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JUL
THE
TRANSMUTATION OF BACTERIA
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THE
TKANSMUTATION OF BACTEKIA
BY
8. GURNEY-DIXON, M.A., M.D. (CANTAB.)
M.R.C.S. (BNG.), L.R.C.P. (LOND.)
"Ogni primaio aspetto ivi era casso:
due e nessun V imagine perversa
parea,...
Cosi vid' io la settima zavorra
mutare e trasmutare; e qui mi scusi
la novita, se fior la penna abborra."
Dante: Inf. XXV.
CAMBRIDGE
AT THE UNIVERSITY PRESS
1919
Ulb.
PREFACE
r I THIS essay is based upon notes and observations which I
-"- collected previous to the year 1913. It was only partly
written when, in August 1914, I proceeded on active service.
I was able, however, to complete it in the following summer
while serving with a Field Ambulance in France, and in the
autumn of the same year (1915) I submitted it in the form of
a Dissertation for the degree of M.D. at the University of
Cambridge.
The difficulties in carrying out work of this character
while serving at the Front remote from libraries and amidst
" alarms and excursions " which break up one's scanty leisure
are sufficiently obvious and I trust may excuse some
of its defects. Some valuable materials which I had hoped to
utilise, including chapters on Viability and Agglutination
Reactions, were buried by a shell explosion and could not
be replaced.
The claims of Army work have also precluded any attempt
on my part to bring the work up to date by reference to
papers published since the beginning of the war. I particu-
larly regret having learnt too late to include any mentio'n
of it in the following pages of the valuable research carried
out by Dr Thiele and Dr Embleton on the part played by
the body ferments in the pathogenicity of bacteria.
Though I have endeavoured to suppress all irrelevant
matter, I am only too conscious of the discursiveness of this
essay. The topic is one of absorbing interest and at every step
one is tempted to digress. In the words of Dante, which I
have quoted on the title page, "The novelty must be my
excuse if my pen has wandered at all/'
vi PREFACE
I have only touched the fringe of the subject. An inex-
perienced sailor in my " piccioletta barca," I have been tossed
about in the breakers of this uncrossed sea. To others, better
equipped by knowledge arid training than I am to explore it,
I would say
" Metier potete ben per 1' alto sale
vostro navigio,...
Quei gloriosi che passaro a Colco
non s' ammiraron, come voi farete,
quando Jason vider fatto bifolco."
(Dante : Par. II.)
S. G.-D.
CONTENTS
PAGE
PREFACE v
SYNOPSIS ix
INTRODUCTION 1
CHAP.
* I. THE SCOPE OF THE ENQUIRY ... 3
II. CONDITIONS MODIFYING THE CHARACTERS
OF BACTERIA 13
III. A CONSIDERATION OF THE EVIDENCE . 28
IV. VARIATIONS IN MORPHOLOGY ... 37
V. VARIATIONS IN FERMENTING POWER . 50
VI. VARIATIONS IN VIRULENCE . . . . 71
VII. VARIATIONS IN PATHOGENIClTY . . . 94
VIII. THE POSSIBLE OCCURRENCE OF TRANS-
MUTATION IN THE LIVING BODY . . 107
IX. SUPPOSED INSTANCES OF TRANSMUTATION
BROUGHT ABOUT EXPERIMENTALLY . .116
X. SUMMARY 140
XL THE ENZYME THEORY OF DISEASE . . 153
XII. CONCLUSIONS 170
APPENDIX. REFERENCES . 171
ao
SYNOPSIS
INTRODUCTION
CHAPTER I
THE SCOPE OF THE ENQUIRY
DEFINITION OF TERMS. Transmutation not evolution evolution in bac-
teria its stages. Natural variation "Spontaneous" and "impressed." Varia-
tion easily studied in bacteria unicellular organisms method of generation
rapidity of generation environment easily modified. Natural selection.
Artificial selection. "Transmutation of Species" apparently contradictory
meaning of "species" based on characters. Arbitrary nature of distinction
between species illustrated by streptococci classified according to food-
stuffs and haemolytic power, adhesiveness, staining, cultural characters,
virulence and pathogenicity, agglutination, fermenting power. "Species"
not a rigid term.
A CONSIDERATION OF THE POSSIBILITIES. 1. Simple variation. 2. Varia-
tions in different directions associated. 3. Development of intermediate
forms. 4. Slight changes in closely allied organisms. 5. Complete change in
characters. (Pages 3 12)
CHAPTER II
CONDITIONS MODIFYING THE CHARACTERS OF BACTERIA
1. Spontaneous variations. Pleomorphism. Unexplained variations.
2. Geographical distribution. 3. Prolonged cultivation extends survey
permits natural selection influence of saprophytism. 4. Conditions of culti-
vation, (a) lowered vitality, (6) crowding of colonies, (c) temperature, (d) at-
mospheric pressure, (e) oxygen, (/) sunlight. 5. Ultra violet rays. 6. Elec-
trolysis. 7. Age of culture pleomorphism other variations. 8. Culture
medium (a) age of medium, (&) reaction of medium, (c) nature of medium
natural secretions pathological exudations water, (d) chemical sub-
stancescarbolic acid, antiseptics, boric acid, potassium bichromate, sodium
benzoate, glycerine, iodine trichloride, lactic acid. 9. Prolonged contact
with particular foodstuff. 10. Artificial selection method sometimes in-
effective. 11. Symbiosis lichens nitrifying organisms parasitism anaer-
obes. Symbiosis may confer new powers may have no effect. Methods of
studying symbiosis mixed growth, adjacent colonies, criss-cross planting,
surface and deep growths, double celluloid sac, successive growth. 12. Para-
sitism, (a) transmission through alimentary canal, (6) passage, (c) celioidin
sac in body cavity, (d) residence in living tissues, (e) during disease, (/) in
"carriers." (1327)
x SYNOPSIS
CHAPTER III
A CONSIDERATION OF THE EVIDENCE
1. Contamination. Growth from single bacterium. 2. Mixed infection
error due to (a) unequal growth of two strains, (6) incomplete recognition.
Proof of continuity necessary. 3. Secondary invasion. Bacteria in healthy
organs. Post mortem invasion. 4. Repetition of experiment. 5. Constancy
of new feature meaning of "permanent." 6. Perseverance necessary.
7. Faultless technique (e.g. agglutination) and accurate observation (e.g.
staining) required. 8. Methods may require to be improved, (a) irregular
results due to media e.g. sugars, may be impure, contaminated by glass,
affected by sterilisation, deteriorate age of medium composition re-
action, (b) age of culture, (c) time allowance. 9. Clinical observation im-
portant, e.g. Widal's test in jaundice effect of drugs pre-existing disease.
(2836)
CHAPTER IV
VARIATIONS IN MORPHOLOGY
A. ZOOGLEIC FORMS not fortuitous B. radicicolaBeggiatoa versatilis.
Zoogleae not in strict sense individuals analogy of regiment of soldiers and
crowd of pitmen typical formations assumed not separate individuals like
a tree formations not invariable may simulate each other this does not
imply transmutation. I. Zoogleic forms occurring spontaneously stages
in life history or variations. B. rubescens other examples. II. Zoogleic
forms artificially produced, 1. Due to chemical substances salt, sewage,
urea, saliva, bile, acid, caustic soda, /3 naphthol, alcohol, potassium bi-
chromate, boric acid, nitrates, antiseptics, tartaric acid. 2. Temperature.
3. Absence of oxygen. 4. Ultra violet rays. 5. Growth in animal body.
B. VARIATIONS IN INDIVIDUAL ORGANISMS. I. Pleomorphism B. ru-
bescens other examples. 1 1. Variations due to environment. 1 . Geographical
distribution. 2. Prolonged cultivation. 3. Crowding of colonies. 4. Changes
in medium reaction. 5. Chemical substances urea, urine, carbolic acid,
creosote, nitrogenous substances. 6. Ultra violet rays. 7. Electrolysis.
8. Symbiosis. 9. Growth in living tissues.
C. VARIATIONS IN COLONIES. 1. Colonies of the same organism vary.
2. Different organisms produce similar colonies. 3. Addition of various
substances to medium affects colonies. 4. Influence of heat. 5. Effect of
"passage."
Variation in other morphological characters. (37 49)
SYNOPSIS xi
CHAPTER V
VARIATIONS IN FERMENTING POWER
THE FERMENTATION OF CARBOHYDRATES its stages. Different types of
variation. I. Different strains may possess different fermenting properties.
II. The same strain may vary spontaneously. III. Fermenting properties
modified by conditions of growth. 1. Temperature. 2. Oxygen. 3. Atmo-
spheric pressure. 4. Age of culture. 5. Age of medium. 6. Composition
of medium effect of carbolic acid, sodium benzoate, monochloracetic acid.
7. Influence of source milk, urine, ascitic fluid. IV. Symbiosis. V. In
"carriers." VI. After "passage." VII. In disease. VIII. Prolonged con-
tact with a particular sugar. IX. Artificial selection method often in-
effective.
THE SIGNIFICANCE OF VARIATIONS IN SUGAR REACTIONS. 1. Fermentation
due to enzymes which are destroyed by antiseptics. 2. Distinct enzyme for
each different sugar. 3. Different enzyme for each different stage in fer-
mentation. 4. Distinct enzyme for forming each acid. 5. Distinct enzyme
for producing gas from each acid. 6. New fermenting power an adaptation
to environment. 7. Such adaptation advantageous to organism. 8. En-
couraged by natural selection. 9. Explanation of incubation period oc-
cupied by preparatory changes ? this disproved, interval before variation
appears? this disproved, time required for variants to predominate?
does not explain definite length of period. 10. Reason for shortening of
period by subculture subculture hastens reproduction. 11. Artificial
selection. 12. Reversion. 13. Variations apparently spontaneous possibly
due to contamination of medium or to impure sugar. No explanation of
spontaneous variation.
THE VALUE OF THE SUGAR REACTIONS unsatisfactory as tests. 1. Time
allowance not fixed. 2. Reactions vary with temperature and other con-
ditions. 3. Media often unreliable sugars impure altered by sterilisation
contaminated by glass vessel deteriorate on keeping acid reaction
masked. 4. Tests inconstant. 5. Positive or negative reaction a matter of
degree only. 6. Different carbohydrate groups yield different classification
if designed to correspond with other tests useful for identification only.
Comparison between fermentation and agglutination tests fermentation
tests may vary while agglutination constant fermentation and agglutination
properties may both be altered they yield a different classification two
tests not related but may supplement each other.
THE VALUE OF VARIATIONS IN THE SUGAR REACTIONS IN THE IDENTIFICA-
TION OF BACTERIA variations themselves constitute a test may be specific
(cf. morphology of B. diph.} may identify source of strain. (50 70)
xii SYNOPSIS
CHAPTER VI
VARIATIONS IN VIRULENCE
Bacteria pathogenic and non-pathogenic. Pathogenic character due to
two factors parasitism nature of activity in tissues. Most bacteria cannot
invade tissues activities of some invaders harmless actual invasion not
essential. Effects of bacterial invasion due to (a) their metabolism, (ft) their
disintegration, (c) their mechanical action, (d] response of living tissues.
Viability pathogenesis virulence.
VARIATIONS IN .VIRULENCE. 1. At different stages of epidemic possibly
explained by unequal resistance met with. 2. Sporadic cases of infectious
disease imply weakened virulence. 3. Endemic diseases become less viru-
lent possibly explained by acquired immunity. 4. Epidemics vary in
severity with date and locality. 5. Intensity of infection by same specific
organism varies. 6. Virulence altered by abnormal conditions of cultiva-
tion, (a) temperature possibly protective influence of fever disproved,
(b) presence of antiseptics carbolic acid, potassium bichromate, iodine tri-
chloride, saliva, (c) oxygen, (d] sunlight, (e) reaction of medium. 7. Virulence
altered by prolonged cultivation outside the body. Results due to several
factors, (a) chemical composition of media blood media pathological exu-
dations urine, (&) physical character of artificial media, (c) response of
tissues, (d} purity of culture. 8. Virulence increased by growth in patho-
logical secretions. 9. Symbiosis affects viability also affects virulence.
10. Virulence altered by "passage" passage alternating with culture more
effective. 1 1. Simultaneous inoculation with another organism intensifies
results even when symbiosis of same organism outside the body ineffective.
Simultaneous subcutaneous and sub-peritoneal inoculations with different
organisms also effective. Exalted virulence is towards species used for
passage not necessarily towards others.
THE SIGNIFICANCE OP VARIATION IN VIRULENCE. Evolution of bacteria
virulence is latest property acquired and first to be lost. Its re-acquire-
ment an example of the survival of the fittest "fittest" not necessarily most
robust, but most capable of defence. Virulence results from adaptation
and is not due to increased robustness, (a) increased virulence to one species
of animal does not apply to another, (b) most virulent not always most robust
contrary true of pneumococcus, (c) analogy suggests adaptation, e.g.
increased resistance to antiseptics, (d) increased virulence accompanied by
other changes obviously adaptive, e.g. growth at body temperature. Diffi-
culties in accepting natural selection as developing virulence, (a) Intra-
cellular toxins only set free after death of organism may nevertheless be
of advantage to strain their effect perhaps purely physiological and not
the result of adaptation, (b) Why are common infective diseases not of
deadly virulence ? death of host involves death of organism, (c) Virulence
established by single "passage" virulence possibly results from sudden
change in metabolism, (d) Toxic saprophyte assists non-toxic as well as
SYNOPSIS xiii
itself nevertheless may benefit strain. Invasion of tissues by virulent
saprophyte involves change in foodstu/s relationship between altered
metabolism and acquirement of toxicity possibly a change in excretion
following change in assimilation experimental evidence, (a) B. coli does not
attack proteid if carbohydrate present, (6) B. diph. does not yield toxin if
much carbohydrate present suggest toxins may result from alteration in
food material. Altered metabolism of saprophyte facilitates invasion of
tissues this supposed alteration in metabolism does not always confer
toxicity toxins may be regarded as an excretion or as a secretion or as
product of enzyme activity of enzyme may be due to adaptation, encouraged
by natural selection.
THE VALUE OF VIRULENCE IN CLASSIFICATION. Classification according to
virulence inconsistent. Non-virulent B. diph. in "carriers" regarded as
lineal descendant of virulent Klebs-Loeffler bacillus other non-virulent
B. diph. provoke antitoxin, therefore same species as Klebs-Loeffler bacillus.
Non-virulent and virulent pneumococcus regarded as varieties of same
species. Non-virulent and virulent B. coli communis thought by some to be
different species cf. amoeba coli. Non-virulent "B.anthracoides" described
as different species from virulent B. anthracfs. S. erysipelatis and S.
pyogenes formerly regarded as distinct species. Virulence not a specific
character. (7193)
CHAPTER VII
VARIATIONS IN PATHOGENICITY
Pathogenicity is power to produce in certain animals certain symptoms
and certain lesions quite distinct from virulence and other characters.
Generally regarded as more fixed than other characters constitutes final
appeal in doubtful cases, e.g. Hofmann's bacillus and Klebs-Loeffler bacillus
gonococcus and meningococcus gonococcus does not cause meningitis
nor meningococcus urethritis. Pathogenicity a variable character in all
three aspects.
I. VARIATION IN KIND OF ANIMAL AFFECTED.
II. VARIATION IN SYMPTOMS CAUSED.
(1) Same organism causes different symptoms in different cases.
Symptoms may depend upon organs affected cf. lead poisoning this
determined by route of infection and vitality of organs also by patho-
genicity of organism e.g. tubercle bacillus causes phthisis, osteitis, arthritis,
lupus unlike lead poisoning types remain distinct skin rarely infected by
tuberculous sputum contrast between gonococcus and meningococcus no
greater than between different strains of tubercle bacilli. Fallacy due to
pre-existing disease, e.g. nephritis in cerebrospinal fever.
(2) Pathogenicity can be artificially modified, e.g. that of B. anthracis
by ultra violet rays.
(3) During epidemic different cases exhibit different symptoms.
xiv SYNOPSIS
S. scarlatinae causes scarlet fever in some cases and puerperal fever in
others. M. catarrhalis produces symptoms of many diseases common cold,
influenza, scarlet fever, diphtheria, typhoid fever, cerebro-spinal fever.
(4) In different epidemics different types of disease presented
B. influenzas causes epidemics simulating coryza, rheumatic fever, typhoid
fever, cerebrospinal fever.
(5) Same train of symptoms follows infection by different organisms
typical rabies due to B. diph. typical scarlet fever, cerebrospinal fever
and influenza due to M. catarrhalis typical cerebrospinal fever due to
B. typhosus and to Klebs-Loeffler bacillus symptoms resembling diphtheria
due to pneumococcus typical typhoid fever due to B. coli.
III. VARIATION IN LESIONS PRODUCED studied in two ways lesions pro-
duced during disease and by artificial inoculation in animals. 1. Variations
in lesions produced during disease. In many cases characteristic not
invariably so lesions typical of one infection may be produced by a different
one lesions influenced by other factors than species of organism e.g. age
of patient, route of invasion, secondary infection, treatment, etc. possibility
of excluding such factors by inoculation. 2. Variation in lesion caused by
artificial inoculation. Method "standardises" lesion lesions said to be
invariable under these conditions. B. pseudo-dip ht her iae distinguished
from B. coli avian tubercle bacillus distinguished from human type
S. mastitidis distinguished from S. anginosa and S. pyogenes pneumo-
coccus of lobar pneumonia distinguished from that of lobular pneumonia.
Are the lesions cawed by artificial means invariable? certainly very
constant e.g. tubercle bacillus but not absolutely fixed e.g. a strain of
B. diph. causes lesions of rabies a strain of S. mastitidis loses its power
to cause typical lesion various types of tubercle bacilli fail to cause their
typical lesions. Two strains causing different lesions arise from single strain
during cultivation D. lanceolatus capsulatus isolated from different organs
causes different lesions type of lesion altered if organism first grown
anaerobically. Every aspect of pathogenicity subject to variation.
Other characters of bacteria equally variable. (94 106)
CHAPTER VIII
THE POSSIBLE OCCURRENCE OF TRANSMUTATION
IN THE LIVING BODY
Organisms closely resembling each other except in pathogenicity often
found associated can one of these be a derivative of the other? e.g.
B. anthracis and B. anthracoides, in hides of cattle. Other instances in
the human body. B. coli and B. typhosus. Klebs-Loeffler bacillus and
Hofmanris bacillus pathogenesis fermenting properties seasonal pre-
valence during convalescence from diphtheria recent work. Staph.
epidermidis and staph. pyogenes pathogenesis fermenting properties
SYNOPSIS xv
pigment formation. The meningococcus and M. catarrhalis morpho-
logy fermenting properties pathogenesis mixed infection habitat. The
meningococcus and the pneumococcus symptoms produced common com-
plications seasonal prevalence age incidence and mortality distribution.
Such transition less credible than it appears but not less credible than
instances known to occur saprophy tic and parasitic types of pneumococcus.
Conclusions. (107115)
CHAPTER IX
SUPPOSED INSTANCES OF TRANSMUTATION BROUGHT
ABOUT EXPERIMENTALLY
I. MAJOR HORROCKS'S EXPERIMENTS (Journal of R.A.M.C. Vol. xvi).
Importance of the claims made by him. Criticism. Possibilities to be
considered purity of original strain, peritoneum possibly not sterile,
possible contamination from skin, possible invasion from gut before or after
death, continuity of strain not confirmed by reversion or presence of
intermediate forms, different results obtained on repeating experiments,
results possibly explained by variation. Criticism. Conclusions.
II. RELATIONSHIP BETWEEN PARATYPHOID ORGANISMS.
A. Schmitfs experiments. Experiment I. "Flugge" type given to calf
in food, injected beneath skin second strain isolated from blood. Experi-
ment II. Second strain injected into nasopharynx of second calf third
strain isolated from blood and injected into vein, fourth strain recovered
after death later strains resembled B. Gaertner in agglutination. Possible
fallacies, (a) contamination in original strain ? (6) contamination in bodies
of calves ? not absolutely excluded by agglutination tests B. Gaertner in
intestines of healthy calves, possible increase in numbers and virulence due
to local inflammation, might lead to systemic invasion, (cf. saprophytes in
inflamed uterus) and fresh agglutination reactions of blood serum.
. Experiments of Miihlens, Dahm and Furst. Mice fed on infected
meat faeces of some contained B. Aertryck, of others B. Gaertner due
to transmutation ? Possible fallacies, (a) contamination of original source ?
(&) contamination in bodies of mice? control B. Aertryck in healthy
mice, presence possibly overlooked ? their appearance favoured by in-
flammation and disturbed function of bowel.
C. The Author's experiments. Experiment I. Guineapigs given B.
Gaertner in food B. Gaertner and B. Aertryck isolated from faeces at
different times control two organisms transmutable ? intestinal bacteria
undetected if few in number disturbed function of bowel reveals their
presence multiply in inflamed intestine (cf . B. coli in cholera) such factors
may explain result of experiment qualification proof that in disordered
intestine unsuspected organisms make their appearance. Experiment II.
Faeces of six guineapigs examined B. proteus found in one case guinea-
xvi SYNOPSIS
pigs given unwholesome food faeces again examined B. proteus found in
four cases. Conclusions suggest presence of secondary invaders in experi-
ments quoted possible error from identifying organism by agglutination
variable agglutination of paratyphoid organisms. Application to experiments
quoted. Results no evidence of transmutation. (116 139)
CHAPTER X
SUMMARY
All characters of bacteria show variation "spontaneous" or "impressed"
modifying influences already discussed (Chap. n). Variation may be ap-
parent only. Apparently spontaneous variation may be due to unrecognised
influences. Variation itself may be specific (morphology of B. diphtheriae
and S. scarlatinae). No one character specific variation need not imply
loss of specific character (morphology of B. coli}. Analogy of regiment of
soldiers and crowd of pitmen. Variation may be specific because it indicates
racial character. Many variations represent past stages in evolution (Mor-
phology, Chap, iv) others represent new steps in evolution (Fermenting
Power and Virulence, Chaps, v and vi).
TRANSMUTATION DIFFERS FROM VARIATION IN DEGREE ONLY different
species derived from a common stock differentiation more, advanced in
some than others reversion therefore differently interpreted necessity of
regarding characters as a whole and their stability danger of relying upon
one character alone already shown (Chap, iv-vn). Analogy of human race
groups. Variation may indicate recent environment and so reveal source
of particular strain (streptococcus from milk general coli infection from
biliary passages).
STABILITY OF VARIATIONS. "Spontaneous" variations. (1) Imperfect
development tend to disappear. (2) Senility or lowered vitality tend to
persist. (3) Atavistic tendencies tend to recur. (4) Fresh stage in evolu-
tion therefore unstable. Two variations constantly associated both due
to lowered vitality both due to higher evolution both due to imperfect
development or degeneracy. "Impressed" variations may lapse when in-
fluence withdrawn may persist for a time may appear permanent danger
of assuming variation is permanent examples ability to ferment sugar or
produce pigment inability to ferment sugar or display virulence. Duration
of impressed variation. (1) If only part of strain varies it may appear to
revert danger of assuming reversion has occurred. (2) If readily acquired
is long retained if slowly acquired quickly lost (ability of B. typhosus to
ferment) not true of spontaneous variations. (3) The longer the training
the more lasting the effect (streptococcus at different stages in disease-
ability of B. typhosus to ferment). Same principle governs development of
races. Absence of reversion does not imply inability to revert (pigment
SYNOPSIS xvii
production by B. ruber mycelial development of B. diph.}. Tendency to
revert does not imply loss of specific character. Variation differs from
transmutation in degree alone.
TRANSMUTATION DIFFERS FROM EVOLUTION IN DEGREE ALONE. Analogy of
different branches of family. Possibility of transmutation. Saprophytic and
parasitic pneumococcus. Other examples already discussed (Chap. vm).
Experiments suggesting transmutation already discussed (Chap. ix). 1st
series, strains not guaranteed pure, results explained by variation. 2nd
series, results explained by variation, secondary invasion not excluded.
Transmutation improbable. Enzyme theory of disease. (140 152)
CHAPTER XI
THE ENZYME THEORY OF DISEASE
Predicates disease not due to bacteria but to their ferments. (1) Ac-
quisition and loss of pathogenic powers. (2) Different organisms may cause
same type of disease rabies due to B. diph. (3) Same organism causes
different types of disease in different epidemics (B. influenzae) cages
differ in same epidemic scarlet fever and puerperal fever M. catarrhalis
infection simulating other diseases, coryza, influenza, scarlet fever, diphtheria,
typhoid fever, cerebrospinal fever. (4) Same conditions influence virulence
and fermenting power, (a) antiseptics, (b) oxygen virulence of cholera,
toxicity of B. diph., fermenting power of B. dysent., of streptococcus,
(c) temperature optimum temperature digestive enzyme in cold blooded
animals germ barley marine enzymes fermenting power of B. coli
virulence of B. diph., B. tetani, B. anthracis, etc. enzymes killed at 60 C.
and virulence destroyed, (d) sunlight, (e) symbiosis tetanus and pyogenic
cocci, B. coli and B. dentrificans. (5) Virulence due to "passage" through
an animal and fermenting power due to growth in a sugar, (a) specific,
(b) repeated inoculations or subcultures more effective, (c) power readily
acquired is easily maintained, (rf) if recently lost is quickly regained.
(6) Intra- and extra-cellular toxins intra- and extra-cellular enzymes, yeast,
digestive enzymes emulsion of gland or bacteria more potent. (7) Virulence
associated with fermenting properties M. catarrhalis, gonococcus and
meningococcus, Hofmann's bacillus and Klebs-Loeffler bacillus, B. coli both
due to adaptation? (8) Living tissues defended by enzymes. (9) Other
functions of bacteria due to enzymes influenced by same conditions as
virulence, e.g. pigment formation. (10) These ferments separable from
bacteria enzyme which liquefies gelatin survives bacteria passes filter
soluble. (11) M. ureae enzyme separable. (12) Isolation of pathogenic
enzymes (pneumococcus). (13) Bacteria deprived of a pathogenic function
by environment same conditions influence ferment activity, (a) ultra violet
rays pathogenesis of B. anthracis, (b) oxygen power of pneumococcus to
xviii SYNOPSIS
produce skin lesion, (c) growth in milk fermentation by B. coli, rash in
scarlet fever, (d) effect of substances added to media and of drugs in disease
sod. benzoate and B. coli sod. salicylate and acute rheumatism.
(14) Different symptoms due to different enzymes? analogy with sugar
ferments of bacteria complexity of action association with particular
vegetable and bacterial cells possibility of complete dissociation of patho-
genic enzymes? (15) Two results obtainable organisms deprived of patho-
genic functions (B. typhosus) functions maintained in absence of organism,
e.g. filter passers. (16) No enzyme isolated which forms toxins outside the
body true also of other recognised ferments artificial media differ from
vital fluids. (17) The enzyme theory and transmutation suggests transfer
of function possible certain conditions essential. Analogy of ships at sea.
Conclusion. (153169)
CHAPTER XII
CONCLUSIONS (170)
APPENDIX
REFERENCES (171179)
INTRODUCTION
THE mediaeval alchemists conceived the idea of the trans-
mutation of metals and dreamt of changing the baser metals
into gold. The task which baffled them the scientists of our own
generation seem destined to achieve. The transmutation of
bacteria is a problem of more recent date but it bears a certain
resemblance. If silver and gold are the currency of wealth
by means of which it changes hands, bacteria represent the
currency of disease by means of which this also is passed from
one person to another. The resemblance, however, goes much
deeper than this, for just as the metals have hitherto been
regarded as "elements" of matter so the functions of the
unicellular organism have been thought to represent the
"elements" of life. The physicist has learnt that the so-called
"elements" of matter are themselves composed of infinitely
small particles or "ions" ; the pathologist is learning that the
functions of bacteria in many cases result from the activity of
ultra-microscopic bodies, of the nature of "enzymes." The
occurrence of transmutation in the case of bacteria would
prove as revolutionary in our conception of disease as its
occurrence in the case of certain rare metals is already proving
in our conception of matter.
The idea of the permanence of characters in the animal
world is at least as old as the question "Can the Ethiopian
change his skin or the leopard his spots?" but it is only in
recent times that the fixity of animal species has been
scientifically demonstrated.
Amongst the less highly organised structures of plant life
variation is of more frequent occurrence and, though it is
not possible to "gather figs from thistles," it is generally
acknowledged that "species" in the case of plants are less
rigidly defined than in the animal world.
In the realm of bacteriology still simpler forms are met
with in which are recognised the beginnings of both animal
D. 1
2 INTRODUCTION
and vegetable life, and amongst these variation is of still
greater frequency. This fact, confirmed by personal observa-
tion and by a perusal of the literature of the subject, suggested
to the writer the question whether actual transmutation of
species might not occur amongst bacteria, and it was in the
hope of answering this question that the investigation here
recorded was undertaken.
An endeavour was made in the first instance to collect
the published records of all experiments in which transmuta-
tion was alleged to have occurred. These were found to be
few in number. In the second place, a series of experiments
was carried out by the writer with the object of disproving
the contention put forward in one of these cases. Thirdly,
with a view to criticising the claim made in the remaining
cases, and in the hope that it might throw some light on the
problem of transmutation as a whole, a study of the subject
of variation amongst bacteria was undertaken. The material
on which it is based has been collected from the scattered
literature of the subject. With a few exceptions, only papers
written in English have been consulted.
In Chapter I the scope of the enquiry is outlined. In
Chapter II the conditions which modify the characters of
bacteria are enumerated and in Chapter III the value of the
evidence adduced in proof of such modification having
occurred is considered. Examples of variation are then studied
in detail and their significance is discussed, reference being
made more particularly to morphological characters, fer-
menting properties, virulence, and pathogenesis (Chapters
IV VII). In Chapter VIII the possibility of transmutation
occurring in the animal body is considered. In Chapter IX
instances of supposed transmutation are examined. In Chapter
X the subject is reviewed at length and the results of the
investigation summarised. In Chapter XI the Enzyme theory
of disease is discussed, together with its bearing upon the
subject of transmutation. In Chapter XII the author's conclu-
sions are briefly stated. References are given in the Appendix.
CHAPTER I
THE SCOPE OF THE ENQUIRY
DEFINITION OF TERMS
THE phrase "transmutation of bacteria" is not synonymous
with "evolution of bacteria." "Evolution" is the gradual de-
velopment of new species and tends towards further differentia-
tion. "Transmutation" is the changing of members of one
recognised species into those of another and, if proved, would
tend towards unification by undermining existing barriers.
There is no reason to doubt and abundant evidence to
support the opinion that in this field of life, as in others, the
forces of natural selection and the survival of the fittest have
been at work and have resulted, in the course of ages, in the
evolution and differentiation of the various types of bacteria
which we recognise and distinguish today.
Andre wes, in the Horace Dobell Lecture for 1906, "traced
the evolution of streptococci from the condition of harmless
mineral-feeders, through that of saprophytism in the alimen-
tary canal, to the development of weak powers of parasitism
which have culminated, in certain instances, in the fully
developed property of aggressive parasitism seen in the
streptococcus pyogenes."
He showed how, at different stages, natural selection and
the survival of those best adapted to the environment in which
they found themselves, resulted in the permanent acquisition
of new characters, such as the ability, when they had once
entered upon a saprophy tic career in the alimentary canal, to
flourish most vigorously at the body temperature of their host
and to utilise the foodstuffs available in their new habitat ; to
resist desiccation during the intervals between their discharge
from one host and their reception by another ; and later still
to support themselves in the actual living tissues of the host
12
4 THE SCOPE OF THE ENQUIRY [CH. i
and to defend their position there by the manufacture of
haemolysins and toxins.
This process of evolution is, no doubt, going on continually
in bacteria as in higher forms of life. It is rendered possible
in both cases by the occurrence of natural variation. This
variation in bacteria is of two kinds, namely, spontaneous or
intrinsic variation between the individuals of a pure culture
that is to say, bacteria derived from a single organism, and
impressed variation, the effect of special environmental con-
ditions upon a succession of bacterial generations, due either
to the direct reaction of the bacterial protoplasm to the
environment, or to selection acting upon slight spontaneous
variations and producing a cumulative effect.
It is reasonable to expect that amongst the bacteria natural
variation would occur with greater frequency than amongst
higher forms of life for, being unicellular organisms, changes
in their environment can operate directly upon the germ plasm.
Moreover the common method by which bacteria multiply,
namely the division of the parent cell into two daughter cells,
ensures the ready transmission of any acquired character from
parent to offspring. The variation in character may be said to
be retained by the daughter cells rather than transmitted to
them. Such retention of parental characters by the daughter
cells is not, however, invariable. For example, McDonald
(1908) has published photographs of a young culture of the
meningococcus showing diplococci in which one member is
stained while the other is not. Thirdly, such variations would
be more readily noted in their case since as many as 30 or
40 successive generations may be observed in the course of
24 hours. In the case of some bacteria division may occur as
frequently as once every 17 minutes (Barber, 1908). Yet a
fourth factor might be mentioned, namely the ease with which
the environment of any strain of organisms can be modified in
any direction and to any extent.
As a matter of fact examples of such variation, as we shall
show, are innumerable, no matter what particular property or
character of bacteria we investigate. Differences occur in size
and shape, in staining properties, in power of growth on various
CH. i] THE SCOPE OF THE ENQUIRY 5
media, in viability, in virulence, in the power to ferment sugars,
and so on.
The environment in which bacteria grow and multiply
tends, in the course of time, to "fix" some of these variations
by offering to the possessors of them a better chance of survival
or perpetuation, so that they ultimately become characteristic
of a new species. This is evolution through natural selection.
By a similar process of artificial selection, as will be shown,
we can encourage variation in almost any direction we choose.
"Within certain limits the simple forms of life are able to adapt
themselves to their surroundings and the adaptation cannot
be ascribed to chance for, with a given environment, the one
particular alteration in properties surely results." (Adami,
1910.) If we so vary the characters of a member of one species
that it comes ultimately to possess all the characters of a
member of another species, that is "transmutation." The
question is, how far can we go in this direction, and to what
extent are the recognised species of bacteria really fixed in
their characters?
THE MEANING OF SPECIES.
The objection may be raised at this point, that the phrase
"transmutation of species" involves a contradiction in terms,
since the very definition of "species" excludes the possibility
of transmutation. This leads us to a further question, namely,
what do we mean by the word "species" as applied to
bacteria? in other words, what determines our present
classification ?
The distinction between different species of bacteria and
their recognition depends upon the observation of their charac-
ters morphological, biological, chemical, physiological and
pathological. Briefly enumerated these are as follows : the
naked eye appearance of colonies and of a stab culture:
microscopic appearances, size, shape, motility, adhesiveness:
method of generation and life history, involution forms : power
to produce pigment : staining properties : cultural characters,
power of growing on different media, in the presence or absence
of oxygen, and under different conditions of temperature and
6 THE SCOPE OF THE ENQUIRY [CH. i
moisture, with or without production of gas or odour ; power
to liquefy gelatin, to reduce neutral red, to clot milk, to ferment
various carbohydrate substances : power to form agglutinins
and susceptibility to agglutination : viability under different
conditions : virulence or the nature of the toxins they produce :
pathogenicity or the nature of the lesions they cause and the
kind of animal susceptible to their invasion.
It is seen from this list that the characteristic qualities of
bacteria are very numerous and it would be thought that their
classification would on this account be very thorough and
complete. But, as will be shown in the course of this enquiry,
every one of these characters is liable to variation and the
occurrence of these variations renders the task of classification
very difficult and in many cases uncertain.
If certain characteristics were invariable, even though
others varied, a definite criterion would be afforded, but where
all alike are subject to modification the division into species
is necessarily an arbitrary one. Amongst the higher animals,
where sexual production prevails, mutual fertility or sterility
offers a guide in determining the limits of species. Here no
hard and fast line can be drawn. Nevertheless we see exhibited
amongst bacteria, in the words of De Bary, " the same periodi-
cally repeated course of development within certain empirically
determined limits of variation," which is considered to justify
the recognition of a species.
Many species of bacteria do show characters apparently
quite fixed and rigid. The anthrax bacillus and the tetanus
bacillus are quite as good species in the natural history sense
as any that can be found amongst flowering plants. But the
classification of others is still a matter of dispute. This can
be illustrated by reference to the streptococci.
THE CLASSIFICATION OF THE STREPTOCOCCI.
Marmorek held the opinion that the human streptococci
constituted one species. "He based his chief argument on the
observation that bouillon in which one sort of streptococcus
had grown would not serve afterwards as a culture medium
for any other streptococcus, and that the same haemolytic
CH. i] THE SCOPE OF THE ENQUIRY 7
power was possessed by them all." (Andrewes and Border,
1906.)
In 1891 von Lingelsheim (ibid.) proposed a division into two
groups according to the length of chains formed: "strepto-
coccus brevis" and "streptococcus longus." Andrewes and
Horder (1906) with a view to further classification on the same
lines suggested the adoption of the terms brevissimus, brevis,
medius, longus, longissimus, conglomeratus. The quality of
cohesiveness by itself was, however, considered too trivial a
character to base a fundamental classification upon.
The power of retaining stains was found to offer no means
of differentiation, since all stained well.
Minute differences in their mode of growth on different
media were found to be too inconstant to be of any value,
though Schottmuller (quoted by Muir and Ritchie) attempted to
classify the streptococci according to the appearance of colonies.
Classification according to pathogenicity and virulence
appeared to have the advantage of being practical and signifi-
cant from a clinical standpoint. Virulence, however, was
likewise found to be an inconstant character, being lost and
regained with great readiness by these organisms. It was lost
after a few days on certain culture media. On the other hand,
after a few "passages" through a susceptible animal, a strepto-
coccus of feeble virulence might become intensely pathogenic.
Clinical experience confirms this variability in virulence. " One
and the same strain of streptococcus may at different stages
in its career produce now a rapidly fatal septicaemia, now a
spreading erysipelas, now a localised suppuration and now no
effect at all" (Andrewes and Horder, 1906), so that the degree
of virulence was an uncertain aid to classification. The
streptococcus erysipelatis, for instance, is no longer considered
on account of its virulence to be a distinct species from the
streptococcus pyogenes.
Agglutination tests have not been found to be sufficiently
specific.
Marmorek's contention, therefore, for the unity of species of
the human streptococci continued to hold the field successfully
until the introduction of Gordon's tests.
8 THE SCOPE OF THE ENQUIRY [OH. i
Gordon (1903-4) isolated from human saliva 300 strains of
streptococci. He tested separately the power of these different
strains to ferment various substances, consisting of 14 carbo-
hydrates, 13 glucosides, and 6 polyatomic alcohols. Many of
these test substances proved of no differential value as regards
streptococci, either because they were uniformly attacked by
all or because no streptococci could ferment them, but he was
led to select a series of seven substances namely saccharose,
raffinose, inulin, salicin, coniferin and mannite as being of
special value as tests for streptococci, and to these he added
two further tests the clotting of milk and the reduction of
neutral red under anaerobic conditions. By such means he
was enabled to distinguish 48 chemical varieties.
Houston (1903-4) applied the same tests (with the omission
of one the action on coniferin) to 300 strains of streptococci
derived from human faeces and was able to distinguish
40 chemical varieties amongst them.
Gordon demonstrated that these chemically different strains
were remarkably constant in their reactions and this was
confirmed later by the work of Andre wes and Horder (1906),
who tested his strains and, in addition, some 200 new strains
derived from foci of disease in human beings. " Gordon himself
was careful to abstain from claiming specific value for his
different chemical types and he did not venture to propose any
reasoned scheme of scientific classification based upon his
tests." Andrewes and Horder attempted to do this. They
collected from various sources particulars of the behaviour of
over 1200 different strains of streptococci when subjected to
Gordon's tests. As a result they found that these 1200 strains
fell into some half a dozen main groups. By adding one
further test the power of growth in gelatin at 20 C. and by
taking into consideration also the morphological characters
and pathogenesis, they were able to define five varieties of
streptococci which they regarded as of "approximately specific
value" though connected by a multiplicity of intermediate
varieties. They named these S. anginosa, S. salivarius,
S. faecalis, S. pyogenes and 8. pneumococcus.
Though confirming, on the whole, the stability of the
CH. i] THE SCOPE OF THE ENQUIRY 9
reactions constituting the tests, these observers noticed that
variation in virulence was sometimes accompanied by changes
in chemical behaviour. They also acknowledged that slight
differences in the composition of the media might possibly
affect the series of reactions to some slight extent.
Subsequently Ainley Walker (1911) offered evidence to
show that greater differences in the media do actually affect
the reactions to a remarkable extent, so much so as, in his
opinion, to invalidate any claim that they should be re-
garded as specific, and he fell back on the position held by
Marmorek.
Still later Jensen and Holth, after a prolonged investiga-
tion, came to the opposite conclusion, that is to say in favour
of the stability of the differences brought out by Gordon's
tests, but they also showed that these differences were in no
way closely related to virulence or pathogenic action so that
the method of classification founded on them was imperfect.
We have thus demonstrated that the term "species" when
applied to bacteria must be interpreted much more loosely
than in the case of plants or the higher animals and the phrase
"transmutation of species" is thereby absolved of the accusa-
tion of being self- contradictory.
The aim of this paper is to show how far transmutation
does occur, and what is its significance.
It will be obvious, from what has already been said, that
in considering the evidence of transmutation it will be neces-
sary to consider also the evidence of variation. The difference
between the two is one of degree only. A member of one
"species" of bacteria is distinguished from a member of
another " species " by its morphological and other characters.
If these characters become altered, within certain limits, the
process may be regarded simply as variation ; if outside these
limits it must be regarded as transmutation.
We have first of all to consider then, what are the possi-
bilities in the direction of such alteration in character.
10 THE SCOPE OF THE ENQUIRY [CH. i
A CONSIDERATION OF THE POSSIBILITIES.
The various possibilities to be considered are five in
number. It will be seen that the first three are instances of
variation and the remaining two, instances of transmutation.
1. Simple variation. Modifications may occur in the
characters of an organism, involving either the loss of some
feature previously regarded as characteristic or the acquisition
of some other feature not hitherto considered to be so, such
modifications not being so numerous, however, or so funda-
mental, as to lead to any doubt as to the proper identification
of the organism. For example, Twort (1907) succeeded, in the
course of two years, in training a strain of B. typJiosus to
ferment lactose, and both Twort (1907) and Penfold (1910 A)
produced pure strains capable of fermenting dulcite, which
the usual variety of typhoid bacillus is practically unable to
do. These new strains retained qualitatively all the other
properties of the B. typhosus unchanged. Similarly Miss
Peckham (1897) induced indol formation in numerous strains
of B. typhosus.
2. Variations in different directions associated. The
acquirement of some fresh character may be associated with
the loss simultaneously of some other character previously
possessed or, with the acquisition of a second new character,
and in some cases this association may prove to be invariable
under the same modifying conditions (vide p. 146). For
example, Eyre and Washbouru (1899) observed that a non-
virulent strain of the pneumococcus growing readily at 20 C.
could by "passage" be converted into a highly virulent strain
which was then unable to grow at a temperature below 37 C.
The reverse change showed the same relation between the
virulence of the organism and the temperature at which it
would grow.
Jenner (1898) was able to revert B. coli capsulatiis to an
unencapsulated form by cultural methods and found that the
new variety had lost the power to coagulate milk, and instead
of being highly pathogenic to white mice had become much
less so or even non-pathogenic.
CH. i] THE SCOPE OF THE ENQUIRY 11
The acquirement of power to ferment a certain carbo-
hydrate may coincide with the loss of fermenting power in
other directions.
Penfold (1910-11) has shown that the development of new
fermenting powers on the part of B. typhosus towards lactose
and dulcite is frequently associated with the formation of
papillae on its colonies. Diminished gas-production in glucose
media on the part of certain coliform organisms (B. Grunthal,
etc.) was likewise associated with papillae formation.
The same observer found that colonies of B. typhosus which
had lost the property of fermenting glycerine showed impaired
agglutinability also, though typical fermenting colonies on
the same plate were normal as regards agglutination.
Adami, Abbott and Nicholson (1899) found that the as-
sumption of coccic and diplococcic forms by B. coli in the
organs of healthy animals was associated with a loss of power
to ferment carbohydrates and to produce indol.
Gordon (1900-1) observed that the tendency of the strep-
tococcus of scarlet fever to assume a bacillary form was
abolished by "passage" and at the same time its virulence
was increased.
Rosenow (1914) obtained a strain of streptococci from the
throat in a case of scarlet fever, which yielded on blood agar
two distinct kinds of colonies. These displayed marked differ-
ences in their fermenting power and also in their pathogenicity.
Many other instances might be given.
3. The development of intermediate forms, i.e. the possible
derivation from one or other of two known species of forms
intermediate between them in their characters. For example,
W. J. Wilson obtained from the urine of a supposed typhoid
carrier (1910), and also from the urine in certain cases of
cystitis and pyelitis (1908), coliform organisms intermediate
in their characters between B. typhosus and B. coli communis
and derived presumably from B. typhosus in one case and
from B. coli in the others. Many other observers have
described organisms resembling both B. typhosus and B. coli
communis in their characters. Klotz (1906) has described
such an organism, isolated from water, and called by him
Bacillus perturbans. Mcnaught (1905) described two varieties,
12 THE SCOPE OF THE ENQUIRY [CH. i
also derived from water, under the term Bacillus typhosm
simulans.
One organism, for example, described by Wilson (1910),
resembled B, typhosus in forming acid without gas in glucose
and in failing to ferment lactose at 37 C. ; but it failed to
agglutinate with typhoid serum, and it resembled B. coli in
producing acid and much gas from mannite and in fermenting
lactose at 22 C.
The Bacillus perturbans of Klotz was agglutinated by
high dilutions of typhoid serum (1-1550 in 15 minutes) and
it produced slight acidity in milk without coagulation ; but it
differed from B. typhosus in fermenting both lactose and
saccharose, in giving the neutral red reaction and forming
indol, and in other ways.
Major Hor rocks obtained from the urine of a patient
convalescent from typhoid fever a typical strain of B. typhosus
which however on subculture gave rise to an organism inter-
mediate in its characters between B. typhosus and B. coli
(vide p. 118).
4. Slight changes in closely allied organisms, i.e. the
possibility in the case of closely allied organisms of a modifica-
tion in the few distinguishing features they possess, so that
they may appear to change, the one into the other. For
example Schmitt (1911) concluded from his experiments that
paratyphoid bacilli of the 'Fliigge' type and of the 'Gaertner'
type, generally regarded as distinct species, could be trans-
formed from one into the other in the animal body.
5. A complete change in characters, i.e. the possibility of
the occurrence, more or less suddenly, of a complete change
simultaneously of all the characters of an organism, or at least
of all the fundamental ones by which it was distinguished.
For example, Major Horrocks (1911) concluded that he had
been able gradually to modify a strain of B. typhosus, by
changes in its environment, to such an extent that it assumed
eventually the characteristics of a Gram-positive coccus having
the cultural characters of Streptococcus faecalis.
Before these several possibilities are studied more fully,
the conditions which modify the characters of bacteria will be
mentioned and examples given under each head.
CHAPTER II
CONDITIONS MODIFYING THE CHARACTERS
OF BACTERIA
THE factors which appear to influence the growth and de-
velopment of bacteria and to produce modification in their
characters are many in number and diverse in nature, and it
is often impossible to state with certainty which of these
various factors is the one primarily responsible for the
modification observed in a particular case.
1. Many variations appear to be spontaneous, not due,
that is to say, to any external agency, but the result of
developmental or atavistic tendencies inherent in the organism
itself. We know as little of the nature of these tendencies to
variation as we know of the nature of those which control
normal development and, in the vast majority of cases, prevent
variation occurring.
Some spontaneous variations are examples of "pleomorph-
ism" and represent stages in the life history of the individual
organism or of the race. Others cannot be explained in this
way. For example, one component of a diplococcus may retain
a stain while its fellow fails to do so. In such a case faulty
technique cannot be held responsible, nor can the variation be
attributed to differences in environment. Moreover in the
case of the meningococcus it has been found to persist after
animal passage (McDonald, 1908). The variation would appear
to date from the cell division which constitutes the "birth"
of the organism. Denny (1903) observed the same inequality
in staining properties in different segments of a segmented
form of B. Xerosis.
Many other examples of spontaneous variation will be
found in later pages.
2. Differences in characters are sometimes associated with
differences in geographical distribution. Thus Schultz (1909)
14 CONDITIONS MODIFYING [OH. n
found that in Cleveland U.S.A., during the 12 months covered
by the investigation, "barred" forms of the diphtheria bacillus
had almost disappeared ; during the same period, in Boston
and Providence, another observer noted that "barred" forms
were unusually common while "granular" forms were very
rarely met with.
3. Many organisms after prolonged cultivation on artificial
media display variation in character.
In some of these cases the length of the period of cultiva-
tion is not the cause of the modification, it merely extends the
survey over a large number of generations and so enables the
observer to detect variations spontaneously occurring.
In other cases the length of iime permits" natural selection"
to play its part and produce modifications which, in a shorter
interval, would not have advanced far enough to be apparent.
In other cases, again, the prolonged exclusion from animal
tissues does lead directly to a modification in character which
is proportionate, as regards its extent and its permanence, to
the duration of such exile, but disappears when the organism
is again "passed" through the body of an animal. This is true
more particularly of the property of virulence (q. v.).
As an example of the influence exerted by prolonged
cultivation in modifying the character of an organism may be
cited the statement of Mohler and Washburn (1906) that
a strain of bovine tubercle bacilli after cultivation for 11 years
was found to have become modified in morphological and
cultural characters to the human type.
Lentz (quoted by Bahr, 1912) found that a "Flexner"
type of B. dysenteriae after 9 years' laboratory cultivation
completely lost the power to ferment maltose. Arkwright
(1909) mentions a strain of the meningococcus which when
first isolated did not ferment glucose but after ten months'
artificial cultivation developed power to do so. Rettger and
Sherrick (1911) describe the gradual loss of power to produce
pigment on the part of an old stock culture of B.pyocyaneus
after 5 years' artificial growth.
Prolonged cultivation in a medium containing a particular
carbohydrate may develop in a strain of bacteria the ability
CH. n] THE CHARACTERS OF BACTERIA 15
to ferment that carbohydrate. B. typhosus for example after
two years' growth in a medium containing lactose acquires the
power to ferment this sugar (vide p. 58).
4. In other cases again the length of the period of cultiva-
tion is of less importance than the conditions under which
such cultivation takes place.
(a) Conditions which lower the vitality of a strain may
modify its characters. Such conditions include starvation (for
example, growth in pure water), acidity of the medium, want
of oxygen, the presence of antiseptics, exposure to sunlight,
high or low temperatures, symbiosis, etc.
One example will suffice. A strain of B. ruber of Kiel if
heated to a temperature just below that known to kill the
organism loses its power to produce pigment (Adami, 1892).
(b) The crowding together of the organisms on the surface
of the medium may lead to a diminution in pigment produc-
tion in the staphylococcus aureus (Andrewes and Gordon,
1905-6) and an earlier appearance of granular staining forms
of the diphtheria bacillus (Denny, 1903).
(c) The temperature at which organisms grow is respon-
sible for certain variations. Laurent (1890) found that
a selected strain of B. ruber which had grown for 12 months
at a temperature of 25 35 C. without exhibiting a trace of
colouration, yielded its characteristic pigment when the tem-
perature was lowered to 18 C. An apparent staphylococcus
"albus" growing at 37 C. may become a vivid "aureus" at
22 C. (Andrewes and Gordon, 1905-6).
The virulence of B. anthracis is greatly modified by growth
at 43 C. and that of B. diphtheriae may be destroyed by
subjection to a similar temperature (Hewlett and Knight,
1897).
Wilson (1910) describes an atypical B. typhosus which
fermented lactose at a temperature of 22 C. but failed to do
so at 37 C. Coplans (1909) found that dulcite was more quickly
fermented by certain colon bacilli at 20 C. than at 37 C.
Rodet (quoted and confirmed by Adami, Abbott and
Nicholson, 1899) found that at a temperature of 45 C. B. coli
developed in a few hours into long filaments. The same
16 CONDITIONS MODIFYING [OH. n
agency will abolish the power of some bacteria such as
B. anthracis to form spores, and may modify the character
of the colonies it forms (Bairibridge, 1903).
Bacteria of the paratyphoid group agglutinate much less
readily after being heated (Sobernheim and Seligmann, 1910).
(d) Differences in atmospheric pressure may modify the
activity of certain organisms.
B. coli yields formic acid and gas from glucose at ordinary
atmospheric pressure. If the pressure is raised the yield of
gas diminishes but the yield of formic acid increases (Harden,
1901).
A great pressure of carbon dioxide is said to deprive B.
anthracis of its power to form spores though it has no effect
on the vitality of the organism (Muir and Ritchie).
Certain organisms which do not readily lose their virulence
on artificial media do so rapidly if grown in an atmosphere of
compressed air (ibid.).
(e) The presence or absence of oxygen is another factor
of importance. Strains of B. typhoid and B. coli growing in
water maintain their viability better if plentifully supplied
with oxygen (Whipple and Mayer, 1906).
In the absence of free oxygen B. pyocyaneus ceases to
produce pigment (Adami, 1892) though the spirillum rubrum
produces it more plentifully (Muir and Ritchie).
Torrey (1905) observed that by alternate aerobic and
anaerobic culture a certain type of dysentery bacillus had its
power to ferment maltose greatly augmented.
Andrewes and Horder (1906) found that a certain strepto-
coccus which refused to ferment lactose, under ordinary con-
ditions of cultivation, did so readily when deprived of oxygen.
Kruse (quoted by Glenn, 1911) found that a staphylococcus
which, similarly, refused to liquefy gelatin did so at once
under the same altered conditions.
Anthrax bacilli in the absence of oxygen may develop
torula zoogleic forms (Wood, 1889). Noguchi (1910) discovered
that B. biftdus communis only exhibited the bifurcating
phase under anaerobic conditions and in the absence of
oxygen became less pathogenic.
CH. ii] THE CHARACTERS OF BACTERIA 17
It is well known that B. diphtheriae produces toxins more
plentifully in a free supply of air (Clark, 1910).
The bacillus of malignant oedema is said to lose virulence
when grown aerobically (Harass, 1906) and that of cholera to
gain virulence when grown anaerobically (Hueppe, quoted by
Adami, 1892).
Foa (1890) describes how a strain of the pneumococcus
could by anaerobic growth be deprived of its property of
causing a characteristic inflammatory oedema of the skin
when injected into an animal.
Wood (1889) attributed the diminished infectivity of
virulent organisms discharged from the bowel in many
diseases to the fact that in the bowel they are practically
deprived of oxygen.
(f) Bright sunlight destroys the virulence of some patho-
genic organisms (Marshall Ward and Blackmail, 1910) and
leads to the loss of pigmentation in others (Laurent, 1890).
The bacterium mycoides, on the other hand, will only produce
its red pigment in the dark (Scholl, quoted by Wood, 1889).
5. Exposure to the ultra violet rays has recently been
shown by Madame Henri (1914) to effect a startling change
in both the morphology and the pathogenicity of B. anthracis.
Cocci and filamentous forms were produced which differed
from the original bacilli in their power of retaining stains,
of forming spores, of liquefying gelatine and coagulating milk,
and which gave rise, on injection into an animal, to symptoms
quite unlike those produced by normal anthrax bacilli. The
new forms did not revert after daily subculture for over two
months.
1 6. Electrolysis. Electrolysis may produce changes in the
morphology and staining properties of bacteria. Russ has
observed the production of elongated forms of B. coli, with
altered reaction to Gram's stain, in urine (within the human
bladder and outside the body) as a result of the passage of
a galvanic current of T 1 5 th m.a. strength for one hour. The
modification persisted for many months.
7. The age of the culture is of importance in the case of
many pleomorphic organisms. For example, B. megatherium
D. 2
18 CONDITIONS MODIFYING [CH. n
and B. subtilis pass in a few hours from a bacillary motile
stage with cilia, to one of filamentous growth preceded by the
casting off of cilia (Marshall Ward and Blackman, 1910).
This factor influences the characters not only of bacteria
known to be "pleomorphic" but of others also. Young tubercle
bacilli are said not to be "acid-fast" (Hamer, 1900).
Young cultures of B. diphtheriae more often show branched
and clubbed forms (Kanthack and Andrewes, 1905) and solid
staining bacilli (Denny, 1903) than do older cultures; at the
same time they are unable to ferment glycerine and lactose
though older cultures usually ferment both (Muir and Ritchie).
A young culture of B. coli does not yield indol (MacConkey,
1909). Wood (1889) found that an old culture of cholera failed
to liquefy gelatin but did so readily after subculturing, and
that a young culture of the same organism was much more
susceptible to the action of antiseptics than a younger one.
Old cultures of pigment forming bacteria are often colour-
less (Adami, 1892).
Arkwright (1909) found bacillary forms of the meningo-
coccus in old cultures. The tubercle bacillus also in old
cultures displays elongated and even branched forms.
8. The character of the culture medium employed may
influence bacteria in many ways.
(a) The age of the medium. S. pyogenes normally does
not ferment saccharose, raffinose or salicin, but if old media be
used this organism will ferment all three substances (S. Martin,
1908-9). On the other hand B. diphtheriae, which in fresh
beef serum gives its characteristic "sugar" reactions, fails to
do so if this medium is old (Fisher, 1909).
(b) Changes in the reaction of the medium employed is in
many cases accompanied by changes in the morphological
characters of organisms growing in it, bacilli giving place to
cocci and diplococci, and vice versa, and pigment formation
being modified or lost (Adami, 1892). The reaction also affects
the vitality of many bacteria (Wood, 1889), and their virulence
(Peckham, 1897).
(c) The nature of the medium is important. Individual
morphology, the appearance of colonies, fermenting power,
CH. IT] THE CHARACTERS OF BACTERIA 19
indol formation, pigment production, and virulence, all vary
with the kind of medium used.
Gordon (1900-1) states that the streptococcus of scarlatina
may form, on serum, rods which closely resemble B. diph-
theriae but in a liquid medium it grows in a typical strepto-
coccal form.
B. diphtheriae does not form toxins readily if there is much
carbohydrate present in the medium (Fisher, 1909).
Many media contain substances derived from the living
body such as serum, or blood, and to this extent are "natural"
rather than "artificial" media and the alteration in the
character of organisms growing in them, particularly as regards
virulence, is possibly to be attributed to this factor.
Penfold (1914) mentions the fact that vaccination with a
plague strain grown on agar will protect rats against itself
but not against the same strain grown on serum.
If the ordinary artificial media are replaced by the natural
secretions of the body the modifications in character on the
part of the organisms growing in them may be even more
marked.
Rosenow (1912-13) found that a streptococcus which
presented certain morphological and cultural characters on
ordinary media, underwent a profound modification in respect
to both as a result of growth in unheated milk.
Horrocks (1911) found that a strain of B.typhosus, obtained
from the urine of a "carrier," lost virulence on ordinary media
(broth, and agar) in a few days but maintained it for over a
year in urine.
Both in milk and in urine B. eoli may form a dense network
of branching filaments (Re vis, 1908, Wilson, 1908) and give
atypical fermenting reactions (ibid.).
In the presence of saliva B. coli yields leptothrix forms
(Adami, Abbott and Nicholson, 1899), while S. mastitidis is
deprived of virulence (Savage, 1908-9).
Diplococcic forms of B. coli occur in bile (Adami, Abbott
and Nicholson), while in ascitic fluid the same organism
undergoes profound changes in respect both to its morphology
and its fermenting power (ibid.).
22
20 CONDITIONS MODIFYING [CH. n
Pathological exudations influence the characters of
bacteria growing in them to an even greater degree than
the natural secretions. This is particularly true of virulence
(vide p. 77).
Harris (1901) examined 15 strains of B. coli from u natural"
sources such as sewage, water, milk, shellfish and also
11 strains from "diseased" sources, that is to say from in-
flammatory exudations. Of the former, only two were virulent ;
of the latter only one was non-virulent.
Growth in water also influences bacteria. B. coli in river
water, where they are practically deprived of proteid food,
appear to lose their power of producing indol (Peckham,
1897). The same organism isolated from drinking water was
found by Savage (1904) to form less typical colonies than when
isolated from sewage or faeces, while Jenner (1898) describes its
morphological appearances as being different, bacilli isolated
from water being less thick and opaque a distinction which
disappeared when the strain was grown in milk.
(d) The addition of various chemical substances, such as
antiseptics, to the media used for cultivation profoundly
modifies the development of bacteria. In the presence of
carbolic acid typhoid bacilli assume the form of non-motile
cocci and diplococci (Adami, 1892), the bacillus anthrax loses
virulence and also the power to form spores (Roux, 1890),
many bacteria no longer liquefy gelatin (Wood, 1889), while
others lose their power to ferment carbohydrates (Penfold r
191 IB). Under the influence of antiseptics B. prodigiosus
forms spirillae and ceases to produce pigment (Wasserzug,
1888). The bacillus of blue pus normally a small short
bacillus yields, on the addition of a trace of boric acid to
the medium, S-shaped forms and close spirals, and on the
addition of potassium bichromate, long undulating filaments
(ibid.). The presence of sodium benzoate inhibits gas pro-
duction in the case of B. coli (Herter, 1909). The addition of
glycerine prevents the liquefaction of gelatin by bacteria
(Adami, 1892) and also inhibits the formation of indol (Wood,
1889). A virulent B. diphtheriae is promptly attenuated by
the addition of iodine trichloride to the medium (Mohler and
CH. n] THE CHARACTERS OF BACTERIA 21
Washburn, 1906) and a non- virulent bacillus of Blackleg
rendered virulent by the addition of lactic acid (ibid.). In-
creased resistance to antiseptics may be developed by pro-
longed exposure to their action (Rettger and Sherrick, 1911 :
Penfold, 1911 c).
Many other examples are given in later pages.
9. Unusual fermenting properties on the part of bacteria
are frequently acquired after prolonged growth on special
sugar or peptone containing media. The length of time
required varies in different cases, within wide limits. For
example, B. typhosm can be "trained" to ferment dulcite in
less than two weeks (Penfold, 1910 A) but cannot be trained
to ferment lactose in less than two years (ibid.). The method
is not invariably successful, but this may be due to the fact
that the trial in many cases is not sufficiently prolonged.
Unusual proteolytic powers may be developed in a strain
of organisms by an analogous process. Miss Peckham (1897)
induced the power to form indol on the part of many strains
of B. typhosm by cultivating them in a medium rich in peptone.
10. By artificial selection, through a number of genera-
tions, it is often possible to develop variation in a particular
direction.
Goodman (1908) by this method obtained from a strain of
B. diphtheriae with moderate power of producing acid in
dextrose broth, two strains possessing respectively a greatly
augmented and a greatly diminished power of acid production.
Rettger and Sherrick (1911) in the same manner obtained
from a slightly pigmented strain of B. prodigiosus two strains
showing in one case brilliant colouration and in the other
complete absence of colour. They also obtained by selection
a strain of staphylococcus aureus unusually resistant to the
action of corrosive sublimate.
Conn (quoted Glenn, 191 X) obtained by the same method
strains of a micrococcus with high and low powers of liquefy-
ing gelatin.
This method, however, may prove unsuccessful. Rettger
and Sherrick failed to modify pigment production in B. ruber
balticus and they quote Buchanan and Traux as having been
22 CONDITIONS MODIFYING [CH. n
unable to establish high and low acid producing races of
streptococcus lacticus. Glenn (1911) failed to produce high
and low acid producing strains of B. proteus. Other observers
have failed in attempts by selection to develop a particular
morphological type of the diphtheria bacillus (Clark, 1910)
and to modify the agglutination reactions of B. typhosus
(Moon, 1911).
11. Symbiosis is known to influence the behaviour of
bacteria and has in many cases a marked effect on the character
of one or other of the organisms growing together. The
phenomenon of symbiosis is a familiar one in vegetable life.
The individual struggle for existence is observed to give place
occasionally to a permanent partnership between two organisms
for their mutual benefit. An example of such cooperation is
furnished by the Lichens each of which is a dual organism
composed of a fungus and an Alga. Vegetable life as a whole
is dependent upon the activity of nitrifying organisms in the
soil and in some cases a definite alliance is formed between
the two parties, as in the case of the Leguminosae and the
nitrifying bacteria which take up their residence in the root
nodules of these plants.
All forms of mutual parasitism are in reality examples of
symbiosis. One species of bacteria may, however, be dependent
for its growth upon another species without necessarily being
parasitic. Thus Pasteur advanced the theory that aerobic
bacteria by exhausting the supply of oxygen gave anaerobic
bacteria a chance of growing.
Allen (1910) found that a strain of B. inflmnzae, which
could not be grown on ordinary media, grew luxuriantly
on sterilised media in which the staphylococcus albus had
previously grown. Neisser was able to cultivate the same
bacillus on plain agar for several generations by growing the
Xerosis bacillus with it, though a dead culture of the latter
had not the same favouring effect (Muir and Ritchie).
In other cases the growth of one species is inimical to the
growth of another. B. typhosus will not grow in a filtered
broth culture of staphylococcus albus, nor in that of many
other organisms (Freudenriech, 1888). The meningococcus is
CH. IT] THE CHARACTERS OF BACTERIA 23
inimical to the growth of the Klebs-Loeffler bacillus (Smirnow,
1908).
Prescott and Baker (1904) describe a similar antagonism
between streptococci and B. coli and they attribute the
extinction of the latter when the two are grown together to
the greater sensitiveness of B. coli to the lactic acid produced
by both combatants.
Klein (1903-4) found that a strain of B. typhosm was
killed by B. coli in the peritoneal cavity and Horrocks (1911)
found that the same thing happened in water which contained
both organisms. Jordan, Russell and Zeit (1904) observed
that B. typhosm quickly died out in polluted water.
Symbiosis is observed to influence not only the viability of
bacteria but their virulence, their morphology, their fermenting
powers, and other characters. The presence of the strepto-
coccus is said to be inimical to the growth of the diphtheria
bacillus (Smirnow, 1908) but it increases the virulence of the
latter during "passage" (Muir and Ritchie, 1910) while on
artificial media it induces changes in the morphology of the
bacillus, "granular" forms appearing earlier than in a pure
culture (Denny, 1903). Smirnow (1908) observed that the
same bacillus grown on agar in the presence of a bacillus
isolated from an acute rhinitis, assumed a coccic form
retaining, however, its virulence unimpaired. The meningo-
coccus produced a similar change.
Lesieur (1901, quoted Clark, 1910) claimed that the pseudo-
diphtheria bacillus may assume the morphological characters
of the Klebs-Loeffler bacillus as a result of symbiosis with
aurococcus aureus.
Horrocks (1911) found that a typical strain of B. typhosus
lost its power to ferment "sugars" when grown in the presence
of a strain of B. coli derived from a typhoid carrier (vide
p. 119).
In some cases bacteria growing together are able to produce
results which neither can do alone. For example, neither
B. coli nor B. dentriftcans alone can reduce nitrates, but if
allowed to act on sodium nitrate together they bring about the
escape of free nitrogen (Marshall Ward and Blackman, 1910).
24 CONDITIONS MODIFYING [CH. n
In other cases the prolonged growth of two species together
appears to produce no change whatever in either of them.
Williams (1902) grew a virulent streptococcus and a virulent
diphtheria bacillus together, transplanting every three or
four days for 90 such "generations" without influencing the
characters of either organism. Horrocks (1911) grew B. typho-
sus and B. fluorescens non-liquefaciens together for a period
of four months. Examinations made at intervals of one week
throughout this period revealed no alteration in the character
or agglutination properties of the B. typhosus.
The methods of studying the effect of symbiosis are various.
Simultaneous growth can be studied by "sowing" different
species of bacteria together indiscriminately on the surface of
the medium ; or distinct colonies may be grown on a plate so
that at first a considerable space intervenes between colonies
of the different species, the interval gradually lessening as the
colonies extend until it is finally obliterated ; a third method
is that of "criss-cross " planting, the effects of symbiosis being
seen at the intersection of the lines of growth ; or fourthly,
one species may be grown on the surface of the medium and
another deep to it in the form of buried colonies ; or fifthly,
a double celluloid sac may be utilised in which the products
of bacterial growth can diffuse from one compartment to the
other; finally, successive growth offers a further means of
investigation, the medium being sterilised after the growth
of one species and then planted with the other.
12. The methods of modifying bacteria which remain to
be described all involve the agency of the living tissues and
may be regarded as forms of parasitism.
(a) Transmission through the alimentary canal is said in
some cases to bring about modifications in character. It has
been suggested that the bacillus of Aertryck may assume
the characters and agglutinative properties of B. enteritidis
Gaertner after transmission through the intestine of the
mouse.
Bahr (1912) found that the fermenting powers of certain
strains of dysentery bacilli were modified after they had been
passed through the intestine of the fly, although for nine
CH. n] THE CHARACTERS OF BACTERIA 25
months previously the strains had repeatedly given normal
sugar reactions.
(b) "Passage " through an animal, or series of animals by
successive injections into the blood, or into the peritoneal
cavity, and subsequent re-cultivation from the heart's blood
or peritoneal fluid is known to modify the characters of
bacteria in many cases.
Fermenting power, by such means, may be greatly modified
(vide p. 57). Virulence, again, may in this way be markedly
increased towards some animals and diminished towards others
(vide p. 81).
It is claimed that the pseudo-diphtheria bacillus can be
converted into the Klebs-Loeifier bacillus by passage through
rabbits (Lesieur, 1901), or through guineapigs (Ohlmacher,
1902).
Adami, Abbott and Nicholson (1899) injected typical B. coli
into the circulation of a rabbit and obtained diplococcic forms
of the organism from the liver after death. They isolated from
ascitic fluid in another case similar diplococci which stained
irregularly and were non-motile, did not ferment sugars or
produce indol, and formed colonies on agar closely resembling
those of S.pyogenes. By intraperitoneal passages through three
guineapigs these organisms were converted into typical B. coli.
Schmitt (1911) claims to have so modified a strain of
B. paratyphosus (Fliigge) by passage through a calf that it
afterwards gave the agglutinative reactions of B. enteritidis
Gaertner.
Calves, monkeys, rabbits, guineapigs, rats, mice and birds
may all be used for this purpose.
(c) A modification of the last-named method consists in
growing organisms in a ceUoidin sac within the body cavity
of an animal . Martin ( 1 898) increased the virulence of a strain
of B. diphtheriae by growing it in a celloidin sac in the peri-
toneal cavity of a rabbit.
(d) Growth in the living tissues of an animal host is
another method of inducing variation. It is only in the animal
body that the actinomyces produces its characteristic rays or
clubs (Bowlby and Andre wes, 1913).
26 CONDITIONS MODIFYING [CH. n
Ohlmacher (1902) claimed to have changed typical B.
diphtheriae into Hoffmann's bacillus by 48 hours subcutaneous
growth in a rat previously immunised.
The "solid-staining" type of B. diphtheriae has been
inoculated into a guineapig and the "granular" type has
been recovered subsequently from the site of the inoculation
(Denny, 1903).
Such a method is not invariably successful. For example,
Baldwin (1910) grew the human type of tubercle bacillus in
the living tissues of the cow for nineteen months, in the hope
of modifying its characters to those of the bovine type, but
without success.
(e) During the course of a disease the organism responsible
is not infrequently observed to undergo modification with
respect to one or another character. Thus in diphtheria, as
convalescence is reached, the "granular" forms of the bacillus
give place to "solid-staining" types (Gorham, 1901).
In the chronic stages of cerebrospinal fever the meningo-
coccus isolated from the spinal fluid is found to have lost in
some cases its power to ferment dextrose (Connal, 1910).
Ark wright (1909) found bacillary forms of the meningococcus
in the spinal fluid in several cases of cerebrospinal meningitis.
Adami, Abbott and Nicholson (1899) isolated from the
ascitic fluid in cases of cirrhosis, strains of B. coli (already
described, vide p. 25) possessing unusual morphological,
cultural and fermenting characters. Similar variants of B. coli
were obtained from an inflamed gall-bladder.
Foa (1890) injected the pneumococcus into a rabbit and
after its death isolated strains from the lung and from the
spinal fluid which produced lesions of two distinct types when
injected into other rabbits. He proved by experiment that
the difference between the two strains was due to differences
in the amount of oxygen available for them in the lung and in
the spinal canal.
Rosenow (1912-13) describes a certain streptococcus,
isolated from cases of epidemic sore throat which exhibited
unusual and distinctive morphological and cultural characters.
"The strains isolated from the peritoneal exudate and blood
CH. n] THE CHARACTERS OF BACTERIA 27
showed them to a greater degree than those isolated earlier in
the attack or from the tonsils at the same time. After cultiva-
tion on blood-agar it was noticed that the strains from the
tonsils soon lost any distinctive peculiarities, whilst those from
the exudate retained them longer." He concludes that the
unusual character of the organism was directly due to residence
in the body fluids and was accentuated as the disease advanced.
Leutscher (1911) found that pneumococci isolated from
the lung in acute pneumonia after the crisis were more virulent
than those isolated earlier in the illness.
(/) In some cases the so-called "carriers" after all
symptoms of a disease have subsided, the particular organism
concerned resists all attempts to eradicate it and continues
for an indefinite period to grow and multiply at the site of
the original lesion. In such cases the organism may become
modified in the course of time. This is true more especially
of its virulence, but other characters may be involved. Wilson
(1910) mentions a strain of B. typhosm, isolated from the
urine of a typhoid carrier, which had acquired the power to
ferment lactose.
CHAPTER III
A CONSIDERATION OF THE EVIDENCE
BEFORE discussing in detail instances of variation and of
" transmutation," it is necessary to consider the value of the
evidence offered in support of them and the possible sources
of error, in the way of both observation and deduction.
1. First and foremost must be considered the possibilities
of contamination. A single colony, even after repeated
subculture and replating, may not represent an absolutely
pure culture and cannot be proved not to contain a single
bacterium of another species, the appearance of which in
greater numbers at a later stage of the experiment might
suggest variation.
The importance of contamination as a source of error is
so obvious that efficient precautions are taken in almost all
cases to eliminate it.
Barber (1908) has described a method by which a strain
can be grown from a single organism thus ensuring the
purity of the culture. By means of a glass pipette possessing
an extremely fine aperture no larger than the diameter of
a yeast cell a single organism is removed under the micro-
scope from a culture which has been repeatedly diluted.
2. The original infection, however, with which the in-
vestigator is dealing may itself be a " mixed " one and this
fact may be overlooked in two ways :
The conditions of cultivation may favour the growth of
one organism and inhibit that of another, so that the first
may be present in such overwhelming preponderance that
the second is for the time being completely submerged, as
it were, and undetected. If the conditions change, as a result
either of the activity of the organisms themselves or the
intervention of the investigator, the balance may be restored
and may even swing in the opposite direction, so that the
CH. in] A CONSIDERATION OF THE EVIDENCE 29
second organism now predominates in numbers to such an
extent that the first one is lost sight of. Such a train of
events might be misinterpreted and the assumption made
that variation or even transmutation had occurred. Horrocks
(1911) investigated the urine of a typhoid carrier which "in
certain dilutions always gave practically pure cultures " of
B. typhosus, although B. coli was present in small numbers.
When the urine was diluted, however, an enormous increase
in the B. coli occurred and the B. typhosus rapidly disappeared.
Klein (1903-4) observed the same sequence of events in the
peritoneal cavity on injecting a strain of B. coli which
contained typhoid bacilli.
Smirnow (1908) quotes experiments in support of the
opinion that streptococci inhibit the growth of the Klebs-
Loeffler bacillus, but only for a time and he explains in this
way the appearance in some cases of the bacillus in what, a
few hours previously, had appeared to be an almost pure
culture of streptococci.
Two other instances may be given. The sputum of a
patient suffering from pneumonia and a swab from the throat
in a case of diphtheria may contain, in addition to the virulent
organisms which cause these diseases, avirulent organisms
closely resembling them the saprophytic pneumococcus and
the pseudodiphtheria bacillus respectively. The saprophytic
bacteria in both cases will grow at a temperature of 20 22 C.
though the virulent types are both unable to do so. It is
evident that, other things being equal, the temperature of
the incubator will decide which of the two types, the virulent
or the avirulent, will predominate in the culture. The other
one, although actually present, may then easily be overlooked
unless an alteration in the temperature gives it, in turn, the
ascendancy. In the latter event the change in virulence and
in other characters would have the appearance of a " variation "
brought about by change of temperature. It would actually
be due to a "contamination" which had been previously
overlooked.
A second organism may, again, escape detection because
its recognition is made dependent upon some one character
30 A CONSIDERATION OF THE EVIDENCE [CH. in
alone such as its morphology where naked eye and micro-
scopic appearances are relied upon, or its virulence in the
case of animal inoculation, or its fermenting power when the
culture is tested by being "put through the sugars." The
organism may be atypical in respect to the particular character
the observer depends upon for its detection, and its subsequent
discovery will then lead to erroneous conclusions. A knowledge
of the extent to which organisms may be atypical in one or
other character is the best safeguard against such an over-
sight.
The most thorough identification is demanded at the con-
clusion of an experiment no less than at its commencement, and
the strictest rules must be observed before the continuity of
two forms differing from each other is regarded as established.
Such continuity may be impossible to prove even when we
are dealing with a " pure culture." A certain number of
organisms in a pure culture may undergo variation while the
rest of the strain remain true to type. From time to time,
as the conditions of growth change, now the variants may
predominate almost to the exclusion of the original stock,
and now the original stock may predominate almost to the
exclusion of the variants, so that, following the variation,
reversion may appear to take place, and yet there may
actually be no continuity in the latter case between the
variants which are dying out and the original stock which is
again asserting itself.
3. In the living tissues the possibility of secondary in-
vasion must be borne in mind. For example, the leptothrix
forms which McDonald (1908) describes in the spinal fluid
in cerebrospinal fever, as this writer himself recognises,
cannot be regarded as morphological variants of the meningo-
coccus without definite proof of identity.
Again, the pathogenic effects in a given case must not be
attributed to an organism isolated from the tissues unless
adequate proof is forthcoming of its being in fact the cause
and not a secondary invader.
Forbes in 1903 drew attention to the frequency with which
diphtheria bacilli were to be found in the ear discharges of
CH. in] A CONSIDERATION OF THE EVIDENCE 31
patients suffering from scarlet fever. The bacilli were present
in 32 out of a series of 40 cases examined and sometimes
greatly outnumbered the other organisms present.
Lustgarten's bacillus (1884) in syphilis and Sanarelli's
Bac. icteroides (1897) in the case of yellow fever may be
quoted as examples of secondary invaders to the presence of
which diseases were wrongly attributed, and many other
instances might be given.
Bacteria may be present in healthy organs. Ford (1900)
examined the liver and kidneys of healthy animals after death
with the most stringent precautions against contamination
and found that at least 80 per cent, contained bacteria of
various kinds.
Dudgeon (1908) states that staphylococcus albus can be
cultivated from the great omentum in many healthy animals
and quotes many examples to show that pathogenic organisms
can exist in the body for long periods without giving rise to
any symptoms. Savage (1907-8) has recorded the presence of
B. Gaertner in the intestines of healthy young calves. Zwich
and Weichel (1910) found that out of 177 healthy mice, 28
contained B. Aertryck in their faeces.
Post mortem invasion must be guarded against, for after
the death of an animal secondary infection is extremely likely
to occur. Dudgeon and Sargent (1907) record a case of
pneumococcal peritonitis in man, in which the peritoneal
exudate one hour after death gave a pure culture of pneu-
mococci, whereas 26 hours later B. coli alone could be
recognised in the same exudate.
4. The repetition of an experiment with an identical
result as regards the variation produced is valuable con-
firmatory evidence, particularly in the hands of different
investigators. Inability to repeat the phenomenon, though
by no means disproving its original occurrence, does to some
extent discredit it.
5. The constancy of the new feature, particularly on
subculture, is of importance both as enabling one to exclude
various errors of observation and as indicating the funda-
mental character of the change. On the other hand a tendency
32 A CONSIDERATION OF THE EVIDENCE [CH. in
to revert more or less quickly to the original type on the
removal of the modifying influence indicates racial stability
in character and minimises the significance of the modification.
This aspect of the problem will be referred to later (vide p. 144).
It is necessary, however, to emphasise at this point the danger
of assuming a change in character to be permanent because
reversion has not occurred within a certain period, even a
lengthy one. A strain of B. ruber, for example, may show no
trace of colour for 12 months together under certain conditions
and yet retain undiminished its power to produce pigment
in more favourable circumstances (Laurent, 1890). In other
cases reversion occurs without any modification in the con-
ditions of growth but apparently spontaneously and this after
long periods of time have elapsed.
6. The necessity for perseverance in following a particular
line of investigation needs no less emphasis. Twort (1907)
took two years to train a particular strain of B. typhosus to
ferment lactose a result which Penfold (1910 A) failed to
achieve in the case of over a dozen strains after a 15 months'
trial. Coplans (1909) grew a strain of B. tetani on a gelatin
medium for 90 days before liquefaction occurred. Eyre and
Washbourn (1899) found that to raise a particular strain of
avirulent saprophytic pneumococci to full virulence by animal
"passage" no less than 53 successive inoculations were
required. Goodman (1908) in his attempts to modify by
artificial selection the acid production in a strain of diphtheria
bacilli, made 18 transfers before any result was perceptible.
In all these cases, if the experiments had concluded earlier,
negative results might have been obtained and a claim based
on this evidence for stability in character which a more
prolonged investigation would have shown not to be justified.
7. Faultless technique is essential to accuracy in results.
In carrying out agglutination tests, for example, the utmost
care and patience is demanded even from a practised observer
if his conclusions are to be of any real value, and much of
the confusion which at present exists on the subject of
agglutination is no doubt to be attributed to bad workman-
ship.
CH. in] A CONSIDERATION OF THE EVIDENCE 33
A ccurate observation is of no less importance. For example,
the particular constituent of the bacterial protoplasm which
retains a stain may be unevenly distributed throughout the
cell. A " solid-staining " type of bacillus may thus give rise
to one exhibiting "polar staining" as in the Klebs-Loeffler
bacillus and also, under certain conditions, B. coli and B.
typhosus. If the demarcation between the staining and the
non-staining material be very definite a bacillus showing
polar staining may closely resemble a diplococcus andconfusion
arise unless careful observation be made.
A deceptive appearance may in the same way be produced
by the uneven staining of a bacterial filament. Wilson (1906)
found that B. coli under the influence of urea developed
filamentous forms. The staining material in these filaments
under certain conditions became segmented, although the
organism as a whole showed no sign of segmentation, with
the result that the filament presented the appearance of a
chain of cocci. Ainley Walker and Murray (1904) had previously
observed the same phenomenon in the filamentous forms of
B. typhosus produced under the influence of methyl violet.
Treatment with silver nitrate may render more apparent
the division of a diplococcus or a filament into individual
cells.
8. In other cases where the actual technique is perfect
and the recognised method is carried out in every detail, the
method itself may be at fault ; conflicting results in such
circumstances would not be due to any variation in the
character of the organism concerned but to such factors as
the composition of the medium, the age of the culture, the
time- allowance made for a "positive result" to declare itself,
and so on.
A few examples will suffice to show the importance of
such factors.
(a) The composition of the medium. Sugar containing
media may be unsuitable on account of impure commercial
sugars being used in their preparation or from their being
sterilised in vessels made of certain kinds of glass (W. B. M.
Martin, 1911); they may undergo decomposition during the
D. 3
34 A CONSIDERATION OF THE EVIDENCE [CH. in
process of sterilisation or they may deteriorate if kept for
some time before being used, and failure to guard against
these sources of error may lead to discordant results.
The streptococcus pyogenes normally fails to ferment both
saccharose and raffinose in broth, but it produces acidity in
old media containing either of these sugars (Martin, 1908-9).
Fisher (1909) on the other hand found that diphtheria and
diphtheroid bacilli which gave fermentation tests readily in
fresh beef serum, failed to do so if the serum were old.
This observer and Theobald Smith (1899) both state that
even virulent diphtheria bacilli may fail to yield toxin if the
medium in which they are growing contains more than a
trace of sugar ; while Williams (1902) found many strains,
which were non-pathogenic when inoculated from ordinary
broth, were highly toxic when inoculated from serum culture
or ascitic broth.
The neutral red reaction in the case of B. coll not
infrequently fails but Moore and Re vis (1905) claim that if
lactose is substituted for glucose in the broth a positive result
is invariably obtained with this organism.
Glenn (1911) observes that the acidity produced in a
medium by the fermentation of its carbohydrate constituents
inhibits the production of indol and may account for the
failure of the indol test.
Wood (1889) has stated that the presence of glycerine in
a medium will prevent the liquefaction of gelatin by organisms,
not by interfering with their power of fermentation but by
offering them a pabulum they prefer.
Again, if the medium used in the case of fermentation
tests is itself markedly alkaline, the production of acid in
small quantities may be completely masked, since it merely
results in a diminution in alkalinity and this requires special
means of detection. Miss Peckham (1897) quotes Timpe to
the effect that all albuminous bodies give an alkaline reaction
to litmus and it is well known that alkaline products are
formed by the breaking down of peptone, so that the use of
litmus as an indicator in peptone holding material makes the
alkaline reaction prominent even when a considerable quantity
CH. in] A CONSIDERATION OF THE EVIDENCE 35
of free acid is really formed. Clark (1910) states that Hofmann's
bacillus produces slight but definite acidity in dextrose broth
if phenol-phthalein be used as an indicator, whereas if litmus
is used the reaction always appears alkaline.
(b) The age of the culture is also a factor of importance.
MacConkey (1909) finds that the indol reaction in the case
of B. coll is not given by a 2 or 3 days' culture ; the latter
should be nearly a week old in order to give a positive result.
A young culture of B. diphtheriae is unable to ferment
glycerine and lactose though an older culture will usually do
so (Muir and Ritchie). An old culture of cholera will not
liquefy gelatin (Wood, 1889). Graham Smith (1906) has
pointed out that many strains of diphtheria bacilli do not
grow well in broth when first isolated from the throat and
therefore do not produce acidity at once.
(c) The time alloivance. In the case of many sugar
fermenters an incubation period of 48 or even 72 hours is
required before acidity becomes apparent, and in the case of
other organisms a similar " latent period " may elapse before
the appearance of pigment.
Still longer observation is sometimes necessary. Klotz
(1906) describes a coliform organism which did not produce
indol until the 20th day. Petrusky (1889-90) showed that in
the case of B. typhosus a certain slow fermentation of lactose
does take place in litmus whey although the organism is
regarded as a non-fermenter of lactose. Penfpld (1910 B)
states that the same organism ferments dulcite a property
usually denied to it if the experiment is prolonged for 2 or
3 weeks. Bahr (1912) describes a dysentery bacillus of the
"Flexuer" type which, after 4 days incubation, only fer-
mented mannite but fermented maltose and saccharose also,
after 15 days' incubation. Wilson (1910) states that he has fre-
quently isolated bacilli from the intestine which required from
9 to 21 days to produce acidity in lactose litmus broth and
several more days to produce gas.
In other cases the change in the reaction is reversed after
an interval. Thus, Bahr mentions another "Flexner" bacillus
which produced a feeble acid reaction in mannite at the end
32
36 A CONSIDERATION OF THE EVIDENCE [CH. m
of 24 hours but after ten days incubation gave a definitely
alkaline reaction.
It is obvious from these facts that the question of the time
allowance is of great importance. This point will be considered
further in connection with variations in fermenting power
(vide p. 66).
9. Finally, pathological research and clinical observation
must go hand in hand. The former, if it is divorced from the
latter, is beset with dangers.
A certain patient's blood, in the laboratory, may give at
one time a positive Widal's test and at another some weeks
later fail to do so. The knowledge that on the first occasion
the patient was the subject of jaundice would suggest a simple
explanation (Griinbaum, 1896) for a phenomenon otherwise
difficult to elucidate.
Similarly the knowledge that sodium benzoate was being
administered to a patient suffering from cystitis would afford
an explanation of the fact that the strain of B. coli contained
in the urine of the patient showed a greatly diminished power
of gas production in dextrose (Penfold, 1911 A).
Again, altered pathogenicity may be falsely attributed to
a strain of organisms if clinical observation is neglected.
A certain disease may be latent in a patient that is to say,
present without giving rise to any noticeable symptoms. The
constitutional disturbances arising from infection by the
organism in question may "light up "this pre-existing disease
and the symptoms of the latter then be incorrectly credited
to the invading organism.
CHAPTER IV
VARIATIONS IN MORPHOLOGY
VARIATIONS in morphology will be considered under three
heads: (A) zoogleic forms, (B) individual organisms, (C)
colonies.
A. ZOOGLEIC FORMS.
One remarkable feature of the bacteria or schizomycetes
is the tendency they show when multiplying to become massed
together, not indiscriminately but in an orderly arrangement,
to form "zoogleae." These forms display an extraordinary
diversity of shape and structure. Thus, a single bacillus as
a result of alternate elongation and division in a transverse
plane may give rise to a long filament consisting of a row of
cylindrical cells placed end to end. In other cases a number
of organisms may be crowded together in a round gelatinous
mass, their swollen cell walls fusing to form a mucilaginous
matrix in which they lie embedded for an indefinite period.
The shape and structure of these zoogleae are not fortui-
tous but appear to be designed in many instances to attain
some definite object of advantage to the organism, and may
thus form a stage in its life history. This is the case for
example in the Bacterium raditicola, the nitrogen-fixing
organism found in the nodules on the roots of leguminous
plants. It first enters the root hair from the soil; it then
assumes a filamentous form and in a manner comparable to
the downward growth of the pollen tube from the stigma to
the ovary pushes its way along the interior of the hair as
a long slimy thread until it penetrates the tissues of the root
itself.
Again the Beggiatoa versatilis, a vegetation often seen at
the mouth of drain pipes, may be observed to send out from
a whitish gelatinous ground mass, long oscillating filaments
38 VARIATIONS IN MORPHOLOGY [CH. iv
which emerge after sundown and the next day split up into
innumerable little bacteria rods (Kerner and Oliver).
The zoogleae in which bacteria are massed together are to
be regarded as a resting stage in their life history. The swollen
envelope or matrix in which they are embedded, and which
in some cases becomes hard and chitinous, being protective in
character.
It is important however to recognise the fact that these
zoogleae are merely conglomerations of a number of organisms
and are not, strictly speaking, individuals themselves. Too
great stress must not be laid, therefore, on their formation
and the changes they undergo as evidence of variation on the
part of the individuals composing them.
A regiment of soldiers during manoeuvres is composed of
a number of individuals, all of the same kind, comparable to
pathogenic bacteria. It assumes various forms from time to
time that of serried ranks when marching, a filamentous form
when advancing in single file, a "square" when awaiting a
cavalry charge and yet another appearance during its resting
stage when bivouacked for the night. Again, a mass meeting
or "demonstration" of coal miners, composed of a different
type of individual all again of the same kind, comparable
to a harmless pigment-producing organism shows quite a
different formation, namely that of an irregularly shaped crowd,
the units of which are arranged somewhat concentrically.
There is sometimes a tendency to the formation, at the peri-
phery, of smaller collections or nodules showing a similar
concentric arrangement. There is a constant tendency on the
part of a regiment or a miners' "demonstration," wherever we
find them, to reproduce exactly the forms described as typical
of each. A regiment of soldiers or a crowd of pitmen may be
regarded as a separate entity in one sense, but neither is an
individual in the sense that a tree, composed of vegetable cells,
is one. There is no interdependence of one part upon another
in a body of troops or a crowd. They are temporary and can
be dispersed, the individual units surviving though separated
from each other. Moreover the forms they assume are not
invariable. A regiment of soldiers, if a certain controlling
CH. iv] VARIATIONS IN MORPHOLOGY 39
influence is removed, or in response to a particular stimulus-
such as the attraction of a boxing-match may assume the
form of an irregular crowd concentrically arranged. A certain
controlling influence in the case of the miners, or a common
spontaneous impulse, may result in their marching in military
formation. In other words, a collection either of the pathogenic
organisms or of the harmless pigment producers, may assume
temporarily a formation rightly regarded as characteristic of
the other; but we should be mistaken in supposing on this
account that the soldiers were being transformed into miners,
or vice versa.
The development of zoogleic forms may occur spontane-
ously, or it may be brought about artificially.
I. ZOOGLEIC FORMS OCCURRING SPONTANEOUSLY.
These may represent a regular phase in the life history
of the organism; on the other hand, they may occur quite
irregularly as an occasional variation either in cultures on
artificial media or in the living tissues in which case one must
regard the change as representing a phase in the life history
of the organism at an earlier stage in its evolution.
Perhaps the earliest account of zoogleic forms occurring
in the life history of a micro-organism was that given by
Ray Lankester in 1873, with reference to the non-pathogenic
Bacterium rubescens. The units of this bacterium were
observed to become aggregated into a multitude of forms,
protean in their variety stellar, globose, massive, arborescent,
eaten ular (or chain-like), reticular, tessellate and so on. (Dia-
grams of each of these forms are appended to the original
article.)
The tubercle bacillus indicates its relationship to the
streptothrices by forming in old cultures a branching filament,
sometimes with "clubbed" ends, while in the living tissues,
under certain conditions, it gives rise to a radiating structure
similar to that of the actinomyces (Muir and Ritchie).
The bacillus of glanders, similarly, on artificial culture may
exhibit short filamentous forms, and under certain conditions
40 VARIATIONS IN MORPHOLOGY [CH. iv
in the living tissues show branching filaments and "clubbing"
(ibid.).
The Klebs-Loeffler bacillus in young cultures, on serum and
agar-agar, likewise shows clubbed and branched forms.
The bacillus of anthrax, both on artificial media and in the
living tissues, forms leptothrix-like chains or filaments. These
may be observed in a three hours' culture of the bacillus in
a drop of aqueous humour (Marshall Ward and Blackman,
1910).
Adami (1892) describes B. typhosm as forming long fila-
ments when grown on potato. Many observers have recorded
the same phenomenon in cultures of B. coli. For example,
Ohlmacher (1902), Revis (1908) and Wilson (1908) isolated
leptothrix forms of B. coli from the heart's blood in a case
of septicaemia, from milk and from urine respectively, the
organism in each case forming a dense network of branching
filaments.
Ritchie (1910) isolated leptothrix forms of B. influenzae
from the cerebrospinal fluid in certain cases of meningitis, and
similar forms of the "pseudo-influenza" bacillus from the lung
in pneumonia. The causal factor in two of these instances
B. coli isolated from urine and B. influenzae from the spinal
fluid in meningitis was probably the presence, in both these
fluids, of urea. Connal (1910) showed that in meningitis the
spinal fluid may contain as much as "5 per cent, of urea, and
Wilson (1906) showed that urea provokes the development of
leptothrix forms in many organisms.
II. ZOOGLEIC FORMS ARTIFICIALLY PRODUCED.
1. The addition of various chemical substances to the
culture medium in which an organism grows, leads to the
development in many cases of leptothrix forms.
Peju and Rajat (quoted by Wilson, 1910) observed that
salts, and Almquist (ibid.) noted that seivage, had this effect on
B. typhosm. Walker and Murray (1904) showed that certain
dyes, particularly methyl violet, had the same action on
B. typhosm, B. coli and the cholera organism. Wilson (1906),
by adding urea to the culture media, obtained leptothrix forms
CH. iv] VARIATIONS IN MORPHOLOGY 41
of B. typhosus, B. coli, B. pyocyaneus, B. enteritidis Gaertner,
and B. pneumoniae Friedlander ; Adami, Abbott and Nichol-
son, by the addition of human saliva, or a trace of bile,
obtained the same result with B. coli. Growth in an acid
lactose containing medium also developed filamentous forms
of this organism. They quote Schmidt's observation that
growth in caustic soda broth had the same effect.
Adami (1892) observed that B. pyocyanem took the form
of a filament or, in some cases, developed into close spirals and
S-shaped forms under the influence of /9. naphthol, alcohol,
potassium bichromate, boric acid ; and Pakes (1901) noted that
the nitrates of sodium, potassium, ammonium, and lithium
developed in the same organism filamentous forms which
showed spurious branching and resembled a cladothrix.
Wasserzug (1888) found that B. prodigiosus formed long
bacilli and spirilla if grown in the presence of antiseptics.
Tartaric acid had the same effect and by prolonged growth
in media containing this acid and subjection to a temperature
of 50 C. subsequently for a few minutes, a race of long bacilli
was obtained which retained its new character "permanently"
that is to say on its return to ordinary media.
2. The formation of zoogleae may be provoked by alteration
in temperature. Rodet (quoted and confirmed by Adami,
Abbott and Nicholson, 1899) found that a culture of B. coli
at a temperature of 44-45 C. developed within a few hours
very long filaments.
3. The absence of oxygen may have the same effect. Wood
(1889) observed "torula" forms of the cholera bacillus when
grown in bouillon anaerobically. Noguchi (1910) found that
B. bifidus communis only exhibited its bifurcating phase in
anaerobic culture.
4. Exposure to the ultra violet rays leads to the formation
of long filaments in B. anthracis (Henri, 1914).
5. Growth in the animal body develops on the part of the
actinomyces its characteristic rays or clubs. These are not
seen in artificial cultures (Bowlby and Andre wes).
42 VARIATIONS IN MORPHOLOGY [OH. iv
B. MORPHOLOGICAL VARIATIONS IN INDIVIDUAL
ORGANISMS.
Morphological variations in individual bacteria may occur
as normal phases in its life history, or they may develop in
response to changes in their environment.
I. PLEOMORPHISM IN THE LIFE HISTORY.
While many bacteria are only known under certain forms
and are regarded as a micrococcus, a bacterium, a bacillus or
a spirillum, others are known which in the course of their
development pass through several such forms and are called
"pleomorphic."
The non-pathogenic Bacterium rubescens already men-
tioned (Ray Lankester, 1873) affords a good example of the
various phases an individual organism may pass through in its
life history forms described as spherical, biscuit-shaped, rod-
like, filamentous and acicular succeeding each other in turn 1 .
Amongst pathogenic organisms that of cholera affords a
good example, the characteristic comma-shaped vibrio or
spirillum giving place to a coccus or to a straight thread
(Haffkine, 1895).
In old cultures of the meningococcus bacillary forms make
their appearance (Arkwright, 1909). Young cultures of B. coli
show not only typical bacilli but also small oval rods and tiny
coccus-like forms (Gordon, 1897). In very young cultures of
B. diphtheriae "solid" types largely predominate, but in a few
hours these give place to "granular" types (Denny, 1903).
The variations in morphology displayed by B. diphtheriae
are many of them so characteristic as to be of value in the
identification of the organism, and with this object have
been classified by Westbrook, Wilson and McDaniel (1900).
Similarly Gordon (1900-1) observed that the streptococcus
of scarlatina was characterised by a tendency to take the form
1 Miss M. C. W. Young has recently reported some observations of great
interest in this connection, revealing a similar pleomorphism in bacteria which
extended, in her experiments, over a cycle of 14 days. (Brit. Med. Journ. 1914,
11, p. 710.)
CH. iv] VARIATIONS IN MORPHOLOGY 43
of a spindle or rod, in which case it was difficult to distinguish
it from B. diphtheriae. At the same time its tendency to
assume a bacillary form afforded a valuable means of distin-
guishing it from S. pyogenes.
II. MORPHOLOGICAL VARIATIONS DUE TO ENVIRONMENT.
1. These maybe associated with differences in geographi-
cal distribution. For example, Schultz (1909) found that in
Cleveland, U.S.A., during the twelve months covered by the
investigation, "barred" forms of the diphtheria bacillus had
almost disappeared; during the same period in Boston and
Providence another observer noted that "barred" forms were
unusually common while "granular" forms were very rarely
met with.
2. Prolonged cultivation may influence morphology.
Mohler and Washburn (1906) found that bovine tubercle
bacilli after 11 years' cultivation had become changed into the
human type.
3. The crowding together of colonies on the surface of the
medium also influences morphology. In cultures of B. diph-
theriae, under these conditions, the change from the "solid" to
the "granular" type takes place much earlier than usual
(Denny, 1903).
4. Changes in the medium employed may lead to changes
in morphology. Gordon (1900-1) published photographs
showing that the streptococcus associated by Klein and him-
self with scarlet fever may form, on serum, rods which closely
resemble the diphtheria bacillus, though in a liquid medium
it grows in typical streptococcal form. This fact may afford an
explanation of the observations, recorded by Duncan Forbes
(1903) and others, as to the prevalence of B. diphtheriae in
the ear discharges of patients suffering from scarlet fever,
without giving rise to symptoms of diphtheria and uninfluenced
by antitoxin.
Ohlmacher (1902) and other observers have called attention
to the fact that streptococci from the throat in cases of
tonsilitis may, on Loeffler's serum, assume the form of bacilli
closely simulating B. diphtheriae.
44 VARIATIONS IN MORPHOLOGY [CH. iv
Rosenow (1912-13) describes a streptococcus which de-
veloped unusual morphological and cultural characters as the
result of growth in unheated milk.
B. coli in ascitic fluid and in bile may assume diplococcic
form (Adami, Abbott and Nicholson, 1899). Jenner (1898)
found that B. coli isolated from water was less thick and
opaque than normal B. coli, this distinction disappearing,
however, after growth in milk.
Changes in the reaction of the medium may bring about
changes in morphology, bacilli giving place to cocci and diplo-
cocci and vice versa (Adami, 1892).
5. The addition to the medium of various cliemical sub-
stances influences morphology. The presence of urea converts
Micrococcus prodigiosus into a bacillus (Wilson, 1906), and
Bacillus Pestis into a coccus grouped singly or in pairs or in
short chains (ibid.). B. enteritidis Gaertner on urine-agar
develops into a coccus (ibid.), while B. typhosus and B.
pyocyaneus grown in carbolic acid (1 in 600), and creosote
(1 in 1000), assume the forms of non-motile cocci or diplococci
(Adami, 1892).
Deceptive appearances are sometimes produced by the
unequal distribution of the staining material in an organism
under conditions such as those we are discussing. A bacillus
may under the microscope appear to be a diplococcus and
a filament resemble closely a chain of cocci.
Haslam (1898) found that the shape of B. coli communis
depended upon the composition of the medium in which it was
growing. If the composition of the medium were changed
every 24 or 48 hours the shape of the organism changed with
it. He found that the bacillus was longest (in proportion to
its breadth) in media rich in nitrogenous substances, such as
proteid and ammonium tartrate, and shortest in glucose media
to which little of such nitrogenous material had been added.
6. Exposure to ultra violet rays in the case of B. anthracis
has been shown to change the bacilli to cocci and diplococci
(Henri, 1914).
7. Electrolysis. Electrolysis may produce changes in the
morphology and staining properties of bacteria. Russ, for
CH. iv] VARIATIONS IN MORPHOLOGY 45
example, has noted the production of elongated forms of B. coli
in urine, with altered reaction to Gram's stain, as a result of
the passage of a galvanic current of ^th rn.a. strength for
one hour. The modification was produced in B. coli present
in the human bladder in a case of cystitis and also in a speci-
men of urine outside the body, and it persisted for many
months.
8. Symbiosis may affect morphology. The presence of
streptococci in a young culture of B. diphtheriae hastens the
appearance of "granular" types of the latter (Denny, 1903).
Smirnow (1908) found that symbiosis of B. diphtheriae on
culture media with (a) a streptococcus, (b) the meningococcus,
and (c) an unidentified bacillus derived from a case of acute
rhinitis, led in all three cases to the appearance of coccoid
involution forms of the diphtheria bacillus. The experiment
in the case of the unidentified bacillus was repeated in another
way, the two organisms being grown in the two compartments
of a double celloidin sac which was inserted into the peritoneal
cavity of a rabbit. In the place of the diphtheria bacillus he
found a Gram-positive coccus which, however, on Loeifler's
blood serum reverted in 24 hours. A repetition of the experi-
ment gave exactly the same result.
Lesieur (1901, quoted by Clark, 1910) claimed that the
pseudo-diphtheria bacillus may assume the morphological
characters of the Klebs-Loeffler bacillus as a result of
symbiosis with aurococcus aureus.
9. Growth in the living tissues will sometimes modify the
morphology of organisms. Gorham (1901) observed that, as
convalescence from diphtheria advances, "granular" types of
the bacillus give place to "solid" types, and he attributes the
change to the action of the body fluids of the now immune
patient.
Adami, Abbott and Nicholson (1899) describe forms of
B. coli, isolated from the liver in normal and diseased animals
(cow, sheep, rabbit, guineapig) and in man, which resembled
diplococci in many cases, and in others short chains of three
or four cocci. Similar forms were obtained from the bile and
from ascitic and peritoneal fluids, and were produced on adding
46 VARIATIONS IN MORPHOLOGY [CH. iv
guineapig bile to culture media, and they attribute the
modification to the action of the body fluids. Further, they
injected B. coli into the circulation in rabbits and found
subsequently enormous numbers of this diplococcic form of
the organism in the endothelial cells lining the hepatic vessels
and also in the cells of the liver, in the bile and in the kidneys.
These were detected within 30 to 60 minutes of the injection.
In some instances the modification was so marked that it was
not possible by passage or other means to obtain complete
reversion to type. In other cases after passage through
guineapigs reversion took place. The change was associated
with irregular staining, and with loss of motility, of fermenting
power and of power to produce indol. They found that
B. typhostts underwent a similar modification under the same
circumstances.
Jenner (1898) found that the difference in morphology
already mentioned in the case of B. coli isolated from water
disappeared after passage.
Ark wright (1909) and other observers have described a
micrococcus closely resembling the meningococcus, found in
almost pure culture in the spinal fluid in many cases of
meningitis, which is characterised by a tendency to assume a
bacillary form.
Ohlmacher (1902) inoculated a guineapig with a long
" granular " type of B. diphtheriae but the organism recovered
later from the site of the inoculation proved to be of the
short "solid " type. The experiment was twice repeated with
the same result. In two other experiments with different
strains of B. diphtheriae the same observer found that during
"passage" through a guineapig the reverse change occurred,
a short "solid" type of organism being injected into the
animal and a long granular type recovered from the spleen
and liver after death.
Mohler and Washburn (1906) claim that, by prolonged
growth in the living tissues of a suitable animal host, one type
of tubercle bacillus can be so modified with respect to its
morphological characters as to become indistinguishable from
another type. Baldwin (1910), however, grew a strain of the
CH. iv] VARIATIONS IN MORPHOLOGY 47
human tubercle bacillus in the tissues of a cow for 19 months
without effecting any change in it.
Gordon (1900-1) found that the tendency exhibited by
S. scarlatinae to assume bacillary form on certain media was
suppressed after passage through the guineapig but was
increased in some cases after passage through the mouse.
C. VARIATIONS IN COLONIES.
A given organism when grown on the same kind of medium
and under the same conditions always tends to produce a
colony of a particular size, form and appearance. Such a
colony is regarded as " typical " of the organism in question
and considerable importance is attached to its character as a
means of isolating the particular organism and identifying it.
The substitution of a macroscopic for a microscopic appearance
possesses such obvious advantages that the former is frequently
made use of instead of the latter and the more constant its
features are found to be, the more reliance is placed upon it.
The question therefore arises, how far can the appearance of
its colonies be trusted as a means of identifying an organism?
MacConkey (1909) in speaking of colonies makes two state-
ments. (1) The colonies of an organism may vary even on the
same plate. (2) Organisms of quite different character may
produce colonies almost identical. Both of these statements
may be confirmed by reference to a common organism such
as B. coli.
1. It is recognised that an organism will yield different
types of colonies when grown upon different kinds of media.
The character of the colony depends upon the composition of
the medium. If, therefore, the material of a culture plate or
tube should present slight differences in composition in
different parts of its surface, it is reasonable to expect slight
corresponding differences in the colonies of an organism
growing upon it. Such an explanation is, however, inadequate
to explain the wide differences frequently observed.
Savage (1904) carried out an elaborate investigation in
order to ascertain to what extent colonies of B. coli on
gelatin conformed to the character commonly accepted as
48 VARIATIONS IN MORPHOLOGY [CH. iv
typical of this organism. He examined 72 strains, derived
from half a dozen different sources. Out of this number 50
strains formed typical colonies but many of them on further
cultivation gave rise to atypical colonies, which later, however,
reverted to the common type. The remaining 22 strains
(30'5 per cent.) all yielded atypical colonies. Many of these
colonies bore no resemblance whatever to the common type
and showed no tendency to revert to it ; moreover, they
differed as much from each other as from the typical colony.
(Photographs showing the different appearances are to be
seen with the original article.) One strain (No. 160) replated
after varying intervals eight times in the course of several
months, gave rise to no less than 14 distinct types of colony,
all of them atypical. In other respects the organisms proved
to be in every case' typical B. coli in pure culture. In his
opinion the material from which the organism was isolated
considerably influenced the type of colony formed.
2. The second statement is confirmed by the same in-
vestigator who observed that colonies whose appearance was
absolutely typical of B. coli, might be composed of different
organisms altogether. Many years previously Klein (1899-00)
pointed out that it was not safe, from their appearance alone,
to regard particular colonies on gelatin as those of B. coli or
its varieties. "Such colonies," he remarked "could not without
animal experiment be declared not to be the bacillus of
pseudo-tuberculosis. Moreover they might be neither B. coli
nor its varieties nor the bacillus of pseudo-tuberculosis but
some totally different organism."
W. B. M. Martin (1911) has published photographs showing
the different appearances presented by colonies of the gono-
coccus grown from the same strain and on the same media.
3. In the third place it is known that the addition of
various substances to a culture medium will modify the
character of bacterial colonies growing on it. Thus, Penfold
(1911 B, c) observed that the addition to an agar medium of
certain carbohydrates developed papillae on the surface of
the colonies of many organisms. B. typhosus exhibits this
papillae formation on agar containing lactose, dulcite, or
CH. iv] VARIATIONS IN MORPHOLOGY 49
isodulcite. He found that on raffinose agar B. paratyphoid
B strains produced papillae but B. Aertryck strains failed
to do so, and that this difference between the two organisms
was sufficiently constant to be of value in distinguishing
between them. The formation of papillae indicated, in certain
cases, the acquirement on the part of some members of the
strain of power to ferment the carbohydrate added to the
medium.
4. Heating a strain of organisms before subculturing
them has been observed to modify the characters of the
colonies formed (Bainbridge, 1903).
5. Finally, as a result of passage, the type of colony
formed by a strain of bacteria may be modified. Thus Adami,
Abbott and Nicholson (1899) injected into a rabbit a strain
of typical B. coli. The organism recovered formed on agar
colonies closely resembling those of S. pyogenes. By intra-
peritoneal passage through three guineapigs typical B. coli
were obtained once more.
VARIATON IN OTHER MORPHOLOGICAL CHARACTERS.
Detailed reference has not been made to variation in
other morphological characters, such as motility, pigment
formation, the development of capsules and staining properties,
since these are well known to vary greatly at different times
and under different conditions. A few examples of such
variation will be found in the earlier sections (vide Chap. 11).
D.
CHAPTER V
VARIATIONS IN FERMENTING POWER
THE FERMENTATION OF CARBOHYDRATES.
THE process of fermentation in the case of themono-saccharides
or glucoses the compounds most readily fermented by the
action of bacteria consists of two stages, (i) the splitting of
the "glucoses" with the formation of acids (formic acid,
lactic acid) and (ii) the conversion of the acid, by a process
of hydration, into simpler substances most of them gaseous
(carbon dioxide, hydrogen, methane, etc.). In the case of the
di-saccharides (lactose, maltose, saccharose) an earlier stage
must first be completed, namely the "inversion" of these
substances into glucose. This preliminary change is not
easily recognised, but the two final stages of the process are
sufficiently indicated by the formation, respectively, of acid
and of gas.
The power possessed by certain bacteria to bring about
the fermentation of carbohydrate compounds is subject to
variation to a remarkable degree. Different strains of the
same organism may differ from each other in their " sugar "
reactions. The same strain may vary from time to time
during cultivation, apparently spontaneously. In other cases
the effect can be traced to the conditions of growth and is
found to depend upon such factors as the temperature, the
presence of oxygen, the atmospheric pressure, the age of the
culture, the age of the medium and its composition. The power
to produce fermentation may be modified by symbiosis. It
is sometimes altered in the case of carriers, after animal
"passage" and in the course of a disease. New fermenting
properties may be developed SiS&remltofprolongedcultivation
in a particular" sugar," or bya process of "artificial selection"
CH. v] VARIATIONS IN FERMENTING POWER 51
I. Different strains may possess different fermenting
properties.
The typical pneumococcus ferments saccharose (a di-
saccharide), mannite (a polyatomic alcohol) and inulin (a
starch). Its power to ferment inulin is a feature upon
which reliance is placed in differentiating the organism from
other members of the streptococcus group. Eyre, Leatham
and Wash bourn (1906) found that out of 14 different
strains examined by them 4 failed to ferment inulin, an equal
number failed to ferment mannite and 3 failed to ferment
saccharose.
Strains of the meningococcus obtained from epidemic
and from sporadic cases of meningococcal meningitis exhibit
differences in their fermenting properties (Batten). Arkwright
(1909) describes several strains of the meningococcus which
failed to ferment any sugars in some cases " permanently,"
and in other cases for varying periods after their isolation.
Wilson (1908) analyses the "sugar reactions" in the case
of 44 gas producing coliform' organisms obtained from the
urine of patients suffering from cystitis and pyelitis. The
various strains showed an extraordinary diversity in their
fermenting properties.
Many observers have recorded marked differences in fer-
menting properties between different strains of dysentery
bacilli. One example will suffice. Bahr (1912) collected 28
different strains of dysentery bacilli in Fiji. He tested the
power of these strains to ferment six sugars (dextrose,
dulcite, maltose, saccharose, lactose and mannite) with the
result that the 28 strains formed no less than 7 distinct
groups. The addition of further sugars for test purposes
would no doubt have revealed still more varieties.
Arkwright (1909) mentions a strain of gonococciis, isolated
from the urethral discharge in a case of acute gonorrhoea,
which fermented glucose and maltose but not saccharose,
thus resembling most strains of the meningococcus and
differing from the typical gonococcus which is a non-fermenter
of maltose. W. B. M. Martin (1911) describes an atypical
strain of the gonococcus, isolated in pure culture from the
42
52 VARIATIONS IX FERMENTING POWER [CH. v
knee joint, which did not ferment glucose, levulose, maltose
or saccharose.
Gordon (quoted Martin, 1911) when testing the "sugar
reactions" of 25 strains of micrococcus catarrhaUs, discovered
3 strains which fermented glucose, maltose, galactose and
saccharose none of which sugars are normally fermented by
this organism.
Strains of B. diphtheriae are very variable in their action
on lactose and saccharose (Graham Smith, 1906).
Klotz (1906) describes a coliform organism, quickly ag-
glutinated by high dilutions of typhoid serum, which differed
from B. typkosus in being able to ferment glucose, lactose
and saccharose.
Many other examples might be given of differences in
fermentation properties displayed by different strains of the
same organism.
II. The same strain may vary spontaneously during
cultivation.
Arkwright (1909) mentions a strain of the meningococcus
which when first tested fermented no sugars : subsequently,
throughout a period of many months, it fermented maltose
only ; finally, after 10 months artificial culture, it fermented
both maltose and glucose. Another of his strains, on the
other hand, at first fermented both maltose and glucose but
later fermented neither.
Rosenow (1914) obtained a strain of haemolysing strepto-
cocci from the throat in a case of Scarlet fever. A culture
on blood agar yielded two distinct kinds of colonies, (a)
colonies of a haemolysing organism which fermented mannite
but failed to ferment maltose and saccharose, (b) green
producing colonies of a non-haemolysing organism which
would not ferment mannite but fermented maltose and
saccharose. The two strains differed also in their patho-
genicity.
Andrewes and Gordon (1905-6) found that i% an undoubtedly
pure" strain of staphylococcus pyogenes aureus, which yielded
a brilliantly pigmented culture, produced on subculture
CH. v] VARIATIONS IN FERMENTING POWER 63
colonies some of which were coloured and others white. In
one experiment the coloured strain formed acid in salicin
and coniferin, which neither the original strain nor the white
colonies was able to accomplish.
Klotz (1906) has described a coliform organism which did
not ferment lactose or saccharose when first isolated from
water but fermented both after 48 hours' growth on the media.
Horrocks (1911) describes a strain of B. typhosus which
after 3 days' growth on bile-salt-glucose-litmus-agar gave
typical fermentation tests but a week later was found to have
acquired the power to ferment lactose, dulcite and salicin.
Normally B. typhosus ferments glycerine. Penfold (1910 A)
found that an old laboratory culture of this organism on agar,
when plated out, gave some colonies which fermented glycerine
and others which failed to do so even after five successive
subcultures had been made into peptone water.
Many observers have recorded instances of dysentery
bacilli acquiring during cultivation the power of fermenting
sugars which previously they were unable to attack such as
(in the case of the "Shiga" organism) mannite (Torrey, 1905),
the di-saccharides (Kruse, quoted by Bahr). Bahr (1912)
records the loss, on the part of an atypical "Shiga" organism,
of power to ferment saccharose after 6 months subculture,
and maltose after 7 months subculture ; the loss on the part
of an atypical "Flexner" organism of power to ferment
lactose after 4 months, accompanied by a temporary loss of
the power to ferment maltose. He quotes records of a similar
loss of power to ferment lactose after 4 years (Morgan), and
maltose after 9 years (Lentz). Another strain of the "Flexner"
type (after a month's subculture) produced a feebly acid
reaction in mannite at the end of 24 hours but after 10 days
further incubation the medium became definitely alkaline.
After further cultivation for some weeks it produced acidity
in mannite in 24 hours. Its action on maltose varied greatly.
Sorenson (1912, quoted by Dobell) has recorded the case
of a patient, suffering from glycosuria who also developed
pneumaturia. The gas formation was found to be due to a
peculiar bacillus, " B. pneumaturiae" which had gained
54 VARIATIONS IN FERMENTING POWER [CH. v
access to the bladder. This organism was isolated and on
cultivation was found to ferment glucose, lactose and sac-
charose with the production of much gas. After two years
the patient recovered spontaneously from the pneumaturia,
though the organism was discovered still to be present in the
bladder. Cultures, however, failed to yield gas on sugar
media. About a year later the strain, which had been sub-
cultured throughout this interval, suddenly re-acquired the
property of producing gas, and "shortly after" the patient
commenced to suffer again from pneumaturia.
The clock-like precision with which these two strains, one
on artificial media and the other in the human body, are
stated to have exhibited this spontaneous variation, after the
same interval of time and in spite of the difference in their
respective environment, may perhaps excuse some incredulity.
One suspects that further investigation would have shown
the modification to exist in the sugar media employed rather
than in the bacteria.
III. Fermenting properties may be modified by the
conditions of growth.
1. The influence of temperature. Wilson (1910) isolated
B. typhosus from the urine of a " carrier." At a temperature
of 22 C. this organism fermented lactose litmus-agar in 2 days
but at a temperature of 37 C. no acidity was produced after
a month's incubation. The absence of acidity at the higher
temperature might be accounted for on the supposition that
the products of the proteid decomposition, at this temperature,
neutralised the acid formed during the process of fermen-
tation. Wilson proved that this was not the true explanation
by estimating the amount of lactose present and showing
that it had not been attacked.
Coplans (1909) mentions some strains of B. coli which
showed the reverse phenomenon, fermenting dulcite more
readily at 37 than at 20 C.
Adami has described an alteration in the fermenting pro-
perties of B. coli communis after subjection to a high tem-
perature in the presence of peritoneal fluid.
CH. v] VARIATIONS IN FERMENTING POWER 55
2. The influence of oxygen. Torrey (1905) found that
the power of a certain dysentery bacillus to ferment maltose
was augmented by alternate aerobic and anaerobic culture.
Wilson describes an atypical B. typhosus which slowly
fermented lactose in a litmus-broth tube but produced
fermentation in 3 days when the same medium was poured
into a Petri dish.
Andre wes and Horder (1906) mention a streptococcal
strain which failed to ferment lactose under ordinary con-
ditions but did so readily when grown anaerobically.
3. The influence of atmospheric pressure . Harden (1901)
has shown that the amount of formic acid produced by B.
coli from glucose, at the ordinary pressure of the atmosphere,
is very small. Under greater pressure the yield of acid is
increased, while at the same time the amount of gas formed
is diminished. In other words, the final stage in the process
of fermentation, which consists in the conversion of the acid
into various gases, is inhibited.
4. The age of the culture. Older cultures of B. diphtheriae
usually ferment both glycerine and lactose ; a young culture
of the same organism can attack neither of these substances
(Muir and Ritchie).
5. The age of the medium. The streptococcus pyogenes
normally does not ferment saccharose, raffinose or salicin,
but if old media be used this organism will ferment both the
first two substances, and even the last named in the course of
a week (Martin). On the other hand B. diphtheriae which
gives its characteristic " sugar reactions " on fresh beef serum,
fails to do so if this medium is old (Fisher, 1909).
6. The composition of the medium. The addition of
carbolic acid in small quantities to the media used destroys
the natural fermenting properties of many bacteria (Penfold,
1911 B).
The presence of sodium benzoate inhibits the power of
B. coli to produce gas from dextrose, one of the most stable
and fundamental differences separating the coli from the
typhoid-dysentery group (Herter, 1909). Penfold (1911 c)
found that many intestinal organisms (B. coli, B. enteritidis
56 VARIATIONS IN FERMENTING POWER [CH. v
Gaertner, B. paratyphosus B, etc. ) by growth on monochlor-
acetic-acid-agar, were deprived of their power to form gas
from glucose and other sugars ; they retained, however, their
power to ferment the corresponding alcohols. He found that
the variation in character was maintained, even on daily
subculture, for many " generations."
7. The source of a strain, that is to say the nature of the
medium from which it is isolated, may determine certain
variations in fermenting power. Thus, Revis (1908) found that
strains of B. coli cultivated from milk were frequently able
to ferment saccharose. Moreover a strain obtained from
cow dung, which was unable to ferment saccharose, acquired
the power to do so after being grown in milk. Wilson (1909)
found that many coliform organisms isolated from urine had
lost the property of forming gas from dextrose. Adami,
Abbott and Nicholson (1899) describe strains of B. coli grown
in ascitic fluid as being unable to ferment glucose or dextrose
broth this power being only partially regained after three
passages through the guineapig.
IV. Symbiosis may influence fermenting power.
Major Horrocks's experiments (1911) are of interest in
this connection. He found in two experiments that a typical
strain of B. typhosus lost its power to ferment " sugars " when
grown in the presence of a strain of B. coli derived from
the urine of a "typhoid carrier." In one case reversion in
character took place on further subculture. He found, in
a third experiment, that a strain of B. typhosus derived
from the urine of a " carrier," lost both its fermenting and
its agglutinating properties when grown in the diluted, filtered
urine of another "typhoid carrier."
V. Variation in fermenting power is found in organisms
isolated from " carriers."
The strain of B. typhosus isolated by Wilson from the
urine of a " typhoid carrier '' was found to have acquired the
power to ferment lactose and saccharose, and the power also
to produce "much gas" from mannite and maltose.
CH. v] VARIATIONS IN FERMENTING POWER 57
VI. Fermenting power may be altered by "animal
passage"
Klotz (1906) isolated from water an atypical organism of
the B. coll group. This organism, after a residence of 144
days' duration in a celluloid sac within the peritoneal cavity
of a rabbit, showed a temporary loss of power to ferment
glucose, saccharose and lactose. The loss was most marked
in the case of lactose which was, however, again fermented
in the 4th subculture into lactose broth, and also by the 8th
subculture on ordinary media (agar).
'Peckham (1897) introduced B. coli into the peritoneal
cavity in sufficient numbers to set up a fatal inflammatory
process. The organism, recovered on the death of the animal
4 days later, showed slight changes in fermenting power.
Horrocks (1911) describes a strain of B. typhosus which,
in the course of cultivation, acquired the power to ferment
lactose, dulcite and salicin. " Passage " through 4 guineapigs
destroyed the power to ferment lactose and dulcite but after
4 further passages the power was regained.
Bahr (1912) describes experiments in which flies were fed
on dysentery bacilli (both of the "Shiga" and of the
" Flexner " type) and states that the organism recovered from
the intestinal tract in several cases, " undoubtedly derived
from the bacillus originally fed to the flies," gave different
sugar reactions. The sugar reactions had, for 9 months
previously, remained constant on repeated trials. One " Shiga "
organism had acquired the power to ferment maltose. In the
case of a "Flexner" organism, the power of fermenting
mannite (upon which the distinction between the acid and
non-acid types depends) was diminished, fermentation only
occurring after 4 days' incubation. Other organisms of the
"Flexner" type had acquired the power to ferment maltose
and saccharose. Both types of organism on subculture re-
verted to their original characters in the course of several
months.
Adami, Abbott and Nicholson (1899) obtained from human
ascitic fluid an atypical B. coli which had completely lost its
fermenting power. This power was restored after a series
58 VARIATIONS IN FERMENTING POWER [CH. v
of three intra-peritoneal passages through the guineapig.
Another similar strain from ascitic fluid after several passages
was still unable to produce gas from glucose or dextrose.
VII. Variation in the fermenting power of organisms
may arise during the course of a disease.
Connal (1910) has shown that in the later, chronic, stages
of cerebrospinal fever the meningococcus isolated from the
spinal fluid has lost its power to ferment dextrose.
Adami, Abbott and Nicholson (1899) in describing diplo-
coccic forms of B. coli, isolated from the ascitic fluid in
cases of hepatic cirrhosis, mentions that they had lost the
power of fermenting glucose and lactose broths.
VIII. The power to ferment a "sugar" may be acquired
by bacteria after prolonged growth in a medium containing
that sugar.
Hiss (1904) has described a bacillus of the dysentery group
which acquired the power to ferment maltose after being
grown for some time in a maltose medium.
Twort (1907) found that dysentery bacilli (both the
"Flexner" and the "Shiga" type) which did not normally
ferment saccharose, did so after cultivation in a medium
containing this ingredient, and by similar means the true
" Shiga Kruse " organism was induced to ferment lactose.
In the same way he found that members of the paratyphoid
group, after prolonged cultivation on a saccharose medium,
all acquired the power to ferment it, while a strain of B.
typhosus acquired the power to ferment lactose, but only
after two years' "training." The same organism could be
made to ferment dulcite in a very shorter period, 2 or 3 weeks
being sufficient.
Penfold (1910) trained B. typhosus to ferment dulcite in
a period of 10 days, arabinose in 2 or 3 months and isodulcite
after varying intervals. He failed however to develop new
fermenting powers on the part of the same organism towards
lactose after a 15 months' trial, or towards saccharose and
other substances after 9 months.
CH. v] VARIATIONS IN FERMENTING POWER 59
Burton Bradley (1910) repeated these experiments with
success as regards dulcitol and arabinose.
Penfold states that in the case of B. typhosus the acquire-
ment of new fermenting properties on the part of certain
individuals of the strain is indicated, in some instances, by
the formation of papillae on the colonies. He observed this
to take place in a medium containing dulcite or sorbite. In
his opinion considerable permanency in the new fermenting
power was indicated if the papillae formation arose early and
without subculture.
IX. Variation in fermenting power may be brought
about by a process of Artificial Selection.
Goodman (1908) made a series of cultures of B. diph-
theriae in dextrose broth. From this series he selected the
tube giving the greatest acidity, when titrated against a
standardised soda solution, and the tube giving the lowest
acidity. From each of these two tubes he made a fresh series
of cultures and, after 3 days' growth at 37 C., he chose out of
the more acid series the tube giving the greatest acidity, and
out of the less acid series the tube giving the lowest acidity.
With these two tubes he made a fresh double series of
cultures and repeated the process of selection. After repeating
this 36 times he obtained one culture which produced intense
acidity in dextrose, and a second culture which failed to pro-
duce acidity in dextrose at all and in fact made it more
alkaline. These two strains were then tested with other
sugars and it was found that, in both cases, the power to
ferment maltose was diminished while the power to ferment
saccharose was increased. Their action on dextrin was not
affected.
It is to be noted that the difference in fermenting power
between these two selected strains was as great as that
normally existing between B. diphtheriae -and B. pseudo-
diphtheriae.
This process of continued selection in opposite directions
does not necessarily succeed in developing strains of extreme
type. For example, Buchanan and Traux (quoted by Rettger
60 VARIATIONS IN FERMENTING POWER [CH. v
and Sherrick, 1911) failed to develop high and low acid
forming strains of streptococcus lacticus and Glenn (1911)
records a similar failure in the case of B. proteus.
THE SIGNIFICANCE OF VARIATIONS IN THE
"SUGAR REACTIONS."
A consideration of the action of bacteria on carbohydrates
and a comparison with the similar action of other (vegetable)
cells, such as yeast, justify certain conclusions.
1. The splitting up of the carbohydrate is undoubtedly
effected through the agency of enzymes or " organised
ferments," the functions of which are inhibited or destroyed
by antiseptics, such as carbolic acid, sodium beuzoate,
monochlor-acetic acid.
2. The fermentation of a particular carbohydrate is
dependent on the activity of a particular ferment, so that the
power to ferment one carbohydrate is quite independent of
the power to ferment another.
This is well illustrated by Goodman's experiments. The
two strains obtained by him from a culture of B. diphtheriae,
one with greatly increased fermenting power towards dextrose
and the other with almost complete absence of such power,
both showed an augmentation of their power to ferment
saccharose and a diminution of their power to ferment
maltose. Penfold succeeded in modifying strains of B. coli,
B. enteritidis Gaertner and B. Grilnthal by growing them
in the presence of a certain antiseptic, with the result that
they lost the power to produce gas from the sugars while
still retaining the power to produce gas from the corresponding
alcohols.
3. The three stages in the process of fermentation,
namely the preliminary stage of " inversion " (in the case of
the di-saccharides) and the two final stages in which acid is
first formed and then split up into gases, are due to the
activity of three different enzymes. Failure to produce gas
may be due to the absence or the inhibition of any one of
these three enzymes. Failure to produce acidity may be due
CH. v] VARIATIONS IN FERMENTING POWER 61
to the absence or inhibition of either the " inverting " ferment
or the " acid-forming " ferment.
That the several steps in the process of fermentation
result from the activity of different enzymes is suggested by
the following considerations. Typhoid and dysentery bacilli
never, of themselves, produce gas and cannot be made to do
so by training or selection ; both however produce acidity in
dextrose and mannite and can be "trained" to do so in
lactose.
B. coli is known to produce formic acid from glucose and
then to split up the formic acid into gases ; Penfold has
shown that growth on mpnochlor-acetic-acid-agar may inter-
fere with the "formic acid-forming" property of this organism
without affecting its " formic acid-splitting " power, that is to
say its power to form gas from formic acid.
4. This observer has gone still further and has shown that,
during the stage of add production, more than one kind of
acid may be formed by an organism but that the formation
of each acid is the work of a special enzyme. The inhibition
of the " formic acid-forming " enzyme in the case of B. coli
did not interfere with the production of acidity in dextrose.
5. Penfold likewise showed that the splitting up of each
acid, with the formation of gases, was the work of a special
enzyme and that an enzyme which could produce gas from one
acid could not do so from another. The strain of B. coli which
was deprived of its power to form formic acid was, on this
account, deprived of its power to produce gas, for, although the
organism could produce other acids (as shown by the reaction
of the medium) it could not split these up. If however sodium
formate was added to the medium the organism at once yielded
gas ; or, again, if the organism were grown in dextrose
with B. typhosus (which possesses the power of producing
formic acid from dextrose but cannot split the acid up) it
once more yielded gas. It is obvious, then, that in the case of
B. coli its " acid-splitting " enzyme is only capable of splitting
up formic acid and cannot form gas from other acids.
6. The development by a strain of bacteria in contact
with a certain sugar, of the power to ferment that sugar is an
62 VARIATIONS IN FERMENTING POWER [OH. v
example of adaptation to environment. If a slow fermenter
of dulcite is grown in a medium containing some other sugar,
such as dextrose, its power to ferment dulcite is not increased.
7. The ability to split up the sugar is apparently an
advantage to the organism concerned. That this is actually
the case is confirmed by the observation of Penfold that the
appearance of acidity coincides with a very rapid and a very
marked increase in the number of organisms present. So
constant did he find this association of events that he regards
a count of the organisms as sufficient by itself to indicate the
occurrence of the variation. He found, moreover, that the
addition of dulcite to peptone water containing a dulcite-
fermenting strain of B. typhosus, rendered the medium capable
of supporting a population many times greater than it was
able to support alone. The addition of other sugars which the
organisms could not ferment did not lead to any increase in
their numbers.
The ability to ferment the sugar, even if in some cases it
were not of actual benefit to the fermenting organism, might
still prove of advantage to it indirectly. Marked acidity ot
the medium is known to be unfavourable, as a rule, to
bacterial growth ; but it might be expected that the acid
producing individuals in a strain would be unusually resistant
to the products of their own activity and that their growth
would, on this account, be inhibited to a less degree than
that of the non-fermenters.
8. It is easy to understand how natural selection will
cause any character to predominate which gives the possessors
of it an advantage over their fellows. This is the obvious
explanation of the development by a strain of bacteria, when
grown on a certain sugar, of the power to ferment that sugar.
Two phases of the phenomenon, however, call for further
explanation, namely the prolonged incubation period and the
shortening of this period by subculture.
9. The incubation period may be explained in one
of three ways.
In the first place it may be regarded as a " latent period "
during which changes occur in the organisms as a result of
CH. v] VARIATIONS IN FERMENTING POWER 63
their contact with the new sugar, such changes being pre-
paratory to the acquisition on their part of new fermenting
powers. The observation already referred to, that a very
rapid increase in the number of fermenting organisms occurs
simultaneously with the appearance of acidity, tends to
support this hypothesis. Penfold has, however, disproved it
by experiment. He observed that B. typhosus when grown
in dulcite broth gradually acquired the power to ferment the
sugar. After several days had passed, plating out on dulcite
agar showed that 95 per cent, of the strain were dulcite fer-
menters. He found that subcultures from the non-fermenting
colonies into dulcite broth took the same length of time as the
original stock to produce fermentation, thus showing that
they had not undergone any preparatory change during the
first incubation period.
In the second place, if the development of the new fer-
menting power is dependent in the first instance upon the
occurrence of a spontaneous "fluctuating" variation in the
required direction, and such variations are infrequent, an
interval of uncertain length must necessarily intervene be-
tween the commencement of the experiment and the appear-
ance of the variant which is to give rise to the new strain.
The fact that the length of the incubation period, whenever
a certain organism is " trained " to ferment a certain sugar, is
fairly constant, disposes of this argument.
In the third place, the original non-fermenting strain might
contain a very few fermenting individuals, in insufficient
numbers, however, to give any evidence of their presence.
These few "fermenters" would possess an advantage over the
"non-fermenters" and multiplying more rapidly would, in time,
outnumber the latter, but a certain period would necessarily
elapse before they gained the ascendancy. Inasmuch, however,
as the original strain can be made in the same way to ferment
a number of different sugars, the strain must, on this hypo-
thesis, contain at one and the same time fermenters of each of
these different sugars. Even if this be granted the hypothesis
offers no explanation of the fact (illustrated by the behaviour
of B. typhosus in dulcite and in lactose respectively) that in the
64 VARIATIONS IN FERMENTING POWER [CH. v
presence of one sugar the fermenters of that sugar invariably
gain complete ascendancy in the course of a few days, while in
the presence of another sugar the fermenters of it invariably
require many months to do so.
1 0. The shortening of the incubation period on subculture
is more easily explained. When a few bacteria are inoculated
into a tube of broth they multiply with amazing rapidity.
There is a limit, however, to the number of organisms a certain
volume of the medium will support owing not only to the
using up of the food but also to the accumulation of waste
products so that after a time multiplication takes place much
more slowly. Subculture into fresh medium gives a new
impetus to reproduction. The "fermenters" in a mixed strain
gain the ascendancy by virtue of their capacity to utilise the
sugar, which enables them to multiply more rapidly than the
"non-fermenters." Any factor therefore which accelerates
the rate of increase of both, hastens the ultimate mastery of
the more rapidly multiplying, that is to say, the "fermenters.'*
11. "Artificial selection" appears to be an even more
powerful factor in developing a particular fermenting power
than "natural selection." For example, Goodman obtained
by artificial selection a strain of B. diphtheriae which had
practically lost its power to ferment dextrose, although it
had been subcultured from one dextrose media to another
repeatedly over a long period.
12. When an organism has been deprived by passage or
by other means, of its power to ferment a particular sugar,
"reversion" in character occurs subsequently on ordinary
media, that is to say in the absence of the particular sugar in
question. (The addition of the latter hastens "reversion," a&
Klotz has shown, but its presence plays only a subordinate
part in the process). The sequence of events in such cases
suggests that the enzyme is temporarily inhibited in its action
and not destroyed.
13. The sudden acquisition on the part of a strain of bac-
teria of power to ferment a certain sugar with which it has not
been in contact, is more difficult to explain particularly so
when such a variation occurs after long periods, even years, of
CH. v] VARIATIONS IN FERMENTING POWER 65
cultivation in one medium, during which repeated examina-
tions revealed no change in fermenting properties.
The possibility must always be considered that the strain
may have been subcultured into fresh media in which the new
sugar was accidentally present as an unrecognised impurity,
and that the bacteria "learnt" to ferment this impurity after
a preliminary "training." They would be more likely to do
this if the process of subculturing were only carried out at
long intervals (as might easily happen in the case of a stock
culture) for this would afford time for the bacteria to exhaust
the normal sugar of the medium and their survival would then
depend upon their power to utilise traces of any other sugar
that might be present.
The possibility also suggests itself that in a medium con-
taining a di-saccharide (lactose, maltose, saccharose) inversion
might occur to a slight extent, with the formation of traces of
a simpler mono-saccharide (dextrose, galactose) which the
bacteria growing in the medium would then, in the same way,
"learn" to ferment.
Yet a third possibility is that the particular specimen of
the "sugar" used to test the fermenting properties of a strain
of bacteria may not be pure. This possible source of error is,
however, more easily guarded against.
Finally even if it be admitted that a variation in ferment-
ing power represents an adaptation to different foodstuffs we
are forced to conclude that different members of a strain differ
from one another in their powers of adaptation ; for, when an
apparently spontaneous variation in fermenting power occurs
during cultivation, in a strain derived originally from a single
bacterium, fermenting and non-fermenting individuals may
be found side by side on the medium. This variation between
the organisms of one and the same strain we are quite unable
to explain.
THE VALUE OF THE SUGAR REACTIONS.
The unsatisfactory nature of the "sugar reactions," both
as a means of identification and as a basis for classification,
will be apparent from the following considerations.
D. 5
66 VARIATIONS IN FERMENTING POWER [CH. v
1. In the first place, there is the question of the time
allowance to be made. When a particular organism only
produces acidity in a certain sugar at the end of an incubation
period lasting several days, one is in doubt whether to regard
the organism in question as a slow fermenter of that sugar or
as a non-fermenter of it which has acquired a new character
as the result of "training."
2. Secondly, many conditions, as we have shown, modify
the normal sugar reactions and may lead to erroneous con-
clusions. A strain of bacteria may ferment a sugar at a
temperature of 22 C. and fail to do so at 37 C. ; an old
culture may ferment substances which a young culture is
unable to do, and so on.
3. In the third place, the composition of the medium may
be responsible for conflicting results. It is almost impossible
to obtain many of the carbohydrates in a pure form, and yet
these are used and conclusions are based on the reactions they
give. Others can be obtained pure but are then too costly
for general use and the commercial "sugar" is substituted.
Different specimens of the same carbohydrate, even when
reasonably pure, msty give different results. This is the case
with the starch inulin, the fermentation of which is an import-
ant distinction between the pneumococcus and other members
of the streptococcus group. The process of sterilisation is a
difficult one. If subjected to too high a temperature, particu-
larly in the presence of alkaline material, the sugar may
undergo a change in composition. If the temperature is not
raised sufficiently sterilisation may be incomplete. If the
vessels holding the medium are not made of the best Jena glass,
there is a danger of the glass yielding a considerable amount
of alkali to the medium during sterilisation (W. B. M. Martin,
1911). If the various media are kept for any length of time
before use, the carbohydrates may deteriorate and lead to
apparently abnormal sugar reactions. Examples of this have
already been given. Finally, if the medium is very alkaline in
the first instance, or if it is rendered so by the decomposition
of the peptone present, the acid reaction may be masked.
4. Even if the composition of the medium is beyond re-
CH. v] VARIATIONS IN FERMENTING POWER 67
proach, the reactions are not necessarily constant. There is
a great deal of evidence to show that spontaneous variations
frequently occur.
5. In any case, the classification of bacteria according to
their action on certain carbohydrates is a very artificial one.
Twort emphasises the fact that the difference between one
organism which produces such slight acidity that the alkaline
reaction of the medium completely masks it, and another
organism which produces a slight but definite acid reaction,
is no greater than the difference between the latter organism
and a third which produces marked acidity. In the same way
the difference between an organism which yields acidity in
24 hours and one which requires 48 or even 72 hours to do so,
cannot be regarded as a fundamental one. It is merely a matter
of degree.
6. Further, the decision as to which group of carbohydrate
compounds (the sugars, the glucosides, the starches, etc.) shall
constitute the test substances is a purely arbitrary one,
depending not infrequently upon their cheapness and the
facility with which they can be obtained. If one group of
carbohydrates be chosen a certain classification will follow ; if
another group be selected an entirely different classification
may result.
The only justification for founding a classification upon
one series of experiments rather than upon the other is the
fact that the classification so obtained corresponds more closely
to differences brought out in other ways, such as differences
in agglutination or pathogenicity.
If these other differences are inconstant and distinctions
based on them have been found to be unreliable, then a series
of fermentation tests designed to correspond with them is at
once suspect and cannot be trusted as a final appeal. If on
the other hand these other differences (in agglutination, patho-
genesis, etc.) are constant and have been found to justify a
certain division into "species," a series of fermentation tests
which correspond to them may afford a very much simpler
method of deciding to which of these species a certain
organism belongs, and also of separating one species from
52
68 VARIATIONS IN FERMENTING POWER [OH. v
another by "plating out" a mixed culture on the appropriate
medium.
It is this that constitutes the real value of the "sugar
reactions." In other words the tests are of more use for
purposes of identification than of classification.
How far is this conclusion borne out by a study of the
relation between fermentation tests and agglutination re-
actions ?
In some cases, alteration in fermenting powers is not
accompanied by any disturbance in the agglutination pro-
perties, which remain constant.
For example, Twort and Penfold have both shown that the
altered fermenting power on the part of B. typhosus towards
lactose is not accompanied by any alteration in agglutination
properties. The organism described by Klotz as differing from
B. typhosus in its capacity to ferment lactose and saccharose,
and in other characters, nevertheless was agglutinated by
typhoid serum in high dilutions.
Bahr, in describing the altered fermenting power of dy-
sentery bacilli, following transmission through the intestine of
the fly, states that the variants displayed no alteration in
agglutination properties. Lentz mentions a "Flexner" strain
which after seven years' laboratory cultivation lost its power to
ferment maltose but still retained its agglutination properties
unchanged.
In other cases, alteration in fermenting power sis associated
with loss of agglutination properties. For example, Wilson,
in describing an organism isolated from the urine of a "typhoid
carrier" and considered by him to be a derivative of B. typho-
sus, states that not only were the fermenting properties altered
but the agglutination tests no longer corresponded with those
of B. typhosus. Penfold found that colonies of B. typhosus
which had lost the property of fermenting glycerine, showed
impaired agglutinability also, though typical fermenting
colonies on the same plate were normal as regards agglutina-
tion.
Horrocks observed that a strain of B. typhosus derived
from the urine of a carrier, when grown in the diluted filtered
CH. v] VARIATIONS IN FERMENTING POWER 69
urine of a second " typhoid carrier," was deprived not only of
its power to ferment but also its property of agglutinating in
the presence of typhoid serum.
Thirdly, classifications according to fermenting powers and
according to agglutination proper ties give altogether different
results.
Ohno (1906), in his elaborate investigations on 74 strains
of dysentery derived from different sources, found that a
classification based on their different powers of fermentation
did not correspond with a classification according to their
agglutination reactions. Torrey describes dysentery bacilli
possessing different fermenting properties but giving the same
agglutination reactions. The pneurnococcus is distinguished
from other members of the streptococcus group by its different
fermenting properties, particularly with reference to inulin;
nevertheless the pneumococcus and other streptococci tend to
originate common group agglutinins.
It would appear therefore that the power of producing
fermentation bears no relation to agglutinability, and in the
final resort, if reliance is to be placed, for purposes of classifica-
tion, on one test the other must necessarily be discredited.
As regards identification, however, there is this to be said
organisms immediately after their isolation from the tissues
or excretions are, as a rule, more typical in their fermenting
properties than the same organisms after even a short period
on artificial media, whereas the reverse frequently holds true
as regards their agglutination reactions. In deciding, there-
fore, which of the two series of tests is to be relied upon in
cases where they conflict, the length of the period of cultivation
is of great importance ; the fermentation tests will be found
most reliable in the identification of an organism in those
cases where agglutination tests are least so.
It is seen then that the fermentation tests, though for
purposes of classification they may prove at variance with
the agglutination reactions, as a means of identification may
supplement the latter.
70 VARIATIONS IN FERMENTING POWER [CH. v
THE VALUE OF VARIATIONS IN THE SUGAR REACTIONS
IN THE IDENTIFICATION OF BACTERIA.
There are ways in which the actual variations in the sugar
reactions may be of value in identifying an organism.
In the first place, the variations observed may themselves
be specific in character and so far from obscuring the identity
of an organism may in some cases actually contribute to its
recognition, in the same way that the morphological variations
of B. diphtheriae help to establish its identity.
In the second place the variations may help to identify
the source of the organism by indicating the nature of its
recent environment. Thus, if "passage" is found to modify
the fermenting power of a particular species of bacteria in
a particular direction, then such modification when found to
exist in a member of that species may indicate a recent animal
host. Again, if growth in a certain material is known to lead to
certain modified "sugar reactions" on the part of a particular
species, then such modification, when it is found, may furnish
a clue to the source of the organism in question. For example,
it has been pointed out by several observers (Re vis, 1908) that
the saccharose-fermenting type of the colon bacillus is more
often isolated from milk than from cow-dung and MacConkey
has stated (quoted, ibid.) that the more prolonged the sojourn
in the former medium is, the more prolonged is the power to
ferment saccharose on the part of the organism. Revis also
describes a strain of bacteria which failed to ferment saccharose
when isolated from cow-dung, but did so after being cultured
in milk. Again, strains of B. coli exhibiting variation in gas
production are more commonly of urinary than of intestinal
origin.
This question will be further considered in a later chapter
(vide p. 144).
CHAPTER VI
VARIATIONS IN YIKULENCE
THE pathologist is apt to forget that the vast majority of
bacteria are non-pathogenic, that is to say, they are harmless
to man. Not only so, but the activities of many of them are
as beneficial to him as those of the pathogenic bacteria are
the reverse. The purification of sewage and the mineraliza-
tion of dead vegetable matter, to mention only two instances
of bacterial action, are processes which contribute to the
health and survival of the human race no less than the
processes of disease conduce to its decay.
The power to cause disease depends upon two factors. In
the first place, it depends upon the ability of the organisms
to become parasitic, that is to say, to invade the living
tissues and live and multiply there, and, in the second place,
it depends upon the result of their activity, more especially
as regards the formation of poisons or toxins.
The harmless nature of most bacteria is due to the fact
that they have not acquired the power of becoming parasitic.
In some instances, where bacteria do succeed in invading
the tissues, the result of their activity within the body is
apparently harmless. Ford (1900) examined the liver and
kidneys of healthy animals after death, with the most stringent
precautions against contamination, and found at least 80 per
cent, contained bacteria of various kinds. In other cases,
though organisms fail to invade the living tissues, they
nevertheless produce symptoms of disease by manufacturing
toxins which are absorbed, as, for example, in puerperal
sapraemia.
The results produced by the presence of bacteria in the
tissues are due to a variety of causes, which include (a) the
metabolism of the living organisms, that is to say, the
72 VARIATIONS IN VIRULENCE [CH. vi
substances assimilated and excreted by them ; (b) the dis-
integration of the dead organisms ; (c) the mechanical effect
of their presence whether living or dead ; (d) the response
made by the living tissues to these various stimuli.
The first factor in the production of disease the "in-
vasiveness " of the organism is dependent upon the degree
of "mobility" the particular organism possesses under various
conditions.
The second factor the result of their activity within the
body may be considered under two heads : (1) the produc-
tion of particular lesions in the tissues and the exhibition
of a certain train of symptoms, both more or less peculiar to
the particular organism giving rise to them phenomena which
are discussed under the term " pathogenesis," and (2) the
production of a condition of "toxaemia," resulting in a
general impairment of health and leading eventually to the
death of the body, which we now propose to consider under
the term "virulence."
The virulence of organisms is known to vary under different
circumstances within wide limits.
1. Thus, cases of disease which occur at the end of an
epidemic are frequently, though not invariably, less severe
than those at the beginning, the virulence of the infecting
organism having gradually diminished during the course of
the epidemic. Thomson mentions an epidemic of cerebro-
spinal fever comprising 30 cases, all of which were admitted
to hospital and received the same treatment ; of the first 16
cases admitted all died except two, of the last 14 admitted all
lived except two.
This difference in virulence might of course be apparent
and not real, the lessened intensity of the disease being
accounted for by a difference in the resistance of those
attacked by it. The weakest individuals, who are the first to
succumb to the infection, also offer the poorest defence,
whereas in the later stages the less fit have already been
weeded out and the more robust alone remain to be attacked,
and these^offer a more stubborn resistance. Such an explana-
tion might hold good in the case of a strictly localised
CH. vi] VARIATIONS IN VIRULENCE 73
outbreak for example on board a ship or in a military
camp but in a widespread epidemic in a crowded city, for
instance, both weak and strong individuals are exposed
equally to infection as the disease extends.
2. Again, the same disease may either take the form of
an epidemic or occur sporadically in the form of isolated
cases, apparently unconnected with each other. In speaking
of meningococcal meningitis, Koptik attributes the diminished
infectivity of the sporadic types to the senility and weakened
virulence of the organism concerned.
3. A similar diminution in virulence is observed in the
case of some endemic diseases in the course of many genera-
tions. Bahr (1912) quotes evidence to show that in Fiji,
dysentery 25 years ago was a much more virulent disease
than it is at the present time. The virulence of the specific
virus of syphilis has been modified considerably in those parts
of Europe in which it has been prevalent for centuries, such
as Spain.
Here again the development, by those exposed to infection,
of increased powers of resistance or "immunity," no doubt
plays a part, for when the infection is introduced from places
where it has long been prevalent to places where previously
it has never been met with, the disease may from the first
assume a virulent type.
4. Epidemics of a particular disease vary in virulence at
different times and at the same time but in different places.
Typhoid fever is, as a rule, much less severe in England than
the same disease in the tropics or in the temperate regions
of South America, although the organisms, apart from the
question of virulence, appear to be identical.
5. Again a particular species of organism may produce,
at different times, diseased conditions widely differing in
their intensity. The classical example of this is the strepto-
coccus pyogenes which may produce at one time merely a
local suppuration, at another a spreading erysipelas and at
another a rapidly fatal septicaemia,
6. Many pathogenic organisms when grown outside the
body, under various abnormal conditions, lose their virulence.
74 VARIATIONS IN VIRULENCE [OH. vi
(a) Pasteur showed 30 years ago that B. anthracis,
if grown at a temperature of 43 '5 C., lost its virulence in 3
or 4 weeks. Hewlett and Knight (1897) destroyed the viru-
lence of a strain of diphtheria bacilli by subjecting it for 17
hours to a temperature of 45 C. Muir and Ritchie state that
a broth culture of the diphtheria bacillus if exposed for only
one hour to a temperature of 65 C. is rendered much less
toxic while a culture of the tetanus bacillus under the same
conditions is deprived altogether of toxicity. The bacillus of
" blackleg " can likewise be rendered innocuous by exposure
to a high temperature (Mohler and Washburn, 1906).
It has been thought that recovery from infectious diseases,
such as the exanthemata, might be due to the effect produced
in this way on the infecting organisms by the continued fever
which their presence provokes. That the increase in tempera-
ture may be a protective measure on the part of the body is
suggested by the experiments of Lowey and Richter (1897),
in which the resistance of rabbits to infection by the pneumo-
coccus, the diphtheria bacillus and the hog cholera bacillus
was artificially increased by injury to the corpus striatum
and a consequent rise in temperature, before inoculation.
These observations do not prove that the rise in tempera-
ture lessens the virulence of the organisms. Indeed this
opinion has been proved to be erroneous, in some cases at
least, by the work of Leutscher (1911), who tested the
virulence of pneumococci isolated from the affected area of
the lung at different stages of an acute lobar pneumonia. He
found that the virulence of the organisms isolated at the
period immediately preceding the crisis was even greater
than that of those isolated in the early stages of the disease.
Other observers, working along different lines, have shown
that a comparatively high temperature is not necessarily
inimical to virulence. Eyre, Leatham and Washbourn (1906)
quote the observations of Kruse and Pansini, which their
own work confirms, to the effect that the virulence of the
parasitic pneumococcus is often associated with inability to
grow at a temperature much below that of the body 37 C.
A slightly virulent strain which would grow readily at 20 C.
CH. vi] VARIATIONS IN VIRULENCE 75
could, they found, be converted by "passage" into a highly
virulent strain which would not grow at a temperature below
37 C. By artificial culture the reverse change could be
brought about. In one experiment a single inoculation
into an animal sufficed to bring about the conversion of
one type into the other, the relationship between virulence
and the temperature at which growth would occur being
constant.
(6) Another abnormal condition of growth which tends
to modify the virulence of organisms is the presence of weak
antiseptics. Thus Chamberland and Roux (quoted, Muir and
Ritchie) found experimentally that B. anthracis lost virulence
if grown on a medium to which carbolic acid had been added,
in the proportion of 1 to 600, or a minute quantity of Pot.
bichromate. A virulent strain of B. diphtheriae is promptly
attenuated by the addition of iodine trichloride to the medium
(Mohler and Washburn, 1906).
In the living body certain secretions play a similar r61e.
Leutscher (1911) proved that the saliva had a bactericidal
effect on the pneumococcus and he attributes the diminished
virulence of the pneumococci found in the mouth to this
agency. Savage showed, by experiment on himself, that the
streptococcus mastitidis, which causes mastitis in cows, had
its virulence greatly reduced by 2 or 3 days' residence in the
mucous membrane of the human pharynx.
On the other hand the addition to a medium of certain
substances may cause heightened virulence. The bacillus
of "blackleg," rendered avirulent by exposure to a high
temperature, has its virulence completely restored if lactic
acid is added to the medium in which it is growing (Mohler
and Washburn, 1906). Many organisms, also, which lose their
virulence rapidly on ordinary culture media, maintain it for
long periods when grown in the secretions of the body for
example, in urine (vide p. 19).
(c) The presence or absence of oxygen is another factor
of importance. For example, Haffkine (quoted Hankin, 1892)
found that the cholera spirillum lost virulence considerably
when grown in a current of sterile air, while Hueppe (quoted
76 VARIATIONS IN VIRULENCE [CH. vi
Adami, 1892) observed that its virulence was heightened by
anaerobic growth.
Pasteur, when investigating cultures of chicken cholera,
found that their virulence gradually disappeared, but he
discovered that it was maintained if he grew the organisms
in sealed tubes so that oxygen was excluded. By this
procedure loss of moisture was likewise prevented and this
fact may possibly have been of no less importance than the
exclusion of air.
Other organisms which, normally, do not readily lose viru-
lence, do so rapidly if grown in an atmosphere of compressed
air (Muir and Ritchie).
Harass (1906) succeeded in growing certain "anaerobic"
bacteria in the presence of air and he found that under these
conditions the bacillus of malignant oedema lost virulence
though the bacillus botulinus retained it. It is well known
that the bacillus of diphtheria produces toxins more plentifully
when there is an abundant supply of air (Clark, 1910).
(d) In other cases the loss of virulence on artificial
cultivation is to be attributed to the influence of sunlight,
which is known to have this effect on B. antliracis and other
pathogenic bacteria (Marshall Ward and Blackman, 1910).
(e) The reaction of the medium may also influence viru-
lence. Miss Peckham (1897) found that the addition of an
alkali to the medium increased the virulence of a strain of
B. coli. Undue acidity of the medium may result from the
action of the organisms themselves in splitting up the
carbohydrate present.
7. Practically every organism becomes less virulent when
cultivated for any length of time outside the body, that is to
say, on artificial media, even under the most favourable
conditions. The common pus cocci and the pneumococcus
afford good illustrations of this. The loss of virulence occurs
even when the growth is abundant and it persists on sub-
culture.
Possibly some of the factors responsible for this change
are those just mentioned, namely differences in temperature,
the presence of oxygen, exposure to sunlight, the increased
CH. vi] VARIATIONS IN VIRULENCE 77
acidity of the medium. Other contributing factors are found,
no doubt, in the nature of the medium itself both as regards
its chemical composition and its physical properties.
(a) The difference in chemical composition between the
body fluids and laboratory media must necessarily profoundly
influence the metabolism of organisms transferred from one
to the other. The more closely the artificial medium used
resembles chemically the body fluids, the less influence will
this factor exert. For example, on solidified blood serum, or
media to which blood has been added, or ascitic fluid, viru-
lence is maintained for a greater length of time than on other
media. Eyre and Washbourn (1899) found that the parasitic
pneumococcus kept its virulence, undiminished for a couple
of months on blood agar but not on ordinary media. Anne
Williams (1902) found many strains of diphtheria bacillus,
which were quite non-pathogenic when inoculated from broth,
were highly toxic when inoculated from serum culture or
ascitic broth.
In the case of pathological exudations the question of
chemical composition is of even greater significance, for the
composition of these fluids is dependent on processes of great
complexity and differs widely from that of healthy excretions,
and it is apparently by virtue of this very difference that
pathological exudations possess the power of maintaining
the virulence of organisms growing in them. It is found in
practice that the best fluid in which to preserve organisms
from a pathological source unchanged for subsequent examina-
tion is normal saline to which a considerable quantity of the
infected material itself has been added, whether it be blood
or pus or fluid from a serous cavity or some other secretion.
Horrocks took the urine of a "typhoid carrier" which was
loaded with virulent bacilli and kept it for 12 months in
flasks exposed to the light and frequently opened to the air.
At the end of that time he found that the bacilli were as
virulent as when first examined, though, when subcultured
on to agar or into broth, virulence was rapidly lost. Thus,
two strains of virulent typhoid bacilli from the urine of
" Carrier /" and " Carrier S" failed to kill a guineapig, when
78 VARIATIONS IN VIRULENCE [CH. vi
injected into the peritoneal cavity, after being cultivated on
agar for periods of two weeks and three weeks respectively.
A striking example of the influence exerted by the culture
medium is afforded by the observation that vaccination with
a plague strain grown on agar will protect rats against itself
but not against the same strain grown on serum (quoted
Penfold, 1914).
(b) The artificial or "unnatural" physical conditions
under which laboratory cultures grow are no less important.
The difference between a medium of solidified blood serum
and the blood circulating in the living body is not only one
of chemical composition. When grown on solid media the
organisms are crowded together and there must necessarily
be an accumulation and concentration of acids formed from
the medium, and also of excreted toxins in their immediate
vicinity, which may inhibit their power to produce more of
the latter substances.
If in this case the metabolism of the organisms could be
" damped down," the possibly inhibitory influence of such an
accumulation might be prevented. Shiga (quoted by Bahr,
191 2) found that the virulence of his dysentery bacillus could
be maintained for as long as 12 months by keeping the
cultures at freezing temperature. Whether decreased meta-
bolism is the true explanation in this case or not, it is
impossible to say without further investigation.
In the living body, on the other hand, the products of the
metabolism of the organism are dissolved and carried away
and absorbed and excreted.
(c) Moreover, in the living tissues the presence of toxins
provokes the formation or liberation of " immune bodies " as
a protective measure on the part of the body, and it has been
shown by Ainley Walker (1903) that the influence of these
" immune bodies " whatever their exact nature may be is
to heighten the virulence of the organism which led to their
production. This observer states, with regard to B. typhosus,
that " the result of growing the bacillus in its immune serum
was a diminution in its agglutinability, a heightening of its
virulence and an increase in its resistance to serum protection."
CH. vi] VARIATIONS IN VIRULENCE 79
Substances which increase the virulence of an organism
may be formed in the tissues as a result of infection by an
organism of a different species. For example, the staphylo-
coccus aureus produces more extensive local lesions if, with
the organism, is injected a small quantity of the serum from
a case of spreading cellulitis; if the local exudate from a
cellulitis is substituted for the serum no such effect is produced,
showing that the phenomenon is due to bodies formed not by
the bacteria which cause the cellulitis but by the tissues.
Moreover the serum from a case of spreading cellulitis has
the same effect in the case of other kinds of infection due
to the pneumococcus, B. typhosus, the tubercle bacillus and
cholera (Hektoen).
(d) Yet a fourth contributing factor to the loss of virulence
on artificial media is the fact that other organisms are, as far
as possible, excluded. The object of the investigator is to
obtain a pure culture, by the use of selective media or by
subculturing. In pathological secretions the primary infecting
organism grows in the presence of many other saprophytic
or parasitic bacteria. A typhoid stool contains a multitude
of organisms in addition to Eberth's bacillus and pneumonic
sputum often supplies evidence of a mixed infection. The
influence of symbiosis on the virulence of organisms will be
referred to later, but it is noteworthy, in this connection, that
many organisms which are more prone than others to lose
virulence when grown on artificial media are likewise more
often found in pathological conditions associated with other
organisms.
Whatever the explanation may be, the fact remains that
cultures on artificial media tend to lose virulence. On the
other hand if bacteria grow in pathological secretions, and
particularly if they successfully invade the body of an animal
and multiply in that animal's tissues, their virulence often
becomes greatly increased.
8. Pathological secretions possess the power not only of
preserving virulence but of actually developing that property
on the part of organisms growing in them. For example, the
comparatively harmless B. coli communis, present in large
80 VARIATIONS IN VIRULENCE [CH. vi
numbers in the healthy intestine, develops in many inflam-
matory conditions of the intestinal mucous membrane the
property of virulence. Sanarelli (1894) caused the colon
bacillus to become virulent in experimental typhoid fever
by producing an inflammation with typhoid toxin. Acute
peritonitis due to B. coli, following strangulation of the gut
a sequence frequently observed clinically would appear to
depend upon something more than a lowering of the vitality
of the tissues, for De Klecki (quoted by Peckham, 1897) found,
by experimenting on dogs, that the colon bacillus acquired
virulence in the lumen of a strangulated coil of intestine.
Dreyfuss has described the increased virulence of B. coli in
intestinal disease, and Fermi and Salto (quoted, Peckham)
a similar increased virulence in inflamed conditions of the
intestines due to cold, bad food, etc.
Harris (1901) tested the toxicity of 29 strains of B. coli
communis derived from various sources. Out of 15 strains
obtained from "natural sources'"' (healthy faeces, sewage,
water, milk, shellfish) only two were virulent (one of these very
slightly so) whereas out of 11 strains derived from pathological
secretions (pus and the stools in epidemic cholera, cholera
nostras and summer diarrhoea) only one (from the last-named
source) was non-virulent.
The acquirement of virulence by saprophytic organisms in
the cavity of an inflamed uterus during the puerperium, is
another case in point.
9. In many cases the development of virulence by organisms
growing in pathological exudations is due not only to the
presence of inflammatory products or to the absence of sub-
stances normally found in the healthy secretions but also to
some extent, as we have already said, to the presence of other
bacteria and their products.
In discussing the effect of symbiosis on organisms, reference
was made (vide p. 22) to the fact that substances excreted by
one species of bacteria may markedly influence the growth of
another species. An example was there given of a strain of
B. influenzae which would grow on a sterilised medium
previously impregnated with the products of a staphylococcal
CH. vi] VARIATIONS IN VIRULENCE 81
growth but which could not be made to grow otherwise
(Allen, 1910).
That symbiosis is an important factor in determining not
only the growth but also the virulence of a strain of bacteria,
is abundantly proved both by experiment and observation.
It is said that a dog will not succumb to the infection of
tetanus unless it is infected simultaneously with pyogenic cocci
and in man it is recognised that the prognosis is more serious
if the tetanus gains access to the body by means of a sup-
purating wound. Some authorities explain these facts by
assuming that the tetanus bacillus can only multiply in the
body in the presence of pus-forming cocci (Marshall Ward
and Blackman, 1910), but analogy with other phenomena of
the same kind certainly suggests that it is a question of altered
virulence.
Sanarelli observes that B. coll communis in typhoid stools
was highly virulent. Muir and Ritchie state "guinea-pigs
may resist the subcutaneous injection of a certain dose of the
typhoid bacillus, but if at the same time a sterilised culture of
the bacillus coli be injected into the peritoneum they quickly
die of general infection." These authors attribute the pheno-
menon to the diminished vitality of the animal, but here
again analogy suggests that increased viability or heightened
virulence on the part of the typhoid bacilli may be factors of
no less importance.
Other examples of symbiosis influencing virulence will be
given in discussing "passage."
10. The successful invasion of the animal body by bacteria,
leading to their increased virulence, may be brought about
experimentally.
Pasteur was the first to discover that virulence could be
"exalted" by " artificial passage" through an animal or series
of animals. Rabbits or guinea-pigs are those commonly em-
ployed for the purpose and inoculation may be made into the
blood stream or the peritoneal cavity and a cultivation made
subsequently from the heart's blood or the peritoneal fluid ;
or the. organisms may instead of being introduced directly
into the peritoneum be shut up in a closed sac which is then
D. 6
82 VARIATIONS IN VIRULENCE [OH. vi
inserted into the body cavity and allowed to remain there for
a certain time.
By the last named method, for example, Martin (1898)
increased the virulence of a slightly virulent strain of B.
diphtheriae.
Eyre and Washbourn (1899) showed that the saprophytic
pneumococcus, found in the mouths of healthy persons, could
by "passage" be made as virulent as the parasitic type isolated
from an acute lobar pneumonia. The number of inoculations
required varied considerably in different cases. In most of the
experiments a series of eight or ten rabbits sufficed. In one
case virulence only reached its height after no less than
53 passages. In another case a single inoculation was sufficient
to convert an avirulent organism into a highly virulent
one. They noted that strains in which virulence was easily
raised were able to maintain their exalted virulence on
suitable media for a long time, while those strains which
very slowly acquired virulence, quickly lost it on artificial
culture.
Mohler and Washburn (1906) mention that the virulence
of the virus of rabies is increased by passage through rabbits
and that the cholera organism, after passage through guinea-
pigs, becomes much more virulent towards pigeons.
Other animals than those named may be utilised for the
purpose of "passage." For example, Salter (1899), by means
of five successive passages through the goldfinch, raised the
virulence of the "pseudo-diphtheria bacillus" sufficiently to
render it fatal to guineapigs.
The process of "passage" may be even more effective if
it be made to alternate with culture on ordinary media.
Marmorek (quoted, Muir and Ritchie) showed that the viru-
lence of the streptococcus was enormously increased by grow-
ing it alternately in a mixture of human blood serum and
bouillon and in the body of a rabbit.
11. Inoculation with a living or dead culture of some
other organism in many cases intensifies the result. Thus,
Klein (1903-4) states that the virulence of the diphtheria
bacillus is greatly increased by inoculation into an animal if
CH. vi] VARIATIONS IN VIRULENCE 83
the streptococcus pyogenes is inoculated with it, but not to
the same degree if the diphtheria bacillus is inoculated
alone.
In this connection Miss Williams' experiments (1902) are
of interest. She grew two strains of avirulent (but morpho-
logically typical) diphtheria bacilli with a strain of virulent
streptococci in broth, transplanting every three or four days
for 90 successive generations, without producing any change
in the virulence of either organism.
The virulence of the bacillus of malignant oedema is
markedly increased if this organism is inoculated together
with B. prodigiosus, and that of the streptococcus if it is
inoculated with B. coli communis (Muir and Ritchie).
Klein (1903-4) also found that the injection of many
organisms subcutaneously (B. coli, B. Gaertner, B. enteritidis
sporogenes, and others) enhanced the virulence of organisms
such as B. typhoms or F. cholerae growing simultaneously in
the peritoneal cavity.
The exalted virulence thus produced applies to the par-
ticular species of animal employed for passage and may, as
we have shown, apply to another species also but this is not
necessarily the case. Indeed, the virulence towards other
species may be markedly diminished. For example, the virus
of rabies becomes attenuated by passage through monkeys
(Mohler and Washburn, 1906). The bacillus of swine erysipelas,
isolated by Loeffler, after passage through the rabbit shows
exalted virulence towards this animal but attenuated virulence
towards pigs (Adami, 1892). Duguid and Burdon-Sanderson
found that the virulence of anthrax bacilli for bovine animals
was diminished after passage through a number of guineapigs.
Pasteur found that, if swine plague were inoculated from rabbit
to rabbit, the organism became more virulent for the rabbit
but less virulent for pigs (Muir and Ritchie). Streptococci, on
being inoculated through a series of mice, acquire increased
virulence for these animals but become less virulent for rabbits
(Knorr, ibid.).
Many other examples might be given. A familiar one is
the preparation of the calf lymph for "vaccination" where
62
84 VARIATIONS IN VIRULENCE [OH. vi
advantage is taken of the same phenomenon to attenuate the
virus of smallpox.
THE SIGNIFICANCE OF VARIATION IN VIRULENCE.
There is a mass of evidence, therefore, to show that the
virulence of bacteria is very variable. What is the explanation
and significance of these observations?
Andrewes, in the Horace Dobell Lecture already quoted
(vide p. 3), described the evolution of bacteria from harmless
mineral feeders into animal saprophytes and finally into
parasitic organisms and showed that virulence was probably
the latest property to be acquired in the process. It is an axiom
in the study of evolution that the latest characteristic or
function to be acquired is the most unstable and the first to
be lost if retrogression occurs. Each new function acquired
indicates a higher degree of specialisation. The more highly
specialised the activity of an organism becomes the more
intricate are the processes upon which it depends and the
more readily is it like a complicated mechanism "put out
of gear." It is recognised, for example, by alienists that in cases
of mental degeneration the highest faculties, which, from their
absence or rudimentary character in lower animals and on
other grounds, are regarded as having been the last to be
evolved, are the first, and often the only ones, to show
signs of impairment. The loss of virulence by bacteria, often
unaccompanied by any other alteration in the character of
the organism is another illustration of the same phenomenon.
The process by which this property of virulence is regained
by a strain of bacteria is exactly similar to the process by
which it was originally acquired by the race, but accelerated,
that is to say it is a case of the survival of the fittest.
The fate however of a band of sojdiers raiding an enemy's
country depends not only on their numbers and strength but
also on their resourcefulness, so that the "fittest" in this
connection must be interpreted to mean not necessarily the
strongest or most robust but those best able to protect them-
selves ; and since the most successful method of resistance
CH. vi] VARIATIONS IN VIRULENCE 85
is to attack, certain of these minute organisms acquire the
power of manufacturing toxins which weaken the defence and
counteract the opposition of the living tissues and so enable
them to gain a firmer foothold there. It is the individuals
which thus accommodate themselves to the exigencies of their
surroundings that are perpetuated by a process of natural
selection.
That it is a case of particular adaptation to environment
and not merely a question of vitality or robustness is shown by
the following observations.
(a) "Passage" through a certain species of animal while
increasing the virulence for that species may actually diminish
it for another species. If the process merely selected the
strongest, the strain of organisms resulting should show
heightened virulence for other animals also.
(6) The most virulent organisms are not necessarily the
most robust. Eyre and Washbourn (1899) found, as Kruse and
Pansini had done previously, that in the case of the pneumo-
coccus the exact contrary was true. "The most virulent strains
were those which were most delicate and sensitive in artificial
cultivations and the less virulent ones were much less delicate
and could grow under conditions in which the virulent ones
were unable to flourish." The parasitic type required a certain
reaction and temperature and special media. It would not
grow if the reaction was even faintly acid or at a temperature
much below that of the body and rapidly died out on agar or
in broth. The saprophytic type grew luxuriantly either at
37 C. or at 20 C., in broth, agar, potato or gelatin, whether
acid or alkaline, and retained its vitality for many months.
(c) Analogy with other processes of adaptation lends
further support to the view put forward. Thus, Rettger and
Sherrick (1911) have shown that by artificial selection a strain
of organisms can be made unusually resistant to the action of
an antiseptic such as corrosive sublimate. Penfold (1911 c)
showed that natural selection might in the same way develop
a special power of resistance to an antiseptic in this case
chlor-acetic acid. The last-named observer went further and
demonstrated that the particular strain of organisms which
86 VARIATIONS IN VIRULENCE [CH. vi
showed increased powers of resistance to chlor-acetic acid
failed to show any similar increase in their power of resisting
other antiseptics such as carbolic acid or formaldehyde. In
other words it was a case of adaptation and not merely
increased robustness.
(d) The acquirement of virulence during the process of
passage is sometimes accompanied by other changes in the
character of the organisms which are only to be accounted for
by the theory of adaptation to a particular environment. For
example, the parasitic pneumococcus isolated from the human
lung in an acute lobar pneumonia (Eyre and Washbourn) was
characterised by inability to grow except at a temperature
near that of the human body. Again the avian tubercle
bacillus the particular race of tubercle virulent to birds
grows best at their body temperature which is higher than
that of man, namely 43*5 C., a temperature at which human
tubercle dies.
Certain difficulties are nevertheless presented by this
theory of the development of virulence by natural selection.
(a) The first to suggest itself is the fact that toxins are
"intracellular" as well as "extracellular" and, inasmuch as the
intracellular toxins are not liberated until the death of the
organism and its disintegration, their nature and potency can
obviously have no influence on that organism's survival or
perpetuation. We are not concerned, however, with one
isolated bacterium's struggle for existence so much as with
the fate of a host of bacteria invading the tissues, and it is no
less obvious in their case that if their intracellular toxins are
destructive of the vitality of the tissues the living bacteria
will receive assistance from their dead and disintegrated
comrades which they would not otherwise do, and this fact may
determine the success of their invasion and consequently their
perpetuation.
It is possible that in the case of some bacteria the toxic
action of the substances set free on their disintegration is
a purely physiological one comparable to the effects produced
by the absorption of extravasated blood and not due to
a special adaptation. The protective action, however, of
CH. vi] VARIATIONS IN VIRULENCE 87
tuberculin and modern vaccines against specific diseases
suggests that the role of the intracellular toxins is of greater
significance than this hypothesis would admit.
(6) Another question that suggests itself is this: why,
if successive passages increase virulence, do not infectious
diseases, which are continually undergoing this process of
passage, become of deadly virulence?
One explanation is that an obligatory parasite which kills
its host sinks the ship it is sailing in and thereby sacrifices its
own chance of survival, so that the most virulent organisms
are weeded out and destroyed. Many infectious diseases,
however, have not reached such a high degree of virulence
that this factor can have become operative in their case.
(c) Yet a third difficulty in accounting for the development
of virulence by natural selection is the occurrence of pheno-
mena such as that described by Eyre and Washbourn in which
an avirulent pneumococcus acquired a high degree of virulence
after a single passage through an animal. The virulence so
acquired was maintained for a couple of months on artificial
media, much longer, that is to say, than it was found possible
to maintain the same character when developed more slowly.
It is difficult to conceive how, in the course of so few
generations comparatively, natural selection could cause the
character of virulence to predominate to such an extent.
Moreover, if it did so one would expect the character to be
lost with equal readiness outside the body.
The explanation may be that when avirulent and virulent
pneumococci grow side by side on artificial media there is no
selective action and the avirulent, being the more robust as
these writers have shown, soon greatly outnumber the virulent ;
the latter, after subculture has been carried out several times,
may be so few in number that they give no evidence of their
presence. "Passage" in this case, by eliminating the avirulent,
would very soon select out a pure strain of virulent organisms.
Another explanation, however, suggests itself. The change
has more the aspect of an alteration in metabolism occurring
as a direct response to the change in the food material pro-
vided. It is easy to imagine that, once established, such
88 VARIATIONS IN VIRULENCE [CH. vi
altered metabolism might persist outside the body for some
time if the substances provided for assimilation remained the
same. In the case quoted, Eyre and Washbourn found that
virulence was maintained only on media containing blood
(blood agar) just as Anne Williams found that the virulence of
diphtheria bacilli was maintained only on serum or ascitic
fluid. (The possible relationship between toxicity and
altered metabolism will be discussed later.)
(d) Another difficulty is the development of the property
of virulence by organisms outside the living tissues, as, for
example, by saprophytic bacteria in the intestine or in the
cavity of the uterus during the puerperium. What part can
adaptation or selection play in the case of these ?
It is true that a saprophyte which has acquired the power
of excreting toxins has thereby acquired the power also of
lowering the vitality of the living cells exposed to their
action, or even of killing these in cases where the superficial
tissues have been injured previously. The toxic saprophyte
by such means is enabled to procure fresh food-stuffs for its
own use, but since it is forced to share the spoils with its
non-toxic brethren this accomplishment is less a private gain
than a public advantage, and hardly conduces more to its
own survival than it does to theirs. It is evident, neverthe-
less, that a strain of saprophytes which developed toxic pro-
perties might survive and multiply under conditions in which
a strain of non-toxic saprophytes would die out, so that the
strain of saprophytes possessing the greatest toxicity would,
other things being equal, stand the best chance of being per-
petuated.
A saprophyte, e.g. in the uterus during the puerperium,
may not only develop extreme toxicity but may actually
invade the living tissues and become parasitic. Here ob-
viously other questions are involved. One of these concerns
the part played by the food-stuffs of bacteria and the effects
of changes in these.
The fortunes of an invading army depend as much upon
its successful victualling as upon its armament ; if the former
breaks down the latter is of no avail. A non-toxic saprophyte
CH. vi] VARIATIONS IN VIRULENCE 89
which develops into a virulent parasite, invading the living
tissues, undergoes a twofold adaptation, for it must neces-
sarily acquire the faculty of nourishing itself upon unac-
customed food-stuffs as well as the faculty of excreting toxins.
The second would be useless without the first.
What is the relation between these two faculties? Both
imply altered metabolism ; one involves a change in assimi-
lation, the other a change in excretion ; one necessitates the
assimilation of highly organised materials, in the shape of
proteid or extractives, the other consists in the excretion of
complex substances which in some cases have been proved to
be proteid in nature the toxalbumins and in others are
more akin to extractives.
The possibility naturally suggests itself that the second
phenomenon may be dependent upon the first, that, in some
cases at least, the excretion of toxins is the direct result of the
altered assimilation, comparable to the increased toxicity of
the urine of a man on a certain diet.
This hypothesis would go far towards explaining the
development of toxicity by saprophytic organisms growing
in material of a highly albuminous nature and rich in extrac-
tives, in an inflamed uterus or in pathological exudations
wherever found, without any actual invasion of the living
tissues by the organism taking place.
Two observations are of interest in this connection.
(a) Miss Peckham (1897), in speaking of coliform organ-
isms, expresses the opinion that the carbohydrate con-
stituents of the culture medium are always attacked by the
organisms present in preference to the proteid material and
it is only when the supply of carbohydrate is exhausted that
the proteid is made use of. She showed that if B. typhosus
were repeatedly subcultured in peptone solution which con-
tained no carbohydrate it acquired the power to split up the
proteid and produced indol. She also quotes Pe"r4 to the
effect that the appearance of the indol reaction (which
depends upon the breaking up of the proteid by the organisms)
is proof of the absence of carbohydrate, an opinion she herself
confirmed by experiment. She found that indol formation
90 VARIATIONS IN VIRULENCE [CH. vi
which followed the elimination of the sugar (lactose) by the
bacteria was inhibited by its further addition.
Glenn (1911) sought to ascertain by experiment whether
this inhibition in indol formation was due to the acid pro-
duced by the splitting up of the sugar. He found, however,
that even when the acid was neutralised indol was not formed
until all the sugar had been eliminated.
It is interesting to note that, as long ago as 1889, Cart-
wright Wood explained the fact that indol formation did not
take place in the presence of glycerine, on the ground not
that the glycerine interfered with the activity of the bacteria
but that the glycerine offered them a pabulum which they
preferred.
(b) The second observation is that in the case of some
organisms for example B. diphiheriae (Theobald Smith
1899, Fisher 1909) toxins are formed in a culture only if the
amount of the sugar in the medium is very small not more
than a " trace."
In one case, therefore, we find that organisms do not split
up proteid material as long as they can subsist on other food-
stuffs such as carbohydrates, and in the second case we find
that some organisms, at any rate, do not elaborate toxins in
the presence of much carbohydrate material. Both these
observations lend support to the theory that the development
of toxicity may result from an alteration in metabolism brought
about by a change in the kind of food-stuffs available.
If once this altered metabolism is established the step is
a short one from a saprophytic existence to a parasitic one.
The organism now trained to feed on "vital" material has
only to cross the border line between the dead and living
tissues to become a virulent parasite.
The invasion of the living tissues, however, on the part of
an organism, although it may necessitate the altered assimi-
lation to which we have been attributing toxicity, does not
always confer on the organism the property of virulence. For
example, in a case recorded by Pansini, staphylococci were
repeatedly subcultured from the blood over a period of years
without there being any evidence of toxaemia.
CH. vi] VARIATIONS IN VIRULENCE 91
We are forced, therefore, to the conclusion that if in some
cases the formation of substances which are toxic in their
action is purely a physiological process of excretion following
on altered assimilation, in other cases this result is due to a
special adaptation, the toxin being a secretion rather than an
excretion.
The explanation of this and other kindred phenomena is,
however, unsatisfactory and the suggestion has been made in
many quarters that the property of virulence may be due
to the action of something more or less distinct from the
organism itself but grafted on or attached temporarily to it
something in the nature of a ferment or enzyme. This
theory we shall discuss later (vide chap, xi) but in connection
with it one observation will be made at this point, namely,
that if the liberation or acquisition of a ferment by the
organism is of advantage to it in its life struggle, it may still
be regarded as an adaptation to environment and natural
selection will in time cause the characteristic to predominate.
THE VALUE OF VIRULENCE IN CLASSIFICATION.
In concluding this section it only remains to say one word
as to the value of classification according to virulence.
Differences in a character so variable as we have shown
virulence to be cannot, alone, be regarded as sufficient to
justify a separation of bacteria into distinct "species." The
inconsistency to which such a classification gives rise can
best be demonstrated by examples.
For instance, we find in the throats of some patients con-
valescent from diphtheria, the so-called "carriers," bacilli
indistinguishable from the Klebs-Loeffler bacillus, but in
many cases non-virulent. In such cases the organism is
almost certainly the lineal descendant of the original
virulent infecting organism and cannot be regarded as a
distinct species.
In other cases, in which no history of diphtheria can be
elicited, bacilli again are found in the throat morphologi-
cally and culturally indistinguishable from the Klebs-Loeffler
92 VARIATIONS IN VIRULENCE [CH. vi
bacillus but non-pathogenic. The weight of evidence is against
the possibility of rendering such strains virulent by passage.
A filtered broth culture, however, of these organisms will
provoke the formation of diphtheria antitoxin in the horse
(Ark wright, 1909) so that they must be regarded as true
diphtheria bacilli although non-pathogenic.
The pneumococcus is found as a virulent organism in acute
lobar pneumonia. It is normally present in the mouths of
healthy persons in a form which is non-virulent but which
can be made virulent by passage through an animal and in
this case is indistinguishable from the pathogenic variety.
These two forms are regarded only as varieties of the same
species.
The comparatively harmless B. coli communis, normally
present in the human intestine in health, gives place in many
unhealthy and inflamed conditions of the intestine to a
virulent organism which in every other respect is identical.
In this case opinion is divided, some authorities regarding the
toxic or pathogenic B. coli as a distinct species from the non-
toxic B. coli although they possess no other distinguishing
feature apart from virulence and this in the case of the
former can be diminished by artificial culture and in the case
of the latter increased by " passage."
The discovery of the amoeba coli in the intestines of
healthy individuals in the tropics, where amoebic dysentery
is rife, presents a similar problem for solution. Bahr (1912)
found the amoeba coli in 30 per cent, of Fijians examined by
him and Ash burn and Craig discovered it in 72 out of 100
soldiers examined in the Philippines though none of these
72 had diarrhoea or dysentery at the time or had ever been
on the sick list with either of these diseases.
The Bac. anthracoides, discovered by Andrewes and de-
scribed by Bainbridge (1903), was distinguished from the true
B. anthracis by slight differences in the appearance of gelatin
and agar cultures, in rate of growth and in motility and by
its non-virulence. The observations of Savage and MacConkey,
already quoted, as to the frequency with which atypical
colonies of some organisms occur on gelatin, shows how
CH. vi] VARIATIONS IN VIRULENCE 93
misleading such a distinction may be, and in this case, more-
over, the difference in the appearance of colonies on agar
could by the adoption of certain precautions be entirely
eliminated. The rate of growth of organisms is always subject
to variation. Slight motility was therefore the only distin-
guishing feature to which any importance could be attached,
apart from the question of virulence. As regards the latter,
experiments indicated that this property could be increased
by passage. Nevertheless these observers regarded the dif-
ference in virulence as sufficiently fundamental to justify
their description of the organism as a new species quite dis-
tinct from the B. anthracis, although spores of the latter
organism were found with it.
The Streptococcus erysipelatis was formerly considered, on
account of its greater virulence and certain minor differences,
to be a distinct species from S. pyogenes\ further investi-
gation has however shown this opinion to be untenable.
Many other examples might be given but these will suffice
to show the difficulties that arise from regarding virulence as
a " specific " character.
CHAPTER VII
VARIATIONS IN PATHOaENICITY
WE have shown that morphology, fermenting properties and
virulence are all variable features. There remains to be
considered one other character of bacteria which is of great
value in their classification, namely their " pathogenicity " or
their power to cause specific disease.
Under this head are to be considered, firstly, the kind of
animal in which a particular organism can develop disease,
secondly, the kind of symptoms caused, and thirdly, the kind
of lesions produced by that organism's invasion of the living
body.
The pathogenicity of an organism is something quite
distinct from its other characters. Two organisms may possess
the same morphology, the same fermenting power and the
same degree of virulence and yet show a wide divergence in
their pathogenicity, giving rise in the body to a totally
different train of symptoms and lesions.
This character of pathogenicity derives particular value
and importance from the fact that it is generally regarded as
being more " fixed " than the other characters we have been
discussing. Great reliance is, for this reason, placed upon
resemblances and differences in pathogenicity in determining
whether two organisms do or do not belong to the same
species, in fact it is regarded as constituting a final appeal
in doubtful cases.
For example, Clark (1910) maintains that Hofmann's
bacillus and the Klebs-Loeffler bacillus represent different
species on the ground that the former when rendered virulent
gives rise to different symptoms in the body.
Again, the gonococcus and the meningococcus show close
resemblances in many of their characters but are readily
CH. vn] VARIATIONS IN PATHOGENICITY 95
distinguished by their pathogenicity. W. B. M. Martin (1911)
writes in this connection : " so far, in spite of the prevalence
of gonorrhoea and the periodical occurrence of great epidemics
of cerebrospinal fever, there is no satisfactory evidence that
the gonococcus ever causes a meningitis or the meningococcus
a urethritis. This is the more remarkable in that, on the one
hand, gonorrhoeal metastases are common enough elsewhere
and that, on the other, meningococci can frequently be isolated
from the urine of cases in which there is not the slightest
evidence of genito-urinary inflammation." He maintains that
the explanation must be in differences in " pathogenicity " on
the part of the two organisms and that such differences justify
our regarding them as distinct species.
A study, however, of the pathogenicity of bacteria reveals
the fact that this, like every other character they possess, is
subject to variation with respect to (i) the kind of animal
affected, (ii) the kind of symptoms caused and (iii) the kind
of lesions produced.
I. As regards the first of these we have already shown
when speaking of virulence that the degree to which a
particular organism can cause disease in different species of
animals is subject to variation and can be artificially modified
(vide p. 81 et seq.).
II. With regard to the second, variability in the symptoms
caused is displayed in several ways.
1. In the first place, the same species of organism may
give rise in different cases to a totally different train of
symptoms.
In many instances the explanation is obvious. The symptoms
naturally depend, to some extent, upon the particular organ,
either primarily or solely, affected in each case. The toxic
action of lead furnishes an analogy. Its absorption into the
body is followed by symptoms of arterio-sclerosis or of
peripheral neuritis or of renal disease according to whether
the blood-vessels or the nerves or the kidneys are primarily
affected. The pneumococcus, for example, may attack the
meninges, the lungs, the pericardium, the peritoneum or a
synovial membrane, and the difference in the symptoms in
96 VARIATIONS IN PATHOGENICITY [CH. vn
each case is attributable to anatomical differences in the
parts affected.
The determining factor may be in one case the route of
infection, in another the lowered vitality of the particular
organ attacked. In a third case neither explanation appears
adequate and we are forced to conclude that the organism
itself has some influence in determining the site of the
disease.
Such an hypothesis is required, for example, to explain
the fact that different strains of the tubercle bacillus, morpho-
logically and culturally indistinguishable from one another,
may produce in one patient phthisis, in another a tuberculous
osteitis or arthritis, and in a third lupus. Here the analogy
with lead poisoning or pneumococcal infection fails, for in
both the latter conditions, although one system or organ may
be for a time affected almost alone, the disease shows a
tendency to extend to other regions of the body and to
produce characteristic symptoms as each fresh region becomes
involved. In the case of tuberculous infection there appear
to be certain limitations. A patient with phthisis may develop
meningitis or peritonitis or general tuberculosis, but it is rare
for a phthisical patient to develop lupus or a tuberculous
joint, or for one suffering from a tuberculous ostitis to develop
either lupus or phthisis facts which are significant when one
considers how widespread are these diseases.
Nield and Dunkley (1909) quote an instance of a phthisical
patient who moistened a scratch on her arm with her own
saliva and developed lupus at that spot. Examples have been
given by various observers (quoted Stel wagon, 1910) of lupus
occurring on the hands of women employed in washing the
clothes of patients with phthisis and of the same disease
following such operations as ear piercing, tatooing and cir-
cumcision when performed by operators themselves suffering
from phthisis, but such instances are sufficiently uncommon
to be regarded as exceptional.
The contrast already referred to between the gonococcus
and the meningococcus, in respect to the organs they par-
ticularly one might almost say exclusively attack and the
CH. vn] VARIATIONS IN PATHOGENICITY 97
lesions to which they give rise, is no greater than the contrast
existing between different strains of Koch's bacillus in the
same respect ; if we ascribe the contrast in the former
case to specific differences in pathogenicity one is forced to
ascribe it in the latter case to the same factor and acknowledge
that different strains of the tubercle bacillus exhibit marked
differences in pathogenicity.
There is one not unlikely fallacy which needs to be
guarded against before we can with confidence attribute to
an organism any unusual symptoms which appear to follow
its invasion of the body. A certain disease may be latent in
the patient, that is to say present without giving rise to any
noticeable symptoms. The constitutional disturbances arising
from infection by the organism in question may " light up "
this pre-existing disease and the symptoms of the latter may
then be incorrectly credited to the invading organism.
Such a sequence is well illustrated by a case recorded by
Roberts and Ford under the title " A case of Cerebrospinal
Fever simulating Acute Nephritis with uraemic convulsions."
The patient suffered from typical symptoms of acute nephritis
and uraemia dropsy, the passage of scanty urine loaded with
albumen, convulsions and coma and showed marked im-
provement as a result of the treatment usually adopted for
these conditions. The meningococcus was, however, isolated
from the spinal fluid and the symptoms on this account
attributed to that organism. In this case a pre-existing
nephritis might, conceivably, have given rise at the onset of
the illness to the symptoms characteristic of that disease
before those characteristic of cerebrospinal fever had had
time to develop ; or the symptoms of the first disease might
have completely masked those of the second.
2. In the second place, one and the same strain of an
organism can by artificial means be so modified as to cause
an altogether different type of disease. For example, Madame
Henri (1914) found that the pathogenicity of B. anthracis
was changed to a remarkable degree by exposure to the
ultra-violet rays. Its subsequent injection into an animal
produced symptoms quite unlike those caused by the normal
D. 7
98 VARIATIONS IN PATHOGENICITY [OH. vn
anthrax bacillus and it did not revert after daily subculture
for two months afterwards.
3. In the third place, a contagious disease passed from
one case to another during the course of an epidemic may be
characterised in different cases by widely different symptoms.
Andre wes and Horder (1906) have recorded an example
of this. A woman (A), admitted for her confinement to a
maternity home, was attended by a nurse (B) who developed
tonsilitis on the day the patient was delivered, and three
days later was notified as a case of scarlet fever. The woman
(A) developed puerperal fever and was removed to a hospital
where she died. Three days after her death a nurse (C) who
had attended her at the hospital developed scarlet fever. At
the maternity home another nurse (D) took nurse B's place
and had attended the woman (A) for a day or two before the
latter's removal to hospital. Ten days later this nurse (D),
who herself remained well, attended another confinement case
(E) in the district. The woman (E) died of septicaemia on
the fifth day after delivery. Nurse B's room, having been
disinfected by the Sanitary Authority, was scrubbed by a
charwoman (F). On the following day this charwoman became
ill but the case was not recognised to be scarlet fever until a
day or two later when her two children (G) and (H) developed
typical scarlet fever and all three were removed to a fever
hospital. A scheme will make the sequence of events clearer :
^ Child (G)
Maternity nurse (B) Charwoman (F) / Scarlet fever
Scarlet fever Scarlet fever \ Child (H)
Scarlet fever
Patient (A) ^ Maternity nurse (D) > District case (E)
Puerperal fever well Puerperal fever
Hospital nurse (C)
Scarlet fever
The original case of scarlet fever infected two other people,
one with scarlet fever and the other with puerperal fever ;
this case of puerperal fever also infected two other people,
one with puerperal fever and the other with scarlet fever.
An even more remarkable instance is recorded by Dunn
CH. vn] VARIATIONS IN PATHOGENICITY 99
and Gordon (1905). They describe an epidemic in Hertford-
shire characterised by an extraordinary diversity of symptoms
in different patients. In some cases there were sneezing,
coryza, and the ordinary symptoms of a common cold. In
other cases patients had "aches and pains all over," stiff neck
and suffered subsequently from great debility ; such cases
had all the appearances of influenza. In others, again, the
illness closely simulated scarlet fever ; it began with sore
throat, rigors, vomiting, headache, fever and rapid pulse, and
was accompanied by a punctate rash at the end of the first
24 hours (followed later by desquamation), the " strawberry "
tongue, circumoral pallor, enlarged cervical glands which in
some cases suppurated, and, in some patients, complications
such as nephritis, arthritis and otorrhoea. A fourth type
resembled diphtheria and exhibited a suspicious membrane
on the tonsil. A fifth type was notified in some cases as typhoid
fever and was characterised by epistaxis, melaena, prostra-
tion and, in some cases it is stated, a positive Widal reaction.
Finally, a number of cases, particularly amongst children,
resembled cerebrospinal fever and were so diagnosed ; these
were characterised by profuse nasal discharge, pain in the
back of the neck, headache, photophobia and irritability,
dilatation of one or both pupils, persistent vomiting, drowsi-
ness, head retraction, paralysis, coma and, sometimes, con-
vulsions and death.
Sometimes these widely divergent types were exhibited
by the different members of a single family or household
struck down by the disease, either simultaneously or con-
secutively. After a thorough investigation these observers
were convinced that the outbreak of these various types of
illness was due to the prevalence and spread of only one
disease and not a number of different diseases, and a bacterio-
logical examination of a large number of cases by Gordon
showed that the disease was due to infection by an organism
closely resembling, if not identical with, M . catarrhalis.
4. In the fourth place, the same species of organism may
give rise in different epidemics to widely different types of
disease. For example, strains of B. influenzae may give rise
72
100 VARIATIONS IN PATHOGENICITY [CH. vn
to epidemics of "influenza" characterised by symptoms
resembling in one epidemic a simple coryza, in another
rheumatic fever, in a third typhoid fever, and in a fourth
cerebrospinal meningitis.
5. In the fifth place, the train of symptoms characteristic
of infection by one organism may develop as a result of
infection by a totally different organism.
A striking instance of this is recorded by Head and
Wilson (1899) who proved that a supposed case of rabies was
actually due to infection by the diphtheria bacillus. The
diagnosis of rabies was founded on the history and clinical
symptoms. " The well authenticated history of a bite on the
cheek by an unknown animal, the two months' incubation
period, the onset with extreme pain and numbness in the
region of the scar, the development of the characteristic
laryngeal and respiratory spasms on attempting to take
liquids, the spasm at first being slight but later more pro-
nounced and towards the close again feeble or absent, the
insomnia, the absence in the beginning of fever which later
in the illness became pronounced, the rapid pulse at all
stages, the attacks of violent delirium interspersed with
periods of calm and complete rationality, the absence of all
symptoms pointing towards any other simulating disease and
the fatal termination all serve to make an almost complete
picture of rabies." The Klebs-Loeffler bacillus was isolated
from the ventricular fluid and detected in the nerve cells of
the medulla. The recognition of this organism was complete
and beyond doubt. " Not less suggestive of rabies than the
clinical history were the results of subdural inoculations of
rabbits with emulsions prepared from the medulla of the
patient. There occurred the long period of incubation (20
and 21 days) followed by phenomena similar to those in
experimental rabies of rabbits, and other rabbits inoculated
subdurally with the medulla of the first rabbits behaved in
a similar manner." B. diphtheriae was demonstrated after
death in the medulla of the rabbits. By a thorough investiga-
tion, full details of which are given, infection by the virus of
rabies was definitely excluded.
CH. vii] VARIATIONS IN PATHOGENICITY 101
Dunn and Gordon (1905, vide supra p. 99) have described
almost typical cases of scarlet fever, of cerebrospinal
fever and of influenza, which proved to be due to infec-
tion by the micrococcus catarrhalis. Gordon has described
elsewhere typical cases of cerebrospinal fever due to B.
typliosm.
Nash has recorded a remarkable case of malignant en-
docarditis characterised by fever, constipation, headache,
drowsiness and delirium, photophobia, strabismus, head re-
traction and the appearance of a petechial rash. The illness,
in fact, presented all the clinical features of cerebrospinal
fever. A copious growth of a pure culture of the Klebs-
Loeffler bacillus was obtained post mortem from the spinal
fluid and a similar growth from the heart's blood. There was
a history of a discharge from the ear at the beginning of the
illness but no history of sore throat.
Thomson (1911) has recorded his own experience of an
acute inflammation of the throat simulating diphtheria in
producing, in the fourth week of the illness, temporary para-
lysis of the tongue, arms and legs, but proved to be due to
pneumococcal infection.
Colman and Hastings (1909) state their conviction that
some strains of B. coli are capable of causing a disease clini-
cally identical with typhoid fever.
III. The pathogenicity of bacteria presents yet another
aspect, namely the character of the lesions produced by them
in the living tissues.
This can be studied in two ways. Firstly, by observing the
lesions produced in the body at various stages in the course
of an infective disease ; and secondly, by observing the lesions
produced by the artificial inoculation of organisms into
animals, both at the site of inoculation and elsewhere.
1. The lesions produced in the course of disease and ob-
served post mortem not infrequently enable one to identify
the infecting organism. For example, tuberculous ulceration
of the intestine, tuberculous consolidation of the lungs, and
tuberculous invasion of the skin, present altogether different
features from typhoid ulceration of the intestine, pneumococcal
102 VARIATIONS IN PATHOGENICITY [OH. vn
consolidation of the lung and streptococcal invasion of the
skin, respectively.
It is however common experience that even in the post
mortem room a certain diagnosis of the nature of the infec-
tion cannot always be made. Sydney Martin, in speaking of
tuberculosis, says "There is, with the exception of the presence
of the tubercle bacillus, no element in the structure of the tu-
berculous lesion which is diagnostic of the disease." In other
words the lesions regarded as characteristic of infection by
one species of organism may be produced by infection by a
totally different species.
Such departures from what experience has taught us to
regard as the normal or characteristic lesion in the case of a
given organism may be accounted for by the influence of other
factors beside the nature of the organism itself such factors,
for example, as the age of the patient, the route of invasion,
the presence of a secondary infection, the effect of treatment,
and many others. The question arises, how far, if it were
possible to exclude such disturbing influences, would the
lesions retain their specific character ?
2. This leads us to a consideration of the second method
of studying the question by observing the lesions produced
by artificial inoculation of animals, both at the site of inocu-
lation and elsewhere. Such a method enables one to, so to
speak, "standardise" the lesion. A healthy animal of the
same species, age and weight can be utilised at each experi-
ment, the inoculation made in the same manner, at the same
site, with the same number of organisms and these of the same
degree of virulence, and the animal can be killed after the
same interval of time.
Many investigators maintain that under such conditions
the lesions produced by a certain species of organism are
constant in their appearance that, however much the other
characters of an organism may vary, this character at any
rate is invariable and will establish beyond dispute to which
of two species a doubtful organism actually belongs.
Thus, Klein as long ago as 1899 in describing the "bacillus
of pseudo-tuberculosis " stated that in cultural and morpho-
CH. vn] VARIATIONS IN PATHOGENICITY 103
logical characters this organism showed certain resemblances
to B. coli. The two organisms could be distinguished from
each other most certainly by animal inoculation. Subcutaneous
inoculation of the first named into the guineapig gave rise to
typical nodular, necrotic, purulent changes in the lymphatic
glands, omen turn, pancreas, liver, spleen, and lung, an effect
which B. coli and its varieties did not produce.
Again, Shattock (and others, 1907) regards the avian tu-
bercle bacillus and the human tubercle bacillus as two distinct
species on the ground that, whereas the former when inoculated
into guineapigs produces merely a local or a local and glandu-
lar disease, the latter produces visceral disease as well.
Savage (1908-9) has recorded some interesting experiments
illustrating the value of animal inoculation in revealing differ-
ences in pathogenicity. He found that streptococcus mastitidis,
which causes mastitis in the cow, was non-virulent to mice
and other rodents but possessed to a marked degree the power
to produce mastitis in goats when inoculated into the mammary
ducts, and was thereby differentiated from streptococcus an-
ginosa (isolated from human sore throat) which, though virulent
to mice, did not possess the power to produce mastitis in goats.
Continuing his experiments with pyogenic streptococci derived
from many sources, he found that, although in their cultural
properties and their virulence to mice they displayed wide
differences, they all resembled each other in their inability to
produce mastitis in goats. One streptococcus, for example,
isolated from a fatal lymphadenitis in a boy, after it was in-
oculated into the teat of a goat survived for seven months as
a harmless saprophyte in the milk passages.
One other example will suffice. We recognise clinically
two types of pneumonia, lobar or croupous pneumonia and
lobular, catarrhal or broncho-pneumonia. Both types may
result from infection of the lung by the pneumococcus. The
invading organism is apparently identical in the two cases,
judged by the ordinary cultural and morphological tests, and
the difference in the results produced are therefore attributed
to differences in the age and vitality of the patient and the
route of infection.
104 VARIATIONS IN PATHOGENICITY [CH. vn
Eyre, Leatham and Washbourne (1906) endeavoured by
the method of animal inoculation to ascertain whether the
difference in the lesions caused depended upon specific differ-
ences in the pathogenicity of the infecting strains. They
found that strains of the pneumococcus isolated from cases of
lobar pneumonia when inoculated subcutaneously into the
guineapig almost invariably gave rise to a local inflammatory
exudation of a fibrinous type, whereas strains isolated from
cases of broncho-pneumonia, when similarly inoculated, almost
invariably gave rise to a local inflammatory exudation of a
celMar type, easily distinguished from the other. A number
of strains of pneumococci obtained from a " neutral " source,
such as the mouth, likewise showed differences in the nature
of the inflammatory reaction they provoked at the site of in-
oculation, some belonging to the "fibrinous" type and others
to the " cellular " type. They further showed that this feature
was not associated with any other differences between the
strains as regards morphology or cultural characters or fer-
menting properties and was quite independent of their degree
of virulence. They therefore regarded it as a specific character.
If the lesions produced in the body during the course of
an infective disease are subject to variation, are those which
result from the artificial inoculation of animals any more
constant?
The materials from which to form an opinion on this point
are somewhat scanty. That the feature in some cases is very
constant was shown by Shattock (and others, 1907) by means
of the following experiment. They grew a strain of human
tubercle bacilli for eight weeks in the spleen of a pigeon. The
subsequent inoculation of the organisms into a guineapig gave
rise, not as might have been expected to the lesions charac-
teristic of avian tubercle, but to those characteristic of the
human type. Baldwin (1910) likewise grew the human type
of tubercle bacillus for 19 months continuously in the bovine
tissues without in any way affecting its pathogenic powers to-
wards rabbits and guineapigs.
On the other hand, we have quoted in an earlier paragraph
an instance of a certain strain of the diphtheria bacillus which
CH. vn] VARIATIONS IN PATHOGENICITY 105
not only gave rise to atypical symptoms and lesions (namely
those of rabies) in the human body in the course of disease
but produced no less atypical lesions when inoculated into a
rabbit (vide p. 100).
Again, Savage (1908-9) found in further experiments that
a virulent strain of the streptococcus mastitidis from the udder
secretion in a case of bovine mastitis, under certain conditions
(namely 3 days' residence in the human pharynx), was almost
deprived of its characteristic power to produce mastitis in
goats.
Again, Mohler and Washburn (1906) claim that the various
types of tubercle bacilli human, bovine, avian can be readily
converted one into another, by prolonged residence in a suitable
animal host, so as to be indistinguishable by the ordinary in-
oculation tests.
Rosenow (1914) obtained a strain of haemolysing strepto-
cocci from the throat in a case of scarlet fever. A culture on
blood agar yielded two distinct kinds of colonies, (a) non-ad-
herent colonies of a haemolysing organism which fermented
mannite but failed to ferment maltose and saccharose,
(6) adherent, green-producing colonies of a non-haemolysing
organism which would not ferment mannite but fermented
maltose and saccharose. When injected into a rabbit, the
former attacked primarily the joints while the latter showed
a predilection for the heart valves. In other words, the original
strain on artificial cultivation gave rise to two strains which
differed in their pathogenicity.
Finally, may be mentioned Foa's experiments (1890). He
inoculated a rabbit with the diplococcus lanceolatus capsulatus
with a fatal result. From this dead rabbit he inoculated two
others, the first by injecting organisms derived from some of
the fresh fibrinous pneumonic exudate in the lung, and the
second by injecting organisms derived from the cerebrospinal
fluid. He found that the disease set up in these two rabbits
differed. The first rabbit showed, for example, an inflammatory
oedema of the skin ; the second did not show this. He found,
however, that if the strain isolated from the lung were grown
anaerobically and then injected into a rabbit the effects it
106 VARIATIONS EST PATHOGENICITY [OH. vn
produced were indistinguishable from those produced by the
strain isolated from the spinal fluid.
Whatever aspect of pathogenicity, therefore, we study, the
same feature becomes apparent namely, that this property
of bacteria is, like others, subject to variation.
VARIATION IN OTHER CHARACTERS OF BACTERIA.
In the foregoing pages variations in morphology, ferment-
ing power, virulence and pathogenesis have been discussed in
detail. There remain many more characters of bacteria to be
considered such as their viability, their staining properties,
their power to produce indol and to liquefy gelatin, their ag-
glutination reactions and many others. It would be easy
to illustrate the variations these characters also undergo
under different conditions. Many examples will be found in
Chapter II.
CHAPTER VIII
THE POSSIBLE OCCURRENCE OF TRANSMUTATION
IN THE LIVING BODY
THE significance of the variations recorded in the foregoing
sections, with reference to the question whether actual trans-
mutation of bacteria can be brought about artificially or not,
will be dealt with later. It is proposed, at this point, to consider
another aspect of the problem, namely the possibility of
transmutation occurring in the tissues of the living body.
In certain regions of the body one finds growing side by
side two strains of organisms closely resembling each other
in every respect save one namely their pathogenicity. One
strain is capable of causing a definite train of lesions and
symptoms ; the other, as a rule, does not give rise to any signs
of disease. The suggestion that one strain may be in some way
a derivative of the other offers a tempting hypothesis to explain
both their resemblance and their proximity to each other.
An illustration will, perhaps, make this clearer. In the hides
of cattle may sometimes be found non-virulent bacilli closely
resembling B. anthracis. Such an organism was discovered
by Andre wes and described by Bainbridge (1903) under the
name B. anthracoides (vide p. 92). The organism was
stated to differ from B. anthracis in the appearance of its
colonies, in its rate of growth, in possessing slight motility and
in being non-virulent. By slightly modifying the conditions of
growth, colonies on agar could be made to assume the typical
appearance of anthrax colonies, while its virulence proved
capable of increase by "passage." The differences in character
between this organism and B. anthracis were deemed sufficient
by these observers to justify them in classifying it as a distinct
species, but it is difficult to resist the conclusion either that
108 THE POSSIBLE OCCURRENCE OF [OH. vm
the non-virulent organism was a direct derivative of the true
anthrax bacillus or that it would be capable of giving rise to
the latter under suitable conditions. Such a supposition is
favoured, firstly, by the admission that the bundle of horse hair
from which the B. anthracoides was isolated contained also
the spores of true anthrax, and, secondly, by the discovery of
Hueppe and Wood some years before (1889) of a similar non-
virulent saprophytic anthrax-like organism in earth, which
however on injection into a mouse rendered the animal immune
to anthrax.
Similar examples of association between non-virulent and
virulent organisms, otherwise closely resembling each other,
may be found in the human body in the intestine B. coli and
B. typhosus, in the throat Hofmann's bacillus and the Klebs-
Loeffler bacillus, in the skin the Staphylococcus epidermidis
albus and the Staphylococcus pyogenes aureus, in the naso-
pharynx the micrococcus catarrhalis and the meningococcus.
The exact relationship in each case has never been satis-
factorily determined. Over twenty years ago Adami (quoted
by Arloing, 1891) put forward the suggestion that B. coli might
give rise in the presence of fermenting faecal matter to
B. typhosus, a theory which has been recently revived by
Tarchette (1904) and others (quoted by Hamer, 1909).
The precise relationship between the virulent Klebs-Loeffler
bacillus and Hofmanns bacillus is still a matter of controversy.
The latter is a harmless saprophyte not infrequently found in
the pharynx of healthy persons. It is distinguished from the
true diphtheria bacillus by the somewhat different appearance
of its colonies on artificial media, by slight and, according to
some observers, inconstant differences in its morphology and
staining, by its inability to ferment glucose and other sugars,
and by being non-pathogenic to man and to the guineapig.
It has not been found possible to produce immunity against
true diphtheria by inoculation with Hofmann's bacillus, and
the injection of a filtered broth culture of the latter does not
give rise to antitoxin formation in the horse (Petrie, 1905)
though the filtrate in the case of even avirulent Klebs-Loeffler
bacilli will do so (Arkwright, 1909). Nevertheless many in-
CH. vin] TRANSMUTATION IN THE LIVING BODY 109
vestigators claim to have converted the Klebs-Loeffler type
of organism into the Hofmann type by prolonged cultivation
(Lesieur, 1901), by growth at a high temperature (Hewlett and
Knight, 1897), by growth in the subcutaneous tissues of an
immune rat (Ohlmacher, 1902) and other methods and main-
tain that the reverse change can be brought about by "passage"
(Lesieur, 1901, Hewlett and Knight, 1897, Ohlmacher, 1902,
etc.). Salter (1899) has stated that, by five successive passages
through goldfinches, he was able to convert four strains of
typical Hofmann's bacilli into no less typical Klebs-Loeffler
bacilli, the transformation being complete as regards virulence,
morphology and acid production, and in the power to form
a toxin neutralised by diphtheria antitoxin.
Thiele and Embleton claim to have converted Hofmann's
bacillus into a bacillus morphologically indistinguishable from
the diphtheria bacillus and capable of secreting an exotoxin
which can be neutralised by diphtheria antitoxin. This was
accomplished by inoculating a succession of guineapigs with
an emulsion of Hofmann's bacillus containing a certain pro-
portion of gelatin, the organism being recovered from the
peritoneal cavity after each passage.
As regards the fermenting properties of the two organisms,
Clark (1910) has shown that Hofmann's bacillus does produce
slight acidity in dextrose broth; while Goodman (1908), by a
process of selection, obtained strains of the true diphtheria
bacillus which exhibited differences in fermenting power as
wide as those naturally existing between this organism and
Hofmann's ; and he concluded that the fermenting power was
a poor guide in determining whether an organism was a
pathogenic one or a harmless saprophyte.
Finally, Boycott's statistics demonstrate (Muir and Ritchie)
that the period of maximal seasonal prevalence of Hofmann's
bacillus immediately precedes that of true diphtheria, and
Hewlett and Knight (1897) have offered evidence in support
of the opinion that Hofmann's bacillus is present in increasing
numbers in the throats of diphtheria patients during recovery
from the disease.
Recent work by Graham Smith and others, and the inability
110 THE POSSIBLE OCCURRENCE OF [CH. vin
of these observers, on repeating the experiments of earlier
investigators, to obtain the same results, somewhat invalidates
the conclusions of the latter, so that the question of the possi-
bility of a mutation between the two species remains sub
judice.
Several species of staphylococci are recognised,
epidermidis albus, S. pyogenes albus, S. pyogenes aureus.
The distinction between these three rests on their inequality
in virulence, on their different powers of fermenting carbo-
hydrates, and, as their names imply, on their dissimilarity in
the production of pigment.
As regards virulence, the first-named organism is normally
present in the skin of healthy persons and is non-pathogenic ;
the second possesses slight virulence, producing mild local
inflammatory conditions ; while the third is a virulent organism
found in pathological conditions such as suppurative cutaneous
and subcutaneous lesions, acute bone infection and septicaemia.
Staphylococcus epidermidis albus may however assume a
certain degree of virulence and give rise to a stitch abscess
or mild inflammation (Dudgeon and Sargent, 1907) and plays
an important role in peritonitis (ibid.). Andre wes and Gordon
(1905-6) isolated it in pure culture in one case of otitis media
and also from a boil. The Staphylococcus pyogenes albus can
be made much more virulent by artificial passage. It has been
known to become parasitic, invading the human body and
circulating in the blood stream (Panichi, 1906, Southard, 1910).
In the second place, as regards their fermenting properties,
Gordon (1904-5) has shown that strains of Staphylococcus
albus isolated from the skin of healthy persons show very
great diversity in their fermenting power. In an earlier paper
(1903-4) he describes two strains, one a Staph. albus derived
from the skin and the other a Staph. pyogeiiies aureus derived
from pus which, when "put through" no less than 20 carbo-
hydrate substances, revealed different fermenting power in
one only, namely mannite.
In the third place, as regards pigment formation, it has
been proved by many investigators (Neumann, Dudgeon, 1908,
Andrewes and Gordon, 1905-6) that non-pigmented cocci can
CH. vin] TRANSMUTATION IN THE LIVING BODY 111
be obtained on culture from pigmented ones, and that cocci
which fail to produce pigment under certain conditions will
<lo so readily if the conditions are altered (vide p. 15). Dudgeon
(1908) cites one experiment in which a Staphylococcus aureus
was injected into an animal and a Staphylococcus albus was
recovered from the spleen at its death. In the last case
Gordon's tests were identical in both instances, showing that
the character of pigment production was the only one to
undergo modification, but it does not require a great stretch
of imagination to suppose that just as the virulent parasitic
pneumococcus and the avirulent saprophytic variety may
undergo mutation (vide p. 82) so the highly virulent "aureus"
and the less virulent "albus" might under certain circum-
stances be converted the one into the other.
Many more hypotheses of the same nature, and based on
similar evidence, might be put forward with varying degrees
of plausibility. One other example will suffice, namely the
question of the relationship of the meningococcus to two
other diplococci Mic. catarrhalis on the one hand and the
pneumococcus on the other.
The Micrococcus catarrhalis which is frequently present
in the mouths of healthy persons, especially children, is an
organism resembling in many respects the meningococcus, but
of low virulence. The two organisms are, as a rule, easily
distinguished by important differences existing between them.
Thus the meningococcus is much smaller than Mic. catarrh-
alis: its colonies are also smaller and their outlines more
regular ; it liquefies blood serum and forms acid in dextrose,
maltose and galactose which Mic. catarrhalis fails to do ;
it is more virulent also and gives rise to a different train of
symptoms. The difference in size, however, is not invariable,
an organism no greater than the meningococcus occasionally
proving on examination to be Mic. catarrhalis (Hachtel
and Hay ward, 1911), while the appearance of a colony is a
character of bacteria liable, as we have shown, to undergo great
modification (vide p. 47).
The power of the meningococcus to produce fermentation
in sugars is subject to variation. Arkwright (1909) found that
112 THE POSSIBLE OCCURRENCE OF [OH. vm
9 per cent, (out of 36 cultures) failed to produce acid in
dextrose. Summers and Wilson (1909) state that out of
80 strains "nearly all" fermented the usual sugars but a
few gave the fermentation reactions of Mic. catarrhalis. The
organism isolated from sporadic cases of meningococcal menin-
gitis shows such marked differences in its sugar reactions
when compared with a typical meningococcus that some
writers regard it as a distinct species (Batten). Arkwright
(1909), though he refutes this, acknowledges that the sporadic
type is less uniform in its fermenting powers. Some of his
strains of meningococcus permanently failed to ferment any
sugars; others, which failed to do so when first examined,
gradually acquired the power in the course of many months ;
others, again, which did ferment sugars, completely lost this
property after cultivation for a certain time. Another interest-
ing fact, in this connection, is mentioned by Andrew Connal
(1910), namely that in the late, chronic stages of cerebrospinal
fever the meningococcus isolated from the cerebrospinal
fluid is found to have lost its power to break up sugar.
Mic. catarrhalis on the other hand may acquire power to
ferment sugars. Gordon (quoted Martin, 1911) found that,
out of 25 strains examined by him, three fermented dextrose,
saccharose, galactose and maltose.
The meningococcus and Mic. catarrhalis differ in virulence
but this property in the latter can be artificially raised by
"passage."
As regards pathogenesis, this distinction, again, between
the two organisms sometimes breaks down, symptoms typical
of infection by one organism being in reality due to infection
by the other. The symptoms attributable to Mic. catarrhalis
infection differ widely. Thus it may cause an acute pharyn-
gitis (Gordon, 1906) or a tonsilitis; it may cause a "common
cold" or give rise to an infective cold and sore throat spreading
from person to person (Allen, 1908); it may set up otitis
media and a secondary meningitis (Barker, 1908), or, again, a
primary meningitis (Arkwright and Wilson) or, finally, an
epidemic so closely resembling cerebrospinal fever in its
symptoms that this disease has actually been diagnosed until
CH. vni] TRANSMUTATION IN THE LIVING BODY 113
a bacteriological examination demonstrated the absence of
the nieningococcus and the presence of Mic. catarrhalis
(Dunn and Gordon, 1 905). Conversely, the nieningococcus may
cause a simple coryza (20th century Diet, of Med.). Sometimes
a "mixed infection" occurs; thus, Arkwright (1909) describes
a case in which Mic. catarrhalis was isolated from the
heart's blood after death although the typical nieningococcus
was proved to be present before death in the cerebrospinal
fluid.
Prof. McDonald (1908) has commented upon the frequency
with which, in cerebrospinal fever, leptothrix forms are
found in the spinal fluid and compares this with the similar
frequency of leptothrix forms in the pharynx. He considers
these leptothrices to be merely secondary invaders but regards
their presence as confirmatory of the opinion, now generally
held, that the route of invasion of the nieningococcus in
cases of cerebrospinal fever is from the nasopharynx. If the
appearance of these "camp followers" tends to support the
opinion as to the locality from which the regiment was
drawn, still further light is thrown on the question by the
presence of "disbanded soldiers" in the form of non- virulent
meningococci in the nasopharynx of healthy persons. Out of
a total of 810 healthy persons, examined by different observers
all over the world, the nieningococcus was isolated from the
nose in 164 cases (Hachtel and Hay ward, 1911). We have
already referred to the fact that organisms normally non-
pathogenic may become pathogenic when growing and multi-
plying in inflammatory exudations (vide p. 79). Cerebro-
spinal fever is a disease more particularly of young children
and it is in children that Mic. catarrhalis is most often
discovered as an inhabitant of the pharynx in health. It has
been observed that an attack of cerebrospinal fever very
often commences with a purulent nasal discharge. The question
arises, does this area of suppurative inflammation in the
vicinity of its natural habitation afford a training ground, so
to speak, for the peaceful Micrococcus catarrhalis preparatory
to its entry upon a military career in the uniform of the
nieningococcus?
D. 8
114 THE POSSIBLE OCCURRENCE OF [CH. vm
The oft mooted question of the relationship of the meningo-
coccus to the pneumococcus is prompted by clinical rather than
by bacteriological evidence. Pneumococcal meningitis, like all
pneumococcal infections, is characterised by certain features
which are also observed in meningococcal meningitis (Preble),
namely an acute onset, a polymorphonuclear leucocytosis,
a diminution in the chlorides in the urine, and herpes. In the
second place, certain complications are common to both, namely
endocarditis, pericarditis, arthritis and otitis media. In the
third place, Preble observes that there is an extraordinary
similarity in the seasonal distribution of the two diseases.
On these grounds he suggests that the meningococcus is a
variant of the pneumococcus. Certain differences between the
two diseases exist. The petechial eruptions which formerly
gave a name to one disease are rare in the other, but this haem-
orrhagic tendency is altogether absent in some epidemics of
"meningococcal" meningitis. Again, " meningococcal" menin-
gitis is a disease more especially of childhood and frequently
ends in recovery; "pneumococcal" meningitis is a disease
more commonly of adult life and is invariably fatal. The
differences in age incidence and mortality are however com-
patible with the view that the causal organism is the same
but of different virulence.
Finally, the sporadic nature of meningococcal meningitis,
which is difficult to'explain if one admits the meningococcus
to be an independent organism, ceases to be so if one assumes
it to be a modified form of the ubiquitous pneumococcus.
It may be argued that, although each of the several differ-
ences in character which distinguish the organisms we have
been comparing, when considered by itself, may appear* trivial
and may prove to be variable, nevertheless all these differences,
if taken together and viewed as a whole, represent a degree
of divergence in type which cannot be so lightly dismissed.
A series of surmises, no matter how credible these may be
made to appear, does not constitute a proof. It is in our
power to prove, however, that in other cases differences no
less diverse in character and no less marked in degree, differ-
ences moreover which, taken together and viewed as a whole,
CH. vin] TRANSMUTATION IN THE LIVING BODY 115
might be thought to represent no less wide a divergence in
type, may disappear entirely under certain conditions con-
ditions, be it noted, precisely analogous to those which we
have surmised might bring about a similar result in the cases
we have been considering namely, invasion of the living body.
The experiments of Eyre, Leatham and Washbourn (1906)
with strains of the pneumococcus furnish an example. These
observers describe the virulent, parasitic pneumococcus as re-
quiring for its growth a certain reaction and temperature, and
particular media (blood agar) ; it would not grow if the reaction
were even faintly acid or at a temperature much below 37 C.
and rapidly died out on agar or in broth. It would not liquefy
gelatin and in broth formed a dust-like deposit. The avirulent
saprophytic variety, on the other hand, grew luxuriantly at
temperatures ranging from 37 to 20 C. on agar, gelatin,
potato or in broth, whether acid or alkaline, slowly liquefying
gelatin and producing a uniform turbidity in broth. It retained
its vitality for many months. It also exhibited differences in
its morphology, " instead of isolated diplococci and strepto-
cocci large masses of cocci and diplococci were found and
forms dividing into tetrads were common." Nevertheless this
avirulent saprophytic pneumococcus could, by a single " pas-
sage " through a rabbit, be converted into a typical parasitic
pneumococcus of high virulence.
Such a remarkable transition, if it did not actually happen,
would seem to us quite as improbable as a transition from,
let us say, the micrococcus catarrhalis to the meningococcus.
The purpose of this section is to suggest that a change in
character, comparable to that brought about in the case of the
saprophytic pneumococcus by a single animal passage, might
be brought about in the case of other saprophytic organisms
by an analogous process, namely by their invasion of the living
body when the lowered vitality or the inflamed condition of
the tissues enable them to gain a foothold therein.
82
CHAPTER IX
SUPPOSED INSTANCES OF TEANSMUTATION
BROUGHT ABOUT EXPERIMENTALLY
I. MAJOR HORROCKS' s EXPERIMENTS. (Journal of R.A.M. C.
Vol. XVI.)
In March, 1911, Major Horrocks published the records of
a series of experiments of great interest. The results of these
experiments may be briefly summarised as follows :
(a) From a strain of B. typhosus (derived from the
urine of a carrier) he obtained, by subculture, an organism
intermediate in its characters between B. typhosus and
B. coli.
(b) From a second (laboratory) strain of B. typhosus, by
symbiosis with .R coli, he obtained an organism which produced
slight acidity in mannite but fermented no other sugars, and
which later reverted to J5. typhosus.
(c) From a third (laboratory) strain of B. typhosus, by
symbiosis with the same strain of B. coli, he obtained an or-
ganism closely resembling B./aecalis alcaligenes.
(d) From a fourth strain of B. typhosus (derived* from the
urine of another carrier), after injection into the peritoneal
cavity of a guineapig, he obtained a Gram-positive coccus
resembling streptococcus faecalis.
(e) From a fifth strain of B. typhosus (derived from the
urine of a third carrier), after injection into the peritoneal
cavity of a guineapig, he obtained in three different ex-
periments a coliform organism which differed widely in its
fermentation and agglutination properties from B. typhosus.
(/) From a sixth strain of B. typhosus (derived from the
stool of a fourth carrier), after growth in the diluted and
filtered urine of another carrier "S," he obtained B./aecalis
OH. ix] SUPPOSED TRANSMUTATIONS 117
alcaligenes. The latter organism in one experiment, after
three passages through the guineapig, gave rise to B. coli
which however reverted subsequently to B. faecalis alcali-
genes.
(g) From the second (laboratory) strain of B. typhosus
referred to above (b\ after exposure to the same conditions,
he obtained in two different experiments B. faecalis alcali-
genes. The latter organism in two later experiments (in the
first after 5 months' further growth and in the second after
8 passages through the guinea-pig) gave rise to streptococcus
faecalis ; while in a third experiment (after 18 successive
passages through the guinea-pig) it gave rise to B. coli which,
after the 19th passage, reverted to B. faecalis alcaligenes and
this, after further "passages," in two different experiments
yielded the streptococcus faecalis.
(h) From the same (laboratory) strain of B. typhosus, after
growth in the diluted and filtered urine a different carrier
" I," he obtained again B. faecalis alcaligenes.
To summarise these results even more concisely, it appears
that Major Horrocks was forced to the conclusion that not
only had an organism arisen from a strain of B. typhosus in-
termediate in character between B. typhosus and B. coli, but
that other strains of B. typhosus, derived from three distinct
sources, had in no less than five of his experiments undergone
mutation into B. faecalis alcaligenes as a result of changes
in their environment ; and, further, that the B. faecalis alcali-
genes so obtained had later, in two instances, become changed
into B. coli (reversion taking place in both cases, however,
subsequently) and, in four instances, become changed into
streptococcus faecalis, once after prolonged cultivation and
three times as the result of passage ; and, finally, that one
of the original strains of B. typhosus had undergone a similar
change into streptococcus faecalis after passage.
Major Horrocks's statements are so startling and, if sub-
stantiated, would prove so revolutionary in character that they
demand careful examination.
It may not be possible to disprove either his facts or his
inferences, but it is not necessary to do so. The onus of proof
118 SUPPOSED INSTANCES [CH. ix
rests with the claimant. If it is possible to show that he has
failed to exclude a single possible source of error, a verdict of
not proven must be returned.
When considering the value of evidence adduced in sup-
port of supposed instances of variation or transmutation (vide
Chap. Ill) we mentioned various sources of error. Bearing these
in mind, and also the wide limits within which we have found
variation may occur (vide Chaps. IY-VII) we will now consider
in detail the processes by which Major Horrocks obtained the
results he claimed and the value of the evidence he brings
forward to support his contentions.
(a) The alteration of B. typhosus to an organism inter-
mediate between B. typhosus and B. coli.
(Page 246.) A strain of B. typhosus was isolated from
the urine of a typhoid carrier " TS " from whose blood a pure
culture of B. typhosus had previously been obtained. After
3 days' incubation on bile salt glucose litmus agar the strain
gave the typical reactions of B. typhosus. At the end of a
week, however, the following characters were displayed : lac-
tose, salicin and dulcite were rendered slightly acid, broth
gave a marked indol reaction, the neutral red reaction yielded
a slight yellow colouration, the organism appeared only slightly
motile and was not agglutinated by anti-typhoid serum. The
organism, however, gave rise to typhoid agglutinins when
injected into a rabbit, and this rabbit's serum deviated com-
plement in the same manner as a known antityphoid serum,
and the organism further had the power of absorbing the
specific agglutinins from a known typhoid serum.
After 4 passages through the guineapig the organism lost
its lactose fermenting property and only differed from the
original B. typhosus by forming a trace of acid in salicin.
After 4 further passages it reverted to the unusual fermenting
type described.
The urine of carrier " TS " from which the strain was origin-
ally derived was again carefully tested but only typical typhoid
organisms were obtained.
Criticism. We have already quoted (vide p. 11) instances
of the occurrence of organisms, derived in some cases from
CH. ix] OF TRANSMUTATION 119
the urine of typhoid carriers, intermediate in character between
B. typhosus and B. coli. Wilson (1910) described such an
organism as fermenting glucose and mannite but, unlike B. ty-
phosus, fermenting lactose also at 22 C. (but not at 37 C.)
and failing to agglutinate with typhoid serum. The Bacillus
perturbans of Klotz (1906), though agglutinated by high dilu-
tions of typhoid serum, fermented lactose and saccharose,
gave the neutral red reaction and produced indol.
Examples have also been given (vide Chapter V) to show
the variability of organisms with respect to their power to
ferment sugars and their ability to acquire fresh fermenting
properties. B. typhosus, for example, may acquire the power
in a few days to ferment dulcite.
The organism described here by Major Horrocks is another
example of temporary variation in character with respect to the
power to ferment sugars and to produce indol, associated with
some modification also in agglutination properties.
(&, c) The change from B. typhosus to B. faecalis alcaligenes
due to symbiosis with B. coli.
(Page 233, exp. 1.) The strain of B. typhosus used was
a stock laboratory strain " R," from which stock vaccines were
prepared a strain, that is to say, of unimpeachable character.
The strain of B. coli was derived from the urine of a typhoid
carrier ("Bomb S "). The two organisms were added to 1 c.c. of
sterilised tap water and the suspension plated. 10 days later,
examination showed typical typhoid colonies and others white
and opaque. The latter were planted on the usual media and
in 48 hours yielded slight acidity in mannite only ; no other
sugars were fermented in 7 days. The original strain of B.
typhosus used was replanted on agar and the resulting growth
gave the typical reactions of this organism.
(Page 234, exp. 3.) The experiment was repeated, a dif-
ferent typhoid strain ("Bombay ") being used. After an interval
of two months, 1 c.c. of the inoculated water was added to
MacConkey's bile salt broth and this plated on lactose bile
salt litmus agar. A few blue colonies were seen consisting of
bacilli which resembled B. faecalis alcaligenes in not ferment-
ing any sugars and producing an alkaline reaction in milk.
120 SUPPOSED INSTANCES [OH. ix
No B. typhosus could be isolated and at later examinations
only B. coli was recovered.
Criticism. Conditions of growth inimical to the life of an
organism might be expected to deprive it gradually of its
functions. A strain of typhoid bacilli whose vitality is at its
lowest ebb would hardly be likely to ferment sugars vigorously,
if at all. In both these experiments two factors were at work
inimical to the life of B. typhosus, namely the presence of
B. coli, and growth in water a non-nutrient medium. After
10 days, in the first experiment, slight fermenting power
persisted. After two months, in the second experiment, all
fermenting power was lost. No attempt was made to resusci-
tate the strain of organisms on ordinary media to ascertain
whether with returning vitality fermenting power would be
restored.
That this explanation is the true one and that no new race
of organisms was produced is suggested further by the obser-
vation that no organisms giving the ordinary reactions of B.
typhosus survived side by side with the non-fermenters and
that, at a later stage, the strain was found to have died out
altogether.
(d) The change from B. typhosus to Streptococcus faecalis
in the peritoneal cavity of the guineapig.
(Page 230, exp. 4.) The urine of a typhoid carrier "S"
was plated and found to contain typhoid bacilli. One colony
was subcultured on agar and a standard loopful of a 24 hours'
growth was injected into the peritoneal cavity of a guineapig.
The animal was found dead in the morning. No typhoid ba-
cilli were found in the peritoneal fluid which contained a pure
culture of a Gram-positive streptococcus giving the reactions
of S. faecalis.
(e) The change in the peritoneal cavity of a guineapig
from B. typhosus to a coliform organism giving atypical re-
actions.
The urine of a typhoid carrier "I" was plated after being
kept 12 months in a flask. Two colonies of B. typhosus were
planted on agar and labelled IB l and IB 2 respectively.
(e i) (Page 230, exp. 6.) One standard loopful of a 24 hours'
CH. ix] OF TRANSMUTATION 121
growth from the culture IB l was injected into the peritoneal
cavity of a guineapig. The animal was found dead the next
morning and a pure culture of B. typhosus was obtained from
the heart's blood. From the peritoneal fluid and spleen was
obtained, in addition to B. typhosus, a coliform organism
possessing the following characters : a gram-negative motile
bacillus, forming acid and gas in glucose, mannite, lactose and
dulcite, but producing no change in salicin or cane sugar but
giving rise in the neutral red medium to gas and fluorescence,
not liquefying gelatin or forming indol in broth and giving an
acid reaction in litmus milk without any clotting. A broth
culture from the original agar slope was carefully tested but
typical B. typhosus alone found. The broth culture was
planted on agar and the experiment repeated with a loopful
of this growth. The animal did not die and a pure culture
of B. typhosus was recovered from the peritoneal cavity.
(e ii) (Page 230, exp. 6.) One standard loopful of a 24 hours'
growth from the culture IB 2 was then injected into the peri-
toneal cavity of a guineapig in the same manner. The animal
was found dead next morning and a pure culture of B. typhosus
was obtained from the heart's blood and spleen. From the
peritoneal fluid was obtained, in addition to B. typhosus, a
coliform bacillus.
(e iii) (Page 231, exp. 7.) The urine of the typhoid carrier
" I " was again plated after having been kept over 14 months
in a flask. A colony was again planted on agar and a standard
loopful of the growth again injected into the peritoneal fluid
of a guineapig. The animal was found to be dying the next
morning and was killed with chloroform and a pure culture
of B. typhosus was obtained from the heart's blood. From the
peritoneal fluid and spleen a pure culture of a coliform or-
ganism was obtained. The latter organism failed to produce
any typhoid agglutinins when injected into a rabbit, or to ab-
sorb agglutinins from a known typhoid serum.
The last experiment was repeated, the same strain being
used (" I ") after 14 days further growth on agar. The injection
did not prove fatal to the guineapig and from the peritoneal
fluid a pure culture of B. typhosus was obtained.
122 SUPPOSED INSTANCES [CH. ix
Criticism. The last four experiments (d, e i, e ii, and e iii)
may be discussed together.
In one experiment (d) B. typhosus derived from a carrier
apparently gave rise, in the peritoneal cavity, to a Gram-positive
coccus. In three experiments (e i, e ii, and e iii) B. typhosus,
derived from another carrier, apparently gave rise, in the peri-
toneal cavity, to atypical coliform organisms.
The questions to be discussed are two whether the strain
of organisms isolated from the peritoneal cavity were derived
from the original strain of B. typhosus injected in each case,
and whether, if such continuity is established, the alteration
in character is to be regarded as a temporary variation or a
transmutation.
The possibilities to be considered are (1) whether the
original strain of B. typhosus was pure ; (2) whether the peri-
toneal cavity in each case was sterile before the injection was
made ; (3) whether it was contaminated from the skin at the
time the injection was made ; (4) whether it was invaded from
the gut after the injection was made or after the death of the
animal ; (5) whether the later strain was linked up with the
original one by the occurrence of reversion or the discovery
of intermediate forms; (6) whether a repetition of the ex-
periments confirmed the results; (7) whether the alteration
in character falls within the recognised limits of variation
discussed in the earlier part of this work.
(1) There are grounds for viewing the original cultures with
suspicion. They were not made in any instance from a single
organism. The urine of carrier "S" and of carrier "I," from
which they were isolated, admittedly contained streptococci,
B. coli, bacilli closely resembling B. faecalis alcaligenes and
other coliform organisms. These other organisms were present
in comparatively small numbers. In one instance it is stated
that B. coli and B. typhosus were present in the proportion
of 1 to 30,000. Such disparity in numbers might easily account
for the less common organisms being overlooked. It is men-
tioned that a change in the character of the medium, brought
about by simple dilution with water, enabled the associated
microbes to multiply so much more rapidly than the B. ly-
CH. ix] OF TRANSMUTATION 123
phosus that the latter organism was soon "swamped," as it
were, and disappeared altogether. If the strain of B. typhosus
injected contained one or two specimens of a streptococcus or
coliform organism, might not growth in the peritoneal cavity
yield a similar result? not before, however, some of the ty-
phoid bacilli had succeeded in escaping from the peritoneum
into the blood vessels and setting up a systemic infection. In
two instances, in which the experiments were repeated, the
injection of the original culture of B. typhosus into the peri-
toneal cavity did not kill the animal although a pure culture
of B. typhosus was recovered from it. The original culture,
therefore, apparently contained strains differing from each
other in virulence. They may conceivably have possessed
other differences.
(2) No control experiments were carried out to prove that
the peritoneal cavity before the experiment was sterile.
Dudgeon (1908) states that in healthy animals the omentum
may normally contain the staphylococcus albus. There is
evidence to show that even in healthy animals the internal
organs may contain both pathogenic and non-pathogenic
bacteria. Ford (1900) showed by experiments, in which rigid
precautions against contamination were adopted, that the
kidneys, liver and spleen of healthy animals, in a large majority
of cases, contained organisms such as the staphylococcus,
mesentericus, colon and paracolon bacilli, B. subtilis and
proteus. In rabbits 66 per cent, of the organs examined
contained bacteria, in cats over 77 per cent, in dogs over 88
per cent. In guineapigs the percentage was 77 per cent, of
the organs examined and the organisms that predominated
were B. subtilis ( staphylococci and the colon bacillus. Adami,
Abbott and Nicholson (1899) found in the livers of healthy
animals (cows, sheep, rabbits, guineapigs) diplococci and
chains of 3 or 4 cocci, which on culture yielded B. coli in
many cases.
(3) No control experiments were conducted to exclude
the possibility of skin contamination at the site of the
inoculation, but such a supposition is inadequate to explain
all the results obtained.
124 SUPPOSED INSTANCES [CH. ix
(4) The injection of organisms into the peritoneal cavity
would have a threefold effect, it would make the animal ill,
produce a more or less marked peritonitis, and finally kill
the animal. All three events would favour the invasion of
the peritoneal cavity by organisms. If the vitality of the
body and consequently of the peritoneum is lowered, organisms
can penetrate it from the gut even in the absence of any
definite lesion or inflammation. Ford (1900) noted that in
animals whose vitality was lowered, by fasting or unhealthy
conditions, bacteria were more abundant in the internal
organs, and that this applied particularly to bacteria of the
colon type. Dudgeon and Sargent (1907) have shown that at
the earliest stage of peritonitis the staphylococcus albus
(either normally present on the surface of the gut or pene-
trating from within it) increases with enormous rapidity.
Miiller (1910) remarks that when organisms (e.g. typhoid
bacilli) are injected into the peritoneal cavity they at first
decrease in number owing to the bactericidal effect of the
body fluids but later on increase again. It is during this
early stage when the injected organisms are decreasing rapidly
that the staphylococcus albus (and possibly, in animals, the
bacteria present in the internal organs) are increasing rapidly.
A culture removed during this period might well convey the
impression that a mutation had occurred.
Dudgeon and Sargent (1907) mention that the staphylo-
coccus albus is often quite non-pathogenic in the peritoneal
cavity of the guineapig.
After death there is a rapid invasion of the peritoneal
cavity by organisms, particularly by B. coli from the gut, so
that the true nature of the infection becomes obscured. In
illustration of this point, Dudgeon and Sargent (1907) record
a case of pneumococcal peritonitis in which the peritoneal
exudate one hour after death gave a pure culture of pneumo-
cocci, whereas 26 hours later B. coli alone could be recognised
in the same exudate.
It is not possible, therefore, to exclude in Major Horrocks's
experiments the possibility of an invasion of the peritoneal
cavity from the gut, following either the inoculation or the
death of the animal.
CH. ix] OF TRANSMUTATION 125
(5) In none of these four experiments was any attempt
made to test the new strain by subculture or passage to
ascertain whether it would revert. In two experiments the
original strain of B. typhosus had, within a few hours of its
injection, completely disappeared and in none of the experi-
ments were any intermediate forms observed which might be
regarded as linking up the new strain with the original one
and suggesting a transmutation. All the organisms were
apparently of the same type. Moreover the agglutination
reactions betrayed no sign, in the only instance in which they
were tested, of any connection between the new strain and
the original B. typhosus. The continuity, therefore, of the
two forms cannot be regarded as proved. Moreover if the
variants were really derived from the original strain of
B. typhosus one would have expected them to be present, like
the typhoid bacilli, in the blood stream as well as in the
peritoneal cavity. One possible explanation is that the change
was dependent upon the influence of some agent existing in
the peritoneum but absent elsewhere. This will be referred
to again (vide p. 126).
(6) A repetition of the experiments was made in only two
cases (e\ and ciii) and without yielding similar results
indeed the results differed from those first obtained in such a
way as to suggest that the original cultures, in both cases,
contained strains of bacteria differing widely, at any rate in
their virulence.
(7) If the continuity of the two different strains were
established in each case, what would be the significance of
these changes ? The transition from B. typhosus to an atypical
coliform organism may be regarded as a variation on the part
of the typhoid strain, probably temporary in character and
of the same nature as those discussed in an earlier part of
this work (vide p. 11). The transition from B. typhosus to a
Gram-positive coccus is more difficult to explain but the
observations of Adami and others would suggest that, in this
case also, a temporary variation and not a true transmutation
might have been brought about. Adami (1892) observed that
the addition to a medium of substances inimical to the life
126 SUPPOSED INSTANCES [CH. ix
of B. typhosm for example, carbolic acid or creosote
made this bacillus in such a medium assume temporarily the
form of non-motile cocci or diplococci. Again, Adami, Abbott
and Nicholson (1899) obtained from the bile in guineapigs
and also from the peritoneal fluid in man, under certain
conditions, coccic forms of B. coli. These were present as
diplococci or short chains of 3 or 4 cocci ; they were non-motile,
non-fermenting and did not produce indol ; their growth on the
surface of agar at first closely resembled that of a strepto-
coccus, the colonies were white and opaque. Intraperitoneal
inoculation into a guineapig increased their fermenting power
and, after 3 passages, yielded normal B. coli. They found
evidence that B. typhosm yielded similar modified coccic
forms when acted on by peritoneal and other fluids. They
describe coccic forms of B. typhosm in the mesenteric and
retroperitoneal glands. They mention that in some cases the
action of ascitic and peritoneal fluids in this respect is so
marked that it was difficult to obtain complete reversion to
type. They found, also, that B. coli injected into the blood
stream in a rabbit appeared in these coccic forms within half
an hour in the liver and the bile, though similar forms were
not found in the systemic circulation. If the modification in
a strain of B. typhosm which Major Horrocks describes was
due to the same agency, one can understand why the variant was
only found in the peritoneal cavity and not in the heart's
blood. In his later experiments, however, cocci possessing the
characters of S. faecalis were obtained not only in the
peritoneal cavity but during culture on artificial media. In
the account of these experiments, however, there is much to
suggest that the original strain was not a pure one.
(f) The change from B. typhosus to B. faecalis alcaligenes
after growth in the diluted and filtered urine of a typhoid
carrier and the further changes from B. faecalis alcaligenes
to B. coli on passage.
(Page 237, exp. I.) The urine of a typhoid carrier " S "
was diluted 1 in 10 with tap water and allowed to stand 11
days. It was then filtered through a Pasteur candle (F) with-
out pressure and shown to be sterile by "prolonged incubation"
CH. ix] OF TRANSMUTATION 127
at 37 C. after plating. The filtrate was then inoculated with
a 48 hours' growth of B. typhosus isolated from the stool of a
carrier " C " and a week later plated out on bile salt neutral
red lactose agar. Two or three colonies were examined. One
gave the typical reactions of B. typhosus but two other
colonies failed to agglutinate with typhoid serum and cor-
responded in their reactions to B. faecalis alcaligenes. One
week later the latter organism alone was found and it
persisted unchanged for several months afterwards.
Criticism. The same criticism applies to this experiment.
The original strain was not grown from a single organism.
"A particle," we are told, of the growth was added to the
solution, sufficient to yield 480 million bacteria to each cubic
centimetre. The purity of such a strain cannot be guaranteed.
The original culture was derived from a stool and it is said
to have yielded an organism closely resembling B. faecalis
alcaligenes. The experiment was however repeated twice
over with a laboratory strain with the same result a fact
which considerably discounts this objection. The new strain
might, again, be regarded as a variant of the original B.
typhosus which, as a result of growth under conditions
inimical to its vitality, had suffered a loss of power with
respect to its fermenting properties and also its property of
agglutinating with typhoid serum. Its other agglutinative
characters were not examined, but a similar organism obtained
in the same manner from a laboratory strain was found to
possess slight power of absorbing agglutinins from antityphoid
serum. The new strain was, however, further tested (" culture
33 " page 242) by being alternately passed through the peri-
toneal cavity of a guineapig and subcultured on agar. In one
experiment, after the 3rd passage, the peritoneal fluid removed
at the end of 6 hours contained B. coli, but the fluid removed
at the end of 12 hours contained not B. coli but the new
strain of B. faecalis alcaligenes which had been injected.
Only three passages were made the investigation, that is
to say, was not persisted in long enough to decide whether
the new strain was capable of reverting or not.
The recovery of B. coli after the 3rd passage Major
128 SUPPOSED INSTANCES [CH. ix
Horrocks considers may have been due to an invasion by this
organism from the gut but was more probably due, in his
opinion, to the still further modification of the strain injected,
inasmuch as " reversion " apparently took place a few hours
later. The disappearance of the B. coli might however be
explained on other grounds. The strain of B. faecalis
alcaligenes undergoing passage may have been contaminated
with B. coli between the second and third passages ; the
former organism after its two passages might be expected to
be more resistant to the body fluids which destroyed the
latter.
(g) The change from B. typhosus to B. faecalis alcaligenes
after growth in the diluted and filtered urine of a typhoid
carrier and the further change from B. faecalis alcaligenes
to Streptococcus faecalis.
(Page 238, exp. II.) The sterile filtrate from the urine
of a typhoid carrier "S" was again inoculated with B.
typhosus the strain used, on this occasion, being a stock
laboratory strain "R" of unimpeachable character. The
result was similar to that in the previous experiment. Side
by side with colonies of typical B. typhosus were found
colonies of an organism (" 35 A. Col. 2 ") which corresponded
with B. faecalis alcaligenes but possessed a slight power of
absorbing agglutinins from antityphoid serum (p. 242). Later
the latter organism alone was found and it persisted unchanged
for many months. The experiment was repeated in exactly
the same way and with the same result, B. faecalis al-
caligenes emerging (p. 240, exp. IV).
These two experiments are simply a repetition of the
experiments already discussed, a laboratory strain of B.
typhosus being used instead of a carrier strain.
When the inoculated filtrate used in the first experiment
(p. 238, exp. II) was 6 months old, a loopful of it was added
to a broth tube and a 48 hours' growth plated on MacConkey's
medium (p. 239). Colonies of B. faecalis alcaligenes were
again found but with them smaller colonies of a streptococcus
closely resembling 8. faecalis.
The strain of B. faecalis alcaligenes obtained in the
CH. ix] OF TRANSMUTATION 129
first experiment (p. 238, exp. II) known after this as "35 A "-
was further tested (p. 243) by successive passages through
the peritoneal cavity of the guineapig. The fluid removed
after the 8th passage when subcultured into broth showed
short-chained cocci and diplococci but when subcultured on
to agar gave, in addition to these cocci, the original bacillus
" 35A." The latter in broth again yielded the short-chained
cocci and these on agar gave a bacillus once more but this
time not " 35 A " but a fermenting coliform organism.
The original strain of B. faecalis alcaligenes (obtained
in Exp. II, p. 238) was a second time tested in the same way
(p. 243). It showed no change in character until the 18th
passage when it gave rise to a fermenting bacillus of the B.
coli type which on the 19th passage reverted to B. faecalis
alcaligenes. This last-named organism, after 7 more passages
in one experiment and 8 more passages in another, gave rise
to a fermenting B. coli type of organism " in pure culture "
and this, on planting in broth, yielded B. faecalis alcaligenes
once more but, with it, cocci corresponding to S. faecalis.
The latter after 3 passages remained unchanged.
Criticism. The transition from the non-fermenting B.
faecalis alcaligenes to the fermenting coli type and back
again, may be regarded as no more than another example of
variation similar to those quoted in an earlier section of this
work. Again, in the " passage " experiments, one or other
type may have been an invader from the gut, the apparent
reversion at a later passage merely representing the de-
struction of the invader which had not been " hardened/' so
to speak, by previous passages. Only one guineapig was used
for each " passage " experiment in a series of passages. If
more than one had been used some check would have existed
on possible errors due to this cause. The repeated transitions
from B. faecalis alcaligenes to & faecalis and vice versa
are more difficult to explain, for 8. faecalis appeared
not only after passage but during cultivation also on artificial
media. One is almost forced to the conclusion that Major
Horrocks was dealing with a mixed stram of the two organisms
and that changes in the conditions of growth at one time
D. 9
130 SUPPOSED INSTANCES [OH. ix
fostered the growth of the first organism almost to the
exclusion of the second, and at another time fostered the
growth of the second almost to the exclusion of the first.
If, after each appearance of S. faecalis, the strain had
been guaranteed pure by the method of successive plating
and growth from a single organism, the results obtained
would have had more weight.
It is worth noting that the strains of B. faecalis al-
caligenes used in all these experiments, and supposed to
have been derived from B. typhosus in the first place, showed
no tendency to revert to B. typhosus. It is also interesting
to compare these experiments with those of Adami, Abbott and
Nicholson (1899) -who obtained from B. coli grown in peri-
toneal fluid cocci which did not ferment sugars or form indol,
and yielded colonies which were white and opaque and
resembled those of a streptococcus. After 3 passages through
the guineapig these cocci yield short bacilli which however
were still unable to ferment sugars. They state that B.
typhosus was modified in much the same way under similar
conditions.
(h) The change from B. typhosus to B. faecalis alcaligenes
after growth in the diluted and filtered urine of a typhoid
carrier.
(Page 239, exp. III.) This experiment was practically a
repetition of the last, the same strain of B. typhosus being
used (laboratory stock "R") but the urine was that of a
different typhoid carrier "I". The result was the same,
colonies of typical B. typhosus being found at first but after
a month's interval colonies of a non-fermenting and non-
agglutinating coliform organism being alone found. To this
experiment the same criticism applies. 19 other experiments
were made but with negative results.
Summary. The evidence that Major Horrocks brings
forward in support of the claim that in the course of his
experiments transmutation occurred is inconclusive. He is
unable in any case to guarantee as pure the culture with
which he was dealing. He is unable to exclude definitely, in
some of his experiments, the occurrence of a secondary
CH. ix] OF TRANSMUTATION 131
invasion in the living body. Lastly, even if the evidence
established the continuity between his original strains and
the new ones he obtained, the changes may possibly have
been merely examples of variation no greater in degree than
many that have been recorded by other observers. In other
words, the strains of atypical B. coli and those closely
resembling B. faecalis alcaligenes or S. faecalis, may con-
ceivably have been variants of his original strains of B.
typhosus whose true identity would have been disclosed by
more prolonged efforts to obtain reversion or more thorough
investigation with regard to their agglutination reactions.
^ II. THE RELATIONSHIP BETWEEN MEMBERS OF THE ENTERITIS
GROUP OF BACILLI B. ENTERITIDIS " GAERTNER " AND THE
PARATYPHOID BACILLUS OF THE " AERTRYCK " OR " FLUGGE "
TYPE.
The question of the specific character of these organisms
has been much discussed. They are generally recognised as
distinct species but evidence has been brought forward from
time to time suggesting that they may be transmuted one
into the other.
A. Schmitt's experiments.
Schmitt (1911), for example, claims that in certain experi-
ments conducted by him strains of the paratyphoid bacillus
of the Fliigge type became changed within the animal body
into the Gaertner type of bacillus. The details of the experi-
ments are briefly as follows.
Experiment I. On July 17th, 21st and 28th he fed a
young calf on milk to which had been added (in amounts
varying from 1 to 50 c.c.) a broth culture of a Fliigge type of
organism without apparent effect, except that the blood
serum which previously did not agglutinate the organism now
did so in dilutions of 1 in 35.
On August 3rd the same strain of organisms was injected
subcutaneously into the calf, with the result that the calf
became ill. An organism ("Pgst I") was isolated from the
92
132 SUPPOSED INSTANCES [OH. ix
calf s blood on the same day. It was found to be agglutinated
by the animal's blood serum in dilutions of 1 in 50 60
although, again, serum which had been taken from the calf
before the experiments began failed to agglutinate it. (A
calf serum, immunised against a Gaertner strain from cattle,
was on the same day injected intravenously but only made
the calf more ill.) A broth culture of this organism, Pgst I,
was injected into the same animal on August 7th and again
on August 10th, giving rise only to a slight febrile reaction
on each occasion.
Experiment II. On August 25th, the strain already
mentioned (Pgst I) as having been isolated from the first
calf s blood was suspended in normal saline and sprayed into
the nose of another calf without apparent effect. On August
28th a similar suspension was injected into the animal's
mouth with the result that the calf became ill. An organism
(Pgst II) was isolated from the calf s blood. On September
4th a saline suspension of this organism was injected intra-
venously into the same calf, which died a few hours later. From
the blood, intestines, muscles and bone marrow was obtained
an organism Pgst III. The " Schluss-serum " was found to
agglutinate the original Fliigge type and also the strains
Pgst I, II, and III in dilutions of 1 in 30004500.
The great interest of the experiments lies in the obser-
vation that these later strains isolated from the blood of the
calf were found to correspond in their agglutination reactions,
not with the original Fliigge type but with the Gaertner type
of bacillus a conclusion confirmed by absorption tests.
Schmitt maintained, therefore, that passage through the calf
modified the agglutination properties of the human para-
typhoid bacillus (" Fliigge ") so that it came to resemble the
calf paratyphoid bacillus (" Gaertner ").
Two possible fallacies at once suggest themselves. The
type of organism which made its appearance later in the
experiment might have been present as a contamination either
(a) in the original strain or (b) in the bodies of the animals
inoculated.
(a) As regards the first alternative, it is conceivable that
CH. ix] OF TRANSMUTATION 133
if bacilli of the Gaertner type were present in the original strain,
but in such scanty numbers as to escape detection, these few
bacilli might in the living tissues multiply with such rapidity
as to become ultimately the predominant organism. The pre-
cautions taken to secure the purity of the original culture
would presumably exclude such a source of error.
(6) In the second place, bacilli of the later or Gaertner
type might conceivably have been present, before the inocu-
lations were made, in the bodies of the calves themselves.
This possibility appears at first sight to be excluded by the
fact that the serum of the calf before inoculation failed to
agglutinate the organism recovered afterwards, although the
serum after inoculation was able to do so. This observation
is not, however, final.
Savage (1907-8) and other observers have recorded the
presence of B. Gaertner in the intestines of healthy young
calves. Its presence in the intestines of the calves inoculated
in these experiments was not definitely excluded.
The repeated administration in the food of as much as
50 c.c. of a young broth culture of a pathogenic organism would
be likely to cause an inflammatory reaction in the bowel
and such inflammation might lead, not only to an enormous
increase in numbers on the part of any other pathogenic
organism present, but also to an exaltation in their virulence.
We have referred elsewhere (vide p. 80) to such a sequence in
the case of B. coli in the intestine during an attack of typhoid
fever and in inflammatory conditions resulting from improper
food.
Such an increase both in numbers and in virulence on the
part of the organism we are discussing might well pave the
way for its invasion of the system as a whole and lead to its
appearance in the blood and internal organs, in a manner
analogous to the invasion of the body by saprophytic organ-
isms of heightened virulence in the cavity of an inflamed
uterus. At the same time the blood serum would acquire
agglutination properties which it did not possess when the
bacilli were few in number, of low virulence and restricted to
the lumen of the intestine.
134 SUPPOSED INSTANCES [CH. ix
B. The experiments ofMiihlens, Dahm and Filrst.
These writers (1909) have recorded experiments which
suggest a similar transmutation. They fed a large number of
mice on meat which was thought to have been infected
although a preliminary bacteriological examination proved
negative. Over 50 per cent, of the mice died.
A bacteriological examination of the faeces of 56 mice was
made. In 20 cases none of the paratyphoid group of bacilli
were detected. Aertryck's bacillus was found to be present in
24 and Gaertner's bacillus in 13 cases.
These results suggest that one type may have arisen from
the other within the animal body.
As in the experiments already discussed, two possibilities
have first to be excluded namely, the possibility of con-
tamination (a) in the original strain and (b) in the bodies of
the mice used for the experiment.
(a) All that can be said by way of excluding the first of
these alternatives is that the simultaneous presence of both
the Aertryck and the Gaertner type of organism in infected
meat is contrary to experience and was thought by other
writers (Zwich and Weichel, 1910) to be highly improbable
in this instance.
(b) With regard to the second alternative, no preliminary
bacteriological examination of the mice used in the experi-
ment was made. The faeces of 40 control mice were examined
and B. Gaertner was discovered in one case only. Zwich and
Weichel (1910), on the other hand, found that out of 177
healthy mice 28 gave B. Aertryck in the faeces.
The bacteriological examination of the 40 control mice
with a negative result in 39 cases is, again, open to the
criticism that paratyphoid organisms might have been
actually present at the time but in such small numbers that
they escaped detection. Any such organisms present, in
equally small numbers, in the other mice would find a nidus
for their growth in the unhealthy and inflamed condition of
the intestine which would result from feeding the mice on
infected meat, while the disturbed functions of the bowel, by
CH. ix] OF TRANSMUTATION 135
hastening its evacuation, would bring about the subsequent
appearance of these organisms in the faeces.
C. The author's experiments.
The following experiments were suggested to the writer
by Professor F. A. Bainbridge as likely to throw some further
light on this aspect of the question and were carried out at the
Lister Institute under his kind supervision.
Experiment /. 24 healthy guineapigs were chosen and 12
of these were confined in 3 cages. On August 13th one of the
four guineapigs in each cage was removed and given in its
food 1 c.c. of a 48 hours' broth culture of B. enteritidis
Gaertner. Every precaution was taken to prevent, as far as
possible, any external contamination of the guineapigs with
the food and they were then returned to their respective
cages. This culture of B. Gaertner was made from a labo-
ratory strain the agglutination reactions of which had been
repeatedly tested and had been found to be constant.
A bacteriological examination of the faeces of the 12
guineapigs was made subsequently on three occasions, namely
on August 23rd, September 19th and October 9th bacilli of
the paratyphoid group being identified by agglutination tests.
During this period each cage was kept separate from the
others and none of the guineapigs were removed from their
cages except for the necessary examination on the three
dates mentioned. Up to the time of the first examination on
August 23rd all the guineapigs remained well, but subse-
quently three of them died and the remainder exhibited
varying degrees of malaise and intestinal disturbance. The
following scheme shows the type of organism found in the
faeces at each examination.
N.B. Guineapig No. 1 in each cage was given 1 c.c. of a
broth culture of B. enteritidis Gaertner on August 13th. " "
indicates that neither the Gaertner nor the Aertryck type of
organism was isolated.
The faeces of the remaining 12 guineapigs, which had
been kept quite apart from the others, were carefully investi-
136
SUPPOSED INSTANCES
[CH. IX
gated at the date of the first examination (August 23rd).
Gaertner's bacillus was not found in any case and Aertryck's
bacillus in one case only.
Cage
Guineapig
1st examination
August 23rd
2nd examination
Sept. 19th
3rd examination
Oct. 9th
A
No. 1
No. 2
No. 3
No. 4
Aertryck
Gaertner
Gaertner
Dead
Aertryck
Dead
P. M. Gaetner
Aertryck
Gaertner
Gaertner
B
No. 1
No. 2
No. 3
No. 4
Aertryck
Aertryck
Gaertner
Dead
(no faeces)
C
No. 1
No. 2
No. 3
No. 4
Gaertner
Aertryck
Aertryck and
Gaertner
Aertryck
Gaertner
(no faeces)
(no faeces)
These experiments appeared to lend further support to
the theory that the two types of organisms were capable of
transmutation. It is necessary, however, again to emphasise
the fact that a negative result in the case of all but one of the
animals examined as a control, does not prove the absence of
organisms it only proves their scarcity. A subsequent in-
crease in their numbers might at once have revealed their
presence. Such an increase might be apparent only, due to
a simple disturbance of the functions of the bowel, such as
diarrhoea, which would dislodge the organism from its usual
habitat, carry it to a lower part of the bowel and hasten its
evacuation. The increase in numbers might, on the other hand,
be a real one, brought about by a lowered vitality of the
body as a whole and local inflammatory changes. It is to such
factors that we attribute the enormous number of B. coli
found in the stools of patients suffering from cholera.
All these factors were, no doubt, operative in the case of
the three guineapigs which were actually fed with the culture
of B. Gaertner and may explain the subsequent discovery of
CH. ix] OF TRANSMUTATION 137
B. Aertryck in the faeces of all of them. The remaining
guineapigs may have been infected by B. Aertryck from the
faeces of these, at one stage or another, owing to their food
becoming contaminated. The same factors would explain, in
their case, the later appearance of B. Gaertner.
It is, however, to be noted that at the 1st examination
(August 23rd) of the guineapigs in cages A and B, while both
those which had been fed with the Gaertner culture were
passing B. Aertryck in their faeces, three of the six animals
which had not been fed with the Gaertner culture were
passing B. Gaertner. To explain this on the grounds that the
food of these three had been contaminated by the faeces of
the Gaertner fed guineapigs, we must assume that the latter
had, at some time previous to the examination, been also
passing B. Gaertner in their faeces and that this organism
had only later given place to B. Aertryck.
That the factors we have been discussing do actually lead
to the detection in the faeces of organisms which previously
did not appear to be present, the writer endeavoured to prove
by further experiment.
Experiment II. On August 25th six apparently healthy
guineapigs were chosen and labelled Nos. 1 to 6. A frag-
ment of the faeces from each guineapig was shaken up in
malachite green broth and after incubation the latter was
plated out, two sterile plates being used for each guineapig.
Both plates from No. 3 showed white colonies. A culture
from these colonies was grown in broth for 48 hours and then
passed through the sugars by which means it was identified
as B. proteus. In the case of the remainder the five pairs of
plates all proved to be sterile.
The guineapigs were then for several days fed on green
vegetables and bran soaked in castor oil a diet designedly
unwholesome and calculated to make the animals ill and also
to set up some intestinal catarrh. On August 30th a fragment
of faeces from each guineapig was again shaken up in
malachite green broth and this, after incubation, plated out
as before on two sterile plates.
In the case of No. 1 and No. 5 no growth was apparent in
138 SUPPOSED INSTANCES [CH. ix
the tubes and both pairs of plates remained sterile. In the
other four tubes growth took place with gas formation and
the four pairs of plates all showed colonies. Broth cultures
were made from these colonies and yielded strains which gave
the sugar reactions of B. proteus.
The writer failed to demonstrate the presence in any case of
organisms of the paratyphoid group. The experiment however
was successful in demonstrating that bacteria which failed to
give evidence of their presence in the faeces of a healthy
guineapig might make their appearance in the faeces of the
same animal after it had been given for a few days unwhole-
some and irritating food.
This conclusion lends weight to the suggestion already
made that the results obtained by Schmitt, and also by
Miihlens, Dahm and Fiirst, might possibly be explained by the
presence of a secondary invader.
Their experiments are, in both instances, open to one
further criticism. The identification of the paratyphoid or-
ganisms was made to depend solely upon their agglutination
reactions. If it is admitted that the power to form and to
absorb specific agglutinins on the part of an organism is
subject to variation it must be recognised that such tests
alone are insufficient to establish the identity of the organism.
In other words, it is within the bounds of possibility that only
one type of organism was actually present, but that its agglu-
tination properties varied.
Such a contingency would be likely to arise in the case of
two organisms so closely allied as B. Aertryck and B. Gaertner.
An elaborate investigation into the agglutination properties
of these two organisms was conducted by Sobernheim and
Seligmann (1910). Pure colonies of numerous strains were
secured by the Indian ink method. They found that colonies
derived from the same strain and growing side by side differed
in their agglutination reactions. The same strain differed at
different times. The agglutination reactions, in some instances,
became altered after passage through the mouse and after a
culture had been heated. In some instances the injection of
living bacilli yielded a serum which was much more variable
CH. ix] OF TRANSMUTATION 139
in its agglutinative powers than a serum obtained by means
of a dead culture. Some strains gave doubtful reactions. The
power of the same strain to form agglutinins and to bind
agglutinins appeared in some cases to differ. They therefore
concluded that the agglutination reactions did not constitute
a specific test.
We may interpret these results in one of two ways. We
may decline to recognise the two types as representing dis-
tinct species ; or we may continue to regard them as distinct
species and acknowledge that their agglutination properties
are liable to variation. In either case the experiments
quoted are deprived of all significance as examples of
transmutation.
CHAPTER X
SUMMARY
IT will be evident from the foregoing pages that practically
every character of bacteria is liable to vary at different times
and under different conditions. These variations are of two
kinds, spontaneous or "intrinsic" that is to say due to
tendencies inherent in the organism itself and impressed
as a result of external influences. These modifying influences
have been enumerated (Chapter II) and examples given of
the variations they produce.
In many cases an organism may appear to vary although
no variation actually takes place, and in other cases what
appears to be a "spontaneous" variation is actually an "im-
pressed" variation due to external influences which have not
been recognised by the observer. These various sources of
error have been enumerated and discussed in Chapter III.
A tendency to vary in a particular way either spontane-
ously or in response to external stimuli may be so charac-
teristic of a certain organism as to be in itself almost specific
in character, and so far from confusing its identity may
actually make this more apparent. The pleomorphism of
B. diphtheriae, the tendency of S. scarlatinae to assume
a bacillary shape, the tendency of B. paratyphoid B to form
papillae on raffinose agar, will serve as examples.
No single property of bacteria can be regarded as specific
nor does the occurrence of variation in respect to any one
quality or function, or to several of them simultaneously,
necessarily imply a loss of specific character on the part of the
organism concerned. This is well illustrated by the mor-
phology of bacteria. A certain appearance may be spoken
of as "characteristic." This does not mean that it is in-
variable but merely that the organism shows a tendency to
CH. x] SUMMARY 141
present such an appearance rather than another. B. coli in
the peritoneal cavity in the case of ascites may take the form of
a diplococcus; in milk or in urine it may develop into a
dense network of branching filaments resembling B.anihracis,
but these changes in form do not imply any obliteration of the
specific character of the organism itself.
We have already referred in this connection (vide p. 38)
to the analogy of a regiment of soldiers at manoeuvres and
a mass meeting of miners at the pithead. The various military
formations assumed by the first are as characteristic as the
concentrically arranged crowd formed by the second so much
so that an observer at a distance might from the appearance
of these "zoogleic forms" state with confidence the character
of the units composing them although too far away to identify
the latter. A crowd of pitmen on strike might, however, march
in military formation and a regiment of soldiers at a boxing
match take the form of a crowd concentrically arranged each
reproducing, that is to say, the appearance regarded as typical
of the other. This would not indicate that the pitmen were
changing into soldiers or the soldiers into pitmen. It is true,
nevertheless, that the arrangement most frequently observed
in one or the other case does indicate a tendency on the part
of the individual unit and may, therefore, afford a clue to
its identification. The behaviour of a civilian under certain
circumstances may furnish evidence of a military training and
deserters from the colours are not infrequently recognised by
such means. In a similar way, the occasional assumption by
the bacillus of diphtheria of clubbed and branched forms,
while helping us to identify it, also provides us with a clue to
its mycelial ancestry (Kanthack and Andrewes, 1905).
Many of the variations exhibited by bacteria do in fact,
represent steps in the evolutionary process by which, in
the past, they have become differentiated the individual
organisms living over again, as it were, the life history of the
race. This would appear to be the explanation of many
variations in morphology (Chapter IV). Others again repre-
sent the advance along new lines of this same evolutionary
process, leading to further specialisation and differentiation.
142 SUMMARY [OH. x
This aspect of the subject has been considered at length in
the sections dealing with Fermenting Power and Virulence
(Chapters Y and VI).
Since we have no absolute criterion as to what constitutes
a "species" amongst bacteria, dissimilarity in the several
characters they present is our sole guide to classification.
In other words the distinction between a "variety" and a
"species" depends simply on less or greater divergence in
character. The difference, therefore, between variation and
transmutation is one of degree only ; or, looking at the matter
from another standpoint, we may say that the same degree
of deviation from type may be interpreted in one case as
variation and in another as transmutation. This will be readily
understood if it be borne in mind that the various types or
"species" of bacteria which we are able to distinguish have
developed from a common stock. In the case of some of them
the differentiation dates from a remote past and the specific
characters are comparatively fixed. In the case of others
differentiation is of more recent date and the newly acquired
characters are less permanent and "reversion" in one or other
character is more frequent. In yet a third class the groups
of closely allied organisms the gradual process of differentia-
tion is only now taking place and it is not yet clear which
characters are of specific value. During the process of evolu-
tion, in all its stages, there is a tendency shown on the part
of the organism to revert towards the original type. Such
reversion in one or more characters, although of no greater
significance in the case of one class or another of the three
we have described, is likely to be differently interpreted. If
differentiation is well advanced, a partial reversion in character
will merely present itself as an unimportant variation. If
differentiation has not progressed very far, a reversion no
greater in degree may confuse the identity of the organism
concerned sufficiently to suggest the possibility that trans-
mutation has occurred. If the process of differentiation is
still incomplete, a reversion in character even smaller in degree
may entirely obliterate the faint lines of division that we have
been able to trace out. The error of assuming too hastily that
CH. x] SUMMARY 143
transmutation has occurred will be prevented by a proper
consideration of, firstly, the biological characters of the
organism in question as a whole and, secondly, the question
of the stability of the characters which distinguish it.
With regard to the first of these questions, we have shown
in the sections dealing with Morphology, Fermenting power,
Virulence and Pathogenesis (Chapters IV- VII) the danger of
relying upon any one of these characters alone for the purpose
of identification or of classification.
In the case of widely divergent types a single character
may sometimes suffice to distinguish one organism from
another but even in such a case, if that character is liable
under any circumstances to variation, it obviously cannot be
trusted as an infallible guide.
The pathologist is in the same boat, in this respect, with
the ethnologist. Certain "race groups," e.g. the Teutonic, the
Mongolian, and the Negroid, though conceivably derived from
a common anthropoid stock, are sufficiently differentiated to
be readily distinguished by a single character. For example,
the flaxen hair of the German, the matted black hair of the
Negro and the straight black hair of the Jap are sufficiently
characteristic of their respective race-groups. Such a dis-
tinction, however, breaks down between the races within
the groups themselves and other characters must then be
considered in addition. In some cases, again, the process of
differentiation is still incomplete, individuals approximating
now to one and now to another recognised type, and a con-
sideration of all the characters may still leave the observer
in doubt as to the correct classification.
The ethnologist has learnt, moreover, that certain char-
acteristics are not to be regarded as racial in character. For
example to return to our previous illustration the pigtail
of the Chinaman and the shaven poll of the Thibetan priest,
the flowing locks of an Italian impressario and the tonsured
crown of a Romish monk, are not racial characters at all but
artificial modifications. They do, however, signify a certain
environment and training and this is precisely the case with
many of the variations which the pathologist meets with
144 SUMMARY [CH. x
amongst bacteria. In other words, a study of such variations
in a given case may afford valuable and trustworthy informa-
tion as to the source from which the particular strain of
organisms has been derived.
This subject would repay further investigation. One
or two instances may be given here to demonstrate its im-
portance.
Rosenow (1912-13) found that the ordinary streptococcus
pyogenes, if grown in unheated milk, became modified in its
morphology, its cultural properties and its virulence. He had
previously isolated from several cases of epidemic sore throat
a streptococcus which possessed precisely similar modifications
in character. The epidemic had been recognised as "milk-
borne" but, had its origin been in doubt, the unusual char-
acters of the organism concerned would obviously have pro-
vided a clue.
Ohlmacher (1902) isolated branching filamentous forms of
B. coli from the heart's blood in a case of septicaemia. He
quotes various observations to the effect that residence in the
biliary passages develops this unusual morphology in B. coli,
and he therefore considers that the original source of the
systemic infection in this case was in the region of the gall
bladder or bile ducts.
Moreover the degree to which the modifications persist on
subculture is a measure of the time during which the organism
was subjected to the modifying influence. This brings one to
the second question, the stability of the variations produced.
Remarkable differences are to be observed in the degree
of permanence exhibited by a variation in different cases and
it is difficult to decide upon what factors these differences
depend.
We have already referred to the fact that variations may
be either "spontaneous" in character or "impressed" upon
the organism by external agencies. Spontaneous variations
may be of several kinds and the nature of the variation may
itself decide its degree of permanence.
1. Some variations represent an early stage in the life
history or are due to imperfect development, and are seen
CH. x] SUMMARY 145
in young or backward cultures. We have spoken of the atypical
morphology of a young culture of the Klebs-Loeffler bacillus,
which renders it difficult to distinguish it from Hofmann's
bacillus (vide p. 42), and of its inability to ferment glycerin and
lactose (vide p. 55). Such differences are comparable to the
juvenile features and unskilled hands of a class of schoolboys
and tend to disappear of their own accord as the strain grows
and develops.
2. Other variations represent senile changes or are due to
lowered vitality, and are seen in old or worn out strains. The
loss of motility, or of pigment production, in an old culture
will serve as an example. Such variations are comparable to
the slow steps and grey hairs that characterise a party of old
men and will tend to become more and more developed unless
some external influence intervenes and, by effecting a radical
change in the conditions of growth, contrives to rejuvenate
the strain.
3. Others again are degenerative in character or are due
to atavistic tendencies such as, for example, the appearance
of branched and clubbed forms of the tubercle bacillus. These
variations are comparable to some forms of mental impairment
in a family, or to defects such as harelip. They may be passed
on from father to son and so persist, or they may disappear,
but in the latter case they tend to recur in a later generation.
4. Others, finally, are evolutionary in character and repre-
sent a higher specialisation on the part of the organism such
as, for instance, the development by B. typhosm, after a long
training, of power to ferment lactose, or the acquisition on
the part of a feebly pathogenic organism of the quality of
extreme virulence. Such changes are analogous to the de-
velopment of a national genius for literature or conquest.
The more highly specialised a function is the more easily does
it become deranged and a character, therefore, of this kind, is
readily lost. For example, however permanent other newly
acquired characters in bacteria may appear to be, variation
in the direction of increased virulence seldom is so and almost
invariably proves unstable.
It is easy to see how, in every one of the four classes we
D. 10
146 SUMMARY [OH. x
have mentioned, two or more variations may be constantly
associated. In some cases the association is explained by the
fact that both variations are due to lowered vitality. For
example, the loss of power to produce pigment may be
associated with the loss of power to liquefy gelatin or to grow
on certain not very favourable media all these functions
being dependent upon the vitality of the organism.
Again, the evolution or higher specialisation of an organism
may involve simultaneous modification in two or more direc-
tions. These modifications may all represent a casting off of
saprophytic characters by the organism in question on its
entry upon a parasitic career. For example, a saprophyte
may derive its vital energy from the sunlight by means of a
pigment, comparable to the chlorophyll of a vegetable cell, or
from carbohydrate food through its ability to ferment it. When
it becomes parasitic, and in many cases pathogenic, it is cut
off from sunlight and must subsist on the body fluids. We may
find therefore that the acquirement of virulence is associated
with the loss of power to form pigment and to ferment sugars.
In the same way, the constant association between two
different variations may be due to the fact that the young
strains which show them have not developed their adult powers,
or to the fact that the variations are both signs of degeneracy
or atavism.
"Impressed" variations show even greater differences in
their degree of permanence. In some cases a variation is only
maintained while the influence which caused it continues to
act. In others the variation persists for a shorter or longer
period after that influence is withdrawn. In others again the
variation is apparently permanent and persists under normal
conditions of growth indefinitely. We use the expression
"apparently permanent" for it is impossible in any case to
guarantee the permanence of the characters exhibited by a
strain of bacteria. This has been shown both by observation
and by experiment. Mention has been made elsewhere (vide
p. 14) of a strain of bacteria which after nine years' cultivation
lost its power to ferment maltose, and of another strain which
after five years cultivation lost its power to produce pigment.
CH. x] SUMMARY 147
Twort found that B. typhosus grown in a lactose medium
retained its character as a non-fermenter of lactose for two
years before variation occurred. Eyre and Washbourn found
that to raise a particular strain of an avirulent saprophytic
pneumococcus to full virulence by animal passage, no less than
fifty-three successive inoculations were required. Characters
which persisted for periods of two, five and nine years, and
withstood a series of over fifty passages through an animal
body, might well have been regarded as "permanent." They
were, however, only "apparently" so.
Certain principles which govern the stability of impressed
variations can, however, be discerned.
1. The variation may affect all the members of a strain
or only certain of them. In the latter case an apparent
reversion is obviously more likely to occur. The rapidity with
which this apparent reversion takes place will depend upon
the comparative rate of growth of the unaltered organisms
and the variants. If the new character is of advantage to the
organism it will enable the variants to multiply more quickly
and they will gradually get the upper hand. Apparent re-
version will not take place as long as the new character
continues to confer an advantage upon its possessors but when
this ceases to be the case the organisms possessing the new
character may disappear and reversion to the original type
appear to take place. For example, the acquirement of viru-
lence by some members of a non-virulent or feebly-virulent
strain, when this is injected into the living body, gives these
variants an advantage as long as they are in the body. If the
mixed strain is grown on artificial media the advantage is
done away with and the unaltered bacteria, other things being
equal, have now as good a chance as the variants of increasing
their numbers and the variants may disappear.
We have used the expression " apparent reversion " for it
is evident that, unless every member of a strain acquires the
new character, the loss of that new character by the strain may
be brought about by the dying out of the variants without a
single organism having actually " reverted." This fallacy can
be readily excluded if care be taken at each step to ensure that
102
148 SUMMARY [CH. x
the strain of bacteria under observation is a pure one that
is to say, derived from a single organism by the methods sug-
gested by Barber and others.
2. The more readily a new character is "impressed " on an
organism the longer it is retained conversely, the more slowly
and reluctantly an organism takes on a new character the more
easily is that character lost. The behaviour of B. typhosus is
a good illustration. This organism can be trained to ferment
dulcite in a few days and will then retain the power for many
weeks in the absence of that sugar. It cannot be trained to
ferment lactose in less than two years and then loses the power
in a few days if the lactose is withdrawn.
It must be understood that we are here speaking of " im-
pressed " variations. In the case of " spontaneous " variations
the reverse holds true. Bacteria which vary spontaneously
with great readiness often revert with equal facility, while
those that are tenacious of their normal characters often
prove tenacious of any new character they may spontaneously
develop.
3. The longer an organism which has undergone a varia-
tion continues to be exposed to the influence which caused it,
the longer will the variation persist after that influence has
been withdrawn.
For example, Rosenow (1912-13) isolated a streptococcus,
from a number of cases of general infection, possessing unusual
morphological and cultural characters which, however, showed
reversion on cultivation outside the body. He found that
strains isolated from the peritoneal exudate and blood at a
later stage in the disease showed these modifications in char-
acter to a greater degree than those isolated earlier in the
attack.
The behaviour of B. typhosus again illustrates this point.
If a strain is grown on dulcite medium it acquires, in a few
days, the power of fermenting that sugar and this power is
retained for some weeks after the strain has been removed
from the dulcite medium. Reversion then occurs and the
power is lost. If however the strain is grown on a dulcite
medium continuously for three months the power to ferment
CH. x] SUMMARY 149
dulcite is found to persist afterwards, on ordinary media,
" permanently."
This observation, based on the results of laboratory experi-
ments, provides a clue, as Adami observes, to the nature of the
process by which new races of bacteria are developed. In the
laboratory organisms can be exposed to certain modifying
influences for many months or even years and the new char-
acters developed by such means are found to persist for long
periods before reversion takes place. In nature agencies which
possess the power of modifying the characters of bacteria may
exert their influence for an indefinite period and the process
of reversion in this case may be indefinitely postponed. In
other words the new characters developed may appear to be
permanent. A variant, however, may retain its new characters
indefinitely and show no tendency whatever to revert under
ordinary conditions of growth and yet it may still be capable
of reverting immediately under suitable conditions. Examples
of this are common in the laboratory and may be found in
nature. Laurent describes a decolourised strain of B. ruber
which was grown for 12 months at a temperature of 25-35 C.,
being subcultured 32 times in this period, without once show-
ing any trace of pigment. On lowering the temperature to 18C.
pigmentation at once reappeared. Again, the diphtheria ba-
cillus is far removed from its mycelial ancestry but under
suitable conditions will still display a partial reversion to a
mycelial structure. We do not on this account deny the title
of " species " to the diphtheria bacillus, for we recognise that
the idea of absolute permanence in character is not essential
to our conception of a species in the case of bacteria. It is not
permanence in character but the degree of resistance to al-
teration in character displayed by an organism that determines
our opinion of its specific nature. In spite of the many minor
variations they display there is exhibited by most species of
bacteria a resistance to modification a " vis inertia " which
constitutes true racial stability.
We have seen, then, that the difference between variation
and transmutation is one of degree alone. It is a question of
the extent of the modification and the degree of permanence
150 SUMMARY [CH. x
it exhibits. It is no less true that the process of transmutation
only differs in degree from the process of evolution. Here it
is a question of the rapidity of the change.
Let us take by way of illustration the case of a family of
ancient lineage, the members of which hold high office in the
State and are remarkable for their wealth and erudition.
Such a family may have sprung 500 years ago from humble
origin and, while the fortunes of one branch have steadily
prospered and successive generations have gradually acquired
fame and amassed wealth, the original yeoman stock from
which it sprang has continued to be represented throughout
the centuries, in some corner of the kingdom, by men chiefly
remarkable for their deficiency in the riches and learning and
reputation for which the others are distinguished. It is con-
ceivable that a son of the older and less distinguished branch
of the family, seizing a favourable opportunity, might, by the
exercise of the same faculties of industry and thrift displayed
by the others, raise himself in the space of a single life-time to
a position of wealth and power equal to theirs. We can trace
the steps by which, in the course of time, a virulent and highly
specialised race of bacteria has been evolved from a less
virulent and less highly organised race. We find the two races
living still side by side. The question arises whether it is pos-
sible under unusually favourable conditions for the process of
adaptation and specialisation to take place with such rapidity
as to suggest a sudden transmutation.
The conversion of the saprophytic pneumococcus into the
parasitic pneumococcus by Eyre, Leatham and Washburn
(vide p. 115) appears to offer an example. These observers
describe the virulent parasitic pneumococcus as requiring for
its growth a certain reaction and temperature and particular
media (blood agar); it would not grow if the reaction were
even faintly acid or at a temperature much below 37 C. and
rapidly died out on agar or in broth. It would not liquefy
gelatin and in broth formed a dust-like deposit. The avirulent
saprophytic variety, on the other hand, grew luxuriantly at
temperatures ranging from 37 to 20 C., on agar, gelatin,
potato or in broth, whether acid or alkaline, slowly liquefying
CH. x] SUMMARY 151
gelatin, producing a uniform turbidity in broth, and it retained
its vitality for many months ; it also exhibited differences in
its morphology, "instead of isolated diplococci and strepto-
cocci, large masses of cocci and diplococci were found, and
forms dividing into tetrads were common." Nevertheless this
avirulent saprophytic pneumococcus could, by a single " pas-
sage" through a rabbit, be converted into a typical parasitic
pneumococcus of high virulence. The occurrence of such a
remarkable transition would be regarded as more significant
if it were not that both organisms bear the same name and
are considered in spite of the many differences existing be-
tween them to be variants of each other.
If we consider the possibility of a similar transition in the
case of two races of bacteria less closely associated with each
other, we find little direct evidence in proof of its occurrence
and this often of doubtful value but a great deal of cir-
cumstantial evidence in favour of the supposition that it may
occur. We have discussed at length (Chapter VIII) such a
possibility in the case of organisms found in close association
in the body, such as Hofmann's bacillus and the Klebs-Loeffler
bacillus, Staphylococcus epidermidis and Staphylococcus pyo-
genes, Micrococcus catarrhalis and the meningococcus, and
others.
Finally, we have discussed in detail (Chapter IX) the re-
cords of certain experiments in the course of which bacteria
became so changed in character as to suggest that they had
undergone transmutation.
In the first series of experiments those of Major Hor-
rocks the results seem to be capable of explanation on other
grounds. In the first place adequate precautions do not appear
to have been taken to guarantee the purity of the strains at
different stages of the experiment. In the second place, many
of the changes in character stated to have been observed may
be regarded as examples of temporary variation only, similar
to those recorded by many other observers.
The second series of experiments those of Schmitt, and
of Miihlens, Dahm and FUrst, and of the writer which
suggest the occurrence of transmutation between different
152 SUMMARY [CH. x
members of the paratyphoid group of bacilli, are open to the
same criticism. In the first place, a temporary variation in one
character alone namely in agglutination properties would
sufficiently explain the results obtained. In the second place,
these results may have been due to a secondary invasion in
other words, it is conceivable that there may have been a pre-
existing but unrecognised infection in the animals utilised for
the experiments. This hypothesis we have shown, from the
records of other investigators and by analogy with other pro-
cesses of infection, to be not improbable ; while the writer's
experiments further demonstrate the ease with which such a
secondary invasion may be overlooked.
In none of these experiments, therefore, can the occurrence
of transmutation be regarded as proved, nor, on close ex-
amination, does its occurrence appear probable.
A theory which we propose to discuss in conclusion suggests
a via media by means of which organisms might conceivably
exchange many of their characters and functions without them-
selves undergoing transmutation. This is the Enzyme theory
of disease.
CHAPTER XI
THti ENZYME THEORY OP DISEASE
IT is impossible to leave this subject without some further
mention of a theory, to which passing reference has already
been made more than once in the foregoing pages, namely the
Enzyme theory of disease.
This theory predicates that the results which follow, and are
regarded as characteristic of, infection by a certain organism
including both the pathological lesions produced and the
train of symptoms observed clinically are caused not only
(if at all) by the activities of the micro-organism itself, but
by the activities of ultra microscopic bodies of the nature of
enzymes which are associated in each case with a particular
bacterial cell in the same way that the ferments of yeast are
associated with a particular vegetable cell.
If the scattered references to this theory in the foregoing
pages be collected together they will be found to constitute
a by no means negligible weight of evidence in favour of it.
The considerations which lend support to the theory are the
following.
1. In the first place, there is the observation that a sapro-
phy tic organism incapable at one time of giving rise to disease,
even after it has invaded the living tissues, may suddenly ac-
quire pathogenic powers and give rise in the living body to
definite lesions and a definite group of symptoms. Harmless
organisms such as the saprophytic pneumococcus, the Micro-
coccus catarrhalis and B. coli, for example, may mysteriously
acquire the power to produce respectively pneumonia, menin-
gitis and enteric fever. On the other hand, virulent pathogenic
organisms such as the Klebs-Loeffler bacillus, the meningo-
coccus and B. typhosus may as mysteriously become deprived
of their power to produce respectively diphtheria, meningitis
154 THE ENZYME THEORY OF DISEASE [CH. xi
and typhoid fever. Eyre and Washbourn (1899) showed in the
case of the pneumococcus that such an alteration in character
could be brought about, in one direction, by a single passage
through an animal and the reverse change with almost equal
facility. We have no explanation of the processes upon which
such changes in character depend but we know that many of
the conditions which bring them about are precisely those
which foster or destroy other properties in organisms which
we believe to depend on ferment action (vide infra).
2. In the second place, there is the observation that the
pathological lesions and clinical symptoms resulting from, and
characteristic of, infection by a certain organism may be faith-
fully reproduced as a result of infection by a totally different
organism. For example, we have noted (vide pp. 99 et seq.)
some of the lesions and symptoms of diphtheria to be caused
by the pneumococcus, those of scarlet fever and of influenza
by M. catarrhalis, those of cerebrospinal fever by the Klebs-
Loeffler bacillus, by M. catarrhalis and by B. typhosus, and
those of rabies by the Klebs-Loeffler bacillus.
The description of the last example given a case of
rabies due to infection by the bacillus of diphtheria will
bear repetition. It was recorded by Head and Wilson (1899).
The diagnosis of rabies was founded on the history and
clinical symptoms. " The well authenticated history of a bite
on the cheek by an animal, the two months' incubation
period, the onset with extreme pain and numbness in the
region of the scar, the development of the characteristic
laryngeal and respiratory spasms on attempting to take
liquids, the spasm at first being slight but later more pro-
nounced and towards the close again feeble or absent, the
insomnia, the absence in the beginning of fever which later
in the illness became pronounced, the rapid pulse at all
stages, the attacks of violent delirium interspersed with
periods of calm and complete rationality, the absence of all
symptoms pointing towards any other simulating disease and
the fatal termination all serve to make an almost complete
picture of rabies. " The Klebs-Loeffler bacillus was isolated
from the ventricular fluid and detected in the nerve cells of
CH. xi] THE ENZYME THEORY OF DISEASE 155
the medulla. The recognition of this organism was complete
and beyond doubt. "Not less suggestive of rabies than the
clinical history were the results of subdural inoculations in
rabbits with emulsions prepared from the medulla of the
patient. There occurred the long period of incubation (20
and 21 days) followed by phenomena similar to those in
experimental rabies of rabbits, and other rabbits inoculated
subdurally with the medulla of the first rabbits behaved in a
similar manner." B. diphtheriae was demonstrated after
death in the medulla of the rabbits. By a thorough investiga-
tion, full details of which are given, infection by the virus of
rabies was definitely excluded.
Such phenomena become intelligible on the supposition
that both the lesions and the symptoms of a disease result
from the activity of particular enzymes which are usually
associated with one particular organism but are capable of
being associated, under certain conditions, with an altogether
different organism.
3. In the third place, representatives of one specific
organism, morphologically and culturally indistinguishable
from one another, may give rise in the living body to entirely
different lesions and symptoms. Indeed, the contrast between
the train of lesions and symptoms produced in one case and
that produced in another may be as marked as the contrast
between the lesions and symptoms produced by two organisms
representing two distinct species.
For example, different epidemics of the same disease may
present altogether different features. Thus, strains of B.
influenzae, morphologically and culturally indistinguishable
from one another, may give rise to epidemics of " influenza "
characterised by symptoms resembling in one epidemic a
simple coryza, in another epidemic rheumatic fever, in a third
typhoid fever, and in a fourth cerebrospinal meningitis.
Not only do different epidemics present different types of
disease but individual cases occurring in the course of one
and the same epidemic, and undoubtedly due to infection by
the same organism, may exhibit a totally different train of
symptoms. We have mentioned elsewhere, the account given
156 THE ENZYME THEORY OF DISEASE [CH. xi
by Andre wes and Horder (1906) of a number of cases of
contagious disease, obviously passed on from one patient to
another, of which some presented the symptoms of scarlet
fever and others those of puerperal fever (vide p. 98).
Another remarkable instance (recorded by Dunn and
Gordon, 1905) has been already alluded to but is of sufficient
interest, in this connection, to warrant a second description.
They mention an epidemic in Hertfordshire characterised by
an extraordinary diversity of symptoms in different patients.
In some cases there were sneezing, coryza and the ordinary
symptoms of a common cold. In other cases patients com-
plained of aches and pains all over and stiff neck, and suffered
subsequently from great debility ; such cases had all the
appearance of influenza. In others, again, the illness closely
resembled scarlet fever ; it began with sore throat, rigors,
vomiting, headache, fever and rapid pulse, and was ac-
companied by a punctate rash at the end of the first 24 hours
(followed later by desquamation), the "strawberry" tongue,
circum-oral pallor, enlarged cervical glands which in some
cases suppurated, and in some patients by complications such
as nephritis, arthritis and otorrhoea. A fourth type resembled
diphtheria and exhibited a suspicious membrane on the
tonsil. A fifth type was notified in some cases as typhoid
fever and was characterised by epistaxis, melaena, prostration
and, in some cases, it is stated, a positive Widal reaction.
Finally, a number of cases, particularly amongst children,
resembled cerebrospinal fever and were so diagnosed ; these
were characterised by profuse nasal discharge, pain in the
back of the neck, headache, photophobia and irritability,
dilatation of one or both pupils, persistent vomiting, drowsiness,
head retraction, paralysis, coma, arid sometimes convulsions
and death.
Sometimes these widely divergent types were exhibited by
the different members of a single family or household struck
down by the disease, either simultaneously or consecutively.
After a thorough investigation, these observers were convinced
that the outbreak of these various types of illness was due to
the prevalence and spread of only one disease and not a
CH. xi] THE ENZYME THEORY OF DISEASE 157
number of different diseases, and a bacteriological examination
of a large number of cases by Gordon showed that the disease
was due to infection by an organism closely resembling, if
not identical with, M. catarrhalis.
Such contrasting groups of symptoms inevitably suggest
to our minds that something beside the mere presence of the
organism is responsible for them.
4. In the fourth place, one can trace a remarkable
resemblance between the conditions which influence the
development and the loss of pathogenic power on the part of
micro-organisms and the conditions which influence the
development and the loss of their power to ferment carbohy-
drates.
(a) The addition, in small quantities, of an antiseptic
such as carbolic acid to the culture medium deprives organ-
isms growing in it of virulence (vide p. 75). The same agency
will destroy the power of organisms to ferment carbohydrates
(vide p. 55).
(b) The influence of oxygen. Pasteur, 30 years ago, found
that the virulence of the organism of chicken cholera was
better maintained in the absence of oxygen. Anaerobic
growth similarly increases the virulence of the cholera spirillum
(Hueppe, quoted Adami, 1892). On the other hand, B. diph-
theriae and other organisms become less toxic if deprived
of oxygen. The same factor influences the activity of ferments.
In some cases the absence of oxygen inhibits their functions,
in other cases it appears to augment them. This is exemplified
by the sugar splitting ferments associated with bacteria. For
example, anaerobic growth may increase the power of the
dysentery bacillus to ferment maltose (Torrey, 1905). Andre wes
and Border (1906) mention a strain of streptococcus which
failed to ferment lactose under ordinary conditions but did
so readily when deprived of oxygen.
(c) Changes in temperature. It is characteristic of
enzymes that each one has an optimum temperature at which
its activities are most effective and also higher and lower
limits of temperature beyond which its activities altogether
cease. The digestive enzymes in man act most rapidly at the
158 THE ENZYME THEORY OF DISEASE [CH. xi
temperature of the human body those of cold blooded animals
at much lower temperatures. The diastatic ferment of germ
barley is most effective at 60 C. a temperature at which
most enzymes are destroyed. The phosphorescence sometimes
observed in sea- water is produced by the Micrococcus phos-
phorescens through the agency of an enzyme the optimum
temperature of which is that of the sea.
The enzymes which are associated with bacteria and bring
about the fermentation of carbohydrates show a similar
behaviour. We find that a strain of bacteria which will
ferment a certain " sugar " at one temperature will not do so
at another. For example, Wilson (1910) describes a strain of
B. typhosus which at 22 C. would ferment lactose within two
days but at 37 C. failed to do so in a month. Coplans (1909)
observed certain strains of B. coli which exhibited the reverse
phenomenon, fermenting dulcite more readily at 37 C. than
at 20 C.
The property of virulence in pathogenic bacteria is like-
wise governed by temperature. Organisms which are virulent
when growing at one temperature lose their virulence when
grown at another. For example, B. diphtheriae, B. tetani,
B. anthracis and many others (vide p. 74) lose their virulence
at temperatures much above that of the body. The fact that
no bacterial disease in cold blooded animals is communicable
to man may possibly be explained on such grounds.
Furthermore, just as the enzymes which ferment carbo-
hydrates are destroyed at temperatures much above 60 C., so
we find the property of virulence may be completely removed
by subjecting an organism to high temperatures, even though
the organism itself survives. The tetanus bacillus is deprived
altogether of toxicity by growth at 65 C. for one hour (Muir
and Ritchie).
(d) Exposure to sunlight is another factor which influences
both the fermenting power and the virulence of organisms.
(e) Finally symbiosis is not without influence. Diseased
conditions may result from a mixed infection which neither
of the organisms concerned is capable of producing alone. It
is said that a dog will not succumb to the infection of tetanus
CH. xi] THE ENZYME THEORY OF DISEASE 159
unless it is infected simultaneously with pyogenic cocci. In
the same way, processes of fermentation may be brought
about by two different organisms growing together in a
certain medium which neither can accomplish by itself. For
example, neither B. coli nor B. dentrificans alone can reduce
nitrates but if allowed to act upon sodium nitrate together
they bring about the escape of free nitrogen.
5. There is, furthermore, a remarkable correspondence
between the acquirement of virulence by "animal passage"
and the acquisition of fresh fermenting properties by prolonged
growth in a medium containing a particular sugar :
(a) The virulence acquired by "passage" through a certain
animal applies to that particular species of animal ; virulence
towards another species may be increased at the same time
but towards a third species it may actually be diminished. The
fresh fermenting power resulting from prolonged growth in a
sugar concerns that particular sugar ; the capacity of the
organism to ferment another sugar may be increased simul-
taneously while in respect to a third sugar the fermenting
power may be diminished.
(b) The method of "passage" is more effective in con-
ferring virulence if repeated inoculations are made through a
series of animals at short intervals. The prolonged growth in
a particular sugar is more successful in developing fermenting
power if repeated subcultures are made at frequent intervals
on to media containing the sugar.
(c) If virulence is readily acquired on "passage" it is
easily maintained and is found to persist for a long time on
artificial media ; on the other hand if it is very slowly
developed by "passage" it is quickly lost outside the body.
It is so, also, as regards fermenting power. In cases where
the property is rapidly developed, by x growth on a particular
sugar, it is retained for long periods on ordinary media ; on
the other hand, where it is very slowly acquired it is found
that a return to ordinary media is soon followed by reversion
in character.
(d) Where virulence has been lost only for a short time
by a strain of organisms it is quickly restored by " passage " ;
160 THE ENZYME THEORY OF DISEASE [CH. xi
the power to ferment a particular sugar, if it has only recently
failed, is rapidly regained in the presence of that sugar.
6. Bacterial toxins, again, are considered to be of two
kinds extra-cellular toxins, secreted by the bacterial cell
into the surrounding medium, and intra-cellular toxins elabor-
ated within the body of the cell and liberated only when the
cell itself is disintegrated. The same may be said of the
enzymes which ferment carbohydrates. The ferment of yeast,
"invertin," which transforms cane sugar into dextrose and
levulose, can be separated from the yeast cell. The breaking
up of the dextrose into alcohol and other products is a
property of the yeast cell itself and the ferment responsible
for this second stage can only be extracted when the actual
cell body is expressed (S. Martin, 1904). The ferments of the
alimentary canal may be distinguished from each other in the
same way. One stage in digestion is brought about in the
lumen of the intestine by extra-cellular ferments present in
the secretions. Another stage is effected within the actual
cells of the intestinal wall by intra-cellular ferments acting
upon the foodstuffs as they are absorbed.
An emulsion of even a small portion of a glandular organ
may possess far more power than its actual secretion, for the
former contains the intra-cellular as well as the extra- cellular
enzymes. An emulsion of pathogenic bacteria is likewise far
more potent than a culture containing the same number of
organisms. For example, the smallest fatal dose to a bovine
animal of a culture of tubercle bacilli contains 20,000 million
organisms. The smallest fatal dose of an emulsion of the
bacilli contains only 5000 (Report of English Tuberculosis
Commission).
7. In the seventh place, it may be observed that the
property of virulence is in many instances associated with
the power of producing fermentation. If we study two closely
allied organisms, one of them virulent and the other non-
virulent, the former will often be found to be the sugar
fermenter while the latter has no action in this respect.
For example, the Micrococcus catarrhalis is comparatively
non-virulent and ferments no sugars ; the gonococcus and
CH. xi] THE ENZYME THEORY OF DISEASE 161
meningococcus are virulent and ferment sugars. Again,
Hofmann's bacillus is non-virulent and non-fermenting while
the Klebs-Loeffler bacillus is virulent and ferments. B. coli
communis is a sugar fermenter and readily acquires virulence.
We may explain the association between the two properties
on the ground that both are examples of adaptation and that
an organism which possesses unusual power of adaptability
in one particular direction may be expected to show a similar
power of adaptability in another direction ; but the associa-
tion between virulence and fermenting power lends some
support to the supposition that the former may depend upon
a process which we have every reason to believe is responsible
for the latter, namely ferment action.
8. Bacterial invasion is met, on the part of the body, by
measures calculated to destroy the organisms and to counter-
act their toxins. These measures consist in the elaboration,
by the fixed cells of the body as well as by the leucocytes, of
various enzymes (Osier and McCrae). The class of weapon
forged by the tissue cells for purposes of defence might,
perhaps, be thought to give some indication as to the class of
weapon it is designed to meet.
9. Many other functions of bacteria, besides the fermen-
tation of carbohydrates, are attributed to ferment action ; for
example, the formation of indol, the coagulation of milk
(Savage, 1910), the liquefaction of gelatin, the production of
pigment (Adami) and the development of agglutinins (Du-
claux). Moreover these other functions of bacteria, like their
power of fermenting carbohydrates, appear to be governed in
many instances by the same conditions which we have already
mentioned as influencing their virulence. Thus, the presence
or absence of oxygen, high and low temperatures, exposure
to and protection from sunlight, the presence of antiseptics,
are all conditions which markedly aflect the production of
pigment by bacteria (Adami, 1892).
10. Many of these ferments are separable from the bac-
teria with which they are associated. Twenty-five years ago
it was proved (Bitter, 1887, quoted Wood) that the lique-
faction of gelatin by bacteria was due to a ferment which
D. 11
162 THE ENZYME THEORY OF DISEASE [CH. xi
was independent of the bacteria and survived when the
latter were killed by subjection to a temperature of 60C.
Sortinin (ibid.) showed that a culture fluid after it had
been passed through a Chamberland filter, which removed
all the bacteria, still retained the power to liquefy gelatin.
Brunton and McFadyean (1889) found that the gelatin lique-
fying ferment could be isolated by suitable solvents, in the
same way that the inverting ferment of yeast can be extracted
with ether.
11. Instances may be cited of chemical processes, taking
place in the body fluids, which are invariably associated with
the presence of certain micro-organisms but which neverthe-
less have been proved to be brought about by the activity
not of the organisms in question but of ferments associated
with these organisms and yet separable from them. Such an
instance is to be found in the action of the micrococcus ureae.
In the presence of this organism the urea of the urine is split
up with the formation of ammonium carbonate. In the
absence of this organism the process does not take place and
if the process has begun the removal of the organism at
once stops it. An ethereal extract, however, of the micrococcus
ureae has the power of accomplishing all that the presence of
the organism itself can effect in this direction. In other words,
the results brought about by its presence are due not to its
own activities but to those of a ferment " urase " which is in
some way associated with it but which can be dissociated
from it without any loss of function.
12. If it were possible to discover a parallel instance of
dissociation, not between an organism and its chemical func-
tions but between an organism and its pathological functions,
the discovery would give great weight to the theory we are
discussing. Now such a parallel can actually be traced in the
action of the pneumococcus. Rosenow (191213) has recently
shown that the artificial injection into the body of the toxins
manufactured by the pneumococcus may bring about the
death of an animal in one of two ways. It may produce an
acute bronchial spasm which proves fatal in a few hours. If
the dose of the toxin is small the bronchial spasm may not
CH. xi] THE ENZYME THEORY OF DISEASE 163
be sufficiently acute to cause death and other symptoms and
lesions follow which result in the death of the animal in a few
days. He discovered that the particular constituent of the
toxin responsible for this bronchial spasm could be removed
altogether from a suspension of the organism by the addition
of blood charcoal, so that the subsequent injection of the
filtered fluid failed to cause the bronchial spasm, although it
still produced the other symptoms and lesions and led to a
fatal termination in a few days. He, likewise, discovered that
the same constituent of the toxin could like the sugar-
splitting ferment of the yeast cell and the urea-splitting
ferment of M. ureae be extracted with ether and, further,
that if a normal saline solution, to which this ethereal extract
had been added, were injected into an animal, the typical
bronchial spasm was developed in the complete absence of
the organism itself.
The force of this analogy is somewhat weakened by the
knowledge that this acute bronchial spasm is by no means
pathognomic of the pneumococcus, many poisons producing
the same result on injection into an animal. The analogy is,
however, suggestive.
13. The dissociation brought about artificially in the
laboratory by this investigator may be observed to take
place naturally in response to certain kinds of environment.
Thus, a strain of pathogenic bacteria may lose its power to
produce a certain lesion or to cause a certain symptom in the
body. Further, the conditions which appear to deprive it of
such functions are comparable to those which, we have seen,
influence ferment activity. A few examples will suffice.
(a) The quality of light to which a culture of bacteria is
exposed may modify their power to produce pigment. Ex-
posure to the ultra-violet rays is found to alter profoundly
the lesions and symptoms caused by B. anthracis (Henri,
1914).
(b) The presence or absence of oxygen influences pigment
production and the fermentation of sugars by bacteria. Foa
^1890) isolated strains of pneumococci from the lung and from
the spinal fluid of a rabbit which had died after inoculation
112
164 THE ENZYME THEORY OF DISEASE [CH. xi
with this organism. The strain from the lung possessed the
property of causing, when inoculated into another rabbit, an
inflammatory oedema of the skin ; the strain from the spinal
fluid failed to do so. The strain from the lung, however, when
grown anaerobically was deprived of its power to cause this
inflammatory oedema of the skin.
(c) Growth in a certain vehicle may alter the fermenting
powers of one organism and the pathogenic powers of another.
The fermentation properties of a strain of B. coli isolated from
cowdung become altered after growth in milk. A milk-borne
epidemic of scarlet fever is not infrequently characterised by
the partial or complete absence of the usual rash.
(d) An analogy may also be traced between the action of
chemical substances added to culture media and the effects
of drugs administered in disease. For example, the presence
of sodium benzoate inhibits the power of B. coli to produce
gas from dextrose one of the most stable and fundamental
differences separating B. coli from the typhoid-dysentery
group without in any way affecting its other fermenting
reactions. The administration of sodium salicylate in rheu-
matic fever eliminates the symptoms of pain and fever
the two most characteristic symptoms of this disease with-
out apparently affecting any other of its symptoms and
lesions in a great many cases.
14. If the foregoing considerations suggest that the
symptoms of disease are due to zymotic action they likewise
imply that each separate symptom is attributable to the
activity of a distinct enzyme. Such a conclusion postulates
the existence of innumerable pathogenic enzymes each one
concerned in the causation of some particular symptom of
disease, and requires us to conceive of different groups or
combinations of enzymes associated with different pathogenic
organisms and responsible in the case of each organism for the
train of symptoms that follow its invasion of the living
tissues.
Analogy with the sugar-fermenting properties of bacteria
renders such a complex picture of the causation of disease
less fanciful than, at first sight, it appears. As we have shown
CH. xi] THE ENZYME THEORY OF DISEASE 165
(vide p. 60) it can be proved that not only is the fermentation
of different carbohydrates effected by distinct and appropriate
ferments but each of the several stages in the fermentation
of a single carbohydrate such as the formation of acids and
the production from these acids of gas is carried out by its
distinct and appropriate ferment. Moreover different carbo-
hydrates yield on fermentation different acids and each
different acid requires to be acted on by a special ferment
before it becomes split up into gaseous products. If such a
comparatively simple result as the production of acid and
gas in various carbohydrate media requires the co-operation
of so many distinct ferments, the extremely complex and
diverse results of the bacterial invasion of the body would
appear to demand proportionately greater complexity and
diversity in the zymotic agents causing them.
We have seen that the enzymes concerned with the fer-
mentation of particular carbohydrates are definitely associated
with certain vegetable and bacterial cells but not with others.
For example, many yeasts are able to invert sugar but only
three yeasts are known which are able to ferment lactose.
Proteolytic ferments are, likewise, associated only with
certain vegetable cells, such as the papain. Proteid-splitting
and lactose-splitting ferments are associated with certain
bacteria of the typhoid-coli group but not with others. It is
conceivable that, in precisely the same way, the agencies
responsible for certain definite symptoms in disease might be
definitely associated with some bacterial cells but not with
others.
A further suggestion occurs to one at this point. If the
enzyme responsible for one particular symptom of a disease
can be dissociated from the specific organism of that disease,
should we not expect to be able, by suitable methods, to
dissociate from the organism not one only but all the enzymes
causing the various symptoms of the disease in question ?
15. If such a complete dissociation were practicable it
should be possible to accomplish two things ; in the first
place, to deprive an organism of its power to produce a single
one of the symptoms of the disease associated with it and, iu
166 THE ENZYME THEORY OF DISEASE [CH. xi
the second place, to reproduce faithfully the complete train
of symptoms and lesions characteristic of a disease in the
entire absence of the specific organism to which the disease
is commonly attributed. Both these results have actually
been observed. As regards the first, numerous examples have
already been given of virulent organisms, normally capable of
giving rise to a complex and characteristic train of symptoms
and lesions in the living body (e.g. the Klebs-Loeffler bacillus,
B. typhosus} being deprived of their power to produce a
single one of these symptoms or lesions although, in every
other respect, retaining their character and properties un-
changed (vide " Virulence," " Pathogenesis ").
It is well recognised, for instance, that infection with B.
typhosus may occur without any of the clinical manifestations
of typhoid fever. Dudgeon (1908) quotes three cases of
patients whose stools contained enormous numbers of typhoid
bacilli and whose blood agglutinated these organisms in
dilutions of 1 in 200, who nevertheless failed to exhibit a
single symptom of typhoid fever.
With regard to the second, examples may be cited of
diseases associated with the presence of certain bacteria but
now generally recognised as being due to "filter passers."
Hog cholera, for instance, is a highly contagious disease
associated with a certain bacillus, the " hog cholera bacillus."
A pig suffering from the disease can infect other healthy
pigs ; the latter develop the same symptoms and are found to
be invaded by the same organism and they are capable, in their
turn, of infecting other healthy animals in precisely the same
way. It has been shown, however, that a broth culture of
the hog cholera bacillus, from an infected animal, after it has
been passed through a Chamberland filter a process which
entirely removes any bacilli present nevertheless retains its
power to "infect" a healthy animal with hog cholera, the
disease running the same course as usual and exhibiting
precisely the same lesions and symptoms.
Such a sequence affords a precise analogy to the ex-
periment of Sortinin, a quarter of a century ago, which led to
his discovery that after a culture of certain bacteria had been
CH. xi] THE ENZYME THEORY OF DISEASE 167
passed through a Chamberland filter, a bacteria-free filtrate
was obtained which nevertheless retained the power of the
original culture to liquefy gelatin.
16. One objection may be urged at this point, namely,
that it has not hitherto been possible to separate from
any pathogenic organism an enzyme capable of producing,
outside the living body, the toxins characteristic of that
organism. It is, however, equally impossible in many cases
to isolate from bacteria agents which will bring about
other of their functions which we recognise to depend on
ferment action. Moreover, we have discussed under the head
of virulence (vide p. 77) some of the qualities in which
artificial media differ from the vital fluids of the body and
such differences may well prove an insuperable obstacle to
the performance by an enzyme of its usual functions. A pick-
pocket may ply his "trade" vigorously in a busy crowded
thoroughfare and yet a few hours later, in a workhouse ward,
give no sign of his peculiar abilities. In the latter situation
certain things are lacking the incentive which normally
stimulates him (that is to say, the "struggle for existence"), the
materials he seeks to gain, the conditions essential to his
work and this fact may render difficult if not impossible
any display of his customary activities.
The enzyme theory of disease is not at the present stage
of our knowledge capable of proof. The above considerations,
however, lend some measure, if not of certainty at least of
probability to the supposition that the organisms associated
with certain diseases are not themselves the causal agents of
those diseases but merely act as carriers of ultra-microscopic
bodies, possibly parasitic in character, which have hitherto
eluded detection but which are the real causal agents of the
lesions and symptoms produced.
17. If such an hypothesis should ultimately prove to be
correct, how would it affect our ideas as to the possibility of
transmutation occurring amongst bacteria? Obviously, if it is
possible for the enzyme or enzymes which produce a certain
disease to become dissociated from the organism to which that
disease is commonly attributed and to become attached to some
168 THE ENZYME THEORY OF DISEASE [CH. xi
other organism, the effect, though not the actual process, of
transmutation would be brought about.
A transference of this kind would present certain diffi-
culties. The enzymes if such be their true nature of disease
would appear to depend, to some extent, for their activity
upon the structure and metabolism of the cell body to which
they are attached and if they are to be transferred from one
organism to another without loss of function the second host
must possess those characters in the way of structure and
metabolism which are vital to the activity of the enzymes.
This implies certain, and possibly rigid limitations. The pro-
blem can best be illustrated by analogy with more familiar
things.
We are able to distinguish at sea, a fleet of fishing smacks,
a line of battleships, a couple of pleasure steamers, a solitary
four masted barque in full sail. We distinguish these different
types of vessels readily from one another by characters analo-
gous to the " morphology " of bacteria, that is to say their
size, shape, motility and grouping. We have, however, another
way of distinguishing them, namely by observing the effects
produced by their arrival at a port, analogous to the effects
of bacterial " invasion.' 3 The arrival of the fleet of fishing
smacks is followed by a rush of people from their houses to
the shore (comparable to the exudation of leucocytes), a
silvery deposit on the quay-side as they empty their fish, re-
placed in a few hours by a brownish membrane as the nets are
spread out to dry. The train of " symptoms " is invariable and
becomes associated in our minds with the entry into port of
this type of vessel. So, too, with the others. The appearance
of gunboats may be followed by the destruction of a town
(comparable to necrosis). The arrival of the pleasure steamers
may be greeted with a display of fireworks (comparable to
pyrexia), that of the tall barque with its cargo of spirits may
give rise to general intoxication (comparable to delirium).
Such a sequence, however, is not invariable. For example,
the fishing smacks might be employed in smuggling and land
a cargo of spirits, giving rise to intoxication on shore. The
gunboats might be employed by Royalty on a pleasure cruise
CH. xi] THE ENZYME THEORY OF DISEASE 169
and their arrival be greeted with fireworks. A couple of in-
nocent looking steamers might be engaged in piracy and open
a destructive fire from their guns. The tall barque might con-
ceivably land a cargo of fish. In other words each type of
vessel might give rise to a train of events rightly regarded as
characteristic of an altogether different type, for the effects
they produce depend not on the activities of the ships them-
selves, which are merely carriers, but on those of their occu-
pants.
At the same time the function of each different type of
vessel, though dependent upon its occupants, is also to some
extent governed by its structure and the equipment it carries
(comparable to the structure and metabolism of a micro-
organism). A mere exchange of crews would not necessarily
effect an exchange of function. For example, a party of
fishermen sent to sea in an ironclad would be as unlikely to
land a catch of fish as a force of naval officers and seamen
embarked in fishing smacks would be to bombard a town.
In one respect our analogy fails. Hitherto we have spoken
of the enzyme as something grafted on to the micro-organism,
in the nature of a parasite, but there is much to suggest in
the evidence we have quoted that it is, in reality, a body ela-
borated by the organism itself, comparable to one of Ehrlich's
*' side-chains." Such a conception of its nature would go far
towards explaining the apparent dependence of the "enzymes"
of a particular disease upon a particular organism. But every
argument in favour of such a supposition in the case of the
enzymes which cause disease applies equally to our conception
of the nature of those which ferment carbohydrates. The pur-
pose of the arguments here presented has not been to explain
the precise nature of these ultra-microscopic bodies but merely
to show that the lesions and symptoms of disease may with
some confidence be attributed to the action of the same class
of body as that to which we unhesitatingly attribute the fer-
mentation of sugars.
115
CHAPTER XII
CONCLUSIONS
1. Variation occurs in every character of bacteria.
2. These variations may be either " spontaneous " or " im-
pressed " by conditions of environment.
3. The recognition of " species " amongst bacteria must,
therefore, depend upon a consideration of their biological
characters as a whole and upon the stability these characters
display.
4. Transmutation differs from variation in degree alone ;
it is a question of the extent of the modification and the de-
gree of permanence it exhibits.
5. Transmutation differs from evolution in degree alone ;
it is a question of the rapidity of the change.
6. The occurrence of transmutation between closely allied
organisms in the human body is not capable of proof but is
suggested by circumstantial evidence.
7. Supposed instances of transmutation, brought about by
experimental inoculation of animals, are shown to rest on in-
conclusive evidence.
8. The Enzyme theory of disease suggests a means by
which bacteria may exchange many of their characters and
functions without themselves undergoing transmutation.
APPENDIX
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p. 349.
"On the Variability of Bacteria and the Development of Races."
The Medical Chronicle, 1892, vol. xvi, p. 366.
ALLEN, R. W. "The Bacillus Influenzae and Symbiosis." Lancet, 1910, vol. i,
p. 1263.
"The Common Cold, its Pathology and Treatment." Lancet, 1908,
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ANDREWES, F. W. The Evolution of the Streptococci. "Horace Dobell"
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and GORDON, M. H. Report on the Biological Characters of the Staphy-
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APPENDIX 179
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