organisms is frequently termed the intes-
tinal group because many, although not all, of the organ-
isms belonging to it are of intestinal origin, or find the best
conditions for growth in the intestines of man or higher
animals and sometimes of birds or fishes. The group is im-
portant because belonging to it are several organisms
capable of causing disease in man and in animals, and
because the detection of certain bacteria belonging to it
constitute the most satisfactory method for the detection of
pollution in water.

The genera Bacterium and Proteus, that is, the organisms
belonging to the intestinal group of bacteria are rod-shaped,
Gram-negative, aerobic or facultative forms which do not
produce spores. Some species are motile, others nonmotile,
some produce capsules, others do not. Frequently there is
considerable power of fermentation of carbohydrates devel-
oped.

The genera Bacterium and Proteus are very closely
related. The organisms belonging to the genus Proteus in
general liquefy gelatin more or less rapidly, while only a
few members of the genus Bacterium are capable of lique-
fying gelatin. These may be differentiated from members
of the genus Proteus by the fact that the latter are never
able to produce gas from the sugar lactose.

The genus Bacterium is much more important than the
genus Proteus in the production of disease, and attention
will, therefore, be concentrated upon the former. Certain

331

332 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

species of Bacterium (such as the Bacterium lactis viscosum
producing ropy milk) will not be discussed in this section
as they do not produce disease and are not of particular
pathologic significance.

The true intestinal bacteria belonging to the genus Bac-
terium are divided usually into three principal sub-groups,
the separation being made upon the basis of the ability to
produce acid and gas in the sugars lactose and glucose. The
bacteria which can produce acid and gas from both lactose
and glucose belong to the first or colon subgroup. Those
which can produce acid and gas from glucose, but not ?rom
lactose, belong to the intermediate or enteritidis subgroup.
Those which produce no gas or acid from lactose, no gas
from glucose, and may be either positive or negative as to
acid production from glucose, belong to the third or
typhoid-dysentery subgroup.

Each of these subgroups has been differentiated into a
large number of species — about thirty altogether being well-
characterized and recognized in the literature. It will be
noted that in the differentiation of the subgroups the
first shows high fermentative power and for the most part
the organisms belonging to it are nonpathogenic. The
second group is intermediate both in power of fermentation
and in power of disease production. The third subgroup
has the lowest fermentative power and has belonging to it a
considerable number of highly pathogenic bacteria.

THE COLON OR COLON AEROGENES SUBGROUP OF BACTERIA

The most important species belonging to this subgroup
are Bacterium coli, Bacterium acidi-lacticif Bacterium com-
muniorf Bacterium neapolitanumf Bacterium coscorobaf
Bacterium cloacce and Bacterium aerogenes.

The Bacterium coli and the Bacterium aerogenes are
more important than the other species and are the only
ones which will be discussed in detail. The differentiation

THE COLON-TYPHOID SEEIES 333

of the other species may be determined by reference to the
key given below.1

BACTERIUM COLI

This organism is taken as a type of the entire colon group.
The other organisms belonging to the subgroup may be
differentiated from it by means of physiological reaction.
Inasmuch as most of them are intestinal forms, differentia-
tion is usually not desired or necessary. In recent years
differentiation of Bacterium aerogenes from Bacterium coli
has been found to be of some importance in water analysis,
inasmuch as the former seems to be much more widely dis-
tributed in nature and is not therefore as delicate an index
of fecal contamination.

Synonym. — Bacillus coli communis. Colon bacillus.

Distribution. — Bacterium coli is normally present in the
alimentary tract, particularly in the colon, of man and
most animals. It is apparently not widely distributed in

1 KEY TO THE MORE IMPORTANT SPECIES OF THE COLON SUBGROUP OF

THE GENUS BACTERIUM

A. Ratio of CO2/H2 = 1/1, Voges-Proskauer test negative, methyl red
(Clark and Lubs test) positive.

1. No acid or gas from sucrose.

a. Acid and gas from salicin.

1. Bacterium coli.

fe. No acid or gas from salicin.

2. Bacterium acidi lactici.

2. Acid and gas from sucrose.

a. Motile.

3. Bacterium communior.
Z>. Nonmotile.

(1) Acid and gas in salicin.

4. Bacterium neapolitanum.

(2) No acid or gas in salicin.

5. Bacterium coscoroba.

Ratio of CO2H/2 = 2/l, Voges-Proskauer test positive, methyl red
test negative.

1. Motile, liquefying gelatin, no gas or acid from starch or
glycerol.

6. Bacterium cloaca.

2. Nonmotile, not liquefying gelatin, gas and acid from starch
-and glycerol.

7. Bacterium aerogenes.

334 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

nature except in places or localities fouled by the excretions
of the body. It is very easily cultivated in the laboratory
and it is probable that under certain conditions in nature it
may multiply for a brief period of time outside the body.
This, however, does not commonly occur, at least not in vir-
gin soil or in unpolluted water. It is so abundant in feces
that it has come to be looked upon generally in England
and America as an indication, of fecal contamination when
found in water. It is not because Bacterium coli itself is
pathogenic, but because it is an indicator of the presence of

Pie. 66. — BACTERIUM COLI.

such fecal pollution that water containing it is generally
condemned for domestic use.

Morphology. — The organism is a plump rod 0.4-0.7/u,
X 2-4ju. When growing rapidly it may be coccuslike. It
rarely occurs in chains. Spores and capsules are not pro-
duced. It is actively motile, stains readily and is Gram-
negative.

Cultural Characters. — The organism grows readily upon
most culture media. Gelatin is not liquefied. Bouillon is
clouded, sometimes with the formation of a pellicle. The
growth upon potato is moist and spreading, the potato
becoming darkened, sometimes almost black. Milk is coag-
ulated with the formation of lactic acid and gas; the curd

THE COLON-TYPHOID SEEIES 335

is not digested. Upon agar plates the colonies are moist,
opaque, becoming darker and more coarsely granulated.
Gelatin is not liquefied.

Physiology. — Bacterium coli grows readily at blood heat
and also at room temperature. Acid and gas are produced
from dextrose and lactose. The typical Bacterium coli does
not produce gas from sucrose but there are closely related
species which show this reaction. It is readily destroyed
by heat. The gas produced from carbohydrates is a mix-
ture of equal volumes of carbon dioxide and hydrogen. In
a suitable glucose peptone medium sufficient acid is pro-
duced so that methyl red is changed to a deep red color.
Indol is usually produced in Dunham's solution. The
Voges-Proskauer test is negative. The organism is aerobic
and facultative anaerobic.

Pathogenesis. — Although Bacterium coli is usually re-
garded as nonpathogenic, there is reason to suppose that
occasionally virulent varieties are developed which may
produce disease in animals, particularly the so-called calf
scours or calf diarrhea.

Recognition. — The physiological and cultural characters
of this organism make it comparatively easy to recognize
when present in the water and to isolate from sewage. The
methods used will be discussed at greater length under the
heading of Bacteriology of Water and Sewage.

INTERMEDIATE OR ENTERITIDIS SUBGROUP

Several species of bacteria belonging to this subgroup are
of economic importance because of their association either
as primary cause or secondary invaders with several dis-
eases of man and animals. Among them are the Bacterium
paratyphi and Bacterium schottmiilleri causing paratyphoid
fevers, the Bacterium enteritidis associated with food
poisoning, Bacterium morgani which has been found asso-
ciated with a typical dysentery in man. Bacterium pul-

336 AGRICULTUKAL AND INDUSTRIAL BACTERIOLOGY

lorum is the cause of the so-called white diarrhea of chicks.
Bacterium cholercB-suis has been found frequently associ-
ated with hog cholera but is evidently not the cause of the
disease, in most cases at least. The following key will
differentiate the species.1

BACTERIUM CHOLER^E-SUIS

Synonyms. — Bacillus suipestifer; Bacillus cholerce-suis,
Bacillus salmoni.

This organism was first described in 1885 by Salmon and
Smith as the probable cause of the disease named by them
swine plague. A revision of terminology with reference to
the swine diseases a little later caused them to describe this
as the cause of the hog cholera. It was isolated from a large
number of hogs affected with this disease. Much work was
done with it in the next two decades in an attempt to
produce vaccine and sera for use in treatment or prevention
of the disease, hog cholera. In general these proved
unsuccessful. It was not until De Schweinitz and Dorset in
1904 studied an outbreak of hog cholera in which they were
unable to isolate this organism that the causal relationship

1 KEY TO THE PRINCIPAL SPECIES OF THE INTERMEDIATE SUBGROUP OF
THE GENUS BACTERIUM

A. Producing gas from mannitol.

1. Producing gas from xylose.

a. Lead acetate medium not blackened. Arabinose and
dulcitol fermented but slowly if at all.

1. Bacterium cholerce-suis.

&. Lead acetate medium blackened. Arabinose and dulcitol
fermented promptly.

(1) Inosite not fermented.

2. Bacterium enteritidis.

(2) Inosite fermented.

3. Bacterium schottmulleri.

2. Not producing gas from xylose.

a. Nonmotile.

4. Bacterium pullorum
&. Motile.

5. Bacterium paratyphi.

B. Not producing gas from mannitol.

6. Bacterium morgani.

THE COLON-TYPHOID SEKIES 337

of this organism was brought into question. They proved
that the disease could be produced by the injection of blood
(from diseased animals) which had been filtered through
fine grained porcelain filters. This procedure was known
to remove -the larger bacteria such as the "hog cholera
bacillus" and demonstrated the disease to be due to one of
the so-called filter-passers. Subsequent work showed quite
conclusively that Bacterium cholerce-suis is not the cause of
hog cholera generally, although it may be an important
secondary invader and possibly in young pigs produces a
disease somewhat resembling hog cholera. The organism

FIG. 67. — BACTERIA. 1. Bacterium cholerae suis. 2. Bacterium
enteritidis.

can be differentiated from other members of the group only
by its physiological and serological reactions.

BACTERIUM ENTERITIDIS

Synonym. — Bacillus of Gartner.

This organism has been repeatedly isolated since its dis-
covery in 1888 by Gartner in outbreaks of food poisoning,
particularly of meat poisoning in Saxony, and likewise from
the uncooked flesh of a cow which had been responsible for
the infection. It is possible that either this organism OP
forms closely related are responsible for many cases of calf
diarrhea. Mohler and Buckley have also observed fatal

338 AGETCULTUEAL AND INDUSTEIAL BACTEEIOLOGY

infection by this organism in cattle. It is responsible for
many of the falsely termed ptomaine poisonings in the
United States.

The organism can be differentiated from other members
of the group only by careful physiological and serological
studies.

The Bacillus enteritidis, when grown in suitable culture
media, produces an unusually soluble and potent endotoxin.
This endotoxin differs from true toxin in being quite resist-
ant to heat. It is therefore possible for food, particularly
meat which has been fairly thoroughly cooked, to give rise
to toxic symptoms when it is eaten. It is probable that this
endotoxin is responsible for the symptoms which develop
promptly when people are poisoned by eating infected food.
The disease produced by infection by the living organisms
in man is very similar to that caused by infection by para-
typhoid bacilli, in fact, the organisms themselves are very
closely related.

Meat and milk from animals which show severe diarrhea
or gastrointestinal disturbances should not be used for
human consumption, as infection in the human has been
traced to such in several cases. It is quite probable that
most of the cases of meat poisoning originate from the use
of flesh of diseased animals, but it is possible that in some
cases, the organism gains access to the meat after the animal
has been slaughtered.

BACTERIUM PARATYPHI AND BACTERIUM SCHOTTMULLERI

Synonyms. — Bacillus paratyphosus A and Bacillis para-
typhosus B.

These organisms produce in man the disease which has
come to be known in recent years as paratyphoid fever.
This disease closely resembles typhoid fever in most of its
clinical aspects, although the mortality is somewhat lower.
Bacterium paratyphi was originally discovered from the

THE COLON-TYPHOID SERIES 339

fact that in certain cases, clinically typhoid, the blood serum
of the patient did not acquire the property of agglutinating
true typhoid bacteria, but that organisms could be isolated
from the feces of the patients which would be agglutinated
specifically by their blood serum. In recent years there have
been recorded many instances of epidemics caused by the
paratyphoid bacilli. The disease is transmitted in the same
manner as is typhoid fever and methods of prevention are
identical. These will be discussed at greater length under
the heading of typhoid.

The bacteria causing paratyphoid fever of the two types
can be differentiated from each other and from other mem-
bers of this subgroup only by their physiological and sero-
logical characters.

BACTERIUM PULLORUM

This organism has been described as the specific cause
of the disease white diarrhea in young chicks. It is similar
in all but a few of its physiological and serological reactions
to other members of this group. It is a nonmotile rod.

The disease is characterized in chicks by emaciation and
white diarrhea. The organism may be isolated in pure
culture from the internal organs, particularly from the
liver. It has been found by Rettger that pullets which
recover from this disease frequently develop into hens
which are bacillus carriers, that is, the organisms remain in
the ovaries and are present in the eggs laid. The chick,
therefore, in such cases is infected before hatching. A few
such chicks among those hatched by an incubator or present
in a brooder will rapidly infect the other chicks. Infected
hens may frequently be detected by means of the agglutina-
tion reaction, that is, their blood serum contains the agglu-
tinin specific for the organism and the precentage of loss
among chicks may be materially reduced by weeding out
hens which react. The disease has been the occasion of very

340 AGKICULTUEAL AND INDUSTKIAL BACTERIOLOGY

considerable losses to the poultry industry in the eastern
states.

TYPHOID-DYSENTERY SUBGROUP

At least ten to fifteen species of bacteria are known which
belong to this subgroup. Only two among them, however,
are important from the standpoint of disease production,
Bacterium typhi and Bacterium dysenteric, the latter with
numerous varieties. Two of the species, Bacterium gal-
linarum, the cause of fowl typhoid, and Bacterium abortus,
the cause of infectious abortion in cattle, are of importance
in disease production in the lower animals.

BACTERIUM TYPHI

Synonyms. — Bacillus typhosus, Bacillus typhi, Bacillus
typhi-abdominalis, Eberth bacillus, Eberth Gaffky bacillus.

This organism was discovered by Eberth in 1880 in the
spleens of those who had died of typhoid fever, and has
since been conclusively proved to be the cause of this dis-
ease. It is one of the most widely distributed of the organ-
isms producing disease in man, the disease being more or
less prevalent in most sections of the United States.

Morphology. — This organism is a short, plump rod,
usually from .5 to .8/x, in diameter and from 1 to 3/* in
length, motile by means of numerous flagella. It is non-
capsulated and does not produce spores. It is Gram-
negative and stains readily with the ordinary aniline dyes.

Cultural Characters. — Bacterium typhi grows readily
upon most of the cultural media, the growth being some-
what more delicate, usually, than that produced by Bac-
terium coli.

Physiology. — Bacterium typlii grows best at blood heat,
but develops readily at room temperature. It is aerobic and
in the presence of suitable sugars a facultative anaerobe.
Acid, but no gas, is formed from dextrose, and neither acid

THE COLON-TYPHOID SERIES 341

nor gas from lactose or sucrose. Milk is not coagulated and
proteolytic enzymes are not produced.

The cultural and physiologic reactions make it possible
to isolate the typhoid bacillus directly from the stools of
infected individuals and, by a process somewhat more
arduous, from infected water or sewage. Many special dif-
ferential media have been developed to permit the ready
isolation and identification of the organism. When pure
cultures have been secured, the final test usually employed
is agglutination by means of a specific anti-typhoid serum.

Disease Produced. — Typhoid fever is a disease charac-
teristic of man and is not produced readily by either injec-
tion or by feeding of cultures to laboratory animals. The
organism apparently develops first in the contents of the
intestine, then invades the intestinal wall and the Peyer's
patches. These latter frequently become ulcerated and
occasionally perforation of the intestinal wall may result.
The organisms are present in considerable numbers in the
blood stream ; in fact, the disease is frequently characterized
as a bacteremia. The spleen is considerably swollen. Infec-
tion of the gall bladder is not infrequent.

The typhoid bacteria leave the body almost exclusively
with the stools and occasionally with the urine. The bac-
teria do not multiply to any extent (if at all) in nature out-
side of the body; in fact, they die out with considerable
rapidity after leaving the alimentary tract. Infection of
another can occur only by swallowing typhoid germs. It is
evident, therefore, that the prevention of typhoid fever
depends upon preventing the ingestion of typhoid bacilli
which have been voided comparatively recently by those
having the disease or by so-called bacillus carriers. A
bacillus carrier is a person who harbors pathogenic organ-
isms such as typhoid bacilli without showing symptoms of
disease. Usually those who have typhoid fever eliminate
the organisms completely and do not show them in their

342 AGKICULTUBAL AND INDUSTRIAL BACTEEIOLOGY

stools within a few weeks after the beginning of con-
valescence. Occasionally, however, the bacterium may per-
sist for considerable periods of time. This is particularly
true when there is an infection of the gall bladder. Such
individuals have been known to retain the organisms for
many years; they are, of course, much more dangerous in
a community even than those who have the disease typhoid
in clinical form, inasmuch as they have much greater oppor-
tunity to infect drinking water, food, milk, etc.

Transmission. — It is evident that typhoid fever must
most commonly be contracted from drinking water, milk
and other foods which have been contaminated. It is prob-
able that not infrequently flies may carry the bacteria from
the stools of typhoid patients to food.

Recognition of the Disease. — Typhoid fever is a disease
which is not readily differentiated from other diseases in its
earliest stages and with difficulty at any time in mild cases.
As an assistance to the physician in recognizing the disease,
the agglutination or Widal reaction has come into common
use. A drop of blood or blood serum from the typhoid
patient will usually agglutinate the typhoid bacillus in
dilutions of at least 1 to 40, frequently in a dilution of one
to many thousand. The disease may also be recognized by
planting blood drawn from a vein in broth.

Prevention of Disease. — The prevention of typhoid fever
is accomplished by preventing the access of fecal material
to water and food, by the recognition and proper care of
patients and carriers, and by the use of vaccines. The latter
are necessary probably only where there is danger of the
individual drinking water or eating food which has not
been properly protected. The vaccine is prepared by grow-
ing typhoid bacilli on the surface of agar, washing them off
with physiological salt solution, killing by heat, and stand-
ardizing to proper dosage. In some cases the bacteria are
dried, ground and mixed with oil to make the so-called

THE COLON-TYPHOID SEEIES 343

lipovaccine. Vaccination has proved quite successful in
preventing a high incidence of typhoid fever in large
groups of individuals, such as soldiers exposed to infection.

BACTERIUM DYSENTERIC

A considerable number of closely related species of bac-
teria are here grouped together. They produce diseases
quite similar clinically, but the bacteria are specific and
may be differentiated from each other by their fermentation
and serologic reactions. The organisms all closely resemble
the typhoid bacillus ; they are, however, nonmotile. Methods
of isolation are very similar to those used in typhoid.

In dysentery in man the intestine, particularly the colon,
becomes inflamed and sometimes ulcerated. The stools are
usually thin and watery. In most cases the bacteria (unlike
typhoid) do not invade the general blood stream, that is, the
disease does not develop into a bacteremia.

Transmission. — Dysentery is spread in quite the same
manner as is typhoid and methods of prevention in general
are identical.

BACTERIUM ABORTUS

Synonyms. — Abortion bacillus of Bang, Bacillus abortus,
Bacillus abortivus.

This organism has been repeatedly isolated as the prob-
able cause of infectious abortion in the cow. The disease is
prevalent in many places in Europe and in the United
States in herds of dairy cattle. It should be noted that
other organisms besides the one discussed here are found
occasioning abortion in cattle and in other animals.

Morphology. — The organism is a small rod .S-.S/iXl-^M.
It is somewhat polymorphic in culture media, and involu-
tion forms are not infrequent. It is nonmotile, does not
produce capsules or spores, and stains readily with aniline
dyes. Frequently polar granules may be observed. This

344 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

has caused some investigators to place the organism in the
genus Pasteurella.

Cultural Characters. — The isolation and cultivation of
this organism are somewhat complicated by its peculiar
relationship to oxygen. When in shake culture in serum
agar, the organism grows in a layer several millimeters
below the surface, that is, while not a strict anaerobe, it does
not grow well (at least upon first cultivation) in the pres-
ence of an atmospheric pressure of oxygen ; in other words
it is micro-aerophilic. After cultivation for a time in the
laboratory, it may be grown readily in the presence of oxy-
gen in the ordinary laboratory media. It grows best with
the addition of serum to the medium. The colonies develop
at 37° as small, usually transparent dots. Growth is very
slow at room temperature.

Pathogenesis. — Experiments have shown that this or-
ganism will cause abortion in pregnant cows, both by
injection and by feeding. Injection into the rabbit or into
guinea pigs is followed by specific changes in the liver and
spleen and by the development of arthritis.

Immunity. — Cows that have aborted one or more times
frequently become immunized against disease. Various
methods of vaccination and serum treatment have been
attempted but have not been attended with general success.

The organism is not infrequently present in milk from
infected cows. The disease is probably frequently trans-
mitted by ingestion, perhaps also by coition.

Abortin prepared in an analogous manner to tuberculin
has been used to some extent for diagnosis. More fre-
quently the agglutination test is used and has come to be
the recognized test for diseased animals.

CHAPTER XXVI

HEMORRHAGIC SEPTICEMIAS AND PLAGUE— THE GENUS
PASTEURELLA

THE genus Pasteurella includes all of these bacteria which
are aerobic, nonmotile, Gram-negative rods, not producing
spores which show usually a decided tendency toward polar
staining and possess a comparatively slight power of fer-
mentation. The organisms belonging to this genus are
closely related to certain forms of the genus Bacterium and
in some cases it is quite difficult to differentiate between the
two genera. The first organism described belonging to this
group was that of chicken cholera, studied by Pasteur.
Other organisms closely related were found later and
Lignieres created the genus Pasteurella to include them all.
The term pasteurellosis is a designation of the disease caused
by any member of this group.

Bacteria of this group have been found associated with
a great variety of diseases in animals and in man. The most
important are the hemorrhagic septicemia of cattle and of
sheep ; the septic pleuropneumonia of calves ; fowl cholera;
swine septicemia and swine plague, and bubonic plague in
man. The organisms causing these diseases in various ani-
mals are so closely related and so difficult if not impossible
to differentiate in the laboratory, that some authors have
regarded all as belonging to a single species and have
designated as varieties merely those forms of causing dis-
eases in the various animals.

In the present discussion the hemorrhagic septicemia of
birds caused by Pasteurella cholerce-gallinarum, of cattle
caused by Pasteurella boviseptica, of swine caused by Pas-

345

346 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

•

teurella suiseptica and of bubonic plague in man caused by
Pasteurella pestis will be considered.

The organisms causing hemorrhagic septicemia in the
lower animals and birds, as already mentioned, are so much
alike that a description of one is satisfactory as a descrip-
tion for all except for pathogenic characteristics.

THE HEMORRHAGIC SEPTICEMIAS

It is probable that organisms belonging to this genus are
in some cases nonpathogenic and that there has developed
in literature some difficulty because of confusion of such
nonpathogenic forms with the disease producers.

Pathology. — The pasteurellas are nonmotile rods, usu-
ally about 0.5 X IM- The usual aniline dyes, particularly
when the organisms are stained from body fluid, as blood,
generally cause more intensive staining at the poles, the
central portion of the cell scarcely staining if at all. Some
forms when grown in culture media show frequently great
variation in size and appearance, and the polar staining is
much more difficult to demonstrate from such cultures than
from tissues. The bacteria are Gram-negative and do not
produce spores.

Cultural Characters. — These bacteria are readily culti-
vated in artificial media, producing upon gelatin and agar
round, flat and grayish colonies, finally becoming grayish
white and opaque. They do not grow well upon potato. In
broth they may produce clouding with clearing by sedi-
mentation; frequently a pellicle develops. There is no
appreciable change in milk. The organisms show very
slight fermentative powers. There has been an attempt to
differentiate species on the basis of acid production from
certain sugars, but this has not proved entirely successful.

Physiological Characters. — The organisms grow best at
blood heat, but fairly well at room temperatures. They are
aerobic, gas is never formed, but volatile acids may be.

HEMORRHAGIC SEPTICEMIAS AND PLAGUE 347

Pathogenic Characteristics. — Practically all species of
the genus Pasteurella quickly prove fatal when injected
into the ear vein of a rabbit. The animal dies as a result of
a septicemia; small hemorrhages usually may be noted in
the membranes of the digestive tract. Practicafly all the
species are pathogenic to sparrows. The various races show
some differences in their ability to affect other species of
animals. For example, fowls, in general, will succumb to
various cultures of the organisms associated with swine
plague, but not of the organisms causing hemorrhagic
septicemia in cattle.

PASTEURELLA CHOLER^E-GALLINARUM

Synonyms. — Bacillus avisepticus, Bacillus cholerce-gal~
linarum, Bacillus cholerce, and Bacterium avicidum.

This organism is the specific cause of the disease chicken
cholera. This disease has a wide distribution over Europe
and America. It was first studied by Pasteur in 1880. He
succeeded in growing the causal organism in artificial
media and made use of it in making studies on immuniza-
tion. By growing this organism in artificial media he suc-
ceeded in attenuating certain strains so that they might be
used as a vaccine. This work was of great historical im-
portance, as it marked the first step in the study of experi-
mental immunity, particularly the production of immunity
by means of injections of pure cultures of microorganisms.

Apparently there are many different strains of the fowl
cholera organism, showing marked differences in virulence.
Some, when injected in considerable quantities into fowls,
cause the development of a high degree of immunity ; others
bring about practically no immunity. The pigeon is very
susceptible and is frequently used in a study of this organ-
ism in the laboratory.

Fowls which have died of chicken cholera usually show
congestion of the heart and an accumulation of serum in

348 AGRICULTURAL AND INDUSTRIAL BACTEKIOLOGY

the pericardium. The liver is usually congested and shows
small hemorrhages. The spleen is enlarged and softened
and usually numerous petechial hemorrhages are to be
found in the internal organs and the mucous and serous
membranes.

Practical methods of immunization against the disease
have not been developed, although there has been much
experimental work done. The disease apparently is trans-
mitted from bird to bird by ingestion of food or water
fouled with excretions which contain the specific causal
organism. The only practicable method of control appar-
ently is careful sanitation.

PASTEURELLA BOVISEPTICA

Synonyms. — Bacterium bovisepticum, Bacillus lovisepti-
cus, Bacillus bipolare multicidum, Bacillus bovicida, Bacil-
lus vitulisepticus.

The disease hemorrhagic septicemia is one of the most
widely distributed in the United States. Similar diseases
have been described in European countries, both in domestic
cattle and wild animals, particularly the deer. The disease
in cattle is characterized by a fever due to the development
of the causal bacteria throughout the blood stream and in
the tissues. Upon post mortem, the animal generally shows
large numbers of hemorrhagic spots or points in the internal
organs and in some cases under the skin. These hemor-
rhages are particularly characteristic of the serous mem-
branes. In some cases the hemorrhages in the subcutaneous
tissues may be quite extensive and cause darkening of the
skin.

It is possible to immunize animals by the injection first, of
nonvirulent and later of virulent organisms, and the serum
from such animals is known to have the power of producing
passive immunity. It is evident, however, that there are
many different strains of bacteria belonging to this species

HEMORRHAGIC SEPTICEMIAS AND PLAGUE 349

and immunity against one is not necessarily potent against
another. An attempt has been made to use a polyvalent
serum, that is, one secured by injecting many strains of
organisms into the animal producing the serum, or by
mixing the serum secured from several animals immunized
each against several strains. In some cases the serum has
appeared to be effective in curing the disease in its early
stages. Bacterins, that is, killed cultures of this organism
have been quite extensively used in preventing the disease.
The disease, however, is sporadic and appears and disap-
pears with such suddenness that it is very difficult to evalu-
ate the advantages of such treatment.

The disease is particularly apt to appear among young
animals in first class condition.

PASTEURELLA SUISEPTICA

Synonyms. — Bacillus suisepticus, Bacterium suicidum,
Loffler-Schutz bacillus and Bacillus suicida.

This organism was usually described until recently as
the specific cause of the disease swine plague. In the early
history of the disease there was much confusion between
swine plague and hog cholera. The discovery that hog
cholera is caused by a filterable virus made necessary
critical examination of the disease swine plague. Undoubt-
edly in many instances, this organism is a secondary invader
in true hog cholera. However, there seems to be ample
evidence that there is a specific disease which may be known
as swine plague or swine septicemia caused by this organism
and distinct from hog cholera,

HEMORRHAGIC SEPTICEMIAS IN OTHER ANIMALS

Bacteria belonging to the hemorrhagic septicemia group
have been found to cause diseases of this general type
among many animals other than those already enumerated.
Rabbit septicemia or rabbit plague is caused by Pasteurella

350 AGBICULTUBAL AND INDUSTRIAL BACTERIOLOGY

cuniculicida. Hemorrhagic septicemia has been described in
the sheep and the horse, likewise an infectious pneumonia
of goats, a pasteurellosis of buffalo, and pasteurelloses of
many species of birds, including geese and ducks.

PASTEURELLA PESTIS

Synonyms. — Bacterium pestis, Bacillus pestis bubonicce.

This organism is the specific cause of the disease plague
in man and in rodents. It was discovered independently by
Yersin and by Kitasato in 1894. The disease is one which
is apparently endemic in certain parts of Asia, particularly
in northern and western China, and has at various times
spread over the entire civilized world. Bubonic plague has
been reported as present from the ports of entry of prac-
tically eveiy continent in recent years.

Pasteurella pestis resembles closely the other organisms
belonging to this group. It grows rather readily upon cul-
ture media.

The disease plague in man is usually contracted from dis-
eased rodents, particularly rats. It seems to be conclusively
demonstrated that the disease is primarily one of rodents
transmitted to man by means of the rat flea. If it attacks
man, it may develop in one of the three forms. Bubonic
plague results from cutaneous infection as a result of a
bite from a flea or of scratching the dejecta of the flea into
the skin. The organism enters the lymph channels and
causes ulceration of the lymph nodes, that is, it forms so-
called buboes. These may ultimately heal and the patient
recover; more frequently, however, the organism finally
invades the blood stream and the patient dies from the
resultant septicemia. When the organism is more virulent
it may enter the blood stream promptly after entrance into
the body and produce a typical hemorrhagic septicemia.
Hemorrhages develop under the skin and large areas
become discolored. This is the so-called " black death"

HEMOKKHAGIC SEPTICEMIAS AND PLAGUE 351

which devastated many parts of Europe at the close of the
Middle Ages. The disease may also be communicated to
man by inhalation, developing as a very fatal type of
pneumonia. Inasmuch as the organism is killed by drying,
transmission in this manner usually takes place when
patients and others come in contact in damp, ill-ventilated
quarters.

It is possible successfully to immunize man against
plague by injections of killed or attenuated bacteria or
their products. It is also possible to immunize horses and
use their sera in combating the disease. Inasmuch as there
is little transmission to man except from the rat, it is neces-
sary to- conduct campaigns against these rodents wherever
there is danger of an outbreak of a disease. In the United
States, outbreaks have been controlled in this fashion in
San Francisco, New Orleans and other ports. In some cases,
as in San Francisco, it was found that the disease eventually
attacked other rodents such as ground squirrels and wood
rats. When once the rodents native to these regions become
thoroughly infected, it becomes increasingly difficult, there-
fore, to stamp out the disease.

CHAPTER XXVII

THE SPOEE-BEAEING EODS— THE GENEEA BACILLUS AND
CLOSTEIDIUM. ANTHEAX, BLACKLEG, MALIGNANT
EDEMA, TETANUS, BOTULISM, AND GASEOUS EDEMA.

THE bacteria belonging to this group are all rod-shaped
organisms capable of producing endospores. They differ
considerably in size, cultural characteristics, shape and
position of spores, motility and fermentative reactions.
The group includes practically all of the spore-bearing
bacteria.

The genus Bacillus includes those organisms which are
aerobic or facultative anaerobic, the genus Clostridium
those which are anaerobic and micro-aerophilic. Most of
the bacteria, but not all, belonging to the group are Gram-
positive, although there is considerable variation in the
rapidity with which decolorization will take place.

One member of the genus Bacillus, Bacillus anthracis
(the cause of the disease anthrax in cattle), and several
members of the genus Clostridium will be considered. The
most important of the species of Clostridium are Clostrid-
ium tetani, causing tetanus or lockjaw in man amd animals,
Clostridium chauvcei, causing blackleg in cattle, the organ-
ism causing botulism, Clostridium botulinum, and a group
of organisms found associated with gaseous edema, the most
important of these being Clostridium welchii.

BACILLUS ANTHRACIS

Synonym. — Bacterium anthracis.

This organism is the specific cause of the disease known
as anthrax or splenic fever in cattle. In man the disease is

352

THE SPORE-BEARING RODS 353

sometimes termed malignant carbuncle, hide-handler's dis-
ease and woolsorter's disease. The anthrax appears most
commonly in sheep, swine and cattle and somewhat more
rarely in horses and in man. The bacillus of anthrax is
of importance from a historical point of view as it was
the first of the pathogenic bacteria to be accurately de-
scribed. Pollender in 1855 stated that he had observed tiny
rods in the blood of animals sick with the disease anthrax
in 1849. In a publication in 1876, Koch completed the
demonstration of the cause and relationship of this organ-
ism to the disease. This is of importance because it is the
first record of the cultivation in the laboratory of an organ-
ism capable of causing disease and of the successful produc-
tion of disease by the use of pure cultures. It is of interest
further because Pasteur with it made the first successful
application of the principle of vaccination by means of
attenuated or weakened cultures of bacteria.

Distribution. — Bacillus anthracis is a strict parasite,
that is, it does not grow widely distributed in nature. There
are records of the occurrence of anthrax from the earliest
times. Outbreaks have been noted throughout Europe and
both Americas. In the United States there are still a few
localities where the disease is enzootic.

Morphology. — The bacillus of anthrax is a straight rod,
occurring in chains, the individual cells usually showing
truncate ends. The bacteria are usually 1-1.25 X 4.5-10/z.
When examined in tissues or blood the chains usually are
short, when grown in culture media the chains are long. The
organism is nonmotile. Capsules are present in body tissues
and in blood but are not readily demonstrated in culture
media. Spores are produced when the organism grows in
the presence of sufficient oxygen, but are not commonly
found in the blood or tissues of animals with the disease.
Each cell produces a single spore, oval or spherical in shape,
which is located in the center of the cell and is of almost

354 AGEICULTUEAL AND INDUSTRIAL BACTEEIOLOGY

the same diameter. The vegetative rods readily stain by
the aniline dyes and are Gram-positive.

The organism culturally and morphologically very closely
resembles the nonpathogenic and saprophytic species of
the genus Bacillus as Bacillus subtilis and Bacillus
mycoides.

Culture. — The Bacillus anthracis can readily be secured
in pure culture from the blood or internal organs, particu-
larly the spleen or liver, of infected animals. The colonies
on gelatin or agar, when observed microscopically under the
low power, are seen to consist of long chains of bacilli which
resemble bunches of curled hair. This appearance is quite
characteristic, but is not very unlike that observed in cul-
tures of some of the saprophytic bacteria. In gelatin stabs
filaments develop from the sides, giving rise to a character-
istic spiking. The gelatin is slowly liquefied. Milk is first
rendered slightly acid, then curdled and the casein finally
digested. In broth a pellicle is developed which readily
settles to the bottom and the medium is not usually clouded.

Physiology. — The organism is aerobic and facultative
anaerobic. It grows best at blood heat, although growth
may be secured at room temperatures. The maximum
growth temperature is about 45° c. The rods are destroyed
by heat, but the spores are more resistant, requiring a tem-
perature approximately that of boiling water for some min-
utes. The latter are also very resistant to drying and have
been kept alive for years dried in soil or upon threads. They
are among the most resistant of the spores of the patho-
genic bacteria. When pastures, therefore, have been
infected with this organism, they may remain capable of
transmitting the disease for a considerable period of years.
Gelatin is digested.

Pathogenesis. — The organism is remarkably patho-
genic for many of the laboratory animals. Mice are par-
ticularly susceptible, guinea pigs hardly less so, rabbits are

THE SPORE-BEARING RODS 355

slightly more resistant. In most cases these animals will
die from the result of infection in from 24 to 48 hours.
Dogs and cats are somewhat less susceptible. Cold-blooded
animals are refractory. Fowls are relatively immune.

Anthrax in cattle is usually marked by acute fever with
not many external symptoms. Difficulty in respiration,
bloody urine, and bloody discharges from the various body
openings is common. The disease is usually fatal, the ani-
mal dying within a short time after the first visible symp-
tom. Sheep are quite susceptible and the disease proves
fatal very quickly. Horses having the disease in acute form
also die within a few days. Occasionally in the horse,
infection will occur through the skin and lead to the devel-
opment of a so-called malignant carbuncle. Swine are
somewhat more resistant and carbuncles and localized
glandular infections are more common.

Anthrax in man is varied in character, dependent upon
the method of infection. The so-called woolsorter's disease
is a pulmonary infection with anthrax resulting from the
breathing in of the spores by those who handle pelts from
sheep which have died of this disease. This type of disease
is always fatal. Cutaneous, or skin anthrax or malignant
carbuncle is the more common infection in man. Such
infection may result from contact with animals having the
disease and has appeared repeatedly among those who
handle hides, particularly hides which have not been ade-
quately disinfected. During the great war there were many
cases of anthrax in the army, both British and American,
due to anthrax spores on shaving brush bristles. Apparently
the horse hairs used for shaving brush manufacturers were
secured from China and had not been adequately sterilized
to insure the destruction of the anthrax spores which were
present. The lesion at first resembles a boil, but the tissues
round it soon begin to show infiltration, and not infre-
quently invasion of the blood and death from septicemia

356 AGEICULTURAL AND INDUSTRIAL BACTERIOLOGY

follows. Radical intervention and the removal of the car-
buncle is usually carried out.

In animals which have died of anthrax there is usually
to be noted a considerable enlargement of the spleen and a
decided softening of its pulp. Usually there are also hemor-
rhages and hemorrhagic exudates. Hemorrhages usually
appear in the liver and lungs.

Immunity. — As previously noted immunization against
anthrax was first developed by Pasteur. This investigator
found that when the organism was grown in pure culture at
temperature decidedly above the optimum, usually from 42°
to 43° c. for varying lengths of time, the ability to produce
disease gradually diminished. Cultures could be finally
secured which when injected into rabbits would no longer
kill them, but which were sufficiently virulent to kill guinea
pigs and mice. Still longer cultivation at this high tem-
perature will attenuate or weaken the organism still more,
until guinea pigs are only occasionally killed by injection.
Vaccination in sheep and cattle is most commonly pe.r-
formed by the injection of a highly attenuated culture;
after the subsidence of all reaction, a culture somewhat less
attenuated is injected as the second vaccine. As a result
of the methods introduced by Pasteur, the disease anthrax
has ceased to be a serious menace to the live stock industry
of France and other European countries.

It is possible to secure an active antiserum by the hyper-
immunization of animals by the injection of attenuated,
then of more and more virulent cultures.

Transmission. — Anthrax is usually transmitted from
animal to animal with feed, more rarely through lesions in
the skin. Infection by inhalation and through the skin are
most common in man. Animals which have died of anthrax
should be completely destroyed by burning or by deep bury-
ing. This organism is peculiar in that it does not form
spores within the body away from the air, but spores may

THE SPORE-BEARING RODS 357

form in bacteria thrown off in bloody exudate or in the
feces. In some cases, particularly in the southern United
States, bloodsucking flies have been found to be of impor-
tance in transmitting the disease by direct inoculation.

CLOSTRIDIUM TETANI

Synonyms. — Plectridium tetani, Bacillus tetani.

This organism is the cause of disease tetanus or lockjaw
in man and many species of domestic animals. It was first
observed by Nicolaier in 1885, who isolated it from animals
that had died as the result of the injection of garden soil.
The organism was cultivated in pure culture by Kitasato,
who noted that it was an obligate anaerobe, that is, it will
not grow in the presence of free oxygen.

This organism is not an obligate pathogen. It appears
that it, in common with other members of the genus
Clostridium, grows in the alimentary tract of animals, par-
ticularly of the horse, and only under exceptional conditions
is able to produce disease. It has long been noted that the
Clostridium tetani is found most frequently in soils which
have been heavily manured. It is possible that it may main-
tain a saprophytic existence and even multiply to some
extent in soil under favorable conditions.

Morphology. — Clostridium tetani is a slender rod .5ju X
2-5/x in length, with rounded ends. Usually it occurs in
short chains; it is actively motile in young cultures by
means of numerous peritrichic flagella. Spores are pro-
duced in abundance. The spore is terminal, spherical or
approximately spherical and greater in diameter than rod
which produces it. This gives a characteristic drum-stick
appearance. There are but few other bacteria, and these
closely related forms, which can be confused upon micro-
scopic examination with this organism. It stains readily
with the usual aniline dyes and is Gram-positive.

358 AGRICULTURAL AND INDUSTKIAL BACTEKIOLOGY

Cultural Characters. — The fact that Clostridium tetani
is an obligate anaerobe makes its isolation in pure culture
somewhat difficult. Colonies grown in gelatin are usually
woolly. Bouillon is clouded and a sediment is formed.
Blood serum is liquefied and milk more or less completely
digested. Brain medium is blackened as a result of the
development of hydrogen sulphide in the presence of iron.

Physiology. — The organism grows best at body tem-
perature but readily likewise at room temperatures. It
requires for the best development almost complete exclusion
of oxygen. It will grow readily in mixed cultures with
some organism which is an obligate aerobe. The spores are
quite resistant and will withstand drying indefinitely. High
temperatures maintained for considerable periods of time
are requisite for certain destruction of the organism. Gas
and acid are produced in small quantities from carbo-
hydrates.

Pathogenesis. — The tetanus bacillus usually produces
disease as a result of its inoculation into the tissue of a
wound under conditions favoring the exclusion of air. The
fact that it is not an obligate pathogen is well illustrated by
the fact that when its washed spores are injected into the
tissues, they usually do not produce tetanus, but are even-
tually destroyed by the body. It is usually only when this
organism is associated with dirt 'and is to some extent pro-
tected that it multiplies and produces its characteristic
toxin and symptoms of disease. It will also produce fatal
results when injected in pure culture together with a little
of the medium (containing toxin) in which it has been
growing.

The Clostridium tetani when growing in a wound pro-
duces a powerful toxin. This travels apparently through
two channels to the central nervous system. Some of the
toxin passes into the blood stream and is absorbed from it
by the central nervous system. Other portions travel along

THE SPORE-BEARING RODS 359

the nerves. The symptoms of the disease are not apparent
until the nerve cells have been injured as a result of the
absorbed toxin. These injured cells stimulate the muscles
(particularly the muscles about the area infected) to spas-
modic contraction (tetanus). In man and some animals
the nerve centers governing the muscles of the face are par-
ticularly apt to be affected and the contraction of the
muscles causes the characteristic " locking " of the jaw.

It is apparent that an incubation period is necessary
between the inoculation of organisms into the wound and
the development of the symptoms. This is the time neces-
sary for the formation of the toxin and the injury to the
central nerve cells. In man this incubation period averages
about 9 or 10 days, in horse it varies from 4 to 20 days or
even more.

Immunity. — The characteristic toxin of the tetanus
organism may readily be prepared in broth in which it is
grown under anaerobic conditions. In commercial manu-
facture of antitoxin, this toxin is first titrated by deter-
mining the smallest amount which will certainly kill a 350
gram guinea pig in 2 to 4 days. This is called the unit of
toxicity. At first small and then increasing doses of
toxin are injected at suitable intervals into the horse;
eventually the blood serum will be found to contain the
specific antitoxin in considerable quantities. The animal is
bled and the serum obtained in much the same fashion as
has been previously discussed for diphtheria antitoxin.

The tetanus antitoxin is an excellent prophylactic, that is,
when injected soon after the organism has been introduced
into the body, it will neutralize the poison as it is formed
and prevent the development of the symptoms of tetanus.
When once, however, the symptoms have developed, the
antitoxin is not always able to prevent a fatal result. In
other words, antitoxin cannot undo the damage already
done to the central nerve cells by the toxin taken up. It

360 AGEICULTUEAL AND INDUSTRIAL BACTERIOLOGY

can, however, neutralize other toxin as rapidly as it is
developed.

Infection occurs almost invariably directly through 'an
injury in the skin. Nail punctures in a horse are particu-
larly apt to result in tetanus as they introduce the organism
deep into the tissues and after the superficial healing with
the exclusion of air which takes place, conditions become
right for rapid multiplication. The statement that a rusty
nail is particularly apt to produce tetanus is in the main
true, inasmuch as it has much more opportunity to come in
contact with dirt; furthermore, particles of rust will gain
entrance to the wound and will not be brushed off by the
skin as the nail enters.

Occasionally, particularly in the horse, there are in-
stances of so-called cryptic infection in which it is not pos-
sible to demonstrate the wound through which the organism
producing the disease has entered. It is possible that the
organisms may circulate occasionally in the blood stream
and be able to localize and grow only when the tissues have
been injured in some fashion. This would explain, for ex-
ample, the development of tetanus following a bruise or a
fractured bone when the skin has not been broken.

CLOSTRIDIUM CHAUV^I

Synonyms. — Bacillus chauvcei, Bacillus feseri, Bacillus
anthracis symptomatici.

This organism is the specific cause of the disease black-
leg, symptomatic anthrax, quarter evil or quarter ill in
cattle.

Morphology. — The Clostridium chauvcei is a relatively
large organism, rod-shaped with rounded ends, rarely in
pairs, usually single, 0.5 to 0.6 X 3-5/x. The organism,
even in tissues, practically never occurs in chains. This is
of some value in differentiation from the rather closely
related Clostridium o&dematis. The organism is motile by

THE SPORE-BEAKING EODS 361

means of peritrichous flagella. Under suitable conditions in
cultural media spores are produced, sometimes centrally
located but more frequently near the poles. The spores are
elliptical in shape and cause the cell to swell some-
what. The organism is easily stained and usually Gram-
positive.

Cultural Characters. — Culture is somewhat difficult
because the organisms grow only under anaerobic condi-
tions. Cultures grow best in a medium that is somewhat
alkaline. Gas is produced from dextrose. Bouillon be-
comes clouded, and in the presence of carbohydrates, gas is
^produced.

Physiology. — The organism grows best at blood heat
but readily also at room temperatures. It is strictly ana-
erobic; the spores are resistant to drying, having been
found to live for years in this condition.

Pathogenesis. — Blackleg occurs most frequently in
young cattle, but rarely in calves or in adult animals. The
disease is characterized by swelling, softening, and gas pro-
duction in the muscles and hemorrhagic edema of the parts
which have been infected. It occurs most frequently in
the shoulder or hind quarter. The presence of gas is usu-
ally demonstrable by the crackling sound which is produced
when the thumb is drawn quickly across the skin of the
infected parts. After the death of the animal the tissues
may be generally invaded and the body become swollen
due to the development of gas. When the tissues infected
are sectioned they are found to be blackened and hemor-
rhagic. The disease is usually fatal.

Immunity. — According to some authors, the Clostridium
chauvcei produces a true toxin, for which an antitoxin may
be prepared. It has been found, however, that the antitoxin
is of relatively little use in the immunization of animals.
Much more commonly practiced is active immunization by
means of vaccine. The standard method of preparation

362 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

adopted by the U. S. Department of Agriculture is to
secure fresh muscle tissue in which the blackleg organism
has been growing, grinding this in a mortar and finally
squeezing out the fluid. This contains many of the organ-
isms, together with spores. This is spread in a thin layer
and dried to a brownish scale at blood heat. The vaccine is
prepared by mixing one part of this material with two parts
of water and placing in a hot air oven at a temperature of
from 95° to 99° c. for six hours. This heating attenuates
the organisms so that they may be injected without fatal
results into susceptible cattle. The vaccine in some cases is
suspended in water and is injected under the skin of ani-
mals to be immunized by means of a hypodermic syringe.
In other cases it is made up into tablets which are inserted
under the skin. In some cases, the vaccine is soaked on
threads which are drawn into tissues by means of a needle.
In general, vaccination against blackleg has proved satis-
factory.

The complete life history of Clostridium chauvcei in
nature is not well understood. It seems probable that it
may under certain circumstances multiply in the alimentary
tract of suitable animals. It is certain that the organism
is present in some soils and not present in others. In some
cases the pastures of one farm or one part of the farm may
harbor the organisms, and such farms be subject to attacks
of blackleg, and adjoining farms may be entirely free. In
European countries it is well known that certain alpine pas-
tures cannot be grazed by young cattle which have not been
previously protected by vaccination against blackleg.

Infection is believed usually to occur through wounds
and the disease is rarely if ever transmitted directly from
one animal to another. Commonly it is difficult to deter-
mine the manner in which the organism has gained en-
trance, ttfat is, cryptic infections are comparatively com-
It is believed that in such cases the organism may

THE SPOKE-BEAEING KODS 363

have gained entrance to the blood stream from the intestinal
tract and have localized in some tissue which has been
injured or bruised.

CLOSTRIDIUM WELCHII

This organism is taken as the type of a group of bacteria
which in recent years have become very prominent because
of their apparent relationship to gaseous and malignant
edemas of various types in man and in animals. These
bacteria are relatively common in many soils and when they
gain access to wounds, produce a variety of lesions.

Clostridium welchii has been isolated repeatedly from
men dying from gaseous edema.

Morphology. — The organism is a large rod about
IM X 2-6 /*. It is frequently found in chains but may occur
in pairs or groups. It is nonmotile. Spores may be pro-
duced under certain conditions in the laboratory upon
culture media but are not produced in the presence of
sugars. These spores are centrally located and the cells
become considerably swollen and spindle-shaped. Capsules
are produced in the body fluid and in some of the artificial
media. The cells are usually Gram-positive.

Cultural Characters. — Colonies produced upon gelatin
or agar plates are round, grayish and semiconfluent, resem-
bling in many ways those of Clostridium tetani. Gelatin is
usually slowly liquefied, bouillon clouded with the forma-
tion of abundant precipitate. Milk is quickly coagulated
with the formation of gas and acid.

Physiology. — The organism grows best at blood heat.
The thermal death point for organisms without spores, that
is, for vegetative rods, is 50° c. for 10 minutes. Spores
require boiling temperature for at least 15 minutes. An
abundance of gas is produced from many of the carbo-
hydrates including dextrose, lactose and sucrose but not
from mannitol.

364 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Pathogenesis. — This organism together with the Clostri-
dium cedematis, Clostridium Ghon-Sachs and the Clos-
tridium sporogenes and others may be present in soils, par-
ticularly cultivated and manured soils, whence they may
gain access to wounds. In man these organisms are of
special significance, because of their influence on war
wounds, particularly "dirty wounds/' Some species
spread rapidly through the tissues, destroying them as they
develop, causing hemorrhage, but not producing gas ; others,
as the Clostridium welchii, cause hemorrhages and produce
an abundance of gas as well.

Immunity. — Certain of these species produce a specific
toxin for which antitoxin may be prepared. Practicable
methods of immunization have not yet been worked out,
however. This is particularly difficult as bacteriological
study including microscopic examination is necessary before
one can know what particular species is causing edema in
the particular individual and in consequence what serum
should be used.

Diseases of this same general type have repeatedly been
observed among animals, particularly sheep and swine.

CLOSTRIDIUM BOTULINUM

Synonym. — Bacillus botulinus.

This organism is one of the frequent causes of meat and
food poisoning in man. The disease is generally termed
botulism from the Latin botulus meaning sausage. The
organism was first isolated by Van Ermengen in 1896 from
meat which he believed to be the cause of an outbreak
of food poisoning. Poisoning caused by this organism
should not be confused with that caused by Bacterium
enleritidis or with the so-called ptomaine poisoning.

Distribution. — Outbreaks of poisoning of the type of
botulism have been repeatedly reported from Europe, par-

THE SPORE-BEARING RODS 365

ticularly in meat foods and sausages. In the United States
practically all of the cases reported have been from the
eating of imperfectly preserved canned foods. A consider-
able variety of these have been found to be at fault. Cases
of poisoning from canned asparagus, canned beans and ripe
olives have been reported. Most of the cases have been
from the Pacific coast.

Morphology. — The Clostridium botulinum is a relatively
large bacillus, usually single, occasionally in pairs or chains.
It is '.9-1. 2/z X 4-6/4. It is actively motile by means of
from 4 to 8 peritichous flagella. Oval or elliptical spores
are produced, one near the end of each cell. The organism
stains readily and is Gram-positive.

Culture. — Care must be taken to exclude oxygen in the
growth of Clostridium botulinum. It grows fairly readily
in ordinary cultural media.

Physiology. — The organism is an obligate anaerobe.
Some strains grow best at room temperatures, others at
body temperature. Apparently there are several distinct
varieties of this organism which show minor differences in
physiological reactions Gas is generally produced from
dextrose but not from sucrose or lactose.

Pathogenesis. — When growing under favorable condi-
tions, the Clostridium botulinum produces a highly potent
toxin. Usually this is accompanied by the formation of
malodorous substances, particularly butyric acid. Usually
food in which this organism has been growing can be
detected by having an "off" odor. Such foods must be
carefully avoided as even a small portion of such food,
sometimes not more than a mere taste, has proved sufficient
to be fatal.

The toxin produced by growing the organism in culture
media is quickly fatal to laboratory animals when fed or
injected. It is also fatal frequently to man. In man the

366 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

symptoms include lack of coordination of the eyes (that is,
double vision) and difficulty in control of the muscles gov-
erning swallowing.

Until recently it has been believed that the organism itself
(free from toxin) when introduced into the animal body
is nonpathogenic, but more recent work seems to show that
the organism when injected or fed to certain animals is
capable of producing serious or fatal results.

Limber neck of chickens may be caused by feeding food
containing the organism or its toxin. The birds are par-
tially paralyzed, resting the tip of the bill upon the ground
and showing lack of ability to hold the head erect.

According to Graham and his coworkers, this organism is
one of the common causes of so-called forage poisoning in
cattle and in horses. It has been found that this organism
could be isolated from silage and fodder, producing the
disease called "blind-staggers" in the horse, and that the
disease could be produced by feeding horses upon fodder
inoculated with pure cultures of the organism.

Inasmuch as this organism produces a toxin, it is possible
that the corresponding antitoxin could be used in treating
the disease. In animals it has been used effectively in
several instances and very possibly it may be of value in
man. Recent work, however, indicates that there are at
least two distinct strains of Clostridium 'botulinum whose
toxins differ somewhat, and it is necessary therefore in suc-
cessful treatment to have an antitoxin specific for the
organisms which produce the infection.

Inasmuch as this organism has been found to infect man
most frequently from canned foods, it is of interest to note
that the disease has resulted both from the eating of com-
mercially canned foods and home canned products. It is
important that canned foods be adequately sterilized and
care should be used not to consume foods which are "off"

THE SPOKE-BEAKING HODS 367

in odor. Boiling will destroy the potency of the toxin and
long continued boiling will usually destroy the organism
and its spores. Recently cooked foods, therefore, are prob-
ably never dangerous.

CHAPTER XXVIII

THE GROUP OF BAY BACTEEIA, LUMPY JAW AND BELATED
INFECTIONS— THE GENUS ACTINOMYCES

ORGANISMS belonging to the genus Actinomyces are very
abundant in soil. Many different species have been de-
scribed. A few only are known which under certain con-
ditions are capable of producing disease in animals and
occasionally in man. It is not at all improbable that these
are really saprophytic forms which are able to adjust them-
selves to parasitic conditions.

The organisms belonging to this group are intermediate
in most of their characteristics between the bacteria and
the molds, and by many investigators they are included
with the latter. They resemble the molds in producing
chains of spores in many cases and also in producing a
branched mycelium. In many forms these hyphae may
break up into short lengths and function in some cases at
least, as spores. Only one species, the form producing
lumpy jaw in cattle (Actinomyces bovis) is to be discussed
here, although several other species have been described
from somewhat similar lesions both in animals and in man.

ACTINOMYCES BOVIS

Synonyms. — Streptothrix bovis, Discomyces bovis.

This organism and perhaps some closely related species
are the specific causes of the disease lumpy jaw and wooden
tongue or actinomycosis in cattle and probably in some cases
in other animals. The disease lumpy jaw is known from
many areas in Europe and North and South America.

368

THE GROUP OF RAY BACTERIA 369

Morphology. — When the purulent material from a case
of lumpy jaw in cattle is examined there will be found
many minute yellowish granules sometimes large enough
to be observed by the unaided eye. Microscopic examina-
tion of these tiny grains show them to be made up of com-
pact masses of the organisms arranged more or less radially.
When the edge of the granule is examined carefully it will
be found to be made up of club-shaped enlargements. The
filaments in the middle of the granule are usually half a
micron in diameter.

The organism stains readily with the common aniline
dyes and is Gram-positive. When growing upon artificial
media it consists of interlacing branched threads, forming
a relatively compact mass.

Culture. — In broth the organism forms distinct spherical
mulberrylike masses, generally near the bottom of the tube.
Agar cultures, particularly those containing glycerin, show
the development of colonies, which resemble tiny drops of
amber, which may enlarge and may remain discrete or
colorless, or form a distinctly wrinkled membrane, some-
times rather dusty in appearance.

Pathogenesis. — The organism does not appear to be
pathogenic for most of the laboratory animals. In most
cases the lumpy jaw or wooden tongue of cattle is primarily
due to the presence of a splinter or grass awn which has
worked its way into the tongue or the gums around the
roots of the teeth. These evidently carry with them the
spores of the Actinomyces which is then aided in invading
the tissues. The swelling or tumorlike mass develops at the
side of the infection. This may soften and ultimately dis-
charge a yellowish pus. When the tongue is the primary
seat of infection, it becomes enlarged, indurated or hardened
and may protrude from the mouth.

The disease apparently is quite noncontagious— it is not
at all readily transmitted from one animal to another.

370 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Methods of immunization against the disease are not known
or have not been developed.

RELATED ORGANISMS

Microorganisms closely related to Actinomyces bovis have
been found to cause one type of bovine farcy, actinomycosis
in goats, in the dog, and Madura foot in man.

CHAPTER XXIX

THE ACID FAST GKOUP— THE GENUS MYCOBACTERIUM—
TUBERCULOSIS, PARATUBERCULAR DYSENTERY AND
LEPROSY.

THE organisms belonging to this group are all slender,
nonsporing rods, which do not generally produce capsules,
are Gram-positive, and nonmotile. They also have in com-
mon the character of acid fastness, that is, they are rela-
tively difficult to stain with the aniline dyes, but when once
stained they retain the stain with avidity, not losing it even
in the presence of relatively strong solutions of mineral
acids. All of the species belonging to this group, further-
more, show a decided tendency under certain cultural con-
ditions to produce elongated, more or less branched threads.
In some respects they are intermediate between forms like
Actinomyces and the other bacteria.

Organisms belonging to this group are widely distributed
in nature, in soil, in manure, etc. Three species are of
importance because of their ability to produce disease, that
is, they are primarily pathogenic and parasitic. These are
the Mycobacterium tuberculosis, the cause of the disease
tuberculosis, Mycobacterium paratuberculosis, the cause of
paratubercular dysentery in cattle, and Mycobacterium
leprce, the cause of leprosy in man.

MYCOBACTERIUM TUBERCULOSIS

Synonyms. — Bacillus tuberculosis, Bacterium tubercu-
losis.

This organism, together with its several varieties, is the
specific cause of tuberculosis in man, birds and various

371

372 AGKICULTTJKAL AND INDUSTRIAL BACTERIOLOGY

animals. Inasmuch as tuberculosis is a disease which may
attack many parts of the body, the clinical symptoms may
vary in different cases and the appearances on post mortem
examination may differ widely. In consequence until mod-
ern times tuberculosis of different types were regarded
as different diseases. Tuberculosis of the skin, tuberculosis
of the lungs, tuberculosis of the peritoneum and tubercu-
losis of the bones and joints were regarded as distinct
diseases. It was only after the discovery of the specific
organism in 1884 by Robert Koch that the essential identity
of the various types of the disease was established. He
succeeded in showing the characteristic organism always to
be present in nodules of the disease, and succeeded further
in growing the organism in pure culture in the laboratory
and reproducing the disease upon experimental inoculation.
The organisms isolated from man, cattle and birds appeared
so nearly alike that at first they were regarded as identical,
that is, it was assumed that tuberculosis in all animals was
caused by the same germ. In 1896, Theobald Smith ob-
served that there were certain differences to be noted in the
culture, morphology and physiology of the bacteria isolated
from cases of human and bovine tuberculosis. This was
further elaborated by Koch who in 1901 concluded that the
organism causing tuberculosis in man was quite distinct from
that of tuberculosis in cattle. At the present time at least
four distinct varieties of Mycobacterium tuberculosis have
been recognized. The variety most frequently causing
tuberculosis in man is variety hominis, the type pro-
ducing disease in cattle, variety bovis, the type com-
monly producing disease in birds, particularly domestic
fowls, variety avium, and the type producing disease in
cold-blooded animals, such as fishes and turtles, variety
piscium. It seems to be well demonstrated that it is difficult
if not impossible to transform one variety into another.
The preceding statement should not be misinterpreted; it

THE ACID FAST GROUP 373

is not impossible to produce disease in humans, for example,
by means of bovine bacilli. The facts will be considered
below.

Tuberculosis is usually the cause of more deaths in man
in the United States annually than any other disease. The
disease in cattle is widely distributed, particularly in dairy
animals both in Europe and America. Swine, particularly
those which run with the cattle, are often infected. The
disease occurs occasionally in the horse and in the sheep.
Tuberculosis of the domestic fowl is common in certain dis-
tricts in the United States.

Morphology. — The Mycobacterium tuberculosis is a
slender rod, frequently somewhat bent and with rounded
ends. Not infrequently the protoplasm stains somewhat
irregularly, giving a beaded appearance to the cell. Spores
and capsules are not produced. It is Gram-positive and is
nonmotile. The cells vary from .2 to .5/i in diameter and
1.5 to 3.5/x in length. When grown upon certain culture
media elongated and branched forms are not at all uncom-
mon. The organism is best stained with one of the more
powerful analine dyes, such as carbol fuchsin. Apparently
the acid fastness of this organism is due to the presence of
a waxlike material in the cell walls.

There is some difference of opinion as to whether there
are noteworthy differences in the morphology of the
varieties of the tubercle bacillus. It has been claimed that
organisms from bovine cases are somewhat shorter,
straighter and thicker than those from human cases and
are somewhat less apt to show the granular staining char-
acteristic. The varieties, however, are much more satis-
factorily differentiated by the use of cultural, physiological
and pathological tests.

Cultural Characters. — The tubercle bacillus, at least
when first isolated from the body, requires special media
for its growth. Pure cultures may be secured by rubbing

374 AGEICULTUEAL AND INDUSTRIAL BACTERIOLOGY

nodules secured under aseptic precautions from infected
animals over the surfaces of solidified blood serum or egg
medium. It is sometimes necessary to keep at blood heat
for one to two weeks before visible growth develops. When
the organism is not in pure culture as is the case in milk,
feces, etc., isolation is frequently attended with difficulty.
It may be accomplished by inoculating the material from
which the culture is to be secured into a suitable animal
such as a guinea pig, allowing the disease tuberculosis to
develop, and securing the organism in pure culture from
the lesions. It may also easily be secured by dissolving the
organic material and other bacteria in antiformin, washing
and inoculating a culture tube.

After cultivation in the laboratory the organism will
grow somewhat more readily upon culture media, particu-
larly those which contain blood serum, egg or similar
proteins or to which glycerol has been added.

The colonies upon blood serum or upon glycerin agar
appear first as tiny grains barely visible to the naked eye.
In cultures where they grow readily, the colonies become
confluent and cover the surface of the medium with a dry,
wrinkled growth. In old cultures this may become creamy
or brown in color. In glycerin broth there is usually
formed a more or less heavy wrinkled scum which readily
sinks to the bottom when shaken.

Organisms isolated from human sources usually adapt
themselves much more quickly and readily to culture media
than those from bovine and always grow much more luxuri-
antly. In typical cases it is not difficult to tell which strain
an organism belongs to by the differences in the growth
appearance. The bovine organism usually forms isolated
colonies while those from human cultures form a continuous
wrinkled growth over the surface of the medium.

Physiological Characters. — The organisms from human
and bovine sources grow best at blood heat and their growth

THE ACID FAST GROUP 375

temperature range is relatively narrow. It is difficult to
get growth below 30° c. or above 40° c. The bacteria are
readily killed by heat, the thermal point being given as
60° c. for 20 minutes. Desiccation destroys the organisms
slowly, although they have been known to remain alive in
dried sputum for several months. Certain physiological
characters have been noted as of assistance in differentiating
the human and bovine tubercle bacillus. It was noted by
Theobald Smith that when the human bacillus is grown in
glycerin broth somewhat acid to phenolphthalein, it brings
about a permanent acid reaction; while with the bacillus
of bovine tuberculosis the acidity finally diminishes and the
reaction eventually becomes alkaline.

Pathogenesis. — Tuberculosis is typically a chronic dis-
ease. It is characterized, no matter what part of the body
is affected, by the development of nodules having eventually
the same structure. When the tubercle bacillus lodges in a
tissue it causes a multiplication of the connective tissue
cells. These become surrounded by a more or less delicate
layer of epitheloid cells while at the center there are so-
called giant cells containing numerous nuclei. The whole
mass is termed a tubercle or a miliary tubercle. Eventually
autolysis of some of the cells in the interior of the nodule
takes place and it undergoes what is termed caseation, that
is, the interior becomes cheeselike in consistency. This is
particularly apt to happen when nodules are found in
large masses. In many cases the tissues surrounding the
nodules develop a firm fibrous capsule, and this may even-
tually become infiltrated with lime, that is, calcified. Such
granules will persist indefinitely in tissues which have been
tubercular even after the disease has been entirely cured.
The organisms are particularly apt to invade lymph nodes
and glands through the lymph channels.

The different varieties of the tubercle bacillus show
marked variations in their pathogenesis for animals. In

376 AGKICULTUBAL AND INDUSTKIAL BACTEEIOLOGY

general the bovine bacillus is the most pathogenic, the
human next and the avian least except for birds. Inocula-
tion in guinea pigs with bovine bacillus usually leads to
death in less than 30 days, while those inoculated with
human bacillus will live for several months. Injection of
the rabbit with bovine bacillus usually kills the animal
within three weeks, while similar injection of the human
bacillus usually does not prove fatal for several months
and in some cases the animals may recover. Calves inocu-
lated with bovine bacilli succumb rather rapidly to the
disease, while similar inoculatidh with human bacilli pro-
duces only local lesions which eventually heal.

In man tuberculosis is most common as a disease of the
lungs (so-called consumption). Next in frequency come
tuberculosis of the lymph glands of the neck (scrofula), of
the bones and joints (bone and articular tuberculosis), of
the covering of the brain (tubercular meningitis), abdom-
inal tuberculosis and occasionally tuberculosis of the skin
(lupus). In cattle the disease generally attacks the mesen-
tery causing the development of the so-called pearly disease
characterized by the nodules on the membrane lining the
peritoneal cavity. Tuberculosis of the lungs is also com-
mon. Frequently in the cattle the lymph nodes become
enlarged. Tuberculosis of the udder is sometimes noted in
tubercular cows. Swine generally show infections of the
lymph glands in the neck, frequently also abdominal and
pulmonary lesions. In the domestic fowl the tuberculosis is
in general abdominal, the lesions and nodules showing par-
ticularly in the liver and spleen and somewhat more rarely
in the lungs.

Immunity. — Practicable methods of immunizing against
tuberculosis have not been developed although many at-
tempts have been made and there has been an immense
amount of systematic work done in this field. It is possible
to immunize cattle against bovine tuberculosis by injection

THE ACID FAST GROUP 377

of nonvirulent strains of the human type. The immunity,
however, does not last for more than a year.

Diagnosis of the Disease. — Tuberculosis is most com-
monly recognized by taking advantage of the fact that
animals affected with the disease show marked hypersus-
ceptibility or anaphylaxis, that is, when an extract of the
tubercle bacillus is injected into an animal having the
disease a marked reaction generally results.

Such an extract of tubercle bacilli used for diagnosing
the disease is known as tuberculin. The .standard tuber-
culin most commonly used in the testing of cattle, the
so-called Koch's old tuberculin, is prepared by inoculating
a flat-bottomed flask containing 5 per cent glycerin broth to
a depth of 2 to 3 centimeters with a culture of Mycobac-
terium tuberculosis. Careful inoculation of the surface
from a pure culture and incubation at blood heat results in
the growth of a film or pellicle. This is well developed in
a few weeks. The broth is then evaporated in a porcelain
dish on a water bath until the glycerin concentration
becomes about 60 per cent, when it is filtered through
paper or a porcelain filter. It is evident that the extract
contains the growth products of the tubercle bacillus to-
gether with glycerin, salt and various substances present
originally in the broth. Purified tuberculins and tubercu-
lins made from the portions only of the tubercle bacillus
have been prepared in a great variety of ways.

Diagnosis of tuberculosis in cattle is most frequently
effected by the subcutaneous injection of tuberculin. The
dose given is about one-fourth cubic centimeter of the
Koch's old tuberculin. Before the injection of the animal
the normal temperature should be determined. After six
or eight hours the temperature is again taken and there-
after every two hours during the test. Animals in heat or
advanced in pregnancy or suffering from other diseases
should not be tested. In general a positive test will be

378 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

recognized by a rise of at least one and one-half degrees
above the previous maximum recorded temperature. Usu-
ally the temperature begins to rise within 8 hours after
injection and reaches its maximum in 18 hours, then grad-
ually subsides.

In addition to the generalized tuberculin reaction local
reactions are frequently used in disease diagnosis. Most
common of these is the so-called intradermal test used in
cattle. In this method a small amount of tuberculin is
injected into the skin (not under the skin) ; most commonly
this injection is made into the skin showing no hair at one
side of the tail. The reaction consists of a circumscribed
swelling about the size of a hulled walnut within twenty-
four hours. A somewhat similar method may be used in
swine, the injection being made into the ear lobe. An
ophthalmic or eye reaction is occasionally used. In this
test a tuberculin which has been prepared free from
glycerin and other irritant substances must be utilized. It
is dropped into the corner of the eye. The eye within
twenty-four hours will become inflamed and a marked con-
junctivitis will develop. This will disappear within three
days.

The tuberculin test has been proved to be a remarkably
reliable method of diagnosing tuberculosis in cattle. It has
been used to some extent in diagnosing the disease in other
animals as well. The tests used in general resemble those
used in cattle. In man, particularly in children, it has been
noted that rubbing tuberculin or tuberculin ointment into
the skin vigorously will result in the development of a
marked reddening at the site of the inunction in tubercu-
lous individuals.

While the tuberculin reaction appears to be a reliable
method of recognizing tuberculosis in cattle when carefully
carried out, it should be noted that an injection of tubercu-
lin may interfere with the thermal reaction of the animal

THE ACID FAST GROUP 379

to tuberculin for a few days thereafter, or in some cases
for a longer period. This fact has been made use of by dis-
honest cattlemen in securing certification of freedom from
tuberculosis in animals. The veterinarian who makes the
test, however, may overcome the results of such previous
injection by the use of larger doses of tuberculin or by the
use of the intradermal test.

It should be noted furthermore, that in some cases, ani-
mals that are in the last stages of the disease or are con-
siderably emaciated may fail to show the reaction. The
presence of large numbers of organisms constantly in the
body has made it impossible for the animal to react nor-
mally to the injection of the tuberculin. Naturally the fact
that animals showing very marked symptoms of the disease
may occasionally fail to show the reaction while animals
having only slight lesions, perhaps only an obscure nodule
in a lymph node, may show marked reaction, has led to
considerable skepticism on the part of practical farmers
with reference to the test. When properly understood,
however, it may be regarded as reliable.

Transmission. — Tuberculosis in man is transmitted most
commonly by inhalation of infectious droplets thrown off in
coughing, through contaminated drinking vessels and in
some cases undoubtedly by the eating of food containing
the tubercle bacillus. The bacteria leave the body with the
sputum or feces and occasionally with the urine.

Tuberculosis exists in animals and man either as a closed
or an open type. In closed tuberculosis the bacteria are
present in organs or tissues that have no direct communica-
tion with the surface of the body. The presence of tubercle
bacilli, for example, in the lymph nodes, would not neces-
sarily lead to their being discharged by the body. In open
tuberculosis the bacteria are growing in tissues which com-
municate readily with the surface of the body, in the lungs,
in the liver or in the walls of the alimentary tract. In

380 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

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THE ACID FAST GKOUP 381

open tuberculosis the organisms are being thrown off more
or less constantly by the body. In the closed type, the bac-
teria are not leaving the body and the disease is in this
condition, noncontagious.

Cattle are probably most commonly infected by ingestion,
perhaps occasionally by inhalation. Hogs generally show
tuberculosis when they are following tubercular cattle and
infection is undoubtedly by ingestion. The same apparently
is true of tuberculosis in fowls.

Transmission of Bovine Tuberculosis to Man. — It has
been noted that the bovine and human tubercle bacilli are
relatively distinct varieties, and that the human tubercle
bacilli do not readily infect cattle. The conclusion was
reached by some investigators, therefore, that bovine
tubercle bacilli do not affect man. In recent years, how-
ever, it has been shown that in certain types of cases, and
particularly in children, bovine tubercle bacilli produce a
considerable proportion of the cases. Evidently tubercu-
losis can be transmitted to man, therefore, by milk from
tuberculous cows. The chart given on page 380 adapted
from Park and Krumweide summarizes the types of
tubercle bacillus producing disease in man in 1511 cases.
An examination of this table would seem to prove quite
conclusively that the tuberculosis of bovine origin is fre-
quent enough in children under sixteen years to justify
all reasonable precaution against the ingestion of infected
meat and milk. On the other hand, the bovine tubercle
bacillus is very rare as a cause of disease in adults.

MYCOBACTERIUM PARATUBERCULOSIS

Synonyms. — Bacillus paratuberculosis and Bacillus of
Johne's disease.

This organism is the cause of a peculiar chronic dysentery
in cattle. The early investigators concluded the organism

382 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

to be identical with the tubercle bacillus which causes dis-
ease in fowls. It seems to be well established, however,
that the organism is distinct. The disease has been
noted in various parts of Europe as well as in the United
States.

Morphology. — The organism closely resembles the Myco-
bacterium tuberculosis. In fact the two are indistinguish-
able under the microscope.

Cultural Characters. — The organism grows with diffi-
culty and only upon special media.

Pathogenesis. — The disease in cattle is characterized by
progressive emaciation and by a persistent chronic diarrhea,
usually proving fatal. Post mortem examination shows the
lesions to be confined largely to the intestines. In these the
mucous membranes show thickening and wrinkling. Prac-
tical methods of immunization have not been developed. It
is probable that the disease is transmitted by ingestion.

MYCOBACTERIUM LEPR.E

Synonyms. — Bacterium leprce and Bacillus leprce.

This organism is the specific cause of leprosy in man, a
disease common 'in Asia, in certain sections of northern
Europe and some of the Pacific Islands. Occasionally it
has been found in the United States. A very closely related
disease sometimes occurs in rats. The organism in many
respects resembles the tubercle bacillus. The organisms
may multiply in the nerves, causing loss of sensation or the
so-called anesthetic leprosy, or they may multiply in the
subcutaneous tissues, causing the formation of tubercles or
nodules closely resembling those of tuberculosis. The dis-
ease is a chronic one and the individual may survive for
many years. The disease apparently is nontransmissible to
any of the lower animals.

THE ACID FAST GROUP 333

Apparently leprosy is not highly contagious. The exact
method of transmission is not known, it is probably accom-
plished as a result of direct contact. The organism does
not grow readily upon artificial media.

The disease is successfully treated in many cases with
Chalmoogra oil.

CHAPTER XXX

DIPHTHEEIA AND GLANDERS GROUP— THE GENERA
CORYNEBACTERIUM AND PFEIFFERELLA

CORYNEBACTERIUM

THE bacteria belonging to the genus Cory neb act erium are
rods, Gram-positive, nonmotile, aerobic and facultative, not
producing spores and characterized by the possession of
metachromatic granules. They may also show marked
tendency to the development of threads and more or less
irregular branching and club forms. The most important
organism belonging to the group is the Coryneb act erium
diphtherice, the cause of the disease diphtheria. Another
organism, the C or ynebact erium pseudotuberculosis, has been
found to produce a considerable variety of infections in
domestic animals, the organism in sheep giving rise to the
formation of enlarged lymph glands and producing some-
what similar diseases in cattle, horses, rabbits and other
animals. In addition there have been described a consider-
able number of organisms belonging to this group, associated
with other diseases in man but not definitely proved
to have a causal relationship to them. Bacteria belonging
to this group furthermore have been isolated repeatedly
from the throat and mouths of healthy individuals. The
only species which will be discussed is the organism causing
diphtheria.

CORYNEBACTERIUM DIPHTHERIA

Synonyms. — Bacillus diphtherice, Bacterium diphtherice,
Mycobacterium diphtherice.

This organism is the specific cause of the disease diph-

384

DIPHTHERIA AND GLANDERS GROUP 385

theria in man. The term diphtheria was originally used
by physicians to indicate a characteristic inflammation of
the mucous membrane together with formation of a false
membrane in the throat or nose. This particular type of
infection was said to be diphtheritic. Inasmuch as the
organism under discussion is the most common cause of con-
ditions of this type in the throat, the term diphtheria is
now generally recognized as applying only to lesions pro-
duced by this particular organism.

Morphology. — Young cultures of Corynebacterium diph-
theria show rods predominantly 0.4-1.4/z in diameter and
1.5-3.5^ or more in length. They show marked variation
in size and staining characteristics, depending upon the
type of medium upon which they are growing and upon the
age of the culture. Not infrequently they may stain uni-
formly, but more commonly there are deeply-stained bands
or granules within the cells. Frequently the cells are
slightly curved or pointed and club-shaped. Occasionally
branched cells have been noted.

Cultural Characters.— The organism may be readily
secured in culture by rubbing a swab first over the inflamed
throat surfaces or upon the false membrane of the diph-
theria patient and then over the surface of coagulated
blood serum. When grown at blood heat upon this medium,
development is very rapid; usually colonies may be ob-
served in 12 to 18 hours as small pin-pointlike translucent
dots, which later 'become opaque and grayish. It grows
best on media containing blood serum or glycerin. It
gradually accustoms itself to growth upon artificial media
and develops more luxuriantly after many transfers. When
grown in broth a delicate scum forms. It will be noted that
this pellicle formation is essential in the manufacture of
diphtheria toxin already discussed.

Physiological Characters. — The organism is aerobic and
facultative anaerobic. The fact that it does not produce

386 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

spores makes it relatively nonresistant to drying at a high
temperature. Gelatin is not liquefied. It is readily de-
stroyed at the temperature of pasteurization.

Pathogenesis. — The disease diphtheria is one of the best
examples of a true toxemia, that is, a disease in which a
microorganism produces a powerful poison without itself
invading the tissues to any great degree. It localizes usually
in the throat, less commonly in the nose and still less fre-
quently on other mucous surfaces and in wounds. Diphtheria
as it attacks the throat produces a poison which causes the
death of the epithelial cells of the mucous membrane. These
are thrown off, leaving a raw surface, plasma is exuded,
coagulation of the fibrinogen takes place and there is
gradually built up a relatively thick membrane. In most
cases the severe reaction in diphtheria is due to the absorp-
tion of the toxin. In some cases asphyxiation due to devel-
opment of the false membrane may occur. In this membrane
conditions are favorable for the growth of the microorgan-
ism and unless the body produces antitoxin rapidly or anti-
toxin is introduced artificially, the case will probably prove
fatal.

A discussion has already been given of the method of
manufacture of the diphtheria toxin and the antitoxin
under the general heading of " Immunity. " It is well
known that persons that have had diphtheria once do not
frequently contract the disease a . second time ; in other
words, such individuals have an acquired immunity. An
immunity conferred by the injection of the diphtheria anti-
toxin does not last for more than a few months at the most.

The disease diphtheria is usually diagnosed by means of
cultures taken from the throat of the patient. Usually the
medium employed is Loffler's blood serum. Mounts made
from such slants after incubation for 8 to 18 hours,
stained with methylene blue show the characteristic meta-
chromatic granules. Occasionally it is possible to grow

DIPHTHERIA AND GLANDERS GROUP 387

the organism in practically pure cultures in smears taken
directly from the throat.

Transmission. — Diphtheria is most commonly trans-
mitted by the use of common drinking vessels or by passing
objects infected in the mouth of one individual to that of
another. It is possible that occasionally the disease may be
transmitted by the inhalation of infectious droplets.

Disease carriers (or bacillus carriers so-called) are par-
ticularly important in this disease. In any outbreak or
epidemic of diphtheria, a considerable proportion of chil-
dren and other people who come in contact with diseased
individuals may show no symptoms of the disease and yet
harbor the organism in the nose or throat. Such individuals
can transmit the disease to those who are susceptible quite
as readily as those showing serious lesions. Such a diph-
theria carrier in a community may readily constitute the
starting point of a serious epidemic.

GENUS PFEIFPERELLA

The organisms belonging to the genus Pfeifferella re-
semble those of the diphtheria group in that they are
slender rods sometimes showing branching, but they are
Gram-negative. The most important species of the group
is the Pfeifferella mallei, the cause of the disease glanders
and farcy in horses and other equines.

PFEIFFERELLA MALLEI

Synonyms. — Bacillus mallei, Bacterium mallei, Myco-
bactcrium mallei.

The disease glanders in the horse and other solipeds is
widely distributed over the surface of the earth. It is
important not only because of the losses occasioned in
domestic animals but also because of the danger of trans-
mission of the disease to man.

388 AGKICULTUKAL AND INDUSTEIAL BACTERIOLOGY

Morphology. — The glanders bacillus is a short usually
straight rod, sometimes somewhat curved, with rounded
ends. It occurs rarely in short chains, usually it is single.
In the stained mounts made from infected tissues the cells
occur frequently in pairs. Involution forms, enlarged and
clubbed cells, filamentous and branched forms are not
infrequently observed. In this respect it resembles the
organisms belonging to the genus Corynebacterium. The
rods usually vary from 0.5-ljuby 1.5-5 /u. They are non-
motile, do not produce spores or capsules. They stain well
with aniline dyes, are not acid fast and are Gram-negative.

Cultural Characters. — The organism is ordinarily found
in the nasal discharges in glanders and in the pus from
farcy ulcers in the horse. In order to secure the organism
in pure culture, it is customary to inject intraperitoneally
into a male guinea pig. Within two to four days, the
organism may be isolated in pure culture from lymph glands
or from the testes. The orchitis developing is quite charac-
teristic.

The organism grows readily upon the ordinary media,
particularly when they contain glycerin or blood serum.
Upon agar and glycerin agar the colonies are usually cir-
cular, glistening and whitish or yellowish. Growth upon
potato medium is among the most characteristic. Within
two days at blood heat a yellow, honeylike, transparent
growth which finally turns brownish or amber in color is
developed, while the potato itself becomes a greenish-brown
color.

Physiology. — The organism is aerobic and facultative
anaerobic, growing best at blood heat but fairly well at
lower temperatures. It is readily destroyed by heat.

Pathogenesis. — The organism is primarily an invader
of the lymph glands of the horse. The first lesions are
usually in the nose, producing the disease type termed
glanders. When the subcutaneous lymph glands are

DIPHTHERIA AND GLANDERS GROUP 389

affected, the disease is termed farcy. The disease may be
either acute or chronic. The former is more common in the
ass and mule, the latter in the horse. The chronic type of
disease may not show anything particularly characteristic
in the early stages, but the lymph nodes become enlarged.
The lesions are generally present on the nasal mucosa in the
lungs and in the lymph glands. When these occur in the
nose they break down, form pus and develop into chronic
ulcers which eventually heal with the formation of a star-
shaped scar which is quite characteristic. In farcy or
cutaneous glanders nodules form below the skin, the lymph
glands and ducts become swollen and appear like strings of
beads. When these nodules become enlarged they may
break through to the surface of the skin and ulcerate. In
man the organism commonly gains entrance through a
wound in the skin or by inhalation and is generally fatal.

Immunity. — Practicable methods of either active or pas-
sive immunization against glanders have not been devel-
oped, although many attempts have been made.

Diagnosis. — The disease glanders is particularly dan-
gerous not only because of the high mortality among horses
affected, but also because of its transmissibility to man. In
most states in the United States animals known to have
glanders must be killed and the body completely destroyed
by burning. Inasmuch as the disease is difficult to recog-
nize clinically in chronic cases, several methods of diagnosis
have been worked out for this disease. It is possible to use
the agglutination test. In general horse serum will not
agglutinate 'glanders bacilli in dilutions of one to four
hundred or more unless the animal is diseased. Usually
diseased animals will show agglutination in dilutions of
serum of one to two thousand. Fixation of complement has
also been used as a satisfactory method of diagnosis of the
disease. Most commonly used, however, is the so-called
mallein test. Mallein is prepared by growing the organism

390 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

in a flask containing glycerin bouillon, then killing by heat
and evaporating to about one tenth of the volume. Such
glycerin extract of the glanders bacillus (mallein) when
injected subcutaneously into animals suffering from glan-
ders will cause a rise in temperature usually beginning
between the fourth and eighth hour. An area usually the
size of a dinner plate about the site of the injection becomes
inflamed and swollen. As in tuberculosis the test is some-
times made by dropping purified mallein into the eye. In
animals which react the conjunctiva becomes inflamed, the
lids are swollen and a purulent discharge collects at the
inner corner and on the hair below.

Transmission. — The disease is transmitted readily from
one animal to another through infected foods, drinking
troughs and mangers, occasionally through wounds or skin
lesions. Veterinarians and horse men sometimes become
infected through the skin, rarely by inhalation.

CHAPTER XXXI

SPIEAL BACTEEIA— ASIATIC CHOLERA— THE GENUS
VIBRIO

THE organisms belonging to the genus Vibrio have been
isolated repeatedly from water, sewage and the alimentary
canal of man and animals. A few species are known which
are associated with disease. One, the Vibrio metchnikovii
produces a septicemia of fowls in Europe and the Vibrio
cholera, the disease Asiatic cholera in man. The latter dis-
ease is the only one which has invaded the United States
and it alone will be discussed.

VIBRIO CHOLERA

Synonyms. — Spirillum comma, Spirillum cholerw asiaticw,
Microspira comma.

This organism is the specific cause of the disease Asiatic
cholera in man. It was first discovered by Robert Koch in
1883 who found it in the rice-water stools of patients having
the disease. Apparently this disease is constantly present
in certain parts of Asia, particularly in China. From this
region it has spread in epidemics over Europe and United
States during the past century.

Morphology. — The cholera vibrio is a short, slightly
curved rod frequently known as the "comma bacillus." It
is motile by means of a single polar flagellum. Elongated
cells and involution forms are commonly produced in cul-
ture media. No spores and no capsules can be demon-
strated. The organism is Gram-negative and stains readily
with the usual aniline dyes. Isolation of the organism from
the tissues of an Asiatic cholera patient is usually accom-

391

392 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

plished by placing fecal material in a flask of broth kept
at blood heat. The organisms of various types multiply,
but, the cholera and other vibrios are most abundant near
the surface of the medium, that is, they are attracted to the
higher concentration of oxygen. A loop full of the surface
film examined microscopically will usually show vibrios if
they are present. The organism may then be isolated by use
of an alkaline egg medium which will inhibit the growth of
other types of bacteria. The organism is easily destroyed
by heat. It produces acid from dextrose but no gas. Indol
is formed. The Vibrio cholerce liquefies gelatin. Milk is not
coagulated.

Pathogenesis. — The cholera vibrio is not markedly
pathogenic for laboratory animals. The disease in man is
acquired in much the same manner as is typhoid fever. The
bacteria are excreted in the stools and the disease is trans-
mitted only by ingestion of such material in water, milk or
food.

The disease may be characterized as an extremely acute
type of diarrhea. The organisms multiply in the alimen-
tary tract, producing a poison which causes more or less
desquamation of the lining membrane of the intestines.
This appears in the watery stools as white particles, some-
what resembling grains of rice, whence the name rice-water
stools. The disease is usually fatal.

Vaccination against the disease by the use of cultures
killed by heat or considerably attenuated has been success-
fully practiced.

CHAPTER XXXII

PATHOGENIC FUNGI, MOLDS AND YEASTS— THE GENERA
BLASTOMYCES, ASPERGILLUS, SPOROTRICHUM, TR1-
CHOPHYTON, MICROSPORUM, ACHORION, OIDIUM.

A CONSIDERABLE number of molds and a few yeasts are
known which are capable of producing disease in man
or animals. For the most part these are not common but
are deserving of brief mention.

PATHOGENIC YEASTS

The Genus Blastomyces. — Blastomyces is a generic name
given to those yeasts capable of producing disease. Two
species have been described. One from Europe and the
United States, one from Europe and Japan, the latter,
Blastomyces farciminosus, causing a disease of the horse
closely resembling farcy, the former, Blastomyces derma-
titidis, causing disease in man. It is probable still another
disease of man, termed coccidioidal granuloma caused by a
Blastomyces has been recorded in the United States and
South America. These diseases are comparatively rare and
are interesting in this connection largely because they illus-
trate the possibility of yeastlike organisms producing dis-
ease.

PATHOGENIC MOLDS

Genus Aspergillus. — Many species of the genus Asper-
gillus have at one time or another been reported as as-
sociated with disease in animals. One species only has
been commonly isolated from such infections, the Asper-
gillus fumigatus, causing the so-called aspergillosis of birds
and pneumomycosis in man and many different species of

393

394 AGRICULTUBAL AND INDUSTRIAL BACTERIOLOGY

animals. Aspergillus fumigatus is a common mold growing
upon decaying vegetable matter such as corn. It forms
in culture media greenish or biuish-green, later brownish
masses. The conidiophores are abundant, short, club-shaped
and the sterigmata are terminal.

Most cases of aspergillosis have been reported from birds.
In these the lesions are generally located in the lungs, in
the air sacs and in the hollow bones communicating with
them. In man and in animals (particularly the horse)
infection of the lungs is most commonly observed. The
organism may spread through the blood stream or possibly
through the lymph channels to other organs of the body. In
the lungs the air sacs and bronchioles are filled by the
growth of the fungus.

It is probable that this organism grows on decaying
organic matter outside of the body. The feeding of moldy
grain or fodder may give ample opportunity for infection
by inhalation. This is undoubtedly especially true for
chicks. It has long been known that the feeding of moldy
grain to chicks frequently was followed by pneumomycosis.

Genus Sporotrichum. — Organisms belonging to the
genus Sporotrichum are molds producing hyphae which are
usually creeping and irregularly branched. Definite conidi-
ophores are not developed. The spores are produced on the
sides or ends of short branches singly or in clusters. They
are usually quite numerous, oval or spherical in shape and
transparent or very light-colored. One species has been
found to produce disease in both man and animals in the
United States. It is the Sporotrichum beurmanni. It is
described from multiple abscesses in man. In the horse it
is known to produce one of the types of epizootic lymphan-
gitis. The disease is characterized in the horse by the devel-
opment of nodules from the lymph glands lying under the
skin. These nodules are generally spherical. They enlarge
but do not open directly. Purulent material collects near

PATHOGENIC FUNGI, MOLDS AND YEASTS 395

the center of the nodule. The skin above becomes thinned
and softened. Serum is exuded from the surface and the
hair becomes loosensd and a crust holding the hair together
is formed. Finally the ulcers become crater-shaped and
contain a little creamy pus. The disease is not common but
has been recognized at several points in the United States,
particularly in Pennsylvania.

The Genus Trichophyton. — Many different species of
this genus of molds have been described. Most of them were
isolated from cases of disease known as herpes tonsurans
and ringworm. In every case they are molds which grow
into the hairs, compactly filling the interior with a mass
of parallel hyphae extending longitudinally. These may
be readily seen by examining hairs from diseased areas
under the microscope after immersing for a few moments in
a warm solution of caustic potash. The organisms may be
grown readily upon suitable cultural media. Ringworms
have been described from horse, cat, cattle, sheep, swine and
dog. In many of these cases the disease has been known to
be transmissible to man.

The Genus Micrpsporum. — Species of this genus are
closely related to members of the preceding genus of
Trichophyton and like themrthey invade the hair. In all
cases the organisms grow over the surface of the hair and
encase it for several millimeters from the skin with a deli-
cate white sheath. Four different species are known which
produce skin disease in man and some seven species have
been described from animals including the dog, the horse
and the cat.

The Genus Achorion. — The organisms belonging to this
genus likewise closely resemble the preceding genera
Trichophyton and Microsporum. In man the Achorion
schonleinii is the common cause of favus, a disease of the
scalp characterized by the destruction of the hair and the
formation of a crust or shieldlike layer over the surface. A

396 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

similar disease in mice and other rodents have been ob-
served in Europe, in America and in Australia. This
disease is transmissible to man as a skin disease. It has
been observed, for example, that those who have handled
sacks gnawed by mice affected with this disease contracted
a skin disease or f avus of the exposed parts of the body and
not of the scalp. A somewhat similar disease caused by
Achorion gallince is the favus in the domestic fowl. This
is a disease in which the fungi form a fungous collar
around the base of the feathers and may attack the head,
the comb, the wattles and ear lobes. It is transmissible to
other animals and to man.

CHAPTER XXXIII

THE SPIROCHETE GROUP— THE GENERA TREPONEMA AND
LKPTOSPIRA, THE DISEASES SYPHILIS AND YELLOW
FEVER.

THE organisms which belong to this group are very
slender spiral forms, usually motile by means of sinuous
movements of the body. In many respects they closely
resemble the protozoa and it is a disputed point as to
whether they belong to this latter group or to the bacteria.
The group as a whole has not been studied until recent years
because of the difficulties in observation. It is only by
means of the dark field illuminator and by the use of
special stains that in the last two decades many of the
species belonging to the group have been recognized and
studied.

Many diseases of man and animals are known to be
caused by organisms belonging to this group. Two will be
considered here. Syphilis caused by Treponema pallida
and yellow fever caused by Leptospira icteroides. In addi-
tion to the diseases just enumerated, there are the so-called
relapsing fevers and tick fevers of several types, caused by
organisms of this group in man. A similar disease in
chickens and geese is caused by a spirochete and is trans-
mitted from one bird into another by means of a fowl tick.
Similar tick-transmitted diseases have been described in

cattle, sheep and horses.

•

TREPONEMA PALLIDA

Synonyms. — Spirochccta pallida, Spirillum pallidum.
This organism, the specific cause of the disease syphilis
in man, was first described by Schaudinn and Hoffman in

397

398 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

1905. It escaped observation by earlier investigators of
the disease because of its slenderness and the difficulty
experienced in getting it stained.

The disease syphilis is widely spread among civilized
people. It has been known from ancient times.

Morphology. — Treponema pallida is a slender organism
less in diameter than 0.5/x and varying in length from 4 to
20/x. In this organism the spirals are regular and vary in
number from three to forty. The spirals are pointed at
the tip, very possibly continued into a minute flagellum
When observed in a suitable medium under the microscope,
the organism is found to be actively motile. It is probable
that in most cases the cells divide by transverse fission,
although longitudinal division has been claimed. It does
not stain well by the ordinary aniline dyes. It is best
observed by use of the Giemas 's stain or by impregnation of
the tissues with solution of a silver salt, followed by the use
of a developer as in photography.

Culture. — The organism was first secured in pure cul-
ture by Noguchi, who succeeded in growing it in medium
consisting of a mixture of agar and ascitic fluid into which
a bit of living tissue had been dropped. A stab culture of
the organism in this medium kept under anaerobic condi-
tions leads to the development of a hazy growth along the
line of the inoculation, particularly near the tissue at the
bottom of the tube.

Pathogenesis. — This organism may always be found in
the primary and secondary lesions of syphilis. It is pos-
sible to reproduce the disease in other animals by inocula-
tion of the proper tissues. In man the primary lesion
usually takes the form of a hard chancre, which appears in
about three weeks after infection. It usually appears on
or near the external genitalia. The organism then invades
neighboring lymphatics and causes enlargements of the
lymph nodes usually in about six weeks after the appear-

THE SPIROCHETE GROUP 399

ance of the primary lesions. The organism then apparently
invades the blood stream and causes the lesions of secondary
syphilis. These usually appear as localized eruptions, fall-
ing of the hair and symptoms of generalized infection, such
as fever. The length of time which the secondary symptoms
may last is variable and depends upon the individual
infected. The disease may persist in latent or chronic form
throughout life of an individual. It leads to a great variety
of disorders, such as paresis (softening of the brain), de-
generation and production of tumors in the liver and other
organs of the body, diseases of the bones and of the blood
vessels.

The disease is so important, so difficult to cure except by
the most careful and painstaking of medical treatment
extending over a period of years, that much .study has been
devoted to it. It is most frequently recognized in indi-
viduals not showing the active symptoms of the disease by
the use of the complement fixation reaction or Wassermann
test. Methods of satisfactorily immunizing against the
disease have not been developed.

Syphilis is one of the most dreaded and loathsome of
diseases. It is most frequently transmitted by coition,
rarely through infected drinking vessels, toilets and by
direct inoculation as in surgical work. The disease may
be transmitted from mother to child, possibly even to the
third generation.

LEPTOSPIRA ICTEROIDES

It is probable that this organism is the specific cause of
the disease yellow fever in man.

This disease is one of the most important of the tropical
plagues of man and has during the warm season frequently
invaded the temperate zone, particularly the Mississippi
valley in the United States.

The organism is a slender, irregular spiral found in the

400 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

blood and various body organs, particularly the spleen and
liver. It was first described by Noguchl in 1919.

Pathogenesis. — Yellow fever is a disease primarily of
man, although Noguchi has shown that the leptospira can
be injected into guinea pigs and will produce a disease
resembling yellow fever. It is of particular interest because
of its method of transmission. Yellow fever is not trans-
mitted by contact, that is, it is not a contagious disease.
In order to be transmitted, a mosquito belonging to the
genus Stegomyia must suck the blood of a person who is
suffering from the disease. The organism then passes
through a period of incubation and probably passes a part
of its life cycle in the body of the insect. Ten days later
if the mosquito bites a healthy individual, it will, after the
puncture of the skin, inject a sufficient number of the
organisms to produce the disease. It is evident that the
disease is one to be eradicated finally by proper screening,
and by oiling or draining the breeding grounds of the
mosquito, that is, by the eradication of the mosquito plague
itself.

CHAPTER XXXIV

BACTERIA WHICH CAUSE PLANT DISEASES— THE GENERA
ERWINIA, PSEUDOMONAS AND LACTOBACILLUS

IN recent years it has come to be recognized that a com-
siderable number of plant diseases are due to the activity of
pathogenic bacteria. Practically all of the forms producing
disease in plants belong to two genera, Erwinia and Pseudo-
monas.

The genus Pseudomonas is characterized by being made
up of rod-shaped, short organisms, usually motile by means
of polar flagella, rarely nonmotile. They are aerobic and
facultative anaerobic. Frequently they are capable of
liquefying gelatine ; they do not produce spores. The Gram
stain is variable, usually negative; usually there is com-
paratively slight fermentation of carbohydrates. Many of
the species produce a water soluble pigment, green, blue,
purple, brown, etc. Among the plant pathogens, a nondif-
fusible yellow pigment is frequently produced. Some
species are white.

The genus Erwinia is very closely related to the genus
Bacterium, that is, to the organisms of the colon typhoid
series of bacteria which have already been discussed. All
of them are plant pathogens. The growth upon culture
media is usually whitish and often slimy. Acid is usually
formed in certain carbohydrate media and occasionally gas.
The cells are rod-shaped, without endospores, Gram-neg-
ative and either motile by means of peritrichic flagella or
occasionally nonmotile.

ERWINIA AMYLOVORA

Synonyms. — Bacillus amylovorus.

This organism is the cause of a specific disease, pear

401

402 AGRICULTURAL AND INDUSTEIAL BACTERIOLOGY

blight, so destructive to the pear and affecting also others
of the pomaceous fruits such as the apple. The disease is
widely spread over the United States.

Morphology. — The organism is a short rod, motile by
means of flagella ; from 0.5 to 1/z in diameter and 1 to l.S/*
in length. It stains readily with the ordinary aniline dyes
and is Gram-negative.

Cultural Characters. — The growth upon artificial media
is quite uniformly good; upon agar it produces a grayish
white, buttery growth. Slight liquefaction in gelatin occurs
after several weeks. There is some production of acid from
dextrose but no gas is developed.

Pathogenesis. — It is believed that this organism is trans-
ferred from plant to plant by bees, by plant lice and by
other insects. The leaves turn brownish or black, the stem
turns black and the young twigs show a blackened bark.
The name "fire blight" is often given to this type of infec-
tion because the infected branches have a scorched appear-
ance. It may also invade the larger branches and trunk of
the tree, producing the so-called body blight.

The disease is combated by pruning out all diseased wood.
Care must be used to remove all infected twigs and
branches, the cutting should take place some distance below
the visible infection inasmuch as the bacteria ordinarily,
at least, early in the season, are a considerable distance in
advance of the blackened area. The wood cut away should
be burned. Instruments used in cutting out wood should
be carefully disinfected, otherwise they may aid in spread-
ing the disease from tree to tree.

ERWINIA SOLAN ACEABUM

Synonym. — Bacillus solanacearum.

This organism is the cause of a wilt attacking many
plants belonging to the family Solanacece, particularly the
tomato, egg plant, pptato and tobacco.

BACTERIA WHICH CAUSE PLANT DISEASES 4Q3

Morphology. — The organism is about 0.5X1-5M, motile
by means of peritrichous flagella; stains readily with
ordinary aniline dyes and is Gram-negative.

Cultural Characters. — In broth there is abundant dirty
white sediment, the reaction usually becomes alkaline. Milk
is rendered alkaline, casein is dissolved without precipita-
tion. On agar the growth is smooth, slightly viscous, first
whitish, then yellowish and finally brown. Gelatin shows
little or no liquefaction ; on potato the growth is white and
later brownish to black on a browned medium. Neither
acid nor gas are produced from the carbohydrates.

Pathogenesis. — The disease is characterized by sudden
wilting or shriveling of the foliage, the softer parts of the
plant also wilt rapidly. The fibrovascular bundles of the
stem become stained brownish and microscopically are
found to be filled with bacteria. The wilting is most evi-
dent in the young plants. Older woody plants may turn
yellow and die without wilting. The organism may invade
the parenchymatous tissues and cause rotting. The potato
tubers infected with the organism show brownish discolora-
tion in the ring of fibrovascular bundles.

EBWINIA TRACHEIPHILA

Synonym. — Bacillus tracheiphiliis.

This organism is the specific cause of a wilt disease of
cucurbits, particularly of cucumber, squash, cantaloupe and
pumpkin. In this disease the foliage shows a sudden wilt-
ing and shriveling because the fibrovascular bundles are
stopped by the large masses of white, sticky, rod-shaped
organisms.

Morphology. — The organism is a rod, motile by means
of peritrichous flagella, about 0.£/x in diameter and from
1.2-2.5/x in length. It stains readily and is Gram-negative.

It is probable that the disease is spread largely through
insects, particularly by the cucumber beetle. The organism

404 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

gaining entrance into a leaf causes first a localized wilted
area, the organisms invade the larger vessels of the fibrovas-
cular bundles and the entire leaf wilts. Upon invasion of
the main stem the organisms spread rapidly, the vessels are
occluded and the entire plant wilts. When the stem is cut
the sticky white bacterial mass oozes from the cut bundles,
and when touched frequently shows its glutinous character
by drawing out in tiny threads.

ERWINIA CAROTOVORA

Synonym. — Bacillus carotovorus.

This organism causes a soft rot of a considerable number
of root crops, particularly carrots and turnips.

Morphology. — The organism is about .8X2.0/1, motile
by means of peritrichous flagella. The other characters
are those common to the genus.

Culture and Physiology. — The organism grows readily
upon the usual culture media, generally abundantly to
luxuriantly. On agar the growth is smooth, glistening and
white. Gelatin is liquefied relatively rapidly. A scum
forms in broth. Milk is coagulated and slowly digested.
Small amounts of gas and acid are produced in dextrose,
lactose and saccharose broth and acid from glycerol.

Pathogenesis. — This organism usually gains access to
the tissues through a wound or injury. It produces an
enzyme pectinase capable of dissolving the middle lamella
of the cells. The cells then fall apart and are themselves
finally invaded and destroyed. The disease is, therefore, a
typical soft rot.

ERWINIA MELONIS

Synonym. — Bacillus melonis.

This is the cause of a soft rot of the fruit of musk-
melons.

BACTERIA WHICH CAUSE PLANT DISEASES 405

Morphology, Culture and Physiology. — It is much like
the preceding organisms and typical of the genus. Acid and
gas production are peculiar in that no gas is produced in
sugar broth, though growth occurs and acid develops, while
some gas is said to develop in asparagin broth and in milk,
the gas consisting of carbon dioxide.

Pathogenesis. — The organism apparently invades the
melon from the soil through cracks or injuries. Like the
preceding organism it is capable of dissolving the middle
lamella and of producing thereby a typical soft rot.

ERWINIA PHYTOPHTHORA

Synonym. — Bacillus phytophthorus.

This organism has been described from Europe and
America as the cause of the basal stem rot of the potato.
It also causes rotting of the tubers. It closely resembles
the other members of the genus in its morphology and
culture.

LACTOBACILLUS TEUTLIUS

Synonym. — Bacterium teutlium. i

This organism is described as the specific cause of a soft
rot of the sugar beet. It is apparently culturally and
morphologically most closely related to the lactic acid bac-
teria, particularly those members of the genus Lactobacillus
most important in the production of acid in the fermenta-
tion of ensilage.

Morphology. — The organism is a nonmotile, Gram-
positive rod, .8 X 1-5M, without capsules or spores and
staining readily with the ordinary aniline dyes.

Cultural and Physiological Characters. — On media not
containing sugar, growth is usually scant, slow, white and
not viscous. On agar it tends to grow into and penetrate
the medium. In the presence of cane sugar the develop-
ment is much more rapid and abundant, becoming watery

406 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

or viscid. Gelatin is not liquefied. No growth occurs on
potato. There is no gas, but acid, from dextrose and sac-
charose, and no visible growth in milk.

Pathogenesis. — The bacteria apparently gain access to
the root through injuries, particularly those produced by
nematodes. The portion of the beet below ground decays
and becomes filled with areas containing an acid, viscous or
slimy liquid.

PSEUDOMONAS CAMPESTRIS

Synonym. — Bacillus campestris.

The black rot of cabbage and related cruciferous plants
and its causal organisms was first described by Pammel in
Iowa, later it was found in many other portions of the
United States and of Europe.

Morphology. — The organism is a Gram-negative rod,
slender, 0.4-0.5 X 0.7-3.0/x. In young cultures, the cells are
motile by means of a single polar flagellum. No capsules
and no spores are produced.

Cultural and Physiological Characters. — Growth is
abundant upon most culture media. Cultures on solid
media are markedly yellow, older cultures are brownish.
Growth is moist, slimy and shining. Milk is made alkaline.
Gelatin is liquefied slowly. No gas is produced from carbo-
hydrates. Indol is developed.

Pathogenesis. — The bacteria apparently usually enter
the leaf through the marginal water pores. The fibro-
vascular bundles are invaded and turn dark. Gradually
most of the surrounding tissues are destroyed or disorgan-
ized. The disease has proved very destructive in many
localities.

PSEUDOMONAS TUMEFACIENS

Synonym. — Bacterium tumefaciens.

This organism is the specific cause of crown gall, soft gall
and hairy root in most of the species of orchard fruits,

BACTERIA WHICH CAUSE PLANT DISEASES 4Q7

grapes, raspberries, blackberries, the Paris daisy and many
other plants.

Morphology and Culture. — The organism ,is a Gram-
negative rod, motile by 1 to 3 polar flagella. Growth
differs from most of the other members of the genus Pseudo-
monas in that it is white instead of yellow. Milk is made
alkaline. Gelatine is not liquefied. No gas is produced
from carbohydrates.

Pathogenesis. — The organisms gain access to the plant
tissues through a wound. It is most common at the junc-
ture of graft and scion in grafted trees. If the meristematic
tissues giving rise to medullary rays or soft tissues are
invaded there is an enormous proliferation of cells, and a
soft gall is formed which usually ultimately disintegrates.
It the tissues invaded are those which give rise to the woody
tissues, the tumor developed is hard. Frequently the roots
are stimulated to abnormal development producing masses
which give rise to the name hairy root. Artificial inocula-
tion gives rise in suitable host plants to the development of
primary and secondary tumors. In many respects the
tumor in the plant simulates a cancer. The disease results
in severe loss to nursery men. Certain apples in particular
are very difficult to secure free from crown gall. It is prob-
able that infection occurs commonly at the time of grafting.
Trees grown from galled plants are frequently defective,
weakened or stunted in their development.

PSEUDOMONAS STEWARTI

Synonym. — Bacterium stewarti.

This organism is the cause of a wilt of sweet corn. It
has been found in several sections of the United States,
though first reported from Long Island.

The organism is a typical yellow pseudomonad in
morphology, culture and physiology. It invades the fibro-
vascular bundles, cutting off the supply of water, in con-

408 AGRICULTUEAL AND INDUSTRIAL BACTERIOLOGY

sequence of which there is prompt wilting of the leaves.
Yellow, slimy masses of bacteria ooze from the surface of
the cut bundles, which are made yellow. Apparently the
bacteria may enter the plant throug1! the roots or the water
pores and stomata.

PSEUDOMONAS JUGALANDIS

This organism causes a serious blight of English walnut
in parts of California. It is a typical yellow pseudomonad.
It produces discolored or black cankers on the nuts, injur-
ing them commercially. It also attacks the young shoots.

PSEUDOMONAS MEDICAGINIS

This organism is the cause of stem blight of alfalfa, a
disease of the first crop of this plant in certain parts of the
western United States. It is described as producing watery,
semitransparent yellowish or blue-green appearance on one
side of the stem. The bacterial slime oozes from the diseased
area. This dries, giving the stems a varnished appearance.
Occasionally the crown of the plant may be involved.
Apparently the bacteria gain entrance through cracks
caused by freezir>s> and thawing in the spring of the year.

The organism diners from most of the other pseudo-
monads pathogenic for plants in that it produces a fluores-
cent green pigment when grown on agar.

CHAPTER XXXV

PROTOZOA CAUSING DISEASE— THE GENERA BABESIA,
PLASMODIUM, ENTAMCEBA, COCCIDIUM AND TRYPANO-
SOMA.

THE protozoa are the one-celled organisms belonging to
the animal kingdom; they occupy the same general rela-
tionship to the higher forms of animals as the bacteria do
to the higher forms of plants. They live throughout their
lives as single celled individuals or in colonies of single
cells. That is to say, the cells do not unite at any time to
form tissues or organs and the various species never become
multicellular. It was previously noted that there are many
intergradations between the bacteria and the protozoa, such
as Treponetna.

The protozoa in general, though they are single-celled,
are much more complex in structure and in life histories
than are the bacteria. The cells of many forms have been
greatly modified and different portions of the cell differen-
tiated for particular purposes. These specialized parts are
termed the organella of the cell.

Classification of the Protozoa. — All the members of the
protozoa may be grouped into two great subdivisions. The
first of these includes those forms which can move about
either by means of pseudopodia or flagella, the second in-
cluding those motile by means of numerous cilia. Cilia
differ from flagella in that they are shorter, more numerous,
generally blunt, and move the organism about by striking
the water in unison, resembling oars in their action. The
first subdivision is termed Pla-smodroma, the second sub-
division Cilioplwra. The group Plasmodroma is divided
into three classes: 1. Mastigophora includes those forms

409

410 AGEICULTUEAL AND INDUSTEIAL BACTERIOLOGY

which are motile by means of flagella ; 2. Rhizopoda includes
those motile by means of pseudopodia ; 3. Sporozoa includes
forms that are variously motile and greatly reduced
through parasitism. At some time in their life apparently
they all produce numerous spores.

None of the group of Ciliophora, will be considered.

One genus belonging to the Mastigophora, Trypanosoma,
includes organisms which are the cause of a variety of dis-
eases in man and animals. One genus belonging to Rhizo-
poda, Entammba, is the cause of amebic dysentery in man,
and three genera belonging to the Sporozoa, Babesia, Plas-
modium and Eimeria, cause Texas fever in cattle, malaria
in man and coccidiosis in fowls respectively.

GENUS TRYPANOSOMA

A trypanosome is a protozoan possessing an elongated
cell usually more or less spindle-shaped, occasionally almost
as broad as long but tapering at the ends. Practically all
of the forms which are of importance from the standpoint
of disease production are longer than broad when they are
observed in the blood. The posterior end of the cells ter-
minate in a flagellum, the length depending upon the
species. Along one side of the cell extending from one
end to the other is a thin membrane. The flagellum extends
along the membrane and forms its outer edge. The flagel-
lum finally enters the cells near the anterior end and
terminates in a granule (the blepharoplast ) which may
stain readily when treated with aniline dyes.

The cells multiply by a splitting first of the blepharo-
plast, later followed by a division of the nucleus and a
longitudinal splitting of the cell. It is probable that some
at least, of the trypanosomes pass through a regular life
cycle. Some have been successfully cultivated in culture
media by mixing rabbit blood in melted agar and preparing
a slant in a test tube. The water of condensation is planted

PROTOZOA CAUSING DISEASE 411

with a small amount of blood containing the trypanosomes
to be cultivated. They multiply in this water of condensa-
tion in some cases quite abundantly.

Disease Produced. — The only disease occurring in the
United States caused by a trypanosome is the dourine of the
horse, sometimes termed horse syphilis. The disease is also
known from various parts of western and southern Europe
and northern Africa. It has been introduced at various
times with imported animals and cases have been known
from several of the states in the Mississippi Valley and in
Canada. Animals which are infected gradually become
emaciated and swellings appear upon the genitals and
whitish chalklike areas (plaques) develop in the skin and
mucosa of the external genitalia. The disease is usually
chronic in form and recovery does not frequently occur.
The disease is probably transmitted by the stallion. It is
claimed by some authors that the disease can also be trans-
mitted by means of the stable fly but this probably does not
occur frequently.

Disease produced by trypanosomes (so-called trypanoso-
miases) are of great importance in tropical countries and
in the southern hemisphere. There are large areas in
Africa, for example, where it is quite impossible to keep
horses because of the presence of the so-called nagana or
tsetse fly disease. T^e organism is found among certain of
the wild animals and is inoculated into the horse by the
bite of the tsetse, a fly somewhat resembling a horsefly.
In South America a disease known as mal de caderas or
rump evil of the horse is known. In this disease there is
progressive emaciation of the horse, the hind quarters
become weak, the horse when walking will scarcely raise
the hoofs off the ground. The method of transmission is not
certainly known. Another trypanosome causes the Gam-
bian horse sickness in Africa, another the so-called baleri
of horses, cattle, sheep and goats in the French Congo.

412 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

Human trypanosomiasia or sleeping sickness is caused by
the Trypanosoma gambiense. This disease has made
uninhabitable a considerable section ' of north, central and
eastern Africa. It is transmitted by means of one of the
tsetse flies. It is probable that the organism is present in
certain of the wild animals in this region. In the first stages
of the disease the organisms can be found in the blood.
Later pains in the back develop and the patient becomes
drowsy, sinks into a coma from which he cannot be readily
aroused and finally dies. The disease is chronic ; sometimes
several years elapse before it proves finally fatal.

A disease known as murrina in the horse in Panama,
Venezuela, and Central America has also been traced to a
Trypanosoma.

THE GENUS ENTAMCEBA

An ameba which is found as a normal inhabitant of the
intestinal tract in man is known as Entamceba coli. An
organism somewhat resembling it and causing so-called
amebic dysentery is termed Entamceba histolytica. The
genus Entamceba differs from Amoeba in the absence of con-
tractile vacuole and by the formation of multinucleated cysts.

The Entamceba histolytica, the cause of amebic dysentery
may be observed in the stools of those who are suffering
from the disease as a mass of protoplasm without definite
cell wall and possessing a definite nucleus. It moves about
by means of the protrusion of blunt pseudopodia. It is
either colorless or slightly tinged with green due to the
presence of hemoglobin from blood corpuscles which have
been engulfed. The inner portion of the protoplasm ( endo-
plasm) and the outer portion (ectoplasm) may be readily
differentiated in that the latter is hyaline, glasslike and
more refractive. Reproduction of the organism is accom-
plished in two ways. In the first method the nucleus frag-
ments into a number of nuclei which gradually collect under

PROTOZOA CAUSING DISEASE 413

the ectoplasm, develop into mature nuclei and, together
with some protoplasm, are finally pinched off as spores or
buds. The disease is apparently contracted by drinking
water or eating food which has been fouled by the excre-
tions of those who harbor the organisms. The disease itself
is a chronic dysentery, frequently associated with intestinal
ulceration and not infrequently with abscesses in the liver.
The disease blackhead in turkeys is due to an organism
named Amoeba meleagridis. This organism is abundant in
the ca>ca of the bird. The characteristic lesions are
thickening and ulceration of the walls of the caeca.
Apparently it is a secondary parasite and occurs in
birds in which the ca?cal walls have been injured by the
attack of the intestinal worm Heterakis. It kills young
birds rapidly and in some sections of the country has made
the raising of turkeys almost impossible.

THE GENUS BABESIA

The organisms belonging to this genus are parasites in
the blood corpuscles of various species of animals. They
cause the diseases equine biliary fever, a disease noted from
India and South Africa in horses, hemoglobinuria or red
water in sheep in Roumania, jaundice of the dog in various
parts of Asia, Europe, Africa and possibly in the United
States. The most important species is the Babesia bigemina
causing red water, Texas fever or tick fever in cattle.

Babesia bigemina. — This organism was discovered by
Theobald Smith in 1889 during his work with Texas fever
in cattle. The disease is known from the southern United
States, Australia, South America, Europe, India, Philip-
pines and Africa. When blood from animals affected with
the disease is examined microscopically, red blood cells are
in part found to be invaded by the protozoan. The cells of
the parasite are usually pear-shaped, one end being rounded
and the other somewhat pointed. Usually two cells are

414 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

found within a single blood corpuscle. The organisms are
from 0.5-2/i X 2~4M- The disease in cattle is characterized
by fever and by the presence of hemoglobin in the urine.
There is considerable destruction of the red blood cor-
puscles. In acute cases death often intervenes in from 5
to 8 days after the first symptoms. Most animals recover
but usually harbor in their bodies organisms for a long
period of time, though showing no symptoms of the disease.
The injection of blood from immune animals is widely
practiced as a method of vaccination in nonimmune cattle.
It results in a mild infection which immunizes against the
severe type when transmitted in the regular manner.

The disease is normally transmitted only by the bite of
infected cattle ticks. The mature female tick gorges on the
blood of an animal, then falls to the ground and after a
time the eggs are laid. These hatch after from 19 days to
5 or 6 months, depending upon conditions, whereupon the
young ticks (called seed ticks) crawl up the stems of grass
and shrubs. They attempt to attach themselves to passing
animals. If they do not succeed, they die of starvation.
Ticks that have come from a mother that has fed herself on
blood from an infected animal are themselves capable of
transmitting the disease to the animal whose blood they in
turn suck.

The disease is gradually being eradicated in the United
States. Within the last few years the line which marks
the northern border of the tick quarantine district has been
moved south until some of the southern states which for-
merly were in the quarantine district have been relieved
entirely. Within a few years the disease will probably have
been completely eradicated.

THE GENUS PLASMODIUM

The organisms belonging "to the genus Plasmodium are
responsible for the disease malaria in man. Three species,

PROTOZOA CAUSING DISEASE 415

at least, are known. They differ in certain characteristics of
morphology and life cycle but closely resemble each other
on the whole. These organisms are Plasmodium vivax, the
cause of tertian malaria in man, Plasmodium malarice, the
cause of quartan malaria, and Plasmodium falciparum, the
cause of malignant or tropical malaria.

The organism is present in the blood stream of individuals
infected with the disease. It attaches itself to and probably
penetrates the red blood corpuscles and develops in the
interior of the cell so the corpuscle is practically entirely
filled with the organism, causing it to become somewhat
swollen. The organism then segments to form a rosette of
bodies which round off to form small spores called
merozoites. These become free by the breaking to pieces
of the red blood cell and in turn attach themselvs to other
cells and begin the cycle again. This may be repeated over
and over. It is termed the asexual part or phase of the
life cycle. The organism may be taken in with blood from
a patient by a mosquito belonging to the genus Anopheles
(or to a related genus), whereupon it passes through
several distinct stages of development. In the body of the
mosquito two types of cells are formed from the spores
taken in with the blood, male cells and female cells, called
respectively microgametes and macrogametes. Fertilization
of the macrogamete by fusion with a microgamete occurs,
the resultant cell being termed a fertilized egg or ookinete.
This makes its way by boring into the gut wall of the
mosquito, where it becomes encysted and enlarges to form a
tumorlike swelling. The contents then break up to form a
considerable number of spherical bodies known as sporo-
blasts, which in turn subdivide and produce within them-
selves great numbers of delicate filaments called sporozoites.
These are eventually freed by breaking of the cyst and pass
out into the body cavity of the mosquito. Some of them
eventually make their way to the salivary or poison gland

416 AGRICULTURAL AND INDUSTRIAL BACTEEIOLOGY

of the mosquito, when they are inoculated into the person
bitten by the mosquito. It requires from 8 to 10 days from
the time the mosquito bites a person having the malarial
parasite in his blood until the mosquito can in turn trans-
mit the disease to another individual.

The disease is commonly known as ague or chills and
fever. It is characterized by chills followed by fever at
intervals of 48 hours. During the period between attacks
of chill and fever the temperature is normal. It is interest-
ing to note that the chill and fever occur at the time when
the merozoites are breaking out of the red cells and attack-
ing new red blood corpuscles.

The description given applies to Plasmodium vivax. In
quartan malaria the interval between paroxysms of chills
and fever is 72 instead of 48 hours. In the tropical type of
malaria, two forms are known, one in which the life cycle is
completed in 24 hours and fever consequently appears
daily, and a tertian type in which the fever occurs every
48 hours.

Inasmuch as the disease can be transmitted only by the
bite of infected mosquitoes, it can be eliminated only by
preventing the breeding of these insects or by preventing
their transmitting the germ from infected individuals to
those who do not have the disease. Since mosquitoes breed
in stagnant water the disease has been eradicated in many
localities by drainage, by the use of oil on the surface of
water and by the introduction of fish which feed upon the
larvae of the mosquito.

THE GENUS EIMERIA (COCCIDIUM)

The organisms belonging to this group that infect animals
or man are intestinal parasites. The adult cells are oval
or spherical and not motile. The life history is quite com-
plex and varies with the different species. The most im-
portant form in North America probably is the Eimeria

PROTOZOA CAUSING DISEASE 417

(Coccidium) avium, the cause of coccidiosis in domestic
fowls and in birds. The disease is widely distributed over
the United States and Europe, but has not been extensively
studied. The organism is taken into the body with food in
the form of a cyst which ruptures and allows the escape
of spindle-shaped protozoa. These burrow into the epi-
thelial cells of the intestinal walls or other membranes as in
the caeca. Upon entering a cell the spore rounds up into a
sphere and grows rapidly in size. The nucleus of this cell
breaks into a number of pieces and the protoplasm itself
divides into a considerable number of spindle-shaped cells.
These break away from the mother cell and in turn invade
new cells. This procedure may be repeated several times.
Eventually some of them develop as sexual reproductive
cells. Some of these are larger than the remainder, the
so-called macrogametes or eggs and the microgametocytes
which resemble macrogametes at first but divide into a
large number of very slender threadlike cells termed micro-
gametes. These are freed by the rupture of the cell and
the macrogametes are fertilized by the microgametes to
form an oocyte. This continues to enlarge and secretes a
firm wall (becomes encysted). When completely mature,
the contents of the cyst divide to form several spherical cells
or bodies. These elongate and become spindle-shaped, and
in each, two small, slender spores develop. When the cyst
is mature it is passed out with the feces of the bird. If
these cysts come in contact with food, they may be ingested
by a suitable host, whereupon the cycle begins again.

Organisms somewhat resembling the form described are
known to produce coccidiosis in the rabbit, in cattle, in
sheep, and in the dog and cat.

CHAPTER XXXVI

INFECTIOUS DISEASES WHOSE CAUSAL ORGANISMS ABE
NOT CERTAINLY KNOWN

A CONSIDERABLE number of diseases regarded as infectious
are known in which the causal organisms have not been
discovered or are not certainly known. It may be asked
why should a disease be termed infectious, that is, caused
by some organism, when the organism itself is not known?
How can one be sure that an organism is responsible for a
disease before such has been proved to be the cause? The
reasons may be summarized as follows: All the diseases
discussed are more or less contagious, that is, more or less,
readily transmitted from one individual to another. It is
evident that something must actually pass from one indi^
vidual to the next. This something is termed a virus. In
many of the cases the cause originally described as a virus
has been discovered, studied and proved to be a specific
microorganism. It is assumed that the virus in all cases is
a microorganism of some kind.

The reasons why the causal organisms have not been
isolated and described for all diseases listed below can
scarcely be determined in advance. Probably in some cases
the organism will not grow in any of the media which have
been tried; in other cases the microscopic examination of
the lesions of the disease show bodies, which may or may not
be microorganisms, but which have not been cultivated as
yet. In other cases the organism, at least at certain stages
in its life cycle, may be ultramicroscopic. It is possible that

418

DISEASES OF UNKNOWN ETIOLOGY 419

in other cases suitable methods of staining or demonstration
have not yet been developed.

Several of the diseases discussed here below are exanthe-
mata, that is, they are marked by an eruption of the skin.
Such, for example, are the poxes (smallpox, chicken pox
and the poxes of the various animals, scarlet fever and
typhus fever). The diseases which come under the cate-
gory of those whose causes are not certainly known are the
following: Smallpox, cowpox, sheep pox, chicken pox,
horsepox, measles of various kinds, scarlet fever, infantile
paralysis, foot and mouth disease, rabies, hog cholera,
influenza, swamp fever, typhus fever and trench fever.

SMALLPOX

The disease smallpox in man has been described in
medical literature for at least a thousand years. It is also
known as variola. It is among the most contagious of
human diseases and can be transmitted to some of the lower
animals. Particularly characteristic of the disease are
the skin eruptions which develop as vesicles and finally
contain pus. These, upon healing, develop scars of variable
extent. Smallpox has an incubation period of about 12
days.

The disease is of particular interest because it was the
first for which a practicable method of vaccination was
developed. It was shown by Edward Jenrier in 1798 that
vaccination by means of material taken from lesions of cow-
pox in cattle will result in an immunity in man to smallpox.
Naturally the question at once arises, are cowpox and small-
pox identical, that is, caused by the same organism. There
is good reason to believe that they are. Several investi-
gators have found it possible to develop the disease cowpox
in calves by inoculation with the virus of smallpox secured
from human cases. Apparently this is not readily accom-
plished, however, showing that there is some difference in

420 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

the microorganisms concerned. The lesions produced by
the two diseases are very similar and it has been found pos-
sible in monkeys to immunize against smallpox by inoculat-
ing with cowpox and against cowpox by immunizing with
smallpox.

Smallpox vaccine is usually prepared from heifer calves
from 2 to 4 months old. They are inspected carefully to
see that they are not diseased. The animal to be used for
the preparation of vaccine is carefully washed and cleaned.
The posterior portion of the abdomen and the inside of the
thighs are carefully shaved and washed. Shallow cuts are
then made both longitudinally and crosswise over a large
area. Vaccine from a suitable source is then rubbed intu
each one of these incisions. The area is then covered with
cotton to protect from dirt. The vaccine is usually ready
for collection in five or six days. The inoculated area is
then cleaned and the hardened crust removed. The soft
material remaining is scraped off and placed in a suitable
container. It is then mixed with glycerin (50 per cent),
and triturated in a mortar until it is homogeneous. One-
half per cent of phenol is also added. The vaccine is ready
after standing for some weeks. Any bacteria which have
gained access during preparation have usually been de-
stroyed and the virus is ready to be used as a vaccine after
having been tested for potency. It may be then used for
vaccination in man. At a result of the widespread use of
vaccination, the disease has lost most of its terrors for civil-
ized people. Usually the immunity resulting from vaccina-
tion lasts for some years.

Stained sections from the lesions characteristic of small-
pox show peculiar cell-like inclusions. These are termed the
vaccine bodies. Some have contended that these vaccine
bodies are artefacts, others that they are living organisms,
probably protozoan in nature. Some investigators have
even worked out the probable life cycle of the organism.

DISEASES OF UNKNOWN ETIOLOGY 421

CHICKEN Pox

The organism responsible for the disease chicken pox has
not been determined. It is probably related to the organism
which causes smallpox, although, of course, entirely distinct.
The eruptions of chicken pox are more superficial and do
not result in the pitting and scars so characteristic of small-
pox.

MEASLES

Three, possibly four, distinct types of measles are known.
The most common are the so-called little red measles and
the German measles. The causal organisms of these dis-
eases are not known. They are highly contagious. The
incubation period is usually 9 or 10 days. The symptoms
begin with headache, fever and sore throat or "cold in the
head," with a rash which develops about the fourth day.
Characteristic spots appear in the mucous membrane of the
mouth. Methods of immunization against the disease are
not known. The disease is passed on by more or less direct
contact.

SCARLET FEVER

This is an acute, highly contagious disease showing a
diffuse erythematous skin eruption. There is usually a
catarrhal, croupous or a gangrenous inflammation of the
throat, and fever. The causal organism is not known.
Streptococci are very common as secondary invaders to the
disease and many of the sequelae of the disease are due to
these organisms.

INFANTILE PARALYSIS

This disease, known also as acute anterior poliomyelitis
or the Heine-Medin disease, has been known for nearly a
hundred years in Europe and has caused several epidemics
in recent years in the United States. Flexner and Lewis in
1909, showed the causal organism to be a filterable virus,

422 AGEICULTUEAL AND INDUSTRIAL BACTEEIOLOGY

very probably ultramicroscopie. The disease is one in which
the spinal cord is primarily infected, leading to a total or
partial paralysis of the limbs. The disease has been trans-
mitted in typical form to monkeys. It is probable that the
disease is spread by ingestion of infected materials. Several
organisms have been reported as commonly associated with
the disease, but adequate proof as to their actual causal
relationship is as yet lacking.

FOOT AND MOUTH DISEASE

This is an acute contagious disease infecting cattle, sheep,
goats, swine, deer, occasionally horses, dogs and cats. It
may be transmitted to man. It has been known for over a
century from Europe and has caused several epizootics in
the United States. Certain investigators have claimed the
causal organism to be a protozoan which they have named
Aphthomonas infestwns. Stauffacher claims to have suc-
ceeded in cultivating the organisms and that the disease
may be transmitted by the use of pure cultures. The report
lacks adequate corroboration at the present time.

The disease is characterized by an acute fever, the appear-
ance of vesicular eruptions in the membranes of the mouth
and on the feet, particularly between the toes. The disease
is usually not fatal, but is so highly contagious and leads
to such losses in milk, that it is among the most dreaded of
the diseases of cattle. Cases have been reported in man
due to the drinking of milk coming from diseased animals.

In parts of Europe vaccination has been extensively prac-
ticed with the idea of producing the disease and ' l having it
over with/' Apparently the organisms gain entrance
through contact of healthy animals with saliva or other
excretions of infected animals. The disease has been dealt
with in the United States almost exclusively by the stamp-
ing-out process. There have been some instances of trans-
mission of the disease through the use of blood from hogs

DISEASES OF UNKNOWN ETIOLOGY 423

hyperimmunized against hog cholera, infected accidentally
with the foot and mouth disease, the serum being used for
the immunization of healthy swine. One of the outbreaks
in the United States was due to the introduction of the virus
with smallpox vaccine from calves infected with the disease.

RABIES OR HYDROPHOBIA

This disease is known as hydrophobia in man and rabies
or lyssa in animals. The causal organism of the disease is
questionable. It has been claimed by a group of investi-
gators headed by Negri that certain bodies which may be
demonstrated in the larger ganglion cells of the Ammon's
horns of the brain are the specific organisms capable of
causing the disease. These have been named the Negri
bodies. These bodies are so constantly present in the brains
of animals having the disease that they are regarded as
diagnostic. Whether they are specific protozoa or degenera-
tion products of brain cells has not been definitely proved.

Rabies is a disease primarily of dogs and closely related
carnivora, transmissible to a wide variety of animals and to
man through direct inoculation.

The virus enters the body through a wound, usually the
bite of a rabid animal. After an incubation period of
several weeks the symptoms of the disease are manifested.
Apparently this time is required for the causal organism to
pass from the site of initial infection to the central nervous
system.

The disease is of particular historic importance as it was
the first of the diseases of man in which vaccination proved
effective, with the exception of smallpox. The Pasteur
treatment is carried out by vaccination with an attenuated
virus. Tests made upon experimental rabbits have shown
that virus secured from different diseased animals, the so-
called " street virus'* is decidedly variable in its ability
to produce disease. By passage through a number of

424 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

rabbits consecutively, the virulence is increased until it will
kill rabbits regularly in from six to seven days. This is then
termed a " fixed " virus. The vaccine is prepared by remov-
ing under aseptic precautions, the spinal cord from a rab-
bit dead as a result of an injection of fixed virus. This is
carefully dried in a desiccator at a constant temperature of
23° c. for a period of two weeks. The vaccine is an emul-
sion of this cord in physiological salt solution. The dry-
ing has so attenuated the virus that it may be injected with
impunity into man or animals. Later other injections are
made with emulsions from spinal cord dried for a shorter
period of time. Inasmuch as the disease has a relatively
long incubation period, the time elapsing between the bite
of a rabid dog and the normal appearance of symptoms may
be used for immunization. This method, first used by Pas-
teur, has proved highly successful.

HOG CHOLERA

This disease of swine is one of the most important affect-
ing the live stock industry. There was much difficulty in
the earlier study of the disease due to confusion with other
swine diseases, and particularly because from 1885, when
Salmon and Smith described the organism Bacterium
chblerce suis as the cause of the disease, until 1904 the
scientific world labored under a misunderstanding of the
cause. In the latter year De Schweinitz and Dorset demon-
strated that the typical hog cholera in the United States
is due to a filterable virus. This discovery led to the devel-
opment of satisfactory methods for immunization against
the disease. The organism which causes hog cholera is
classed as a filterable virus, inasmuch as it will pass readily
through porcelain filters. The organism in the blood serum
of hogs will remain virulent for many weeks. It may be
destroyed by disinfectants, but is relatively resistant.

Hog cholera usually manifests itself as an acute disease,

DISEASES OF UNKNOWN ETIOLOGY 425

occasionally assuming a chronic form. Post mortem exam-
ination of acute cases usually show congestion and acute
swelling of the internal organs and hemorrhages on the
serous and mucous membranes and not infrequently a
serous transudate in the pericardium. In the chronic type
necrotic and ulcerated areas are commonly found in the
intestines, frequently associated with pneumonia in the
lungs. It has not proved possible to produce the disease in
animals other than swine. The organism Bacterium cholerce
suis is undoubtedly an important secondary invader in the
disease and possibly may in some cases have the ability to
produce a choleralike disease in the entire absence of the
virus.

The work of Dorset, MacBryde and Niles in the United
States showed that the blood serum from a hog that has
recovered from hog cholera will confer some degree of im-
munity when injected into susceptible animals. This fact
has been made use of in the development of a practicable
method of prevention of the disease. An animal is first
secured that has already been immunized or has recovered
spontaneously from hog cholera. This animal is hyper-
immunized by injection of blood from a so-called virus pig,
that is, one suffering from acute attack of hog cholera as a
result of the injection of virus. Usually about 5 cubic
centimeters of this defibrinated virulent blood is injected
for each pound of body weight of the animal to be im-
munized. Some days later this animal is bled either from
the carotid or by cutting off the tip of the tail. The blood
thus secured is defibrinated and preserved by the addition
of one-half per cent of phenol. The animal may again be
hyperimmunized and bled several times; eventually all of
the blood is removed from the body. The potency of the
serum thus secured is tested upon young pigs usually weigh-
ing from 50 to 60 pounds. A serum sufficiently potent
should in a dose of 15 cubic centimeters or less, protect

426 AGKICULTURAL AND INDUSTRIAL BACTERIOLOGY

such an animal when injected with 2 cubic centimeters of
virulent blood.

In practice the hog cholera antiserum is used either for
conferring a temporary passive immunity or combined with
the injection of virus for the conferring of an active im-
munity. In the latter method, the antiserum together with
the virulent blood are injected into the animal to be im-
munized. The immunity thus secured is relatively per-
manent, while immunity secured by the injection of anti-
hog cholera serum alone is relatively temporary, lasting
only from 4 to 6 weeks. As a result of the utilization of
this method the losses from hog cholera have been greatly
reduced.

INFLUENZA

The disease influenza is an acute infection of man which
has. swept in a great pandemic around the world in recent
years. The ordinary form shows an onset of severe head-
ache, accompanied by pains and aches in the back, by fever
and by general prostration. The fever continues in such
cases from 3 to 5 days and finally leaves the patient
exhausted.

Pfeiffer in 1892 described an organism which he believed
to be the cause of the disease influenza. This has been
generally assumed to be the cause until investigation in the
past few years failed to prove any causal relationship
between this organism and the disease. While much work
has been done upon the disease, the causal organism has
not yet been isolated with certainty.

The disease is important not only in itself but because it
is followed in a considerable proportion of cases by severe
and frequently fatal pneumonia.

SWAMP FEVER OR INFECTIOUS ANEMIA

This disease has been known for many years in Europe
and more recently has been studied in North America. It

DISEASES OF UNKNOWN ETIOLOGY 427

is a disease of the horse caused by a filterable virus found
in the blood, in the urine and the feces of infected animals.
Blood sera from animals showing symptoms of the dis-
ease inoculated into susceptible individuals produce the
disease after 5 to 9 days or more. The first symptom is
fever. The disease may be characterized as an acute or
chronic anemia. There is a great destruction of blood cells.
Together with this there is degeneration of kidneys and
liver and changes in the blood vessels. The spleen becomes
enlarged and frequently degenerates and the bone marrow
shows profound degeneration. The disease generally proves
fatal. Methods of transmission of the disease have not been
satisfactorily worked out.

TYPHUS FEVER

This is an acute infectious disease of man. It has been
recorded in the United States under the name, Brill's dis-
ease in mild form in the city of New York, and as tabardillo
in Mexico. The disease is transmitted from man to man
by means of the body louse, occasionally by the head louse.
The disease may also be transmitted to the monkey.

SECTION VI
SANITARY BACTERIOLOGY

CHAPTER XXXVII

BACTERIOLOGY OF WATER AND SEWAGE— SEWAGE
DISPOSAL

BACTERIA are normally present in most natural waters.
Occasionally springs or wells may be found from which
the water is practically sterile, but these are uncommon.
Unpolluted waters from various sources, therefore, may be
regarded as having each its characteristic flora.

Many distinct kinds of bacteria have been recorded from
natural waters. Frequently the pigment-producing forms
are relatively abundant, particularly the chromogenic
micrococci and sarcinae, yellow, orange and red.

The number of bacteria normally present per cubic cen-
timeter in water varies greatly with the source and the
conditions under which the water has been kept. It has
already been noted that occasionally waters may be found
which are practically sterile. At the other extreme we may
find waters such as those heavily contaminated with sewage
in which bacteria are present by the hundreds of thousands
.or even millions per cubic centimeter. Whenever there are
considerable quantities of organic matter present in water,
there' is naturally a rapid multiplication of bacteria, pro-
viding temperature and other conditions are satisfactory.

ECONOMIC IMPORTANCE OF IMPURE WATER

An impure water may be defined as one which contains
disease-producing bacteria or which contains an excessive
quantity of organic material susceptible to putrefaction
or decay.

A number of diseases of man and animals are trans-
mitted through drinking water. The most common of these

431

432 AGEICULTUEAL AND INDUSTEIAL BACTEKIOLOGY

in man are typhoid fever, paratyphoid fever, Asiatic
cholera and dysentery (both amebic and bacillary). In
every case the organisms causing these diseases are forms
that multiply in the intestines of those who are infected and
can contaminate water only when the body excretions, that
is, the feces and urine of those harboring the organisms,
gain access. It is evident, therefore, that to a very great
extent these diseases are transmitted only by water which
contains excreta from those having the disease.

Diseases of animals are not so frequently transmitted
through water. It is probable that occasionally animals,
drinking from brooks or streams which have, farther up in
their course, passed through farms where there are diseased
animals, may contract such diseases as hog cholera or an-
thrax. Certain diseases of the alimentary tract in some
respects closely resemble the paratyphoid fever of man and
may also be transmitted in the same fashion.

The contamination of natural waters with considerable
quantities of putrescible material, particularly sewage, is
important also in other ways. The water of the streams
containing a large percentage of sewage of factory wastes
may become so polluted as to stop the development of the
normal water fauna. It has been recognized, for example,
that most of the so-called game fish will not live or develop
in water containing any considerable admixture of sewage.
Some fish can live in larger proportions, such as the carp.
The presence of sewage also prevents the growth of insects
and Crustacea which constitute the food for these fish. It
may also prevent the growth of the fresh water mussels or
clams so important as a source of supply of material for
manufacturing.

Large quantities of sewage or other organic material, such
as factory wastes in the water of running streams, may also
create a nuisance as a result of putrefaction. The presence
of such organic matter in the water soon leads to the using

BACTERIOLOGY OF WATER AND SEWAGE 433

up of all of the dissolved oxygen, the organic matter then
under these conditions undergoes putrefaction and malodor-
ous gases may be thrown off.

METHODS OP DETERMINING PURITY OF WATER

The degree of desirable purity in water varies according
to circumstances. A water which is to be used for drinking
purposes must naturally have a much higher degree of
purity than the water of some stream in which it is neces-
sary only to prevent undesirable odors and putrefaction,
that is, the development of a nuisance. The methods for
determining whether or not water is suitable for drinking
purposes and the methods for determining whether or not
water is so heavily contaminated as to injure the water
fauna such as fish and mussels or create a nuisance are
quite different. They will be considered in order.

Methods of Determining the Potability of Water. —
When methods of determining numbers of bacteria by
means of plate counts were first introduced, it was assumed
that this would give ft reliable test as to the suitability of
water for drinking purposes. In course of time, however,
it developed that there is not always a direct relationship
between the numbers of bacteria present and the potability
of the water. The amount of food material required by
microorganisms is very small indeed in some cases. The
water from a well, for example, which contains not more
than 8 or 10 bacteria per cubic centimeter, when allowed
to stand in a warm place for twenty-four hours or more,
may have bacteria present by the thousands. These micro-
organisms may all be harmless and do not seriously injure
it for drinking purposes. Although the determination of
numbers is therefore not a reliable test, it is in many cases
very helpful. In general it is true that natural waters do
not contain large numbers of bacteria unless they contain
considerable amounts of organic material. Any natural

434 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

water containing large numbers of bacteria, therefore, is
suspicious and should not be used until investigation has
shown that the bacteria present are not derived from any
source which might prove harmful or injurious.

Many attempts have been made to formulate a standard
for potability of water, using the number of bacteria pres-
ent per cubic centimeter as an index. These have in general
proved impracticable. However, it may be said that water
containing less than a hundred bacteria per cubic centi-
meter is usually, although not always, potable, and when
bacteria are present in numbers greater than a thousand
per cubic centimeter it usually indicates a water which is
not suitable for drinking, certainly a water which should
be investigated carefully before it is used.

Inasmuch as the bacteria which transmit disease through
water are organisms present in the alimentary tract, it is
evident that if organisms can be identified which are char-
acteristic of sewage, that is, of human and animal feces,
their presence in water will prove the presence of sewage in
the 'water and the unsuitability of the water for drinking
purposes, providing, of course, it can be shown that these
same organisms do not exist in nature. A large amount
of work has been done in recent years on this problem.
Obviously, if water upon examination is shown to contain
a particular disease microorganism such as the typhoid, it
is dangerous. It is evident, however, that any water con-
stantly contaminated by sewage will sooner or later contain
typhoid bacilli or that it at least is open to pollution from
the excretions of typhoid patients. While not impossible, it
is very difficult to isolate typhoid and similar bacteria from
water in which they are present because they are generally
found in numbers much smaller than other intestinal forms.
At the present day attempts are therefore rarely made to
isolate such organisms from infected water supplies. Atten-

BACTEEIOLOGY OF WATER AND SEWAGE 435

tion is directed almost entirely to the recognition of organ-
isms which are characteristic of sewage.

The organism usually sought to determine the potability
of water is the Bacterium coli, and in some cases certain of
its close relatives. Bacterium coli is a normal and constant
inhabitant of the alimentary tract of man and of most, if
not all species of warm-blooded animals. It is present in
enormous numbers in the stools of man. While certain
closely related forms such as Bacterium aerogenes may be
found not infrequently in soil, Bacterium coli itself appar-
ently does not live outside the human or animal body for
any great period of time. Its presence in water accordingly
is an indication of comparatively recent contamination with
sewage. It has come, therefore, generally to be regarded
as the most satisfactory indication of fecal contamination
of water.

Many methods, both qualitative and quantitative, have
been devised for determining the presence of Bacterium coli
in water.

Presumptive Tests. — In bacteriological analysis of
water to determine potability, it is customary first to make
what is termed a presumptive test. This is a test which will
readily differentiate waters which are quite certainly good
from those which are suspicious. It is not customary, how-
ever, to condemn a water entirely upon the basis of the
presumptive test. It is based upon the fact that there are
not many species of bacteria which are capable of producing
gas in fermentation tubes from the sugar lactose, quite cer-
tainly not in the presence of certain inhibiting agents such
as ox bile. In making the test, varying amounts of water
are added to a series of fermentation tubes containing either
lactose bile or lactose broth. "Water which does not induce
gas production, even when used in quantities as great as
one hundred centimeters, is quite certainly free from

436 AGEICULTUEAL AND INDUSTRIAL BACTERIOLOGY

sewage bacteria. If gas is produced in any fermentation
tube it is highly probable that intestinal bacteria are pres-
ent, although this does not constitute proof. It is custom-
ary to inoculate certain differential media from test tubes
showing gas and by isolation of pure cultures from
separated colonies identify Bacterium coli.

If Bacterium coli is shown to be present in quantities as
small as ten cubic centimeters of water, it is evident that
there has been more or less recent pollution by sewage. It
is possible that in some cases confusion will arise due to the
presence of organisms such as Bacterium a'erogenes not
uncommon in soils which simulate in many respects the
characteristics of Bacterium coli. Condemnation of surface
waters, particularly when only small numbers of gas-pro-
ducing bacteria are present, should be made with caution.

It should be emphasized that water containing Bacterium
coli is not condemned for drinking purposes because this
organism is capable of causing disease when ingested, but
because its presence indicates the presence also of sewage
and opportunity for other microorganisms, such as the
typhoid bacillus, to gain access to the supply.

Chemical Studies of Water to Determine Pollution. —
Organic matter in sewage ordinarily will not become a
nuisance, that is, give off disagreeable odors, providing
there is sufficient aeration of the water in the stream. When
large quantities of organic matter are present, the dis-
solved oxygen is quickly used up and changes which take
place in the sewage, therefore, are necessarily anaerobic in
nature. Anaerobic composition or putrefaction is apt to
give rise to malodorous gases. The absence of oxygen, fur-
thermore, prevents the growth of most forms of animal life
in the streams. It is possible, therefore, by chemical analy-
sis and comparison of organic matter found by such analy-
sis with the amount of dissolved oxygen to determine some-
thing concerning the "putrescibility" of the water. In

BACTERIOLOGY OF WATER AND SEWAGE 437

many cases it is found necessary for cities not wholly to
purify their sewage so that no disease-producing bacteria
are present, but simply to purify it so that it will be no
longer putrescible. Gross pollution of water may also be
studied by chemical analysis for the determination of the
amount of dissolved organic material (so-called albuminoid
ammonia) the presence of nitrates and nitrites, and free
ammonia.

SANITARY QUALITIES OF VARIOUS WATERS

As noted above, the sanitary quality of any water
depends directly upon whether or not it is receiving sewage
or other wastes which would make it harmful either to
health of the user or to animal life contained.

Water in flowing streams is potable providing it does not
receive sewage or factory waste. Most streams in the more
thickly settled districts in America are polluted. There are
some exceptions to this. Some of our larger cities use water
from streams whose watersheds have been carefully pro-
tected and do not permit any sewage to gain access.

Lake water is in most cases somewhat less apt to be
polluted than water in streams. Wherever it receives
sewage, however, it is polluted.

Water from springs usually is of a high quality. Most
springs receive water which has filtered through a consider-
able depth of soil and in some cases through rock. Some
springs, however, particularly intermittent springs, are
simply the opening of the channel of an underground
stream which has direct communication with the surface of
the earth. In limestone regions, for example, so-called sinks
are found frequently. These are openings in the ground
through which surface water passes. These communicate
quite directly with these underground streams which, when
they appear again upon the surface on the hillside, may
contain all the contaminating materials present in the

438 AGEICULTUEAL AND INDUSTKIAL BACTEEIOLOGY

streams which entered the sink. Such springs are apt to
be muddy following rainy weather, showing the surface
communication. Springs of this character, of course, are
dangerous.

Water from cisterns carefully protected from dirt and
adequately filtered is usually satisfactory for domestic use.
There is very little opportunity in most cases for human
excretions to reach such water.

Wells constitute the source of water supply for most
rural communities and for farms. Studies which have been
made upon the water in farm wells in different parts of the
United States would seem to indicate that in general their
character is relatively poor. In one survey made in Iowa,
for example, about sixty per cent of samples secured from
well water supplies were found to contain Bacterium coli.
An examination of the surroundings of wells shown thus
to be polluted will usually reveal the manner in which the
contamination reaches the well. Many wells are located in
barnyards and are not satisfactorily protected from surface
water. It will be recalled that Bacterium coli is abundant
in animal excretions as well as in human dejecta. The
presence of such an organism in a farm water, therefore,
does not necessarily mean that human excreta are finding
their way into the supply, but that barnyard manure is
gaining access. There is no reason to suppose that water
containing diluted barnyard manure will produce disease
in man unless it also contains human excretions. Any one
who has had experience upon a farm will know, however,
that water receiving drainage from the barnyard will con-
tain not only organisms derived from animal sources but
from human sources as well. In other words, while a water
containing large numbers of Bacterium coli because of sur-
face drainage from the barnyard, may be used for years
without causing disease, nevertheless, inasmuch as it is
subject to contamination, it is a source of constant danger.

BACTERIOLOGY OF WATER AND SEWAGE 439

Wells sunk to a sufficient depth and properly protected
from surface wash practically always will yield pure water,
provided there are no cracks or crevices in rock through
which surface drainage may reach the water in the well.
The latter do not frequently occur except in limestone
regions. Wells in sand, particularly if clay has been pene-
trated in digging, usually will contain water of high sani-
tary quality. Care must be exercised, however, to see that
privy vaults and cesspools are some distance removed. It
is true that passing polluted water through sand or even
gravel will rapidly remove the bacteria present by filtra-
tion ; nevertheless constant passage of heavily contaminated
or polluted water may in some cases cause microorganisms to
cross considerable distance and eventually lead to pollution
of wells.

METHODS OF MAKING IMPURE WATER POTABLE

It is evident that the most satisfactory method of making
and keeping water suitable for drinking purposes is entirely
to prevent initial contamination. As has already been
noted this sometimes is done for streams by protecting their
watersheds, being sure that no sewage of any kind enters
the stream. The same is true of lakes and other natural
sources of supply. In some cases, however, it is necessary
for cities and communities to make use of a supply of water,
such as a stream or river, which receives sewage at some
point farther up on its course. The water, therefore, is
more or less contaminated and must be purified before it
can be used. Purification is generally brought about in one
of four ways: first, by the self-purification which is con-
stantly going on in streams and lakes; second, by coagula-
tion and sedimentation; third, by a chemical treatment;
and fourth, by filtration.

Self Purification of Water. — It is a matter of common
observation that streams, although they may be heavily

440 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

polluted at some portion of their course, will eventually
apparently regain their purity at some distance down
stream. The factors which have to do with such purification
have been carefully studied in connection with several of
the rivers and streams in this country. The most important
of the factors which have to do with self-purification are
as follows:

First, the oxidation and removal of organic material
through the activity of the decay and putrefactive micro-
organisms. This occurs relatively rapidly, particularly if
the water in the streams is sufficiently agitated to receive an
abundant supply of oxygen.

Second, antibiosis. Microorganisms which develop most
abundantly in the water of streams and sewage are more or
less unfavorable for the life, .certainly for the development,
of the pathogenic bacteria such as the Bacterium typhi. As
a result the latter organisms die off more or less rapidly.
This is an example of what has been termed antibiosis, the
antagonistic influence of one form of life upon another.

Third, sedimentation. Microorganisms are constantly fall-
ing to the bottom of the stream and remaining there,
usually dying off rather rapidly. This sedimentation occurs
particularly rapidly when there is more or less sediment in
the stream which settles out. Bacteria show a decided ten-
dency to cling to the surface of solid particles in suspension.
As these settle out, the bacteria would of .course pass down
with them.

Fourth, the antagonistic action of protozoa. It is an
easily demonstrated fact that many of the protozoa live
upon bacteria. A sample of sewage, for example, allowed
to stand for a few hours usually will be found to swarm
with protozoa feeding upon the bacteria. These protozoa
are quite abundant in the water of polluted streams and
assist materially in its purification.

Fifth, filtration. The water in any stream constantly

BACTERIOLOGY OF WATEB AND SEWAGE 441

encounters obstacles larger or smaller which produce a cer-
tain amount of filtering action. The water in a stream
which is passing slowly through a wide bed of water plants
or algae is quite effectually filtered. Microorganisms tend
to cling to the surface of the plant stems, sand, etc., with
which they come in contact.

Sixth, food and temperature conditions are in general
unfavorable to the growth of pathogenic bacteria and these
die off relatively rapidly in consequence.

The distance which any water which has become con-
taminated must flow in order to become adequately puri-
fied differs with the conditions. Rapidity of flow, temper-
ature, amount of dilution, and amount of contamination
will all have their influence. In some cases evidence of
sewage contamination will disappear within a few miles;
under other conditions it will exist for some hundreds of
miles down stream.

Coagulation and Sedimentation. — When certain chem-
icals such as iron salts, alum, etc., are added to waters con-
taining sediment or organic material, the hydroxides which
are more or less gelatinous in nature are generally formed.
These cause coagulation or agglutination of the particles in
suspension and lead to relatively rapid sedimentation. Most
of the microorganisms are removed with the suspended
material in this process. Usually, however, mere coagula-
tion and sedimentation is not trusted to remove all harmful
bacteria from polluted water, but it is an essential pre-
liminary process in the purification of water of most
streams used as a source of supply by cities.

Chemical Treatment. — In some cases where it has not
been found practicable absolutely to eliminate harmful
bacteria by other processes, the water supply of the city is
treated with some chemical which will, on the one hand,
destroy such harmful microorganisms, and on the other not
injure the flavor of the water, prove poisonous, or injurious

4A2 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

in any way to consumers. Within recent years the most
common chemicals to be employed have been bleaching
powder, efficient because of its ability to release free chlor-
ine, and free chlorine gas itself. These usually disappear
relatively quickly by combining with various bases. As a
result there is a temporary but efficient disinfecting
action.

Filtration. — Many cities purify their water (usually
after coagulation and sedimentation) by a process of nitra-
tion. It has been found by experience that if water is
allowed to settle through a bed of sand, bacteria are quite
largely eliminated. It must not be supposed that the open-
ings between the sand grains are so small as mechanically
to prevent the passage of the bacteria. Experience has
shown, however, that when water is being filtered in this
fashion, the grains of sand soon become coated with a gelat-
inous covering of microorganisms which oxidize the organic
materials of the water with which they come in contact.
These gelatin-coated sand grains have large powers of
adsorption, and the microorganisms coming in contact with
them usually cling to their surfaces. It is evident that sand
filters become more efficient after a moderate amount of use.
Eventually, of course, the surface coatings may be so heavy
as to obstruct the water and it becomes necessary to clean
them.

Efforts are sometimes made in the home to purify water
by passing it through porcelain filters. When entirely free
from cracks and freshly cleaned, these are relatively
efficient, but like the sand grains, the pores in the filter are
in most cases large enough eventually to permit the passage
of microorganisms. They are, therefore, not usually advised
for permanent installations for the purification of water.

Emergency purification of water can be quickly and
easily effected by boiling. All of the disease-producing
bacteria are destroyed at this temperature.

BACTERIOLOGY OF WATER AND SEWAGE 443

SEWAGE DISPOSAL

In addition to the diseases already enumerated as trans-
mitted through water contaminated by sewage, there are
some other diseases transmitted quite directly from human
excretions. The most important of these in the United
States is the hookworm disease or uncinariasis. The hook-
worm lives during the latter part of its life history in the
intestines of man. The eggs are passed out with the feces,
hatched and gain access to the human body again only
through the skin, usually the skin of the bare foot. It is
evident, therefore, that proper disposal of the body excreta
will stamp out this disease completely. The disease is
primarily a rural disease in the south and has drawn atten-
tion to the necessity of better methods of disposing of the
body excreta than are frequently used.

The rural and city problems of disposal of sewage are
somewhat different. The presence of large numbers of
people in a community necessitates the disposal of the
sewage in order to prevent the creation of nuisances and to,
safeguard health. In practically all cases in America this
is effected by the use of water. The farm and rural problem
is complicated by the fact that expert advice as to disposal
of sewage is usually not available. Fundamentally the
reasons for proper sewage disposal are quite as potent in
rural districts as in the city. Human excretions must be
disposed of in such a manner that they will not come in
contact with human beings or find their way into water or
food.

The Rural Problem. — The necessity of disposing prop-
erly of human excretions of the farm can hardly be stressed
too strongly. They must be so disposed of that they will not
be a nuisance because of their odor ; they must be so placed
that they cannot come in contact, directly or indirectly with

444 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

human beings; they should as far as possible be protected
from flies and vermin which might carry disease organisms
present to the food of man; and particular care should be
used not to allow them to gain access to drinking supply.

Probably in most rural communities the privy vault is
most used for this purpose. Wherever there is any danger
of contamination of wells, it should be water tight and
arranged so that it is possible to clean at intervals. The
addition of lime or bleaching powder in some cases will
reduce odors. Proper covers should be provided so that flies
cannot gain access and breed in the excreta.

The modern farm is rapidly becoming equipped with the
same sanitary conveniences as the city home. In most cases
this means the use of water-flushed closets and water-borne
sewage. The installation of such necessitates the building
of some adequate sewage disposal plant. In some cases, it
is necessary simply to pass the sewage into a suitably located
concrete cistern or septic tank in which the solid materials
settle out and gradually decompose, finally for the most part
dissolving in the water. The overflow from this septic tank
may be treated in a variety of ways. In some cases
it is found practicable to pass it through drainage tile laid
in sandy soil, so that the liquid will rapidly seep away into
the ground. In other cases filter beds, usually covered and
constructed of sand, have been used, with tile laid below.
As the effluent from the septic tank spreads out over the
sand, it filters through, passes into the tiles below and is
carried away to a convenient stream or emptied into some
drainage tile. In some cases it is found expedient to install
an additional chamber beyond the septic tank which will
automatically flush at intervals or when filled to a certain
height. Sewage disposal plans show a great variety of
types. Directions for their construction and operation may
be found in various government and experiment station
bulletins. When properly constructed, they probably con-

BACTERIOLOGY OF WATER AND SEWAGE 445

stitute the ideal solution for the disposal of the sewage and
waste from the country home.

The City Problem. — Rural communities are in some
respects quite as much interested in the disposal of sewage
from the cities as are the cities themselves. There is a gen-
eral tendency wherever possible for cities to empty their
unpurified sewage directly into streams or lakes or into the
ocean. This pollution of natural waters, of course, interests
those who live upon farms, through whose pastures the
streams pass, or which border upon lake, bays, etc. which
may be heavily polluted. It should be noted that in Amer-
ica at least, city sewage is practically universally water
carried. In general it is diluted when it is emptied into
streams. In some cases it is impounded in large reservoirs
and there treated with certain chemicals, such as alum, or
with acids which precipitate the solid materials as sludge.
This latter is eventually dried and may be used for ferti-
lizer. The supernatant liquid is allowed to pass without
further treatment into streams. In some cases where sewage
has been disinfected by the addition of chlorine or by the
addition of acid, the microorganisms which are present are
largely destroyed. Where the city is not so large as to
make it impracticable, septic tanks and filter beds of various
types have been employed. In recent years a modification
of this has appeared in the use of the so-called activated
sludge method. It has been found by experience that if
sewage is drawn into a large tank and air bubbled through
it, it can be rapidly purified. The more insoluble materials
settle out in the form of a sludge and the clear super-
natant liquid is allowed to discharge into a stream. Con-
tact beds and sprinkling filters have also been used for pur-
poses of purification.

CHAPTER XXXVIII

BACTEEIOLOGY OF MILK

THE following table summarized from Van Slyke's anal-
yses will demonstrate that milk is an exceedingly complex
mixture of substances :

[Water 87.1 rAlbumen. .0.7

MILK . . . i f Fat 4.0

I Solids. . . 12.9 ^ Solids not f Proteins 3.3^

I fat 8.9-^ Milk-sugar. . 4.9 I Casein. . . .2.6

I Ash (salts) . . 0.7

100. 12.9 8.9 3'3

Some of the constituents probably are in true solution.
Such, for example, are some of the salts and the milk sugar.
Others, such as the proteins, are in colloidal solution, and
the fat in the form of microscopic globules forms an emul-
sion. It will be noted that about 87 per cent of the milk is
made up of water and 13 per cent solids, of which about
4 per cent should be fat and the remainder milk sugar,
protein and ash.

Fermentative and Other Changes in Milk. — Common
observation of the changes which occur in milk when it is
allowed to stand after milking, show that there are several
which follow each other in sequence. Ordinarily there are
differentiated the stages of slight souring, curdling and
eventual digestion of the curd. Careful laboratory studies
show that the number of stages is really greater than those
usually enumerated. They may be listed as follows :

1. Bactericidal stage.

2. Development of lactic acid.

3. Neutralization and fermentation of lactic acid.

4. Decomposition of the milk proteins.

In addition to the stages listed, milk frequently undergoes

446

BACTERIOLOGY OF MILK 447

other changes as well, such as sweet curdling, ropiness,
soapiness and discoloration by pigment. The stages
enumerated above will be discussed in order.

Germicidal Stage. — Usually milk contains some bac-
teria when drawn from the udder. Others rapidly find
their way into it from the milking utensils, from dust, dirt,
the milker, etc. Careful observation of the apparent num-
bers of bacteria present in this milk indicate that there is
a period of diminution. The numbers of bacteria show a
tendency to decrease rather than to increase. It has been
assumed, therefore, that freshly drawn milk has a certain
amount of germicidal power. Experimentally it may be
readily demonstrated that freshly drawn milk, when inoc-
ulated with considerable numbers of many different kinds
of bacteria, will show a decided diminution in the numbers
upon plating. This action continues for a longer period
when the milk is held at a low temperature and disappears
more quickly at a higher temperature. There has been a
considerable amount of argument in the scientific world as
to whether this so-called germicidal action really represents
the actual destruction of bacteria, or whether it simply
means the bacteria have clumped together or agglutinated
and upon plating the number of colonies is reduced,
although the actual number of bacteria may even have
increased. The evidence seems to indicate that there is some
actual decrease in the numbers of bacteria. Various kinds
of antibodies have been detected in milk, particularly bac-
teriolysins. Microscopic examination, furthermore, reveals
considerable numbers of white corpuscles or leucocytes.
These do not cease their activity for some time after the
milk is drawn and it is possible that they may continue
phagocytic action and destroy some microorganisms. This
germicidal action never leads to the complete sterilization
of the milk. It may be that the action is to a certain degree
specific, some species of bacteria being destroyed while

448 AGRICULTURAL AND INDUSTRIAL BACTERIOLOGY

others are not injured. This ability to destroy microorgan-
isms is lost when milk is heated above 80° c. It should be
emphasized that the germicidal action in milk, while of
some assistance in holding down the number of bacteria,
can never be depended upon to destroy the disease-pro-
ducing germs which have gained access.

The Development of Lactic Acid. — The organisms re-
sponsible for the formation of lactic acid have been dis-
cussed in the chapter on lactic acid fermentation. In milk
usually these bacteria develop rapidly, particularly if the
milk is not kept chilled. Milk which contains 0.4 per cent
of lactic acid has a decidedly sour taste and the milk curdles
when the acidity reaches about 0.8 per cent.
) Disappearance of the Acid. — Sour milk exposed to the
air soon comes to have a coating or scum composed very
largely of Oospora lactis and other molds. These oxidize
the lactic acid, that is, utilize it as food, forming carbon
dioxide and water. Part of the acid also combines chem-
ically with some of the proteins of the milk, particularly the
caseinogen.

Decomposition of Protein. — Some of the lactic acid
bacteria produce small quantities of proteolytic enzymes
and lead to some digestion of the curd. This usually, how-
ever, is not apparent to the eye. Upon the neutralization or
oxidation of the acid, putrefactive forms become active.
Certain molds growing upon the surface of the milk pro-
duce proteolytic enzymes in considerable quantities. The
proteins, particularly the caseinogen of the milk, are soon
digested and transformed into peptones, polypeptids,
amino acids, etc.

ABNORMAL MILK FERMENTATIONS

Usually when milk is allowed to stand, the lactic acid
bacteria are present in sufficient numbers, so that their
multiplication prevents the development of other forms of

BACTEEIOLOGY OF MILK 449

bacteria. In some cases, however, particularly when there
is unusually heavy inoculation of other forms, the latter
may develop and the milk will not show normal souring.
Milk, for example, which has been heated nearly to the
boiling temperature, will contain no lactic acid bacteria.
The spores of many of the putrefactive and decay-pro-
ducing forms, however, will remain. These will develop
when the milk is maintained at room temperature and lead
to various changes such as sweet curdling. Many of the
organisms belonging to the group of Bacilliis subtilis bring
about this change. The sweet curdling is due to the action
of a rennetlike enzyme produced by the bacteria. In most
cases the microorganisms proceed to decompose the curd,
transforming it into amino acids and ammonia.

Under the discussion of capsule formation by micro-
organisms, it has already been noted that there are many
species which are capable of producing sliminess or ropiness
in milk. Some of these species are aerobic and produce the
sliminess near the surface of the medium in contact with the
air. Other forms apparently are facultative and produce
sliminess throughout the medium.

Pigment-producing bacteria sometimes cause trouble in
milk. Species are known which form blue islands on the
surface of the milk, others change the milk itself to red,
yellow or even brown or black. Other species have been
described which produce undesirable flavors, in some cases
developing bitter milk and soapy milk.

BACTERIAL INFECTION OF MILK

Bacteria gain access to milk in a variety of ways. It is
of considerable practical significance, therefore, to know of
the sources of most importance. It is evident that some
sources of bacterial contamination are important because of
the numbers of bacteria which they will contribute, others
are important rather because of the kinds.

450 AGKICULTUBAL AND INDUSTRIAL BACTERIOLOGY

Udder Infection. — It has previously been stated that
the normal healthy tissues of animals usually do not con-
tain living bacteria. This is true of the tissues of the mam-
mary gland. However, there are apparently some bacteria
which find conditions favorable for development in the milk
cistern and in the larger ducts of the udder. The number
found here differs greatly with different individuals, some
animals normally having large numbers, others compar-
atively small numbers or even none. It is apparent, there-
fore, that the milk first drawn from the udder usually will
contain somewhat larger numbers of bacteria than that
drawn later. Very rarely do the numbers of bacteria
amount to more than 100 per cubic centimeter in the freshly
drawn milk. Animals revealing larger numbers usually are
suffering from some udder infection.

Bacteria from Body Surfaces. — When animals are
milked by hand into an open pail, it is inevitable that
particles of dust and dirt adhering to the skin and hair will
fall into the milk. Usually these are covered with large
numbers of bacteria. If the animal is filthy, the numbers
gaining access to the milk in this way may be considerable.
Under usual conditions, however, the proportion of bacteria
gaining access in this manner is not so large as from some
other sources. It should be noted that this source of bac-
teria is very largely avoided in the use of the milking
machine.

Bacteria from the Dust. — If the air in the stable or barn
in which the cows are being milked is filled with dust, some
will, of course, gain access to the milk and add to the num-
bers of bacteria present. Unless conditions are very bad,
however, the total number of bacteria gaining access in this
manner is not sufficient to be notable. This source of infec-
tion is also, in part at least, guarded against by properly
constructed milking machines. The organisms gaining

BACTERIOLOGY OF MILK 451

access from this source are usually chromogenic cocci or
spore-bearing bacilli.

Bacteria from the Hands of the Milker. — Undoubtedly
bacteria may gain access to the milk from the hands of the
milker. Bacteria from this source are particularly objec-
tionable inasmuch as they are of human origin, and if the
individual is suffering from any disease, there is a possibil-
ity of inoculating the milk with the organisms capable of
causing this disease and thus spreading it through the milk.
Many instances of infection in this manner are on record.
Typhoid fever has been spread as a result of milkers being
carriers of the disease. Diphtheria and scarlet fever have
also been transmitted through milk.

Bacteria from Milking Vessels. — Studies made by sev-
eral of the experiment stations in the United States have
shown that this is probably the most important source of
initial infection by bacteria. Unless all milk utensils are
thoroughly sterilized there is a tendency to add many bac-
teria to the milk. Milking machines in particular are dif-
ficult to keep clean. The tubes should be thoroughly steril-
ized if gross contamination by bacteria is to be avoided.
In the period elapsing between the time when the milk is
drawn from the animal until it is finally delivered to the
consumer, there may be numerous opportunities for con-
tamination by bacteria.

The number of bacteria actually present in the milk at
the time it is delivered to the consumer will vary according
to the relative importance of several factors. In the first
place, the number of bacteria gaining access to the milk
during the process of milking will be important. Milk that
is kept at a low temperature will allow but slow multiplication
of microorganisms. Milk kept at a higher temperature or
room temperature will show very rapid increase in bacteria
present. The temperature, then, at which milk is held, has
very important bearing upon the total number present

452 AGRICULTURAL AND INDUSTEIAL BACTERIOLOGY

when delivered. It is evident also that the number of bac-
teria present will be a function of the time which has
elapsed since the milk was drawn. It will also be affected
by the care with which it has been handled and whether
or not it has been pasteurized.

It should be recalled that milk is an excellent medium for
the growth of many microorganisms, and if given the oppor-
tunity they will multiply until great numbers are present.

Disease Bacteria in Milk. — From the standpoint of the
sanitarian, the fact that there are certain kinds of bacteria
present in the milk may be more important than the num-
bers. LaVge numbers of lactic acid bacteria, for example,
may be without particular significance from the standpoint
of health, but the presence of any disease-producing organ-
isms is apt to cause human infection. Evidently the most
important of the diseases transmitted by milk are various
diarrheas and dysenteries of young children. Whenever
there has been a marked improvement in the character of
the milk supply of our larger cities, there has been coinci-
dently a marked decrease in the death rate, particularly in
the morbidity rate among young children. Apparently the
alimentary tract of the child is more susceptible to an
infection of this kind than that of the adult. Particularly
in summer when bacteria are apt to multiply to large num-
bers in milk there is trouble with so-called "summer com-
plaint. ' ' The only safe procedure when milk does not come
from a source which is entirely above suspicion is to pas-
teurize it.

Among the diseases attacking adults and transmitted by
milk is typhoid fever. A number of epidemics of this dis-
ease has been traced to infected milk. It is usually not diffi-
cult to differentiate epidemics spread by milk from those
which have their origin in contaminated water by a study
of the distribution of the cases and by the fact that in most
such epidemics a considerable proportion of the victims are

BACTERIOLOGY OF MILK 453

children. In some instances it has been found possible by
marking the location of infected families on a map to show
the milk route of some individual. Inasmuch as typhoid
fever is not a disease of cattle it is apparent that the organ-
isms can gain access to milk only after milk has been drawn,
through careless handling, use of contaminated water, or
perhaps more commonly by contact with a typhoid carrier
or one who is just coming down with the disease or has
recently recovered from it. Scarlet fever and diphtheria
have apparently in some cases been transmitted through
milk. Inasmuch as these no more than typhoid are diseases
of animals, it is apparent that the organisms have gained
access only through careless handling.

The more important of the organisms which may be pres-
ent in market milk is the Bacillus tuberculosis which has
already been discussed, as has also the relationship of tuber-
culosis in animals to the disease in man. It seems to be a
well-demonstrated fact that the use of milk from tuber-
culous animals causes a considerable percentage of the cases
of tuberculosis, particularly of the scrofulous type, in chil-
dren. Milk used for human consumption should, if possible,
be secured from animals that have been proved to be free
from disease. If this is impossible then pasteurization
should be resorted to.

Other diseases which have been occasionally transmitted
by the milk of the cow are foot and mouth disease, milk sick-
ness, Malta fever and anthrax. These diseases are very rare
and occur only occasionally in animals in the United States.
They are an unimportant type of disease in man.

Numerous instances in recent years have been recorded in
the United States of cases of so-called septic sore throat,
caused by a species of the genus Streptococcus. It is prob-
able that in some cases the organism has entered the milk
from human carriers, but in other cases it is possible that it

454 AGRICULTUKAL AND INDUSTRIAL BACTERIOLOGY

has been transmitted from animals affected with inflamma-
tion of the udder (mastitis).

CLASSIFICATION OF MARKET MILK

Methods of classifying market milk differ with the local-
ity. In a number of cities milk has been divided into four
grades: certified milk, inspected milk, pasteurized milk,
and uninspected milk.

Certified Milk. — The term certified milk was introduced
originally to designate milk produced by a dairy which had
a regular system of inspection by some board of health or
committee of physicians. The requirements which must be
met when producing milk of this type are numerous and
must be rigidly adhered to. Among the more important
items are that the animals must be shown by continual
veterinary supervision to be free from contagious diseases.
The attendants, particularly the milkers, must be in good
health ; the stables must be sanitary, well lighted, free from
dust; milking vessels must be sterile and every reasonable
precaution used to prevent bacteria getting into the milk.
When milk has been drawn from the animal, it must be
quickly cooled, sealed in bottles and kept cool until it has
been delivered. Furthermore, in most cases a bacterial
standard is imposed and certification is withdrawn if milk is
found consistently to have more than ten thousand bacteria
to the cubic centimeter. In a few instances milk can be
certified when the numbers do not reach more than twenty-
five thousand. Additional requirements over those of the
other grades of milk make it necessary to charge a higher
price for certified than for other types of milk.

Inspected Milk. — Inspected milk is milk that comes
from a dairy where the cows have been inspected and cer-
tified to be free from tuberculosis. It must be drawn and
cared for under sanitary conditions, but the extreme pre-
cautions used in certified milk are not required.

BACTERIOLOGY OF MILK 455

Uninspected Milk. — Even yet in the United States most
milk which is sold to the consumer is uninspected and no
sanitary control whatever is maintained. In a few instances,
cities have established maximum standards for bacterial
count, and withdraw licenses from those who sell milk con-
taining more than the maximum number of organisms.
Such ordinances, however, are difficult to enforce.

Pasteurized Milk. — Pasteurization has already been
defined as heating milk for such a time and temperature
as will destroy any disease-producing bacteria present and
not seriously injure the flavor or creaming qualities of the
milk. In some cases, the term " pasteurized*' has been used
to indicate a milk of supposedly superior quality that has
not been actually heated. This, of course, is misbranding.
Pasteurization is a technical operation, requiring some
intelligence on the part of those carrying out the process.
In some instances the label "Pasteurized" has been used to
mean that milk has passed through an apparatus called a
"pasteurizer" whose efficiency is wholly unknown.

Pasteurization may be carried out in the home. It is best
then to heat the milk in stoppered bottles ip 60° c., hold at
this temperature for at least 20 minutes and cool rapidly.
In commercial pasteurization, two types of pasteurizers are
used. In the so-called flash process, the milk is heated to
the required temperature, usually 80° to 85° c., and main-
tained at that temperature for from 30 seconds to a minute.
It is then cooled and maintained at a low temperature until
distributed. In the holder process, the milk is kept at a
temperature required, 60° to 65° c., for about half an hour.
A combination of the two methods is particularly efficient.
Usually over 99 per cent of the bacteria present in milk can
be destroyed by proper pasteurization. All pathogenic
microorganisms should be destroyed.

INDEX

, 6

Abortin, 343, 344
Abscesses, 318
Acetic acid, as preservative,
Acetic fermentation, 209

types of, 209
Acetobacter, 27, 30, 31, 210,

aceti, 210, 211, 212

orleanense, 212

pasteurianum, 211

schutzenbachii, 212, 216
Acetyl methyl carbinol, 98
Achorion, 395

gallinae, 396

schonleinii, 395
Acid determination, 96
Acid fast group, 371
Acid fast stain, 111
Acidophiles, 140
Acne, 318
Actinobacillosis, 41
Actinobacillus, 41
Actinomyces, 41

bovis, 41, 368

cellulose digestion by, 221
Actinomycetales, 24, 29, 41
Actinomycosis, 368
Aerobic bacteria, 134, 98
Aerotaxis, 132
Agar-agar, 79
Agglutination, 290
Agglutinins, 281, 288
Ague, 416

Albococcus albus, 317
Alcohol determination, 97
Alcoholic fermentation, 183
Aldehyde detection, 97
Ale, 188

Alfalfa blight, 408
Algae, 13

140

211

457

Alternaria, 58

tenuis, 67
Amboceptor, 294
Amebic dysentery, 412
Amidase, 164
Ammonification, 153, 230, 239,

240

Amoeba meleagridis, 413
Amyl alcohol, 231
Amylase, 163, 186
Amylomyces process, 225
Anaerobes, 98, 134
Anaphylactic shock, 301
Anaphylaxis, 300
Anopheles, 415
Anthrax, 352
Antibodies, 277
Antigen, 281
Antiseptics, 137
Antitoxin, 281, 282

diphtheria, 284
Aphthomonas infestans, 422
Arsphenamine, 146
Arthritis, 315
Ascomycetes, 47
Ascospores, 46, 53
Ascus, 46

in molds, 54
Asiatic cholera, 391
Aspergillosis, 393
Aspergillus, 56, 58, 186, 219

albus, 63

calyptratus, 64

candidus, 63

clavatus, 63

flavus, 63

fumigatus, 62, 63, 393

giganteus, 63

glaucus, 62, 63

niger, 62, 64

458

INDEX

Aspergillus, nidulans, 64

ochraceus, 64

oryzse, 62, 63

perithecium, of, 54

pseudoclavatus, 64

versicolor, 63

wentii, 64
Associative action of bacteria,

198

Aurococcus aureus, 317
Autoclave, 74
Autolytic enzymes, 168
Azotobacter, 26, 27, 32

Beyerincki, 261

chroococcum, 261

Vinelandii, 261
Azotomonas, 32

Babesia bigemina, 413
Bacillaceae, 29, 33
Bacillus, 15, 27, 33, 352

abortivus, 343

abortus, 343

anthracis, 352

anthracis symptomatici, 360

amylovprus, 401

avisepticus, 347

bipolare multicidum, 348

botulinus, 364

bovicida, 348

bovisepticus, 348

campestris, 406

carotovorus, 404

chauvaei, 360

cholerse, 347

cholerae-gallinarum, 347

cholerse-suis, 336

coli communis, 333

diphtherise, 384

feseri, 360

Gartner, of, 337

lactis acidi, 193

leprae, 382

mallei, 387

melonis, 404

mycoides, 233

paratuberculosis, 382

Bacillus, paratyphosus A, 338

paratyphosus B, 338

pestis bubonicse, 350

phytophthorus, 405

radicicola, 33, 251

salmoni, 336

solanacearum, 402

suicida, 349

suipestifer, 336

suisepticus, 349

tetani, 357

tracheiphilus, 403

tuberculosis, 371

typhi, 340

typhi abdominalis, 340

typhosus, 340

vitulisepticus, 348
Bacillus carrier, 341
Bacteremia, 307
Bacteria, 14

cell groupings, 17

cell inclusions, 20

cell wall, 19

classification of, 22

conidia, 22

ectoplast, 20

morphology of, 15

multiplication, 16

protoplasm, 20

shape, 15

size, 15

structure of cell, 19

spores, 22

vacuoles, 20
Bacteriaceae, 30, 37
Bactericidal action, 137
Bacterin, 300
Bacteriochlorin, 148
Bacteriology, definition, 3
Bacteriolysins, 282, 292
Bacteriopurpurin, 148
Bacterioses, 274
Bacteriosis, 308
Bacte ium, 28, 38, 197, 331.

aerogenes, 163, 195, 197, 199,
206, 332, 333. 435, 436

abortus, 340, 343

INDEX

459

Bacterium, acidi-lactici, 332, 333

anthracis, 352

avicidum, 347

bovisepticum, 348

cholerae suis, 336

cloacae, 332, 333

coli, 39, 332,[333, 435, 436, 438

communior, 332, 333

coscoroba, 332, 333

diphtheria-, 384

dysenteriae, 340, 343

enteritidis, 335, 337

gallinarum, 340

lactis acidi, 193

lactis viscosum, 332

leprse, 382

mallei, 387

morgani, 335

neapolitanum, 332, 333

paratyphi, 335, 338

pestis, 350

producing lactic acid, 197

pullorum, 335, 339

schottmulleri, 335, 338

stewarti, 407

suicidum, 349

teutlium, 405

tuberculosis, 371

tumefaciens, 406

typhi, 340

Bacterium coli, as index of pol-
lution, 435
Bacteroids, 256
Baled, 411
Bang, 343
Barm, 190
Beer, 188

Beer wort, as medium, 78
Beggiatoa, 267
Billroth, 8
Black death, 350
Blackleg, 360
Black molds, 58
Blanching, 175
Blastomyces, 393

dermatitidis, 393

farciminosus, 393

Blight of English walnut, 408

Blind staggers, 366

Blood serum, as medium, 78

Boils, 318

Botulism, 364

Bovine farcy, 370

Brandy, 188

Bread making, 189

Bread molds, 58

Brill's disease, 427

Broth, 77

Bryophyta, 12, 13

Bubonic plague, 350

Buckley, 337

Buffers, 83

Bulgarian soured milk, 199

Butter starters, 201

Butyric fermentation, 217

Cadayerin, 231
Canning of foods, 174
Capsules, 19, 20
Carbohydrase, 161
Carbon cycle, 155, 263
Carboxydomonas, 30
Carbuncles, 318
Carrier, 341
Cellulose, 161, 162
Cell wall of bacteria, 19
Cheese, acid curd, 235

Camembert, 236, 237

Cheddar, 236

lactic acid in, 203

rennet curd, 235

ripening, 234

Roquefort, 236

sour curd, 201
Chemo taxis, 131
Chemotropism, 132
Chicken pox, 421
Chlamydobacteriales, 24
Chlamydospores, 47

of molds, 55, 56
Cholera, 391

Chromobacterium, 28, 37
Cider, 187
Ciliophora, 409

460

INDEX

Citric acid fermentation, 219
Citromyces, 219
Cladosporium, 58, 66

herbarum, 66
Clark and Lubs, 93
Clostridium, 22, 27, 34, 217

asterosporum, 224

botulinum, 352, 364

butyricum, 218, 224

chauvaei, 352, 360

Ghon-Sachs, 364

oedematis, 364

pasteurianum, 263

pectinovorum, 218, 224

putrificum, 218, 232

sporogenes, 364

tetani, 232, 352, 357

welchii, 352, 363
Coccacese, 29, 34
Coccidioidal granuloma, 393
Coccidium avium, 416
Coccus, 15
Cohn, 6
Cold pack process of canning,

176

Cold shock, 175
Colds, 314

Colloidal solutions, 289
Colon bacillus, 333
Colon subgroup, 332
Colony, 86
Columella, 55
Comma bacillus, 391
Complement, 294
Complement fixation, 295
Conidia of bacteria, 24
Conidiophores, 55, 56
Conidium, 55
Consumption, 376
Contagious diseases, 275
Corynebacterium, 28, 41

diphtheria, 41, 384

pseudotuberculosis, 384
Cowpox, 419
Cream ripening, 201
Cresol, 143
Crown gall, 406

Cultural methods, 71
Culture media, 74
Cycles of carbon, 263

iron, 268

nitrogen, 239

phosphorus, 266

sulphur, 266

the elements, 152
Cystitis, 327
Cytase, 161, 162
Cytolysin, 292

Decarboxylation, 230
Decay, 168

Denitrification, 248, 154
Deodorant, 137
De Schweinitz, 336, 424
Desensitization, 303
Desiccation, effect of, 125
Desulphofication, 156
Diastase, 161, 163
Differential media, 86
Diphtheria, 384
Diplococcus, 18, 27, 36

intracellularis, 328

gonorrhoeas, 325

lanceolatus, 321

meningitidis, 328

pneumoniae, 321
Discomyces bovis, 368
Disease, 273
Disinfectant, 137

acetic acid, 140

alkalies, 141

anilin dyes, 144

benzoic acid, 141, 143

characteristics of ideal, 137

cresol, 143

creosote, 144

copper salts, 142

ethyl alcohol, 142

essential oils, 145

formaldehyde, 142

hydrogen ions, 140

hypochlorites, 144

lactic acid, 140

lime, 141

INDEX

461

Disinfectant, mercury salts, 142

ozone, 144

phenol, 143

potassium permanganate, 145

salicylic acid, 143

salts of heavy metals, 141

silver salts, 142
Distilled liquors, 188
Dorset, 336, 424, 425
Douglas, 298
Dourine, 411
Drying, effect of, 125
Drying of foods, 179
Dunham's solution, 77

Eberth, 340
Eberth bacillus, 340
Ectoplast, bacteria, 20

yeast, 44

Ectotrophic fungi, 259
Ehrenberg, 5
Ehrlich, 277
Eimeria, 416

avium, 416

Electricity, effect of, 123
Endocarditis, 315
Endospores, 22
Endotrophic fungi, 259
Energy relationships, 147

sources of energy, 147
Ensilage, 204
Entamceba, 412

coli, 412

histolytica, 412
Enteritidis subgroup, 332
Enzyme, 158
Epididymitis, 327
Equine biliary fever, 413
Erepsin, 164
Erwinia, 287, 38

amylovora, 38, 401

carotovora, 404

melonis, 404

phytophthora, 405

solanacearum, 402

tracheiphila, 403
Krysipelas, 314

Erysipelothrix, 41
Erythrobacillus, 28, 37

prodigiosus, 37
Esterase, 101, 105
Eubacteriales, 24. 29
Exanthemata, 419

Facultative bacteria, 98, 135
Farcy, 387, 389
Farvus, 395
Fermentation, 167

acetic, 209

alcoholic, 183

butyric, 217

celluloses, 220

citric, 219

glycerin, 227

of fats, 226

of fatty acids, 226

of gums, 220

of hemicelluloses, 220

of inulin, 224

of starch, 224

origin of products, 168

oxalic, 219

pectin, 222
Fistula, 319
Flagella, 21

stain, 112
Flexner, 421
Focal infections, 313
Food deteriorations, 173

poisoning, 364

Foot and mouth disease, 422
Forage poisoning, 366
Fowl cholera, 347
Fumigant, 137
Fungi, 13
Fungicides, 142
Furunculosis, 319
Fusel oils, 191
Fusiformis, 41

Garget, 314
Gartner, 337
Gaseous edema, 363
Gas production, 99

462

INDEX

Gelatin, 79

Generation time, 115

Germ, 3

Germicide, 137

Germ theory of disease, 8

fermentation, 6
Giant cells, 375
Gingivitis, 314
Glanders, 387
Glycogen in yeasts, 44
Gonococcus, 325
Gonorrhoea, 326
Graham, 366
Gram's stain, 111
Granulose, 21

Growth, effect of hydrogen ion
concentration on, 133

rates, 115

temperature range, 122

Hairy root, 406
Hay fever, 322
Heat, effect of, 120

production, 128
Heine-Medin disease, 421
Hemoglobinuria, 413
Hemolysin, 292
Hemophilus, 28, 39

influenzse, 39
Hemorrhagic septicemia, 345,

346

Hepatization, 324
Herpes tonsurans, 395
Hide handler's disease, 352
Hill, 128
Histamin, 230
Hoffmann, 397
Hog cholera, 424
Hookworm disease, 443
Hormodendrum, 58, 66
Hydrogen ion concentration, 80,
92

effect on death rates .140

growth, 133
Hydrogenomonas, 30
Hydrolases, 160, 161
Hydrophobia, 423

Hydrotropism, 132
Hydroxyl ion concentration, 141
Hypersusceptibility, 300
Hypha, 50

Immunity, 276

acquired, 279

active, 279

factors determining, 280

natural, 279

passive, 279
Immunology, 9
Indicators, 82, 92
Indol determination, 102
Infantile paralysis, 421
Infection atria, 306
Infectious anemia, 426

diseases, 9, 273
Inflammation, 309
Influenza, 426

Intermediate subgroup, 332, 33
Intestinal group, 331
Inulase, 161, 163
Invertase. 161, 164
Involution forms, 15
Iron bacteria, 24
Isotonic solutions, 125

Jenner, 419 .
Johne's disease, 382

Kitasato, 357
Koch, 8, 39, 353, 372

Lactacidase, 166

Lactase, 164

Lactic acid as preservative, 140

bacteria in milk, 198

dextro, 193

in bread making, 207

in cheese, 203

in foods, 204, 207

in milk, 201

in yeast manufacture, 207

levo, 193

produced by bacteria, 193

acid fermentation, 192

INDEX

463

Lactobacillus, 28, 39, 193, 195

bulgaricus, 195, 196, 200, 206

caucasicus, 197, 204

lactis acidi, 195

producing lactic acid, 195

teutlius, 405
Leeuwenhoek, 5
Leprosy, 382

Leptospira icteroides, 397, 399
Leptotrichia, 41
Leuconostoc, 27, 35
Lewis, 421
Liebig, 7
Light, effect on bacteria, 118

production, 127
Limber neck, 366
Lime, disinfecting value of, 141
Lipase, 161, 165
Lockjaw, 357
Lumpy jaw, 368
Lupus, 376
Lymphangitis, 394

MacBryde, 425
Madura foot, 370
Malaria, 415
Mai de caderas, 411
Malignant carbuncle, 352

edema, 363
Mallein, 304, 389
Malt, 185, 186
Maltase, 164
Malting, 185
Market milk, 454
Mastigophora, 409
Mastoiditis, 314
Mastitis, 314
Measles, 421
Meat infusion, 77

poisoning, 364, 337
Medium, 75
Meningitis, 314
Meningococcus, 328
Merozoites, 415
Mesophilic bacteria, 121
Motatrophic bacteria, 148, 149
Methanomonas, 30

Metschnikoff, 196, 278, 297
Micro-a?rophiles, 99, 134
Microbe, 3
Micrococcus, 27, 36

albus, 317

aureus, 317

gonorrhceae, 325

meningitidis, 328
Microorganism, 3
Microscope, 5

development of, 5
Microscopic methods, 106
Microspira comma, 391
Microsporum, 395
Miliary tubercle, 375
Milk, as a medium, 78

bacteriology of, 446

infection, 449

lactic fermentation, 198

secondary changes in soured,
203

souring, 201

Minimum lethal dose, 285
Mixed culture, 85
M.L.D., 285
Mohler, 337
Mold bacteria, 24
Molds, 13, 14

classification of, 57

growth, 52

multiplication, 52

pathogenic, 393

reproduction, 53

structure, 50
Mordants, 109
Mother of vinegar, 210
Mucor, 54, 56, 58, 59

alternans, 61

ambiguus, 61

circinelloides, 61

erectus, 61

fragilis, 61

hiemalis, 60

mucedo, ;60

piriformis, 60

plumbeus, 60

racemosus, 60

464

INDEX

Mucor, rouxii, 60
Miiller, 5
Miilleriella, 56
Murrina, 412
Mycelium, 50
Mycobacterium, 27, 41

diphtherise, 384

leprse, 371, 382

mallei, 387

paratuberculosis, 371, 382

tuberculosis, 371
Mycorrhiza, 259
Myxobacteriales, 24

Nagana, 411
Navel ill, 314
Negri bodies, 423
Neisseria, 27, 35

gonorrhoea^ 325

meningitidis, 328
Neurin, 231
Nicolaier, 357
Niles, 425
Nitration, 154, 244
Nitrification, 153, 243
Nitrobacter, 31, 27, 246
Nitrobacteriaceae, 29, 30
Nitrogen assimilation, 154, 247

cycle, 152, 240

fixation, 104, 154, 250
Nitrosation, 154, 244
Nitrosococcus, 31
Nitroso-indol, 103
Nitrosomonas, 31, 27, 244
Noguchi, 398, 400
Noninfectious diseases, 9, 273
Nonsynthetic media, 76

Oidia, 55

Oil immersion objective, 106

Ookinete, 415

Oospora, 58, 61

lactis, 61, 237, 448
Ophthalmia neonatorum, 326
Opsonic index, 299
Opsonin, 282, 297
Orchitis, 327

Organized ferments, 157
Osmotic pressure, effect of, 124

effect on, 129
Otitis, 313

Oxalic fermentation, 219
Oxidases, 160, 166
Oxygen, effect on rate of growth,
134

Pammel, 406
Parachromatium, 32
Paratrophic bacteria, 148
Paratyphoid, 338
Paresis, 399
Pasteur, 6, 7, 347, 356
Pasteurella, 28, 39, 345

boviseptica, 345, 348

cholerse gallinarum, 345, 347

cuniculicida, 350

pestis, 346, 350

suiseptica, 346, 349
Pasteurellosis, 345
Pasteurization, 202, 178

of milk, 202, 455
Pear blight, 401
Pectinase, 161, 162, 223
Pediococcus, 18
Penicillium, 56, 58, 64, 219

brevicaule, 65

cellulose digestion by, 222

Camembertii, 64, 65, 237

digitatum, 65

expansum, 65

italicum, 65

Roquefortii, 64, 65, 236
Pepsin, 164
Peptid, 229
Peptones, 77, 229
Peptone water, 77
Perithecium, 54
Perry, 187
Pfeiffer, 426
Pfeifferella, 28, 42

mallei, 42, 387
Phagocytosis, 297
Phenol, 143

coefficient, 138

INDEX

•Hi",

Phlogistic infections, 308
Phosphorus, cycle of, 266
Photogenic bacteria, 127
Phototaxis, 118
Phototropism, 119
Physical effects upon microor-
ganisms, 115
Physiologic characters, 92

salt solution, 125
Plague, 350

Plants and animals, differentia-
tion, 11
Plasmodium, 414

falciparum, 415

malarise, 415

vivax, 415
Plasmodroma, 409
Plasmolysis, 124
Plasmoptysis, 124
Plating, 85
Plectridium, 22

tetani, 357
Pleomophism, 7
Pneumococcus, 321
Pneumomycosis, 393
Pneumonia, 314, 321
Pollender, 353
Poll-evil, 319
Polypeptid, 229
Precipitation, 291
Precipitins, 281, 288
Preservatives, 137

acetic acid, 140

benzoic acid, 140, 143

lactic acid, 140

salicylic acid, 143
Pressure, effect of, 123
Presumptive test, 435
Protease, 161, 164
Proteolysis, 153, 239, 240
Proteus, 28, 38, 331

vulgaris, 233
Protista, 11

Prototrophic bacteria, 148, 150
Protozoa, 14

classification of, 409

pathogenic, 409

Pseudomonadaceae, 30, 37
Pseudomonas, 27, 37

aeruginosa, 37

campestis, 406

fluorescens, 233, 243

juglandis, 408

medicaginis, 408

radicicola, 251

stewartii, 407

tumefaciens, 406
Psvchrophilic bacteria, 121
Pteridophyta, 12, 13
Ptomaine poisoning, 338
Ptomaines, 230
Pulque, 187
Pus, 310

Putrefaction, 168, 232
Putrescin, 231
Pyogenic bacteria, 309

membrane, 310
Pyorrhea, 314
Pyosalpingitis, 327

Quarter evil, 360

ill, 360
Quinsy, 313

Rabies, 423
Radium, effect of, 123
Reaction of medium, 80
Reductase, 161
Reductions, 101

nitrates to nitrites, 101

pigments, 102

sulphates to sulphides, 101
Red water, 413
Relapsing fever, 397
Rettger, 339
Retting, 222
Rhinitis, 314
Rhizobium, 28, 32

leguminosarum, 251
Rhizoids, 58
Rhizopoda, 410
Rhizopus, 58

japonicus, 59

nigricans, 59

oryzae, 59

466

INDEX

Rhodococcus, 27, 35
Ringworm, 395
Root nodules, 252
Rum, 188

Saccharification of starch, 184
Saccharomyces, 47, 183

cerevisiae, 48

ellipsoideus, 48, 187

pastorianus, 48
Salmon, 424
Salt rising bread, 190
Sanitary bacteriology, 9, 431
Sapremia, 308
Sarcina, 18, 27, 36
Sauerkraut, 206
Scarlet fever, 421
Schaudinn, 397
Schizomycetes, 13, 24

classification, 24
Schizophyta, 13
Schizosaccharomyces, 44, 47,

49

Scrofula, 376
Septicemia, 308
Septum, 50
Sewage disposal, 443
Sheaths, 19, 20
Silicic acid in media, 79
Sinusitis, 314
Sleeping sickness, 412
Slime bacteria, 24
Smallpox, 419

Smith, Theobald, 372, 375, 413
Soft gall, 406
Soft rot of, root crops, 404

muskmelons, 404

sugar beet, 405
Soil bacteria, 238

inoculation, 258
Spermatophyta, 12, 13
Spirillacea?, 30, 39
Spirillosis, 308
Spirillum, 15, 29, 40

cholera? asiatica?, 391

comma, 391

pallidum, 397

Spirochsetales, 24, 42
Spirocha?ta pallida, 397
Spirochsetosis, 308
Spirochetes, 24
Splenic fever; 352
Spontaneous generation, 6
Sporangiophore, 55
Sporangium of bacteria, 23

of molds, 54
Spores, asexual, 52, 54

of bacteria, 22

of molds, 45

of yeast, 45

sexual, 52, 53
Spore stain, 110
Sporoblasts, 415
Sporotrichum, 394

beurmanni, 394
Sporozoa, 410
Sporozoites, 415
Stained preparations, 109
Staphylococcus, 18, 27, 36

albus, 317

aureus, 317

pyogenes albus, 317

aureus, 317

Starch, digestion of, 103
Starter, 190, 201
Stauffacher, 422
Stegomyia, 400
Sterigmata, 61
Sterilization, 72

filtration, 74

heat, 72, 174

light, 74

foods, 174
Stolon, 59
Streaking, 86
Stream pollution, 437
Streptobacillus, 17
Streptococcus, 18, 27, 35

citrovorus, 202

erysipelatis, 311

lacticus, 193, 194, 195, 199,
201, 202, 204

paracitrovorus, 202

pneumonia?, 321

INDKX

407

Streptococcus, producing lactic
acid, 193

pyogenes, 193, 311
Streptothrix bovis, 368
Stricture, 327
Substrate, 75
Sucrase, 164
Sulphofication, 156, 267
Sulphur bacteria, 24

cycle, 155, 266

granules in bacteria, 21
Suppuration, 309
Susceptibility, 276
Swamp fever, 426
Swine plague, 349
Synthetic media, 76
Syphilis, 397

Tabardillo, 427

Taxy, 131, 118

Temperature relationships, 120

Tetanus, 357

Texas fever, 413

Thallophyta, 12, 13

Thermal death point, 122

Thermophilic bacteria, 121

Thiobacteriales, 24, 148

Thiothrix, 267

Tick fever, 413

Tonsillitis, 313

Torula, 47

Toxemia, 308

Toxin, 281, 282

Traumata, 273, 306

Treponema, 42

pallida, 42, 397
Trichobacteria, 15
Trichophyton, 395
Trichothecium, 58, 66

roseum, 66
Trimethylamin, 231
Tropism, 119, 132
True bacteria, 24
Trypanosoma, 410

gambiense, 412
Trypanosomiases, 411
Trypsin, 164

Tsetse fly diseases, 411

Tubercles, 375

Tuberculin, 304, 377

Tuberculosis, 371

Tyndall, 6

Typhoid-dysentery group, 332,

341

Typhoid fever, 340
Typhus fever, 427

Uncinariasis, 443
Unorganized ferments, 157
Urethritis, 327
Urospora, 56

Vaccine, 300

autogenic, 316
Vacuoles of bacteria, 20
Van Ermengen, 364
Variola, 419
Vibrio, 28, 40

desulphuricans, 268

cholera, 40, 292, 391

metschnikovii, 391
Vinegar, 213
Virulence, of legume bacteria,

258

Virus, 418

Viscosity changes, 129
Voges-Proskauer reaction, 98

Wassermann test, 297
Water analysis, 433

in disease transmission, 432

purification, 439

White diarrhea, 339
Widal test, 290
Wilt of, tobacco, 402

egg plant, 402

potato, 402

tomato, 402

sweet corn, 407
Wine, 187

Wooden tongue, 368
Woolsorter's disease, 353
Wright, 298

468 INDEX

X-Rays, effect of, 123

Yeast, 7, 13, 14, 43
bottom,. 48
cellulose, 43
classification, 47
commercial preparation of, 190
false, 47
pathogenic, 393
relationships, 47

Yeast, structure, 43

spores, 45

top, 48

true, 47

vegetative multiplication, 44
Yellow fever, 399
Yersin, 350

Zygospore, 53
Zymases, 161, 166, 183

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